ANALYSIS OF URINE MLS 321

& OTHER BODILY FLUIDS 8438

LECTURE BS MLS 3 PRELIM
M1: RENAL ANATOMY AND PHYSIOLOGY

UNIT 1: RENAL ANATOMY

THE URINARY SYSTEM

● disposes metabolic waste
● regulate blood composition, pH, volume, and pressure
● maintains blood osmolarity
● produces hormones
● composition:
○ kidneys
○ ureter Cross-Section of The Kidney
○ urinary bladder ● superficial layer with granular
○ urethra appearance
● light red
● units of renal columns
Renal Cortex ○ a portion of the renal cortex that
extends into the renal medulla
○ divides medulla into renal pyramids
● location of the glomerulus
● exclusive site for plasma filtration
● middle area
Renal Medulla ● dark red inner layer
● units of renal pyramid
● comprised of renal pyramid, overlying
ANATOMY OF THE KIDNEY
Renal Lobe renal cortex and half of the adjacent
● retroperitoneal region
renal column
○ behind the peritoneum in the abdominal cavity
● reddish, bean-shaped organ
Renal ● functional portion of the kidney
● 135 g - 150 g
Parenchyma ● comprised of renal cortex and medulla
● 10 – 12 cm: length, 5 – 7 cm: wide, 3 cm: thick
● renal hilum
● tip/apex of the renal pyramids
○ indentation at the concave side ● drains urine into a the renal calyx
○ where the ureter, blood vessel, nerves emerges Renal Papilla
○ each papillae has a papillary duct that
opens to the minor calyces
Kidney: Three Layers of Tissue ● minor calyces
● dense irregular connective tissue ○ receives urine from the renal papillae
Renal Fascia ● superficial layer ● major calyces
● anchors the kidney Calyces ○ drains the urine to the funnel-shaped
● fatty layer renal pelvis
Adipose Capsule
● protects the kidney
■ narrows and joins the ureter to
● dense irregular tissue
facilitate excretion of urine
Renal Capsule ● innermost layer
● maintains the shape of the kidney

Transcribed by: Abenes, Aliping, Guarin, Villanueva (2024). 1

 THREE REGIONS OF THE NEPHRON
1. Renal Corpuscle
2. Renal Tubule
3. Collecting Ducts

1) Renal Corpuscle
NEPHRON ● blood is filtered by the glomerulus → initial low
● functional unit of the kidney molecular weight plasma ultrafiltrate is collected in
Bowman’s space → flows into the renal tubules
● 2.6 million in total
○ 1.3 million in each kidney ● network of fenestrated capillaries
Glomerulus ● filters blood
● produces the glomerular filtrate
Two Types of Nephrons
● thin epithelial layer
● location: renal cortex
○ surrounds the glomerulus
● 85% Bowman’s
Cortical ● where renal tubules originate
● major site for filtration Capsule
Nephron ● ⬆hydrostatic pressure allows filtrate to
● primarily removes waste and reabsorb
flow in the tubules
nutrients
● location: closer to the medulla
● 15%
Juxtamedullary
● primarily concentrates urine
Nephron
(⬆ osmolality)
● have a longer loop of Henle

Glomerular Filtration Barrier

● permeable to water and low-molecular weight solutes
○ excludes proteins larger than albumin
● product = plasma ultrafiltrate
○ same composition with blood plasma but is free of
blood cells and proteins
● mesangial cells (contractile cells)
○ contractility helps to regulate renal
Mesangium perfusion (blood flow) in the glomerulus
● capable of phagocytosis & pinocytosis
to remove entrapped macromolecules
● large open pores (50 to 100 nm)
● negatively-charged coating
Fenestrated
○ repels anionic molecules
Endothelium
■ solute selectivity during filtration
● e.g. protein (albumin), blood cells

Transcribed by: Abenes, Aliping, Guarin, Villanueva (2024). 2

 ● podocytes (foot-like structures) 2) Renal Tubule
● interdigitate with each other forming
filtration slit Renal Tubule: Proximal Tubules (Cortex)
● slits are lined with an extracellular structure
● major site for tubular reabsorption and secretion
called slit diaphragm
Visceral ● types
○ filters albumin from plasma
Epithelium ○ proximal convoluted tubules (PCT)
● capable of phagocytosis of trapped
○ straight proximal tubule
molecules
● capable of negativity ● simple cuboidal to low columnar
○ negatively charged glycoproteins cover epithelium
podocytes ○ interdigitate with each other to increase
surface area
● separates the epithelium of the urinary
space from the endothelium of glomerular
capillaries
Basement Proximal
● 3 layers: lamina rara, lamina densa, lamina
Membrane Convoluted
rara externa
● heparan sulfate Tubule (PCT)
○ acts as shield of negativity
● facilitate reabsorption
Microvilli
● ⬆surface area
Mitochondria ● for active transport

Renal Tubule: Loop Of Henle (Medulla)

● descends into the medulla
● forms the Distal tubules
● site for water exchange
● simple squamous epithelium
Thin ● flat & non-interdigitating
Descending
Limb

Thin
● simple squamous epithelium
Ascending
● tall, interdigitating
Limb
Juxtaglomerular Apparatus ● simple columnar to low columnar
● located in the vascular pole epithelium
● responsible for renin production ● tall & interdigitating
● composition Thick ● similar to PCT
○ afferent arteriole Ascending
■ major site for renin synthesis Limb
○ efferent arteriole
○ extraglomerular mesangial cells
○ macula densa
■ detects and responds to changes in glomerular
pressure Renal Tubule: Distal Tubule (Cortex)
■ in response, JC in the arterioles release renin ● predominantly simple cuboidal epithelium

NOTE: Distal
● function of Angiotensin II in urinary circulation: DACE Convoluted
○ Dilating the Afferent arteriole, Tubules
Constricting the Efferent arteriole (DCT)
● latter portion: lined with simple cuboidal
epithelium without interdigitations

Transcribed by: Abenes, Aliping, Guarin, Villanueva (2024). 3

3) Collecting Ducts BLOOD FLOW THROUGH THE KIDNEY
● site for final urine concentration
● fours parts
○ connecting tubule
○ collecting tubule
○ cortical collecting duct
○ medullary collecting duct
● simple cuboidal epithelium
● NO intercellular interdigitation

Collecting Duct: Cells

Polygonal Cells ● small and stubby microvilli
Principal Cells ● ADH and aldosterone receptor
● regulation of blood pH with ADH
Intercalated ○ ADH = spaces between the cells
Cells
dilate (⬆H2O permeability)

RENAL BLOOD CIRCULATION

Renal Artery
● originates from the aorta
Blood Flow: Hormones
● supplies the kidney with O2
● major role in regulating blood flow
○ only organ in which an arteriole subdivides into a
● secrete: juxtaglomerular cells in
capillary bed and becomes an arteriole again
response to:
● 20-25% of blood enters the kidney (from LV of heart)
Renin ○ ⬇ arterial pressure
● 1,200 mL/min ○ ⬇blood flow
Blood Flow Rate
○ per kidney = 600 mL/min ● renin → angiotensinogen → angiotensin
● 600 - 700 mL/min I (ACE; lungs) → angiotensin II
Plasma Flow Rate
○ 300 - 350 mL/min ● constrict the afferent arterioles
Angiotensin II ● dilate the efferent arterioles
Renal Arterioles ● trigger release: aldosterone & ADH
● Terminal; supply blood to specific areas and do not
interconnect. PATHWAY OF URINE FORMATION
● Size help create hydrostatic pressure & maintain 1. Glomerulus
consistency of glomerular capillary pressure and renal 2. Bowman's Capsule
blood flow 3. Proximal Convoluted Tubules
4. Descending Loop of Henle
Peritubular Capillaries 5. Ascending Loop of Henle
● cortical nephrons 6. Distal Convoluted Tubules
○ wrap tubules entirely 7. Collecting Ducts
● juxtamedullary nephrons divide into: 8. Renal Papillae
○ vasa recta (U-shaped) 9. Minor Calyx
■ maintain osmotic gradient (salt concentration) 10. Major Calyx
■ where major exchanges of water and salts 11. Renal Pelvis
tasks place between blood and the medullary 12. Ureter
interstation 13. Urinary Bladder
● proximity with renal tubules allows processing and 14. Urethra
exchange of solutes throughout the nephron 15. Urethral Orifice

Transcribed by: Abenes, Aliping, Guarin, Villanueva (2024). 4

 UNIT 2: RENAL PHYSIOLOGY Glomerular ● 90 - 120 mL/day
Filtration Rate
FUNCTIONS OF THE KIDNEY
Plasma Ultrafiltrate
● regulates homeostasis through regulating:
● free of blood cells and protein
○ acid base balance
○ except for some low molecular weight proteins that
○ electrolytes balance
can pass through the glomerulus
● excretion of waste products
■ e.g. β-2 microglobulin
● maintenance of blood pressure and erythropoiesis
● specific gravity: 1.010
URINE FORMATION ● temporarily stored in the Bowman’s capsule
● primary excretory function of the kidneys ● isosmotic/isotonic urine
● urine with an osmolality that is similar to osmolality of
● play an important role in the:
the initial ultrafiltrate (300 mOsm/Kg)
○ removal of metabolic waste products
○ regulation of water and electrolytes Filtered Plasma ● 180,000 mL /day
○ maintenance of the body’s acid base balance. Final Urine Volume ● 600 - 1800 mL/day
Urine Formation Process Factors Affecting Glomerular Filtration
● solutes in blood plasma → wall of the 1) Cellular Structure of the Glomerulus
A) Glomerular
glomerular capillaries → glomerular
Filtration ● glomerular filtration barrier
space
○ wall that consists of coils including:
● 99% of the water & essential solutes ■ capillary endothelium
B) Tubular are reabsorbed ■ basement membrane
Reabsorption ● returns via peritubular capillaries & ■ filtration diaphragms/slits between podocytes.
vasa recta ○ permeable to water and low molecular weight solutes
● waste products removed by renal ■ < 50,000 Da
C) Tubular tubular cells & duct cells ○ shield of negativity of the endothelium → solute
Secretion ○ drugs selectivity
○ excess ions ■ anions (–) are repelled
■ prevent loss of blood
Renin Angiotensin Aldosterone System (RAAS) ■ accumulation on barrier
● main hormonal system that is responsible for the 2) Filtration Pressure
regulation of renal Na+ excretion ● net pressure that forces substances out of glomerulus
● stimuli = ⬇plasma sodium = ⬇water retention = ○ sum of forces conferred by vascular arrangement,
⬇overall blood pressure fluid and protein in the renal corpuscle
○ ⬇BP is detected by macula densa → ● net filtration pressure = 10 mmHg
Juxtaglomerular cells respond by secreting renin ○ GBHP - CHP - BCOP
○ 55 mmHg - 15 mmHg - 30 mmHg
○ pressure favorable for plasma ultrafiltrate formation
● < GBHP = no urine formation (anuria, oliguria)
● pressure need to force the ultrafiltrate
to the glomerulus
● due to the reduction of the size (lumen)
of efferent arterioles and glomerular
capillaries
Glomerular
● this process continues until the blood pressure is ● forces fluid → Bowman’s space
Blood
corrected and returned to normal ○ pressure of 55 mmHg
Hydrostatic
○ smaller the size, the greater
Pressure
A) GLOMERULAR FILTRATION glomerular hydrostatic pressure
(GBHP)
● glomerulus forms a barrier that specifically allows ● necessary to maintain consistency of
plasma ultrafiltration glomerular capillary pressure and renal
● permeable to water & low-molecular weight solutes blood flow
○ excludes proteins larger than albumin ● opposes capsular hydrostatic pressure
● product = glomerular filtrate / plasma ultrafiltrate and oncotic pressure

Transcribed by: Abenes, Aliping, Guarin, Villanueva (2024). 5

 ● exerted by fluid in the Bowman’s space Types of Tubular Transport
● opposes filtration by 15 mmHg ● requires NO energy
○ represents the resistance of the Passive ● movement of solutes along a concentration
capsule wall and the fluid present in Transport gradient
Capsular the renal tubule ○ high → low solute conc.
Hydrostatic ● water tends to reenter the plasma from ● requires energy
Pressure the Bowman’s space creating a ● movement of solute against the concentration
(CHP) pressure opposite to the direction of gradient
filtration ○ low → high solute conc.
○ rationale: principle of osmosis;
ultrafiltrate has significantly lower ● one substance is
Direct Active
independent of transport
protein content and higher water Active Transport
from another substance
Blood Colloid Transport
● transport of one substance
Osmotic ● exerted by unfiltered proteins in the facilitated by the transport
Pressure Indirect
glomerular capillaries of another
(BCOP) or Active
● opposes filtration by 30 mmHg ○ e.g. glucose reabsorption
Oncotic Transport
● coupled with another
Pressure substance

Proximal Convoluted Tubules (PCT)

● performs majority of the reabsorption (80%)
● Na & Cl = partly reabsorbed
● glucose & proteins = completely reabsorbed
Maximum Tubular Reabsorptive Capacity (Tm)
● ⬆number of solute = ⬆ rate of reabsorption
○ continues to increase until maximum rate of
reabsorption is reached
● expressed in mg/min
● remains constant once Tm is reached
○ any excess amount of solute in the tubule will be
3) Feedback Mechanisms excreted in the urine
● Renin-Angiotensin-Aldosterone System (RAAS) ● solutes that exhibit Tm
● DACE (dilates afferent; constricts efferent) when BP drops ○ glucose ○ phosphates
○ prevent a marked decrease in blood flowing ○ amino acids ○ sulfates
through the kidney ○ proteins ○ uric acid
○ preventing an increase in the blood level of toxic ● Na: not refined by Tm
waste products Renal Threshold
● ⬆BP constricts afferent arterioles ● concentration of solute in plasma at which Tm is
○ prevents overfiltration or glomerular damage exceeded
○ excess amount of solute is excreted in urine
B) TUBULAR REABSORPTION ● e.g. glucose
● occurs in the tubules and demonstrates its ability to ○ renal threshold = 160 - 180 mg/dL
adjust the excretion of water and solutes accordingly ■ >160 mg/dL = detected in urine
● occur simultaneously with tubular secretion ○ Tm = 350 mg/min
● majority of the plasma ultrafiltrate are reabsorbed by the ○ note: glucose is NOT normally found in the urine
peritubular capillaries ● some solutes (e.g. Na) are not influenced by amount
○ surrounds the tubules for reabsorption reabsorbed, but influenced by:
● ability to adjust the excretion of H2O and solutes ○ blood flow rate
○ Na, amino acids, glucose ○ contact time with renal epithelium
● allows substances to return to circulation ○ concentration of other solutes
● differs in terms of the direction at which the substance is ○ transport inhibitors
actively or passively transported ○ hormone levels

Transcribed by: Abenes, Aliping, Guarin, Villanueva (2024). 6

Tubular Reabsorption in Various Nephron Segments Summary of Solutes Reabsorbed and
● fluid exiting the PCT has the same osmolality with the Secreted by The Renal Tubules
initial plasma ultrafiltrate Location Reabsorbed Secreted
Passive:
○ absolute number of solute per kg of water is also ● Water
the same ● Cl–
○ differs only on the type of solute particles ● K+
■ less Na, more Cl ● HCO3–
● Urea ● H+
● osmolality eventually changes as more water and
Active: ● NH3
solutes are reabsorbed ● Na+ ● Urea
○ e.g. fluid that leaves the loop of Henle has lower PCT
● Glucose ● Creatinine
osmolality (hypotonic) than the initial plasma filtrate ● Amino Acids ● Weak Acids
due to loss of Na and Cl ● Proteins ● Weak Bases
● Phosphate
● water is reabsorbed throughout the nephron via
● Sulfate
osmosis or with the help of the tubular epithelium ● Mg2+
● loop of Henle ● Ca2+
○ majority of the solute and water exchange occurs ● Uric Acid
● ascending loop of Henle Passive:
Descending LH ● Water ● Urea
○ selectively impermeable to water ● Urea
○ barrier that prevents diffusion of water into the Passive:
interstitium ● Na+
Thin Ascending LH ● Urea
○ but cell membrane still semipermeable and allows ● Cl–
secretion of ultrafiltrate ● Urea
Passive:
● collecting ducts ● Urea
○ under the influence of ADH, as water leaves the Thick Ascending LH Active: –
CD, the filtrate becomes more concentrated with ● Na+
the solutes left behind = hyperosmotic/hypertonic ● Cl–
Passive:
● Water
Active:
● Na+
Early DCT –
● Cl–
● Ca2+
● Sulfate
● Uric Acid
Passive:
● Water
● H+
Active:
● NH3
● Na+
Late DCT – ● K+
● Cl
● Uric Acid
● Ca2+
● Some drugs
● Sulfate
● Uric Acid
Passive:
● H+
● Water
● NH3
Osmolality Gradient Cortical CD ● Cl–
● K+
● induces the diffusion of water to reach osmotic Active:
● Some drugs
● Na+
equilibrium
● H+
● increasing osmotic gradient = water → medullary Passive:
● NH3
Medullary CD ● Water
interstitium via osmosis → medulla ● K+
● Urea
● water cannot reenter ascending limb since it is ● Some drugs
impermeable to water
○ allows kidneys to conserve water NOTE: Urea
○ reabsorbs substances and eliminate wastes ● considered as salt of urine
regardless of body hydration ● highest component in urine

Transcribed by: Abenes, Aliping, Guarin, Villanueva (2024). 7

 C) TUBULAR SECRETION Secretory Mechanisms Maintaining Blood pH
● simultaneously happens throughout the nephron ● secretion of H+ ions in the proximal
○ primarily in the PCT tubular lumen
● movement of substances from the blood through the ○ reacts with HCO3– (passes through
peritubular capillaries of the tubular lumen is excreted glomerulus) → H2CO3 (formed in tubular
● maintains blood pH lumen)
● elimination of metabolic wastes and other substances ○ H2CO3→ carbonic anhydrase→ CO2 +
not normally found in plasma H2O→ HCO3– + H+
○ passage of substances from peritubular ○ H2CO3 in tubular cells → diffuses
capillaries→ tubular filtrate interstitial fluid & peritubular capillaries
● exogenous substances that are metabolized ■ resupplies blood-buffer system
incompletely, not at all, or not normally present in
plasma cannot readily pass through GFR
Bicarbonate
○ eventually released in tubular lumen for excretion
Recovery
Solutes Example
Metabolized Incompletely ● Thiamine
● Radiographic Contrast Media
Not Metabolized
● Mannitol
Not Normally Present ● Penicillin
Maximal Tubular
● Hippuric acid
Secretory Capacity
Not limited in the
● Potassium
Amount Secreted

● most metabolic waste products are weak acids & bases ● neutral phosphate salts present in the
● normal blood pH: 7.35 - 7.45 tubular lumen
○ constantly affected by endogenous products of ○ neutral phosphate salts
acids from normal dietary metabolism ■ from rapid conversion of phosphoric
● source endogenous acids: acid from diet source in blood
○ production of CO2 ■ transported to kidney
○ catabolism of dietary proteins & phospholipids ○ phosphate salts + H+ = NaH2PO4
○ uncontrollable DM (pathologic) ■ titratable acids & excreted in urine
○ lactic acid w/ exercise (physiologic) ○ directly removes acids from the body
● pH is maintained by: while reabsorbing Na and HCO3– via
○ blood buffering system peritubular capillaries
○ respiratory system Excretion
○ kidneys via
■ restores pH balance by secreting hydrogen ions Titratable
& ammonia Acids
■ alkalosis: retention of h+
■ acidosis: secretion of H+

Transcribed by: Abenes, Aliping, Guarin, Villanueva (2024). 8

 ● ammonia (NH3) secretion and COUNTERCURRENT EXCHANGE MECHANISM
subsequent exchange of Na for ● passively occurs in the vasa recta
ammonium ions (NH4+) ○ diffusion between renal medulla & vasa recta
○ glutamine (from peritubular capillary ● blood osmolality increases as it flow deeper in the renal
blood) → glutaminase → NH3 medulla up to the papillary tips
■ NH3 not ionized & lipid soluble = ○ blood progressively becomes diluted when it
readily diffuses through cell ascends towards the renal cortex
membrane of tubular lumen ● main purpose = provides nutrients to the medulla and
○ NH3 + H+ → NH4+ remove water from the interstitium and distribute it to
■ NH4+ cannot diffuse to tubular cells = the rest of the body
Excretion excreted in urine ○ maintains tonicity of the medullary interstitium
via
Ammonium
Salt
Formation

RENAL CONCENTRATING MECHANISM

● the osmotic gradient of the renal medulla makes it the
only tissue in the human body that is hypertonic with
respect to normal plasma
● maintenance of osmotic gradient is essential for the
final concentration of the filtrate when it reached the CD
○ maintained by two counter current mechanism
● countercurrent = the loop of Henle and vasa recta
parallel to each other with fluids flowing in the
ascending & descending segments in opposite direction

COUNTERCURRENT MULTIPLIER MECHANISM

● actively occurs in the loop of Henle
○ solutes not reabsorbed (except urea)
● development of a gradient of hypertonicity
○ descending limb = osmolality between interstitium
and fluid will eventually become equal
○ ascending limb = Na & Cl actively reabsorbed into
medullary interstitium → tubular tonicity decreases
○ the descending limb fluid & interstitium becomes
more concentrated
● “multiplied” = descending limb
○ tubular fluid concentrates due to water being
passively absorbed in the descending limb →
osmotic gradient within the tubule from the cortex
has been multiplied
● “diluting” = ascending limb
○ tubular fluid is diluted due Na and Cl actively
reabsorbed into medullary interstitium
● end product = fluid slightly hypotonic than the fluid
before entering the loop of Henle
● main purpose = maintain the gradient hypertonicity
of the medullary interstitium and not the tubules

Transcribed by: Abenes, Aliping, Guarin, Villanueva (2024). 9

Final Urine Summary of Values
● produced via concentration and dilution of tubular fluid ● 1,200 mL/min
Blood Flow Rate
in the collecting tubules ○ per kidney = 600 mL/min
○ hormonally controlled by RAAS and ADH secretion ● 600 - 700 mL/min
Plasma Flow Rate
● dilute filtrate in CT + higher osmotic con. of medullary ○ 300 - 350 mL/min
interstitium = passive reabsorption of water Glomerular Filtration Rate ● 90 - 120 mL/day
○ controlled by ADH Filtered Plasma ● 180,000 mL /day
● 1,200 – 1,500 mL daily urine output Final Urine Volume ● 600 - 1800 mL/day
○ ave. 1mL/min excreted as urine Glucose Renal Threshold ● 160 - 180 mg/dL
Glucose Tm ● 350 mg/min
Antidiuretic Hormone (ADH) Net Filtration Pressure ● 10 mmHg
● renders the DCT & CT permeable or impermeable to GBHP ● 5 mmHg
water CHP ● 15 mmHg
○ ⬆ ADH = ⬆ reabsorption of water = ⬇ con. urine BCOP ● 30 mmHg
○ absence of ADH = ⬆ dilute urine Daily Urine Output ● 1,200 – 1,500 mL
● production is determined by state of body hydration Polyuria ● > 2,500 mL/day
○ ⬆ body hydration = ⬇ ADH = ⬆ urine volume Oliguria ● < 400 mL/day
○ ⬇ body hydration = ⬆ ADH = ⬇ urine volume Anuria ● < 100 mL/day (2-3 days)
Isosmotic / Isotonic Urine
● 300 mOsm/Kg
ASSOCIATED DISEASE (initial ultrafiltrate osmolality)
SG of Plasma Ultrafiltrate ● 1.010
Urinary Disorders
● increased urine output
○ > 2,500 mL/day
● px in under controlled hydration
● associated cause:
○ increased water intake
Polyuria
○ diabetes mellitus or insipidus
● secondary cause:
○ hypernatremia,
○ hypertension
○ diuretics
● decreased urine output
○ < 400 mL/day
Oliguria ● associated conditions:
○ tubular obstruction
○ infection (pyelonephritis, UTI)
● absence of urine output
○ < 100 mL/day for 2-3 consecutive days
despite a normal hydration
Anuria
● associated conditions:
○ tubular obstruction,
○ hypotension (⬇ blood flow)
● increased urine output at night
Nocturia ● urinating at night twice or more than 2
occurrences
● associated with polyuria
● unchanging SG (or osmolality)
regardless of hydration
● implies significant renal tubular
Isosthenuria
dysfunction
○ a feature of end-stage renal disease
● associated with polyuria and nocturia

Transcribed by: Abenes, Aliping, Guarin, Villanueva (2024). 10

 UNIT 3: RENAL FUNCTION TESTS Other ● hormones, vitamins,
–
Substances medications
URINE COMPOSITION ● cells, casts, crystals,
Formed
● volume and composition depends on diet, physical – mucus and bacteria (not
Elements
part of the ultrafiltrate)
activity, and health
● substances excreted reflect how well the kidneys are
ASSESSMENT OF RENAL FUNCTION
able to alter the excretion of water & solutes effectively
I. Glomerular Filtration Tests
II. Tubular Reabsorption Tests
Urine Composition III. Tubular Secretion Tests
Composition Amount Remarks
Organic
● 60–90% nitrogenous
material
Urea 25.0 – 35.0 g ● derived from metabolism
of amino acids into
ammonia
● derived from creatine
Creatinine 1.5 g ○ nitrogenous substance
in muscle tissue
● common component of
kidney stones
Uric Acid 0.4 – 1.0 g
● derived from catabolism
of nucleic acid
● benzoic acid is eliminated
from the body in this form
Hippuric Acid 0.7 g
● increases with high
vegetable diet
● CHO, pigments, fatty
acids, mucin, enzymes,
Other and hormones
2.9 g
Substances ● may be present in small
amounts depending on
diet and health
Inorganic
Sodium
● principal salt
Chloride 15.0 g
● varies with intake
(NaCl) Renal Blood Flow
● occurs as chloride,
Potassium ● necessary for kidney to perform its function
3.3 g sulfate, and phosphate
(K+) ● changes in renal blood flow (RBF) affects all functions
salts
Sulfate (SO4-2) 2.5 g ● derived from amino acid of the kidneys
● occurs primarily as Na
Phosphate compounds Renal Plasma Flow (RPF)
2.5 g
(PO4-3) ○ serves as buffers in ● portion of plasma that is filtered by the glomerulus
blood ● determines the amount of plasma ultrafiltrate that is
● derived from protein processed by the nephrons
metabolism from
Ammonium ● directly affects the volume and composition of the urine
+ 0.7 g glutamine
(NH4 ) excreted
● amount varies depending
on acid-base balance ○ e.g. ⬇ renal plasma flow = ⬇ urine excreted
● occurs as chloride,
Magnesium
+2 0.1 g sulfate and phosphate
(Mg )
salts
● occurs as chloride,
Calcium
0.3 g sulfate and phosphate
(Ca+2)
salts

Transcribed by: Abenes, Aliping, Guarin, Villanueva (2024). 11

 I. GLOMERULAR FILTRATION TESTS UREA CLEARANCE TEST
● “clearance tests” ● urea
● measures a substance that can readily pass ○ an endogenous substance produced by the
through the glomerular filtration barrier but will breakdown of proteins
● the earliest test for GFR
neither be reabsorbed or excreted
○ since it’s present in all urine specimen
● can be used to determine extent of nephron damage ● disadvantage:
● tests required prior to administration of certain ○ 40% of urea is reabsorbed by renal tubules
medications ■ thus, normal values were adjusted to reflect
○ i.e. those toxic if not eliminated by the body for a reabsorption
certain period of time ○ directly affected by urine flow rate
■ urine flow rate must be stabilized by 2 mL/min
■ the slower the urine flows (<2 mL/min) the more
GFR: Terminologies
urea is reabsorbed
● ability of the kidney to remove substances ○ diet affects plasma urea levels
from the blood ■ it has constant rate of production but it is
○ substances removed exclusively by the affected by protein intake
glomerulus, can be used in the
Renal
determination of GFR
Clearance CREATININE CLEARANCE TEST
● volume of plasma in ‘mm’ from which a
substance is completely removed/cleared Creatinine
in the kidneys per unit time ● an endogenous substance formed from creatine and
● most common test for glomerular filtration
phosphocreatine as a by-product of muscle metabolism
● measures the ability of the kidneys to
○ waste product; has high concentration in urine
Renal remove a filterable substance from the
blood ● substance of choice for clearance test
Clearance
Test ● standard tests used to measure the ○ produced at a constant rate
filtering capacity of glomeruli ■ thus, constant plasma concentration and urine
Clearance of ● amount of substance removed from the excretion rate
a Substance blood and excreted in the urine ■ not affected by urine flow rate
○ freely filtered
NOTE: ■ it is not bound to plasma proteins
● plasma components that are not filtered ○ not reabsorbed by the renal tubules
○ does not pass through the tubular lumen ● affected by
○ flows in the peritubular capillaries ○ muscle mass
■ where renal tubular cells actively and ■ creatinine clearance values are normalized to
selectively remove solutes for excretion the external body surface area of an average
individual which is 1.73 m2
GFT: Substance of Choice ■ normalization enables the comparison of
1. Neither reabsorbed nor secreted in the tubules. clearance results with those of other individuals
2. Excreted solely by glomerular filtration. regardless of the patient’s body surface area
3. Stable for 24 hours. ○ heavy meat diet
4. Constant plasma level. ● disadvantages
5. Readily available. ○ drugs block tubular secretion = false ⬇
6. Availability of tests both in urine and plasma. ■ cimetidine
■ cephalosporin
Types of Clearance Tests ■ salicylates
Endogenous Exogenous ■ gentamicin
● urea ○ chromogens interfere with picrate method
● creatinine ● inulin ○ prone to bacterial decomposition = false ⬇
● beta-2 microglobulin ● radionucleotides ■ must be preserved
● cystatin C ○ muscle wasting disease = false ⬆
○ diminished renal function = false ⬆
■ increased tubular secretion of creatinine as a
result of elevated plasma creatinine

Transcribed by: Abenes, Aliping, Guarin, Villanueva (2024). 12

Creatinine Clearance Test NOTE:
● routine screening method for GFR ● renal diseases are often detected when there is already
○ addresses the disadvantages associated with extensive nephron damage due to the ability of
exogenous agents while arriving at a measure functional nephrons to compensate for the damaged
comparable to inulin ones
○ assesses the extent of nephron damage
● for monitoring GFR diagnosed with renal disease ● ⬇ Cr = kidneys not removing enough Cr
○ for management of disease ● ⬆ Cr = could be good since Cr is a waste product
○ for prognosis (e.g. treatment response) ○ but could also imply damage to GFB
● NOT performed for early detection of renal disease
○ since GFR is diagnostic when nephron damage is
50-60% Example 1: Normalized CrCl
● key advantage of creatinine clearance is that plasma Given:
and urine creatinine can easily be performed C = ? SA = ?
● disadvantages U = 154 mg/dL W = 60 kg
○ corrected for body surface area P = 1.6 mg/dL H = 160 cm
■ normalize creatinine clearance values to V = 1440 mL (24hr urine)
external body surface area of the body due to
the dependence of creatinine to muscle mass
■ reference values is the same regardless of the
muscle mass since it was normalized
○ not for patients with muscle wasting disease
● specimen
○ 24-hr urine sample
■ due to diurnal variation in the GF of creatinine
○ blood
■ recommended to be collected midway the
urine collection
NOTE:
Creatinine Clearance: Computation
● the glomerulus has to filter 102.79 mL/min to arrive at a
Normalized Creatinine Clearance urine creatinine of 154 mg/dL

Example 2: Normalized CrCl

Given:
C (mL/min) = creatinine clearance
U (mg/dL) = urine concentration of creatinine C = ? SA = ?
P (mg/dL) = plasma concentration of creatinine U = 140 mg/dL W = 50000 g
V (mL/min) = volume of urine used in collection P = 1.2 mg/dL H = 180 cm
1.73 m2 = average body surface area V = 1200 mL (24hr urine)
SA = average body surface area of patient

W = weight in kg
H = height in cm

Reference Value: Creatinine Clearance

Males ● 107 – 139 mL/min
Females ● 87 – 107 mL/min

Steps In Computing
Step 1 ● compute for logSA
Step 2 ● determine antilog of logSA
Step 3 ● convert 24-hour to min for volume
Step 4 ● substitute values to formula

Transcribed by: Abenes, Aliping, Guarin, Villanueva (2024). 13

 Cockcroft & Gault eGFR: IDMS-Traceable

CCR = creatinine clearance GFR (mL/min) = glomerular filtration rate

140 = terminal age
72 = average weight of of a healthy individual NOTE:
● MDRD equation calibrated to an isotope dilution mass
NOTE: spectrometry (IDMS) reference method
● variables ○ modification of diet in renal disease system (MDRDS)
○ age ● does not require weight because the results are reported
○ sex
normalized to 1.73 m2 body surface area.
○ serum / plasma creatinine
○ weight ● recommended to use for patients ≥ 18 years old
● not adjusted for body surface area ● not accurate for pediatric patients
● no longer recommended for use because it has not been ● do not report actual values of GFR results ≥ 60
expressed using standardized creatinine values. ○ report: “equal to or greater than 60 mL/min”
○ only report the actual values of results below 60 GFR

Modified Cockcroft & Gault eGFR: Schwartz & Counahan-Barratt

CCR (mL/min) = creatinine clearance GFR (mL/min) = glomerular filtration rate

140 = terminal age k = proportionality constant
72 = average weight of of a healthy individual
ABW = adjusted body weight “k”
● children to 13 years of age
0.55
● females ≥ 13 years old
0.43 ● Counahan-Barratt Equation
IBW = ideal body weight ● Counahan-Barratt Equation
0.45 = correction factor if aBW is 30% > IBW (optional) 43
○ serum Cr = µmol/L
aBW = actual body weight ● Modified Counahan-Barratt Equation
40
○ serum Cr = µmol/L
Ideal Body Weight (IBW) 0.413 ● IDMS-Traceable Schwartz Equation
● 50 kg + 2.3 kg for each inch of height 41.3 ● height = meters (m)
Males
over 60 inches
● 45 kg + 2.3 kg for each inch of height
Females Reference Value: Modified Counahan-Barratt
over 60 inches
Males ● 80 – 135 mL/min
Females ● 70 – 120 mL/min
NOTE:
● if aBW is less than the IBW = do not adjust; use aBW
● ABW is used to correct other sources of weight aside NOTE:
from muscle mass ● use in infants, children, and adolescents with or without
chronic kidney disease (CKD)
● determines stage and severity of kidney dysfunction in
Creatinine Blind Conditions children
● reduced renal function ● inaccurate in acute kidney injury (AKI), as GFR in AKI
● range where a decrease in GFR starts to occur in the has not reached a steady state
range between 40 and 70 ml/min/1.73 m2 ● k value of 0.413 is more accurate for labs that use
● led to the measurement of low-molecular-weight enzymatic methods to determine serum creatinine levels
proteins, β2 – macroglobulin and cystatin C, as serum
markers of renal function NOTE: Conversion Factors
○ suggest a decreased GFR if these markers have ● 1 kg = 1 g ● 1 cm = 1000 µm
● 1 inch = 2.54 cm ● 1 foot = 12 inches
an increased concentration in serum
● 1 cm = 10 mm ● 1 m = 1 cm

Transcribed by: Abenes, Aliping, Guarin, Villanueva (2024). 14

 Example 3: Modified Cockcroft & Gault CYSTATIN C
Given: ● ⬆ plasma level = ⬇GFR
Age = 21 aBW = 55 ● potential marker for long-term monitoring of renal
Sex = Female IBW = ? function
Cr = 1.2 mg/dL ABW = ? ● produced at a constant rate = constant concentration
H = 62 inch CCR = ? ● readily passes through the GFB
● completely reabsorbed but NOT secreted
○ rationale: catabolized by tubular cells and therefore
does not return to blood circulation attributing to its
constant concentration
● not affected by age, gender and muscle mass
● disadvantages:
○ higher cost for analysis
○ intraindividual variability

INULIN CLEARANCE TEST

● an exogenous substance; fructopolysaccharide
● reference method for determination of GFR
○ administered intravenously
β2 – MICROGLOBULIN ■ extremely stable = not altered by the body
● ⬆ plasma level = ⬇GFR ○ readily passes through the glomerular filtration
● constant concentration in plasma and all body fluids barrier completely
○ rationale: a component of human leukocyte Ag and ○ neither reabsorbed nor secreted
is present on the surface of all nucleated cells ● disadvantage: not recommended for routine
● readily passes through the glomerular filtration barrier ○ there must be a continuous infusion of inulin for a
● reabsorbed completely but secreted constant plasma concentration
● a better marker of reduced tubular reabsorptive function ○ expensive
than of glomerular function ○ not always commercially available
○ rationale: there is an increase concentration of β2 –
microglobulin in urine while a having a normal RADIONUCLEOTIDES
serum concentration ● can be used to visualize glomerular filtration
○ thus, simultaneous testing of serum and urine ● exogenous agents which include radioisotope labels
concentration is recommended ○ e.g. 125I-iothalamate
■ administered intravenously
■ clearance can be observed using an equipment
● a faded or “plasma disappearance” = ⬇ GFR

SUMMARY OF CLEARANCE TESTS

Criteria Urea Creatinine β2-Microglobulin Cystatin C Inulin

Neither ___ nor reabsorbed

___ in the tubules. secreted

Excreted solely by glomerular filtration.

Stable for 24 hours.

Constant plasma level.

Readily available.
Availability of tests both in urine and
plasma.

Transcribed by: Abenes, Aliping, Guarin, Villanueva (2024). 15

 II. TUBULAR REABSORPTION TESTS ● 2 separate samples collected for 24hrs
● assessment of renal concentrating ability of tubules
● expected to have a higher
○ usually the first function that is affected in renal volume but lower SG
diseases 12 Hr
● SG may be variable due to its
● “concentration tests” Day
dependence on hydration status
○ test to determine the ability of tubules to reabsorb or physical activity of px
the essential salts and water that have been non 12 Hr ● expected to be lower in volume
selectively filtered by the glomerulus Night but higher SG ≥ 1.018
○ can be used as an indicator for early renal disease
● abnormalities can be detected by two methods MODERN TUBULAR REABSORPTION TESTS
○ conventional
○ modern a. Osmolality
● often used to express solute concentration of different
Types of Concentration Tests body fluids, including urine
Conventional Modern ● osmoles of particles per kg of solvent (Osm/kg H2O)
● fluid deprivation tests ● osmolality ○ osmolaRity = solute particles per liter of solvent
○ Fishberg ● specific gravity ● milliosmole: reflects the solute con. in body fluids
○ Mosenthal ● free water clearance ○ 1 mmol of urea = 1 mOsm
○ 1 mmol of NaCl = 2 mOsm
■ not always 1:1 due to the dissociation of solute
NOTE:
particles (i.e. 1 Na and 1 Cl)
● urine concentration is largely determined by the body’s
● often measured using osmometers that either observe
state of hydration changes to the sample in either:
● normal kidney will reabsorb only the amount of water ○ freezing point
necessary to preserve an adequate supply of body H2O ○ vapor pressure

CONVENTIONAL TUBULAR REABSORPTION TESTS NOTE: Colligative Properties

● determines the ability of the renal tubules to reabsorb ● inverse relationship with osmolality
○ freezing point
essential salts and water that had been filtered by the ○ vapor pressure
glomerulus ● direct relationship with osmolality
○ boiling point
a. Fluid Deprivation Test ○ osmotic pressure
● patients deprived of fluids for 24 hrs before measuring
● supercool sample below its freezing point
specific gravity (-7ºC) → vibration of sample (i.e. stirring) →
● used to differentiate types of diabetes insipidus produces crystallization → produces heat of
fusion → raises the temp. of the sol’n to its
Neurogenic (True) DI Nephrogenic DI freezing point → detected by thermistor
● ADH deficiency with ● normal ADH but ○ thermistor = temp. water probe
normal ADH receptor abnormal ADH receptor ● thermistor detects ⬆temp → corresponds
○ failure to secrete ADH ○ renal resistance to ADH to the FP of sol’n → converted into mOsm
● obsolete tests ○ 1 mol (1000 mOsm) of a nonionizing
○ SG measurement is most useful as screening substance dissolved in 1 kg of water is
Freezing known to lower the FP 1.86ºC
procedure
Point ● freezing point depression is correlated with
○ quantitative measurement of renal concentrating
Osmometer an increased osmolality
ability is best assessed through osmometry ○ ⬆solute particles = ⬇freezing point
○ e.g. ⬆ urea = ⬇ FP = ⬆osmolality
Two Methods of Fluid Deprivation Test
● osmolarity of unknown sol’n
Fishberg ● 24hr water deprivation → measure SG ○ can be calculated by comparing the FP
Concentration ● normal value: > 1.025 depression of an unknown sol’n with that
Test ○ indicates hyperstenuria of a known molal sol’n
● compares volume and SG of day and ● NaCl sol’n is often used as the standard
Mosenthal
night urine samples reference because a solution of partially
Concentration
● requires no diet restriction and fluid ionized substance is more representative of
Test
deprivation urine and plasma

Transcribed by: Abenes, Aliping, Guarin, Villanueva (2024). 16

 ● “dew point” b. Specific Gravity
○ temp. at which vapor condenses into a ● density of urine to density of an equal vol. of pure H2O
liquid state ● a ratio comparing the mass of the solutes present in
○ proportional to the vapor pressure from urine to pure water
the evaporating sample ○ thus, urine SG will always be greater than 1.000
● samples are absorbed into small filter paper ● depends on no. & density of particles present in a sol’n
disks that are placed in a sealed chamber ● a function of the number and mass of solutes present
Vapor containing a thermocouple → sample in the sample
Pressure evaporates in chamber → forms vapor → ○ small quantities of high MW solutes will affect
Osmometer water condenses when the temp. in the specific gravity measurement but do not affect
chamber lowers → heat of condensation osmolality measurement (e.g., glucose and protein)
raises the temp. of the thermocouple to the ○ ⬆solute + ⬆MW = ⬇osmolality + ⬆SG
dew point temp. ● a crude measurement of patient’s hydration status
● ⬇ dew point = ⬇ vapor pressure ⬆
solutes = ⬆ osmolality ● normal hydration status
● temps. are compared w/ NaCl standards 1.010 - 1.025 ● < 1.010 = overhydrated
● > 1.025 = dehydrated
and converted into mOsm
● physiologically acceptable SG
Urine Osmolality ○ range of SG the kidneys can produce in a
● a measure of urine concentration, reflecting the healthy or disease state
“number” of solutes present in urine ● ask for a recollection if SG beyonds or under
● contributors to urine osmolality 1.001 - 1.040 the range
○ urea (major) ○ beyond 1.040 seen in rare cases of DI/DM
○ NaCl (in plasma) ● 1.040 = not possible
● determination of urine osmolality requires water
○ could be contaminated (e.g. mannitol)
restrictions and determination of urine concentration at
timed intervals ○ reject and recollect

Normal Values for Osmolality NOTE: Specific Gravity and Osmolality

Serum Osmolality ● 275 – 300 mOsm/kg ● physical properties used to assess urine concentration
Random Urine ● express the ability of kidneys to concentrate the plasma
● 50 – 1400 mOsm/kg
Osmolality ultrafiltrate from the Bowman’s space, which is reflected
Urine-Serum Osmolality Ratio by the amount of solute present in the final urine
Normal ● 1:1
Controlled Fluid Osmolality Specific Gravity
● 3:1
Intake
● almost all solutes ● one solute may have a
contribute equally higher contribution
Urine Osmolality Determination ● some ionic solute may ● some ionic solute may
● an important tool in differentiating the types of DI contribute NOT contribute
● DI and DM may present with polyuria, but can be ● some may NOT affect osmolality but CAN affect SG
differentiated by the osmolality of their urine
○ e.g. glucose and protein can increase SG but
○ DI = decreased production or function of the ADH
leading to excessive loss of water in urine osmolality remains constant
○ DM = CHO metabolism disorder caused by inadequate ● preferred for the evaluation ● SG detected by:
secretion or utilization of insulin leading to the loss of of renal concentrating ○ refractometry = reflects
glucose in urine ability the collective number &
■ osmolality of urine from DM px are higher compared
○ since it takes considers mass of solutes in urine
to DI px due to the presence of glucose in urine
the contribution of each ○ reagent strip = detects
*refer to flowchart solute particle to the only ionic solutes
overall value

Transcribed by: Abenes, Aliping, Guarin, Villanueva (2024). 17

Flowchart for Fluid Deprivation Tests

Transcribed by: Abenes, Aliping, Guarin, Villanueva (2024). 18

c. Free Water Clearance Example 1: Free Water Clearance
● volume of blood plasma that is cleared of solute-free Given:
water per unit of time Cosm = ? V = 2 mL/min
○ test used to evaluate the ability of the kidneys to Uosm = 600 mOsm/kg FWC = ?
reabsorb a water free from solutes Posm = 300 mOsm/kg
○ volume of blood plasma = mL cleared of solute free
water
● determine the quantity of water not reabsorbed or
secreted by the kidneys
● assesses how the kidneys respond to the state of body
hydration
● osmolar clearance
○ theoretical volume of plasma per unit time that the
NOTE:
kidney has to filter in order to extract all the solute
● kidneys must excrete 4mL of water per min to produce
out of the plasma volume an osmolality of 600 mOsm/kg
○ it determines how much water must be cleared ● kidneys excreted 2mL less than what is expected
each minute to produce a urine with the same ● hyperosmotic urine
osmolality as the plasma
Example 2: Free Water Clearance
Free Water / Solute-Free Clearance (CH2O) Given:
Cosm = ? V = 10 mL/min
Uosm = 100 mOsm/kg FWC = ?
Free Water Clearance (mL/min)
Posm = 290 mOsm/kg
V (mL/min) = volume of urine
Cosm (mL/min) = osmolar clearance

Cosm (mL/min) = osmolar clearance

Uosm (mOsm/kg) = urine osmolality
Posm (mOsm/kg) = plasma osmolality
NOTE:
● kidneys must excrete 3.45mL of water per min to
Reference: Free Water Clearance produce an osmolality of 100 mOsm/kg
(+) ● excess water excreted ● kidneys excreted an excess of 3.45mL of water
(0) ● no renal concentration or dilution ● hypoosmotic urine
(–) ● less than necessary amount of water excreted
NOTE:
Urine Osmolality ● if plasma osmolality is too low, urine osmolality should
Isosmotic ● total urine volume = clearance volume lower in order to compensate
● urine osmolality < serum osmolality ○ thus the solute free clearance should be negative
● total urine volume > Cosm water (–) since the kidneys will conserve water, producing
Hypoosmotic
● solute free water clearance = (+)
a hyperosmotic urine
● most probably due to diuresis
● urine osmolality > serum osmolality
● total urine volume < Cosm water
Hyperosmotic
● solute free water clearance = (–)
● most probably due to dehydration

Steps In Computing
Step 1 ● compute for osmolar clearance
Step 2 ● compute for free water clearance
Step 3 ● determine urine osmolality

Transcribed by: Abenes, Aliping, Guarin, Villanueva (2024). 19

 III. TUBULAR SECRETION TESTS Renal Blood Flow: p-Aminohippurate Clearance
● utilizes substances that are exclusively removed from
the bloodstream via tubular secretion
● before assessing the ability of the kidneys to secrete
CPAH (mL/min) = clearance of p-amminohippurate
substances → renal blood flow must be established
U = urine PAH concentration
and reported as ‘normal’
P = plasma PAH concentration
○ normal renal blood flow (RBF): 1200mL/min
V = volume of urine
○ normal renal plasma flow (RPF): 600-700 mL/min
● not routinely performed NOTE:
○ tubular secretion cannot be evaluated when renal ● the actual measurement is renal plasma flow rather than
blood flow is impaired and vice versa renal blood flow because the PAH is contained only in
○ exogenous substance mostly used the plasma portion of the blood
○ substance must always be monitored
● also uses “concentration tests” TITRATABLE ACIDS AND URINE AMMONIA
○ ability of tubules to absorb & secrete ● measurement of titratable acids and ammonium salts in
urine can therefore assess how effective these excess
Renal Blood Flow Computation acids are excreted
● RBF function is highly dependent on renal perfusion ● acid secreted per day: 50-100 mmol
● computed using the following equation: ● measures:
○ amount of H+ ions
𝑅𝑒𝑛𝑎𝑙 𝐵𝑙𝑜𝑜𝑑 𝐹𝑙𝑜𝑤 = 𝑅𝑒𝑛𝑎𝑙 𝑃𝑙𝑎𝑠𝑚𝑎 / 𝐹𝑙𝑜𝑤 𝐻𝑒𝑚𝑎𝑡𝑜𝑐𝑟𝑖𝑡 ○ ammonium salts (NH4)2SO4
○ titratable acids
Tubular Secretion = Ideal Clearance Substance ○ titratable secretion
1. Constantly present in the plasma. ○ NH3 production & secretion
2. Removed completely from the plasma by tubular
secretion. Renal Tubular Acidosis (RTA)
3. Must contain a zero concentration in the plasma after ● used for the diagnosis of RTA
secretion. ● tubules fails to acidify urine making it alkaline
○ even despite the presence of metabolic acidosis
PHENOLSULFONPHTHALEIN (PSP) ● inability to produce acid urine due to impaired secretion
● dye excretion test of pH changing substances
○ dye infused in px → con. of dye measured in urine ○ H+ (PCT)
○ less than expected amount = problem w/ secretion ○ NH3 (DCT)
○ expected amount of dye is 0 under normal condition
● not currently performed since ORAL AMMONIUM CHLORIDE TEST
○ standardization and interpretation is difficult ● involves oral administration of ammonium chloride
○ possibility of producing anaphylactic shock ○ metabolized → urea & hydrochloric acid (HCl)
○ HCl produced will induce “acidosis”
p-AMINOHIPPURATE (PAH) ○ kidneys compensate by ⬆excretion of titratable
● exogenous, non-toxic, weak organic acid acids & ammonium salts → acidic urine
○ measures urine & blood spx ● urine is collected 8 -10 hours at 2-hour intervals to
● completely secreted by the renal tubules (PCT) determine pH, titratable acids & ammonium salts
○ thus, the vol. of plasma flowing through the kidneys ○ normal urine
determines the amount of PAH excreted in urine ■ pH = < 5.3
○ it is loosely bound to plasma proteins, which ■ total H+ excretion (sum of titratable acids and
permits its complete removal as the blood passes ammonium salts) = > 60 mmol/min
through the peritubular capillaries
● most commonly used Test Criteria Before Oral Ammonium Chloride Test
○ reference method for RBF ● Bicarbonate measurements = > 20 mmol/L
● urine pH = < 5.3 (normal)
RADIOACTIVE HIPPURATE ● if NOT followed, renal tubular acidosis are unable to
● infused to the px → con. is measured in blood produce an acid urine despite systemic acidosis
specimen to assess RBF
● measures the plasma disappearance of radioactive
substance
● provides a clear visualization of blood flow through
the kidneys

Transcribed by: Abenes, Aliping, Guarin, Villanueva (2024). 20

ANALYSIS OF URINE MLS 321L

& OTHER BODILY FLUIDS 8438L

LABORATORY BS MLS 3 PRELIM
M2: MACROSCOPIC EXAMINATION OF URINE

UNIT 1: URINE SPECIMEN TYPES, ● primary inorganic component

COLLECTION AND PRESERVATION Chloride ● found in combination w/ Na (table salt)
and many other inorganic substances
URINE
Sodium ● primarily from salt (varies by intake)
● ultrafiltrate of plasma
○ used to evaluate & monitor body homeostasis & Potassium ● combined with Cl and other salts
metabolic disease processes Phosphate ● combines w/ Na to buffer the blood
● specimen collection, transport and handling Ammonium ● regulates blood & tissue fluid acidity
○ integral part of routine of routine urinalysis
Calcium ● combines w/ Cl, sulfate, and phosphate
PROPERTIES OF URINE
● pH: 4.5 - 8.0 URINE SPECIMEN TYPES
● specific gravity: 1.001 - 1.040
○ 1.000
■ px may have underlying condition Specimen Types
■ e.g. diabetes insipidus First Morning Urine
○ >1.040
■ px taking up medication ● aka “8hr urine sample”
■ e.g. radiographic contrast media (x-ray dye) ● specimen of choice for routine analysis
■ e.g. mannitol infusion ● most concentrated
● temperature: 32.5°C - 37.5°C
● color: light yellow to amber ● more stable formed elements seen:
● appearance: clear & transparent ○ blood cells
○ red blood cells
Composition of Urine ○ cast
95% Water ● more enhanced due to ⬆ osmolality
● organic solutes ● pH 5-6 (acidic urine)
○ urea (primary) ● px must deliver specimen within 1 hour
○ uric creatinine ● delayed specimen
○ uric acid ○ must be preserved properly (refrigeration)
● inorganic solutes ● for diagnosing postural or orthostatic proteinuria
5% Solutes
○ salts (primary) ○ Mg ○ horizontal → vertical position
○ Cl (major ion) ○ phosphates ○ sudden ⬆ protein concentration
○ Na ○ electrolytes ● for possible diagnosis of non-obvious (early) kidney
○K disease
● for the prevention of false-positive pregnancy tests

Primary Components in Normal Urine Second Morning (Fasting) Specimen

● primary organic component ● 2nd voided specimen after a period of fasting

Urea ● product of metabolism of protein and ● does not contain any metabolites
amino acids ● Glucose monitoring
● product of metabolism of creatine by ○ px with hyperglycemia
Creatinine
muscles
● product of breakdown of nucleic acid in
Uric Acid
food and cells

Transcribed by: Abenes, Aliping, Guarin, Villanueva (2024). 1

 Glucose Tolerance Specimen Timed (24-Hour) Urine
● collected for a predetermined length of time (2hrs,
● not routinely submitted
12hrs, 24 hrs)
● corresponds with the blood samples drawn during
● measuring substances that changes with diurnal
Glucose Tolerance Test (GTT)
variations
● length of test varies:
● activities causing diurnal variations:
○ fasting
○ exercise
○ half-hour
○ meals
○ 1,2,3,4,5,6-hour (max: 10 hrs)
○ body metabolism
● tested for glucose & ketones
● solutes exhibiting diurnal variations:
● reported along with the blood test
○ catecholamines
● correlated with renal threshold for glucose
○ 17-hydroxysteroid
● assess px with possible case of diabetes mellitus
○ electrolytes
○ required along with second morning specimen
○ proteins
2-Hour Post-Prandial Specimen ● lowest concentration: early morning
● highest concentration: afternoon
● used primarily for monitoring insulin therapy for px ● determines urinary urobilinogen (maximal excretion)
with diabetes mellitus ○ collected during a specific time of the day (ex. 2pm
● along with: - 4pm)
○ Glucose Tolerance Specimen ● shorter period collection
○ Second Morning Urine ○ must keep the patient adequately hydrated
● 1st collection ● utilization:
○ px must urinate shortly before consuming a routine ○ fistula evaluation
meal ○ cytology studies
● 2nd collection ○ clearance test
○ collect specimen 2 hours after eating ● px must begin & end the collection period with an
● comprehensive evaluation empty bladder
○ comparing the results of 2-hour post-prandial ● substance concentration in a set period must be
specimen with 2-hour post-prandial blood test calculated from the urine volume produced during
that time
Random Urine
● inaccurate results:
● collected at any time (usually daytime) ○ addition of urine before the start of the collection
○ most common and most convenient ○ failure to include urine produced at the end
● NO px preparation ● 24-hour urine must be mixed & volume must be
● NO fasting accurately measured and recorded
● follows the same routine for First Morning Urine ● do an aliquot:
Collection ○ 50 ml routine test
● factors that affect urine composition: ○ 1 ml quantitative test (creatinine, electrolytes)
○ excessive fluid intake ● ALL specimens should be refrigerated during collection
○ exercise period
● for screening tests in routine urinalysis
● for obvious kidney diseases and UTI
● random “clean catch” urine specimen
○ ideal for cytology studies
○ instruct px to drink 680-900 mL of water each
hour for 2 hours
○ collected daily for 3 - 5 consecutive days
○ exercising for 5 mins (jumping, skipping)
■ increases cellularity
■ enhances detection

Transcribed by: Abenes, Aliping, Guarin, Villanueva (2024). 2

●
 SPECIMEN COLLECTION AND PRESERVATION

SPECIMEN COLLECTION
● urine is a biohazardous substance whose handling
requires observance of the “standard precautions”

● laboratory protocol
○ specimen must be tested within 1-2 hrs
(time sensitive)
● max: 30-50 mL (for additional or repeat
testing)
○ 10-12 mL (routine urinalysis)
Container ○ <12 mL (limit chemical tests)
● clean, sterile dry, clear or translucent (for
color & clarity)
● disposable material
● wide mouth; opening at least 4-5 cm
● capacity of 50-100 mL
● leak proof seal
● all specimens are labeled before or
immediately after collection
● placed directly on the container
Label ● includes:
○ Patient’s full name, age, & sex
○ Type of specimen
○ Date and time of collection
● must match the label
● presents:
○ initial diagnosis
Requisition
○ condition
Form
○ special preparation
● time specimen is received in the lab should
be recorded on the form

SPECIMEN REJECTION
1. Unlabeled or improper labeling
2. Nonmatching requisition and label
3. Contaminated with feces of toilet paper
4. Contaminated exterior
5. Insufficient quantity
○ case to case basis
○ <12 mL- kidney disorders, infections, pediatric &
newborn patients
6. Improperly transported

Transcribed by: Abenes, Aliping, Guarin, Villanueva (2024). 3

●
 Collection Techniques
● insertion of sterile catheter through:
● requires no patient preparation
Routine Void ○ urethral catheterization
● random or first morning specimen
■ urethra→bladder
● more ideal specimen for routine ○ ureteral catheterization
urinalysis ■ directly into ureters
● represents elements and analytes from: ■ determine kidneys involvement
○ bladder Catheterized ● used to determine if the kidneys are
○ ureters involved in a disease process.
○ kidneys ● stored in a plastic reservoir bag
● less contaminated by ● submitted for bacterial culture (UTI)
○ vaginal discharge ○ random or timed specimen
○ epithelial cells ● culture must be first performed before
○ bacteria other test to prevent contamination
● requires additional;
○ patient instructions ● collecting a normally sterile urine directly
○ cleaning supplies from the bladder
○ assistance for elderly patients or ● puncture to the abdominal wall and the
young children distended bladder using a sterile syringe
● safer, less traumatic method Suprapubic
and needle
● patient must wash his hands to prevent Collection
● bladder puncture→ sterile urine
possible contamination ○ bacterial cultures (anaerobic microbes)
○ glans penis of the male or the urethral & fungal culture.
meatus of the female is thoroughly ○ in infants
cleansed and rinsed.
● observed among infants & who
Midstream ○ uses strong bacterial agents→
Pediatric cannot urinate voluntarily
“Clean Catch” cleansing agents.
Collection ○ plastic bag→adhered to skin
■ hexachlorophene
● routine analysis & quantitative assays
■ povidone-iodine
○ mild antiseptic towelettes are ● used to determine prostatic infection
recommended ○ 1st container: first urine passed
○ interior of the container must not collected in a sterile container
come in contact with the patient’s ○ 2nd container: midstream portion
hands or perineal area. collected in another sterile container
○ 3rd container: prostate fluid collected
by massaging the prostate
● quantitative cultures
○ performed on all specimens
Three Glass ○ 1st and 3rd specimens
Collection ■ examined microscopically
Prostatic Infection Determination
● white blood cell count per
high-power field
● bacterial count 10 times of the
3rd Spx
1st specimen
● macrophages containing lipids
may be present
● bacterial count compared from
1st Spx
the 3rd specimen

Transcribed by: Abenes, Aliping, Guarin, Villanueva (2024). 4

●
 ● control for bladder & kidney
infection
2nd
● if (+) for wbc & bacteria
Spx
○ 3rd specimen = invalid
(contaminated)

● 4-glass method: prostatitis screening

○ initially voided urine
■ urethral infection
Stamey-Mears ■ inflammation
Four Glass ○ midstream urine
Localization ■ urinary bladder infection
Method ○ expressed prostatic secretions &
post-prostatic massage urine
■ cultured/examined wbc
■ + result: >10-20 wbc
● screening tool for prostatitis.
● + result: significant bacteriuria (>10x)
Pre and ○ post massage
Post-Massage ● procedure:
Test (PPMT) 1. collect midstream urine
2. collect a second urine sample after
prostate massage
CHANGES IN UNPRESERVED URINE
● urine should be delivered to the lab within 1 hr
SPECIMEN HANDLING ○ other references: up to 2 hrs
● expected delay must be preserved
Specimen Integrity
● refrigeration of the sample at 2 - 8° C
● specimens tested within 2 hours ○ commonly suitable for most specimens
● a specimen that cannot be delivered and tested within 2 ○ ↓ bacterial growth→ suitable culture 24hr
hours should be refrigerated or have an appropriate ○ easiest means of preservation
chemical preservative added ● disadvantages:
○ Induce precipitation of non/pathologic crystals
Specimen Preservation (PO4, SO4)
○ ↑ specific gravity
● refrigeration at 2-8ºC is the most routinely used
● urinometer
○ decreases bacterial growth & metabolism ○ device to measure urine specific gravity
○ should be refrigerated during transit and until
cultured, up to 24 hours Changes In Unpreserved Urine
○ can cause precipitation of amorphous urate & ● color change: altered urine solutes
phosphate crystals Physical ● odor: bacterial growth
● specimen must return to room temp. before chemical Changes ● ⬇ urine clarity: solute precipitation in the
testing by reagent strips form of amorphous material
○ enzyme reaction of strips perform best at RT ● false-negative result
● chemical preservatives added when transported over ● ex. urine nitrite & pH increase in
long distances & refrigeration is impossible Chemical
unpreserved urine
● commercially prepared transport tubes w/ lyophilized Changes
○ nitrate → nitrite & metabolizing urea →
preservative available for transport, testing & storage ammonia
● ideal preservative:
○ bactericidal Microscopic ● disintegration of formed elements
○ inhibit urease Changes ● hypotonic & alkaline urine
○ preserve formed elements in the sediment
○ should not interfere with chemical test
● confirm is appropriate preservative is used when
preserving spx that will be transported to another lab

Transcribed by: Abenes, Aliping, Guarin, Villanueva (2024). 5

●
Potential Changes in Unpreserved Urine

Component Observed Change Mechanism

Physical
Color Darkens ● oxidation or reduction of solutes (urobilinogen, bilirubin)
● crystal precipitation
Clarity Decreases
● bacterial proliferation
● bacterial conversion of urea to ammonia
Odor Ammoniacal (foul smelling)
● bacterial proliferation
Chemical
● bacterial conversion of urea to ammonia
pH Increase
● loss of CO2
Glucose Decrease ● bacterial and cellular consumption
Ketones Decrease ● volatilization and bacterial conversion
Bilirubin Decrease ● photo-oxidation → biliverdin
Urobilinogen Decrease ● oxidation → urobilin
Nitrile Increase ● bacterial conversion of nitrates
Microscopic
● lysis and disintegration (dilute/ hypotonic and alkaline
Blood cells Decrease
urine)
● lysis and disintegration (dilute/ hypotonic and alkaline
Cast Decrease
urine)
● loss of motility
Trichomonads Decrease
● death
Bacteria Increase ● proliferation

MNEMONICS:
Increased OP NaBaCol
PBaON
Decreases BUCK GluT Cells

Transcribed by: Abenes, Aliping, Guarin, Villanueva (2024). 6

●
Urine Preservatives

Type Advantages Disadvantages Use

● routine urinalysis
● precipitates amorphous &
● urine culture
Refrigeration crystalline solutes ● storage before & after testing
● inexpensive
● (urinometer): ↑ specific gravity
● does not interfere
● pH & specific gravity may be
Commercial ● routine urinalysis
altered ● urine transport from off-site to
Transport ● preserves chemicals & formed
● interfere w/ Na, K, hormone laboratory
Tubes elements at room temp
and drug assays

● preserve proteins and formed ● precipitate crystals in large ● keep 6 pH

elements well amount ● bacteriostatic at 18 g/l (not
Boric Acid
● does not interfere except in ● interferes w/ drug and bactericidal)
pH hormone analysis ● culture transport

● preserve sediment elements

● interfere with
& glucose
Thymol protein precipitation test ● sediment preservation
● inhibits bacterial and yeast
● precipitate crystals
growth
● false-negative reagent strip
test for reduction:
○ blood
● excellent cellular preservative ○ urobilinogen ● cytology, especially Addis’
Formalin
(especially casts) ○ glucose count
○ leukocyte
○ esterase
○ copper
● does not interfere with ● floats on surface of
Toluene glucose & protein specimens and clings to
determination materials
● inhibits reagent strip test for:
Sodium ○ blood
● prevents glycolysis ● drug analysis
Fluoride ○ glucose
○ leukocyte
Sacco-
manno’s ● excellent cellular preservative ● potential chemical hazard ● cytology
Fixative
● inexpensive
● stabilizes:
Acids ● quantitative analysis of
○ calcium ● unacceptable for urinalysis
(HCL, Glacial Acetic steroids, hormones and other
○ phosphorous ● potential chemical hazard
Acid) solutes
○ steroids
○ hormones
● inexpensive
● quantitative analysis of
Sodium ● stabilizes
● unacceptable for urinalysis ● porphyrins
Carbonate ○ porphyrins
● porpho- bilinogen
○ porphobilinogen
● does not interfere w/ glucose
Phenol ● odor change
& protein determination

Transcribed by: Abenes, Aliping, Guarin, Villanueva (2024). 7

●
 Commercial Urine Transport Tubes
BD Vacutainer; + BD Vacutainer;
InTac UA System, Therapak
Plastic ConicalUA C&S
Corporation
Preservative Tube Preservative Tube
Product No. 364946 94500 364948 & 364976

Tube ● plastic conical ● plastic conical ● glass tube

● yellow & cherry red marble stopper ● yellow plastic cap ● gray stopper
● Dowicil 200 ● boric acid
Preservative ● chlorhexidine ○ formaldehyde releasing agent ● sodium formate
● ethyl paraben
& Additive ● mannitol ● d-sorbitol
● Na propionate
● polyethylene glycol ● sodium acetate
● urinalysis
● urinalysis ● urinalysis
Use ● bacteriostatic
● bactericidal; ● bactericidal;
● urine culture
○ don't use for urine culture ○ don't use for urine culture
& sensitivity
● stabilize: up to 48 hr
● stabilize: up to 72 hr ● stabilize: up to 96 hr
○ room temp
Comments ○ room temp ○ room temp
● ph adjusted: 6-7
● conical bottom ● no change in ph or specific
● ↑ specific gravity
○ fit kova- pettors gravity
○ ≈0.006- 0.007

INSTRUMENTS & MATERIALS Watch Glass and Beaker

● used for confirmatory chemical tests
Microscope
● primary instrument used in Clinical Microscopy
Oven
● for sterilization of glasswares
Refractometer
Watch Glass Beaker
● a now near-obsolete instrument
● used for determining urine specific gravity
Test Tubes
○ urinometer – also a near-obsolete alternative
● for confirmatory chemical tests
○ reagent strip – more commonly used and best
alternative for urine SG determination ● usually utilized
13 x 100 ● also used as alternative to conical test tubes
for sediment preparation
16 x 150 ● for larger-volumed samples or assays
18 x 150 ● for larger-volumed samples or assays

Refractometer Urinometer Reagent Strip Conical Test Tubes with Graduations

● for sediment preparation & subjected to centrifugation
Serological Pipettes
● for transferring reagents for the performance of Glass Slides and Coverslips
confirmatory chemical tests ● designed to provide accuracy, uniformity and safety
in the microscopic examination of urinary sediment
Pasteur Pipettes
● for transferring urine samples to avoid spillage Evaporating Dish
● for confirmatory chemical tests
Thermometer ● used for the evaporation of solutions and supernatant
● for temperature-sensitive analytes, such as liquids, and sometimes to their melting point
Bence-Jones proteins
Neubauer Chamber and WBC Thoma Pipette
Graduated Cylinder and Erlenmeyer Flask ● instruments in hematology useful for other body fluids
● volumetric glasswares test, such as in semen and CSF

Transcribed by: Abenes, Aliping, Guarin, Villanueva (2024). 8

●
 UNIT 2: PHYSICAL EXAMINATION OF URINE Urine Color Variations Caused by Red Blood Cell
I. Urine Color ● pink (few RBC)
Urine Color
II. Urine Clarity ● red (many RBC)
III. Urine Concentration (Specific Gravity) ●
IV. Urine Foam
● alkaline urine w/ RBC
V. Urine Odor
VI. Urine Volume Urine pH ○ red-brown color
VII. Urine Taste ○ RBC lyse in a hypotonic, alkaline urine
○ releases hemoglobin → methemoglobin
Urine Physical Characteristics ● urine may appear brown or black
Structural
● study of urine ● results from the disintegration of RBC
Form of
● release and oxidation of hemoglobin →
○ oldest clinical laboratory test RBC
methemoglobin
● physical characteristics of urine: color, clarity, specific
gravity, volume, odor
Urine Pigments
● main properties reported:
● pigment responsible for normal color of
○ color urine
○ clarity ● dependent on water
○ specific gravity ● darkens when exposed to light
Urochrome ● px w/ chronic renal failure
I. URINE COLOR ○ excretion of urochrome
● vary from colorless to pale yellow, pink, orange, red, ○ exhibits yellow pigmentation of the skin
○ urochrome gets deposited in
amber, green, purple, blue, brown, black
subcutaneous fat
● normal urine color: yellow/ light yellow ● orange-brown tinge
● technical terminologies: Urobilin ● oxidation of urobilinogen
○ replace “straw & beer brown” → “light yellow &
● pinkish color
amber”
● more evident when it deposits on urate
○ avoid using the term “bloody” → “red” or “pink” Uroerythrin
crystals → producing a precipitate called
● several urine color variations may be observed that can brick dust
indicate metabolic abnormalities
○ ex. red blood cells Urine Color
● color variations can simply result from: ● common cause: recent ⬆intake of
○ excessive physical activity water
○ medications Colorless to ● px with polyuria
○ foods eaten Light Yellow ○ diabetes insipidus
○ stress ○ diabetes mellitus
● px w/ severe tubular disease
● abnormal urine reflecting a pathologic process may not
● concentrated urine
always have an abnormal color ● first morning urine spx
● normal colored urine does not always indicate a healthy ● common cases:
condition as it may contain significant pathologic ○ dehydration
elements ○ strenuous exercise
Dark Yellow/
○ normal urine may contain large amounts of glucose ○ fever
Amber
& protein ● also seen in ⬆amounts:
○ bilirubin (hepatic disease)
○ red colored urine
○ urobilin (oxidation product of
■ may result from ingestion of beets urobilinogen)
■ contaminated menstrual blood ○ acriflavin (antiseptic)
● common among px under medication
○ phenazopyridine
Yellow- ○ phenindione (colorless in acid urine)
Orange ○ warfarin
○ rifampin
● also caused by foods rich in carotene
Yellow-Brown ● px under treatment w/ nitrofurantoin
● px taking multivitamins
Bright Yellow
○ riboflavin

Transcribed by: Abenes, Aliping, Guarin, Villanueva (2024). 9

●
Yellow-Green ● oxidation of bilirubin → biliverdin ● rarely encountered
● indicate presence of melanin
● minimal presence of blood ○ oxidation product of melanogen
● menstrual bleeding ○ px w/ malignant melanoma
● ⬆amount of porphobilin ● presence of homogentisic acid
Pink
○ oxidation of porphobilinogen ○ px w/ alkaptonuria
○ associated with acute intermittent Brown- ● develops when an alkaline urine +
porphyria Black melanin/ homogentisic acid → stand and
● ⬆ amount of blood (RBC, hemoglobin) exposed in the air
● red & cloudy ● px under the medication of:
○ high RBC concentration ○ levodopa
○ hematuria ○ methyldopa
● red & clear ○ phenol derivatives
○ high hemoglobin concentration ○ metronidazole
○ intravascular hemolysis ● px with severe urinary tract infection
○ hemoglobinuria (pseudomonas spp)
○ myoglobinuria ● px with intestinal inception
● may indicate kidney/urinary tract ○ ⬆ indican procession
disease ● px under:
○ glomerulonephritis Blue-Green
○ amitriptylin (anti-depressant)
Red ○ pyelonephritis ○ methocarbamol (treat muscle
○ cystitis spasms)
○ renal calculi & tumors ○ methylene blue (treat
● also occur in: methemoglobinemia)
○ menstrual contamination ● associated with growth of Klebsiella or
○ highly pigmented foods Providencia spp.
○ phenolphthalein Purple
● urine sample stored for a long period in
● consuming large amounts of: urine collection bags
○ fresh beets
● excessive oxidation of porphyrinogen
○ black berries Red-Purple/
and porphobilinogen → porphyrin
○ senna laxatives Port Wine
● improper handling and storage of urine
● intravascular hemolysis Color
specimen
○ evident w/ red plasma after centri.
● indicated presence of hemoglobin or
myoglobin (fresh urine) Evaluation of Urine Color
○ requires urine chemical testing & 1. Mix the specimen well by gentle inversion/ swirling.
evaluation of blood plasma 2. View through a clear container. If the specimen is not
○ myoglobin clear, transfer the sample onto a clear tube/ container.
■ does not affect normal color of 3. View the urine sample against a white background.
plasma 4. Determine color of urine by reporting what is visibly
■ skeletal muscle destruction
(rhabdomyolysis) seen.
○ hemoglobin
Red Brown ■ appear red NOTE:
■ intravascular hemolysis ● there should be adequate lighting
● intact RBC are found in rare cases ● evaluating multiple specimen
○ confirmed by microscopic examination ○ volume of urine must be consistent to the rest of the
● substances produce a positive chemical
patients
test for blood
● cola-colored urine ● color evaluation must not take too much time
○ associated with ⬆methemoglobin
● px under metronidazole
○ treatment of parasitic infection
○ giardiasis & amoebiasis

Transcribed by: Abenes, Aliping, Guarin, Villanueva (2024). 10

●
 II. URINE CLARITY
● describes the transparency or cloudiness caused by
suspended particulate matter that scatters light
● overall visual appearance of a urine specimen
● freshly voided “clean catch” urine specimen is usually
clear
● clear urine is not necessarily normal
● turbid urine does not necessarily indicate a pathologic
process

Urine Clarity Precautions

● normal specimen for female px
○ may appear cloudy
○ contaminated w/ vaginal secretions
● turbid urine
○ may indicate contamination, not necessarily
indicate a pathologic process
● clear urine
○ may contain ⬆ glucose, protein, lysed RBC
(indicators of kidney damage)

Indications of Pathologic Substances

● deterioration of the barrier normally separating the
urinary tract from the blood
● disease process
● metabolic dysfunction
○ RBC in urine → urinary tract damage

dysmorphic RBC ● glomerular damage

RBC in casts ● glomerular / tubular origin

Factors Affecting Urine Clarity

Pathologic Cause
Haziness ● RBC & WBC
● non-squamous epithelial cells
○ renal tubular epithelial cells
○ transitional epithelial cells
● lipids
● chyle
● bacteria
● yeast & trichomonads
○ originate from vaginal infection
Turbidity ● abnormal crystals
○ cystine
○ leucine
● parasite
● semen
● feces
● mucus
● pus
Milky ● excretion of fat / lymph

Transcribed by: Abenes, Aliping, Guarin, Villanueva (2024). 11

●
 Non-Pathologic Cause Terminologies for Describing Urine Clarity
● squamous epithelial cells Clear ● no visible particulates observed
○ improper collection
● if few particles seen if placed against a
● mucus threads Hazy printed background
● precipitated amorphous materials ● print can easily be seen through urine
● radiographic contrast media Cloudy or ● if there are many particulates seen if
○ iatrogenic ‘Slightly placed against a printed background
○ not indicative of a disease Turbid’ ● print is blurred through urine
● talcum powders ● if print from background cannot be seen
Turbid
● vaginal creams through urine
Contaminants
● feces Milky ● if urine is already precipitated or clotted
○ improper collection
or
○ fistula bet. bladder & colon
● spermatozoa
Normal Urine
○ possible indicator of chronic
Components
neurogenic impairments
○ direct damage to nerve paths →
dysfunction of internal sphincter of
urethra
○ indicates traumatic / drug-induced
impairment of internal urethral orifice
of the bladder → fails to close during
ejaculation
● prostatic fluid
● ⬆ bacteria
Turbidity
● prolonged standing of urine sample

Chemical Correlations of Urine

● amorphous urates
Found in Acidic Urine
● radiographic contrast media
● amorphous phosphates
Found in Alkaline Urine ● carbonates

● amorphous urates
Soluble with Heat
● uric acid crystals
● red blood cells
Soluble in Dilute Acetic
● amorphous phosphates
Acid
● carbonates
● white blood cells
Insoluble in Dilute ● bacteria
Acetic Acid ● yeast
● sperm cells
● lipids
● lymph
Soluble in Ether
● fluid
● chyle
Evaluation of Urine Clarity
1. Mix the specimen well by gentle inversion/ swirling.
2. View through a clear container. If the specimen is not
clear, transfer the sample onto a clear tube/ container.
3. View the urine sample against a printed white
background.
4. Determine clarity of urine using the terminologies below
5. Report what is visibly seen.

Transcribed by: Abenes, Aliping, Guarin, Villanueva (2024). 12

●
 III. URINE CONCENTRATION A) SPECIFIC GRAVITY: DIRECT METHODS
● describes the number of solutes present in volume of ● actual or true density of urine, regardless of solute
H2O excreted affected by px’s present
○ diet ● solutes detected and measured
○ physical activity
● affected by temperature
○ Health
● diluted urine
○ fewer solute particles per volume of H2O Solutes Detected and Measured: Specific Gravity
○ color: light yellow/ colorless Urea &
● concentrated urine ● always present in urine
Electrolytes
○ more solutes particles per volume of H2O
○ color: dark yellow Glucose & ● present as a result of disease
● color: crude indicator of urine concentration Protein ● corrections are needed in measuring SG
● for iatrogenic reasons
Urine Specific Gravity Radiographic ● do not reflect actual concentrating ability
● expression of urine concentration in terms of density Media ● other abnormal process unrelated to
(mass of solute per volume of solution)
renal function is present
● ratio of urine density to the density of an equal volume
of pure water
● factors that affect specific gravity: A.1) Direct Method: Urinometer
○ density measurement ● involves the use of a
○ no. of solutes urinometer
○ molecular size
● aka hydrometer
● the greater the urine density = the larger the specific
gravity ○ no longer used in the
lab
Specific Gravity Values ○ inaccuracy of
● urine specific gravity produced by the body measurements
1.002 - 1.040 ● NOTE: it is impossible for the body to ● disadvantages:
excrete pure water (SG= 1.000) ○ requires a large
1.001 ● value of SG possible for newborns volume of urine
1.010 - 1.025 ● SG for random urine and 24-hour urine ○ daily calibration will
● commonly encountered specific gravity not address
1.015 - 1.025 ● average solute & water intake
inaccuracy
● average water excretion
● weighted float
● indicates dilute urine
● ⬆ water intake ○ attached to a scale
● intake of diuretics ○ calibrated in terms of
1.001 - 1.009
● related conditions: urine SG
○ diabetes mellitus ○ designed to sink to a level of 1.000 in distilled water
○ diabetes insipidus ● SG = level to which the urinometer sinks
● concentrated urine ● calibrated to read 1.000 in distilled water at 20ºC
1.025 - 1.035 ● dehydration
● excessive intake of osmotic diuresis
● iatrogenic substances Procedure: Urinometer
1.040 ○ radiographic contrast media ● An adequate (10-15 mL) amount of urine is poured
○ mannitol 1
into the cylinder.
● Isostenuria
2 ● The hydrometer is added with a spinning motion.
○ inability of kidneys to alter the SG of the
urine excreted ● The scale reading is taken at the bottom of the urine
3
○ end stage renal disease meniscus.
● ultrafiltrate that enters the Bowman’s
1.010 space of the glomeruli has the same
specific gravity as protein free plasma
(1.010)
> 1.010 ● hypersthenuria
< 1.010 ● hyposthenuria

Transcribed by: Abenes, Aliping, Guarin, Villanueva (2024). 13

●
 Urinometer: Temperature Calibration A.2) Direct Method: Harmonic Oscillation Densitometry
● 0.001 must be subtracted from the SG reading for every ● uses sound waves to measure urine density
3ºC that the specimen temperature is below the ● during testing:
urinometer calibration temp ○ portion of the urine sample is held in a U- shaped
● 0.001 must be added to the SG reading for every 3ºC glass tube
that the spx measures above calibration temp ■ electromagnetic coil on one end
Example: ■ motion detector on the other end
● Specific gravity reading: 1.035 ● electrical current applied to the coil generates a sound
● Urine Temperature: 23ºC wave of fixed frequency
● Corrected Specific Gravity reading: ● sonic oscillation
○ 1.035 + 0.001 = 1.036 ○ transmitted through the specimen
NOTE: 0.001 is added to the original specific gravity since ○ decrease in frequency is measured
the urine temperature is 3ºC higher than the calibration ● change in frequency (oscillating cycle period) of the
temperature of the urinometer (20ºC) sound wave entering the urine sample = directly
Glucose & Protein Correction proportional to the sample density
● each gram per deciliter (g/dL) of protein present, the ● microprocessor converts the frequency to a
specific gravity is increased by 0.003 corresponding specific gravity value
● each gram per deciliter (g/dL) of glucose, the specific ● temperature affects density
gravity is increased by 0.004 ● thermistor
○ monitors the sample temperature in the tube →
Example: microprocessor for correction
● Specific gravity reading: 1.035
● rarely used today despite its ability to accurately and
● Urine protein: 1 g/dL
● Urine glucose: 1 g/dL precisely determine urine specific gravity with linearity
● Corrected Specific Gravity: 1. 035 - 0.004 - 0.003 up to 1.080
= 1. 028
SPECIFIC GRAVITY: INDIRECT METHODS
Note: 0.003 is subtracted to correct for the presence of 1 ● involve measurement of dimensions
g/dl protein and 0.004 is subtracted to correct for the ○ refractive index & ionic charges to determine urine
presence of 1 g/dl glucose
density
● spx with specific gravity readings greater than the B.1) Indirect Method: Refractometry
urinometer scale can be diluted & retested
● measurement of specific gravity based on the
○ only the decimal portion of the observed specific
gravity is multiplied by the dilution factor refractive index of urine
○ e.g. a specimen diluted 1:2 with a reading of 1.025 ● measures ALL the solutes in a solution (including
would have an actual specific gravity of 1.050 glucose & protein)
● when using the urinometer, if correcting the dilution and ● when light passes from air into a solution at an angle →
presence of sugar or protein is required at the same direction of the light beam is refracted → ⬇speed
time: ● specific gravity scale is calibrated in terms of the angles
○ dilution must be corrected 1st
at which light passes through the specimen
○ 2nd is correcting the reading for the presence of
sugar or protein ○ ⬆no. of solutes = ⬇velocity of light = ⬇angle of
● urinometers must be calibrated daily light refraction
○ calibration will not address the inaccuracy of
measurement generated by using the urinometer

Urinometer: Solutions Used for Calibration

Solution Specific Gravity Readings
Pure Distilled Water 1.000
Sodium Chloride
1.018
Solution (2.5 g/dl)
Sodium Chloride
1.035
Solution (5 g/dl)
Sodium Chloride
1.051
Solution (7.5 g/dl)

Transcribed by: Abenes, Aliping, Guarin, Villanueva (2024). 14

●
● advantages:
○ requiring a small volume of specimen (10 ul) Refractometry: Solutions Used for Calibration
○ temperature corrections are not necessary Solution Specific Gravity Readings
● light beam → temperature-compensating liquid → NaCl, 0.513 mol/L ● 1.015 ± 0.001
specific gravity scale (3% w/v)
● temperature is compensated between 15° C & 38° C NaCl, 0.856 mol/L ● 1.022 ± 0.001
(5% w/v)
● comparison of the velocity or angle of Sucrose, 0.263 mol/L ● 1.034 ± 0.001
Refractive Index refraction of light in a solution vs. with (9% w/v)
that of the velocity of light in air
Concentration of ● determines the velocity and angle at Procedure: Calibration Of Refractometer
Dissolved which light passes through a solution 1 ● The calibrating solution is placed onto the prism.
Particles
● used to direct a specific ● The refractometer should yield the expected specific
2
(monochromatic) wavelength of gravity as per weight over volume of solution.
Prism
daylight against a manufacturer ● If the refractometer does not provide the same
calibrated specific gravity scale 3 reading, the calibration screw is adjusted until the
Concentration of ● determines the angle at which the light desired reading is achieved.
the Specimen beam enters the prism

Refractometry: Temperature Calibration

● refracted light beam from the sample → reservoir
● light beam is corrected to the value that would be
obtained at a temperature of 20°C
● refracted light beam → measuring prism before the lens
focuses it onto the calibrated scale

● direct specific gravity measurements

○ refractometer results are also ⬆ by the presence of
high molecular weight solutes
■ glucose
■ protein
■ mannitol
B.2) Indirect Method: Reagent Strips
■ radiographic media
● ionic specific gravity which reflects the concentrating
○ by refractometry
ability of the kidneys to selectively reabsorb & secrete
■ each gram per deciliter (g/dL) of protein
ionic solutes and water
present, the specific gravity is increased by
● indirect colorimetric method for estimating urine density
0.003
based on the quantity of ionic or charged solutes
■ each gram per deciliter (g/dL) of glucose, the
○ nonionic solutes are not measured
specific gravity is increased by 0.004
Ionic Solutes Nonionic Solutes
Corrections To ● results when protein and glucose are ● sodium ● urea
Urine present ● chloride ● glucose
Refractometer ● same method as that of the urinometer ● potassium ● protein
Dilution ● follows the same method as that of ● ammonium ions ● radiographic contrast media
Correction urinometer
● each strip consists of chemical-impregnated
● calibration assessment absorbent pads attached to a plastic strip
○ checked daily ○ color-producing chemical reaction takes place when
○ determining the specific gravity of distilled water the absorbent pad comes in contact with urine
(1.000) ○ reactions are interpreted by comparing the color
○ one or two additional solutions are analyzed to produced color on the pad with a chart supplied by
ensure calibration across the range of possible the manufacturer
urine specific gravity values

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●
● specific gravity & pH ● If the refractometer does not provide the same
○ reported numerically 3 reading, the calibration screw is adjusted until the
● urine chemicals desired reading is achieved.
○ semiquantitative value of trace ● Upon getting the reagent strip, dip it immediately for
○ 1+, 2+, 3+, or 4+ about 1-2 second onto the urine sample. Allowing
● estimate of the mg/dL present is available for 4 the strip to remain in the urine for an extended period
appropriate testing area of time may cause leaching of reagents from other
Principle: Reagent Strips pads

● based on the change in pKa (dissociation constant) of a ● Remove the excess urine in the strip by blotting the
polyelectrolyte in an alkaline medium edge of the strip on absorbent pad and holding the
○ the polyelectrolyte ionizes, releasing hydrogen ions in strip horizontally. Excess urine remaining on the strip
5
proportion to the number of ions in the solution after its removal from the specimen can produce a
○ reagent test pad (polyelectrolyte) runover between chemicals on adjacent pads,
● ⬆ionic polyelectrolyte → ⬇pH → color change producing distortion of the colors.
○ the higher the concentration of urine, the more ● Allow maximum reaction time to proceed. For most
hydrogen ions are released, thereby lowering the pH reagent strip pads, the maximum reaction
● incorporation of the indicator proceeds for 30-60 seconds, excepts for leukocyte
bromothymol blue on the reagent pad 6
esterase pad which proceeds for 120
measures the change in pH
[Link], specific gravity pad proceeds
pH of the ● as the specific gravity increases, the
indicator changes from blue (1.000 for 45 seconds.
Test Pad ⬇:
[alkaline]), through shades of green, to ● Compare the color change to the color chart
yellow (1.030 [acid]) 7
provided, holding the strip in a horizontal position
● readings can be made in 0.005 intervals by
8 ● Interpret results not longer than two (2) minutes.
careful comparison with the color chart.

● as the number of ions present in the urine ⬆ → more

protons are released from the polyelectrolyte → ⬇ test
pad pH & change in the indicator
● reagents strip specific gravity measurements
○ affected by urine pH
○ acidic urine → falsely increased results
○ highly buffered alkaline urine → falsely
decreased results
● when reagent strips are read by a reagent strip reader,
the specific gravity reading is automatically corrected by
the instrument
● most accurate results are obtained when the urine pH is
7.0-7.5

Procedure: Reagent Strips

● Mix the specimen well. Formed elements such as
1 red and white blood cells sink to the bottom and will
be undetected in an unmixed specimen.
● Uncap the reagent strip canister and get 1 reagent
strip needed for testing. Make sure to recap
immediately as to prevent the other reagent strips
2
from being exposed to air and light.
● Prolonged exposure will affect the sensitivity of the
reagent strips.

Transcribed by: Abenes, Aliping, Guarin, Villanueva (2024). 16

●
 IV. URINE FOAM V. URINE ODOR
● preliminary & supportive evidence for the presence of ● normal urine odor
bilirubin and protein ○ aromatic odor
○ bilirubin ○ faint & unremarkable
○ odorous & ammoniacal
● detected & confirmed during the chemical examination
■ if left to stand in room temp too long
● shaken or agitated normal urine: ■ urea → ammonia (by bacteria)
○ white foam can be forced to develop ● urine in the urinary tract is sterile → urine in urethra is
○ readily dissipates on standing easily contaminated by normal bacterial flora
● urine foam characteristics: ● urine odor may indicate:
○ color ○ spx is old
○ ease of formation ○ spx is not suitable for testing
● px with UTI
○ amount produced
○ produce ammonia-smelling urine
○ bacterial metabolism in the urinary tract
Bilirubin Protein ○ severe UTI
● yellow foam ● stable white foam ■ strongly pungent / fetid aroma from pus,
● ⬆amounts of carotene ● moderate to large amounts protein, decay, bacteria
(readily dissipates) of protein (albumin)
Urine Odor
● observed in diabetes mellitus px
● other causes:
○ starvation
Sweety / Fruity Urine ○ profuse vomiting
○ strenuous exercise
○ diarrhea
○ malnutrition
Mousy / Barny Odor ● observed in phenylketonuria
Maple Syrup Odor ● maple syrup disease
● px w/ routine diet involving
○ garlic
Pungent Odor
○ onions
○ asparagus
● tyrosinemia
Rancid Urine ○ genetic disorder characterized by
problems breaking down tyrosine
● isovaleric
○ body unable to process leucine
Sweaty-Feet Odor ● glutaric acidemia
○ body is unable to process certain
proteins properly
● trimethylaminuria
Rotting / Old Fish
○ body is unable to break down
Odor
trimethylamine
Cabbage like / Hop
● methionine malabsorption
like / Grass like Odor
● may be intentionally added to
interfere with the detection of
Bleach Smell / other prescription or illicit drugs
Cleaning Agents ● household container is used to
collect a spx
● not acceptable for urinalysis

Transcribed by: Abenes, Aliping, Guarin, Villanueva (2024). 17

●
 ● decrease in urine excretion < 400 mL/day
● ⬇ renal blood flow
○ dehydration
○ hypotension
Oliguria ● other causes:
○ urinary tract obstruction
○ renal tubular dysfunction
○ end stage renal disease
○ edematous nephrotic syndrome
● complete lack of urine excretion
● < 100 mL/day for 2-3 consecutive days despite
fluid intake
● conditions that destroys functioning renal
tissue
● decrease the blood supply to the kidneys:
○ heart failure
○ hypotension
Anuria
○ hemorrhage
○ shock
● toxic chemicals and nephrotic antibiotics
○ induce acute tubular necrosis
○ leads to loss of functional renal tissue &
anuria / oliguria
● also caused by hemolytic transfusion reactions
& urinary tract obstruction

VII. URINE TASTE

● historically, urine was tasted to detect the presence of
VI. URINE VOLUME
urinary sugars
● valuable diagnostic aid to assess renal function
● taste of urine of a diabetic px
● normal urine volume: 600 - 1800 mL/day
○ diabetes mellitus (sweet)
○ < 400 mL excreted at night
○ diabetes insipidus (tasteless)
● urine volume is affected by:
○ individual’s diet
○ health condition
○ exercise

Urine Disorders
● excretion of urine at night > 500 mL
● associated w/ chronic progressive renal
Nocturia
failure
○ kidneys fail to concentrate urine
● excretion of > 2.5L urine each day
● diuresis
○ any increase in urine excretion
● can be due to:
○ excessive water intake (polydipsia in DM)
○ diabetes insipidus (DI)
Polyuria
○ diuretic therapy
○ excessive caffeine intake
○ excessive alcohol intake
○ hormonal imbalance
○ renal disease
○ lithium treatment

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●
ANALYSIS OF URINE MLS 321

& OTHER BODILY FLUIDS 8438

LECTURE BS MLS 3 PRELIM
M3: CHEMICAL EXAMINATION OF URINE

UNIT 1: URINE pH AND PROTEIN Acidic Urine: pH 4.5 – 6.9

● high protein diet
pH ● cranberry ingestion
● “potential of hydrogen” ● metabolic acidosis
● numerical reporting ● respiratory acidosis
● kidneys are the major regulators of the acid-base ● urinary system disorders
content in the body ○ UTI caused by acid producing bacteria (e.g. E. coli)
○ secretion of H+ (i.e. ammonium ions, H phosphate, ● medications
weak organic acids) ○ e.g. vitamin C
○ reabsorption of bicarbonate from the filtrate in the
convoluted tubules Neutral or Alkaline Urine: pH 7.0 – 7.9
● metabolic alkalosis
PHYSIOLOGICALLY ACCEPTABLE URINE pH ● respiratory alkalosis
● physiologically acceptable urine pH = 4.5 - 8.0 ● increased vegetables and citrus fruits
○ low CHO diet
First Morning Urine: 5.0 - 6.0 ● urinary system disorders
● ideal sample ○ UTI caused by urease producing organisms
● mostly acidic due to the endogenous production of acid ■ e.g. Proteus, Pseudomonas
○ maintains the integrity of cells ● medications
○ since at alkaline pH ○ sodium bicarbonate
■ cells lyse (i.e. RBCs, WBCs) ○ potassium citrate
■ casts disintegrates

Alkaline Tide
● a situation when the urine pH becomes less acidic
during and after a meal
● due to the increased production of bicarbonates by the
parietal cells

pH CHANGES THROUGHOUT THE DAY

● urine pH normally changes throughout the day due to
diet and physical activity

Acidic to Alkaline Alkaline to Acidic

● prevents stone formation ● prevents stone formation of
of: alkaline-precipitating
○ calcium oxalate solutes
○ uric acid ○ calcium carbonate
○ cystine crystals ○ calcium phosphate
● inhibits the development of
● enhances the excretion of
UTI
various drugs
○ disrupts the alkaline
○ sulfonamides
environment of
○ streptomycin
UTI-causing organisms
○ salicylate
(e.g. Proteus)

Transcribed by: Abenes, Agtarap, Alacar, Aliping, Guarin, Villar, Villanueva (2024). 1
 CLINICAL SIGNIFICANCE: URINE pH PROTEINS
● assessing and managing disease ● protein determination is the most indicative of renal
○ aids in determining systemic acid-base disorders of disease among routine urine chemical tests
metabolic or respiratory origin
● proteins are ideally not excreted
○ aids diagnosis of a particular disease
■ e.g. renal tubular acidosis ○ except ≈ 10 mg/dL low MW proteins
● precipitation of substances associated with stone ● normally excreted = 150 mg/day or 1-14 mg/day
formation depends on urinary pH ○ 1/3 = albumin
○ e.g. calcium oxalate ■ major serum protein in normal urine
■ a frequent constituent of renal calculi, ■ 0.1% of albumin may pass through GFB
precipitates primarily in acidic urine ■ normal urinary albumin content since much of
■ thus, maintaining urine at an alkaline pH
filtered albumin is reabsorbed by the tubules
prevents calculi formation
○ knowledge of urinary pH is important in the ○ 2/3 = globulin
identification of crystals observed during ● 95-99% of all the filtered proteins are reabsorbed
microscopic examination of urine sediment ● most protein originate from the ultrafiltration of plasma
○ patients with stone formation history can use their and from the urinary tract itself
urine pH info to modify their diets if necessary ○ other proteins include
● determine specimen suitability ■ small amounts of serum and tubular
○ acceptable urine = pH 4.5 – 8.0
microglobulins
Not Physiologically Acceptable Urine Ph ■ proteins from prostatic, seminal and vaginal
Less Than pH 4.5 secretions
● possibly contaminated with acid agents Protein From The Urinary Tract
● reject and recollect ● Tamm-Horsfall Protein
Greater Than pH 8 Uromodulin ● synthesized by the distal tubular cells
● improperly preserved ● involved in cast formation
○ delayed transportation ● fibrinolytic enzyme secreted by the
○ refrigeration = most common method of preservation tubular cells
Urokinase
■ expect amorphous materials to precipitate ● plasminogen activator to plasmin for
■ affects clarity and microscopic findings proteolysis/thrombolysis
● adultured specimen = contaminated ● synthesized by the renal tubular
● administration of highly alkaline substance IgA
epithelial cells
● collecting specimens in a container other than the
single-use laboratory-supplied containers may contain
alkaline detergent

Summary of pH Values
Physiologically Acceptable 4.5 - 8.0
First Morning Urine 5.0 - 6.0
Acidic Urine 4.5 – 6.9
Neutral or Alkaline Urine 7.0 – 7.9

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Proteinuria (≥30 mg/dL) RENAL PROTEINURIA
● increased protein (albumin) in urine ● may result from either
● may result from an ○ glomerular damage = most common & clinically
○ increased filtered plasma proteins serious
○ reduced reabsorptive ability of renal tubules ○ tubular damage
● increased amount of protein is presented to the tubules
for reabsorption = tubules randomly reabsorb the Glomerular Proteinuria
protein in a rate-limited process ● damage to the glomerular filtration barrier
○ thus, as the quantities of proteins other than ○ altered shield of negativity
albumin increase, the amount of albumin excreted ○ allows excretion of ALMOST ALL PROTEINS
in the urine also increases since the other proteins ○ 2.5 – 20 g/day of proteins excreted
compete for reabsorption ● albumin is the primary protein increased
● the first indicator of renal disease ○ allows other moderate-MW proteins to be excreted
● categories of proteinuria ■
○ prerenal or overflow proteinuria ● selective glomeruli = able to retard the passage of
○ glomerular proteinuria high-MW proteins even when damaged
○ tubular proteinuria ○ alpha-antitrypsin
○ postrenal proteinuria ○ alpha-acid glycoprotein
○ transferrin
PRE-RENAL OR OVERFLOW PROTEINURIA ● nonselective glomeruli = discrimination is lost and
● caused by conditions affecting the plasma concentration high-MW proteins are allowed into the ultrafiltrate
of protein prior to it reaching the kidneys ● can develop into nephrotic syndrome
○ thus, not indicative of actual renal disease ○ a complication of numerous renal disorders
○ occurs in the absence of renal disease ■ massive proteinuria that exceeds ≈ 3.5 g/day
● due to increased plasma proteins in the blood readily ■ hypoalbuminemia
passing the glomerular filtration barriers into the urine ■ hyperlipidemia
○ LMW plasma proteins ■ lipiduria (increased lipids in urine)
■ hemoglobin = following a hemolytic disease ■ generalized edema
■ myoglobin = after a muscle injury
■ acute phase reactants = associated w/ infection Primary Glomerular Diseases
and inflammation ● glomerulonephritis ● sickle cell anemia
○ Ig paraproteins (Kappa & gamma light chains) ● glomerulosclerosi ● kidney transplant rejection,
■ e.g. Bence Jones protein ● minimal change disease ● infectious diseases
○ minimal amount of albumin ● poststreptococcal ○ malaria
● increased plasma proteins → increased amount of ● glomerulonephritis ○ hepatitis B
proteins filtered → reaches the maximum renal ● diabetes mellitus ○ bacterial endocarditis)
threshold of proteins → excreted ● lupus erythematosus ● preeclampsia
● transient ● amyloidosis ● toxins (e.g. heavy metals)
○ can be corrected once underlying cause is identified ● cancer (leukemia & ● drugs (e.g. lithium)
lymphoma)
Bence Jones Proteins
● abnormal protein in persons w/ multiple myeloma
Functional Proteinuria (<1g/day)
● LMW protein filtered in quantities exceeding the tubular
reabsorption capacity and is excreted in the urine ● caused by mild glomerular or mixed pattern of
● does not coagulate and remain coagulated when proteinuria in the absence of renal disease
exposed to heat like other proteins ● primary mechanisms
○ coagulates at 400ºC – 600ºC ○ changes in glomerular blood flow (due to renal
○ dissolves at 1000ºC vasoconstriction)
● suspected → perform screening test that uses the ○ enhanced glomerular permeability
unique solubility characteristics of the protein ● glomerular changes are transitory and may resolve with
○ appears turbid between 400ºC and 600ºC time and with proper treatment
○ clear at 100ºC ● associated conditions
○ interference due to other precipitated proteins can ○ strenuous exercise
be removed by ○ fever
■ filtering the specimen ○ extreme cold exposure
■ observing the specimen for turbidity as it cools ○ emotional distress
from 400ºC – 600ºC ○ congestive heart failure
○ dehydration

Transcribed by: Abenes, Agtarap, Alacar, Aliping, Guarin, Villar, Villanueva (2024). 3
 Postural / Orthostatic Proteinuria (<1.5g/day) POST RENAL PROTEINURIA
● type of functional proteinuria ● results from an inflammatory process anywhere in the
● urinary excretion of protein only when the individual urinary tract
is in an upright (orthostatic) position/vertical posture ○ renal pelvis
● prolonged standing → increased renal venous pressure ○ ureters
→ causes renal congestion and glomerular changes ○ bladder
● benign; may disappear when the posture is corrected ○ prostate
● persistent proteinuria may develop ○ urethra
● glomerular abnormalities may develop ○ external genitalia
● suspected → submit two specimens ● inflammation of bacterial or fungal origin
○ 1st spx = first morning urine ○ produces secretions containing protein from the
○ 2nd spx = after the px has been in an upright position interstitial fluid
for several hours ○ these proteins are added to the urine as it passes
through the lower urinary tract
Probable Orthostatic ● (+) first morning (+) postural
● contributes to the presence of protein
Proteinuria ● (-) first morning (+) postural
○ presence of blood as a result of injury, hemorrhage
or menstrual contamination
Tubular Proteinuria (<2.5g/day) ○ presence of prostatic fluid, large amounts of
spermatozoa and vaginal secretions.
● defect in tubular reabsorption of proteins
● plasma proteins that are normally reabsorbed are
increasingly excreted in urine but does not approach NOTE:
the level found in glomerular proteinuria ● proteinuria can occur during pregnancy
○ β2-microglobulin ○ usually transient but ALARMING
○ retinol-binding protein ○ excretion may be associated with renal infection or
○ α2- microglobulin possible pre-eclamptic toxemia
○ lysozyme ○ pre-eclamptic toxemia ≈ protein level of 3 g/day
○ albumin ○ normal pregnancy = minor increase of 300 mg/day
● can occur alone or with glomerular proteinuria ● onset of renal complications can be predicted by the
○ e.g. chronic renal disease, with exposure to toxic occurrence of microalbuminuria (30-300 mg/ day)
substances & heavy metals as predisposing factors ○ described as a moderate increase in the level of
albumin in urine
Causes of Tubular Proteinuria ○ presence of microalbumin in urine has been
● acute/chronic ● Fanconi’s syndrome associated with:
pyelonephritis ● hemoglobinuria ■ hypertension
● interstitial nephritis ● myoglobinuria ■ diabetes mellitus
● renal tubular acidosis ● kidney transplant rejection ■ cardiovascular disease
● renal tuberculosis ● strenuous exercise
● systemic diseases ● drugs Summary of Protein Values
○ sarcoidosis ○ aminoglycosides Condition Amount of Protein Excreted
○ lupus erythematosus ○ sulfonamides ● 150 mg/day
○ cystinosis ○ penicillin Normal
● 1-14 mg/day
○ galactosemia ○ cephalosporins
○ Wilson’s disease Low MW proteins ● 10 mg/dL
Proteinuria ● ≥30 mg/dL
Fanconi’s Syndrome Glomerular Proteinuria ● 2.5 – 20 g/day
● proximal tubular dysfunction Functional Proteinuria ● <1g/day
● characterized by Postural Proteinuria ● <1.5g/day
○ aminoaciduria Tubular Proteinuria ● 1<2.5g/day
○ proteinuria
○ glycosuria
○ phosphaturia
● still retains normal glomerular function even if tubular
functions are altered

Transcribed by: Abenes, Agtarap, Alacar, Aliping, Guarin, Villar, Villanueva (2024). 4
 UNIT 2: GLUCOSE, OTHER REDUCING SUGARS ● liver disease
AND KETONES ● pancreatic damage
● stroke
GLUCOSE ● increase epinephrine & glucocorticoids
● glucose test Other ● meal containing a high glucose content
○ included in all physical exams (urine & blood spx) Conditions
○ detection and monitoring of DM ● increased growth hormone
of
○ performed most frequently in urine Acromegaly which contributes to
Nondiabetic
○ reagent strip method hyperglycemia
Origin
■ px are able to monitor glucose at home Hyper- ● increased thyroxine which
thyroidism promotes hyperglycemia
CLINICAL SIGNIFICANCE: GLYCOSURIA Cushing’s ● increased cortisol that also
● glucosuria / glycosuria disease promotes hyperglycemia
○ glucose in urine ≥ 20 mg/dL
● normally, glucose that passes through the GFB → Renal Glycosuria
ultrafiltrate is actively reabsorbed (PCT) ● defective tubular reabsorption
● tubular reabsorption of glucose ○ end-stage renal disease
○ threshold-limited process ○ cystinosis
○ maximum reabsorptive capacity (Tm) ○ Fanconi syndrome
● ultrafiltrate conc. of glucose exceeds the reabsorptive ○ heavy metal poisoning
ability of the tubules → glucosuria ● possible for an individual to have hyperglycemia w/ out
glycosuria
Maximum Reabsorptive Capacity (Tm) ● glucose freely passes through the glomerular filtration
Average 350 mg/min, barriers → damaged GFB → glomerular filtration rate is
decreased
Female 250 - 360 mg/min
○ hyperglycemia can be present, but because of a
Males 295 - 455 mg/min decreased glomerular filtration rate, only limited
Glucose Renal Threshold 160 to 180 mg/dL amounts of glucose are able to pass into the
ultrafiltrate
Pre Renal Glycosuria ● tubules are able to reabsorb all the glucose presented
to them, and glycosuria is not present
● does not involve renal disease yet ● renal arteriosclerosis
○ low cardiac output
Diseases & Conditions Involving Pre Renal Glycosuria ○ result in hyperglycemia w/ out glucosuria
○ Decreases GFR
● most common pre-renal disease that
● also occur in the absence of hyperglycemia when the
causes glycosuria reabsorption of glucose by the renal tubules is
● characterized by hyperglycemia compromised
○ increased plasma glucose
Diabetes ○ ineffective glucose utilization caused by
Mellitus (DM) inadequate insulin secretion or abnormal
insulin action
○ uncontrolled DM px have blood glucose
levels that exceed the renal threshold
level, and thus glycosuria occur
Gestational
● also presents glycosuria during pregnancy
DM (GDM)
Glycosuria
● during pregnancy
NOT
● occasionally seen as a result of a
associated
temporary lowering of renal threshold
w/ GDM

Transcribed by: Abenes, Agtarap, Alacar, Aliping, Guarin, Villar, Villanueva (2024). 5
 OTHER REDUCING SUGARS KETONES
● other sugars such as galactose, fructose, lactose,
maltose or pentose can also appear in urine FATTY ACID METABOLISM INTO KETONE BODIES
● not normally present unlike glucose ● ketones are formed during the catabolism of fatty acids
● one of the intermediate products of fatty acid
Other Sugars Not Normally Present in Urine breakdown is acetyl CoA
● most significant excretion
Galactose
● indicates a severe condition = galactosemia Acetyl CoA & Oxaloacetate
● disaccharide of D-galactose & D-glucose ● 1st step in Krebs: acetyl CoA + oxaloacetate → citrate
● present in milk
● principal dietary source of galactose If fat & CHO degradation ● acetyl CoA enters the citric
Lactose
● may be found in the urine of either are balanced: acid cycle (Krebs cycle)
○ pregnant women ● all available oxaloacetate will
○ premature infants If CHO is not available or
be used to form glucose
● rarely in urine is not being properly
● thus, condensation with
● due to either utilized
acetyl CoA does not occur
○ excessive fruit or honey ingestion If CoA cannot enter the ● it is diverted to the formation
Fructose
○ essential fructosuria Krebs cycle: of ketone bodies in the liver
■ a rare genetic defect involving hepatic
fructokinase deficiency.
Ketone Bodies
● xylose and arabinose
● due to either ● are acetoacetic acid (diacetic acid), beta-hydroxybutyric
○ excessive fruit ingestion acid, and acetone
Pentose ■ e.g., plums, cherries ○ acetoacetic acid
○ rare genetic defect involving deficiency of ■ the first ketone formed from acetyl CoA
enzyme that catalyzes the conversion of ■ the other ketones are formed from acetoacetic
1-xylulose into xylitol acid
○ hydroxybutyric acid
■ formed by reversible reduction and
CLINICAL SIGNIFICANCE: GALACTOSEMIA
○ acetone
● an inherited disorder that begins at birth ■ formed by a slow spontaneous decarboxylation
● inability to metabolize galactose to glucose due to ● acetoacetic acid and beta-hydroxybutyric acid as
the reduction or absence of an enzyme required for sources of energy
galactose metabolism ○ are normal fuels of respiration
● detection in urine among newborn is necessary ○ heart muscle and the kidneys, particularly the renal
cortex prefer to use acetoacetate instead of glucose
○ since galactosemia has serious complications and
○ glucose is still the major fuel of the brain in
is life threatening well-nourished individuals
Galactose ● most common enzyme deficiency ■ even if the brain can adapt to utilize
acetoacetate in the absence of glucose
1-phosphate involved
Uridyl ● resulting to accumulation of galactose
Detection of Ketones
Transferase in the blood since it is not converted to
● normally small amounts of ketones are present in the
(GALT) glucose blood = 2–4 mg/dL
● galactonate accumulates and causes ○ 20% acetoacetic acid
vomiting and diarrhea ○ 2% acetone
○ the infant fails to thrive ○ 78% beta- hydroxybutyric acid
Classical ○ hepatomegaly and jaundice occur ● measurable amounts of ketones do not appear in the
urine in most instances
Galactosemia ● galactitol is also formed
○ since all the metabolized fat is completely broken
○ causes cataract formation down into CO2 and water
● both of these substances may cause ● body stores of fat are metabolized to supply energy
irreversible brain damage when the use of available CHO as the major source of
● galactose can be eliminated from the diet if these energy becomes compromised
○ hence, ketones are formed and detected in urine
conditions are recognized early, resulting in normal
when in excess
growth and development

Transcribed by: Abenes, Agtarap, Alacar, Aliping, Guarin, Villar, Villanueva (2024). 6
 NOTE:
● the pattern of ketone formation is the same regardless
of the initiating condition
● CHO are the primary sources of energy
○ inappropriate use or loss of CHO → fat
metabolism increases to compensate for the energy
requirement of the body → ketones accumulate in
the patient’s blood → decreased pH and
bicarbonate

Increased Excretion of Water

● the body eliminates these ketones through increased
excretion of water
● if this is not corrected, it may cause:
○ large quantities of electrolytes are lost in the urine
causing dehydration
○ a chemical imbalance results in acidosis and
potentially diabetic coma
● this condition is characteristically preceded by
○ polyphagia
○ polydipsia
○ polyuria
○ complaints of fatigue, nausea, and vomiting

CLINICAL SIGNIFICANCE: KETONURIA

● any condition that causes increased fat metabolism can
result in ketonemia and ketonuria
● ketonemia
○ increased ketone concentration in the blood
○ > 3 mg/dL
○ may result to ketonuria when the blood ketone level
exceeds the renal threshold = 70 mg/dL
● ketonuria
○ increased excretion of ketones
○ 20 mg/day

Conditions Causing Increased Fat Metabolism

● when the body is unable to use carbohydrates Summary of CHO Values
○ diabetes mellitus Condition Amount Excreted
● if there is an inadequate carbohydrate intake
Glucosuria / Glycosuria ● ≥ 20 mg/dL
○ starvation
○ alcoholism Normal Ketones ● 2–4 mg/dL
○ strenuous exercise Ketonemia ● > 3mg/dL
○ cold exposure Ketonuria ● 20 mg/day
○ acute febrile illnesses
● if there is loss of carbohydrates Glucose Renal Threshold ● 160 to 180 mg/dL
○ frequent vomiting Ketone Renal Threshold ● 70 mg/dL
○ defective renal reabsorption of ketones
○ digestive disturbances causing increased
absorption of ketones

Transcribed by: Abenes, Agtarap, Alacar, Aliping, Guarin, Villar, Villanueva (2024). 7
 UNIT 3: BLOOD, PORPHOBILINOGEN, Porphyrin Formation
BILIRUBIN AND UROBILINOGEN ● PBG is formed when 2 molecules of δ-ALA condensed
in a reaction catalyzed by ALA dehydratase
PORPHOBILINOGEN ● 4 molecules of porphobilinogen (PBG) condense to
form the → uroporphyrinogen (UPG) [first
HEME SYNTHESIS porphyrinogen] → coproporphyrinogen (CPG) →
● heme: primary component of Hgb that allows it to carry protoporphyrinogen
oxygen ● porphyrinogens can oxidize spontaneously and
irreversibly to form their respective porphyrins
○ UPG oxidized → uroporphyrin
○ CPG oxidized → coproporphyrin
○ protoporphyrinogen oxidized → protoporphyrin
● porphyrins cannot enter the heme-synthetic pathway &
has no biologic function → excreted in urine
● heme-synthesis is regulated so that only trace amounts
of porphyrin precursors (ALA, PBG) are formed
● normal amount excreted in the urine:

ALA <1.4 mg/dL

PBG <0.4 mg/d

● if heme-synthesis is disrupted → porphyrin precursors

(ALA, PBG) accumulate → increase amounts will be
detected in urine

Porphyrin
● porphyrin precursors
○ porphobilinogen
○ δ-aminolevulinic acid [ALA]
● porphyrins
○ uroporphyrin
○ coproporphyrin
○ protoporphyrin)
Porphobilinogen (PBG) ● intermediate compound
● a porphyrin precursor important in the formation of
heme
CLINICAL SIGNIFICANCE: PORPHOBILINOGEN
● a rate-limiting of the synthesis of heme is the reaction
catalyzed by aminolevulinic acid synthase (ALA
synthase) Acute Intermittent Porphyria
● condition characterized by the deficiency of enzyme
Sufficient Heme ● pathway is retarded / inhibited porphobilinogen deaminase
● pathway is stimulated to increase ○ causing PBG & ALA to accumulate
Lacking Heme
heme formation ○ heme is not being formed to inhibit the pathway →
overstimulation of porphobilinogen deaminase →
● if an enzyme required in the pathway is either absent,
overproduction of porphyrin precursors
decreased, defective, or inhibited → compound
● porphyrin precursors
preceding the action of the enzyme accumulates
○ low renal threshold = rapidly removed from
○ e.g. deficiency of ALA dehydratase → ALA will
bloodstream → appear in urine
accumulate because it is not converted to PBG
○ no screening procedure for ALA is currently
available, only for PBG

Transcribed by: Abenes, Agtarap, Alacar, Aliping, Guarin, Villar, Villanueva (2024). 8
 Porphyrin Precursor Screening Methods HEME CATABOLISM
● pink/ red urine = blood/ Hgb in urine ● heme undergoes catabolism to form bilirubin and
● (–) blood = possibly porphyria eventually urobilinogen when released in peripheral
● PBG usually colorless & non fluorescent tissues
○ photooxidation of PBG →
porphobilin (pigment)
Physical ■ reddish appearance
Examination ■ depends on the concentration &
hydration of the px
NOTE:
● other chromogens normally present in
urine can modify the color observed
● screening method that can detect as low
as 2 mg/dL PBG in urine
● based on Ehrlich’s rxn
○ inverse ratio of urine volume to reagent
Hoesch
○ creates a highly acidic mixture
Test
preventing the interference of
urobilinogen
○ deep pink or red color = disperses
uniformly in the urine when shaken
● modification of Erhlich’s reaction
● solubility characteristics of
porphobilinogen w/ respect to pH and
solvent type
● red color is observed identifies the
substance present
● positive (+) for PBG
○ chloroform extraction
■ red color (top)
○ butanol extraction
■ red color (bottom)
Watson-
Schwartz
BLOOD
Test
● even a small increase in urine RBC is clinically
significant even though it may not be visually evident
● quantity of RBC in urine has NO correlation w/ severity
○ cannot pinpoint exact location of bleeding

CLINICAL SIGNIFICANCE: BLOOD

Hematuria
● increased presence of intact RBCs in urine
● commonly observed in cases of
○ kidney or urinary tract diseases
■ glomerulonephritis
■ pyelonephritis
■ cystitis
■ renal stones
■ tumors
○ trauma
○ drug therapy

Transcribed by: Abenes, Agtarap, Alacar, Aliping, Guarin, Villar, Villanueva (2024). 9
 ○ strenuous exercise Other Causes of Hemoglobinuria
○ hypertension
● extensive burns ● long distance running
○ smoking
● may also be due to medications ● malaria ● karate
○ cyclophosphamide ● syphilis ● chemical toxicity
○ anticoagulants ● exertional hemolysis ○ copper, nitrites, nitrates
associated to marching
Hemoglobinuria
● increased presence of Hgb in urine
● pseudohemoglobinuria Differentiating Hematuria vs Hemoglobinuria
○ hemoglobinuria secondary to hematuria Parameter Hematuria Hemoglobinuria
○ intact red cells that pass through the urinary tract ● clear red
● cloudy red
undergoes lysis in the urine, liberating Hgb ○ red sediment
Clarity ○ remains red after
○ hemolysis occurs after passing through glomerulus button after
centrifugation
○ much more common than true hemoglobinuria centrifugation
● true hemoglobinuria Microscopic ● intact RBCs –
○ caused by the presence of free Hgb as a result of ● true = pink to red
intravascular hemolysis Serum Color ● normal; yellow ● pseudo = color not
○ lysis occurs before exiting the glomerulus affected
● conditions causing hemolytic episodes
NOTE:
○ transfusion reactions
○ hemolytic anemia ● it is still best to compare urine and plasma values of
○ paroxysmal nocturnal hemoglobinuria analytes to confirm or monitor hemolytic episodes
Differentiating Mild from Severe Hemolytic Episodes
Presence of Hemosiderin Granules Using Urine and Plasma Components
● yellow-brown granules Chronic (Mild) Acute (Severe)
● one indicator of true hemoglobinuria Test Normal Value Intravascular Intravascular
Hemolysis Hemolysis
● this should not be the sole basis for confirming a Urine
hemolytic episode because urinary hemosiderin excretion Bilirubin (both types) Absent Absent Absent
is intermittent Normal to
Urobilinogen <0.1 mg/dL Increased
● can be detected by Ross-Test Increased
Blood (Hgb) Absent Absent Present
○ Prussian-blue staining test
Hemosiderin Absent Absent Present
● rationale:
Plasma
○ free Hgb is normally bound to available haptoglobin to B2 <0.2 mg/dL Normal Normal
form a hemoglobin-haptoglobin complex that is too B1 0.8-1.0 mg/dL Increased Increased
large to pass through glomerulus & remains in plasma Haptoglobin 83-267 mg/dL Decreased Absent
■ this complex is removed from circulation and Free Hgb 1-5 mg/dL Normal Increased
metabolized by liver
■ when all haptoglobin is already bound, Hgb freely Myoglobinuria
circulates in the bloodstream ● increased excretion of myoglobin in urine
○ free Hgb readily passes through the GFB → presence ○ normal excretion in urine = less than 0.04 mg/dL
in the ultrafiltrate → reabsorbed in the PCT → ● myoglobin is a compound similar to Hgb as it contains
catabolized to form ferritin in renal tubular cells heme to facilitate transport of oxygen in muscles
○ ferritin → denatured into hemosiderin (insoluble storage ○ it readily passes through the glomerular filtration
form of iron) barrier when present in the bloodstream
■ usually occurs 2-3 days after a hemolytic episode ○ released in the circulation as a result of muscle
damage due to either
■ injury = trauma
■ pathology = ischemia, infections, myopathy,
toxins or convulsions
○ more toxic to the renal tubules than Hgb due to
unclear reasons may be associated with
■ glomerular clearance
■ hydration, hypotension, and aciduria

Transcribed by: Abenes, Agtarap, Alacar, Aliping, Guarin, Villar, Villanueva (2024). 10
● examples of muscle injury BILIRUBIN
○ crush injuries, contact sports and surgery ● orange-yellow pigment
○ muscle ischemia ● imparts a characteristic color in urine and plasma
■ due to carbon monoxide poisoning, alcohol
when produced excessively or when accumulated
intake and illicit drug (heroin)
○ seizures or convulsions ● formed from heme-containing proteins
■ due to an imbalance between energy ○ cytochrome
consumption and production ○ myoglobin
○ myopathy ○ hemoglobin (85%)
■ due to steroids and retroviral agents ● formed primarily from senescent RBCs releasing
● myoglobin is more toxic to the renal tubules than Hgb methemoglobin
○ due to unclear reasons but may be associated with
● excreted into the bile and urine as urobilinogen
■ glomerular clearance
■ hydration, hypotension, and aciduria ● trace amount of bilirubin is normally undetectable
○ urine excretion = 1.5 mg/dL ○ since most routine testing methods are inclined to
■ risk of developing acute renal failure detect significant increases in urinary bilirubin
○ thus, it’s necessary to differentiate myoglobin &
Hgb for diagnosis, determining risk for renal failure Unconjugated (B1) Conjugated (B2)
and treatment ● formed in the kidneys
● initial bilirubin formed in the
Differentiating Hemoglobinuria from Myoglobinuria where it is conjugated with
bloodstream due to rapid
Parameter Hemoglobinuria Myoglobinuria glucuronic acid
reduction of biliverdin
Pink, Red, Brown ○ makes it water soluble
Urine Color Pink, Red, Brown ● water insoluble
(25 mg/dL) ● transported to bile duct and
Blood ● bound to albumin
Positive Positive ultimately the small
Reagent Strip ○ prevents deposition in
intestine against a
Serum Color Pink to Red Pale Yellow tissues where it can be
concentration gradient
Serum Chemistry Test toxic
● if B2 re enters bloodstream
Haptoglobin Decreased to Absent Normal ● transported to kidney
○ easily eliminated in urine
Myoglobin Normal Increased ○ site of conjugation
Free Hgb Increased Normal ○ can readily pass GFB
Creatine Increased Increased
Kinase (<10x upper limit) (>10x upper limit)

UROBILINOGEN
● colorless substance formed from the decontamination
of bilirubin in the intestine
● 20% = reabsorbed
○ most is re-excreted in urine (1mg/dL or less)
● urobilinogen and stercobilinogen in the intestine
○ undergoes spontaneous oxidation → urobilin and
stercobilin → imparts orange-brown color of feces
● urobilinogen present in urine
○ undergoes oxidation → contributes to color of urine
○ tea colored urine = increased urinary urobilinogen
● a portion is reduced to form stercobilinogen

Transcribed by: Abenes, Agtarap, Alacar, Aliping, Guarin, Villar, Villanueva (2024). 11
 ○ ineffective erythropoiesis
■ thalassemia
■ pernicious anemia
● in the peripheral tissues, B1 is bound to albumin and
the complex form cannot pass through the GFB
○ thus, cannot be excreted in urine
○ the liver has a large capacity for conjugation and
would compensate by increasing amounts of B2 →
overflow in the intestine → more urobilinogen
CLINICAL SIGNIFICANCE:
reabsorbed → increase urobilinogen in
BILIRUBIN AND UROBILINOGEN
circulation, plasma ultrafiltrate and finally in the
urine
Normal Bilirubin
● 0.02 mg/dL
in Urine
Normal Urobilinogen ● ≤ 1 mg/dL
in Urine ● 0.5-2.5 mg/day

● bilirubinemia and bilirubinuria

○ early indicators of liver disease, even before other
symptoms such as jaundice manifests
● any disturbance in the metabolic pathway will cause the
accumulation of substances depending on where the
disruption occurred
○ an altered bilirubin metabolism usually results to
changes in
■ urine bilirubin
■ urine urobilinogen
■ fecal color

Pre Hepatic Mechanism

● since there is no liver disease, the liver
● involve all conditions that occurs prior to the liver that Normal Urine
can conjugate & excrete the increased
results to the overproduction of unconjugated Bilirubin
bilirubin load
bilirubin from the catabolism of heme
● B1 isn't water-soluble so it can't be
● conditions include
excreted in the urine
○ hemolytic disorders
Increased Urine ● intestinal bacteria convert some of the
■ transfusion reactions
Urobilinogen extra bilirubin into urobilinogen, some
■ sickle cell disease
of which is reabsorbed and is excreted
■ hereditary spherocytosis
by the kidneys
■ hemolytic disease of the newborn

Transcribed by: Abenes, Agtarap, Alacar, Aliping, Guarin, Villar, Villanueva (2024). 12
Hepatic Mechanism Post Hepatic Mechanism
● caused by hepatocellular diseases ● associated with obstructions in the biliary system or
○ e.g. hepatitis and cirrhosis bile duct
○ results to disruption in the uptake, conjugation, ○ results to the accumulation of B2 in the liver
transport and excretion of bilirubin ● B2 would overflow back into the blood while there is
● the amount of bilirubin or urobilinogen in urine is little to no amount of it passing to intestines causing:
correlated with the severity of the disorder ○ an increase in B2 in urine is observed
○ e.g. an increase in B2 in plasma would eventually ○ decreased or absence of urobilinogen
manifest in urine since B2 readily passes through ● absence of urobilinogen in the intestine
the glomerulus no urobilin and stercobilin formed → pale chalky or
■ since B2 is being excreted in the urine, this “acholic” stool
would also affect the re-excretion of
urobilinogen in the intestine
■ a damaged liver cannot remove urobilinogen in
the portal circulation and therefore are excreted
via the kidneys
● the extent of urinary urobilinogen increase depends
on the severity of disease

Transcribed by: Abenes, Agtarap, Alacar, Aliping, Guarin, Villar, Villanueva (2024). 13
 UNIT 4: LEUKOCYTE ESTERASE, ● observed in:
NITRITE, AND ASCORBIC ACID ○ acute interstitial nephritis
○ polycystic kidneys
URINARY TRACT INFECTION Leukocyturia ○ other organisms:
without ■ Trichomonas vaginalis
Bacteriuria ■ Chlamydia trachomatis
Type of UTI ■ Mycoses
● infection that may occur in the lower ■ Mycoplasma
Urethritis
urinary tract involving the urethra ○ viruses
Cystitis ● infection that occurs in the bladder
● infection that occur in the upper urinary
Pyelonephritis tract involving the renal pelvis &
tubules
● gram-negative bacilli
○ most common infectious microorganism that cause
UTI
○ normal flora in the intestinal tract
○ commonly:
■ Escherichia coli
■ Proteus species NITRITE
■ Enterobacter species ● formed as a result of diet / bacterial conversion of
■ Klebsiella species nitrate
● nitrate
NOTE:
○ normally excreted from the diet w/o nitrite formation
● chemical tests for UTI does not replace the need for
● nitrite positive (+) urine
urine culture
○ detection of UTI in conjunction with LE test
Two Pathways Causing UTI ○ success of antibiotic therapy
● bacteria moves from the ● movement of bacteria ■ consistent nitrite negative (-) result after
urethra → urinary bladder from the blood → antibiotic treatment → successful antibiotic
● aka ascending infection kidneys → urinary tract therapy
○ more prevalent type of UTI
Factors Affecting Nitrite Formation & Detection
LEUKOCYTE ESTERASE (LE) Bacteria Involved ● reduces nitrate to nitrite
● enzyme found in all WBC Must Be a Nitrate ● urine is (+) bacteriuria but bacteria
○ except lymphocytes Reducer is not a nitrate reducer = false (–)
● (+) result = ⬆ WBC, except lymphocyte ● to allow bacterial conversion from
● WBC normally present in urine: nitrate to nitrite
○ 0-8/HPF Adequate Time for ○ minimum of 4 hrs
○ 10 WBC/uL Conversion ● first morning urine is more ideal
● dilute & hypotonic urine giving time for bacteria to convert
○ insufficient leukocyte esterase nitrate to nitrite
○ < 10 WBC/uL Adequate Dietary ● nitrite cannot before when there is
Intake deficiency of nitrate
Leukocyturia ● nitrite detection can be reduced by
Conversion to
● ≥ 20 WBC/uL subsequent conversion of nitrite to
Nitrogen
○ indicator of inflammation in the urinary nitrogen
tract & kidneys
● observed in:
○ Pyelonephritis
Leukocyturia ○ Cystitis
with ○ Urethritis
Bacteriuria
NOTE:
● leukocyturia in females may indicate
contamination w/ vaginal secretion
● thus, always correlate leukocyturia w/
bacteriuria in confirming UTI

Transcribed by: Abenes, Agtarap, Alacar, Aliping, Guarin, Villar, Villanueva (2024). 14
ANALYSIS OF URINE MLS 321L

& OTHER BODILY FLUIDS 8438L

LABORATORY BS MLS 3 PRELIM
M3: CHEMICAL EXAMINATION OF URINE

ROUTINE URINALYSIS: CHEMICAL EXAMINATION REAGENT STRIPS: PRECAUTIONS

1. The direction of the manufacturer must be followed
REAGENT STRIPS exactly.
○ accurate timing is essential to provide optimal
REAGENT STRIPS: PROCEDURES results
1. Mix the specimen gently for homogenous distribution. 2. Do not re-use reagent strips.
2. Dip the strip completely into the specimen for about 1-2 3. The reagent strip must be kept in the bottle with the cap
secs. tightly closed to maintain test reactivity.
○ DO NOT dip the strip for more than 2 secs as it may ○ unused reagent strips must remain in original bottle
cause leaching of chemical pads onto the urine ○ do not remove desiccant from the bottle
sample. 4. Do not remove the strip from the bottle until
3. Remove excess urine by withdrawing the strip against immediately before it is to be used for testing.
the rim of the container and by blotting the edge of the ○ replace cap immediately and tightly after removing
strip while maintaining it in a horizontal position to the test strip
prevent “run-over” phenomenon. 5. Do not touch the test areas / reaction pads of the
4. Allow the reaction to proceed for 30-60 seconds (120 test strips.
seconds for Leukocyte Esterase test). 6. To obtain optimal results, it is necessary to use fresh,
5. Compare reaction colors on the reagent pads for pH, well mixed, uncentrifuged urine at RT.
sugar and protein with the Manufacturer’s Chart under 7. Dip test areas in urine completely, but briefly.
a good light source at the specified time while ○ to to avoid dissolving out the reagents
maintaining the strip at a horizontal position. 8. Protection against ambient moisture, light and heat
○ DO NOT interpret results beyond two minutes. is essential to guard against altered reagent activity,
6. Follow the Manufacturer’s guideline in reporting results: 9. Discoloration or darkening of reagent areas may
○ pH: Numerical (6, 6.5, 7) indicate deterioration.
○ sugar and albumin: Negative, Trace, 1+, 2+, 3+, ○ do not use the test strip
4+ 10. Compare the reagent strips to its bottle color chart.
○ do no use other bottles manufactured by others
11. Store the reagent stips at RT (1–30ºC).
○ do not store inside the refrigerator to guard against
altered reagent reactivity.
12. Do not use the product after the expiration date.
13. Do not store bottle in direct sunlight.
14. For quality control, use urine controls (positive and
Urine Analytes Chemically Tested Using Reagent Strips negative) everyday to determine the reactivity of the
● Blood reagent strips.
● Protein
● Bilirubin ○ record all control results and reagent lot numbers
● Nitrite
● Urobilinogen
● Glucose
● Ketone
● pH
● Specific Gravity
● Leukocyte
● Ascorbic Acid

Transcribed by: Abenes, Aliping, Guarin, Villanueva (2024). 1

 UNIT 1: URINE pH AND PROTEIN ● Arkray Inc., Kyoto, Japan

I. TESTS FOR URINE pH

A) REAGENT STRIPS: pH Aution Sticks

● principle: pKa changes of a polyelectrolyte by ions
present

Interfering Factors
Double Indicator System Improper Storage ● with bacterial proliferation
of the Specimen ● falsely INCREASE pH
Contamination of
● falsely INCREASE or DECREASE
the Specimen
pH depending on the agent
Bromthymol ● indicator of alkaline urine Before Collection
Blue ● yellow to blue in the range of 6 - 9 ● causing the acid buffer from the
Improper
● indicator of acidic urine protein test pad to contaminate the
Methyl Red Reagent Strip
● red to yellow in the pH range 4 - 6 pH test area
Technique
● orange (pH 5.0) ● falsely DECREASED pH
Indicator
● green (pH 7.0) ● bet. pH testing area & highly acidic
Combination
● blue (pH 9.0) protein testing area
○ produce false acidic reading in
Commercial Reagent Strips alkaline urine
● measures in 0.5-unit increments ● “runover effect”
(5.0-8.5) ○ excess urine is left on the stick
● reported as: after dipping → acid buffer from the
○ reagent in the protein area runs
○ 5.0, 5.5, 6.0,6.5, onto the pH area
7.0, 7.5, 8.0, 8.5 Runover of Urine NOTE:
Multistix ● runover effect is ● runover effect is often recognized
common due to the edge nearest the protein
● Siemens area will usually change first instead
Healthcare of the pH area
Diagnostics Inc.,
Deerfield, IL ● to avoid runover effect:
○ holding the reagent strip in a
● measure pH in 1-unit increment (pH horizontal position after dipping
5.0-9.0) ○ removing excess urine clinging
● reported as: from the sides of the strip
○ 5.0, 6.0 ,7.0
Chemstrips ,8.0, 9.0
● Roche
Diagnostics,
Indianapolis, IN

● measure pH in 1-unit increment (pH

5.0-9.0)
● reported as:
○ 5.0, 6.0 ,7.0 ,8.0, 9.0
● Iris Diagnostics, Chatsworth, CA
vChem Strips

Transcribed by: Abenes, Aliping, Guarin, Villanueva (2024). 2

 B) pH TEST PAPERS Sensitivity to Protein
Commercial Reagent Strips
● indicator papers that do not add impurities to the urine Concentration
● produces sharp color changes for comparison with a Multistix ● 15 to 30 mg/dL
supplied color chart of pH values Chemstrips ● 6.0 mg/dL
vChem Strips ● 15 mg/dL
C) pH METER
● alternative method for determining the urine pH
● pH meter composition
○ silver-silver chloride indicator electrode with a
pH-sensitive glass membrane connected by a salt
bridge to a reference electrode
■ (1) when the indicator electrode is placed in
urine → difference in H+ activity develops
across the glass membrane
■ (2) difference causes a change in the potential
difference between the indicator and the
reference electrodes
■ (3) voltage difference is registered by a
voltmeter and is converted to a pH reading
● pH measurement is temperature dependent
○ ⬆temperature = ⬇pH

II. TESTS FOR ALBUMIN

A) Reagent Strip
B) Sensitive Albumin Tests
C) Qualitative Tests: Coagulation / Precipitation
D) Quantitative Tests
E) Tests for Bence Jones Proteins

A) REAGENT STRIP: ALBUMIN

● uses the principle protein error of indicators

● when the pH is held constant by a buffer (pH 3.0) →

indicator dyes release H+ as a result of the presence of
proteins (anions) → color change (from yellow to B) SENSITIVE ALBUMIN TESTS
blue-green) ● detect low levels of albumin excretion of about 1-2
● protein acts as a hydrogen receptor mg/dL (microalbuminuria)
● intensity of the color change is directly related to the ● used to monitor:
amount of protein present ○ microalbuminuria
● reagent strip is more sensitive to albumin ○ diabetes
○ hypertension
○ peripheral vascular disease
B.1) Immunodip Test
● immunochemical
● measures the concentration of urinary albumin in a
Undetected Proteins False Positive Readings lateral flow device containing a test strip
● globulins ● extremely alkaline (pH≥9.0)
● myoglobin ● highly buffered urine
● hemoglobin ● highly colored substances
● immunoglobulin light chains ○ phenazopyridine drugs
(Bence Jones Proteins) ○ beets
● mucoproteins

Transcribed by: Abenes, Aliping, Guarin, Villanueva (2024). 3

B.2) Micral Test Method Albumin: Creatinine Ratio
● uses gold-labeled monoclonal antibodies ● expressed as mg of albumin excreted per g of urinary
(immunochemical) creatinine
● detects 15 to 20 mg/L (1.5 to 2 mg/dL) albumin
● reaction time: 1 min
● color produced is stable for ≤5 mins

Normal < 30 mg/g ACR

Microalbuminuria 30-300 mg/g ACR
Macroalbuminuria >300 mg/g

● principle:
○ albumin (urine) binds with → soluble
antibody-enzyme conjugate impregnated on the
reaction pad
○ only albumin-conjugate immunocomplex (moving
by capillary action) passes into reaction zone
○ intermediate zone immobilizes any excess,
unbound conjugate
○ once in the reaction zone, the enzyme
(β-galactosidase) bound to the antibody reacts
with → substrate (chlorophenol red galactoside)
in the reaction zone to produce a red dye

Result Cause of False Result

● oxytetracycline
False Positive (+)
● strong oxidizing agents
False Negative (–) ● urine specimen < 10° C

B.3) Clinitek Microalbumin & Multistix Pro Reagent Strips

● provide simultaneous measurement of albumin/protein
& creatinine
○ permits an estimation of the 24-hour
microalbumin excretion
● compares albumin excretion to creatinine excretion
○ albumin reading can be corrected for overhydration
and dehydration in a random sample
○ albumin test pad → changed to a dye- binding rxn
■ more specific for albumin than the protein error
of indicators rxn on strips measuring protein

Transcribed by: Abenes, Aliping, Guarin, Villanueva (2024). 4

 C) QUALITATIVE TESTS FOR PROTEIN: Results and Interpretation: Heat and Acetic Acid (ALB)
COAGULATION / PRECIPITATION TEST Negative ● Absence of cloudiness
Trace ● Cloudiness is rarely visible
C.1) Heat and Acetic Acid Test
● principle: heat and acid denatures protein, and thus 1+ ● Cloudiness is distinct but not granular
results to precipitation 2+ ● Cloudiness is distinct & granular
● use: detect the presence of other proteins in urine 3+ ● Cloudiness is heavy with distinct flocculi
aside from albumin 4+ ● Cloudiness is heavy with large flocculi
● NOTE: If cloudiness appears, it may be due to
○ protein C.2) Sulfosalicylic Acid Precipitation Test (SSA)
○ phosphates
● a cold precipitation test that reacts equally with all forms
○ carbonates
● if the cloudiness initially formed does not disappear of protein
after addition of acid and reheating process the ● proteins denature upon exposure to extreme pH/
substance present could be: temperature → ⬇ solubility → precipitation
○ albumin ● urine samples must be centrifuged to prevent particles
○ globulin from interfering with the turbidity assessment
○ mucoproteins ● sensitive to 5 - 10 mg/dL of protein
○ other proteins
● adding acid lowers the pH (closer to the isoelectric Result Cause of False Result
point) of proteins cloudiness may increase → after ● penicillin
addition of the acid due to increased precipitation False Positive (+) ● contrast media (crystalline
precipitate)
Result Cause of False Result False Negative (–) ● highly alkaline / buffered urine
● tolbutamide
False Positive (+) ● massive doses of penicillin NOTE:
● radiographic dyes ● SSA test should not be used to confirm a protein result
● highly buffered alkaline obtained by reagent strip.
False Negative (–)
● very dilute urine
Procedure: SSA (ALB)
1 ● Centrifuge urine sample
2 ● Pipette 3mL of clear supernatant
3 ● Add 3mL of 3% Sulfosalicylic Acid
● Invert to mix & stand for 10 mins then mix by inverting
4
twice
● Using ordinary room light, observe the degree of
5
turbidity and/ or precipitation.

Procedure: Heat and Acetic Acid

● Fill a 13x100 mm test tube with 5 ml urine sample
1 (approximately 3/4 full) & centrifuge for 5 mins at
400-450 RCF (2500-3000 rpm).
● Clamp the midportion of the tube then heat evenly the
2 upper 1/3 of the preparation.
● Allow the preparation to boil for about 30 secs
● Observe for the presence of cloudiness at the heated
3
portion against a black background
● Add three to five drops of 5 % or 10 % of acetic acid
4
& boil again for about 30 secs

Transcribed by: Abenes, Aliping, Guarin, Villanueva (2024). 5

 SSA Precipitation Interpretation Result (ALB) C.4) Heller’s Ring Test
Protein ● nitric acid is added
Result Turbidity ● false positive (+) in increased:
Concentration
● No increase in turbidity ○ uric acid
○ view the bottom of the ○ urea
tube through the solution ○ mucin (dissolved by nitric acid at point of contact)
Negative < 5mg/dL ○ nucleo-albumin
from the top
○ a circle formed by the
bottom is visible Procedure: Heller’s Ring Test
● Noticeable turbidity 1 ● Place 0.5 mL of pure nitric acid in a 13x100 test tube
Trace ○ can read newsprint 5 – 20 mg/dL 2 ● Stratify the nitric acid with 2 mL of urine
through mixture
● Distinct turbidity with no Results and Interpretation: Heller’s Ring Test
granulation ● white ring at the point of contact of the two
1+ 30 mg/dL (+) Albumin
○ cannot read newsprint fluids
through mixture
● Turbidity with granulation C.5) Robert’s Ring Test
with no flocculation ● saturated aqueous solution of magnesium sulfate &
○ flocculation is the concentrated nitric acid
2+ 100 mg/dL
association of precipitates
to form small clumps or
Procedure: Robert’s Ring Test
aggregates called floc
1 ● Centrifuge urine if cloudy.
● Turbidity with granulation
3+ 300 mg/dL 2 ● Place 1 mL of Robert’s reagent in 13x100 test tube.
& flocculation
4+ ● Clumps of protein ≥ 500 mg/dL 3 ● Stratify the reagent with 1mL of urine.

Albumin: Reagent Strip vs SSA Results and Interpretation: Robert’s Ring Test
RS SSA Indication ● White ring at the point of contact of the two
(+) (+) Albumin fluids indicates albumin.
(+) Albumin
(+) (–) Albumin ● The larger the ring, the greater is the
(+) F(–) Albumin is Present amount of albumin present.
F(+) (–) Highly Alkaline; No Albumin
(–) (+) Other Proteins
F(+) SSA due to medications, or
(–) F(+)
radiographic dyes.

C.3) Picric Acid Test

● use of 3 % picric acid to precipitate proteins

Procedure: Picric Acid Test

1 ● Filter. Centrifuge freshly voided urine.
2 ● Place 5mL of filtered urine in 16x150 test tube.
3 ● Add an equal amount of saturated picric acid sol’n.

Results and Interpretation: Picric Acid Test

● precipitate forms varying from light cloud -
(+) Albumin heavy flocculence
● depends on the albumin present

Transcribed by: Abenes, Aliping, Guarin, Villanueva (2024). 6

 Other Qualitative Tests For Protein: D) QUANTITATIVE TESTS FOR PROTEINS
Coagulation / Precipitation Test
● similar to the traditional heat and acetic D.1) Esbach’s Test (Tsuchiyua’s Modification)
Purdy’s acid method ● principle that urinary proteins are precipitated when
Modification of ● requires addition of saturated sol’n of Esbach’s reagent is added
Heat and Acetic sodium chloride & glacial acetic acid ○ albumin + picric acid → albumin-picrate complex
Acid Test ● remove interference from ● measurement is based on volume occupied by the
nucleoproteins and mucin protein-picrate after gravity settling at RT for 30 mins
● albumin and globulin are coagulated ○ read from the calibrated tube as grams albumin per
Heat liter of urine
Coagulation by heat at an acid pH
● before performing the procedure check the qualitative
Method by ● sodium acetate provides inorganic salt test for albumin
for protein flocculation and buffering ○ 3+ = dilute urine sample at 1: 5
Henry
capability ○ 4 + = dilute at 1:10
Osgood-Haskin ● 50 % acetic acid & saturated sodium ● barium sulfate
chloride (prevents precipitation of ○ added to increase sedimentation since urine
Test
mucin) samples with high SG will not allow the
● uses glacial acetic acid and 5-10 % of sedimentation of the albumin-picrate complex
potassium ferrocyanide to precipitate ● alkaline urine must also be acidified
proteins ○ so that the acidity (10 % HAC) of the Esbach’s
Potassium reagent is maintained
Ferrocyanide ● faint cloudiness appears at
● 1% picric acid
Test once Esbach’s Reagent ● 2% citric acid
(+) ○ flocculence may occur if ● distilled water
large amount of albumin ● phosphotungstic acid
Tsuchiya’s
present ● 95% alcohol
Reagent
● concentrated HCl
● positive result = white ring at the
point of contact between the reagent Procedure: Esbach’s Test
added and urine sample 1 ● The urine must be filtered until clear.
● white ring ● Dilute with water of the quantitative test of albumin is
○ evidence of precipitation 2 ○ 3+ = 1:5 dilution
Heller’s RIng Test ○ 4+ = 1:10 dilution
● nitric acid is added 3 ● If not acid, add 10% HAC drop by drop to give ph 5.0
Robert’s Ring Test ● Add barium sulfate to graduated conical tube, up to
4
0.5 mark to increase the rate of sedimentation.
Ring or Contact ● magnesium sulfate & concentrated
Tests nitric acid ● Add Esbach’s reagent up to the 10th mark.
5
● Add urine sample up to the 15th mark.
Spiegler’s Test
(Jolles Modification) 6 ● Cover with a rubber stopper and invert to mix.
● Spiegler’s reagent ● Place in a test tube rack, and read sediments after
7
○ mercuric chloride 30 mins.
○ succinic acid ● The readings will give grams albumin/liter of urine.
8
○ sodium chloride ○ divide the values by 10 to get the percentage
○ distilled water ● If dilutions have been made, multiply the final reading
9
● 5 ml of urine + 30 % acetic acid → by the dilution.
stratified w/ Spiegler’s Reagent
● use of 10 % NaOH/KOH & 10 %
Cupric sulfate
Result Protein Present
Biuret Reaction ● albumin
Violet Mixture
● globulin
Rose Colored ● albumin
Mixture ● peptone

Transcribed by: Abenes, Aliping, Guarin, Villanueva (2024). 7

 Other Quantitative Tests for Proteins E.1) BJP: Sulfosalicylic Acid (SSA) Precipitation Test
● involves acidification of an alkaline ● same principle applies as that of the SSA test for
urine with acetic acid and addition of 3 albumin
% SSA to precipitates protein in urine
● turbidity Procedure: SSA (BJP)
Kingsbury’s–
○ measured using photometer with a 1 ● Centrifuge urine sample
Clark Method
standard solution with known amount
(SSA) 2 ● Pipette 3mL of clear supernatant
of protein
3 ● Add 3mL of 3% Sulfosalicylic Acid
● cloudiness
○ due to urates will disappear when ● Invert to mix & stand for 10 mins then mix by inverting
4
warmed twice
● uses Tsuchiya’s reagent to precipitate ● Using ordinary room light, observe the degree of
Shevky and 5
proteins turbidity and/ or precipitation.
Stafford Method
● degree of flocculation is quantified
Kwilecki’s SSA Precipitation Interpretation Result (BJP)
● involves heating a mixture of 10 %
Modification of Protein
ferric chloride & urine at 72°C Result Turbidity
Esbach’s Concentration
● uses ethanolic HCl phosphotungstic Negative ● No increase in turbidity <6
Modified acid Trace ● Noticeable turbidity 6-30
Tsuchiya’s Test ● precipitate proteins at 56°C followed by
● Distinct turbidity with no
spectrophotometry at 540 nm 1+ 30-100
granulation
● use of 50 % acetic acid & 10 %
potassium ferrocyanide added with ● Turbidity with granulation
2+ 100-200
Purdy’s Method 15 ml of urine with no flocculation
● amount of precipitate formed after 10 ● Turbidity with granulation
3+ 200-400
mins is measured & flocculation
4+ ● Clumps of protein >400
E) TESTS FOR BENCE JONES PROTEIN (BJP)
● Bence Jones Protein (BJP)
○ an abnormal proteinThis l
○ low molecular-weight protein = 22,000-44, 000 Da
○ filtered in quantities exceeding the tubular
reabsorption capacity and is excreted in the urine
○ if suspected, a screening test that uses the unique
solubility characteristics of the protein can be
performed
● BJP detected in urine of patients with:
○ multiple myeloma
○ bone tumor
E.2) BJP: Heat & Acetic Method (Harrison Test)
○ chronic leukemia
○ hyperthyroidism ● performed when SSA result is positive
○ amyloidosis ● 3 tubes are prepared wherein 5 ml of urine are
○ fibrotic disease dispensed in each tube
○ adenocarcinoma of stomach ○ 1st tube: 1 drop of 33% acetic acid
○ prostatitis ○ 2nd tube: 2 drops of 33% acetic acid
● precipitation of BJP depends on ○ 3rd tube: no addition
○ urine pH (4.6 – 5.4) ● tubes are heated at 40-60°C
○ ionic strength of the solution ● turbidity is observed which will disappear at 100°C
○ electrolyte composition of the solution
○ protein concentration

Transcribed by: Abenes, Aliping, Guarin, Villanueva (2024). 8

 Procedure: Heat and Acetic Method (BJP) Other Tests For BJP
● Fill the test tube ¾ full with urine. ● performed when Heller’s ring test or
○ if urine is not acidic, acidify with 10% HAc Bradshaw Test SSA is positive (+)
○ check acidity using reagent strips ● involves the addition of HCl
1 ● uses 2M acetate buffer (sodium
1st Tube ● 5 mL urine + 1 drop 33% acetic acid
Putnam Test acetate trihydrate & glacial acetic acid)
2nd Tube ● 5 mL urine + 2 drop 33% acetic acid
to precipitate BJP
3rd Tube ● 5 mL urine + NO addition
● TSA precipitates BJP but does not
● Gently heat the preparation in a beaker of water with a Toluene Sulfonic precipitate albumin
2
thermometer in it. Acid ● unmasks globulin only if present in
● Tubes are heated at 40-60°C high concentrations
3 ● uses 50 % acetic acid & saturated
● Turbidity is observed which will disappear at 100°C Osgood-Haskin
aqueous solution of NaCl
Test
(precipitation as positive result)
Results and Interpretation: Heat and Acetic Method
● uses fresh urine to prevent conversion
● turbid at 40ºC
of proteins to pseudo-BJP or
● flocculent precipitate will form at 60ºC
decomposition of BJP
○ disappears at boiling point (100ºC)
● concentrated HNO3 is overlaid with
(+) BJP ○ reappears at 60ºC
urine and white ring is produced at
○ disappears at 40ºC and below
zone of contact
NOTE: ● indicate presence of:
● albumin can interfere with a positive result ○ albumin
(–) BJP ● if coagulates does not clear ○ proteoses
Jacobson and
○ resinosis drugs
Milner Test
○ BJP.
Confirmatory Test: Heat and Acetic Method
● rings >1-2 mm contact zone are due to
● filter the preparation immediately after boiling
water, urea or bile
○ this removes albumin interference
● positive (+) result:
● turbidity reappears at 60ºC ○ procedure must be repeated by
(+) BJP using concentrated HCl then 25 %
● turbidity begins to disappear at 40ºC
acetic acid.
○ if white ring still appears after
warming at 70°C, BJP is present

Transcribed by: Abenes, Aliping, Guarin, Villanueva (2024). 9

 UNIT 2: GLUCOSE, OTHER REDUCING SUGARS Lowest Concentration
Commercial Reagent Strips
AND KETONES Detected (Sensitivity)
Multistix ● 75-125 mg/dL
TESTS FOR GLUCOSE IN URINE Chemstrips ● 40 mg/dL
vChem Strips ● 45 mg/dL
A) Reagent Strip
B) Copper Reduction Tests
B.1) Benedict’s Copper Reduction Test
B.2) CLINITEST
Result Cause of False Result
C) Qualitative Tests for Glucose ● strong oxidizing agents
D) Quantitative Tests for Glucose False Positive (+) ○ sodium hypochlorite
○ peroxides
Glucose
● sugars are not normally excreted in urine ● ascorbic acid (≥50 mg/dL)
○ glucose (most common) ○ oxidized by H2O2 → oxidation of
○ galactose (part of newborn screening test [< 2yrs potassium iodide → no color
old]) formation
● fasting is recommended False Negative (–) ● high SG
● first morning spx does not always represent a fasting
● ketones
spx
○ glucose from an evening meal may remain in the ● proteins
bladder overnight ● improper preservation
○ px are advised to empty the bladder → collect ● ○ ↓ Temperature
second specimen
● collected in conjunction with the blood samples drawn
during glucose tolerance test
○ diabetes mellitus
○ hypoglycemia

A) REAGENT STRIP TEST: GLUCOSE

● uses a double sequential enzyme reaction, using:
○ glucose oxidase
○ peroxidase
● ONLY detects glucose
● glucose oxidase reaction
○ glucose oxidase impregnated on the reaction pad
→ catalyzes the oxidation of glucose → form
hydrogen peroxide & gluconic acid
○ hydrogen peroxide (H2O2) → oxidizes the
chromogen (potassium iodide) on the pad→ into
iodide in the presence of peroxidase → color
changes (blue to green to brown)

● interpretation:
○ reported as negative & positive using the plus
system (1+, 2+, 3+, 4+)
○ with corresponding quantitative concentrations in
mg/dL or g/dL
● glucose is read after 30 secs

Transcribed by: Abenes, Aliping, Guarin, Villanueva (2024). 10

 B) COPPER REDUCTION TEST ● Drop one CLINITEST tablet into the tube & observe
● depends on the ability of reducing substances to the complete reaction.
convert cupric sulfate → cuprous oxide (alkaline ○ tablet is rapidly dissolved by the action of sodium
environment & heat) → presence of glucose → color carbonate & citric acid (effervescent)
change (blue to green to orange) 3 ● DO NOT shake the tube during the reaction or for 15
○ reducing sugars secs after the boiling has stopped.
■ glucose ■ maltose ○ sodium hydroxide (alkaline medium)→ heat
■ fructose ■ pentose required provided by the reaction of sodium
■ galactose ■ ribose hydroxide with water & citric acid
■ lactose ■ arabinose ● At the end of the 15-second waiting period, shake the
tube gently and then compare with the color chart that
B.1. Benedict’s Copper Reduction Test is provided.
● method that uses the Benedict’s Reagent 4
○ reducing substances in the urine then react with the
○ copper sulfate copper sulfate → reduce the cupric ions to
○ sodium carbonate cuprous oxide
○ sodium citrate buffer
● any color change after 15 secs = IGNORED
○ it no longer indicates the high glucose conc.

Results and Interpretation:CLINITEST

Trace ● 1/4% (500 mg/dL)
1 ● 1/2% (1000 mg/dL)
Result Cause of False Result 2 ● 3/4% (2000 mg/dL)
● ascorbic acid 3 ● 1% (3000 mg/dL)
● tetracycline >5 ● 2% or more (>5000 mg/dL)
● salicylates (–) ● blue; no color change
False Positive (+) ● penicillin
● cysteine
● homogentisic acid
● glucoronates
● radiographic contrast media
False Negative (–)
● sulfonamides

B.2) CLINITEST Reagent Tablet

● tablet version of Benedict’s test
○ performed when Benedict’s method is not available
and for confirmation of reagent strip
● detect a minimum glucose concentration of 200 mg/dL
● self-heating method for the semiquantitative
determination of reducing substances in the urine
● reagents:
○ copper sulfate
○ citric acid
○ sodium hydroxide
○ sodium carbonate Glucose: Reagent Strip vs CLINITEST
RS CT Indication
Procedure: CLINITEST (+) (+) Presence of Glucose
● Place 5 drops of urine into a glass test tube (or use (–) (+) No Sugar; Contaminant
1
0.3 mL) (1+) (–) Small Amount of Glucose
2 ● Add 10 drops of water (or 0.6 mL) & mix by shaking (4+) (–) False Positive (+)

Transcribed by: Abenes, Aliping, Guarin, Villanueva (2024). 11

● pass-through phenomenon C.2) Trommer’s Method
○ high concentrations of reducing substances and is
evidenced by the color of the mixture “passing Procedure: Trommer’s Method
through” all colors possible 1 ● Pipette 1 ml of urine into 13x100 test tube
○ orange (highest concentration) → change back to
2 ● Add 1 ml of 10% KOH
green-brown (low concentration)
○ mechanism:reoxidation of the resultant cuprous ● Add drop by drop 10% Copper Sulfate solution,
oxide → cupric oxide and other cupric complexes 3 shaking preparation after each addition until the
(green) mixture turns blue
○ occur so rapidly that it can be missed if not 4 ● Heat the mixture
observed closely
Results and Interpretation: Trommer’s Method
Result Cause of False Result
● nalidixic acid (+) ● yellow red precipitate
● cephalosporins (–) ● absence of yellow precipitate
● probenecid
False Positive (+) ● ascorbic acid C.3) Nylander’s Method
● urinary preservatives
○ formalin Procedure: Nylander’s Method
○ formaldehyde
1 ● Pipette 5 ml of urine into 16x150 test tube.
2 ● Add 0.5 ml of Nylander’s reagent. Mix the preparation
C) QUALITATIVE TESTS: GLUCOSE
3 ● Allow the mixture to stand for 3-5 minutes.
C.1) Fehling’s Method
Results and Interpretation:
Procedure: Fehling’s Method Nylander’s Method
● Pipette 1 ml each of Fehling’s A and B into a 16x150 (+) ● black color
1
test tube
● Add 4 ml of distilled water and boil the mixture for a Trace ● brown color
few seconds
2
○ N.B. If the mixture remains clear after boiling, the ● absence or black or
(–)
solution may be used; otherwise, discard. brown color
● Continue boiling the solution at the same time, add 1
3 ml of the urine to be tested drop by drop at the rate of C.4) Moore-Heller’s Method
1 drop every 2 seconds.
Procedure: Moore-Heller’s Method
Results and Interpretation: Fehling’s Method 1 ● Pipette 2 ml of urine into 13x100 test tube
(+) ● yellow precipitate 2 ● Add 1 ml of 10% KOH
(–) ● absence of yellow precipitate ● Boil the upper part of the mixture for 2-3 minutes.
3 ○ If phosphates are present in large amount, filter the
preparation and examine the filtrate.

Results and Interpretation:Moore-Heller’s Method

1% or less ● canary yellow
1-2% ● wine yellow
2-3% ● cherry yellow
3-4% ● rum color
>4% ● dark brown or black color

Transcribed by: Abenes, Aliping, Guarin, Villanueva (2024). 12

 Other Qualitative Tests for Glucose
Method Reagent Principle Positive Result
● Fehling’s A ● ability of reducing sugars to reduce
○ cupric sulfate (in distilled alkaline copper sulfate reagent
water) ○ cupric hydroxide→ cuprous
● Fehling’s B hydroxide
Fehling’s Test ○ Rochelle Salt ● Fehling's solution contains blue ● (+) yellow precipitate
■ sodium potassium alkaline cupric hydroxide solution,
tartrate heated with reducing sugars gets
■ sodium hydroxide reduced to yellow or red cuprous
■ distilled water oxide and is precipitated
● Haine’s Reagent ● ability of reducing sugars to reduce
○ copper sulfate alkaline copper sulfate reagent
● (+) yellow to red
Haine’s Test ○ NaOH ○ cupric hydroxide→ cuprous oxide
precipitate
○ glycerol ○ 4mL of reagent boil & add 6-8 drop
○ distilled water of urine
● ability of reducing sugars to reduce
● 10% KOH
Trommer’s Test alkaline copper sulfate reagent ● (+) yellow to red precipitate
● 10% Copper sulfate
○ cupric hydroxide→ cuprous oxide
● (+) black color
● Nylander’s reagent ● (trace) brown color
○ Rochelle Salt (Na or K ● (-) white precipitate
● reduction of bismuth to metallic
Nylander’s Test lactate) ○ presence of phosphates
bismuth in a hot alkaline solution.
■ 10% NaOH or KOH ○ ↑ albumin (similar rxn)
■ bismuth subnitrate ■ removal: boiling &
filtration
● canary yellow: 1% or less
● wine yellow: 1 – 2%
Moore-Heller’s ● based on the caramelization of
● 10% KOH ● cherry yellow: 2 – 3%
Test sugars by strong alkali and heat.
● rum color: 3 – 4%
● dark brown or black: >4%
Phenylhydrazine
● phenylhydrazine ● sugar forms a definite form → contact ● (+) phenylglucosazone
Test (Osazone
● acetate w/ phenylhydrazine and acetate crystals
Test)
○ fine bright yellow
Kowarsky needles
● pure phenylhydrazine ● carbohydrates with free or potentially
Phenylhydrazine ■ in bundles, sheaves or
● glacial acetic acid free carbonyl groups react with
Test (Blumel’s rosettes
● saturated solution of NaCl phenylhydrazine to form osazone
Modification)
● (+) purple in test area
Glucose Oxidase ● based on the glucose ● (-) or false (-) red
● o-toluidine oxidase-peroxidase-chromogen
Paper Strip Test ○ presence of ascorbic
reaction
acid

Transcribed by: Abenes, Aliping, Guarin, Villanueva (2024). 13

 D) QUANTITATIVE TESTS: GLUCOSE Procedure: Benedict’s Method
● principle: reducing sugars reduce copper sulfate to ● Pipette 5ml Benedict’s qualitative reagent into 16x150
cuprous oxide → white copper thiocyanate 1
test tube.
● endpoint: blue color is gone w/ gray remnant
2 ● Add 8 drops of urine sample then mix preparation.
○ all cupric ions are reduced
● 24hr urine sample is required ● Heat preparation over a Bunsen flame for 2 minutes.
● usually used for differential diagnosis and correlation to ● If water bath is used, heat for 5 minutes.
medication 3 ● Remove the preparation from the flame from time to
○ however, reagent strips if sufficient for time to avoid the preparation from spilling out from the
concentration tube.
4 ● Read results immediately after heating process
D.1) Benedict’s Method
● 0.01 gram of glucose → reduces 5 ml of Benedict’s Results and Interpretation: Benedict’s (Qualitative) test
reagent
Negative (-) ● No change from the original blue color.
● Normal Value:
Trace ● Green solution without any precipitate.
○ 0.01-0.03 g/100 mL
○ 0.05-1.5 g/24 hrs ● Greenish yellow solution with yellow
1+
precipitate.
● Yellowish green solution with yellow
2+
precipitate
● Yellowish orange solution with orange
3+
precipitate
● Orange red solution with brick red
4+
Benedict’s Reagent precipitate.
Copper ● pure crystallized
17.3g
Sulfate ● main reagent
● prevent the precipitation
of cupric ions as cuprous
○ carbonate / cuprous
hydroxide→forms loosely
Sodium
bound complex with 173g
Citrate
copper (trisodium citrate:
cupric complex)
○ dissociation → continuous
supply of copper ions
Sodium
Carbonate ● provides alkaline medium 100g Fehling’s test
(Anhydrous) ● 1 ml of Fehling’s solution is mixed with 4 ml of distilled
water and then heated to boiling
Distilled 709.7g
– ● urine is added drop by drop until the grayish white
Water (up to 1 liter)
color is obtained
● precipitates cuprous ions
Potassium
as cuprous cyanide (white –
Sulfocyanate
colored precipitate)
Potassium ● prevent precipitation of
–
Ferrocyanide cuprous oxide

Transcribed by: Abenes, Aliping, Guarin, Villanueva (2024). 14

 Other Quantitative Tests for Glucose
Method Reagent Principle
Benedict’s ● Benedict’s reagent ● glucose and other reducing substances reduce
the copper sulfate to cuprous oxide in the
Fehling’s ● Fehling’s solution presence of potassium thiocyanate
● used to determine whether or not a bacteria
can utilize a certain carbohydrate
Robert Fermentation Test ● pH indicators
● the action of organism on a carbohydrate
○ e.g. phenol red
substrate results in acidification of the medium,
detected by a pH indicator dye
● somogyi-nelson is the amount of deposro oxide
Somogyi Method ● copper reagents deposits that react with arsenomolybdate which
● arsenolmolibdat is reduced to molybdine blue and the blue color
is measured absorbance

TESTS FOR OTHER REDUCING SUGARS

A) Fructose
B) Lactose
C) Pentose (Xylose, Arabinose, Ribose)

A) FRUCTOSE
Seliwannof’s Test

Procedure: Seliwannof’s Test Results and Interpretation: Seliwannof’s Test

● Pipette 3 ml of Seliwanoff's reagent into 13x100 mm ● intense orange – red color and dark
1 (+)
test tube precipitate which dissolves with ethanol
2 ● Add 6 mL of urine sample ● absence of red orange color and dark
(–)
3 ● Heat the preparation to boiling. precipitate

Other Tests for Fructose

Method Reagent Principle Positive Result
● hot HCl converts fructose →
● (+) intense orange with red color
● resorcinol hydroxymethyl furfural → links with
Seliwannof’s and dark precipitate
● concentrated HCl resorcinol → red colored compound
Test ○ > 2% glucose → fructose (by HCl)
(< 12%) ● observe rxn NOT after more than
→ false positive (+)
20-30 secs of boiling
● hot HCl converts fructose →
● resorcinol hydroxymethyl furfural → links with
● concentrated HCl resorcinol → red colored compound
Borchardt’s Test ● (+) red precipitate
● potassium hydroxide ● by heating urine sample w/ HCl &
● acetic ether resorcinol & yellow-colored acetic ether
layer (from KOH + acetic ether)
● (+) red precipitate
● confirmatory test:
● cupric acetate ● cupric acetate is reduced from cupric ○ addition of
Barfoed’s Test ● 38% acetic acid hydroxide → cuprous oxide (by ■ 10 mL water
● distilled water fructose) → red precipitate ■ 1 mL of phosphomolybdic acid
○ (+) very dark, almost opaque,
blackish hue /color

Transcribed by: Abenes, Aliping, Guarin, Villanueva (2024). 15

 B) LACTOSE B.2) Woehkl’s Test
● normal urine excretion: 50 mg/24 hrs
Procedure: Woehkl’s Test
B.1) Rubner’s Test 1 ● Pipet 5 ml of urine into 13 x 100 mm test tube
2 ● Add 2.5 mL NH3
Procedure: Rubner’s Test
3 ● Add 5 drops 15% KOH
1 ● Pipette 3 ml urine into 13x100 test tube.
4 ● Warm the mixture.
2 ● Add 2 ml of ammonia
3 ● Add 8 drops of 10% KOH
Results and Interpretation: Woehkl’s Test
4 ● Warm in water bath (DO NOT BOIL)
(+) ● red color
(–) ● absence of red color

Other Tests for Lactose

Method Reagent Positive Result
● (+) brick red solution with cherry red
● lead acetate ● (+) copper colored precipitate
Rubner’s Test
● concentrated NH4OH ● (-) yellow solution with yellow precipitate =
glucose
● methylamine hydrochloride
Ormsby’s Test ● (+) intense red color
● Na OH
● (+) fine white precipitate of mucic acid crystals
Mucic Acid Test ● concentrated HNO3
○ indicated as increased galactose in urine
● (+) lactosazone crystals
Phenylhydrazine / Osazone ● phenylhydrazine hydrochloride
○ cotton-ball-shaped (hedgehog)
Test ● sodium acetate
● galactose in urine yields the same positive result
● NH3
Woehlk’s Test ● (+) red color
● 15% KOHN

C) PENTOSE (XYLOSE, ARABINOSE, & RIBOSE) C.2) Tauber’s Test

C.1) Bial-Orcinol Procedure: Tauber’s Test

1 ● Pipette 2.0 ml of urine into 13x100 test tube
Procedure: Bial-Orcinol 2 ● Add 2.0 ml of Tauber’s reagent
1 ● Boil 5 ml of reagent 3 ● Boil the preparation vigorously for 2 minutes
2 ● Add 1 mL of urine drop by drop 4 ● Immerse in water and then add 5 ml distilled water

Results and Interpretation: Bial-Orcinol Results and Interpretation: Tauber’s Test

(+) Pentose ● olive green solution in amyl alcohol (+) Pentose ● pink to red color (cherry red)
(–) Pentose ● no olive green solution (–) Pentose ● absence of pink to red color

Transcribed by: Abenes, Aliping, Guarin, Villanueva (2024). 16

 Other Tests for Pentose
Method Reagent Positive Result Note
● Bial-orcinol reagent: ● by heating with HCl →
○ orcinol ● (+) olive green solution in amyl pentose is converted to
Bial-Orcinol Test
○ concentrated HCl alcohol furfural → reacts with orcinol
○ 10% FeCl3 → colored compound
● Tauber’s Reagent:
● (+) pink to red (cherry red)
Tauber’s Test ○ benzidine –
● disregard: any yellowish to brown color
○ glacial acetic acid
● charcoal removes all
● HCl reducing substances except:
Tollen’s Test ● (+) cherry red color
● phloroglucinol ○ pentose
○ formaldehyde
Cole’s Test ● blood charcoal ● (+) green color
● NH3
Woehlk’s Test ● (+) red color –
● 15% KOHN
● glacial acetic acid
● (+) bright red color (chloroform layer)
Aniline Test ● pure aniline ● sensitivity = 0.03%
● glucose fives an interfering green color
● chloroform
Phenylhydrazine / ● phenylhydrazine HCl ● (+) pentosazone crystals
–
Osazone Test ● sodium acetate ○ mass of entangled rootlet

TEST FOR KETONES Commercial

Sensitivity
Reagent Strips
A) Reagent Strip Tests
B) Acetest Multistix ● 5-10 mg/dL of acetoacetate
C) Specific Tests for Ketones Chemstrips ● 50-70 mg/dL acetone
C.1) Acetone
C.2) Diacetic Acid vChem Strips ● 50-70 mg/dL acetone
C.3) ß-Hydrobutyric Acid

Result Cause of False Result

Ketones ● high specific gravity
● valuable monitoring and management tool for patients ● low pH
with type 1 DM ● sulfhydryl containing compounds
● ketonuria ○ 2- mercaptoethane sulfonic acid
○ early indicator of insulin deficiency ○ captopril
● ketoacidosis ○ cysteine
○ develop slowly and progressively False Positive (+)
● levodopa metabolites
○ repeated insufficient insulin doses ● phenylketones
○ cause red-orange coloration
A) REAGENT STRIP TESTS: KETONES ● phthaleins
● rxn of ketones with sodium nitroprusside ○ bromsulphthalein
(nitroferricyanide) impregnated in the reagent pad ○ phenolsulfonphthalein red dyes
● rapid volatilization of acetone at
room temperature
● breakdown of acetoacetate by
● glycine in the reagent pad enables detection of
False Negative (–) bacteria
acetone ● deterioration of the nitroprusside
reagent
○ exposure to moisture, heat, or light

Transcribed by: Abenes, Aliping, Guarin, Villanueva (2024). 17

 Results and Interpretation: Acetest
(+) Ketones ● purple color
Small ● 5–10 mg/dL of diacetic acid
Moderate ● 30–40 mg/dL
Large ● 80–100 mg/dL

B) NITROPRUSSIDE TEST FOR KETONES (ACETEST)

● 10 times more sensitive to diacetic acid than acetone
○ sensitivity:
■ 5–10 mg/dL of diacetic acid C) SPECIFIC TESTS FOR KETONES
■ 20–25 mg/dL of acetone
○ DO NOT react with beta-hydroxybutyric acid C.1. Acetone: Legal’s Test
● acetate tablet:
○ sodium nitroprusside Procedure: Legal’s Test
○ glycine ● Place a few ml of freshly voided urine sample in 16x
○ disodium phosphate (strong alkaline buffer) 1
150 test tube.
○ lactose (enhance color)
● Add enough NAOH or KOH to render the sample
● principle:
2 alkaline.
○ diacetic acid & acetone + sodium nitroprusside &
● Test with litmus paper after each addition of alkali.
glycine (alkaline medium) → purple color
● Add a few drops of fresh Sodium Nitroprusside
○ lactose helps enhance the color 3
solution.
4 ● Add a few drops of concentrated acetic acid.

Results and Interpretation: Legal’s Test

(+) ● purple or violet-red color
Procedure: Acetest
● alcohol or acetic aldehyde and diacetic acid
1 ● Place the tablet on a piece of clean, dry white paper N.B.
gives the same reaction
2 ● Put one drop of urine directly on top of the tablet.
● Compare the color of the tablet with the color chart at
3
30 seconds.
4 ● Report results

Transcribed by: Abenes, Aliping, Guarin, Villanueva (2024). 18

 Specific Ketone Tests: Other Acetone Tests
Method Reagent Principle Positive Result
● (+) red to purple ring at zone of
● decomposition of sodium contact
nitroprusside→ sodium ferrocyanide ● (-) purple ring
and ferric oxide ● false (+)
● ammonium sulfate
Rothera’s Test ○ nitroprusside 20x more sensitive to ○ heavy meal
● sodium nitroprusside
acetoacetate than acetone ■ color persist for 30 secs and
○ do not react w/ fade w/in 3-4 mins
beta-hydroxybutyrate ○ amorphous urates
■ brown to orange color
● KOH ● (+) yellow precipitate of iodoform
Lieben’s
● iodine ○ thin, yellow, hexagonal plates
Iodoform Test
● potassium-iodine sol’n ○ starlike groups of crystals
● involves addition of an excess ● (+) black coloration
Gunning’s Test alcoholic iodine sol’n & ammonia ○ iodoform precipitate is formed
(Modification of ● lugol’s iodine water when acetone is present
Lieben Test) ○ observe for six sided tablets or
stars microscopicallt
● 1 mol of salicylic aldehyde + one 1
mol of acetone → oxybenzol acetone
● KOH (strong alkali) → dioxy-dibenzol
● (+) red-purple color
Frommer’s Test ● 10 % alcoholic sol’n of acetone
○ purplish red ring
salicylaldehyde ● glacial acetic acid and saturated sol’n
of sodium nitroprusside + 5ml of urine
○ overlay 28% ammonium hydroxide
● decomposition of sodium
● aceto acetic acid nitroprusside / sodium ferrocyanide
Legal’s test ● (+) purple color
● acetone ○ sodium ferrocyanide + ferric oxide
→ purple color
● glacial acetic acid
● saturated sol’n of sodium
● (+) purple/purplish red ring at
nitroprusside
Lange Test – junction
● + 5ml urine
● ammonium hydroxide (overlay
28%)
● modification of legal’s test ● (+) few purple-red color
Jackson-Taylor’s
– ● sodium nitroprusside + ml of urine → ● (+) permanganate ring at point of
Test
NH4 sulfate → NH4 hydroxide contact
Diffusion Test for ● acetone being volatile diffuses and
– ● (+) cream-colored precipitate
Acetone fixed → react w/ Nessler reagent
● Scott-Wilson Reagent
Wallhausser’s ○ mercuric cyanide ● urine turns turbid with the addition of a
● (+) turbid
Test ○ silver nitrate drop of Scott-Wilson Reagent
○ NaOH

Transcribed by: Abenes, Aliping, Guarin, Villanueva (2024). 19

C.2. Diacetic Acid: Gerhardt’s Test C.3. ß-Hydroxybutyric: Hart’s
● ferric ions chelate with enol groups of diacetic acid → ● ß-Hydroxybutyrate (ß-HB) reagent is the best test to
bordeaux red or wine-red complex detect ketones to diagnose and monitor diabetic
ketoacidosis and other conditions
Result Cause of False Result
○ aspirin (salicylates)
○ phenol Procedure: ß-Hydroxybutyric: Hart’s
False Positive (+) ● Place 5 ml of freshly voided urine sample in 16x150
○ antipyrine 1
○ sodium bicarbonate test tube
● Add equal volume of water and a few drops of acetic
2
Procedure: Gerhardt’s Test acid
● Place a few ml of freshly voided urine sample in 3 ● Boil the preparation 1⁄2 its original volume
1
16x150 test tube. 4 ● Dilute to 10 ml with distilled water
● Using a pipette, add 10% Ferric chloride drop by drop ● Mix and divide the contents equally into two portions.
2 5
until no further precipitation occurs. Label them I and II
● To tube I, add 0.5 ml hydrogen peroxide. NO addition
6
Results and Interpretation: Gerhardt’s Test of hydrogen peroxide to tube II.
(+) ● bordeaux red color 7 ● Warm both tubes gently and allow to cool.
(–) ● absence of bordeaux red color 8 ● Apply Legal’s test to both tubes
9 ● Stand for few hours.

Results and Interpretation: ß-Hydroxybutyric: Hart’s

● Tube with hydrogen peroxide
(+)
○ red ring at the point of contact
● Tube without hydrogen peroxide
(–)
○ no change in reactions

Specific Ketone Tests: Other Diacetic Tests

Positive
Method Reagent Principle
Result
● concentrated
Confirmatory Test: Gerhardt’s Test ● >0.5% = dark
NH4OH ● conc. of
● heat the precipitation Osterberg purple
● sodium beta-hydroxyb
Test ● <0.5% = light
Bordeaux Red Color DOES ● due to drugs nitroprusside utyric acid
NOT Disappear purple
● distilled water
Bordeaux Red Color ● due to diacetic acid
Disappear

Specific Ketone Tests: Other Diacetic Tests

Method Reagent Positive Result
● 30 % acetic acid ● (-) reddish violet
Lindemann’s
● iodine ○ diacetic is NOT
Test
● chloroform present
● 1% sodium nitrate
● acetophenone
Arnold’s Test solution ● (+) violet color
● ammonia
● concentrated HCl.

Transcribed by: Abenes, Aliping, Guarin, Villanueva (2024). 20

 UNIT 3: BLOOD, PORPHOBILINOGEN, Pseudoperoxidase Activity of the Heme Moiety
BILIRUBIN AND UROBILINOGEN ● causes the:
○ reduction of peroxide
Red-Colored Urine ○ oxidation of the chromogen
● associated with the presence of blood in urine as either: ○ production of a color change on the reaction pad
○ hematuria from yellow to green
■ increased presence of intact RBCs in urine
○ hemoglobinuria
■ urinary presence of hemoglobin
■ commonly thought to arise from the lysis of
intact RBCs in urine due to prolonged standing
Sensitivity of the Test and Reagent Used
of urine
● depends on the brand of reagent strip used
○ true hemoglobinuria
■ associated with free intravascular hemoglobin
that traversed the glomerulus and became part ● uses diisopropyl- benzene
of the plasma ultrafiltrate in the tubules dihydroperoxide tetramethylbenzidine
Multistix
● can detect 6-20 RBCs/uL and 0.02-0.06
mg/dL of hemoglobin
Differentiating Hematuria from Hemoglobinuria
● uses dimethyl- dihydroperoxyhexane
Hematuria Hemoglobinuria tetramethylbenzidine
Chemstrip
ranges from normal, ranges from normal, ● can detect 5-10 RBCs/uL and 0.02-0.03
Color
pink, red, and brown pink, red, and brown mg/dL hemoglobin
cloudy/smoky
Clarity clear
appearance Result Cause of False Result
promote lysis of intact promote hemoglobin ● strong oxidizing agents
Alkaline Urine
RBCs oxidation ● bacterial peroxidases
After ● blood contamination
cell button is observed uniform clear red urine
Centrifugation ○ hemorrhoidal blood
○ menstrual blood
TEST FOR BLOOD ● strong oxidizing agents
○ ex.: sodium hypochlorite
A) Reagent Strip
B) Qualitative Test for Blood ○ oxidizes the chromogen instead of
B.1) Salt Precipitation Method (Blondheim Test) False Positive (+) the heme moiety
B.2) Benzidine Test
B.3) Guaiac Test ● bacterial peroxidases
B.4) Occult Tablet Test ○ produced by organisms such as
[Link]
A) REAGENT STRIP FOR BLOOD ○ catalyzes the reaction of the
● impregnated with tetramethylbenzidine (chromogen) reagent and chromogen despite
and peroxide the absence of the
pseudoperoxidase activity of
heme
● technical errors
○ failing to mix the sample prior to
dipping the reagent strip may not
Homogenous ● results from hemoglobin
Color Change detect the intact cells as they tend
to settle at the bottom of the tube.
Mottled or ● observed when intact red cells lyse False Negative (–)
● presence of ascorbic acid (>25
Speckled Pattern and release their hemoglobin
mg/dL)
○ ascorbic acid tend to react and
consume the peroxidase present
in the reagent pad.

Transcribed by: Abenes, Aliping, Guarin, Villanueva (2024). 21

 ● strong reducing substances B) QUALITATIVE TEST FOR BLOOD
○ other reducing substances tend to
react and consume the B.1) Salt Precipitation Method (Blondheim Test)
peroxidase present in the reagent ● differentiate hemoglobinuria from myoglobinuria
pad
● high specific gravity Procedure:
○ causes intact red cells in urine to ● Add 2.8g of ammonium sulfate to 5 mL of centrifuged
become crenated and resist lysis 1
urine.
which is necessary to detect heme
2 ● Mix and stand for 5 minutes.
● increased nitrite (>10 mg/dL)
● The urine is now 80% saturated with ammonium
3
sulfate–this is optimal for hemoglobin precipitation.
4 ● Filter or centrifuge urine.
5 ● Test supernatant with reagent strip.

Results and Interpretation:

● Hemoglobin is present.
(+) ○ red precipitate
○ supernatant has a negative blood reagent strip
● Myoglobin is present.
(–) ○ supernatant remains red in color
○ the blood reagent strip is positive

B.2) Benzidine Test

Procedure: Benzidine Test

● Place 1 mL clear Benzidine reagent in a 13x100 test
1
tube.
2 ● Add 2 mL of urine previously boiled then cooled.
3 ● Add 1 mL fresh 3% hydrogen peroxide
4 ● Observe color reaction after 5 minutes

Results and Interpretation: Benzidine Test

Trace ● faint green
+ ● green
++ ● greenish blue
+++ ● blue
++++ ● deep blue

Transcribed by: Abenes, Aliping, Guarin, Villanueva (2024). 22

 Other Qualitative Test For Blood
Method Reagent Principle Positive Result
● myoglobin
○ supernatant remains red in
● larger Hgb molecules are
color
Salt Precipitation precipitated by ammonium
○ (+) blood reagent strip
Method (Blondheim ● ammonium sulfate sulfate
● hemoglobin
Test) ● myoglobin remain the
○ red precipitate
supernatant
○ supernatant has a negative
blood reagent strip
● green to blue color
● benzidine powder
○ liberated O2 oxidizes
Benzidine Test ● glacial acetic acid
benzidine to a green or blue
● 3% hydrogen peroxide
color
● green to blue color at the zone
● guaiac gum solution of contact
Guaiac Test ● glacial acetic acid ○ liberated O2 oxidizes the
● 3% hydrogen peroxide phenols present in guaiac to
● hydrogen peroxide is quinones
decomposed to H2O and O2 in ● blue color within 2 minutes
the presence of ○ liberated O2 oxidizes
pseudoperoxidase activity of o-toluidine to a blue color
Hgb or myoglobin ○ red dye is added to mask
● o-toluidine
discoloration of the tablet by
● strontium peroxide
blood
● Ca acetate
Occult Tablet Test ○ Na bicarbonate reacts w/
● tartaric acid
tartaric acid to liberate CO2,
● Na bicarbonate
causing effervescence to
● red dye
facilitate the reaction
○ tartaric acid and Ca acetate
reacts w/ strontium peroxide to
form HsO2
● ortholouidine in 1% pure
methyl alcohol + mixture ● ortholouidine and acid-peroxide ● greenish blue to deep blue color
Orthotoluidine Test
of glacial acetic acid is added to urine sediment that last for 1 minute or longer
● H2O2

Transcribed by: Abenes, Aliping, Guarin, Villanueva (2024). 23

 TEST FOR BILIRUBIN B) QUALITATIVE TEST FOR BILIRUBIN
A) Reagent Strip
B) Qualitative Test for Bilirubin B.1. Diazo Tablet Test (Ictotest Method)
B.1) Diazo Tablet Test (Ictotest Method) ● confirmatory test for bilirubin
B.2) Rosenbach Modification of Gmelin’s Test
B.3) Harrison’s Spot Test ● most sensitive test for bilirubin
○ can detect bilirubin concentration as low as 0.05-0.1
mg/dL
A) REAGENT STRIP FOR BILIRUBIN
Procedure: Ictotest
Principle: Azocoupling Reaction
1 ● 10 drops of urine are added to a special adsorbent
● based on the coupling reaction between bilirubin and pad
the diazonium salt impregnated in the reagent pad in ● Ictotest tablet is placed on top of the pad then 2 drops
acid medium 2
of water.
3 ● The tablet is removed after 30 seconds.

● produces a color change from light tan to beige or pink

Results and Interpretation: Ictotest
(+) ● Purple or blue coloration
(–) ● Red or pink (or any other coloration)

Sensitivity of the Test and Reagent Used

● depends on the brand of reagent strip used

● uses 2,4-dichloroanilinediazonium
Multistix
salt
● uses 2,6-dichlorobenzine
Chemstrip diazonium salt as reagent
impregnated in the pad

Result Cause of False Result

● drugs
○ chlorpromazine and iodine metabolites
■ drugs that can react directly with
diazonium salt
● highly pigmented substances
False
○ can mask the reaction
Positive (+)
○ phenazopyridine (drug) and indican (in
intestinal disorders)
■ results to a highly pigmented urine
B.2. Rosenbach’s Modification of Gmelin’s Test
making the interpretation of the result
difficult Procedure: Rosenbach’s Mod. of Gmelin’s Test
● improper specimen storage 1 ● Pipette 5mL of freshly voided urine in 13x100 test
○ must be protected from light tube.
■ as it causes rapid oxidation of bilirubin ● Tilt the tube a little and overlay with 2mL of nitric acid
2
to biliverdin or hydrolysis to free by allowing the acid to flow on the sides of the tube.
False bilirubin. ● Carefully return the test tube to its original vertical
3
Negative (–) ● ascorbic acid (>25 mg/dL) position
○ interferes with the test by combining with
the diazonium salt, preventing its Results and Interpretation: Rosenbach’s
reaction with bilirubin (+) ● Play of colors - Greenish towards urine layer
● high concentrations of nitrite
(–) ● Play of colors - Without the green

Transcribed by: Abenes, Aliping, Guarin, Villanueva (2024). 24

B.3. Harrison’s Spot Test

Procedure: Harrison’s Spot Test Results and Interpretation: Harrison’s Spot Test
1 ● Pipette 5mL urine into a 16x150 test tube. (+) ● Blue to green color
2 ● Add 5mL of 10% Barium Chloride. (–) ● No blue to green color
\

3 ● Invert tube 3x to mix preparation.

● Filter the preparation.
4
● Save filtrate for urobilin determination.
5 ● Spread the filter paper and allow to partially dry.
● Place the filter paper into an evaporating dish then
6 add a few drops of Fouchet’s reagent at the center of
the filter paper.

Other Qualitative Test For Blood

Method Reagent Principle Positive Result
● azocoupling reaction w/ diazonium
Diazo Tablet Test
– salt ● (+) purple or blue
(Ictotest Method)
● same principle with the reagent strip
● (+) green (biliverdin)
● bilirubin (w/ acid) → varying degrees
Gmelin’s Test ● nitric acid ● (+)blue (bilicyanine)
of oxidation
● (+)yellow (choletelin)
● Barium chloride + sulfate radicals in
● 10% barium chloride
urine → barium sulfate
● fouchet’s reagent
precipitates → bilirubin will adhere
Harrison’s Spot Test (25% trichloracetic ● (+) blue to green color
● ferric chloride, (w/ trichloroacetic
acid)
acid) oxidizes bilirubin → biliverdin
● 10% ferric chloride
→ color change.
Rosenbach’s ● drop of nitric acid containing yellow
● (+) band of colored ring around
Modification of ● nitric acid nitrous acid → applied to a filter
the nitric acid
Gmelin’s Test paper soaked in urine
● 20 % p-toluene
sulfonic acid
Maher Test – ● (+) green color
● 0.1 % solution of
sodium nitrate
● bile pigments + calcium hydroxide
→ precipitated → oxidized → green
Huppert’s Test – ● (+) green compound
colored compound (acid-alcholod
sol’n [HCl & ethyl alcohol]) ]
● bilirubin is concentrated by barium
sulfate & phosphate → oxidized by
Ferric Chloride Test – –
ferric chloride (acid sol’n) →
biliverdin & cholecyanin
● barium chloride + urine → barium
● barium chloride
sulfate ppt, bilirubin absorbed → ppt
solution
Fouchet Test + drop of Fouchet’s ferric chloride ● (+) greenish-blue spot
● Fouchet’s ferric
solution → FeCl oxidize bilirubin →
chloride sol’n
biliverdin (green)

Transcribed by: Abenes, Aliping, Guarin, Villanueva (2024). 25

 TEST FOR UROBILINOGEN Result Cause of False Result
A) Reagent Strip
● drugs
B) Qualitative Test for Urobilinogen ○ phenazopyridine
B.1) Wallace-Diamond Method False ● atypical urine color
B.2) Schlesinger’s Method Positive (+) ○ ingestion of red beets
C) Tests for Bile Salts
C.1) Hay’s Test
○ masks the reaction pad color change
● PBG and Ehrlich’s reactive substances
● improper preservation
A) REAGENT STRIP FOR UROBILINOGEN
○ resulting to the oxidation of urobilinogen
● reagent strip reaction for urobilinogen differs between False
to urobilin
Chemstrip and Multistix more significantly than do other Negative (–)
○ presence of formalin and nitrites
parameter as each brand follows a different principle ■ would also inhibit the reaction

B) QUALITATIVE TEST FOR UROBILINOGEN

B.1. Wallace-Diamond Method (Ehrlich’s Benzaldehyde Test)

Sensitivity of the Test and Reagent Used Procedure: Wallace Diamond

● depends on the brand of reagent strip used 1 ● Place 5 mL of freshly voided urine in 13x100 test tube.
Multistix ● employs Ehrlich’s aldehyde reaction 2 ● Add 0.5 mL of Erlich’s aldehyde reagent.
Chemstrip ● based on azocoupling reaction 3 ● Mix and observe for 10 min.
● Observe color by looking down into the tube held over
4
Multistix Reagent Pad’s Ehrlich’s Reagent a white surface.
● slightly more sensitive
○ can detect urobilinogen levels as low as 0.2 mg/dL Results and Interpretation: Wallace Diamond
○ also due to temperature (+) ● Rose red color
○ principle may react with other Ehrlich’s reactive (–) ● Absence of rose red color
substances
■ such as porphobilinogen, methydopa, indicant Semi-Quantitative Test
etc. 1 ● If the qualitative test is positive, proceed as follows.
■ causes a false-positive reaction ● Prepare 10 mL of 1:10, 1:20, 1:40, 1:80 dilutions at
2
● impregnated with: room temperature
○ Ehrlich’s reagent (pdimethylaminobenzaldehyde), 3 ● Repeat steps 1-4 above
○ a color enhancer, and 4 ● Observe for any color change in each dilution
○ an acid buffer ● Result is expressed in terms of the highest dilution
5
● urobilinogen reacts with Ehrlich’s reagent to form a red giving a definite pink or rose color.
chromophore
B.2. Schlesinger’s Method (Urobilin Test)
○ the reagent pad changes from light pink to dark pink
Procedure: Schlesinger’s
● Pipette 5mL urine free of bile pigments into 16x150
test tube.
1
○ use the barium chloride filtrate from bilirubin
Chemstrip Reagent Pad’s Azocoupling Reaction determination
● less sensitive compared to Multistix 2 ● Add 5 drops of Lugol’s solution.
○ can detect levels as low as 0.4 mg/dL 3 ● Finally, add 5 mL of Schlesinger’s reagent.
○ but principle is more specific to urobilinogen 4 ● Mix preparation by inversion, then centrifuge.
● with diazonium salt (4-methoxy- benzenediazonium- 5 ● Examine clear filtrate after 1 hour against bright light.
tetrafluoroborate)
○ reacts with urobilinogen in an acid medium Results and Interpretation: Schlesinger’s
○ azo dye produces a color change from white to pink
● Greenish fluorescence indicates the
(+)
presence of more than normal
(–) ● Absence of greenish fluorescence

Transcribed by: Abenes, Aliping, Guarin, Villanueva (2024). 26

 Qualitative Test For Blood
Method Reagent Principle Positive Result
● red color

● urobilinogens → condense
Wallace-Diamond
● Ehrlich’s aldehyde rgn’t (Ehrlich’s rgn’t) in acid sol’n →
Method
color rxn

● Lugol’s solution
(Potassium triiodide) ● urobilinogen oxidized to →
Schlesinger’s
● Schlesinger’s reagent urobilin (by iodine) → ● (+) greenish fluorescence
Method
(Zinc acetate in absolute zinc-urobilin complex
alcohol)

C) TEST FOR BILE SALTS

C.1. Hay’s Test

● bile salt lowers the surface tension of urine
○ sulfur sinks in the presence of bile

Procedure: Hay’s Test Results and Interpretation: Hay’s Test

1 ● Cool urine sample by refrigeration for several hours. ≥ 0.01% ● sulfur powder sinks at once
● Once cooled, sprinkled unto the surface small ≥ 0.0025% ● sulfur powder sinks after gentle agitation
2
amounts of sulfur powder. ● sulfur powder remains floating even after
Absence
agitation

Test For Bile Salts

Method Reagent Principle Positive Result
● ≥ 0.01%
● ≥ 0.0025%

● bile salts lower the surface

tension of a liquid causing sulfur
Hay’s Test ● sulfur powder
powder to sink when placed on
the solution

● sucrose (dehydrating agent)

● sucrose
Pettenkofer’s Test → hydroxymethylfurfural → red ● (+) red ring at point of contact
● sulfuric acid
compound (with bile salts)

Transcribed by: Abenes, Aliping, Guarin, Villanueva (2024). 27

 TEST FOR PORPHOBILINOGEN ● Chloroform extraction on aqueous layer
○ 2-5mL chloroform is added in the tube and shaken
A) QUALITATIVE TEST FOR PORPHOBILINOGEN 4 vigorously
○ these solvents are then allowed to separate upon
standing or can be hastened by centrifugation
A.1) Hoesch Test
● Butanol extraction
● screening method and modification of Ehrlich’s reaction
○ perform when red is in the aqueous layer in
● can detect as low as 2 mg/dL of PBG chloroform extraction
● urine with atypical colors make it difficult to interpret 5 ○ aqueous layer is transferred to another tube and an
equal volume of butanol is added
○ the mixture is vigorously shaken and allowed to
separate

Results and Interpretation: Watson-Schwartz

● presence of Ehrlich reactive
Red or Magenta substances
○ perform chloroform extraction
Chloroform Extraction

Red in
Result Cause of False Result Chloroform ● presence of Urobilinogen
● same as those affecting Ehrlich’s Layer (Bottom)
reaction ● presence of PBG and other Ehrlich
● large quantities of drugs and indoles Red in Aqueous reactive substances
False Positive (+) Layer (Top)
○ e.g. phenazopyridine ○ perform butanol extraction
○ thus, Hoesch test is preferred over
Butanol Extraction
Watson-Schwartz

A.2) Watson-Schwartz Test

● modification of Ehrlich’s reaction
● allows differentiation of urine PBG from other Ehrlich
reactive substances (e.g. urobilinogen)
○ Ehrlich’s reaction performed on solvents with
Red in Aqueous
different polarity ● presence of PGB
● the layer in the characteristic red color is observed Layer (Bottom)
identifies the substance present
● PBG is not soluble in chloroform and butanol Red in Butanol ● presence of other Ehrlich reactive
Layer (Top) substances
Procedure: Watson-Schwartz
● Mix equal parts of urine and Ehrlich’s reagent (2mL
1
each).
2 ● Add equal amount of Na acetate to the tube (4mL).
● Color change of red or magenta color = further testing
○ presence of Ehrlich reactive substances
3
○ perform chloroform extraction
○ no color change = no further testing

Transcribed by: Abenes, Aliping, Guarin, Villanueva (2024). 28

 Test For Porphobilinogen
Method Reagent Principle Positive Result
● based on Ehrlich’s reaction but
the ratio of urine volume is ● deep pink or red color develops
inversed and disperses uniformly%
● Ehrlich’s reagent (DMAB)
Hoesch Test ● creates a highly acidic mixture ● intensity of color directly relates
● hydrochloric acid
preventing the interference of to the amount of PBG present in
urobilinogen (except when con. urine
is >20 mg/dL)
● urobilinogen
● based on the solubility ○ red color is only found the in
● Ehrlich’s reagent (DMAB)
characteristics of PBG with the chloroform layer (bottom)
Watson-Schwartz ● sodium acetate
respect to pH and solvent ● PGB and other Ehrlich reactive
Test ● chloroform
● PBG + Ehrlich’s reagent + Na substances
● butanol
acetate → red color ○ red color only in the aqueous
layer (top)

Transcribed by: Abenes, Aliping, Guarin, Villanueva (2024). 29

 UNIT 4: LEUKOCYTE ESTERASE, NITRITE AND Result Cause of False Result
ASCORBIC ACID ● urine contaminated with vaginal secretions
● drugs
LEUKOCYTE ESTERASE TESTS ○ phenazopyridine
False
○ nitrofurantoin
Leukocyte Esterase Positive (+)
● food that may cause a red or pink urine
● derived from granulocytes, monocytes and
color
macrophages
○ beets
○ an increase in WBCs primarily comprised of
lymphocytes will not be detected ● increase urinary protein (500 mg/dL)
● glucose (>3 g/dL)
LEUKOCYTE ESTERASE REAGENT PAD ● high specific gravity
● antibiotics
Principle: Leukocyte Esterase Pad False
○ gentamicin
● incorporated with esters that will be cleaved in the Negative (–)
○ cephalosporin
presence of leukocyte esterase forming an aromatic ● strong oxidizers
compound ○ interferes with reaction pH
○ aromatic compound formed is coupled with ○ can also cause a false negative result
diazonium salt in the reagent pad
○ causes a color change from beige to violet
● often reported as positive or negative
○ quantitative evaluation of WBCs is included in
routine microscopic examination of urine

Screening Test
● initially detects about 10 to 25 WBCs/uL
○ negative result does not rule out the presence of
increased numbers of WBCs
■ indicates that the amount of leukocyte esterase
may not be sufficient to produce a positive test
■ may be from an increase in WBCs primarily
comprised of lymphocytes
○ positive leukocyte esterase result may be
observed despite the absence of WBCs during the
microscopic examination
■ when the urine is significantly dilute causing cell
lysis
● capable of detecting the presence of both intact and
lysed white blood cells
● result of this screening test can give us an idea what to
look for
○ increased leukocyte esterase would:
■ suggest the presence of WBCs vv
■ help select specimens that require further
investigation during the microscopic
examination

Transcribed by: Abenes, Aliping, Guarin, Villanueva (2024). 30

 NITRITE TESTS

NITRITE REAGENT PAD

Principle: Nitrite Reagent Pad

● based on the diazotization rxn of nitrites with aromatic
amine impregnated in the pad → diazonium salt →
azocoupling rxn → color change → white to pink
● results: (+) or (–) with any degree of pink
● there is no correlation between a positive result and
quantity of bacteria present
● negative result does not rule out bacteriuria (nitrate
ASCORBIC ACID TESTS reducing bacteria)

ASCORBIC ACID REAGENT PAD

● rarely included in reagent strips

Principle: Ascorbic Acid Reagent Pad

● action of ascorbic acid to reduce a dye impregnates in
the reagent pad causing distinct color change → blue to
orange
● detect ascorbic acid levels as low as 7mg/dL
Results and Interpretation: Nitrite Reagent Pad
(+) ● pink color
(–) ● absence of pink color
Result Cause of False Result
False Positive (+) ● free sulfhydryl drugs Result Cause of False Result
● substances that color urine red or
pink in an acid pH:
○ phenazopyridine
False Positive (+)
○ beets
● improper handling and storage
●
● chemical tests that involve
False Negative (–) diazonium salt
● high concentrations of ascorbic acid

NOTE:
● Chemstrip reagent strip tests for blood and glucose are
significantly resistant to high ascorbic acid
concentration

Transcribed by: Abenes, Aliping, Guarin, Villanueva (2024). 31

Urinalysis Test Strip Color Chart (Siemens Multistix® 10 SG Reagent Strips)

*Tetramethylbenzine. Hb, Hemoglobin; Mb, Myoglobin

● The test pad is impregnated with a polyelectrolyte, a pH

indicator, and is maintained at an alkaline pH. When the strip is
immersed in urine, the pKa of the polyelectrolyte decreases
proportionally to the ionic concentration of the specimen.
● As the pH of the test pad decreases, the bromothymol blue
indicator changes color from dark blue-green (SGionic 1.000) to
yellow-green (SGionic 1.030). As the number of ions present in the
urine is increased, more protons are released from the
polyelectrolyte, resulting in a decrease in the test pad pH and a
color change in the indicator.

Sigrid Ali. (2024).

Sigrid Ali. (2024).
ANALYSIS OF URINE MLS 321L

& OTHER BODILY FLUIDS 8438L

LABORATORY BS MLS 3 MIDTERMS
M4: MICROSCOPIC EXAMINATION OF URINE

UNIT 1: SEDIMENT PREPARATION & EXAMINATION GUIDELINES ON SEDIMENT PREPARATION

A) Sediment Preparation
Definition of Terms B) Specimen Volume
● specialized centrifuge procedure C) Centrifugation
● produces a monolayer of the D) Sediment Concentration
Cytocentrifugation E) Volume of Sediment Examined
cellular constituents in various body F) Examination of Sediment
fluids (i.e. urine) G) Format of Reporting
Rous Test/
● chemical reaction used to identify
Prussian Blue A) SPECIMEN PREPARATION
the presence of Fe in body fluids
Staining
Uromodulin/ ● glycoprotein that is secreted by the Routine Urinalysis Specimen Guidelines
Tamm-Horsfall renal tubular cells of the thick ● freshly collected or adequately preserved
Ideal
Protein ascending loop of Henle and DCT first morning specimen (midstream clean
Specimen
● cellular pellet remaining after urine catch)
centrifugation Formed ● RBC, WBC, hyaline casts disintegrate
Urinary Sediment ● normal urinary sediment: Elements rapidly in dilute, alkaline urine
○ free of crystals ● may cause precipitation of amorphous
○ contain less cells
urates & phosphates, other
○ very low concentration of proteins
Refrigeration non-pathologic crystals
Birefringent / ● ability of a substance to refract light
● can obscure other elements in the urine
Double Refractile in two proteins
sediment
● circular field observed through a
Field of View ● warming spx to 37°C prior to
microscope Warming
centrifugation may dissolve some crystals
● type of microscopic illumination
● lamp condenser located above the
B) SPECIMEN VOLUME
Kohler Illumination light source → focuses the image of
● recommended urine volume = 30-50 mL
the light source onto the front focal
● sediment preparation volume = 10–15 mL (12 mL)
plane of the condenser
○ representative sampling of urine formed elements
● describes objective lenses that
remain in focus when the user pediatric ● if obtaining a 12 mL urine is not possible,
Parfocal
switches from one objective to samples volume of urine can be reduced to 6 mL
another of different magnification ● note in the report form
● traditional method developed by if < 12 mL ● corrections:
Addis (1926) ○ e.g. if 6 mL of urine is centrifuged,
● standardize the quantification of results are multiplied by 2
formed elements in the urine if < 3 mL is ● urine is examined microscopically, w/out
Addis Count available concentration of the sediment
● uses hemocytometer to count the
no. of RBC, WBC, cast, and
epithelial cells present in a 12-hour
specimen

Transcribed by: Abenes, Aliping, Guarin, Villanueva (2024). 1

 C) CENTRIFUGATION E) VOLUME OF SEDIMENT EXAMINED
● well-mixed urine → poured into a centri tube →
Conventional Glass Slide Method
centrifuged at 400 to 450 x g for 5 mins
● recommended volume = 20ul covered by a 22x22
● DO NOT use the centrifuge break because this will
mm glass coverslip
cause the sediment to resuspend
● 20 ul
○ result: erroneously ⬇ no. of formed elements in the
○ representative sample for detecting formed
concentrated sediment
elements
● centrifuge in capped tubes to prevent biohazard
aerosols F) EXAMINATION OF SEDIMENT
● allows optimal sediment concentration
w/out damaging fragile formed elements Standardized Slide
Centrifugation ● used for the microscopic
Speed ● fragile cellular casts:
○ RBC casts examination of urine
○ WBC casts sediment
● ensure that the same
D) SEDIMENT CONCENTRATION volume of sediment is
presented for viewing
Technique Sediment Concentration each time
Commercial Systems ● 12:1 to 30:1
● 24:1 to 12:1 Commercial Standardized
○ achieved by using a 12 mL Slides
Manual Techniques
urine, leaving a sediment of ● made of molded plastic
0.5 - 1 mL
● built-in coverslip
Sediment Concentration Factor ● used when standardized slides are absent
● relates to the probability of detecting elements present
● urine sediment is presented to a chamber
in low quantities
filled by capillary action
● used when quantifying the number of elements per mL
● technique facilitates uniform distribution of
Disposable
the formed elements throughout the
Transfer
viewing area
Pipette
● e.g. CF of 24 indicates a higher probability of detecting ● ensures the specimen does not flow
elements present in low amounts a compared to a CF outside of the coverslip as it may result in
of 12 the loss of heavier elements (casts)
○ 12 ÷ 0. 5 = 24 ● under both LPO & HPO
○ 24: 1 𝑐𝑜𝑛𝑐𝑒𝑛𝑡𝑟𝑎𝑡𝑖𝑜𝑛 ● (1) first examined under LPF to detect
Minimum of
casts and to ascertain the general
Supernatant Urine 10 Fields
composition of the sediment
● removed by decanting or using a disposable pipette ● (2) switch to HPF to delineate structures
until 0.5 - 1 mL of urine is retained ● have the tendency to locate near the
● urine should be aspirated off rather than poured off Casts
edges of the cover slip
○ maintain uniform sediment concentration
● it is essential that sediments be examined
under reduced light when using bright
Resuspension
Unstained field microscopy
● done by flicking the bottom of the tube
Sediment ○ obtained by partially closing the iris
● essential to provide equal distribution of elements in the
Have the diaphragm and then adjusting the
microscopic examination field condenser downward until optimum
Refractive
● used to gently suspend the sediment by contrast is achieved
Pipette Index (RI)
gently pipetting it through and through ● too much light = unseen structures
Similar to
● e.g. hyaline casts
● cause fragile and brittle formed elements Urine
Too Vigorous ○ have a very low RI
● RBC cast, waxy cast may break into ○ overlooked if the light is too bright or not
Agitation
pieces enough contrast

Transcribed by: Abenes, Aliping, Guarin, Villanueva (2024). 2

Fine ● enable the viewer to see the depth of the ● enumerated by considering the most frequent
Adjustment object & other structures number seen per field in a total of 10 fields
Should Be ● aids in obtaining a complete (average no. in range)
● e.g. if the no. of hyaline casts in 10 different LPF
Continuously representation of the sediment Range
is 0, 2, 2, 0, 0, 2, 2, 0, 1, 2
Adjusted constituents ○ the range reported would be 0-2 hyaline cast/
Sediments low power field since consistently, the counts
Should Be ● may be refrigerated for a few hours if the observed are 0 & 2
Examined examination cannot be performed NOTE:
Within 10-15 immediately ● although a component may be reported using
Mins low-power magnification, high-power magnification may
be needed to specifically identify/categorize it
G) FORMAT OF REPORTING ● absence of the formed elements is reported as:
NEGATIVE
Manual Microscopic Examination Sample Reference Values of Formed Elements in Urine
● amount of components is qualitatively assessed per Component Number
field of view (descriptive / semi-quantitative/ numeric RBC 0-3/HPF
terms WBC 0-8/HPF
Hyaline Cast 0-2/LPF
Qualitative Terms & Descriptions for Fields Of Views Fine Granular Cast 0-2/HPF
Numeric Transitional Epithelial Cell 0-2/HPF (few)
Terms Qualitative Terms & Descriptions
Terms Renal Tubular Epithelial Cells 0-2/HPF (few)
Rare 1+ ● present, but hard to find
Reference Values
● one (or more) present in almost
Few 1+ ● affected by the sediment concentration factor in use
every field of view
● e.g. concentration factor of 30
● easy to find; number present per ○ reference value of WBC is 0-8/HPF
Moderate 2+ field of view varies bit it should be ● e.g. concentration factor of 12
more than few and less than many ○ conventional value of 0-5/HPF
● prominent; large number present in
Many 3+ Average Number of Elements Per LPF/HPF is
all field of view
Converted to the Number Per mL
● field of view is crowded with the
Packed* 4+ ● the area of the field of view for the LPF & HPF must
element
*’packed’ is not routinely used in the laboratory for describing quantities of
1 be determined first using the formula:
formed elements ○ area = 2πr
● calculate the maximum number of LPF or HPF in
Terminology & Manner of Reporting the viewing area by dividing the area of the coverslip
used for viewing with that of the area per field of view.
● differ slightly among laboratories but must be consistent
within a particular laboratory system 2
● formed elements (see Table below) NOTE:
○ reported per LPO / HPO ● 22 mm x 22 mm is the dimension of the ordinary
microscopic slide used in the laboratory; other
Manner of Reporting for Formed Elements standardized microscopic slides have their own area
Manner of Reporting ● calculate the field conversion factor or the number
Formed Element of HPF/LPF per mL of urine testing using the
(in at least 10 fields)
● quantitative concentration factor and the volume of sediment
cast, parasites examined
(average range)/ LPF 3
RBC, WBC, transitional
● quantitative
epithelial cells, renal tubular
(average range)/ HPF* ● vol. of sediment viewed per mL = vol. of sediment
epithelial cells
that is placed onto the slide for microscopic viewing
squamous epithelial cells, ● semiquantitative
crystal & mucus threads (descriptive terms)/ LPF ● calculate the number of formed elements per ml of
● semiquantitative 4 urine
bacteria, yeast
(descriptive terms)/ LPF

Transcribed by: Abenes, Aliping, Guarin, Villanueva (2024). 3

 FORMED ELEMENTS RBCs in Different Urine Concentration
● normal urine sediment (variety of formed elements) Hypertonic Urine
○ RBC ● RBCs become smaller
○ WBC ○ intracellular water is lost by osmosis → crenated
○ epithelial cells ● crenated RBCs
○ casts ○ lose biconcave shape
○ crystalline materials ○ become spheres covered w/ spicules or crenations
○ appear rough microscopically compared to normal
○ opportunistic microorganisms
RBCs
■ bacteria
■ yeast
■ Trichomonads

Where Formed Components Can Originate

Throughout the Urinary Tract ● glomerulus → urethra
● menstrual blood
From Contamination ● spermatozoa
Fig. Crenated RBCs
● fibers
● starch granules
● NOT ALL formed elements indicate an abnormal /
pathologic process
○ normally, few RBCs, WBCs, epithelial cells, &
hyaline casts are observed in the urine sediment of
a normal, healthy individual
● presence of ‘abnormal’ components is already
Fig. Microcytic & Crenated RBCs (x100, LPF)
diagnostically significant
● determine the actual concentration of the Hypotonic Urine
Sediment ● RBCs swell & release their hemoglobin → ghost cells
sediment
Preparation ● ghost cells
● reflects the number of elements that
Methods ○ colorless, empty cells w/ intact membranes but no
may be present in the microscopic field hemoglobin
○ difficult to see using brightfield microscopy
A) RED BLOOD CELLS (RBCS) ○ phase contrast/ interference contrast microscopy is
● anucleated, biconcave, refractile cells recommended
● 8um in diameter & 3um in depth ● alkaline urine
○ promotes RBC lysis & disintegration
● urine sediment is characteristically red in color

Fig. Ghost Cells

Fig. Normal Red Blood Cell in Urine. Fig. RBCs: 2 viewed from above
(x400, HPF) appear as biconcave disks, and one
Dysmorphic / Distorted Erythrocytes
viewed from the side appears
hourglass-shaped (arrows). (x400, ● indicates glomerular origin
HPF) ● present with normal erythrocytes in the urine of healthy
individuals
○ especially after strenuous exercise
● ⬆ in numbers are associated with a pathologic process
● abnormal urine concentration affects RBC appearance
& small no. of dysmorphic cells found in
non-glomerular hematuria

Transcribed by: Abenes, Aliping, Guarin, Villanueva (2024). 4

Acanthocytes ● glomerular disorders Conditions Associated with RBCs In Urine
Sickle Cells ● sickle cell disease ● glomerular membrane damage
Hematuria
● vascular injury w/ genitourinary tract
● glomerular or tubular bleeding may be
indicated
Hematuria w/ ○ glomerulonephritis
RBC casts ○ pyelonephritis
○ cystitis
○ renal calculi
Hematuria w/ ● indicative of the extent of the damage/
Proteinuria injury
Fig. Dysmorphic RBC Fig. Dysmorphic RBCs (x400).
Notice the smaller size and
● bleed is most likely occurring below the
fragmentation. Hematuria kidney
w/out Casts ● may be caused by contamination
or Proteinuria ○ menstrual blood
○ hemorrhoid
● attributed to the occurrence of hematuria:
○ hypertension
○ smoking
○ appendicitis
Non-Renal
○ tumors
Disorders
○ trauma
Fig. Acathocytes Fig. Acanthocytes (arrows), red
blood cells with one or more blebs
○ drugs
protruding from a ring form. Phase ■ anticoagulant drugs
contrast, (x400, HPF) ■ sulfonamides

Interpretation of Tests Results For RBCs

● (1) lysis might have occurred
(+) Chemical Test ○ especially if the sample is
hypotonic/ alkaline
(–) Microscopic ● (2) lysis have already occurred
Examination within the urinary tract before urine
collection
Fig. Dysmorphic (schizocyte, crenated/echinocyte) and normal forms of
● (1) ascorbic acid interference
red blood cells. A single ghost red blood cell is located at top of view.
Phase contrast. (x400, HPF) ● (2) ruled out ascorbic acid
○ formed elements observed are not
RBCs but a ‘look-alike’ component
(–) Chemical Test
■ yeast
■ oil droplets
(+) Microscopic
■ bubbles
Examination
■ monohydrate calcium oxalate
crystals
● identified by alternative techniques
(staining/ polarizing microscopy)
● cause of other substance:
○ myoglobin
(F+) Chemical Test
○ microbial peroxidases
○ strong oxidizing agents

Transcribed by: Abenes, Aliping, Guarin, Villanueva (2024). 5

‘Look Alike’ Elements of RBCs & Methods To Differentiate

Calcium Oxalate Small Bubbles or

RBCs Yeast WBCs
Crystals Droplets of Oil
● microscopic Factors Characteristic
appearance ● microscopic Variation in ● larger than
○ non-nucleated appearance Size RBCs ● microscopic
biconcave disks ○ varies in size ● occur in appearance
○ crenated in ○ spherical or Uniformity in ○ WBC can be
different
Microscopic hypertonic urine ovoid rather Appearance difficult to
– sizes
Appearance ○ ghost cells in than ● higher distinguish from
hypotonic urine biconcave & Refractility crenated RBCs
refractility
○ dysmorphic w/ often (hypertonic
glomerular exhibiting urine)
membrane budding
damage
Sternheimer
-Malbin ● stains RBC ● does not stain ● does not stain –
Stain
FIg. Oil Droplets
● easier to see the
nuclein of WBC
2% Acetic ● not eliminated ● not eliminated
● lyse RBCs
Acid after use after use
(Toluidine Blue
Stain )

Polarized ● do not polarize ● polarize unlike

– –
Light RBC RBC
Fig. Bubbles

B) WHITE BLOOD CELLS (WBCS)

● distribution of WBCs in urine mirrors that of peripheral
blood

Fig. White blood cells in urine Fig. One segmented & one
nonsegmented WBC. (x400, HPF)

Fig. Notice the multilobed nucleoli. (x400, HPF)

Transcribed by: Abenes, Aliping, Guarin, Villanueva (2024). 6

Neutrophil ● may develop and remain within the cell
● easier to identify than RBCs pushing the cytoplasm to one side and
○ contain granules & multilobed/segmented nuclei giving rise to large pale areas
● usually larger in diameter (10-20um)
● in an unstained preparation
○ grayish hue & grainy appearance
● in a dilute, hypotonic, alkaline urine
○ lyse rapidly (almost 50% in 2-3 hrs @ room temp)
○ become spherical balls
● in a hypertonic urine
○ neutrophils & other leukocytes become smaller but
do not crenate Fig. Bleb formation

● Brownian Movement ● finger-like or wormlike projections

○ demonstrated by large swollen cells protruding from their surfaces
○ uncontrolled & fast movement of granules within ● myelin forms (long filaments)
the cytoplasm as a result of collision forces ○ results from the breakdown of the cell
membrane
○ refractile cytoplasmic granules giving the
○ as cells die, additional vacuolization,
descriptive name– “glitter cells” rupturing or pseudopod formation may
Filament be observed
Formation

Fig. Myelin Filament formation

Fig. Glitter Cells

Eosinophil
Degenerative Changes of Neutrophils ● easily identified with their bilobed nuclei & acidophilic
● as neutrophils age and begin to cytoplasmic granules
disintegrate, their lobed nuclei fuse ● diameter: 12-17um
● can make neutrophils difficult to ● not normally seen in urine
distinguish from renal tubular collecting ○ 1% eosinophil is considered significant
duct cells

Resemble
Mononuclear
Cells

Fig. Hansel-stain eosinophil

Fig. WBCs with fused lobe Mononuclear Cells

● vacuoles develop within the cell periphery ● present in small numbers & are usually not identified
or on the outer membranes during urine microscopic analysis
Formation of ● appear to be empty or may contain a few
Blebs small granules
● blebs can detach and become free
floating in urine

Transcribed by: Abenes, Aliping, Guarin, Villanueva (2024). 7

 Mononuclear Cells Conditions Associated with WBCs in Urine
Lymphocytes ● ⬆ pus excretion in urine
Pyuria
● smallest WBCs (6-9um) ● pus = contains white blood cells
● may resemble RBCs ● ⬆ in urinary WBCs
● single, round to slightly oval nucleus & scant clear ● indicates the presence of an infection of
cytoplasm inflammation in the genitourinary system
● occurs in bacterial infections
○ e.g. pyelonephritis, cystitis, prostatitis, &
urethritis
● nonbacterial disorders
○ e.g. glomerulonephritis, lupus
Leuko- erythematosus, interstitial nephritis,
cyturia chlamydia, mycoplasmosis, tuberculosis,
tumors in the urinary tract, trichomonads
and mycoses
Fig. Lymphocyte ○ trichomonads & mycoses
■ often appear in urine of women as
Monocytes contaminants from vaginal secretions
● macrophages / histiocytes ■ when these organisms are present in the
○ derived from monocytes urine from a male, a urinary tract
○ diameter: 30-40 um infection is implied
■ can be as small as 10 um ● eosinophils
■ or as large as 100 um ○ primarily associated with drug-induced
interstitial nephritis
● oval nuclei & azurophilic granules make ● small numbers may be seen with chronic
Small Sized
them difficult to distinguish from UTI & renal transplant rejection
Monocytes
neutrophile ● good predictor of acute interstitial
Eosino-
Large Sized ● irregular, kidney-shaped nuclei & nephritis (AIN) → untreated AIN →
philuria
Monocytes abundant cytoplasm permanent renal damage
○ assoc. with drug hypersensitivity
● macrophages & monocytes
(penicillin)
○ actively phagocytic cells capable of phagocytizing
● acute allograft rejection
bacteria, viruses, Ag-Ab complexes, RBCs, organic &
○ ⬆eosinophils in a kidney biopsy specimen
inorganic (fats & hemosiderin)
is considered a poor prognostic indicator
○ function to defend body against dead/ dying cells &
debris
○ interact immunologically with lymphoid cells to regulate Leukocyte Esterase (LE) Test
immunity ● detects the presence of WBCs in urine
● leukocyturia results to a (+) LE test
● if bacteria are present, nitrite test will also be (+)
○ nitrite test detects presence of bacteriuria
● however, since leukocytes readily lyse in urine,
discrepancies can occur between the number of cells
seen microscopically and the leukocyte esterase (LE)
screening test

(+) LE Test,
Fig. Monocyte Fig. Macrophage ● can occur because of WBC lysis and
disintegration
w/out WBCs
● predominant WBC is most probably
lymphocyte
○ lymphocytes have no leukocyte
(–) LE test,
esterase
● check that the reagent strips are
⬆WBC
functioning properly
● possibly indicate that “look-a- like”
elements such as RTE may be present

Transcribed by: Abenes, Aliping, Guarin, Villanueva (2024). 8

 ● amount of esterase present may be C) EPITHELIAL CELLS
insufficient to produce a positive ● not unusual to find epithelial cells in urine because they
(–) LE Test,
response are derived from the linings of the genitourinary system
● owing to hydration, hypotonic urine ● commonly result from the normal turnover of old cells
10 – 25
could cause the leukocyte esterase to (sloughing) and epithelial damage caused by
WBC/mL
be diluted such that it is below the inflammatory processes/renal disease
detection limit of the LE reaction ● classified as:
○ squamous epithelial cells
Renal Tubular Epithelial (RTE) Cells ○ transition epithelial cells
● primary concern in the identification of WBCs is the ○ renal tubular epithelial cells
differentiation from RTE cells Squamous Epithelial Cell
● usually larger than WBCs and more polyhedral in ● largest (40-60 um) and most common cells found in
shape, with an eccentrically located denser nuclei the urine sediment
● WBCs in the process of ameboid motion ● composition:
○ may be difficult to distinguish from epithelial cells ○ abundant, irregular cytoplasm w/ granulation aka
because of their irregular shape keratohyalin granules (increases as the cell ages)
● monocytes & macrophages are identified more ○ prominent centrally located nucleus about the
easily by size of an RBC (8- 14 um)
● often the 1st structures observed when the sediment
○ using supravital stains on the urine sediment
is examined under LPO appearing as:
○ making a cytocentrifuge preparation followed by ○ thin
Wright’s or Papanicolaou’s stain ○ flagstone-shaped elements w/ distinct cell border
○ acetic acid can be used to enhance nuclear detail ○ can be anucleated or multinucleated
● assume different conformations because their edges
can fold over (possibly resembling a cast) or curl while
they are suspended in urine
● specimens collected using the midstream clean- catch
technique contain less squamous cell contamination
Origination of Squamous Epithelial Cells
● from the linings of the vagina and entire
female urethra
Women
● large numbers of SEC often indicate vaginal
Fig. RTE cells
or perineal contamination
● lower/distal portion of the male urethra
Men ● for uncircumcised men, large numbers
suggest specimen contamination

Fig. Squamous epithelial cells.

Fig. Folded Squamous epithelial Fig. Squamous epithelial cell

cells (x200) embedded with bacilli

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 Squamous EC: Clue Cells
● appearing as squamous epithelial cells covered with the
Gardnerella coccobacillus
● bacteria should cover most of the cell surface and extend
beyond the edges of the cell
● granular, irregular appearance
● routine test
○ performed by examining a vaginal wet preparation Fig. Cluster of Transitional Epithelial Cells (x100)
○ used to assess vaginal infection by the bacterium
Gardnerella vaginalis

Fig. Transitional Epithelial cell (x400) Fig. Transitional & Caudete

Renal Tubular Epithelial Cells

Fig. Clue Cells and Bacterial Vaginosis
● vary in size and shape depending on the area of the
renal tubule
Transitional Epithelial Cell
○ convoluted tubular cells
● smaller than squamous cells
● appear in several forms (ALL have distinct, centrally ○ collecting duct cells
located nuclei): ● most clinically significant of the epithelial cells
○ spherical ● often present as a result of tissue destruction
○ polyhedral ○ nucleus is not easily visible in unstained sediment
○ caudate ● RTE can absorb bilirubin (as a result of liver damage)
● differences are caused by the ability of transitional present in the filtrate and appear deep yellow
epithelial cells to absorb large amounts of water
● originate from ● necrosis of the renal tubules (e.g., acute
○ lining of the renal pelvis ischemic or toxic tubular necrosis)
⬆ RTE
○ calyces
(< 2/HPF) ● heavy metals or drug (aminoglycosides)
○ ureters and bladder
○ upper portion of the male urethra toxicity
● usually present in small nos. in normal urine (0-2/HPF) ● appear turbid
● spherical forms of transitional epithelial cells Physical & ● color varies depending on the etiology.
○ difficult to distinguish from RTE cells Chemical
● positive (+) for blood, protein, LE and
Examination
● invasive urologic procedures nitrite
(catheterization)
⬆ Transitional
● instrument procedures (chemotherapy
Cells
and radiation)
● imply urinary tract infection
Physical & ● appear turbid
Chemical ● positive for blood and protein if
Examination malignancy is considered

Transitional EC: Variations in Size

Fig. Renal Epithelial Cells
● most prevalent transitional epithelial cell
● round or pear-shaped (30-40 um)
RTE Cells: Convoluted Tubular Cells
● dense oval to round centrally located
Superficial ● larger (20-60 um) than other RTE cells
nucleus and abundant cytoplasm
Cells ● oblong or cigar-shaped with a grainy
● nucleus is about the size RBC/ WBC
● peripheral borders of the nucleus and cell cytoplasm
membrane are distinctly outlined PCT ● nucleus: dense chromatin pattern that is
Cells usually eccentric, and can be multinucleated
Intermediate ● smaller than superficial cells
● could easily be mistaken for a granular or fatty
Cells ● appears round (20-30 um)
cast if it absorbed fat globules
Basal Cells ● columnar form
○ difference is that casts do not have nucleus

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 ● smaller (14-25 um) than those from the PCT
● round or oval
● nucleus: small, dense, round, usually eccentric
DCT ● cytoplasm is granular
Cells ● can be mistaken for WBCs and spherical
transitional epithelial cells
○ note: eccentrically placed round nucleus in
DCT

NOTE:
● differentiation bet. proximal and distal convoluted
tubular cells is not necessary, and are collectively
reported as “convoluted” renal tubular cells

RTE Cells: Collecting Duct Cells

● observed as fragments of undisrupted tubular
epithelium (at least three cells must be sloughed
together)

D) OVAL FAT BODIES

● oval fat bodies are RTE cells, monocytes/macrophages
that absorbed lipids/fats present in the glomerular
filtrate
Fig. Small Duct Cells Fig. Large Duct Cells
● appears highly refractile, w/ small no. of glistening
● polygonal or cuboidal (12-20 um) granules & the nucleus being difficult to observe
● nucleus: single, large, moderately dense ● indicate glomerular dysfunction and renal tubular
nucleus that occupies about 60-70% of the cell death
cytoplasm ● stain used in the sediment to confirm oval
● rarely round or spherical fat bodies and is examined using
● flat corner/edge on the membrane of the polarized microscopy
Large Duct cell ● triglycerides and neutral fats
Cells Sudan III or ○ stained orange-red
NOTE: Oil Red O ● cholesterol
● consider that over time, when these cells in Fat Stains ○ unstained
urine absorb water, they become swollen ○ thus polarized microscopy must be
● cells may be mistaken as macrophages or employed for its examination
monocytes if they assume a round or ○ characteristic Maltese cross formation is
spherical shape observed
● measure about 6-10 um
● nucleus: occupying about 90 % of the ● observed in cases of lipiduria caused by:
cytoplasm (6-8 um) ○ nephrotic syndrome
Small Duct ● eccentrically located ○ severe tubular necrosis
Cells ● often observed in cases of: ○ diabetes mellitus
○ pyelonephritis, acute tubular necrosis, ○ trauma that cause release of bone marrow fat from
kidney transplant rejection, malignant long bones
infiltration, and salicylate poisoning

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 ● used primarily in the microbiology for the
differentiation between of bacteria
○ gram-positive (blue)
Gram Stain
○ gram-negative (red)
● role in routine urinalysis is limited to the
identification of bacterial casts
● used to facilitate the visualization of
Fig. Oval Fat Body Fig. Cholesterol with Maltese Cross
hemosiderin
Appearance Prussian Blue ○ appears 2- 3 days after intravascular
Stain hemolysis
● also facilitates free floating or embedded
Oval Fat Bodies: Bubble Cells in epithelial cells & cast
● RTE cells in acute tubular necrosis
● large, nonlipid-filled vacuoles Microscopes
● endoplasmic reticulum has dilated prior to cell death Brightfield Microscopy
● magnified image that appears dark against a bright or
white background
Phase-Contrast Microscopy
● variations in the specimen’s refractive index are
converted into variations in light intensity or contrast
Fig. Bubble Cell ● ideal for viewing low-refractile elements
● e.g., hyaline cast, mucus threads, bacteria) and living
cells (e.g., Trichomonas).
SEDIMENT STAINS & MICROSCOPIC TECHNIQUES
● staining increases the overall visibility of elements Polarized Microscopy
using bright-field microscopy by changing their ● illuminates the specimen with polarized light
refractive index ○ light waves in which vibrations occur in a single plane
● used to identify and classify birefringent substances
Stains ○ substances that refract light in two directions
● most frequently used in urinalysis ○ shine brilliantly against a dark background
● supravital stain, consisting of
● uric acid crystals ● amorphous crystals
○ crystal violet
● monohydrate (urates: strongly;
Sternheimer- ○ safranin
Polarized calcium oxalate phosphates: very
Malbin Stain ● stained (pink to purple)
● fiber (clothing, weakly)
● providing clearer delineation of structure Substances
diapers) ● fatty casts
& contrasting colors of the nucleus and
● plastic fragments ● cholesterol globules
cytoplasm
● starch granule
● metachromatic stain that provides ● bacteria
enhancement of the nuclear details of a Non- ● RBCs
Polarizing ● casts ● cellular debris
cell (membrane
0.5% Solution ● useful in the differentiation between Substances ● mucus phospholipids)
of Toluidine WBCs and RTE cells
Blue ● also used in the examination of cells from Interference-Contrast Microscopy
other body fluids
● nuclear detail is also enhanced by the ● provides a three-dimensional image of an element,
addition of 2% acetic acid either of low or high refractive index, thus enhancing the
● confirm the presence of fats with cellular details
polarizing microscopy Dark-Field Microscopy
● stain orange-red
Oil Red O and ● enhance visualization of specimens that cannot be seen
○ triglycerides
Sudan III
○ neutral fats easily viewed with a bright-field microscope
● polarization ● used for unstained specimens
○ cholesterol ● e.g. identify the spirochete Treponema pallidum

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 Fluorescence Microscopy UNIT 2: URINARY CASTS
● used to detect bacteria and viruses within cells and
tissues through immunofluorescence CAST FORMATION
● urinary casts are formed in the distal and collecting
tubules with a core matrix of uromodulin
(Tamm-Horsfall protein)
● glycoprotein is secreted by the renal tubular cells of the
thick ascending limb of the loop of Henle and by the
DCT
● proteins such as albumin and immunoglobulins are
also incorporated into the cast matrix
● any urinary component (chemical or a formed element)
can be found incorporated into a cast
● formed cast detaches from the tubular epithelial cells →
flushed through the urine
● cylindruria
○ presence of casts in urine

Cast Shape & Size

● vary greatly depending on the diameter and shape of
the tubule
● cylindrical in shape and do NOT have dark edges
● nearly parallel sides and rounded or blunted ends, with
a thick middle portion
● cast becomes convoluted when after formation and
release from a tubule
● the narrower the tubular lumen, the narrower is the
resulting cast
LOH & DCT ● casts with tapered end
Collecting Tubules
● broad casts (2-6x wider
Dilated or Atrophic
than ordinary casts)
Tubules
Cytodiagnostic Urine Testing
● preparation of permanent slides using
cytocentrifugation followed by staining with
Papanicolaou stain
○ performed by the cytology laboratory
● provides an additional method for detecting and
monitoring renal diseases
● frequently performed independently of routine
urinalysis
● for detection of malignancies of lower urinary tract Fig. Convoluted Cast
● provides more definitive information about renal
tubular changes associated with Physiologic Correlation of Casts
○ transplant rejection ● casts are always renal in origin
○ viral, fungal, and parasitic infections
● important indicators of intrinsic renal disease
○ cellular inclusions (pathologic casts)
○ inflammatory conditions ● disorders in which cast may be present include:
● uses voided first morning specimen ○ glomerular damage
○ tubular damage
○ renal inflammation
○ renal infection

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 FACTORS INVOLVED IN CAST FORMATION Dissolution of Casts
1. urinary stasis (marked decrease in urine flow) ● dissolve in alkaline and neutral urine having a specific
2. increased urine acidity gravity of 1.003 or less
3. high solute concentration (sodium and calcium) ● can be retained in the tubule for varying lengths of time
4. presence of abnormal ionic or protein constituents ○ substances enmeshed in their matrices can
disintegrate
Casts in Normal Conditions ● cast matrix itself can undergo changes that become
● Tamm-Horsfall proteins is excreted at a relatively apparent microscopically
constant rate ○ e.g. transition from a granular to a waxy cast
● increase in cases of stress and exercise
Cylindroids
Acidic Urine ● casts are well formed at one end but are tapered or have
● in an acidic environment, gelation of protein and the a tail at the other end
● may result from:
precipitation of solutes are enhanced
○ incomplete cast formation
● acidification and concentration of urine occur in the ○ formation of a cast in a tubule where the lumen width
distal and collecting tubules, these tubules are the differs (naturally or from disease)
sites of most cast formation. ○ cast disintegration
● hyaline cast
Urinary Stasis ○ most common type of cast occurring as cylindroid
● fine granular casts
● due to obstruction from disease processes or ○ also forms cylindroids in rare cases
congenital abnormalities
● promotes the accumulation and concentration of
ultrafiltrate components
○ as the cast forms, urinary flow within the tubule
decreases as the lumen becomes blocked
● internal tension formed account for the wrinkled and
convoluted appearance of older casts
Fig. Cylindroids

Plasma Protein Constituents

CLASSIFICATION OF CASTS
● e.g., albumin, globulins, hemoglobin, myoglobin
● made on the basis of their appearance and the cellular
● ⬆ in the lumen ultrafiltrate
components that they contain
● cast formation is enhanced greatly
● become incorporated into the uromodulin protein A) Casts with Homogeneous Matrix Composition
matrix, along with any cells and cellular or granular 1. Hyaline Casts
2. Waxy Casts
debris
B) Cellular Inclusion Casts
1. RBC Casts
Cast Materials 2. WBC Casts
3. RTE Cells Casts
● materials present in the filtrate at the time of its
4. Mixed Cellular Casts
formation and the length of time it remains in the tubule 5. Bacterial Casts
● any element in the tubular filtrate become embedded C) Casts with Inclusion
in or attached to the cast matrix, including: 1. Granular Casts
2. Fatty Casts
○ cells
D) Casts with Non-Cellular Inclusions
○ bacteria 1. Hemosiderin Granules
○ granules 2. Sulfonamide Crystals
3. Calcium Oxalate Crystals
○ pigments
E) Casts with Pigments
○ crystals 1. Hemoglobin
2. Myoglobin
3. Bilirubin
F) Broad Casts

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Reporting of Casts
● casts are reported according to type and the number Clinical Significance: Increased Hyaline Casts
that is present /LPF Cause Associated Conditions
● ranges reported:
● extreme physiologic
○ 0–2, 2–5, 5–10, 10–20/LPF
conditions ● pyelonephritis
○ NEGATIVE if none is observed
○ strenuous exercise ● glomerulonephritis
(>10/lpf) ● chronic renal disease
A) CASTS WITH HOMOGENOUS ○ dehydration
● congestive heart failure
MATRIX COMPOSITION ○ fever
○ emotional stress.
Hyaline Casts
Waxy Casts

Fig. Hyaline Cast

Fig. Waxy Cast

Fig. Degenerating Hyaline Cast Fig. Fibrillar Matrix of Hyaline Cast

● most commonly observed casts in the urine

sediment Fig. Waxy Cast with Jagged Sides Fig. Waxy Cast in a Supravital Stain
● composed primarily of a homogeneous uromodulin
protein matrix (low refractive index similar to urine) ● homogeneous, with their edges well defined, and
● appearance: often have sharp, blunt, or uneven ends
○ colorless in unstained urine sediment ● cracks/ fissures from their lateral margins or along
○ consisting of normal parallel sides and rounded their axes are often present
ends ● brittle, highly refractive cast matrix
○ cylindroid forms ○ caused by degeneration of the hyaline cast matrix
○ wrinkled or convoluted shapes & any cellular elements or granules
■ indicate aging of the cast matrix ○ often appear fragmented with jagged ends & have
■ accompanying dehydration of the protein fibrils notches in their sides
and internal tension ● often accompanied by hematuria (may be negative),
● seen in phase-contrast microscopy: proteinuria, leukocyturia and other casts
○ fibrillar protein matrix is more apparent and often ● often mistakenly identified with fiber & fecal materials
includes some fine granulation
● homogenous, dark pink
Supravital Stains
Sternheimer- ● diffuse, ground glass appearance
● pink with a more defined edge
Malbin Stain

● occasionally, a hyaline cast may have a single epithelial

or blood cell within its matrix have and NO diagnostic
significance, as well as cylindroids
● NOT detected by the reagent strip test for protein
● often mistakenly identified with mucus thread & fibers

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 Clinical Significance: Increased Waxy Casts Clinical Significance: RBC Casts
Cause Associated Conditions ● can result from tubular necrosis/damage
● acute renal disease ○ e.g., blood leakage into the tubules, as
● indicate prolonged stasis with acute interstitial nephritis
○ acute glomerulonephritis
& tubular obstruction ○ nephrotic syndrome ● after strenuous exercise
● found most frequently in ● tubular inflammation Cause ○ aka athletic pseudonephritis
patients with chronic renal ● malignant hypertension ○ contact sports such as football,
failure basketball, or boxing
● renal amyloidosis
○ frequently referred to as ○ urine sediment returns to normal within
● diabetic nephropathy 24-48 hours
“renal failure casts” ● renal allograft rejection ● acute renal disease
○ acute glomerulonephritis
CELLULAR INCLUSION CASTS ○ nephrotic syndrome
Associated tubular inflammation
●
RBC Casts Conditions ● malignant hypertension
● renal amyloidosis
● diabetic nephropathy
● renal allograft rejection

Blood Casts or Muddy Brown Casts

● RBC casts in massive hemoglobinuria or myoglobinuria
and with sufficient urine stasis
Fig. RBC Cast
● homogenous orange-red or red-brown casts
● contain no distinct RBCs in their matrix → cells have
● fragile, and overly vigorous resuspension of the urine lysed & undergone degeneration
sediment can result in breakage of the casts into pieces ● may also occur in vitro when the urine specimen is old
● diagnostic of intrinsic renal disease and improperly stored
● RBCs are most often of glomerular origin
○ e.g., passage across glomerular filtration barriers
as in glomerulonephritis
● casts must be present in conjunction with other
pathologic findings such as RTE cells and a positive
reagent strip test for blood
● phase-contrast and interference contrast
microscopy Fig. Muddy Brown Casts
○ aid in identification of RBC casts by enhancing the
detail of cells trapped within the cast matrix WBC Casts
○ free-floating RBCs are also present
○ one must ensure that cells are actually within the
cast matrix and are not simply superimposed on its
surface
● microscopic appearance:
○ varies; packed with RBCs or may present
Fig. WBC Cast
principally as a hyaline cast with several clearly
defined RBCs embedded within its matrix ● highly refractile casts consist of leukocytes
○ chunks of casts would be present and may be embedded in a hyaline cast matrix
difficult to identify ○ thus readily apparent identifiable with brightfield
microscopy due
Unstained Urine ● strongly suggested by the presence of increased
● characteristically yellow
Sediments
numbers of WBC free-floating or in clumps
● appear colorless or lavender in a
Sternheimer-Malbin ● WBC casts are mistaken as RTE cell casts
pink homogeneous matrix

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 Clinical Significance: WBC Casts ● composed of a combination of multiple cell types such
● glomerular infections as RBCs, WBC and RTE cells
○ glomerulonephritis Glomerulonephritis ● RBC and WBC
Cause ○ pyelonephritis
● non-bacterial inflammations ● WBC and RTE cell or
Pyelonephritis
○ acute interstitial nephritis ● WBC and bacteria
● presence of WBC casts, along with RTE ● identification of these casts is difficult and thus
casts, indicates inflammation of the upper requires staining and phase microscopy
urinary tract (glomerulus or the tubules) ● primary diagnostic marker = homogenous casts of at
Associated
● RBC casts will also be present and in least one of the cell types
Conditions
greater numbers than white blood cell casts ○ e.g., in glomerulonephritis, the predominant casts
along with bacteriuria, proteinuria and will be RBC
hematuria ○ in pyelonephritis, the predominant casts will be
WBC
RTE Cell Casts ● often reported with a description of the cells involved
○ e.g., “cellular cast – leukocytes and renal tubular
cells”
○ cast is identified using the term that has the
greatest clinical significance when a cast of two
matrix types (half granular and half waxy) is
encountered
■ e.g. enumerated and reported as a “waxy cast”
Fig. RTE Cell Cast

● colorless, highly refractile casts Bacterial Casts

● contain individual renal tubular cells or fragments of the
tubular lining removed intact from the tubule
● some are stained with bilirubin (in cases of hepatitis)
and thus appear yellow

Clinical Significance: RTE Cell Casts

● heavy metal and chemical or drug-induced
toxicity
Cause
● viral infections Fig. Bacterial Cast
● allograft rejection
● contain bacilli both within and bound to protein matrix
● presence of degenerating renal tubular cell
● usually include leukocytes and thus often reported as
casts in urine sediment indicates
“leukocyte casts”
Associated ○ intrinsic renal tubular disease
● often observed in cases of pyelonephritis
Conditions ○ severe urinary stasis
● usually accompanied by proteinuria, ● casts packed with bacteria can resemble granular casts
hematuria and granular casts ○ presence should be considered when WBC casts &
many free WBCs & bacteria are seen in sediment
● confirmation = Gram stain on the dried or
Mixed Cellular Casts
cytocentrifuge sediment
● contrast interference microscopy
○ allows even better visualization of bacteria within
casts
● associated positive reagent strip test results
○ protein
○ LE
Fig. Mixed Cellular Cast ○ nitrite
○ blood

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 CASTS WITH INCLUSIONS Fatty Casts

Granular Casts

Fig. Fatty Cast Fig. Fatty cast loaded with fat that is
often mistaken as a fecal debris

Fig. Granular Casts ● contain highly refractile either

○ free fat globules
○ oval fat bodies
○ both, free and oval
● matrix can be hyaline or granular
● fatty cast loaded with fat that is often mistaken as a
fecal debris

Fig. Fine Granular Cast Fig. Coarse Granular Cast Unstained Urine ● lipid globules may appear light
Sediment yellow or darker
● highly refractile casts
● triglyceride (neutral fat) globules
● granular casts almost always indicate significant Sudan III or Oil within casts stain orange or red
renal disease Red O ○ except cholesterol and cholesteryl
● fine granular casts often mistakenly identified with esters
columnar RTE and clumps of small crystals
Clinical Significance: Fatty Casts
● small, fine granules ● most frequently associated with the
Fine Granules
● gray or pale yellow nephrotic syndrome
● large, coarse granules with a ○ accompanied by significant proteinuria
uromodulin protein matrix ● toxic tubular necrosis
Course Granules ● darker in color ● lipid nephrosis
● appear black because of the density Associated
of the granules ● chronic glomerulonephritis
Conditions
● Kimmelstiel-Wilson syndrome (diabetic
Clinical Significance: Granular Casts nephropathy)
Fine Granules Coarse Granules ● SLE
● granules in finely granular ● crush injuries
casts are by-products of ● degenerative tubular disease
lysosome and protein ● initially, the granules are
metabolism that are large and coarse that CASTS WITH NON-CELLULAR INCLUSIONS
excreted by renal tubular results from ● casts w/ non-cellular inclusions:
epithelial cells degeneration of cellular ○ hemosiderin granules
● further degeneration would casts ○ sulfonamide crystals
then result in formation of ○ but when urine stasis is ○ calcium oxalate crystals
waxy casts prolonged, these ● because crystals can aggregate along mucous threads,
granules break down to it is important that the hyaline matrix is observed and
● may be present in the urine fine granules
for a short time following that it actually encases the crystals
strenuous exercise ● presence of crystal casts indicates crystal precipitation
within the tubules→ damage tubular epithelium→
tubular obstruction
● varying amounts of hematuria usually accompany
crystalline casts

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 CASTS WITH PIGMENTS MUCUS THREADS AND FIBERS

MUCUS THREADS

[Link] stained RTE cell cast Fig. Hemosiderin granules in cast

Fig. Mucus Threads
● pigments such as hemoglobin, myoglobin, or
bilirubin (bile) can be incorporated in a hyaline matrix ● formed elements in urine sediment can be confused
and produce characteristic colors with casts such as mucous threads being misidentified
● highly pigmented drugs, such as phenazopyridine, as hyaline casts
can also characteristically color sediment elements ● appearance:
○ low refractive index
Hemoglobin and ● casts appear yellow to brown ○ ribbon-like
Myoglobin ● accompanied by hematuria ○ ends are not rounded but are serrated
Bilirubin ● appear yellow- or golden-brown ○ irregular (hyaline casts are more formed)
● orange-brown color to urine
Urobilin ● does NOT color the formed elements FIBERS
of the sediment

BROAD CAST

Fig. Hair Fiber Fig. Hemosiderin granules in cast

● fibers, such as cotton threads or diaper fibers, can

resemble waxy casts
● fibers tend to be flatter in the middle and thicker at
Fig.A single “broad” waxy cast and Fig. A long, broad waxy cast their margins
two hyaline casts.
● fibers are more refractile than casts
● indicate cast formation in dilated tubules or in the ● under polarizing microscopy:
large collecting ducts ○ fibers polarize light, whereas casts do not
● due to continued proteinuria and other factors that ● fibers may contaminate the urine at any time, whereas
enhance their formation casts, particularly waxy casts, must be accompanied by
● indicates significant urinary stasis due to obstruction proteinuria
or disease
NOTE: Cast Misidentification
○ since several nephrons empty into a single
collecting duct Squamous ● folded into a tubular shape or scratches on
● when the flow of urine to the larger collecting ducts Epithelial the coverslip surface, may be misidentified
becomes severely compromised, casts form in this area Cells as cast
and appear broad ● amorphous urates and phosphates
● all types of casts may occur in the broad form ● can aggregate together or along a mucous
○ most commonly seen broad casts are granular thread to simulate a cast
and waxy (considering urinary stasis) Crystals ● birefringence identifies them as crystalline
● can be seen in chronic renal diseases in which entities in polarizing microscopy
nephrons have sustained previous damage ● lack of a distinct matrix differentiates
them from a true cast

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Transcribed by: Abenes, Aliping, Guarin, Villanueva (2024). 20
 UNIT 3: URINARY CRYSTALS AND CRYSTALS IN URINE
MISCELLANEOUS ELEMENTS
FACTORS AFFECTING CRYSTAL FORMATION
Normal Crystals in Urine
A) Crystals in Acidic Urine
Concentration of the Solute in the Urine
1. Amorphous Urates
● concentration of urinary solutes increases, solute
2. Acid Urates
precipitation is more likely
3. Monosodium Urates
4. Calcium Oxalate
Urine pH
a. Dihydrate Calcium Oxalate
● provides a means of identifying and differentiating
b. Monohydrate Calcium Oxalate
crystals
5. Uric Acid
● less soluble in neutral or alkaline urine
B) Crystals in Alkaline Urine
○ inorganic salts
1. Amorphous Phosphates
■ oxalate, phosphate, calcium,
2. Triple Phosphate
■ ammonium, and magnesium
3. Calcium Phosphate
○ precipitate out in crystalline form
a. Dibasic Calcium Phosphate
● less soluble in acidic conditions
b. Monobasic Calcium Phosphate
○ form crystals in acidic urine.
4. Magnesium Phosphate
○ organic solutes
5. Ammonium Biurate
■ uric acid, bilirubin, and cystine
6. Calcium Carbonate
Abnormal Urine Crystals Flow of Urine Through the Tubules
A) Crystals of Metabolic Origin ● ↓ urine flow allows maximum concentration of solutes in
1. Bilirubin the ultrafiltrate
2. Cystine
3. Tyrosine Temperature
4. Leucine ● solutes precipitate more readily at low temp.
5. Cholesterol ○ amorphous urates
B) Crystals of Iatrogenic Origin ■ form in refrigerated specimens
1. Hippuric Acid ■ dissolve if specimen is warmed
2. Ampicillin ● crystal formation remained at:
3. Indinavir ○ room temperature
4. Sulfamethoxazole/ Sulfonamides ○ refrigerated prior to testing
5. Radiographic Contrast Media
Miscellaneous Elements NORMAL URINE CRYSTALS
A) Mucus Threads ● urine crystals occur in acidic or alkaline urine
B) Fats ● phosphates represent the majority of the crystals seen
C) Hemosiderin in alkaline urine
D) Sperm Cells
● calcium carbonate
E) Microorganisms Normal Crystals
● ammonium biurate
1. Bacteria
2. Yeast Cells ● amorphous phosphate
Crystals in
3. Parasites ● triple phosphate
Alkaline Urine
F) Contaminants ● calcium phosphate
1. Fibers ● uric acid
Crystals in
● calcium oxalate
Acidic Urine
● amorphous urates
Less Frequently ● calcium sulfate ● leucine
Encountered ● sodium urates ● tyrosine
● hippuric acid ● cholesterol
Crystals ● cystine ● sulfa

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 CRYSTALS IN ACIDIC URINE Monosodium Urates

Amorphous Urates

Fig. Monosodium Urates

Fig. Amorphous Urates
● urate salts precipitate in amorphous or non-crystalline ● distinct form of a uric acid salt
forms, such as: ● microscopic appearance:
○ sodium ○ colorless to light-yellow slender, pencil-like prisms
○ potassium ○ may be present singly or in small clusters
○ magnesium ○ ends are not pointed
○ calcium ● dissolve at 60° C
● microscopic appearance: ● no clinical significance
○ small, yellow-brown granules (much like sand) ● reported as “urate crystals”
○ may appear in clumps resembling granular casts ● seen in synovial fluid during episodes of gout
● usually encountered in spx that have been refrigerated
● produce a very characteristic pink sediment due to Calcium Oxalate
accumulation of uroerythrin
● found in acidic urine with a pH > 5.5
● can be converted to uric acid crystals
○ if concentrated acetic acid is added & time allowed
● rarely occur in a neutral urine
● NO clinical significance
● distinguished from amorphous phosphates
Fig. Dihydrate Calcium Oxalate Fig. Monohydrate Calcium Oxalate
Acid Urates
● most frequently observed crystals in human urine
● can form in urine of any pH
● greatly affected by diet
● solutes normally found in the urine of healthy people
● microscopic appearance:
○ colorless and can vary significantly in size
○ small and require HPF
Fig. Acid Urates ■ can be large enough to be identified under LPF
● sodium, potassium, and ammonium salts of uric acid ○ may cluster together and can stick to mucous
● microscopic appearance: threads
○ small, yellow-brown balls or spheres ■ may be mistaken for crystal casts
○ often misidentified as leucine crystals
○ larger granules & may have spicules similar to the Source of Ca Oxalate Crystals
ammonium biurate crystals seen in alkaline urine Beverages that
● present when the urine pH is neutral to slightly acidic Foods high in oxalic
Approx. 50% are high in oxalic
● frequently are not observed in fresh urine acid or ascorbic acid
acid
○ dissolve at 60°C ● vegetables (rhubarb, ● cocoa
● can be converted to uric acid crystals ● ascorbic acid
tomatoes, ● tea
○ by the addition of glacial acetic acid (vitamin C)
asparagus, spinach) ● coffee
● NO clinical significance ● oxalic acid
● citrus fruits ● chocolate
● reported as “urate crystals”

Transcribed by: Abenes, Aliping, Guarin, Villanueva (2024). 22

 Clinical Associations: Increased Ca Oxalate Crystals Uric Acid
● ingestion of the oxalate ● severe chronic renal disease
precursor ethylene glycol ● diabetes mellitus
(antifreeze) ● liver disease

● as urine forms in the renal tubules, oxalate ions

associate with calcium ions → calcium oxalate
● calcium oxalate occurs in dihydrate and monohydrate
form, the latter being less common
Fig. Rhombic. Uric Acid Crystal. Fig. Rosette. Uric Acid Crystal.

Dihydrate Calcium Oxalate (Weddellite)

● octahedral or pyramidal in shape
○ two pyramids joined at their bases
● aka enveloped crystals
○ appear as squares scribed with lines that intersect in
the center when viewed from one end
● viewer will see the “X” of the crystal popping out of the
field Fig. Barrel Form. Uric Acid Crystal. Fig. Hexagonal crystals are uric acid
since there are surrounding
barrel-shaped uric acid crystals.

● normal urine solute that originates from catabolism of

purine nucleosides (adenosine and guanosine)
○ can appear in urine from healthy individuals
● microscopic appearance:
○ seen in a variety of shapes
Fig. Dihydrate Calcium Oxalate ■ including rhombic or diamond
■ four-sided flat plates (whetstones/cubes)
Monohydrate Calcium Oxalate (Whewellite) ■ barrel
● small and ovoid or dumbbell shaped ■ wedges
● resemble RBCs and may require differentiation by ■ bands
polarizing microscopy to demonstrate the birefringence ■ rosettes (clusters)
of these crystals ○ may also have a six-sided shape, similar to cystine
crystals
○ appear yellow to golden brown
■ intensity of their color varies directly with the
thickness of the crystal
○ uric acid crystals may be colorless when they are
thin or when urine is low in uroerythrin
● use of polarizing microscopy
○ uric acid crystals exhibit strong birefringence and
Fig. Monohydrate Calcium Oxalate produce a variety of interference colors
● present only if the urine pH is less than 5.7
● urate salts
○ ionized form
○ formed at a pH greater than 5.7
● 17x less soluble than urate salt crystals
● if urine with uric acid crystals is adjusted to an alkaline
pH → crystals readily dissolve
● ⬆ no. of uric acid present following:
○ gout and patients with Lesch-Nyhan Syndrome
■ administration of cytotoxic drugs (e.g.,
chemotherapeutic agents)

Transcribed by: Abenes, Aliping, Guarin, Villanueva (2024). 23

 CRYSTALS IN ALKALINE URINE ● ammonium magnesium phosphate
○ normal urine solute
Amorphous Phosphate ○ triple phosphate crystals can be present in urine
from healthy individuals
● has little clinical significance
○ associated with UTIs
○ implicated in the formation of renal calculi

Calcium Phosphate
● present in any urine pH (alkaline, neutral, slightly
acidic)
Fig. Amorphous Phosphate
● dibasic = calcium monohydrogen phosphate
● microscopic appearance: ● monobasic = calcium biphosphate
○ non-crystalline form of phosphates that resemble ● no clinical significance
fine, colorless grains of sand in the sediment ● common constituent of renal calculi
○ granular particles also found in neutral urine
○ no definite shape Dibasic Calcium Phosphate Crystals
○ indistinguishable from amorphous urates ● aka calcium monohydrogen phosphate, stellar
● differentiated from amorphous urates on the basis of phosphates
urine pH, their solubility characteristics, and, to a lesser ● appear as colorless, thin, wedgelike prisms
degree, their macroscopic appearance ● arranged in small groupings or in a rosette pattern
● each prism has one tapered or pointed end, with the
○ large quantities of amorphous phosphates cause a
other end squared off
urine specimen to appear cloudy ● others may appear as long needles arranged in
○ precipitate is white or gray which is in contrast to bundles or sheaves
the pink-orange color of amorphous urates ● dibasic form can be confused with sulfonamide crystals
● soluble in acid ○ when the urine pH is in the neutral range
● do not dissolve when heated to approximately 60° C ● dissolves in dilute acetic acid while sulfonamides do not
● no clinical significance
● microscopic examination difficult if large quantity
● refrigeration enhances their deposition
○ specimens maintained at room temperature and
analyzed within 2 hours of collection minimize
amorphous phosphate formation
Fig. Needle-like. Ca Phosphate Fig. Fern-leaf. Ca Phosphate
Triple Phosphate

Monobasic Calcium Phosphate

● aka calcium biphosphate
● microscopic appearance:
○ irregular, granular sheets or flat plates that can be
large
○ may be noticed floating on the top of a urine spx
○ colorless crystalline sheets can resemble large
Fig. Coffin-lid. Triple phosphate. Fig. Fern-leaf. Triple phosphate.
degenerating squamous epithelial cells
● composed of ammonium, magnesium and ○ weakly birefringent with polarizing microscopy
phosphate materials
● microscopic appearance:
○ colorless crystal that appear in several different
forms, both in alkaline and neutral urine
○ most common forms are 3-6 sided prisms
○ “coffin lids” = oblique terminal surfaces
● prolonged storage cause crystals to dissolve →
Fig. Monobasic Calcium Phosphate sheet or plate.
taking on a feathery form that resembles a fern leaf

Transcribed by: Abenes, Aliping, Guarin, Villanueva (2024). 24

Magnesium Phosphate Calcium Carbonate

Fig. Urine triple phosphate crystals Fig. Calcium Carbonate

Fig. Magnesium Phosphate are composed of magnesium,
ammonium & phosphate.

● microscopic appearance:
○ large, flat, colorless crystals, appearing as
elongated rectangular or rhomboid plates
○ notched and their edges can be irregular or
eroded
Fig. Calcium carbonate. Numerous Fig. Aggregate of calcium carbonate
○ weakly birefringent under polarizing microscopy single crystals. Brightfield, ×400. crystals. Brightfield, ×400
● form from normal urine solutes (neutral, slightly acidic
● microscopic appearance:
or alkaline urine)
○ tiny, colorless granular crystals that assume
● rarely seen
dumbbell, spherical or tetrad shapes
○ slightly larger than amorphous material
Ammonium Biurate
○ resemble amorphous material if in clumps
○ can be distinguished by the formation of gas after
the addition of acetic acid
○ also birefringent (differentiates them from bacteria)
● present primarily in alkaline urine
● not found frequently in the urine sediment
● no clinical significance
● positively identified through the production of carbon
Fig. Ammonium biurate crystals. Fig. Ammonium biurate crystals dioxide gas (effervescence) with the addition of acetic
Spheres and a “thorny apple” form. (×400). Notice the “thorny apple” acid to the sediment
Brightfield, ×200. appearance.

● microscopic appearance:
○ yellow-brown spherical crystals with
striations/projections or spicules on the surface
giving them a “thorny apple” appearance
● normal urine solute that can also form in a neutral urine
● occur most frequently in urine specimens that have
undergone prolonged storage
● form precipitate in fresh urine specimens
○ when following iatrogenically induced alkalinization
and inadequate hydration of the patient
● clinically significant because in vivo precipitation can
cause renal tubular damage
● strongly birefringent and dissolve in acetic acid or on
heating to approximately 60° C
● similar to other urate salts, they can be converted to
uric acid crystals
○ with the addition of concentrated HCl/ acetic acid
● resemble some forms of sulfonamide crystals

Transcribed by: Abenes, Aliping, Guarin, Villanueva (2024). 25

 ABNORMAL URINE CRYSTALS ● in vivo: ⬆ solubility rises exponentially w/ ph
● mostly found in acidic urine and rarely in neutral urine ○ 4x more soluble at ph 8 than at ph 5
● very characteristic shapes; however, their identity ● disintegrating forms of cystine
should be confirmed by chemical tests or by any ○ presence of ammonia
medication of the patient ○ hard to diff colorless uric acid crystals.
● sodium cyanide–sodium nitroprusside test
CRYSTALS OF METABOLIC ORIGIN
○ dissolve in alkali
Bilirubin ○ dissolve in hydrochloric acid (ph <2)

Tyrosine

Fig. Bilirubin Crystals Fig. Bilirubin Crystals

● microscopic appearance: Fig. Tyrosine Crystals

○ appear as yellow-brown small clusters of fine
● microscopic appearance:
needles (20 to 30 μm in diameter)
○ appear as fine, delicate needles that are
○ sometimes in granules and plates and only form in
colorless or yellow
an acidic urine
○ frequently aggregate to form clusters or sheaves
● dissolve when alkali or strong acids are added
but also may appear singly or in small groups.
● classified as abnormal crystals because bilirubinuria
○ needle clusters often appear to be black, especially
indicates a metabolic disease process
in the center,
● confirmed by correlation with a positive bilirubin
■ may take on a yellow color in the presence of
chemical examination
bilirubin
● crystals form in the urine after excretion and cooling
● soluble in ammonium hydroxide and hydrochloric acid
(refrigeration)
but insoluble in acetic acid
● not frequently observed and are not usually reported
● require refrigeration to force them out of solution
Cystine ● often present in the urine of patients:
○ cystinuria
○ tyrosinosis
○ oasthouse urine disease
● have been observed in the urine of patients with
severe liver disease

Leucine

Fig. Cystine crystals with layers or

Fig. Cystine Crystals
lamination

● clear, colorless, hexagonal plates w/ uneven sides

○ refractile, laminated or layered
○ tend to clump
○ have polarizing capability
● primarily in acidic urine (also neutral and alkaline urine)
● found in cystinuria:
Fig. Leucine Crystals
○ form renal calculi, at an early age
○ metabolic disorder = prevents reabsorption of
cystine

Transcribed by: Abenes, Aliping, Guarin, Villanueva (2024). 26

● microscopic appearance: CRYSTALS OF IATROGENIC ORIGIN
○ highly refractile, yellow to brown spheres ● crystals induced in patients as a result of a particular
○ resemble fat globules when observed in acidic treatment (e.g., medications)
urine ○ most medications and their metabolites are
○ concentric circles or radial striations on their eliminated from the body by the kidneys
surface ○ as urine forms within the nephrons, high
● require refrigeration or addition of alcohol to force concentrations of these agents can cause their
them out from the sample precipitation out of solution.
○ tyrosine is found more often in urine because ● if the crystals are forming in vivo, such as in the renal
leucine is more soluble tubules, they can cause kidney damage
● often present in urine of patients with: ● sulfonamides and ampicillin
○ cystinuria ○ 2 common antibiotic drugs
○ severe liver disease (rare) ○ known for their ability to form crystals in urine

Cholesterol Hippuric Acid

Fig. Cholesterol Crystals Fig. Hippuric Acid Crystal

\

● microscopic appearance: ● microscopic appearance:

○ clear, flat, rectangular plates with notched ○ yellow–brown or colorless elongated prisms or
corners plates occurring in clusters
○ present in acidic urine and can be in neutral urine ○ may be thin enough to resemble needles
● soluble in chloroform and ether ● more soluble in water and ether than uric acid crystal
● rarely seen in urine as an indicator of enhanced ● rarely seen in urine
supersaturation and precipitation of increased urine ● no clinical significance
cholesterol
● lipids (such as free-floating fat droplets), fatty casts, or Ampicillin
oval fat bodies and large amounts of protein often
accompany these crystals
● often present in the urine of patients:
○ nephrotic syndrome
○ chyluria
■ rupture of lymphatic vessels into the renal
tubules as a result of tumors,
○ filariasis
○ chronic renal diseases Fig. Ampicillin Crystal

Radiopaque Contrast Media ● microscopic appearance:

Cholesterol ○ long, colorless, thin prisms or needle-like
(X-Ray Dye)
● flat and layered ● flat and layered rectangular crystals
rectangular crystals crystals ● may aggregate into small groupings or, with
● normal SG ● abnormally high urine SG refrigeration, into large clusters
● accompanied by ● not associated with ● indicate large doses of ampicillin
proteinuria and proteinuria/ lipiduria ● rarely observed with adequate hydration
lipiduria

Transcribed by: Abenes, Aliping, Guarin, Villanueva (2024). 27

Indinavir Radiographic Contrast Media Crystals

Fig. Indinavir Crystals Fig. Radiographic Contrast Media Crystals

● crystals of radiographic contrast media following
● microscopic appearance:
retrograde administration
○ slender, feather-like crystals that aggregate into
● microscopic appearance:
wing-like bundles
○ colorless, long, rectangular needles that occur
○ associate into a rosette-like or cross form
singly or clustered in sheaves
● more often observed in neutral and alkaline urines
● administered intravenously, they appear as flat,
elongated rectangular plates
Sulfamethoxazole/ Sulfonamides
● in contrast with cholesterol crystals, these types of
crystals occur in large numbers in one field of view

Fig. Sulfamethoxazole/ Sulfonamides Crystals

● microscopic appearance:
○ highly refractile and birefringent crystals
○ appear as colorless to brown rosettes, spheres,
whetstones or rhombic with irregular radial
striations
○ appear as bundles of needles that resemble
sheaves of wheat
○ constriction of the bundle may be located centrally
or extremely eccentric, resulting in a fan formation
● primary cause of sulfonamide crystallization
○ inadequate patient hydration
● appearance of sulfonamide crystals in fresh urine can
suggest the possibility of UTI or tubular damage if
crystals are forming in the nephron

Transcribed by: Abenes, Aliping, Guarin, Villanueva (2024). 28

 ● microscopic appearance:
○ readily identified by their delicate, wavy,
ribbon-like strands and irregular or serrated ends
○ can be present also as distinct strands or as a
clumped mass
○ some of the wider threads may be confused with
cylindroids or hyaline casts
○ cylindrical composition of casts and their rounded
ends aid in their differentiation from mucus
● present in normal urine in small numbers
● no clinical significance
● very abundant in the presence of inflammation or
irritation of the urinary tract

FATS

Fig. Fat Droplet Fig. Three oval fat bodies stained

with Sudan III stain. Note orange-red
staining triglyceride.

● microscopic appearance:
○ found in urine as free-floating fat globules, within
oval fat bodies, or within a cast matrix
○ highly refractile and appear colorless to
MISCELLANEOUS ELEMENTS yellow-green or even brownish
● chemical analysis does not detect most of these types ● fats include triglycerides and cholesterol
of sediment ● often present in the urine of patients:
● microscopic evaluation of urinary sediment is ○ diabetes mellitus and
important if these structures are to be detected ○ nephrotic syndrome
■ damage to the glomerular filtration barrier
MUCUS THREADS ○ preeclampsia
■ fat is often present and can persist for several
weeks after delivery
○ lipiduria
■ extreme physical exercise (e.g., marathon) and
crush injuries
○ prostatitis
■ urine from men
■ free-floating fat globules
Fig. Mucus Threads
● oils and fats from lubricants, ointments, creams, and
● fibrillar protein (some contain uromodulin) that comes lotions can contaminate urine
from the renal tubules and vaginal epithelium (women) ● contaminating lipids are often indicated by the lack of
● very low refractive index proteinuria, fatty casts, and oval fat bodies
○ better visualized when phase contrast or
interference contrast microscopy is used

Transcribed by: Abenes, Aliping, Guarin, Villanueva (2024). 29

 HEMOSIDERIN MICROORGANISMS
● normal urinary tract is sterile
● consequently, the presence of bacteria, yeast,
trichomonads, or parasites indicates an infection or that
contamination occurred during the collection process

Bacteria

Fig. Hemosiderin Granules

● form of iron that results from ferritin denaturation

● microscopic appearance:
○ yellow-brown granules
○ difficult to distinguish from amorphous crystalline
material in the sediment Fig. Bacteria
● usually found 2-3 days after a severe hemolytic
● most commonly encountered bacteria in urine are
episode such as:
bacilli but coccoid forms can also be present
○ transfusion reaction
● in wet preparations, their motility often distinguishes
○ paroxysmal nocturnal hemoglobinuria
them from amorphous substances
● found free floating or within macrophages, casts, or
● often reflects contamination from these sources that
tubular epithelial cells
normally contain bacteria
● Rous test
○ skin
○ prussian blue reaction
○ vagina
○ identify hemosiderin in the urine sediment and in
○ gastrointestinal tract
tissues
● may also imply UTI or because of a fistula between the
urinary tract and the bowel.
SPERM CELLS
● multiply rapidly in improperly stored urine
● present in urine sediment from males and females
○ presence has clinical significance only if urine
● presence of motile sperm indicates recent
specimen has been properly collected and stored
intercourse or ejaculation
● investigate specimen collection and handling when
● in urine from women, sperm is usually considered a
significant bacteriuria is present without leukocytes
vaginal contaminant
○ could potentially identify sexual abuse in underage Yeast Cells
and other vulnerable females
● in men, sperm can be present owing to nocturnal
emissions, from normal or retrograde ejaculation

Fig. Yeast Cells Fig. Yeast cells and blastoconidia

(budding yeast).

● ovoid, colorless cells, about 10-12 um, that are more

Fig. Sperm Cells
refractile than erythrocytes
● have a characteristic budding form and
pseudohyphae (in severe infections)
● does NOT
○ dissolve in acid
○ stain with supravital stains

Transcribed by: Abenes, Aliping, Guarin, Villanueva (2024). 30

● most often indicate contamination with vaginal Parasites
secretions in women
● UTI resulting from yeasts are possible
● may promote the development of vaginal yeast
infection:
○ pregnancy
○ use of oral contraceptives
○ vaginal moniliasis
Fig. Trichomonas vaginalis in urine.
○ kidney disease
○ immunocompromised patients ● parasites that can be observed in the urine sediment:
○ diabetes mellitus ○ associated with fecal contamination
● most commonly encountered yeast in urine sediment is ■ eggs of Enterobius vermicularis
Candida albicans ■ cysts of Giardia lamblia
○ rarely found = Candida glabrata (not involved in ○ eggs of blood fluke Schistosoma haematobium
pseudohyphae formation) ● Trichomonas vaginalis
● during the microscopic examination, only the presence ○ most common cause of parasitic gynecologic
of yeast can be determined infection in female patients
○ true yeast infection should be accompanied by the ○ appear as turnip-shaped flagellates with
presence of WBCs unicellular bodies averaging 15 um in length
○ identification of the species requires fungal culture ○ transmitted sexually
○ represent an infection of the vagina and/or urethra
■ presence in the urine often indicates
contamination with vaginal secretions
○ in male patients, trichomonad infections of the
urethra are usually asymptomatic
● urine to be analyzed should be freshly collected as old
specimens may no longer demonstrate their motility

Transcribed by: Abenes, Aliping, Guarin, Villanueva (2024). 31

 CONTAMINANTS

Starch Granules

Fig. Cloth Fiber

Fig. Starch Granules

● occur when cornstarch is the powder used in gloves

● granules are highly refractile spheres; dimpled center
● resemble fat droplets when polarized, producing a Fig. Fecal Contaminant
Maltese cross formation
● may also occasionally be confused with RBCs

Fibers (Hair, Cloth, Diaper

● short to long, flat structures often mistaken for casts
● usually have dark edges
○ most of the fibers are flat, most casts are cylindrical
Fig. Pollen with Concentric Circle Fig. Pollen Grain

Fig. Glass Shards.

Fig. Talcum Powder

Fig. Diaper Fiber Diaper fiber resembling a cast.

Notice the refractility (x400)

Transcribed by: Abenes, Aliping, Guarin, Villanueva (2024). 32

 MICROSCOPIC URINE EXAMINATION Bacterial & Yeast/ HPF
ROUTINE ANALYSIS
Negative 0
PROCEDURE Rare 0-10
Few 10-50
● Obtain freshly voided sample; Moderate 50-200
1 ● Check for the proper label of the sample and correct Many >200
requisition form
● Mix well your sample and transfer in a conical tube/
13x100 tube Parasite/ LPF
2
● Record the color and clarity of your sample ● Average count in range
● Perform your reagent strip method
● Compare test areas to the corresponding colors of
3 your parameter bottle (be mindful of the reading time Fungal Hyphae
for each parameter) ● Presence or Absence
● Cover your tube with parafilm and centrifuge your If present,
4 “Positive for fungal hyphae”
sample for 5mins @ 400 RCF report
● Decant your sample leaving 0.5 mL of your urine and
5
sediment
WBC & RBC/ HPF
● Thoroughly re-suspend the sediment by gentle
6 agitation to ensure equal distribution of the elements ● Average count in range
on the microscopic field If negative,
● “0”
7 ● Aspirate about 0.02mL and place it on your glass slide report
● Place a coverslip to the sample on your glass slide.
8
Make sure not to introduce any bubbles
9 ● Put in the microscope for examination Casts/ LPF
● Average count in range
MANNER OF REPORTING
If negative,
● “0”
report
Epithelial Cells (Squamous)/ LPF
Negative 0
Cylindroids
Rare 1-5
● Reported as ‘Casts’
Few 5-20
Moderate 20-100
For Quantitative Reporting
Many >100 ● use the ff. ranges:
● RTE: Average count in range/ HPF ○ 0-1
● Transitional: Average count in range/ HPF ○ 0-2
Mucus Threads/ LPF ○ 1-3
○ 2-5
Rare 0-1
○ 5-10
Few 1-3
○ 10-25
Moderate 3-10 ○ 25-50
Many >10 ○ 50-100
○ > 100
Crystals/ LPF
Reference Intervals for Microscopic Examination
Negative 0
● RBC: 0-3/ HPF
Rare 0-5 ● WBC: 0-8/ HPF
Few 5-20 ● Hyaline/ Fine GRanular Casts: 0-2/ LPF
Moderate 20-100
Many >100

Transcribed by: Abenes, Aliping, Guarin, Villanueva (2024). 33

ANALYSIS OF URINE MLS 321

& OTHER BODILY FLUIDS 8438

LECTURE BS MLS 3 MIDTERM
M5: RENAL DISEASES

UNIT 1: RENAL DISEASES II. CAUSES & GENERAL MECHANISMS OF DAMAGE

I. CLASSIFICATION OF RENAL DISEASES A) PATHOGENESIS OF GLOMERULAR DISEASES

● classified based on morphological component (initial
organ affected) Immune Mediated Mechanism
○ other components: structural & functional ● primary mechanism of glomerular damage
interdependence
● deposited in glomerulus → trapped in
Glomerular Diseases glomeruli → binds complement →
● damage to the glomeruli Deposition of glomerular injury
● immune-mediated Immune ● created in response to endogenous or
○ deposition of immune complexes on the glomerulus exogenous Ag
Complexes ○ endogenous Ag = tumor Ag,
○ results to inflammation / damage
thyroglobulin
● may also result from metabolic disorder or genetically
○ exogenous Ag = viruses, parasites
predisposed
● e.g. anti-glomerular basement
Ab Directly membrane Ab
Tubular & Tubulointerstitial Diseases
Reacting to ● can also react with non-glomerular Ag
● damaged to the tubules Glomerular that reside in the glomerulus
● by infectious & toxic substances taken by the px Tissue ● can originate from drugs and infectious
agents
Vascular Diseases
● damage to the vascular system of the kidney ● exposure to Ag → immune complexes
● affect renal perfusion in the kidneys ● immune complexes are then trapped in the glomerular
● normal renal function is dependent tuft → activate the complement system → cell lysis
(glomerular cells)
Renal Failure ● cross-reaction
● presence of the aforementioned diseases may result to ○ Abs react with the glomerular tissue Ag / Ag that
renal failure currently reside in the glomeruli (drugs, infectious
● may be acute or chronic depending on the severity of agents)
the damage
NOTE:
● sudden loss of RF ● glomerular disease does not results from the
Acute Renal immune complexes but rather from the chemical
● reversible
Failure mediators and toxic substances that they induce
● high mortality rate
○ e.g. complements at the site of attachment can
Chronic Renal ● progressive produce proteases, oxygen derived free radicals,
Failure ● irreversible and arachidonic acid metabolites = can stimulate an
inflammatory response that can further induce
glomerular tissue damage

Transcribed by: Lustre, Aliping, Guarin, Villanueva (2024). 1

Chemical Mediators & Toxic Substance ● also observed in:
● cause clinical features / symptoms that characterize ○ px w/ systemic disease
glomerular damage ○ px taking nephrotoxic agents
● syndrome (group of symptoms) ● NOTE:
● px may either experience nephritic or nephrotic ○ subnephrotic proteinuria (< 3.5g/day)
syndrome
● increased lipid synthesis in the liver
Nephritic Syndrome ● decreased lipid catabolism
● lipids withing renal tubular cells or renal
● indicative of glomerular damage by inflammatory Hyperlipid- casts in urine
process emia & ● elevation in the plasma conc.:
● clinical features: Lipiduria ○ triglycerides
○ hematuria ○ cholesterol
○ hypertensions ○ phospholipids
○ very-low density lipoproteins (VLDL)
○ oliguria
● primarily caused by sodium retention
○ azotemia (high BUN)
○ ⬆sodium & H20 reabsorption in DCT
○ mild proteinuria
● clinical features:
○ mild edema
○ hypoalbuminemia
● primarily seen in:
■ plays a minor role
○ primary glomerular diseases
■ loss of proteins in plasma → reduce
○ glomerular diseases secondary to systemic diseases Generalized
oncotic pressure → movement of
● severity depends on: Edema fluid into interstitial tissues
○ no. of glomeruli involved ○ soft & pitting edema (eyes/ legs)
○ mechanism of injury ○ severe cases:
○ onset of disease ■ pleural effusions (fluid in pleural
space)
Nephrotic Syndrome ■ ascites (fluid within abdomen)
● adverse glomerular changes by non-inflammatory
mechanisms
● due to increased glomerular permeability
● substances elevated in urine:
○ albumin
■ predominant protein loss
○ immunoglobulins
○ complement cofactors
○ coagulation factors
○ lipids
● px becomes susceptible to:
○ infections
○ thrombotic complications, loss of:
■ immunoglobulins
■ complement
■ cofactors
■ coagulation factors
● clinical features:
○ proteinuria (>3.5 g/day)
○ hypoalbuminemia
○ edema
○ hyperlipidemia
○ lipiduria
○ milder hematuria

Transcribed by: Lustre, Aliping, Guarin, Villanueva (2024). 2

 B) MORPHOLOGICAL CHANGES IN III. SPECIFIC RENAL DISEASE
GLOMERULAR DISEASES
● one or more morphological changes occur in the A) GLOMERULAR DISEASES
glomerulus → glomerulonephritis
● Acute glomerulonephritis
Morphological Changes ○ Poststreptococcal
● infiltration of neutrophils & ○ Non Postreptococcal
macrophages ● Rapidly progressive glomerulonephritis
● chemotactic response Primary (Crescentic glomerulonephritis)
Leukocyte
● observed in: Glomerular ● Membranous glomerulonephritis
Infiltration
○ acute glomerulonephritis Diseases ● Minimal change disease (lipoid nephrosis)
● almost always accompanied by Cellular ● Focal segmental glomerulonephritis
Proliferation ● Membranoproliferative glomerulonephritis
● increase in the number of endothelial ● IgA Nephropathy (Berger’s Disease)
cells, mesangial cells & podocytes in ● Chronic glomerulonephritis
the glomerular turf Systemic Diseases
● Diabetes Mellitus
● only a part of each
Segmental ● Systemic Lupus Erythematosus
Cellular glomerulus
Secondary ● Amyloidosis
Proliferation ● only a certain number
Glomerular ● Vasculitis
Focal of glomeruli in the
Diseases ● Bacterial Endocarditis
kidney
Hereditary Disorders
● involves all glomeruli in
Diffuse ● Alport’s Syndrome
the kidneys
● Fabry’s Disease
● enlargement of basement membrane
● caused by deposition of: B) PRIMARY GLOMERULAR DISEASES
○ immune complexes
Glomerular
○ fibrin 1. Acute Glomerulonephritis (AGN)
Basement
○ precipitated proteins ● can either be streptococcal or non-streptococcal
Membrane
● diabetic glomerulosclerosis
Thickening
○ basement membrane thickens without Acute Poststreptococcal Glomerulonephritis
any evidence of deposition of
● common glomerular disease
materials
● occurs 1-2 weeks after a streptococcal infection (skin &
● accumulation of homogenous throat)
eosinophilic material in the glomeruli → ● forms Abs against the M protein
Hyalinization
loss of structural detail ○ delay in the manifestation of glomerular damage
with Sclerosis
● causes glomerulus to become sclerotic ○ from cell wall of some strains of group A
(irreversible) beta-hemolytic streptococci
● immune complexes forms → deposits on the
glomerulus → morphological changes
● px experiences acute nephritic syndrome
● may progress to chronic glomerulonephritis
Non-Streptococcal Glomerulonephritis
● rare type of AGN
● cause:
○ other etiologic agents that shares the same clinical
feature

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2. Rapid Progressive Glomerulonephritis / Crescentic
Glomerulonephritis
● cause:
○ formation of Abs against the glomerular basement
membrane (GBM)
● 90% chance of progressing to chronic glomerulonephritis
● usually develops after infection, as a result of an
underlying systemic disease or idiopathically
● experience sx of acute nephritic syndrome (except
oliguria)

3. Membranous Glomerulonephritis (MGN) Membranous Minimal Change Disease

● major cause of nephrotic syndrome among adults Glomerulonephritis (MGN) (MCD)
● 50% chance of progressing to chronic glomerulonephritis
● cause: Microscopic Appearance
○ deposition of immune complexes & activation of ● small, evenly-spaced
complement → glomerular basement thickening → subepithelial deposits
encloses immune deposits → diffuse lost of foot ○ GBM grows up around ● foot processes are fused /
processes → forming ‘spikes’ obliterated
● possible causes of MGN: ● becomes quite thick
○ idiopathic (85%) ● fine granularity
○ Immune-mediated disease Clinical Features
○ underlying conditions
● idiopathic ● “nil disease”
○ exposure to toxic chemicals (Au, Hg)
● lupus V ● “lipoid nephrosis
○ nephrotoxic drugs (penicillamine)
● hepatitis B/C ● idiopathic
● other clinical features:
● tubulopathies ● treatable kid’s disease
○ hematuria
● experimental Heymann’s ● no immune deposits
○ hypertension
● usually nephrotic ● nephrotic syndrome
● hyalinization & sclerosis reduce the capillary lumen
syndrome ○ selective proteinuria
diameter

5. Focal Segmental Glomerulosclerosis (FSGS)

4. Minimal Change Disease (MCD)
● major cause of nephrotic syndrome among children ● differs from MCD
● responds well to corticosteroid therapy & has better ● has little to no response to corticosteroid therapy
prognosis than MGN ● 50-80% chance of progressing to chronic
● cause: glomerulonephritis
○ dysfunction in T cell immunity → loss of polyanions ● cause:
(heparan sulfate) responsible for the shield of ○ theorized that it is caused by an unknown circulating
negativity systemic factor
○ may recur after transplantation
● clinical features:
Microscopic Appearance of MCD
○ nephrotic syndrome
Routine
● glomeruli looks normal ○ proteinuria (subnephrotic to nephrotic)
Microscopy
○ hematuria
Electron ● loss of podocyte foot processes ○ hypertension
Microscope ● loss of vacuolization of cytoplasm ○ reduced glomerular filtration rate

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6. Membranoproliferative Glomerulonephritis (MPGN) 7. IgA Nephropathy / Berger’s Disease
● slow & progressive disease ● most common cause of primary glomerulonephritis that
● 50% chance of progressing to chronic glomerulonephritis primarily affects children & young adults
● may recur after transplantation ● 30-50% chance of progressing to chronic
● cause: glomerulonephritis
○ deposition of immune complexes or complement ● cause:
activation ○ occurs 1-2 days following a mucosal infection
● clinical features: (respiratory, GIT, urinary tract) → stimulates IgA
○ nephrotic syndrome synthesis → increase IgA in circulation
○ hematuria ○ circulating IgA are trapped / engulfed by mesangial
○ subnephrotic proteinuria (<3.5g/day) cells → IgA complexes activates alternative
complement → glomerular damage

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 C.1) SECONDARY GLOMERULAR DISEASES 5. Good Pasture Syndrome
(SYSTEMIC) ● rare autoimmune disorder
● causes the immune system to attack tissues in the
1. Diabetes Mellitus lungs and kidneys
● metabolic disease with the inability to metabolize glucose ● cytotoxic Ab attaches to the GBM → complement
due to a defect in the metabolism of insulin activation → capillary epithelium damage
○ causes alterations in the metabolism of fat & proteins ● px present with
● clinical features: ○ hemoptysis
○ hyperglycemia ○ dyspnea
○ glucosuria ○ development of nephritic syndrome
○ proteinuria (subnephrotic to nephrotic) ● morphological changes: similar to rapidly progressive
○ hypertension glomerulonephritis

2. Systemic Lupus Erythematosus (SLE) C.2) SECONDARY GLOMERULAR DISEASES

● autoimmune disorder characterized by the production of (HEREDITARY)
autoantibodies
● almost all SLE px with SLE manifest kidney problems 1. Alport’s Syndrome
○ as a result of immune complex deposition & ● causes basement membrane to become lamellated
complement activation ○ resembles thin plates or scales
● clinical features: ● px develop nephrotic syndrome and chronic renal
○ acute nephritic syndrome failure
■ with recurrent hematuria
○ nephrotic syndrome 2. Fabry’s Disease
■ progressing to chronic kidney failure ● causes the deposition of glycosphingolipids
(cerebroside trihexoside) on the glomeruli
3. Amyloidosis ● genetic lysosomal storage disease
● group of systemic diseases that result to the deposition ○ alpha-galactosidase A deficiency
of amyloid on the glomeruli and effectively destroying it ○ form of sphingolipidoses as it involves dysfunctional
● amyloid metabolism of sphingolipids
○ proteinaceous substance
○ 90% fibril protein
○ 10% glycoprotein
● px develop nephrotic syndrome to chronic renal failure

4. Wegener’s Granulomatosis
● systemic disorder
● formation of granuloma
● inflammation of blood vessels primarily of the kidneys
and respiratory system
● antineutrophilic cytoplasmic Ab
○ bind to the neutrophils located in the vascular walls
of the such organs → initiates immune response →
granuloma formation
● px present pulmonary symptoms then later develop
renal symptoms
○ hematuria
○ proteinuria (subnephrotic to nephritic syndrome)

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 IV. TUBULAR DISEASES 1. Ischemic ATN
● focal tubular disease
Tubular Diseases ○ characterized by tubular basement membrane
● either a result of necrosis / pre-existing condition disruption (ex. tubulorrhexis)
causing tubular dysfunction ● resulting from the complete necrosis of tubular cells
● tubular necrosis → exposing the renal interstitium to the tubular lumen
○ decreased blood flow in the kidneys → ischemia / ● found in urine:
direct damage to the tubules (toxic substances) ○ fragments of the renal epithelium from CD
● develops following a hypotensive event → ⬇renal
Tubular Dysfunction perfusion→ renal ischemia
● may result to: ● commonly associated with an increased number and
○ (1) primary renal disease variety of casts
○ (2) secondary → pre-existing condition ○ granular
○ renal tubular cell
● may be a single pathway with only one solute type
○ waxy
affected or may involve multiple pathways ○ broad
● unaffected parts and glomerular function remains
normal 2. Toxic ATN
● common tubular dysfunction ● necrosis in the proximal convoluted tubules
○ Fanconi’s syndrome ○ NOT including the basement membrane
○ cystinosis & cystinuria ● found in urine:
○ renal glucosuria ○ distinctive large convoluted renal tubular
○ renal phosphaturia epithelial cells from the PCT
○ renal tubular acidosis ● manifest clinical features similar to ischemic ATN bec.
of nephrotoxins causing renal vasoconstriction
A) ACUTE TUBULAR NECROSIS (ATN) ● often caused by endogenous or exogenous nephrotoxic
● reversible destruction of the renal tubular epithelium agents
○ 50% of cases result from surgical procedures
● px may recover completely Endogenous Agents
● classified as ischemic or toxic depending on the ● normal substances in circulation that become nephrotoxic
cause of injury & the epithelium involved in higher con.
● renal cast formation in the DCT and collecting ducts can ● hemoglobin
be observed in both types of ATN ○ severe hemolytic episodes
● myoglobin
Phases of Acute Tubular Necrosis
○ rhabdomyolysis
● abrupt after a hypotensive event / ● uric acid
Onset of the subtle after exposure to a nephrotoxic ○ Lesch-Nyhan disease
Disease substance ● Ig light chains
● develop to renal failure ○ multiple myeloma
● clinical features: Exogenous Agents
○ azotemia (high BUN) ● substances ingested, injected, absorbed or inhaled
Renal Failure ○ hyperkalemia ● therapeutic agents (aminoglycosides)
○ metabolic acidosis ● anesthetics (enflurane)
○ oliguria (<400mL urine/day) ● radiographic contrast media
● diuresis (3L/day) ● chemotherapeutic drugs (cyclosporin)
● indicates GFR is returning to normal ● recreational drugs (heroin and cocaine)
prior to the recovery of the tubular ● industrial chemicals (heavy metals)
epithelium ● organic solvents (carbon tetrachloride)
Recovery
● results: ● poisons
○ continued loss of large amounts
of H2O, Na, & K until tubular function
is restored and azotemia is resolved

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 B) TUBULAR DYSFUNCTION
● may result from a primary renal disease or may be
induced secondarily
● dysfunction may involve a single pathway with only one
solute type affected or may involve multiple pathways,
thereby affecting a variety of tubular functions
● common tubular dysfunction includes
○ Fanconi syndrome
■ characterized by generalized loss of function of
proximal convoluted tubules
■ results to amino acids, glucose, water,
phosphates, potassium and calcium not being
reabsorbed and therefore excreted in urine
○ cystinosis and cystinuria
○ renal glucosuria
○ renal phosphaturia
○ renal tubular acidosis. Fanconi syndrome is

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 Types of Renal Tubular Acidosis (RTA) 1. Renal Tubular Acidosis (RTA)
● an autosomal dominant disorder resulting to the inability
● characterized by the inability to maintain the
of the tubules to produce an acid urine despite
normal H+ gradient and increases the
adequate hydrogen ions for secretion & a normal GFR
secretion of ammonia to compensate
Type I 2. Cystinosis
● H+ ion is retained
○ decreased H+ excretion ● autosomal recessive disorder resulting to cystinuria
○ compensation = decreased ammonia which is the urinary excretion of the amino acid cystine
secretion
● characterized by the decreased reabsorption 3. Renal Glucosuria
of bicarbonates in the PCT = negativity more ● a benign inherited condition that lowers the maximal
increased tubular reabsorptive capacity of the tubules resulting to
Type II ○ compensation = decreased excretion of H+ glucosuria
● results to the utilization of H+ to reabsorbed
bicarbonates in the DCT instead of being 4. Renal Phosphaturia (Familial Hypophosphatemia)
eliminated in urine ● is a rare dominant sex-linked disorder
Type III ● combination of type I and type II RTA defects ● characterized by the inability of the DCT to reabsorb
inorganic phosphorus
● characterized by impaired Na-K and H+
● appears to be a two-fold defect wherein the DCT is
exchange in the DCT
hypersensitive to the parathyroid hormone increasing
● usually initiated by decreased aldosterone
Type IV phosphate excretion and a decrease PCT response to
levels = decreased Na reabsorption = H+ must
hypophosphatemia
retained to maintain electroneutrality
● decrease in plasma phosphate level, bone growth and
● hyperkalemia also observed
mineralization are decreased

V. TUBULOINTERSTITIAL DISEASE
& URINARY TRACT INFECTION
● mostly due to the proximity and interconnectedness ○ metabolic diseases
in function of the renal tubules and renal interstitium ○ vascular diseases
● causes ○ irradiation = radiation nephritis
○ infection ○ neoplasms
■ acute pyelonephritis ○ multiple myeloma
■ chronic pyelonephritis ○ transplant rejection
○ toxins ● lower urinary tract infection is closely related to
■ acute interstitial nephritis (AIN) tubulointerstitial disease as the former presents the
■ analgesic nephritis overdose of analgesics mechanism leading to the development of acute
■ *nephritis due to heavy metals pyelonephritis.

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 Higher Prevalence of UTI Among Women
● due to a shorter urethra among women
○ close proximity of the urethra to the vaginal canal
and vaginal canal to the rectum
● cyclical pattern of hormones among women that puts
them at a higher risk
○ estrogen production during menstrual cycle
○ increased estrogen can enhance the adherence of
the bacteria in the lower urinary tract
● absence of prostatic secretion among women
○ prostatic secretion have antibacterial action
● “milking” of bacteria up the urethra during sexual
intercourse
Common Signs and Symptoms of UTI
● lower and upper
○ painful urination (dysuria)
○ frequent urge to urinate (urgency)
● lower UTI
○ occasional low-grade fever and cramping.
○ mental confusion or distress among geriatric px
A) URINARY TRACT INFECTION (UTI) ○ may be asymptomatic
● may involve either the lower or upper urinary tract ● upper UTI
● lower UTI ○ high-grade fever
○ urethra (urethritis) ○ nocturia
○ pain the lower urinary tract
○ bladder (cystitis)
● upper UTI
B) ACUTE PYELONEPHRITIS
○ renal pelvis (pyelitis)
● involves the renal tubules, interstitium and renal pelvis
○ interstitium (pyelonephritis)
as a result of either:
○ ascending infection = movement of bacteria from
Causes of UTI
the lower urinary tract to the kidneys
● bacterial invasion in the lower urinary tract
○ hematogenous infection = localization of bacteria
● in a healthy state, urine from the urinary tract is sterile
from the bloodstream in the kidneys
until it passes through the distal portion of the urethra
● most common cause is an ascending urinary tract
where it gets contaminated by the normal flora
infection from gram-negative organisms that are
○ this normal flora remains isolated in the urethra due
normal intestinal flora
to continual flushing during voiding
○ e.g. Escherichia coli
● commonly caused by bacteria from the GI tract
● bacteria reaching the bladder are usually prevented
○ intestinal bacteria get introduced into the urinary
from ascending the ureters to the kidneys by the
tract, where they proliferate and cause an infection
continual flow of urine into the bladder and by other
● fungi
antibacterial mechanisms
○ more common among women
● presence of bacteria in the interstitium results to
○ usually normal flora from the GIT and vagina
acute inflammation and tubules becoming necrotic
■ e.g. Candida albicans
● presence of bacterial toxins and leukocytic enzymes
○ their proliferation is kept in check by the normal
results in the formation of abscesses.
bacterial flora in these areas
● glomeruli are rarely involved & has a normal function
■ yeasts proliferate and may cause an infection
● px experience
when the bacterial flora is adversely disrupted
○ acute pain in their flank, back or groin
by antibiotics or pH changes
○ nocturia together with typical UTI clinical features
(dysuria and urgency to urinate)
○ high fever, chills, nausea, headache and
generalized malaise may also be experienced

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Predisposing Factors: Acute Pyelonephritis ● signs and symptoms:
● conditions or procedures that causes the proliferation ○ hypertension
and movement of bacteria to the upper urinary tract ○ polyuria
○ e.g. vesicoureteral reflux ○ nocturia
■ abnormal movement of urine from the bladder ○ renal failure (in 10 – 15% of cases)
to the ureter as a result of an inherited
congenital defect D) ACUTE INTERSTITIAL NEPHRITIS
● catheterization ● caused by an allergic response in the interstitium of
● urinary tract obstruction the kidney
● sepsis ○ kidney transplant rejection = most common
● pregnancy cause of allergic response
● diabetes mellitus ● allergic reactions to antibiotic, NSAIDs, antiepileptics
● immunosuppression and allopurinol can also cause AIN
○ usually induce cell-mediated immune response
C) CHRONIC PYELONEPHRITIS causes damage to the
● often develops when there is persistent inflammation ■ interstitium (edema and infiltration)
of the renal tissue resulting to fibrosis and scarring of ■ tubules (necrosis)
the renal calyces and renal pelvis ■ glomeruli and blood vessels are normal
● commonly caused by reflux nephropathies ● clinical features associated with drug-induced AIN:
○ vesicoureteral reflux ○ fever, skin rash and eosinophilia
○ urinary tract obstruction ○ these symptoms may resolve and renal function
○ intrarenal reflux. returns to normal
■ movement of urine back to the collecting duct ● irreversible damage may also occur, if untreated,
and as far as the renal cortex due to a especially among the elders
structural abnormality

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 VI. VASCULAR DISEASE ● 65% of ARF cases (majority)
● dysfunction of the vessels that receives blood towards ● includes glomerular, tubular & vascular
kidneys disease
● atherosclerosis ○ acute glomerulonephritis
○ vascular disease that reduce renal blood flow ○ acute tubular necrosis
Renal
● disease that causes structural changes on the ○ acute pyelonephritis
Mechanism
arterioles & glomerular capillaries resulting to ○ acute interstitial nephritis
ischemia ● cause damage and therefore affecting the
○ hypertensions function implicated structure
○ polyarteritis nodosa ● clinical feature:
○ eclampsia ○ severe anuria
○ diabetes
○ amyloidosis ● 10% of ARF cases
● associated with obstruction to urine flow
VII. RENAL FAILURE ○ renal calculi
Postrenal
● exist in both acute & chronic forms ○ tumors
Mechanism
● progression to end-stage renal disease is characterized ● obstruction causes an:
by: ○ ⬆ hydrostatic pressure in the tubules &
○ decrease GFR (<25 ml/min) bowman’s capsule resulting to damage
○ azotemia (⬆BUN, ⬆CREA)
○ electrolyte imbalance Chronic Renal Failure (CRF)
○ lack of renal concentrating ability → isosthenuric ● irreversible progressive loss of renal function
urine ● GFR slowly but continuously decreases
○ proteinuria ○ clinically recognizable: 80-85% if normal renal
○ glycosuria function is lost
○ ⬆ granular, waxy, & broad casts ○ GRF= 15-20 ml/min
● leads to end-stage renal disease
Acute Renal Failure (ARF) ● “slow & silent”
● sudden loss of renal function with the nephron ○ ability of the healthy nephrons to compensate for
appearing normal histologically damaged nephron
● reversible when prompt diagnosis & treatment is given ● “wear & tear principle”
to the px ○ healthy nephrons become overworked
● higher mortality rate due to: ○ undergo hypertrophy until they lose their function
○ (1) contaminant infection
○ (2) potassium intoxication
● glomerulonephritis
CAUSES OF ARF ● diabetic nephropathy
● 25% of ARF cases Clinical ● chronic pyelonephritis
● cause: Conditions ● hypertension
● systemic lupus erythematosus (SLE)
○ ⬇ renal perfusion → ischemia
● congenital abnormalities
● clinical conditions:
● azotemia
○ ⬇ cardiac output (BP)
● acid-base imbalance
○ hemorrhages Other Clinical ● water
○ burns Features ● electrolyte imbalance
Prerenal
○ surgical procedures ● abnormal calcium & phosphate
Mechanism metabolism
○ diarrhea
○ vomiting ● isosthenuria
Typical
○ septicemia ● proteinuria
Urinalysis
● other clinical features: ● hematuria
Results
● ALL types of cast
○ ⬇urine Na
○ ⬆urine osmolality
○ ⬆BUN:Creatinine ratio

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 UNIT 2: RENAL CALCULI ● gout (hyperuricemia)
Metabolic
● cystinosis (cystinuria)
Conditions
I. URINARY CRYSTALS TO RENAL CALCULI ● primary hyperoxaluria

Renal Calculi or Renal Stones 2. Absence of Natural Inhibitors

● aggregates of mineral salts interlaid with a matrix of ● natural inhibitors
protein & lipids ○ prevents crystal aggregation, nucleation and
● may form in secretory organs of the body: adherence
○ pancreas ● identified inhibitors:
○ gallbladder ○ nephrocalcin ○ calgranulin
○ salivary gland ○ osteopontin (uropontin) ○ bikunin
○ lacrimal gland ○ citrate ○ prothrombin F1 fragment
○ urinary tract ○ Tamm-Horsfall protein ○ heparan sulfate
● in the urinary system, they are commonly found in: (uromodulin) ○ pyrophosphate
○ renal calyces ○ chondroitin sulfate ○ CD 59.8,9
○ pelvis
○ ureter
3. Urinary pH
○ bladder
● important role in the formation of urinary crystals and
therefore its eventual growth to a renal stone
II. RENAL CALCULI FORMATION
● solubility of these chemicals varies, hence the
importance of the acid-alkaline tide to prevent the
A) FACTORS INFLUENCING CALCULI FORMATION
formation or growth of renal stones
● isohyduria
1. Supersaturation of Chemical Salts in Urine
○ urine pH of the patient remains constant and
● stone formation may or may not be associated with a
therefore enhances crystal and stone formation
concomitant increase of ff. solutes in the blood
● renal tubular acidosis
○ 25% of individuals who develop calcium stones do
○ tubules fail to secrete H+ ions
not have hypercalcemia
○ ⬆ Ca excretion to compensate
○ 50% of px with uric acid stones do not have
○ failure to produce an acid urine, to which calcium is
hyperuricemia
soluble to, and hypercalciuria increases the chance
● conditions occur when renal tubular reabsorption
of renal stone formation
mechanisms are dysfunctional
● urea-splitting organisms
○ ⬆urinary excretion of chemical salt
○ causes UTI
○ organisms convert urea to ammonia causing the
● dehydration urine to become alkaline
○ kidney continues excreting solutes with ○ organisms tend to form magnesium ammonium
minimal amount of H2O → concentrated phosphate or struvite stones
concentrated urine ○ stones are usually large, causing:
● diet ■ bleeding (hematuria)
External ○ ⬆ urinary oxalate among vegetarians ■ obstruction
Causes ● increased interstitial absorption ■ Infection w/out stone passage
○ ⬆ urine concentrations of particular ● staghorn stones
solutes ○ when stones in the renal pelvis become so large
○ px with inflammatory bowel disease that they extend into two or more calyces
● Medication ○ assoc. with upper urinary tract infection
○ cytotoxic drugs
● hyperparathyroidism 4. Urine Stasis
Endocrine ○ ⬆ Ca reabsorption ● encourage renal stone formation
Disorders ○ hypercalcemia ● allowing solutes to supersaturate and eventually
○ hypercalciuria precipitate in urine

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5. Nucleation & Attachme 1-2%
● cystine –
● occur on either: (1-3%)
○ renal epithelium ● results from adsorption
○ cell surfaces <1% ● carbonate of CO2 by Ca
○ bacteria phosphate crystals
○ aggregated/denatured proteins (Class Lecture Reference)
● once attachment or deposition occurs, the stone will
continue to grow in size as long as these factors are
present

B) NATURE OF RENAL CALCULI

Uromucoid
● organic matrix of renal calculi composed of lipids and
proteins
● incorporates urinary proteins such as
○ uromodulin (Tamm- Horsfall protein)
○ albumin
○ prothrombin fragments
○ lipids (cholesterol, triglycerides, phospholipids and Size
gangliosides)
● small calculi = sand or gravel type of stones
● presence of these organic compounds suggests the
○ common in the upper urinary tract
involvement of the cell membrane in the nucleation
○ upper renal stones
of crystals
○ can be eliminated with diuretics
● large calculi
Composition
○ bladder stones
● some renal stones are made up of two or more ○ less common
chemicals
○ e.g. calcium oxalate stones, Color and Texture
ammonium-magnesium-phosphate stones
● either simple or compound chemical composition Stone Color and Texture
○ simple renal stones = single chemical component Calcium ● white, pale friable stones
○ compound renal stones = 2 or more chemicals Phosphate ○ “friable” = easily break
■ characterized by distinct concentric rings ● yellow to brown red
around a central nucleus Uric Acid and
● moderately harder than calcium
● majority of renal calculi cases contain calcium mixed w/ Urate
phosphate
other components such as oxalate and/or phosphate
Cystine ● yellow, greasy or oily

Frequency Stone Composition Remarks

● darker, rough surface
Calcium Oxalate
○ one of the hardest stones
● calcium stones
○ calcium ● Ca oxalate precipitates
80% III. CONDITIONS ASSOCIATED WITH
oxalate at an acid or neutral
(75-80%) STONE FORMATION
○ calcium pH
phosphate
A) HYPERCALCIURIA (CALCIUM STONES)
● calcium ● forms calculi at normal
● forms
phosphate urinary pH (6.0)
○ calcium oxalate (both alkaline and acidic)
3-10% ● uric acid ● crystallizes and forms
○ calcium phosphate
(3-15%) ● magnesium-am calculi at low pH
● usually favors alkaline urine
monium-phosph ● forms calculi at alkaline
● one of the leading causes of Ca stones
ate (struvite) pH
● may or may not be accompanied hypercalcemia

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● causes: C) HYPERURICURIA (URIC ACID STONES)
○ increased intestinal absorption of calcium ● increased uric acid in urine which predisposes an
■ expect hypercalcemia individual to develop uric acid stones
■ e.g. vitamin D toxicity ● average adult excretion of uric acid = 500-600 mg/day
○ inappropriate renal tubular reabsorption ● at pH 5.5 it can exist as
○ resorption or loss from bone ○ free insoluble uric acid
■ increase bone resorption = hypercalcemia ○ urate that is more soluble in the presence of Na & K
○ primary hyperparathyroidism (5-10%) ● amount of free uric acid decreases in urine as urine
■ increased reabsorption becomes more alkaline
■ bone resorption ● causes:
■ intestinal absorption ○ diet / lifestyle
■ px often present with stone symptoms and have ■ increased intake of purines
Ca phosphate deposits in the renal tissue, ■ e.g. liver, dried beans, some fish, meat
cornea, and other organs ○ metabolic disorders
○ dietary hypercalciuria ■ gout
■ uncommon cause ■ Lesch-Nyhan syndrome
■ 3-4g/day of Ca = at risk for hypercalciuria ● 20% of px w/ gout form stones of pure uric acid
■ 800 mg/day already sufficient or mixed uric acid and calcium
● may develop uric acid stones (but not all gout px)
● most common Gout ● gouty nephropathy
● may form with excess oxalate and uric ○ not all are associated with presence of stones
Calcium ○ may be due to increased sodium-urate
acid in the urine
deposition
Oxalate ○ uric acid sometimes provide a medium
● an extremely rare metabolic disorder that occurs
Stones for stone formation before birth, mostly in boys
● newly formed aggregates = 20-25µm in Lesch-
● caused by a deficiency of the enzyme
diameter Nyhan
hypoxanthine-guanine
○ smaller than lumen of collecting ducts phosphoribosyltransferase (HGPRT)
● adherence to the epithelial surface allows ○ chemotherapy and irradiation
stones to continue growth rather than be ■ due to nucleotides released from damaged
excreted tumors or cells during therapy
● very hard, dark color, rough surface ■ e.g. leukemia, necrosis
● favored by less acidic urine ○ dehydration
Calcium ○ renal tubular acidosis ○ absence of bacteria degrading uric acid
Phosphate ○ infection ■ e.g. Lactobacillus, Pseudomonas
Stones ○ px who consume large amount of alkali ○ ileostomy
● commonly observed in px w/ primary ■ increased loss of alkaline fluid → acidic
hyperparathyroidism environment → favors uric acid formation
○ even if urine pH is w/in normal range
● px exposed to heat and dehydration can D) CYSTINURIA (CYSTINE STONES)
contribute to its formation ● form in px w/ an inherited amino acid transport
● pale and friable disorder
○ only cystine forms crystals and stones even if
B) HYPEROXALURIA (OXALATE STONES) cystine, ornithine, lysine, and arginine are
● dietary hyperoxaluria subsequently excreted in large amounts in the urine
○ e.g. cocoa, tea, coffee, peanut, spinach ○ heterozygous carriers of the disease have
● bowel disorders = increased reabsorption increased amounts of cystine in urine but do not
○ Crohn’s disease form stones
○ malabsorption with steatorrhea ○ homozygous patients are stone formers
● absence of Oxalobacter formigenes in cystic ● cystine is soluble at a urine pH of 7.4
fibrosis ○ thus, stones form within normal urine pH
● yellow-brown and feel somewhat greasy

Transcribed by: Lustre, Aliping, Guarin, Villanueva (2024). 15

 E) OTHER CALCULI (RARE RENAL CALCULI) Routine Urinalysis for Renal Colic
● pink to red urine
Composition Cause Physical ● NOTE: hematuria is expected when stones
Silica ● prolonged silica ingestion are passed in urine
● relatively insoluble diuretic ● positive for blood, proteins, pH, leukocyte
● may contribute to stone formation esterase, nitrite
● 1-2 mm mustard colored stones ● serum chemistry for calcium, phosphorus,
Triamterene Chemical
● bright blue fluorescence uric acid, and electrolytes may also help
○ when dissolved in butanol and identify patients predisposed to forming
exposure to UV light renal stones
Adenine ● inherited enzyme deficiency disorder and ● intact RBCs
Stone hyperuricemia ● casts are rare
Microscopy
Xanthine ● associated with a genetic disorder with an ● increased WBCs, bacteria
Stone absence of xanthine oxidase ● clusters of transitional epithelial cells
Sulfonamides –
Urine Quantitative Tests
IV. LABORATORY TESTS FOR ● quantitation is important to be able to determine the
DIAGNOSIS OF STONE FORMERS nature of the stone
● 24 hours urine specimen
A) ROUTINE URINALYSIS ○ considers the urinal variation of certain solutes
○ 3 samples needed
● constant finding when stones are present, ○ may help detect supersaturation of solutes and
Hematuria
even when they are asymptomatic reflect stone compositions
● usually not a feature of calculous disease ● serum chemistry
● but with renal tubular damage there may ● ultrasound, CT scan
Proteinuria be increased excretion of LMW plasma
proteins such as B2 microglobulin, and Urine pH Determination
some albumin ● important in determining the kinds of crystals likely to be
Erythrocyte ● usually not found precipitated
Casts ● other casts are unusual ○ e.g. uric acid with low pH (5 to 5.5), triple phosphate
with alkaline urine
● increased when infection is present,
● pH is not enough to determine if the patient has renal
Leukocytes resulting to a positive nitrite and leukocyte
stone but may used to indicate the stone’s composition
esterase reagent strip
Multiple
B) RENAL STONE ANALYSIS
Clusters of ● may be found in urine of patients with
Nonmalignant calculous disease and may be helpful in
Physical Examination
Transitional the diagnosis of unsuspected calculi.
● may not provide a definitive identification but some
Cells
points are worth noting

Renal Colic Gross Examination for Calculi

● pain that occurs when a small stones blocks the urinary Wash the stone(s) free from blood, mucus,
tract and causes an obstruction preservation solution, and so forth.
● formation of renal stones is most often discovered when ● Place stones in a beaker, cover with several
the urinary tract becomes obstructed or the stones thicknesses of gauze held firmly in place with rubber
produce ulceration and bleeding 1 bands, and wash under cold running water.
○ hydronephrosis = a condition where one or both ● Drain, remove gauze carefully, and dry beaker and
kidneys become stretched and swollen as the result stones in an oven.
of a build-up of urine inside them ● Rinse tiny stones with water from a squeeze bottle
● labs tests requested to confirm stone formation (not running water)
○ protein urinalysis = most commonly performed

Transcribed by: Lustre, Aliping, Guarin, Villanueva (2024). 16

2 Record the dimension of the stone. NOTE: Other Methods of Renal Stone Analysis
Describe briefly the color and texture of the stone's ● optical crystallography
● x-ray diffraction
3 exterior surface. The stone may be photographed for
● infrared spectroscopy
record purposes. ● electron beam analysis
Cut, saw, or break the stone so as to examine the ● mass spectroscopy
interior. ● clinical chemistry methods
● Note whether there is a foreign body that may have
4 V. PREVENTION AND TREATMENT OF RENAL STONES
acted as a nucleus for its formation.
● Describe the color and texture of the interior and
layers, if present. A) PREVENTION
● eliminating the factors that enhance renal stone
Reduce small stones to a fine powder by pulverizing
5 formation is key in preventing their occurrence
with a mortar and pestle.
● prevents to supersaturation of solutes in urine and are
If possible, where there is a very large stone, it may be
therefore effective ways of preventing renal stone
6 advisable to make separate analyses of layers that formation and growth
appear to have different constituents
○ increasing fluid intake = 6-8 glasses a day
○ modification of diet
Chemical Examination ● medications
Chemical ○ taking oral medications that enhances tubular
Principle Result reabsorption of some solutes can also prevent their
Group
● manganese oxide supersaturation
acts as an oxidizing ○ some medications also convert these solutes into a
Oxalate Effervescence more soluble form
agent in the
production of CO2
B) TREATMENT
● production of reduced
● several techniques available that can facilitate the
Phosphates oxides of Blue Color
removal or destruction of a renal stone once it is formed
molybdenum
● extracorporeal shockwave lithotripsy
Urates and ● uric acid reduces
Blue Color ○ not invasive
Uric Acid orthophosphoric acid
○ machine generates soundwaves applied to break
● Ca ions in the renal stones in the body
presence of ● cystoscopy
Calcium ammonium oxalate White Precipitate ○ can be used to remove the stone or to place a stent
produces Ca oxalate for drainage if the stone is found in the lower third of
precipitates the ureter or in the bladder
● formation of Mg ● lithotripsy
hydroxide in an ○ surgical procedure
alkaline solution that Blue Color Change
Magnesium ○ out px surgery
and Precipitates
absorbs the azo dye ○ takes multiple days before stones are passed
in the reagent ● percutaneous nephrolithotomy
● ammonia reacts with ○ minimally invasive
double iodide to form Orange-Brown ○ small puncture wound on skin (abdomen area)
Ammonium ○ mostly removes stones more than 2 cm in size near
dimercuric Precipitate
ammonium iodide the renal pelvis
● proteins w/ free ○ only performed if shockwave lithotripsy is not
cysteine group yield a successful
reddish color w/ ● ureterolithotomy
Cystine Beet-Red Color ○ removal of stones in the ureters
sodium nitroprusside
in an ammoniacal ● surgical nephrolithotomy
sol’n ○ rarely necessary or performed due to a variety of
less invasive procedures

Transcribed by: Lustre, Aliping, Guarin, Villanueva (2024). 17

ANALYSIS OF URINE MLS 321

& OTHER BODILY FLUIDS 8438

LECTURE BS MLS 3 MIDTERM
METABOLIC DISEASES

UNIT 1: AMINO ACID DISORDERS PRIMARY AMINOACIDURIA

● inborn errors of metabolism
Metabolic Disorders ● characterized by excess amino acid excretion in urine,
● can be renal or overflow disorders either resulting from or from

● due to tubular disease or renal toxins Classification of Aminoacidurias

Renal ○ affects reabsorption processes ● increased plasma concentration
Disorders ● overflow of excess abnormal metabolites is exceeds the renal plasma
in the urine only threshold, resulting in their overflow
● disruption of the normal metabolic pathway into the urine
Overflow ● diseases include:
due to organ malfunction or toxins, Aminoaciduria
○ affects the complete processing of ○ phenylketonuria
metabolites ○ maple syrup urine disease,
Overflow ● an excess of metabolites occurs in the ○ histidinemia
Disorders ○ tyrosinosis
blood and then follows to the urine
● due to a genetic “inborn error of metabolism ● excessive amino acids in urine
(IEM)” in newborns because there is no normal renal
○ related to a missing or defective enzyme mechanism for reabsorption
○ not limited by renal threshold
○ any increase in the plasma
AMINOACIDURIA concentration of the amino acid
● involved disruption of normal pathway for the synthesis, No Threshold
may result to aminoaciduria, even
Aminoaciduria
distribution, excretion, and utilization of amino acids if renal threshold has not been
● excess amino acids are excreted in urine in the reached
presence of increased plasma amino acids due to ● diseases include:
○ cystathioninuria
metabolic disorders
○ homocystinuria
● hereditary metabolic diseases ○ beta-aminoisobutyric aciduria
○ frequently inherited in an autosomal recessive
● plasma concentration of the affected
manner amino acid is normal to low but
○ usually caused by the absence and/or inactivity of a leaks into the urine
specific enzyme required for normal metabolic ○ due to tubular defect in the
activity Renal reabsorptive capacity of the
■ may either produce a metabolite which is Aminoaciduria tubules
● diseases include:
essential to the body
○ Hartnup disease
■ may be responsible for catalyzing the ○ cystinuria
metabolism of a substance which is toxic ○ glycinuria

Amino Acids Two Causes of Primary Aminoaciduria

● small biomolecules; building blocks of protein ● enzyme deficiency in a specific amino acid metabolic
● filtered through the glomerular membrane pathway due to gene alteration or mutation
● readily reabsorbed in the renal tubules ○ leads to a decrease in the synthesis of the enzyme
○ reabsorption is threshold limited and is facilitated by important for amino acid metabolism
membrane-bound carriers ○ most common cause
● excreted very minimally in the urine ● defective tubular reabsorption of amino acids

Transcribed by: Abenes, Aliping, Guarin, Villanueva (2024). 1

 Overview of Primary Aminoacidurias Classifications of Cystinosis
● Cystinosis ● most common and severe form
Cystine Disorders ● Cystinuria ● deposition of cystine in the lysosomes of
● Homocystinuria proximal tubular cells
● Maple Syrup Urine Disease ○ due to the proximity of PCT to other
Branched-Chain
(MSUD) tubules, it may also affect other
Amino Acid Disorder
● Organic Acidemias
tubules
● Phenylketonuria (PKU) /
○ causes proximal tubular dysfunction
Phenylpyruvic Oligophrenia
Phenylalanine- and development of Fanconi’s
● Alkaptonuria
Tyrosine Disorders syndrome
● Tyrosinuria
● Melanuria ● tubular reabsorption and secretion
● Indicanuria function is affected since tubules can no
Tryptophan Disorders
● 5-Hydroxyindoleacetic Acid longer concentrate urine
○ e.g. distal tubules become involved
Nephropathic
SECONDARY AMINOACIDURIA and patients are unable to concentrate
Cystinosis
● aminoaciduria occurs secondary to a disease and acidify urine
○ does not involve enzyme deficiency ● corneal deposition due to fast
● are induced, most notably by progression
○ severe liver disease ○ may lead to increased light sensitivity
○ galactosemia or blindness
○ Wilson’s disease ○ present at a very early age (2 yrs old)
○ renal tubular dysfunction (e.g. Fanconi syndrome) ● patients may experience polyuria,
isosthenuria and increased excretion of
CLINICALLY SIGNIFICANT AMINO ACID DISORDERS reducing sugars in urine
● may begin and be diagnosed at birth
● Cystinosis ● Alkaptonuria
● Cystinuria ● Homocystinuria ○ no treatment = patients die by the age
● MSUD ● Tyrosinemia of 10 years due to extensive kidney
● Phenylketonuria ● Melanuria
damage
CYSTINOSIS ● less (rare) common and less severe
● autosomal recessive lysosomal storage disease ○ same manifestation as nephropathic
○ since it involves accumulation of cystine in the ● adolescent onset
lysosomes of cells distributed in the spleen, ○ signs and symptoms are manifested
Intermediate
kidneys, bone marrow, and eyes and may impair during adolescence or adulthood
Cystinosis
organ function ● diagnosis may be delayed due to slower
○ preventing the release of cystine into the cellular rate of progression
cytoplasm for metabolism ○ px develop kidney failure by their late
● usually results to gene mutation and only differ in twenties or early thirties
disease severity, age of onset and clinical presentation ● milder form
Ocular
● manifests only ocular impairment
Cystinosis
● due to cystine deposition in the cornea
without involvement of renal damage

Transcribed by: Abenes, Aliping, Guarin, Villanueva (2024). 2

 CYSTINURIA
● caused by a defect in the reabsorption in the PCT
leading to increased urinary excretion of [*COLA]
○ cystine
○ dibasic amino acids
■ arginine
■ lysine
■ ornithine
● defective intestinal absorption of the same amino acids
may occur leading to decreased plasma concentrations
PHENYLKETONURIA (PKU)
Urine Findings: Cystinuria ● autosomal recessive disease
● pathological urinary cystine crystals and stones ● increased urinary excretion of phenylpyruvic acid &
composed of cystine phenylketone metabolites
○ increase urinary excretion of cystine crystals may ○ phenylacetic acid
contribute to renal stone formation ○ phenyllactic acid
● cystine crystals = clear, hexagonal crystals ○ alpha-hydroxyphenylacetic acid
○ differential tests is needed to differentiate cystine ● phenylalanine metabolic pathway
crystals from other hexagonal crystals ○ normally, phenylalanine is hydrolysed and
converted to tyrosine
MAPLE SYRUP URINE DISEASE (MSUD) ○ due to a defect or deficiency of phenylalanine
● rare, autosomal recessive inherited disease hydroxylase, it goes through transamination and
● rare type of inborn error of metabolism; autosomal forms phenylpyruvic acid
recessive ○ PKU can result when a defect or decrease occurs in
● accumulation of branched-chain amino acids in the the enzyme cofactor, tetrahydrobiopterin
blood, urine, or CSF [*MiS U Don’t LIV me] ● baby may appear normal at birth but signs and
○ leucine symptoms may manifest within 24 hrs
○ isoleucine ○ delayed development and feeding difficulties such
○ valine as severe vomiting appear within the infant’s first 2
● due to deficiency in the “branched-chain α-keto acid to 3 weeks of life
dehydrogenase” responsible for their oxidative ● diet restrictions for PKU patients; rich in phenylalanine
decarboxylation to acyl coenzyme A derivatives (e.g., ○ meat
fatty acids) ○ dairy products
● infants with MSUD appear normal at birth, symptoms ○ dry beans
begin to appear within the first few weeks of life ○ nuts
● branched-chain amino acids are neurotoxic = ○ eggs
neurological symptoms
● may result to death in a few months if untreated Clinical Manifestations of Untreated PKU
● eczema
Clinical Findings (Arranged In Progression): MSUD
● hypopigmentation
● distinct urine odor within 24 hrs = caramelized-sugar /
○ due to accumulation of phenylalanine, which is a
maple-syrup odor
strong inhibitor of tyrosinase needed for
○ due to high excretion of keto acids gives
synthesis of melanin from tyrosine
○ mainly due to accumulation of leucine
● seizures
● ketoacidosis
● limb spasticity
○ may result in poor feeding, vomiting, seizures,
● mousy or musty odor of urine, sweat or breath
lethargy
○ caused by the phenylacetic acid in these fluids
● ketonuria
● severe mental retardation
● muscle-spasticity = uncontrolled muscle contraction
○ maximum detrimental effects achieved by nine (9)
● respiratory failure
months of age
● brain injury = mental retardation

Transcribed by: Abenes, Aliping, Guarin, Villanueva (2024). 3

 ALKAPTONURIA NOTE: Black Urine
● rare, autosomal recessive disease characterized by the ● urine darkening is not unique
urinary excretion of homogentisic acid (2,5- ● requires further differentiation from other substances
dihydroxyphenylacetic acid), or “alkapton” that can cause similar changes such as:
● due to the congenital lack of the enzyme homogentisic ○ melanin
acid oxidase ○ indican (indoxyl sulfate)
○ mediates an essential step in the catabolism of ○ gentisic acid (a salicylate metabolite
phenylalanine and tyrosine
○ thus, homogentisic acid is not oxidized into HOMOCYSTINURIA
maleylacetoacetic acid and becomes accumulated ● rare autosomal recessive disorders of methionine
● normally, there is no homogentisic acid present in urine metabolism leading to an abnormal accumulation of
○ “alkali lover” = if present, urine allowed to stand homocysteine and its metabolites
for a period of time (12- 24 hours) ● deficiency of cystathionine-beta-synthase, leading to
○ turns dark as a result of the formation of accumulation of homocysteine
polymerization products of homogentisic acid ● treatable but cognitive function of the patient may be
○ ascorbic acid interferes with this darkening process delayed
● diet modification with reduction in methionine sources
Clinical Findings: Alkaptonuria can help prevent some of these adverse effects
● black coloration of urine upon oxidation
○ exposure to air, sunlight, or alkaline reagent Clinical Findings: Homocystinuria
○ one of the early signs ● nearsightedness (myopia)
○ historically, when infants wore cloth diapers that ● dislocation of eye lens
were laundered using strong alkaline soap, the ● glaucoma
presence of a black pigment on the diaper was ● osteoporosis and other skeletal abnormalities
suggestive of HGA ● increased risk of abnormal blood clotting
● ochronosis [adults]
○ homogentisic acid accumulates in cartilages TYROSINEMIA
○ dark pigmentation of the cartilage ● tyrosine is the metabolic precursor of:
● tendinitis ○ melanin
● kidney and prostate stones ○ thyroxine
● kyphosis or hunchback [adults] ○ catecholamines (epinephrine and norepinephrine)
○ degenerative arthritis ○ homogentisic acid formation
● dark tissue pigmentation (e.g. palm area) ● disorders associated to tyrosine metabolism can either
○ due to accumulation in tissues be hereditary or can be from transient metabolic defects

Transcribed by: Abenes, Aliping, Guarin, Villanueva (2024). 4

 ○ urine findings: MELANURIA
■ tyrosine and leucine ● melanin is a pigment which occurs normally in the skin,
■ p-hydroxyphenylpyruvic acid or hair, and in the choroid of the eye
p-hydroxyphenyllactic acid ○ derived from tyrosine
● increased tyrosine in the blood and may result to ○ normally not present in the urine
tyrosyluria (urinary excretion of tyrosine) if ○ melanogen
uncontrolled ■ precursor substance of melanin
■ readily oxidized to melanin causing black
Classification of Tyrosinemia coloration of urine when exposed to air or
Transient Neonatal Tyrosinemia sunlight for a long period of time
● associated with underdeveloped liver of the newborn ● increased urinary excretion of melanin and colorless
(or premature infants), which is not yet capable of precursors (e.g., 5,6- dihydroxyindole, melanogen)
synthesizing enzymes needed for the metabolism of ○ accumulation may lead to hyperpigmentation and
tyrosine result in melanuria
● transient = can be resolved once liver is capable of ● leading cause is due malignant melanoma
synthesizing the needed enzymes; 4-8 weeks
○ involve hypersecretion of melanin from tumor cells
● rarely causes permanent liver damage
(e.g. melanocytes)
Hereditary Tyrosinemia
○ too much melanin produced = overflows into urine
● usually fatal conditions that result in both liver and renal
● hypomelanosis or albinism
tubular disease producing a generalized aminoaciduria
● based according to enzyme deficiency ○ when inherited defects result in defective melanin
● can be classified into 3 types all producing tyrosylemia production occurs
and tyrosyluria
● fumarylacetoacetate hydrolase (FAH)
○ accumulation of tyrosine in the liver and
Type 1
kidney
○ associated with liver and renal damage
● tyrosine aminotransferase
○ increased deposition in the cornea
Type 2 ○ may lead to blindness
○ deposition of the fingers, palms, and
soles of the feet possible
● p-hydroxyphenylpyruvic acid
dioxygenase
Type 3
○ accumulation in CSF
○ brain injury = mental retardation

Transcribed by: Abenes, Aliping, Guarin, Villanueva (2024). 5

 OTHER AMINO ACID DISORDERS ● deficiency of transporter protein → tryptophan is
converted into indole compounds which is conjugated in
OTHER ORGANIC ACIDEMIAS the liver to form indican → indicanuria
● isovaleric acidemia may be suspected when ● scaly skin rash
○ characteristic odor of “sweaty feet” ● indicanuria “blue diaper syndrome” = blue staining of
○ caused by the accumulation of isovalerylglycine infant’s diapers
due to a deficiency of isovaleryl coenzyme A in
the leucine pathway Mnemonics: HARTNUP
● propionic and methylmalonic acidemias Hereditary
○ result from errors in the metabolic pathway Autosomal R-ecessive; Aminoaciduria; Ataxia
converting isoleucine, valine, threonine, and Tryptophan; Transport Protein Deficiency
methionine to succinyl coenzyme A Niacin deficiency (vitamin B3); NeUtral Protein

INDICANURIA 5-HYDROXYINDOLEACETIC ACID (HIAA)

● indican is formed from the conversion of excess indole ● a second metabolic pathway of tryptophan is for the
in the liver production of serotonin by the argentaffin cells in the
○ indole is normally formed from tryptophan small intestines
○ under normal conditions, most tryptophan that ○ normally, the body uses most of the serotonin, as
enters the intestine is either reabsorbed or is carried through the body primarily by the platelets
converted to indole and excreted in the feces ○ only small amounts of its degradation product HIAA
● increased amounts of tryptophan are converted to are available for excretion in the urine
indole in certain intestinal disorders including: ● due to development of when the argentaffin
○ malabsorption syndromes (enterochromaffin) cells in carcinoid tumors
○ Hartnup disease ○ produce excess amounts of serotonin, resulting in
● indican excreted in the urine is colorless until the elevation of urinary 5- HIAA levels
oxidized to the dye indigo blue by exposure to air ● test to detect 5-HIAA can be performed on a random or
● an early diagnosis of Hartnup disease first morning specimen
● false-negative results can occur
Hartnup Disease ○ 5-HIAA is not produced at a constant rate
● deficiency = sodium dependent transporter proteins throughout the day
○ B0AT1 protein = facilitates the absorption of ○ patients must be given diet instructions prior to the
neutral amino acids in the intestine and collection of any sample to be tested for 5-HIAA
reabsorption in the renal tubules ■ serotonin is a major constituent of foods such
● neutral amino acids as bananas, pineapples, and tomatoes
○ e.g. tryptophan ○ medications, including phenothiazines and
○ normally converted to melatonin, serotonin, and acetanilide, may also cause interference
niacin

Transcribed by: Abenes, Aliping, Guarin, Villanueva (2024). 6

 UNIT 2: OTHER METABOLIC DISORDERS

CARBOHYDRATE DISORDERS
● abnormal distribution or synthesis of carbohydrates
● primarily result from either
○ genetic abnormality in enzyme production
■ galactosemia
○ defect in hormonal function and production
■ diabetes mellitus
● other metabolic disorders may result from the
accumulation of metabolites due defects in enzyme
metabolism (either inherited or acquired)
● levels of these substances are elevated more rapidly in
blood than urine
○ urine may contain additional abnormal substances
not tested for by routine urinalysis

Melutria
● a condition characterized by increased urinary sugar
● most frequently observed in cases of
○ diabetes mellitus Type 3c DM (Pancreatogenic DM)
○ pentosuria, lactosuria, fructosuria ● can develop when the pancreas stops producing
○ galactosemia enough of the hormone called insulin
● most commonly identified causes:
DIABETES MELLITUS ○ chronic pancreatitis
● disorder characterized by hyperglycemia as a result of ○ pancreatic ductal adenocarcinoma
hormonal deficiency or a defect in hormonal action of ○ haemochromatosis
insulin or both ○ cystic fibrosis
○ previous pancreatic surgery
Type 1 DM and Type 2 DM Gestational DM
Type Type 1 DM Type 2 DM ● occurs during pregnancy
● inability to produce ● defective insulin ○ resolved after pregnancy
Description ○ may lead to T2DM if mismanaged after pregnancy
insulin production
● autoimmune ● due to insulin resistance or tolerance
(autoantibody) Other Specific Types of DM
Cause ● insulin resistance
destruction of the
ß-cells Gigantism and ● increased growth hormone increases
● normal production of Acromegaly plasma glucose concentration
insulin → cells are not Cushing’s ● increased cortisol
● absence of insulin
responsive to insulin Syndrome ○ cortisol is a hyperglycemic agent
→ glucose does not
→ increased glucose
enter the cells and
signal is sent to the
cannot be utilized Panel of Tests for Signs and Symptoms
hypothalamus →
Mechanism as an energy Diagnosis of DM of DM
ß-cells keeps
source → ● polyuria
producing insulin →
accumulates in ○ excessive urination
ß-cells dysfunction
blood plasma → ● Fasting Blood Sugar (FBS) ● polyphagia
due to overproduction
hyperglycemia ● Random Blood Sugar ○ excessive hunger
of insulin (overworked
ß-cells) (RBS) ● polidipsia
● 2-Hr Postprandial ○ excessive thirst
NOTE: CDC ● HBA1c (Glycosylated Hgb) ● lethargy
● no longer required to categorize DM according to insulin ● fatigue
dependency and onset ● unexplained weight loss

Transcribed by: Abenes, Aliping, Guarin, Villanueva (2024). 7

 CLASSIC GALACTOSEMIA Other Carbohydrates
● increase of galactose in the ● increased xylitol in urine
○ blood = galactosemia Pentosuria ● ingestion of large amounts of fruit
○ urine = galactosuria ● usually among pregnant women and
● caused by an absence or deficiency with the enzymes Lactosuria
premature infants
that break down the galactose ● associated with parenteral feeding
Fructosuria
● indicates the inability to properly metabolize galactose
to glucose
NOTE:
○ galactose can only be used as an energy source if
● breastfeeding or milk is usually stopped
converted into glucose
● persons with deficiencies in the other two enzymes may
still produce galactosuria but have negative newborn
Type 1: Galactose-1-Phosphate Uridyl Transferase (GALT)
screening tests
Deficiency
● diabetes insipidus is not a carbohydrate disorder
● most common & most the severe fatal symptoms
○ nephrogenic = kidneys does to respond to ADH
● accumulation of galactose metabolites
○ neurogenic = deficiency of ADH
○ galactonate
■ aldose reductase enzyme reduces galactose →
PORPHYRIN DISORDERS
galactitol
○ galactitol
Porphyrin
● signs and symptoms
● intermediate compounds in the production of heme
○ first few days of newborn = “failure to thrive”
● main sites of production:
■ vomiting, poor feeding, diarrhea, fatigue,
○ bone marrow = intermediates in the synthesis of
lethargy
hemoglobin
■ delayed growth and development
○ liver and other tissues = intermediates for other
● disease progression
heme proteins such as myoglobin
○ hepatomegaly
● synthesis of heme can be blocked at a no. of stages
○ jaundice
○ blockage of a pathway reaction results in the
○ may lead to liver cirrhosis
accumulation of the product formed just prior to the
○ cataract formation due to deposition of
interruption and may become elevated in the urine,
galactitol in the cornea → blindness
blood, or feces
○ ascites
● porphyria results from the to accumulation of
○ splenomegaly
○ porphyrin precursors
○ mental retardation
■ delta-aminolevulinic acid (ALA)
● newborn screening programs currently test for the
■ porphobilinogen (PBG)
presence of GALT deficiency
○ primary porphyrins
○ the enzyme is measured in the red blood cells as
■ uroporphyrin,
part of the newborn heel puncture protocol
■ coproporphyrin
■ protoporphyrin
Type 2: Galactokinase (GALK) Deficiency
● can result in cataracts in adulthood (more common)
Common Deficiencies in Heme Synthesis
Type 3: Uridine Diphosphate-Galactose-4-Epimerase Aminolevulinate ● lead poisoning
(GALE) Deficiency Dehydratase
● may be asymptomatic or produce mild symptoms Porphobilinogen
● acute intermittent porphyria
● manifestations Deaminase
○ cataracts Uroporphyrinogen
● porphyria cutanea tarda
○ failure to thrive Decarboxylase
○ liver and kidney disease Ferrochelatase ● lead poisoning

Transcribed by: Abenes, Aliping, Guarin, Villanueva (2024). 8

Porphyria
● collective term of porphyrin metabolism disorders

● much rarer than acquired porphyrias

● caused by failure to inherit the gene
Inherited that produces an enzyme needed in
Porphyria the metabolic pathway
● e.g. congenital erythropoietic
porphyria
● e.g. lead intoxication
○ will alter or decrease the
expression of an enzyme
Acquired ● other causes include
Porphyria ○ excessive alcohol exposure
○ iron deficiency
○ chronic liver disease
○ renal disease

Clinical Findings: Porphyria

● cutaneous lesions (skin rashes with blisters)
○ extensive blistering or bullous lesions
○ porphyrins are photosensitive = exposure and
absorption of light = formation of toxic free radicals
causing cutaneous lesions or burning sensation in
the skin
● neurological symptoms (neuropsychiatric problems)
○ porphyrins are neurotoxic
○ i.e. hysterical disorders, mood disorders,
hallucinations = psychosis may develop
○ seizures
● muscle weakness, hypertension, and tachycardia
● abdominal pain, nausea
○ due to intestinal pseudo obstruction = constipation
● depression
● physical exam: port wine color

Transcribed by: Abenes, Aliping, Guarin, Villanueva (2024). 9

 MUCOPOLYSACCHARIDE DISORDER

Mucopolysaccharides (Glycosaminoglycans)
● proteins with multiple glycan units residing in the
connective tissues
○ e.g. hyaluronic acid, chondroitin sulfuric acids,
chondroitin, keratosulfate, heparin, and heparin
sulfuric acid
● inherited disorders in the metabolism of these
compounds prevent the complete breakdown of the
polysaccharide portion
○ results in the accumulation of the incompletely
metabolized polysaccharide portions in the
lysosomes of the CT cells and in the urine
● enzyme deficiency → accumulates in the blood →
increased urinary expression in the form of by-products
● products most frequently found in the urine are
○ dermatan sulfate
○ keratan sulfate
○ heparin sulfate
● commonly targets males (rarely females)
○ inherited as sex-linked recessive

Common Mucopolysaccharides Disorders

● severe mental retardation
● abnormal skeletal formation
Hurler Syndrome ● accumulation of mucopolysaccharide in
the cornea of the eyes → blindness
● severe mental retardation
● abnormal skeletal formation
Hunter ○ frontal bossing
○ jaw deformity leading to abnormal
Syndrome
growth of teeth
○ scoliosis
○ feet
Sanfilippo ● mental retardation
Syndrome ● does NOT involve skeletal deformity

NOTE: Other Mucopolysaccharides Disorders

● Scheie syndrome
● Morquio syndrome
● Maroteaux– Lamy syndrome

Transcribed by: Abenes, Aliping, Guarin, Villanueva (2024). 10

 PURINE DISORDERS NOTE: Newborn Screening Act of 2004 (RA 9288)
● purine is a precursor of uric acid synthesis ● illustrates a procedure to detect a genetic and
metabolic disorder in newborns that may lead to mental
Lesch–Nyhan Syndrome / Disease retardation and even death if left untreated
● an inherited sex-linked recessive purine overflow ● the rules and regulations implementing the Newborn
disease Screening Act recommended the conditions to be
● excessive excretion of uric acid in urine included in the newborn screening panel of disorders
○ deficiency: hypoxanthine guanine ● the advancement of newborn screening has succeeded
phosphoribosyl transferase in reducing morbidity and mortality associated with
● clinical manifestations certain congenital disorders as it allows timely medical
○ mental retardation intervention for diagnosed patients
○ severe motor defects ● affected children identified through newborn screening
○ gout can lead normal lives with the help of prompt and
○ renal calculi appropriate medical management
○ tendency toward self-destruction
○ orange sand diaper syndrome Newborn Screening: Factors to Consider
■ one of the first symptoms observed of uric acid ● shall be performed after twenty-four (24) hours
crystals of life but not later than three (3) days from
complete delivery of the newborn
● a newborn that must be placed in intensive care in
order to ensure survival may be exempted from
the 3-day requirement but must be tested by
seven (7) days of age
● it shall be the joint responsibility of the parent(s)
and the practitioner or other person delivering the
newborn to ensure that newborn screening is
performed

Transcribed by: Abenes, Aliping, Guarin, Villanueva (2024). 11

 SUMMARY OF METABOLIC DISEASES
Disorder Enzyme Deficient Accumulation Clinical Findings
● nephropathic cystinosis
○ renal tubular acidosis
Cystinosis – ● cystine
● intermediate cystinosis
● ocular cystinosis
● cystine
Cystinuria ● ornithine
– ● renal calculi
(COLA) ● lysine
● arginine
● branched-chain amino acids
● caramelized-sugar odor
MSUD ● branched-chain alpha-keto ○ leucine
● mental retardation
(MiS U Dont LIV me) acid dehydrogenase ○ isoleucine
● muscle spasticity
○ valine
● phenylpyruvic acid
● mousy or musty odor
● phenylketone metabolites
● mental retardation
PKU ● phenylalanine hydroxylase ○ phenylacetic acid
● eczema
○ phenyllactic acid
● hypopigmentation
○ alpha-hydroxyphenylacetic acid
● black urine
● ochronosis
Alkaptonuria ● homogentisic acid oxidase ● homogentisic acid (alkapton)
● kyphosis
● dark tissue pigmentation
● myopia
Homocystinuria ● cystathionine-beta-synthase ● homocysteine and its metabolites ● dislocation of eye lens
● abnormal blood clotting
● fumarylacetoacetate hydrolase ● tyrosine in liver and kidney ● liver and renal damage
● tyrosine aminotransferase ● tyrosine in cornea ● blindness
Tyrosinemia
● p-hydroxyphenylpyruvic acid
● tyrosine in CSF ● mental retardation
dioxygenase
● albinism
Melanuria ● malignant melanoma ● melanin
● hyperpigmentation
● argentaffin cells in carcinoid
5-HIAA ● serotonin ● sleeping disorder
tumor
Indicanuria – ● indole ● Hartnup’s disease
*caused by a tumor

Metabolite Enzyme Deficient Clinical Significance

Insulin ● DM
GALT
Carbohydrate
GALK ● Galactosemia
GALE
delta-Aminolevulinic acid dehydratase ● Lead Poisoning
Porphobilinogen Deaminase ● Acute Intermittent Porphyria
Porphyrin Uroporphyrinogen Decarboxylase ● Porphyria Cutanea Tarda
● Lead Poisoning
Ferrochelatase
● Erythropoietic Protoporphyria
● Hurler Syndrome
Mucopolysaccharide Enzyme needed to break down glycosaminoglycans ● Hunter Syndrome
● Sanfilippo Syndrome
Purine Hypoxanthine Guanine Phosphoribosyl Transferase ● Lesch-Nyhan Syndrome

Diaper Syndromes Mental Retardation

Black Alkaptonuria, Melanuria MSUD
Blue Hartnup’s Syndrome Amino Acid PKU
Sand-Orange Lesch-Nyhan Type 2 Tyrosinemia
Carbohydrate GALT
Purine Lesch-Nyhan
Skin Rash Hunter
Blistering Porphyrin Disorders Mucopolysaccharide Hurler
Scaly (Pellagra) Hartnup’s Syndrome Sanfilippo

Transcribed by: Abenes, Aliping, Guarin, Villanueva (2024). 12

ANALYSIS OF URINE MLS 321L

& OTHER BODILY FLUIDS 8438L

LABORATORY BS MLS 3 FINALS
M7U1: CEREBROSPINAL FLUID

UNIT 1: CEREBROSPINAL FLUID

● brain and spinal cord are surrounded by three
membranes called the meninges

Meninges
● “hard mother”
Dura Mater ● tough outermost membrane that lines
the surface of the bone
● middle layer
Arachnoid
● filamentous inner membrane that
Mater
resembles spider web
● where CSF flows in the brain & spinal
*Subarachnoid
cord
Space
● bet. the arachnoid and pia mater
Pia Mater ● lines the surface of neural tissue

CEREBROSPINAL FLUID
● sterile, clear, colorless fluid
● product of selective secretion from plasma
○ by the choroid plexus → into the ventricles by
ependymal cells
● important functions of CSF
○ (1) provides physical support
■ suspends the brain and decreasing its weight
from 1.5 kg to 50g
○ (2) confers a protective effect against sudden
changes in blood pressure and trauma
○ (3) acts as a medium for the transport of nutrients
○ (4) removal of metabolic waste products
■ via the capillary endothelium w/ tight junctions

PHYSIOLOGY AND COMPOSITION

● produced in the choroid plexuses of 4 ventricles
○ 2 lateral ventricles
○ 3rd ventricle
○ 4th ventricle
● reabsorbed back into blood capillaries via arachnoid
granulations or villae
● 70% of CSF
○ derived by secretion of plasma through the choroid
plexuses and a few from ultrafiltration of plasma
● 30% of CSF
○ ventricular ependymal lining and subarachnoid
space accounts for the remainder

Transcribed by: Lustre, Aliping, Guarin, Villanueva (2024). 1

NOTE: CEREBROSPINAL FLUID ANALYSIS
● CSF is NOT an ultrafiltrate of plasma but a product of
selective secretion from plasma LUMBAR PUNCTURE (SPINAL TAP)
● primarily used to collect CSF sample routinely
● patient is asked to lie in fetal position → puncture site
is cleansed and a local anesthetic is applied→ spinal
needle is inserted in the lumbar interspace and
advanced until the dura mater is penetrated which is
usually indicated by a popping sound

CSF Collection Site

Adults ● 3rd & 4th vertebrae
Children ● 4th & 5th vertebrae
● may be selected if the preferred site is
Alternative Site infected as this procedure may introduce
● blood-brain barrier (BBB) organisms in the CNS
○ unique capillary endothelium that has tight junctions
● data included in the px chart for CSF collection:
○ reduces the extracellular passage of substances
○ (1) initial pressure
between plasma and CSF
○ (2) CSF volume
○ ALL substances that enter or leave the CSF must
○ (3) closing pressure
pass through this

CSF Flow

● obstruction in the flow of CSF can lead to:

○ (1) hydrocephalus
○ (2) ⬆ intracranial pressure
■ brain damage
■ intellectual and developmental disabilities
■ death (if left untreated)

Transcribed by: Lustre, Aliping, Guarin, Villanueva (2024). 2

Initial or “Opening Pressure” Queckenstedt Test
● taken by the physician before CSF is collected ● to confirm for the presence of subarachnoid block
● NOTE: ○ causes:
○ sudden drop in the opening pressure after removal ■ sinus thrombosis
of 1-2 mL is an indication to STOP the withdrawal ■ mass lesion of the spinal cord
● manual pressure is applied to the jugular vein to
Normal Opening Pressure elevate venous pressure
● 180 mmHg
● CSF volume collected: Results and Interpretation: Queckenstedt Test
Adults ○ up to 20 mL (+) Presence
○ 15% of total CSF volume ● when the pressure remains
of a Block
○ 1-2 mL should be removed
(–) Absence
● 10-100 mmHg ● simultaneous increased CSF pressure
Infants & of a Block
● CSF volume collected:
Young
○ proportionate smaller volume can be
Children SPECIMEN COLLECTION AND HANDLING
obtained
● as CSF is collected, it must be dispensed in three (or
more) sequentially labeled sterile collection tubes
“Closing” Pressure
● normally 10-30 mmHg less than the opening pressure

Other CSF Collection Methods

● performed on infants with open fontanels
from which CSF is collected

Ventricular
Puncture

● involves collection of CSF from the sub-

occipital region of the head

CSF Specimen Collection Tubes

Cisternal Storage Temp
Tube Laboratory Section
Puncture (Delayed Test)
● chemical, immunology
and serology tests
● -15°C to -30°C
Tube 1 ● minimal blood contamination
● frozen
during needle insertion does
● collects CSF from the cervical region not affect these tests
● 19°C to 26°C
Tube 2 ● microbiological studies
● room temp
Lateral ● cell counts and cytology ● 2°C to 8°C
Cervical Tube 3
studies ● refrigerate
Puncture

Transcribed by: Lustre, Aliping, Guarin, Villanueva (2024). 3

Small Amount of CSF Collected A) PHYSICAL EXAMINATION
● order of test priority: ● normal CSF physical characteristics:
○ (1) microbiological studies ○ colorless
○ (2) cell count ○ crystal clear
○ (3) chemistry tests ○ same viscosity as water
○ (4) immunologic tests ○ does not clot
● specific gravity = 1.006-1.008
Delayed Testing ● pH = 7.30-7.45
● falsely low cell counts ● causes mucin to be secreted in CSF
○ due to cell lysis ⬆ CSF ● assoc. conditions:
● false lactate increase viscosity ○ cryptococcal meningitis
○ metastatic adenocarcinoma
○ due to glycolysis
○ viable microbial organisms might not be recovered ● assoc. conditions:
○ ⬆ permeability of the choroid plexus
from the sample
⬆ CSF ○ perforation of the BBB
● samples must be stored in a temperature that would volume ○ acute and chronic meningeal congestion
ensure recovery of constituents (see table above) ■ due to ⬆ transport transudation of
plasma through the capillaries
NOTE:
● consider ALL test on CSF as STAT Pleocytosis
● CSF collection causes significant px discomfort and ● refers to an increase in the number of cells present in
puts the px at risk of developing complications CSF resulting to varying degrees of cloudiness
● proper labeling & handling must be observed ● result from the presence
○ cells
Indications of Performing Lumbar Puncture ○ microorganisms
Turbid CSF
● meningitis ○ protein
○ aspirated epidural fats
Infections ● encephalitis
○ radiographic contrast media dye
● brain abscess
Slightly
● subarachnoid ● 200 – 500 WBCs/uL
Hemorrhage Hazy CSF
● intracerebral
Cloudy
● multiple sclerosis ● >500 WBCs/uL
Neurologic Disease Appearance
● Guillain-Barre syndrome
● leukemia Oily
● presence of radiographic contrast media
Appearance
Malignancy ● lymphoma
● metastatic carcinoma
Tyndall’s Effect
● brain
Tumor ● direct sunlight directed on the tube at a 90° angle from
● spinal cord
observer
● headache ● shows SPARKLING or snowy appearance as a result
⬆ Increased
● convulsions of the presence of suspended particles (e.g., <50
Intracranial pressure
● coma WBCs/ul) that scatter the light.
Abnormal CNS ● paralysis
Function ● abnormal reflexes Clot Formation
● indicates a compromised BBB
● leads to ⬆ fibrinogen levels
Contraindications of Lumbar Puncture
● CSF clots
● septicemia
○ fine or delicate
● systemic infections
○ IF refrigerated > 12hrs
● localized lumbar puncture
■ can form a thin film or pellicle on the surface
● assoc. conditions:
○ Froin’s syndrome

Transcribed by: Lustre, Aliping, Guarin, Villanueva (2024). 4

 ■ ⬆ proteins and clotting factors that causes Presence of Blood
clotting of CSF ● blood decreases from the first to the last
Traumatic
○ traumatic lumbar puncture tube
Tap
■ sample is contaminated by blood and plasma ● tendency to clot
proteins ● presence of blood is even in ALL 3 tubes
○ paresis (weakened muscle movement) ● does not have the tendency to clot
■ small clots ● appearance of substances:
Intracranial ○ lysed RBC (after 1-2 hrs)
○ purulent meningitis
Hemorrhage ○ siderophage
■ large clots ■ hemosiderin-laden macrophage
○ tubercular and suppurative meningitis ○ hematoidin crystals
■ (+) web-like pellicle after overnight refrigeration ○ (+) D-dimer Test

Xanthochromia D-Dimer Test

● means “yellow” Results and Interpretation: D-Dimer Test
● term is used to refer to any CSF discoloration including
● microscopic finding of macrophages
pink, yellow and orange
(+) intracranial containing ingested RBCs
● xanthochromia causing samples:
hemorrhage (erythrophagocytosis) or
○ Carotene concentration above 150 mg/dL
hemosiderin granules
○ meningeal melanoma
○ CSF collected 2-5 days after a traumatic tap ● detection of the fibrin degradation
(+) fibrin formation product D-dimer by latex
CSF Discoloration at hemorrhage site
agglutination immunoassay
● usually after centrifugation
Pink
● from oxyhemoglobin
Yellow ● bilirubin
Orange ● oxyhemoglobin & bilirubin
Brownish ● methemoglobin

Fig. Tubes of CSF. Appearance left to right is normal, xanthochromic,

hemolyzed, and cloudy.

Transcribed by: Lustre, Aliping, Guarin, Villanueva (2024). 5

 B) MICROSCOPIC EXAMINATION
● CSF normally has 0-5 WBCs/uL (adults)
○ lymphocytes
○ monocytes
● children (0-10 WBCs/uL) & neonates (30 WBCs/uL)
○ predominantly comprised of monocyte
● RBCs are not normally present in CSF
○ contamination during lumbar puncture

B1. Total Cell Count

● performed on a well-mixed, undiluted CSF sample
● hemocytometer is charged and cells are allowed to
settle for 1 minute
● counted in ALL 9 squares of the Neubauer counting grid
● dilution:
○ IF no. of cells = >200
○ appear overlapping or crowded
○ dilute in NSS
○ alternative dilution method:
■ counting lesser squares and properly adjusting
the calculation according to the actual volume
of the sample used in counting

Dilution by Visual Appearance

Slightly Cloudy ● 1:10
Bloody ● 1:10,000

Total Cell Count Formula

#𝑐𝑒𝑙𝑙𝑠/𝑚𝑚3 = 𝐶𝑒𝑙𝑙𝑠 𝐶𝑜𝑢𝑛𝑡𝑒𝑑 × 𝐷𝐹 × 𝑉𝐶𝐹

𝐷𝐹(𝑑𝑖𝑙𝑢𝑡𝑖𝑜𝑛 𝑓𝑎𝑐𝑡𝑜𝑟) = 𝑡𝑜𝑡𝑎𝑙 𝑣𝑜𝑙𝑢𝑚𝑒 𝑜𝑓 𝑠𝑜𝑙𝑢𝑡𝑖𝑜𝑛 ÷ 𝑣𝑜𝑙𝑢𝑚𝑒 𝑜𝑓 𝑠𝑎𝑚𝑝𝑙𝑒

𝑉𝐶𝐹(𝑣𝑜𝑙𝑢𝑚𝑒 𝑐𝑜𝑟𝑟𝑒𝑐𝑡𝑖𝑜𝑛 𝑓𝑎𝑐𝑡𝑜𝑟 = 𝑣𝑜𝑙𝑢𝑚𝑒 𝑑𝑒𝑠𝑖𝑟𝑒𝑑 (1𝑛𝑙) ÷ 𝑣𝑜𝑙𝑢𝑚𝑒 𝑢𝑠𝑒𝑑

● 𝑣𝑜𝑙𝑢𝑚𝑒 𝑢𝑠𝑒𝑑 = 𝑛𝑜. 𝑜𝑓 𝑠𝑞𝑢𝑎𝑟𝑒𝑑 𝑐𝑜𝑢𝑛𝑡𝑒𝑑 × 𝑣𝑜𝑙. 𝑜𝑓 𝑠𝑞𝑢𝑎𝑟𝑒
● 𝑣𝑜𝑙𝑢𝑚𝑒 𝑑𝑒𝑠𝑖𝑟𝑒𝑑 = 𝑓𝑖𝑥𝑒𝑑 𝑎𝑡 1 𝑛𝑙
● 𝑣𝑜𝑙𝑢𝑚𝑒 𝑜𝑓 𝑠𝑞𝑢𝑎𝑟𝑒 = 𝑙𝑒𝑛𝑔𝑡ℎ × 𝑤𝑖𝑑𝑡ℎ × 𝑑𝑒𝑝𝑡ℎ

Fuchs-Rosenthal Chamber (Phase Microscopy)

#𝑐𝑒𝑙𝑙𝑠/3 = 𝑐𝑒𝑙𝑙𝑠 𝑢/𝐿

B2. WBC Count

● CSF is treated with 3% glacial acetic acid for 3-5 mins
○ to lyse RBCs
○ enhance visualization of WBCs
● methylene blue
○ can also be used to stain WBCs to enhance
visualization and facilitate counting of WBCs

Transcribed by: Lustre, Aliping, Guarin, Villanueva (2024). 6

B3. RBC Count Cells Found in Differential Count
● performing RBC count is only recommended when: Types of Microscopic
○ IF there is a traumatic tap Clinical Significance
Cells Findings
○ IF correction for proteins and WBCs is needed
● normal
● in some institutions, RBC count is derived from:
● late stage of viral
𝑅𝐵𝐶 𝑐𝑜𝑢𝑛𝑡 = 𝑊𝐵𝐶 𝑐𝑜𝑢𝑛𝑡 − 𝑡𝑜𝑡𝑎𝑙 𝑐𝑒𝑙𝑙 𝑐𝑜𝑢𝑛𝑡
● tubercular, fungal, &
● ALL stages of
syphilitic meningitis
B4. Differential Count Lymphocytes development may
● multiple sclerosis
● spx preparation for differential count be found
● Guillain-Barre
○ specimen is concentrated ● lymphoma
○ stained smear is prepared ● drug abuse
● cytocentrifugation
● bacterial meningitis ● granules may be
○ recommended method of concentrating sample
● early stage of viral, less prominent than
○ produces “cytospin” slides that show good cellular
Neutrophils tubercular & fungal in blood
recovery
meningitis ● cells disintegrate
● microscopic scanning
● cerebral hemorrhage rapidly
○ entire slide is then scanned first using aLPO (10x)
providing an overview of the cellularity of the ● normal
specimen and allowing detection of abnormalities ● viral, tubercular, & ● found mixed with
Monocytes
○ higher objectives fungal meningitis lymphocyte
■ identify and classify cells observed in the ● multiple sclerosis
sample ● may contain
phagocytized RBCs
Methods Used for Differential Count
appearing as:
● addition of albumin ● tubercular and ○ empty vacuoles
Cyto-
○ ⬆cell yield and ⬇cell distortions Macrophages fungal meningitis or ghost cells
centrifugation
■ cytoplasmic vacuoles
● RBCs in spinal fluid ○ hemosiderin
Sedimentation ● produce less distortion granules
& Filtration ● NOT routinely used ○ hematoidin
● 5-10 mins crystals
Centrifugation ● smear (Wright stain) ● lymphoblasts
● routinely used Blast Forms ● acute leukemia ● myeloblasts
● monoblasts
Normal CSF Differential Count ● resemble
Lymphoma ● disseminated
Age Lymphocytes Monocytes Neutrophils Cells lymphocytes with
lymphomas
cleft nuclei
Neonates
5-35% 50-90% 0-8% ● metastatic ● seen in clusters
(0-2 mons) Malignant
carcinoma & primary with fusing of cell
Adults Cells
40-80% 15-45% 0-6% CNS carcinomas borders and nuclei
(>18 yrs)
● tuberculous &
syphilitic meningitis ● traditional and
Plasma Cells ● multiple sclerosis classic forms seen
● lymphocyte ● reactive lymphs
reactions
Ependymal, ● observed after
● seen in clusters
Choroidal, performance of
with distinct nuclei
and diagnostic
and distinct cell
Spindle- procedures for CSF
walls
Shaped Cells collection

Transcribed by: Lustre, Aliping, Guarin, Villanueva (2024). 7

 FigNormal lymphocytes. Some Fig. Normal lymphocytes and Fig. Macrophages. Notice the large Fig. Macrophage with RBC
cytocentrifuge distortion of monocytes amount of cytoplasm and vacuoles remnants (×500).
cytoplasm (×500)

[Link] with cytoplasmic Fig. Neutrophils with intracellular [Link] with hemosiderin Fig. Macrophage containing
vacuoles resulting from bacteria and hematoidin(×250). Notice the hemosiderin stained with Prussian
cytocentrifugation bright yellow color. blue

Fig. Neutrophils with intracellular Fig. Nucleated RBCs seen with Fig. Macrophage with coarse Fig. Macrophage containing
and extracellular bacteria bone marrow contamination hemosiderin granules(×500). hemosiderin and hematoidin
crystals.

Fig. Bone marrow contamination Fig. Capillary and tissue fragments

(×1000). Notice the immature RBCs from a traumatic tap (×100). Fig. Choroidal cells showing distinct Fig. Ependymal cells. Notice the
and granulocytes cell borders and nuclear uniformity nucleoli and less distinct cell borders
(×500).

Fig. Broad spectrum of lymphocytes Fig. Eosinophils(×1000). Notice

and monocytes in viral meningitis cytocentrifuge distortion. Fig. Cluster of spindle-shaped cells Fig. Lymphoblasts from acute
(×1000) lymphocytic leukemia

Transcribed by: Lustre, Aliping, Guarin, Villanueva (2024). 8

 ● CSF contamination with
peripheral blood
● Altered capillary endothelial
exchange
● ⬇reabsorption into venous
High
blood
Levels
● ⬆synthesis in the central
Fig. Myeloblasts from acute Fig. Lymphoma cells with nucleoli
nervous system
myelocytic leukemia(×500)
● damage to BBB
● degeneration of neural tissues
○ multiple sclerosis

● NOT synthesized in the CNS

○ results from passage across BBB
● monitor permeability of the BBB
● CSF/serum albumin index
Fig. Burkitt lymphoma. Notice Fig. Medulloblastoma (×1000).
○ used to evaluate albumin
characteristic vacuoles Notice cellular clustering, nuclear
irregularities, and rosette formation ○ ratio of CSF albumin & serum albumin

C) CHEMICAL EXAMINATION 𝐶𝑆𝐹/𝑆𝑒𝑟𝑢𝑚 𝐴𝑙𝑏𝑢𝑚𝑖𝑛 𝑖𝑛𝑑𝑒𝑥

𝐴𝑙𝑏𝑢𝑚𝑖𝑛 𝐶𝑆𝐹 (𝑚𝑔/𝑑𝐿)
Albumin = 𝐴𝑙𝑏𝑢𝑚𝑖𝑛 𝑆𝑒𝑟𝑢𝑚 (𝑔/𝑑𝐿)
C1. Protein
● > 80% of CSF protein CSF/Serum Albumin Index
○ via capillary endothelium Normal ● <9
■ choroid plexus and meninges
Minimal Impairment ● 9-14
● remaining CSF protein
Moderate to Severe
○ comes from intrathecal synthesis ● 15-100
Impairment
● ONLY low-molecular-weight proteins are present in
CSF Complete Breakdown ● >100
○ haptoglobin ● large-molecular-weight protein normally
○ ceruloplasmin present in CSF
○ transferrin ● normal value = approx. 1 mg/dL
○ trace amounts of IgG ● ⬆ IgG may be due to:
■ high MW CHON ○ (1) ⬆ production within the CNS
○ trace amounts of IgA Immuno- ○ (2) ⬆ transport from the blood plasma
Tau Protein ● unique to CSF globulin G ● IgG index
Albumin ● major CSF protein (IgG) 𝐶𝑆𝐹 𝐼𝑔𝐺 𝑖𝑛𝑑𝑒𝑥
𝐼𝑔𝐺 𝐶𝑆𝐹 (𝑚𝑔/𝑑𝐿) 𝐴𝑙𝑏𝑢𝑚𝑖𝑛 𝑆𝑒𝑟𝑢𝑚 (𝑔/𝑑𝐿)
Transthyretin ● prealbumin = 𝐼𝑔𝐺 𝑆𝑒𝑟𝑢𝑚 (𝑔/𝑑𝐿)
× 𝐴𝑙𝑏𝑢𝑚𝑖𝑛 𝑆𝑒𝑟𝑢𝑚 (𝑚𝑔/𝑑𝐿)

Protein Tests For CSF CSF IgG Index

● total amount of protein in the CSF Multiple Sclerosis (MS) ● >0.7
○ varies with the age and site of collection
● tumor marker for Neuro-Bechet’s
○ assess integrity of the BBB
Syndrome
● normal value = 15–45 mg/dL B2 Micro-
Total ● >1.8mg/L
● lumbar region globulin
Protein ○ leptomeningeal leukemia
○ has a generally higher protein content than
○ lymphoma
other sites
C-Reactive ● acute phase reactant protein that is used to
Low ● Increased intracranial pressure
Protein differentiate bacterial from viral
Levels ● CSF leakage due to trauma
(CRP) meningitis especially in children

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 ● large glycoprotein involved in:
○ (1) cellular adhesion
○ (2) phagocytosis
● ⬆fibrinolectin
Fibrino-
○ lymphoblastic leukemia
lectin
○ Burkitt’s lymphoma
○ some metastatic tumor
○ astrocytomas
○ bacterial meningitis
Myelin
● ⬆after a recent destruction of myelin sheath
Basic
○ protect the axons of the neurons
Protein
○ monitor the course of multiple sclerosis
(MBP)

Other Tests For Protein

● measures the change in color
● NH3 sulfate
Nonne-Apelt
● (+) cloudy
● NH3 sulfate
Rose Jones
● (+) white ring
● phenol
Pandy’s C2. Glucose
● (+) bluish-white
Quali ● CSF glucose concentration is maintained via:
● 10% Butyric acid
Nogochi ○ active transport by endothelial cells
● (+) cloudy
○ simple diffusion along a concentration gradient bet.
● +1: blue color the blood plasma and CSF
● +2: purple color ● CSF glucose reflects the plasma glucose
Colloidal Gold
● +3: deeper blue color concentration 30-90 mins preceding collection of
Test
● +4: pale blue color the fluid
● +5: colorless ● fasting plasma glucose
○ collected to accurately interpret CSF glucose values
● precipitation of proteins ○ must be drawn 0-60 mins before lumbar puncture
1. Trichloroacetic acid (TCA) ● normal value = 50-80 mg/dL
Turbidimetric ○ detect albumin & globulin ○ approx. 60-70% of plasma glucose
2. Sulfosalicylic acid (SSA ● ⬆ CSF glucose
○ detect albumin only ○ hyperglycemia
● coomassie blue ○ traumatic puncture
○ most sensitive ● ⬇ CSF glucose
Dye Binding
● mechanism: protein error of ○ hypoglycorrhachia (< 40 mg/dL), assoc. with:
Technique
indicator ■ hypoglycemia
Quanti
○ for smaller samples ■ bacterial meningitis
■ tumors
Nephelometry ● similar to chem

Electro- ● detect
phoresis ○ multiple sclerosis (MS)
(Agarose Gel) ○ multiple myeloma (MM)
● CSF albumin /Serum albumin
Protein
Index
Fractions
● CSF IgG Index

Transcribed by: Lustre, Aliping, Guarin, Villanueva (2024). 10

C3. Lactate C4. Glutamine
● unrelated to its plasma concentration ● used as an indirect measure of CSF ammonia
● normal value = 10-22 mg/dL ● ⬆ glutamine
● ⬆ CSF lactate ○ liver disorder
○ anaerobic metabolism within the CNS, due to: ○ Reye’s syndrome
■ hypoxia C5. Enzymes
■ hydrocephalus
■ cerebral infarction
■ cerebral edema
■ cerebral arteriosclerosis CSF Enzyme
■ meningitis
■ intracranial hemorrhage
● useful in differentiating meningitis caused by bacterial,
LD Isoenzymes
fungal or viral agents
Lactate LD1 & LD2 ● brain tissue destruction
dehydro– LD2 & LD3 ● viral meningitis
Viral
● 25-30 mg/dL genase (LD) LD4 & LD5 ● bacterial meningitis
Meningitis
Fungal
● > 35 mg/dL
Meningitis
Bacterial ● > 35 mg/dL CSF lactate Creatine ● < 17 mg/dL
Meningitis ● low CSF glucose (diagnostic indicator) Kinase ○ can predict recovery from cardiac arrest
(CK) BB after resuscitation

Major Laboratory Results for the Differential Diagnosis of Meningitis

Bacterial Meningitis Viral Meningitis Tubercular Meningitis Fungal Meningitis

WBC Count ⬆ ⬆ ⬆ ⬆
lymphocyte lymphocyte
Differential Count neutrophil lymphocyte
monocyte monocyte
Protein marked ⬆ moderate ⬆ moderate to marked ⬆ moderate to marked ⬆
Glucose marked ⬇ normal ⬇ ⬇ to normal
Lactate > 35 mg/dl normal > 25 mg/dL > 25 mg/dL
● (+) immunologic test
● (+) gram stain
Other Tests ● (+) pellicle formation ● C. neoformans
● (+) bacterial Ag test
○ (+) india test for

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 D) MICROBIOLOGICAL EXAMINATION ● identification of gram (-) organism that
● key role in the diagnosis of and treatment of Limulus Lysate causes meningitis
meningitis Assay ● rapid test for newborns
● sample must be processed ASAP ● uses Horseshoe Crab blood as reagent
● centrifuged at 1500 xg for 15 mins ● MUST procedure before reporting
India Ink
○ smear & culture preparation lymphocytic pleocytosis
preparation
● detect Cryptococcus neoformans
Most Common Causes of Bacterial Meningitis ● Latex agglutination & ELISA
Affected ○ Haemophilus influenzae
Causative Agent Morphology ○ Streptococcus spp.
Population
Immunology ○ Escherichia coli K1ag
Haemophilus ● pleomorphic ● 1 month to 5 Tests ● Reverse Latex agglutination
influenzae gram (-) rods years old ○ C. neoformans
Neisseria ■ more sensitive than BAT
● gram (-) cocci ● 5 to 29 years old ○ RF→ false increase
meningitidis
Streptococcus ● 29 years old & Other Tests for Microbiologic Examination
● gram (+) cocci
pneumoniae older ● tests for tertiary syphilis or neurosyphilis
Escherichia coli ● gram (-) rods ● newborns
● Venereal Disease Research Laboratory test
Streptococcus VDRL ● acute syphilis; CSF by CDC
● gram (+) cocci ● newborns
agalactiae ● method of choice
Listeria ● Fluorescent Treponemal Ab Absorption Test
● gram (+) rods ● newborns
monocytogenes FTA-ABS ● avoid contamination with blood
○ mostly used for neurosyphilis
Microbiologic Examination ● Rapid Plasma Reagin Test
● 60% to 90% sensitive RPR ● screening test
● centri for 15 min first ● ⬇sensitivity & specificity
Gram Stain
● cryptococcus
○ ‘starburst pattern’
Acid Fast ● for tubular meningitis
Blood Culture ● 80% to 90% sensitive
Rapid Bacterial
● NOT sensitive to Neisseria
Antigen Test/
● combined with hema & microbiology
BAT
Fig. India ink preparation of C. Fig. Gram stain of C. neoformans
neoformans (×400). Notice budding showing starburst pattern
yeast form.

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 MULTIPLE SCLEROSIS
Multiple Sclerosis (MS)
● immune mediated inflammatory demyelinating disease
of the CNS
● degradation of myelin that surrounds the axons of
nerves
○ myelin
■ 70% lipid, 30% myelin basic protein
■ detected by immunoassays
■ necessary for proper nerve conduction
● clinical features:
○ muscle weakness
○ optic neuritis
○ Paresthesia

Oligoclonal Bands
● gamma region of CSF protein electrophoresis
associated with MS
● diagnostic indicator of MS, if:
○ (+) CSF oligoclonal bands
○ (-) serum oligoclonal bands

● NOTE:
○ presence or intensity does not correlate with the
particular stage of disease
○ NOT used to predict disease progression
● oligoclonal bands can be associated with myelin basic
protein detection

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Transcribed by: Lustre, Aliping, Guarin, Villanueva (2024). 14
ANALYSIS OF URINE MLS 321L

& OTHER BODILY FLUIDS 8438L

LABORATORY BS MLS 3 FINALS
M7U2: SEMINAL FLUID ANALYSIS

UNIT 2: SEMINAL FLUID ANALYSIS Semen Production

● during ejaculation, semen is produced from a
MALE REPRODUCTIVE SYSTEM concentrated suspension of spermatozoa (about 5 %)
mixed with, and diluted by fluid secretions from the
Reproductive System accessory sex organs
Primary
● produce gametes (e.g. sperm cells) ORGANS NECESSARY FOR
Organs
● organs and structures that are SPERMATOGENESIS → MATURATION
Secondary
necessary for reproduction to occur
Organs
through semen formation Sertoli Cells (Seminiferous Tubules)
● spermatozoa production is regulated by the Sertoli
Male Reproductive System cells in the seminiferous tubules of the testes
● (1) testes ● play a role in supplying substances for normal
Male Primary
○ sex organs that contribute to seminal spermatogenesis, such as:
Organs
fluid formation ○ nutrients
Male ○ hormones
● (1) prostate gland
Secondary ○ other necessary substances
● (2) seminal vesicles
Organs
● (3) bulbourethral glands ● when Sertoli cells release sperm into the lumen of the
(Accessory Sex
● (4) epididymis seminiferous tubules, they are nonmotile and still
Organs)
immature

Fig. A schematic diagram of spermatogenesis from germ cells in the

seminiferous tubules.

Epididymis
● where sperms are stored and concentrated
● location where sperms mature and gain motility until
ejaculation
● the epididymis also adds carnitine and
acetylcarnitine to the lumen fluid
Fig. The male genitalia. Top, sagittal view; bottom, anterior view. ○ NOTE:
■ ⬆excess carnitine & acetylcarnitine may
contribute to infertility

Transcribed by: Abenes, Aliping, Guarin, Villanueva (2024). 1

 SEMINAL FLUID COMPOSITION ● (2) proteolytic enzymes
○ responsible for PFS liquefaction
Semen ● play a role in coagulation of the
Proteins
● highly complex transport medium for sperm ejaculate
● composition: ● added to semen by the prostate gland
○ seminal vesicle alkaline fluid (60-70%) (and also by the testes)
Zinc
○ prostatic fluid secretions (20-30%) ● decreased level is associated with
prostate gland disorders
○ bulbourethral gland fluid (5%)

Bulbourethral Glands
Seminal Vesicle Alkaline Fluid
● located below the prostate
● accounts for approx. 60-70% of the ejaculate
● contribute about 5% of the fluid volume
● have high concentrations of:
● in the form of a thick, alkaline mucus that helps to
○ fructose
neutralize acidity from the prostate secretions and
○ prostaglandins
the vagina (as a result of normal bacterial vaginal flora)
○ flavin
● without this neutralization, sperm motility would be
● alkaline fluid helps neutralize the acidic nature of the
diminished
male urethra and female vaginal canal
● responsible for the secretion of a pre-ejaculate
○ a clear fluid rich in mucoproteins
Seminal Vesicle Alkaline Fluid Composition
○ neutralizes acidity of urine remaining in the urethra
● serves as a nutrient for the sperm cells
● provides energy needed for the flagella
Fructose
to propel them through the female
reproductive tract
● imparts the characteristic gray or
opalescent to light yellow
appearance
● responsible for its green-white
fluorescence under UV light
Flavin
● various proteins (e.g., semenogelin)
○ secreted by the seminal vesicles play
a role in coagulation of the ejaculate
○ forms a gel-like protective layer
around the sperm
● function of prostaglandins remains
under investigation
Prostaglandins ● assumed to lower the female immune
response to the semen, hence
preventing possible sperm malfunction
Other FACTORS AFFECTING SEMEN QUALITY
Substances A) Sample Collected
● contributes to sperm motility
(phosphorous B) Activity of the Accessory Sex Glands
& potassium) C) Time since the Last Sexual Activity
D) Penultimate Abstinence Period
E) Size of the Testis
Prostatic Fluid Secretions F) Manner of Collection
● account for approx. 20-30% of the ejaculate volume ● seminal fluid analysis involves evaluating the nature of
● milky, slightly acidic fluid spermatozoa:
○ vitality
Prostatic Fluid Secretions Components ○ motility
Citric Acid ● acidifies the fluid ○ morphology
● play a role in coagulation of the ○ total number
ejaculate ○ total fluid volume
Enzymes ● (1) acid phosphatase
○ ⬆ high levels of acid phosphatase
can be used to identify semen

Transcribed by: Abenes, Aliping, Guarin, Villanueva (2024). 2

● under given conditions, the results of laboratory of spermatozoa in the ejaculate
measurements depend on different factors affecting ● size of the testis influences the total
semen quality number of spermatozoa per ejaculate
Size of the
● also reflects the level of sperm activity
Testis
Factors Affecting Semen Quality which also affects sperm morphology
● size of the testis = testicular function
● ejaculates produced by masturbation and
Semen Released collected into containers in a room near the
● mainly prostatic secretions rich laboratory can be of lower quality than
in sperm cells those collected from non-spermicidal
● IF not released or collected condoms used during intercourse at
First Manner of
○ volume = ⬇ home
Portion Collection
○ concentration = falsely ⬇ ● difference in collection may be attributed to
○ pH = falsely ⬆ the difference in sexual arousal
○ coagulum = fails to liquefy ● time spent producing a sample by
● dominated by seminal vesicular masturbation also influences semen
fluid quality
Sample ● IF not released or collected
Collected Last
○ volume = ⬇ SEMINAL FLUID ANALYSIS
Portion
○ concentration = falsely ⬆
○ pH = falsely ⬇ A) Sample Collection
A.1) Preparation
○ coagulum = will not form A.2) Collection Proper
A.3) Safe Handling of Specimen
● NOTE: B) Physical Examination
○ losing the first portion of the ejaculate B.1) Liquefaction
B.2) Viscosity
has more influence on the results of B.3) Color & Appearance
semen analysis than losing the last B.4) Volume
portion B.5) Odor
○ pH & coagulum of semen is affected by C) Chemical Examination
C.1) Semen pH
the reduction in prostate secretions D) Microscopic Examination
● affects sperm concentration D.1) Initial Microscopic Examination
Activity of ● large volume of seminal fluid at ejaculation D.1a) Making a Wet Preparation
D.1b) Aggregation of Spermatozoa
the causes: D.1c) Agglutination of Spermatozoa
Accessory ○ enhance sperm motility D.1d) Cellular Elements other than spermatozoa
Sex Glands ○ dilution may occur D.2) Sperm Motility
■ underestimate sperm concentration D.3) Sperm Number
D.4) Sperm Morphology
● better semen quality is obtained when it is D.5) Sperm Vitality
collected: ● involves assessment of semen functions including
○ min of 2 days
○ sperm motility
○ max of 7 days
● specimens collected without a minimum ○ vitality
of two (2) days abstinence ○ concentration
Time since ○ affect sperm number & quality ○ morphology
the Last ● specimens collected following prolonged ● semen analysis is performed for:
Sexual abstinence ○ (a) evaluation of infertility & post-vasectomy cases
Activity ○ tend to have higher volumes & ⬇ for married men
motility
● absence of ejaculation ○ (b) selection of donors for therapeutic insemination,
○ spermatozoa accumulate in the in-vitro fertilization or embryo transfer
epididymis → overflow into the urethra ○ (c) exposure screening to reproductive toxicants
→ tendency to be flushed out in the ○ (d) cryopreservation/sperm banking
urine
● epididymis are NOT completely emptied by
Penultimate one ejaculation
Abstinence ● some spermatozoa remain from the time of
Period the previous ejaculation
● influences the range of age and quality

Transcribed by: Abenes, Aliping, Guarin, Villanueva (2024). 3

● specifically, determining the total number of A.2) Collection Proper
spermatozoa: ● ideal collection of sample should be obtained by
○ essential for assessing the capacity of the testes to masturbation
produce sperm cells ● ejaculated into a clean wide mouthed container
○ assessing the patency of the post-testicular or ○ made of glass/ plastic
ejaculatory duct system ○ confirmed to be non-toxic for spermatozoa
■ patency - the state of being unobstructed ● specimen container should be kept at an ambient
● evaluating the total fluid volume temperature bet. 20°C-37°C
○ provide a picture on the secretory activity of the ○ avoid large changes in temperature
different accessory glands ○ large changes in temp → cause cold shock to
● assessing fertility sperm → affect the spermatozoa after they are
○ 2 or 3 samples within a 3-month period and at ejaculated into it
least 7 days apart should be examined ● sample may be collected at home
● all aspects of semen collection and analysis must be ○ IF collection cannot be done in the laboratory
done accurately, following standardized procedures ○ px is given a pre-weighed container labeled with
■ (1) name
A) SAMPLE COLLECTION ■ (2) identification number
A.1) Preparation
○ px must be given clear instructions that the sample
A.2) Collection Proper should be transported to the laboratory within 1 hr
A.3) Safe Handling of Specimen of collection
○ time of semen production must be recorded
A.1) Preparation ● sample may be collected in a non-lubricated rubber
● px should be given clear written and spoken or polyurethane condom during sexual intercourse
instructions concerning the collection of the sample ○ IF the px cannot collect through masturbation
● emphasize that the semen sample needs to be ○ only special non-toxic condoms designed for semen
complete collection should be used
○ px should report any loss of any fraction of the ○ ordinary latex condoms
sample ■ NOT used
● sample should be collected in a private room near the ■ spermicidal content - contain agents that
laboratory interfere with the motility of spermatozoa
○ limit the exposure of the semen ● coitus interruptus
○ limit fluctuations in temperature ○ NOT a reliable means of semen collection
○ control the time between collection and analysis ○ first portion of the ejaculate may be lost
● sample should be collected after a min. of 2 days and ○ cellular and bacteriologic contamination of the
a max. of 7 days of sexual abstinence sample from the vagina
● IF additional samples are required: ○ low pH (acidic) of the vaginal fluid could adversely
○ no. of days of sexual abstinence should be as affect sperm motility
constant as possible at each visit ● incomplete sample
● there should be a report form that contains: ○ second sample should be collected again after an
○ (1) name of the patient abstinence period of 2-7 days
○ (2) date of birth
○ (3) period of abstinence A.3) Safe Handling of Specimen
○ (4) date and time of collection
● semen samples may contain infectious agents such
○ (5) completeness of the sample
as HIV, HBV and herpes simplex virus (HSV) should
○ (6) any difficulties in producing the sample
therefore be handled as biohazard
○ (7) time interval between collection and the start of
● if the sample is to be processed for bioassay, sterile
semen analysis
materials and techniques must be used:
○ intrauterine insemination
○ in-vitro fertilization
○ intracytoplasmic sperm injection

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 B) PHYSICAL EXAMINATION Liquefaction Treatments
B.1) Liquefaction Dulbecco’s ● addition of an equal volume of
B.2) Viscosity phosphate Dulbecco’s phosphate-buffered saline
B.3) Color & Appearance -buffered saline ● followed by repeated pipetting
B.4) Volume
B.5) Odor ● inhomogeneity can be reduced by
Reducing repeated (6-10 times) gentle passage
● semen analysis should begin within 30-60 mins right inhomogeneity through a blunt gauge 18 or gauge 19
needle attached to a syringe
after liquefaction to prevent dehydration or changes
Digestion by ● a proteolytic enzyme
in temperature from affecting semen quality.
bromelain ● may help to promote liquefaction

B.1) Liquefaction

Fig. Normal macroscopic liquefaction characteristics.

Procedure: Evaluating Liquefaction
● liquefaction can be recognized both macroscopically ● start evaluating the liquefaction @ room temp 15 min
and microscopically after ejaculation
● IF immobilized spermatozoa are observed on ● sample should appear homogeneous and watery with
microscopic examination only small areas of coagulation
○ more time must be allowed for liquefaction process 1
to be completed NOTE:
● continuous gentle mixing or rotation of the
● occasionally, samples may not liquefy completely
sample container in a shaker can help produce a
after 60 minutes, making semen evaluation difficult homogeneous sample
● failure of liquefaction
○ caused by a deficiency in prostatic enzymes ● if liquefaction is not complete, check again after an
○ should be reported 2
additional 15 mins
○ failed liquefaction can be treated by: ● if liquefaction is still not complete, check liquefaction
3
■ additional treatment after an additional 30 minutes
■ mechanical mixing ● Report the time of complete liquefaction in minutes
■ enzymatic digestion
○ NOTE: NOTE:
■ these treatments may affect sperm motility 4 ● semen samples collected at home or by condom will
and morphology; hence, their use must be normally have liquefied by the time they arrive in the
laboratory
recorded

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B.2) Viscosity Results and Interpretation: Viscosity
● refers to the consistency of the fluid and may not (Alternative Reporting)
totally related to specimen liquefaction Normal ● normal, watery specimen
● incompletely liquefied specimen
Slightly Viscous ● thick
○ does NOT necessarily mean it has an abnormal
Moderately Viscous -
viscosity
● ⬆ viscosity may be caused by: ● unable to be aspirated into the
Extremely Viscous
○ high mucus content pipette
○ (+) antisperm Abs
○ oligoasthenospermia B.3) Color & Appearance
■ ⬇ concentration and motility of sperm ● normal color:
○ homogeneous
Procedure: Evaluating Viscosity ○ grey-opalescent appearance
● after liquefaction, the viscosity of the sample can be ○ opacity varies
1 assessed by gently aspirating semen into a wide bore
■ ⬇sperm concentration = ⬇opacity
(approx. 1.5 mm diameter) plastic disposable pipette
■ ⬆sperm concentration = ⬆opacity
● allowing the semen to drop by gravity and observing
2 ● ⬆WBCs = more turbid
the length of any thread

Abnormal Sperm Color

Results and Interpretation: Viscosity
● presence of RBC
Normal ● leaves the pipette in small discrete drops Red-Brown ● indicates hemospermia
Abnormal ● drop will form a thread >2 cm long ● due to the drug pyridium
● can be due to either
● alternative method in assessing viscosity ○ (1) accumulation of flavin
Yellowish ■ due to prolonged abstinence
○ can be evaluated by introducing a glass rod into
Coloration ○ (2) contamination with first morning
the sample → observing the length of the thread urine that is considered toxic to
that forms upon withdrawal of the rod sperm cells
○ methods to reduce viscosity are the same as those ● observed in man with jaundice
for delayed liquefaction Yellow ○ e.g., in cases of Hepatitis
● taking multivitamins
Unliquefied Semen (High Viscosity)
● exhibits homogeneous stickiness B.4) Volume
● its consistency will not change with time ● volume of the ejaculate must be precisely measured
● recognized by the elastic properties of the sample ● semen volume allows assessment of:
● adheres strongly to itself when attempts are made to ○ (1) activity of the accessory sex glands
pipette it ○ (2) calculation of the total number of spermatozoa
● interfere with determination of:
● best measuring method for volume
○ sperm motility
○ sperm concentration ○ weighing the sample in the container in which it was
○ detection of antibody-coated spermatozoa collected
○ measurement of biochemical markers
Procedure: Evaluating Semen Volume
● NOTE: ● collect the sample in a pre-weighed, clean, sterile,
1
disposable plastic or glass container
○ grading viscosity varies among laboratories
2 ● weigh the container with semen in it
Results and Interpretation: Viscosity (Numeric)
3 ● subtract the weight of the container
‘0’ ● normal, watery specimen
● calculate the volume from the sample weight
‘4’ ● gel-like consistency
𝑣𝑜𝑙𝑢𝑚𝑒 (𝑚𝐿) = 𝑤𝑒𝑖𝑔ℎ𝑡 (𝑚𝐿) ÷ 𝑑𝑒𝑛𝑠𝑖𝑡𝑦 (𝑔/𝑚𝐿)
4
● assume, semen density = 1g/mL
○ actual density range = 1.043-1.102 g/mL

Transcribed by: Abenes, Aliping, Guarin, Villanueva (2024). 6

● alternative method Results and Interpretation: Semen Volume
○ volume can be measured directly by collecting the Normal ● 7.2 - 8.0
sample into a clean graduated centrifuge
● < 7.0
tube/measuring cylinder → read the volume from
● with low volume and low sperm numbers
the graduations
● assoc. accessory gland:
Low pH ○ ejaculatory duct obstruction
Results and Interpretation: Semen Volume ○ congenital bilateral absence of the vas
Normal ● 1.5 - 5 mL deferens
● < 1.5 mL ○ increase prostatic secretion
● may indicate: ● > 8.0
High pH
○ obstruction of the ejaculatory duct ● infection within the reproductive tract
○ congenital bilateral absence of the vas
Hypospermia deferens D) MICROSCOPIC EXAMINATION
○ poorly developed seminal vesicles D.1) Initial Microscopic Examination
○ loss during collection D.1a) Making a Wet Preparation
D.1b) Aggregation of Spermatozoa
○ partial retrograded ejaculation D.1c) Agglutination of Spermatozoa
○ androgen deficiency (hypogonadism) D.1d) Cellular Elements other than spermatozoa
D.2) Sperm Motility
● > 5 mL D.3) Sperm Number
● may reflect active inflammation of the D.4) Sperm Morphology
D.5) Sperm Vitality
Hyperspermia accessory glands
● can also be a case of prolonged ● phase contrast microscope
abstinence ○ recommended for all examinations of unstained
preparations of fresh semen
B.5) Odor ○ total magnification of x400 (HPO)
● stained preparation
Normal Odor ● musty, likened to a bleach odor ○ magnified at x1000 (OIO)
● infections modify normal semen odor ● involves evaluation of:
Infections
● change in odor is rarely reported ○ sperm motility
Sperm ○ vitality
● produces the distinct odor
Oxidation ○ concentration
○ morphology
C) CHEMICAL EXAMINATION
D.1) Initial Microscopic Examination
C.1) Semen pH ● done before evaluating sperm motility, morphology,
● pH should be measured after liquefaction number and vitality
● preferably after 30-60 mins ● performed at a total magnification of x100 (LPO) to:
● any delay may ⬆pH ○ (1) provide an overview of the sample (especially
○ due to loss of CO2 that occurs after production the number of spermatozoa)
● pH of semen reflects the balance bet. the pH values of ○ (2) assess the presence of:
the different accessory gland secretions, mainly the ■ mucus strands
alkaline seminal vesicular secretion and acidic prostatic ■ sperm agglutination or aggregation
secretion ○ (3) determine cells other than spermatozoa
■ epithelial cells
Procedure: Evaluating Semen pH
■ round cells
1 ● mix the sample well
■ leukocytes
2 ● spread a drop of semen evenly onto the pH paper ■ immature germ cells
● wait for the color of the impregnated zone to become ● (4) allow the microscopist to determine the
3
uniform (<30 sec)
appropriate dilution and chambers to use when
● compare the color with the calibration strip to read the
4 performing sperm number determination
pH

Transcribed by: Abenes, Aliping, Guarin, Villanueva (2024). 7

D.1a) Making A Wet Preparation D.1b) Aggregation of Spermatozoa
● described as the adherence of:
Procedure: Making A Wet Preparation ○ either immotile spermatozoon to each other;
● mix the semen sample well ○ or motile spermatozoa to mucus strands,
○ allow uniform distribution of the semen components non-sperm cells or debris
especially the spermatozoa ● must be strictly observed and recorded
● well mixed can be achieved by aspirating the sample
10 times using a disposable plastic pipette D.1c) Agglutination of Spermatozoa
1
● specifically refers to motile spermatozoa sticking to
NOTE:
● DO NOT mix vigorously (and in a vortex mixer at each other
high speed) as it will create bubbles and damage the ○ head-to-head
spermatozoa ○ tail-to-tail
○ in a mixed way
● remove an aliquot of semen (10 ul) immediately ● severe agglutination
2 after mixing allowing no time for the spermatozoa to ○ affect the assessment of sperm motility and
settle out of suspension concentration
● transfer the aliquot onto a clean slide and cover it ○ hence, the major type of agglutination, reflecting
with a coverslip (22 mm x 22 mm) the degree and site of attachment, should be
● weight of the coverslip will spread the sample
recorded
● NOTE:
Depth
20 um ● allows the spermatozoa to swim ○ presence of agglutination
freely ■ NOT sufficient evidence to deduce an
< 20 um ● constrains the rotational movement of immunological cause of infertility
spermatozoa ■ suggestive of the presence of anti- sperm
> 20 um ● make it difficult to assess antibodies → further testing is required
(too deep) spermatozoa as they move in and out
of focus Results and Interpretation: Agglutination
3
Grading
NOTE: (degree of Description
● 20-um depth is obtained by dividing the volume of attachment)
sample used by the area which it was spread Grade 1: ● < 10 spermatozoa per agglutinate
● volume of 10 ul semen delivered onto a clean slide Isolated ● many free spermatozoa
and covered with a 22 mm x 22 mm cover slip
provides a chamber depth of 20.7 um Grade 2: ● 10-50 spermatozoa per agglutinate
Moderate ● moderate free spermatozoa
Grade 3: ● > 50 spermatozoa
Large ● few free spermatozoa
Grade 4: ● ALL spermatozoa agglutinated and
Gross agglutinates interconnected
● assess the freshly made wet preparation as soon
as the contents are no longer drifting

NOTE:
● IF the number of spermatozoa per field varies
considerably, the sample is NOT homogeneous
4 ○ semen sample should be mixed again thoroughly
and new slide is prepared
● lack of homogeneity may also result from:
○ abnormal consistency
○ abnormal liquefaction and aggregation
○ agglutination of spermatozoa.

● remix the semen sample before removing replicate

5
aliquots Fig. Sperm Agglutination Grade

Transcribed by: Abenes, Aliping, Guarin, Villanueva (2024). 8

D.1d) Cellular Elements Other than Spermatozoa ● standardized reporting of sperm motility
○ computer-aided sperm analysis (CASA)
Other Cells Seen in Semen Analysis ■ yield reliable assessments
Ciliary Tufts ● seen with efferent duct damage ● sperm motility should be assessed ASAP
Epithelial Cells ● from the GIT ○ after liquefaction of the sample
● immature germ cells ○ preferably at 30 minutes, but in any case, within 1
○ commonly observed when there is hour, following ejaculation, to limit the deleterious
Round Cells testicular damage due to viral effects of:
(Leukocytes & infections and exposure to toxic ■ dehydration
Immature Germ chemicals ■ pH
Cells) ● leukocytes ■ changes in temperature
○ when there is inflammation of the ● procedure may be performed at room temperature or
accessory glands
at 37°C with a heated microscope state (standardized
for each laboratory)
Calculation of the Concentration of Round cells in ● @ 37 °C
Semen ○ sample should be incubated at 37°C and the
● concentration of round cells is calculated relative to that of preparations made with prewarmed slides and
spermatozoa by assessing fixed and stained semen coverslips
smears made from undiluted semen
● if N is the number of round cells counted in the same Procedure: Evaluating Sperm Motility
number of fields as 400 spermatozoa, and S is the
concentration of spermatozoa (106 per ml), then the ● mix semen well
6
concentration of round cells (10 per ml) can be calculated
from the formula: NOTE:
● improperly mixed semen
1
𝑁 ○ separate aliquots may show marked differences
𝐶 = 𝑆 𝑥 𝑇𝑜𝑡𝑎𝑙 𝑁𝑜. 𝑜𝑓 𝑆𝑝𝑒𝑟𝑚𝑎𝑡𝑜𝑧𝑜𝑎
in sperm motility (even in vitality, concentration
● 𝑆 = 𝐶𝑜𝑛𝑐𝑒𝑛𝑡𝑟𝑎𝑡𝑖𝑜𝑛 𝑜𝑓 𝑠𝑝𝑒𝑟𝑚𝑎𝑡𝑜𝑧𝑜𝑎 and morphology)
● 𝑁 = 𝑁𝑢𝑚𝑏𝑒𝑟 𝑜𝑓 𝑅𝑜𝑢𝑛𝑑 𝐶𝑒𝑙𝑙𝑠

● example: ● remove an aliquot (10 ul) of semen immediately using

2
○ sperm concentration (S) = 23 x 106 per ml an automatic pipet & transfer to a slide (Replicate 1)
○ number of round cells (N) = 600 ● remix the semen and remove another replicate aliquot
3
(10 ul) & transfer to another slide (Replicate 2)
6 600
𝐶 = (23 𝑥 10 ) × 400
6
4 ● wait for the sample to stop drifting for 30-60s
𝐶 = (23 𝑥 10 ) × 1. 5
6
𝐶 = 34. 𝑥 10 𝑝𝑒𝑟 𝑚𝐿 5 ● scan under LPO → shift to HPO for examination

D.2) Sperm Motility

Procedure: Scanning for Sperm Motility
● motility
● evaluate atleast 200 spermatozoa in a total of AT
○ one of the most important characteristics of sperm
1 LEAST 5 FIELDS IN EACH REPLICATE
● immotile sperms, even in high concentrations, are ○ to achieve an acceptably low sampling error
unable to reach an ovum ● look for spermatozoa in an area at least 5 mm from
● sperm capable of forward, progressive movement the edge of the coverslip
○ critical for fertility 2
○ to prevent observation of immotile sperm cells due
○ once presented to the cervix → sperm must propel to drying
themselves through the cervical mucosa → uterus ● systematically scan the slide to avoid repeatedly
3
→ fallopian tubes → ovum viewing the same area
● traditional reporting of sperm motility ● assess the motility of ALL spermatozoa within a
○ a subjective evaluation performed by examining an defined area of the field
undiluted specimen ● eyepiece reticle
4
○ sample is most easily achieved when used
○ determining the: ○ an eyepiece with grid limits the area viewed
■ (1) percentage of motile sperm ○ allows the same area of the slide to be assessed
■ (2) quality of the motility

Transcribed by: Abenes, Aliping, Guarin, Villanueva (2024). 9

 during both stages of scoring ● calculate the average percentages and differences
9 between the 2 replicates to the nearest WHOLE
NUMBER
● determine the acceptability of the difference basing on
the percent average and difference of the MOST
10
FREQUENT MOTILITY GRADE

● IF the difference between the percentages is

acceptable
○ report the average percentage of each motility
grade (progressive, non-progressive & immotile)
● IF the difference is too high
○ take new aliquots from the semen sample and
11 repeat the assessment
Fig. Eyepiece reticle / eyepiece micrometer
NOTE:
● scan and count QUICKLY ● difference is acceptable if it is within or below the
○ to avoid overestimating the number of motile acceptable difference set by WHO.
spermatozoa
● when using an eyepiece reticle ● report the average percentage for each motility grade
5
○ the goal is to count all MOTILE spermatozoa in to the nearest WHOLE NUMBER
the grid section instantly
○ avoid counting both those present initially plus NOTE:
those that swim into the grid section during scoring ● (a) report if AGGREGATION AND
● start counting a given field at a RANDOM INSTANT AGGLUTINATION are observed
● do NOT wait for spermatozoa to swim into the field ● (b) report other cells observed aside from sperm
● avoid choosing fields on the basis of the number of 12
cells:
motile sperm cells seen ○ epithelial cells
6 ○ leukocytes
NOTE: ○ immature germ cells
● assess only intact spermatozoa (head+tail) ● results for replicate aliquots should AGREE
● DO NOT COUNT MOTILE PINHEADS before the values are accepted

● counting order:
○ (1) progressive cells Categories of Sperm Movement
○ (2) non-progressive
● spermatozoa moving actively
○ (3) immotile cells Progressive
● linearly or in a large circle, regardless
● to avoid overestimating sperm motility, the order of Motility (PR)
of speed
analysis can be reversed:
○ (1) immotile cells ● all other patterns of motility with an
○ (2) non-progressive absence of progression
○ (3) progressive ● e.g.
● with experience, it may be possible to score all three Non-progressi ○ swimming in small circles
7 categories of sperm movement at one time. ve Motility (NP) ○ flagellar force hardly displacing the
head
○ when only a flagellar beat can be
NOTE:
observed
● IF a count of 200 spermatozoa is achieved before all
motility categories have been scored, counting must Immotile (IM) ● no movement
continue beyond 200 spermatozoa until ALL
categories have been counted Sperm Motility Reference Values
○ to avoid bias towards the motility category scored Lower Reference limit for
first ● 40%
TOTAL MOTILITY (PR & NP)

● tally the number of spermatozoa in each motility Lower reference for PR ● 32%
8 category and calculate the percentages of the
Lower reference for NP ● 8%
different motilities to the nearest WHOLE NUMBER

Transcribed by: Abenes, Aliping, Guarin, Villanueva (2024). 10

 Acceptable Differences bet. 2 percentages D.3) Sperm Number
Acceptable Acceptable
Average % Average %
Difference Difference ● no. of spermatozoa per unit volume
0 1 66-76 9 of semen (eg., per mL)
1 2 77-83 8 ● a function of the number of spermatozoa
emitted and the volume of fluid diluting
2 3 84-88 7 them
3-4 4 89-92 6 Sperm ● related to fertilization and pregnancy
5-7 5 93-95 5 Concentration rates
8-11 6 96-97 4 ● influenced by the volume of the
12-16 7 98 3 secretions from the seminal vesicles and
prostate
17-23 8 99 2 ● not a specific measure of testicular
24-34 9 100 1 function
35-65 10 ● total number of spermatozoa in the
*Acceptable differences between two percentages for a given average entire ejaculate
determined from replicate counts of 200 spermatozoa (total 400 counted) Total Sperm
Number ● obtained by:
○ 𝑠𝑝𝑒𝑟𝑚 𝑐𝑜𝑛𝑐𝑒𝑛𝑡𝑟𝑎𝑡𝑖𝑜𝑛 × 𝑠𝑝𝑒𝑟𝑚 𝑣𝑜𝑙𝑢𝑚𝑒

● for normal ejaculates, when the male tract is

unobstructed and the abstinence time is short
○ total number of spermatozoa in the ejaculate is
correlated with testicular volume and thus is a
measure of the capability of the testes to
produce spermatozoa and the patency of the
male tract
● total number of spermatozoa per ejaculate and the
sperm concentration (per ml) are related to both time of
pregnancy and are predictors of conception
● materials needed in sperm number evaluation
○ 12 x 75 mm test tubes
○ transfer pipets (positive displacement)
○ WBC pipettes (if necessary)
○ hemocytometer with thick coverslip
○ tally counter
○ fixative
■ sodium bicarbonate in 35 % formalin

Procedure: Evaluating Sperm Number

● prepare a wet preparation
1 ● estimate the average number of spermatozoa
seen per HPO in at least 5 fields
● determine the appropriate dilution to be use
Dilution of Sample
Average Dilution Semen Vol Fixative Vol Grids to be
Sperm per required (ul) (uL) Assessed
HPF
>101 1:20 950 ul 5, 4, 6
2 16-100 1:5 200 ul 5, 4, 6
50 ul
2-15 1:2 50 ul 5, 4, 6
<2 1:2 50 ul All

● IF there are too few spermatozoa per field of view at

the recommended dilution, prepare another lower
dilution.

Transcribed by: Abenes, Aliping, Guarin, Villanueva (2024). 11

 ● IF there are too many overlapping spermatozoa NOTE:
per field of view at the recommended dilution, prepare ● improved Neubauer has two separate counting
a higher dilution (e.g., 1:50) chambers, each of which has a microscopic 3 mm x 3
mm pattern of gridlines
3 ● prepare 2 replicates using the recommended fixative ● each grid (all 9 grids) has a dimension of 1 mm x 1 mm
● use positive displacement pipette to dispense the with a depth of 0.1 mm
4 appropriate amount of fixative onto a properly labeled ● grid 5
12 x 75 mm test tube ○ dimension: 0.2 mm x 0.2 mm
○ depth: 0.1 mm
5 ● mix the semen well
● aspirate 50 ul of semen immediately after mixing,
6 allowing no time for the spermatozoa to settle out of
suspension
● wipe the semen off the outside of the pipet tip, taking
7 care not to touch the opening of the tip as this may
aspirate the content of the pipet tip
● dispense the semen into the fixative and rinse the
8
pipet tip by aspirating and expressing the fixative
● mix first dilution by vortex mixer for 10s (or by
9
vigorous mixing for 10s)
● immediately remove approx. 10 ul of fixed
10
suspension, to avoid settling of spermatozoa
● touch the pipette tip carefully against the lower edge
of one of the chambers at the V- shaped groove

11 NOTE: ● count at least 200 spermatozoa in each replicate to

16
● the use of 100 um deep hemocytometer is achieve an acceptably low sampling error
recommended

● depress the plunger slowly, allowing the chamber to

fill by capillary action
12
● coverslip should NOT be moved during filling and
should not be overfilled or under- filled
● mix the second dilution and immediately remove a
second 10 ul aliquot
13
● load the second chamber of the hemocytometer
following the steps above
● examine the hemocytometer and start counting
14
using x400 (HPO) total magnification
● locate the central grid
● start counting on the first square

Fig. Sperm count guide

15
● the boundary of a square is indicated by the MIDDLE
line of the three, thus, all cells within the boundary
line are counted
● cells are counted if most of the sperm head lies
NOTE: between the two inner lines (the orientation of the tail is
● grid 5 is indicated by triple lines on all sides
unimportant)
● cells may be counted if most of the sperm head lies
within the lower or left middle line, which forms an
“L” shape.

Transcribed by: Abenes, Aliping, Guarin, Villanueva (2024). 12

 ○ A and C are counted ● make note of the number of ROWS assessed to
○ B and D are not reach at least 200 spermatozoa
● Cells ARE NOT COUNTED if it lies outside or on the 20 ● same number of rows will be counted from the
upper or right middle boundary line. other chamber of the hemocytometer, even if these
yields fewer than 200 spermatozoa
○ G and E are counted
○ H and F are not ● determine the acceptability difference (see table) by
21 calculating the sum and difference of the two
● count only whole spermatozoa (head + tail). counts
● Poisson’s Law
● IF the difference is acceptable
○ the difference of count in each square should NOT ○ calculate the concentration
be > 14 22 ● IF the difference is too high
○ IF difference of count in each square is > 14 ○ prepare 2 new dilutions
■ recharge and recount sperm cells ○ repeat replicate counts

● continue counting until at least 200 spermatozoa Acceptable Differences bet. 2 counts
17 have been observed and a complete row (of 5 Acceptable Acceptable
squares) has been examined Average % Average %
Difference Difference
● Counting must be done by COMPLETE ROWS; DO 144-156 24 329-346 36
NOT STOP IN THE MIDDLE OF A ROW
157-169 25 347-366 37
NOTE: 170-182 26 367-385 38
● the NUMBER OF GRIDS to be counted on the 183-196 27 386-406 39
hemocytometer will also depend on the dilution 197-211 28 407-426 40
performed 212-226 29 427-448 41
18 227-242 30 449-470 42
Dilution of Sample
Average Dilution Semen Vol Fixative Vol Grids to be
243-258 31 471-492 43
Sperm per required (ul) (uL) Assessed 259-274 32 493-515 44
HPF
275-292 33 516-538 45
>101 1:20 950 ul 5, 4, 6
16-100 1:5 200 ul 5, 4, 6 293-309 34 539-562 46
50 ul
2-15 1:2 50 ul 5, 4, 6 310-328 35 563-587 47
<2 1:2 50 ul All Acceptability difference obtained by calculating the sum and difference of
the two counts
● IF 200 spermatozoa are NOT observed in the five
rows of the central grid NOTE:
○ continue counting in grids 4 & 6 ● the difference is acceptable if it is within or below the
● IF 200 spermatozoa are NOT observed in grids 5, 4, acceptable difference set by WHO
6
○ DO NOT COUNT ON GRIDS 1, 2, 3, 7, 8, 9 ● calculate the total number of spermatozoa per
23
○ the volume of each row in these grids differ from ejaculate
that of the rows in grids 4, 5, and 6.
24 ● report values into TWO SIGNIFICANT FIGURES
● NOT UNLESS an average of less than 2
spermatozoa per HPF is observed and a 1:2
dilution is made, then ALL chambers may be
counted
19
NOTE:
● grids 1,2,3,7,8 and 9
○ 4 rows holding 25 nl per row
● grids 4, 5, and 6
○ 5 rows holding 20 nl per row

● when immediate counting is NOT possible

○ hemocytometer may be stored in a humid
chamber to prevent drying out
■ e.g., on water-saturated filter paper in a
covered Petri dish

Transcribed by: Abenes, Aliping, Guarin, Villanueva (2024). 13

 Calculation and Reporting of Sperm Concentration D.4) Sperm Morphology
● assessment of sperm morphology has helped to define
the appearance of spermatozoa that are potentially
𝐷𝐹(𝑑𝑖𝑙𝑢𝑡𝑖𝑜𝑛 𝑓𝑎𝑐𝑡𝑜𝑟) = 𝑡𝑜𝑡𝑎𝑙 𝑣𝑜𝑙𝑢𝑚𝑒 𝑜𝑓 𝑠𝑜𝑙𝑢𝑡𝑖𝑜𝑛 ÷ 𝑣𝑜𝑙𝑢𝑚𝑒 𝑜𝑓 𝑠𝑎𝑚𝑝𝑙𝑒
capable of fertilizing
● sperm cells that are morphologically incapable of
𝑉𝐶𝐹(𝑣𝑜𝑙𝑢𝑚𝑒 𝑐𝑜𝑟𝑟𝑒𝑐𝑡𝑖𝑜𝑛 𝑓𝑎𝑐𝑡𝑜𝑟 = 𝑣𝑜𝑙𝑢𝑚𝑒 𝑑𝑒𝑠𝑖𝑟𝑒𝑑 (1𝑛𝑙) ÷ 𝑣𝑜𝑙𝑢𝑚𝑒 𝑢𝑠𝑒𝑑 fertilization also results in infertility
● 𝑣𝑜𝑙𝑢𝑚𝑒 𝑢𝑠𝑒𝑑 = 𝑛𝑜. 𝑜𝑓 𝑠𝑞𝑢𝑎𝑟𝑒𝑑 𝑐𝑜𝑢𝑛𝑡𝑒𝑑 × 𝑣𝑜𝑙. 𝑜𝑓 𝑠𝑞𝑢𝑎𝑟𝑒
● 𝑣𝑜𝑙𝑢𝑚𝑒 𝑑𝑒𝑠𝑖𝑟𝑒𝑑 = 𝑓𝑖𝑥𝑒𝑑 𝑎𝑡 1 𝑛𝑙
● 𝑣𝑜𝑙𝑢𝑚𝑒 𝑜𝑓 𝑠𝑞𝑢𝑎𝑟𝑒 = 𝑙𝑒𝑛𝑔𝑡ℎ × 𝑤𝑖𝑑𝑡ℎ × 𝑑𝑒𝑝𝑡ℎ Morphological Abnormalities of the Spermatozoa

Head ● associated with poor ovum penetration

NOTE:
● (a) when the total number of rows will be included as a Neckpiece
denominator in the formula, the “number of squares
Midpiece ● affects motility of the sperm
used” would refer only to the number of squares in
just a single row (which is 5) Tail
● associated with abnormal spermatozoa
● (b) when the total number of rows will be eliminated as ● from a defective spermatogenic process
Excess
a denominator in the formula, the “number of squares ● characterized by large amounts of irregular
Residual
used” would refer to the total number of squares stained cytoplasm,
Cytoplasm
counted in both replicates ● ⅓ or more of the sperm head size
● often associated with defective midpieces

Normal Lower Reference Limit

Sperm Concentration ● ≥15x106/mL

Total Sperm Number ● ≥39x106/ejaculate

Low Sperm Number

● azoospermia
○ IF no spermatozoa are observed in the replicate wet
preparation
○ can only be used if no spermatozoa are found in the
sediment of a centrifuged sample
● cryptozoospermia
○ presence of spermatozoa after centrifugation

● IF the number of spermatozoa per HPF is <4 (approx. 1

x106)
○ it is sufficient for most clinical purposes to report the
sperm concentration as <2 x106/ml

Fig. Normal spermatozoon structure.

Transcribed by: Abenes, Aliping, Guarin, Villanueva (2024). 14

 Normal Spermatozoon Structure Procedure: Evaluating Sperm Morphology
● smooth, regularly contoured, oval-shaped A) Preparation of Semen Smears
● 5 um long 1 ● mix the semen well
● 3 um wide
● remove an aliquot of 5-10 ul immediately, allowing
no time for the spermatozoa to settle out of
● critical to ovum penetration
2 suspension
● located at the tip of the
● NOTE:
head
acrosomal ○ 2 replicates
● encompasses approx. half
cap
of the head (40-70%)
Head ● transfer onto the slide and smear
● covers two thirds of the
sperm nucleus ● do NOT let the droplet of semen remain on the end of
3
● ree of large vacuoles and the slide for more than a couple of seconds before
acrosomal smearing
only contains less than two
region
small vacuoles
post ● does NOT contain any Preparation of Semen Smears
acrosomal vacuole
● semen spreads along the back edge of the
region
angled slide and is pulled forward over the
slide to form the smear
● attaches the head to the tail and the ● do NOT use the slide to push the semen
Neckpiece
midpiece Feathering from behind
● thickest part of the tail Method ● maintain an angle of 45° and a speed of 1s
● surrounded by a mitochondrial sheath (Undiluted
○ produces the energy required by the Semen)
tail for motility
● slender, regular and about the same
length as the sperm head
Midpiece
● major axis is aligned with the major axis of
the head ● drop of the sperm suspension is spread over
● residual cytoplasm the surface of the slide by pushing the
○ may be present horizontal pipette
○ should NOT exceed 1/3 of the sperm Pipette
head size Method
● long, flagellar (Washed
● 45 um long Sample)
Tail ● uniform caliber along its length (may
looped back on itself)
● thinner than the midpiece
● label the slides with the patient’s name
4
○ use pencil lead if slide is frosted
● allow the slides to dry in air and stain using either:
○ Papanicolaou
○ Shorr
○ Wright Giemsa
5 ○ Diff-Quik stain
● however, air drying may cause:
○ changes in sperm dimensions
○ expansion of immature sperm heads
○ loss of osmotically sensitive cytoplasmic droplets

Procedure: Evaluating Sperm Morphology

A) Examination of a stained smear
1 ● examine the slide using oil immersion objective (OIO)
● assess ALL spermatozoa in each field, moving from
2
one microscopic field to another

Transcribed by: Abenes, Aliping, Guarin, Villanueva (2024). 15

 ● evaluate at least 200 spermatozoa in each replicate, Acceptable Differences bet. 2 percentages
3
in order to achieve an acceptably low sampling error Acceptable Acceptable
Average % Average %
● tally the number of normal and abnormal Difference Difference
4
spermatozoa 0 1 66-76 9
● indicate the percentages (%) of normal and 1 2 77-83 8
abnormal forms 2 3 84-88 7
5
○ including percentages (%) of each abnormal 3-4 4 89-92 6
forms
5-7 5 93-95 5
● calculate the percentage average and percentage
difference from both replicates (R1 and R2) 8-11 6 96-97 4
6 12-16 7 98 3
● report the average percentage to the nearest whole
number 17-23 8 99 2
● determine the acceptability of the difference from 24-34 9 100 1
7 basing on the percent average and difference of the 35-65 10
NORMAL FORMS *Acceptable differences between two percentages for a given average
● IF the difference between the percentages is determined from replicate counts of 200 spermatozoa (total 400 counted)
acceptable
○ report the average percentage normal morphology Classification of Abnormal Sperm Morphology
● IF the difference is too high ● human semen samples contain spermatozoa with different
○ repeat the assessment on both replicates kinds of malformations
● defective spermatogenesis and some epididymal
NOTE: pathologies are commonly associated with an increased
8 ● the difference is acceptable if it is within or below the percentage of spermatozoa with abnormal shapes
acceptable difference set by WHO. ● morphological defects are usually mixed

● (a) assess only intact spermatozoa. ● amorphous

○ do NOT count immature germ cells. ● large or small
● vacuolated
● (b) do NOT assess overlapping spermatozoa and Head ● tapered
● small or large
those lying with the head on edge Defects ● pyriform
acrosomal areas
● round
● double heads
● asymmetrical
● sharply bent
Staining insertion of the
Neck and ● abnormally thin
midpiece into the
acrosomal region ● pale blue Midpiece ● ANY combination of
head
Head post- acrosomal ● dark blue these
● thick or irregular
region
● short
Midpiece ● may show some red staining ● sharply angulated
Principal ● multiple
bends, of irregular
Tail ● blue or reddish Piece ● broken
width, coiled, or any
● located behind the head and around the Defects ● smooth hairpin
Excess combination of these
midpiece bends
Residual
● stained pink or red (Pap stain)
Cytoplasm Kruger’s Strict Criteria
● reddish-orange (Shorr stain)
● additional parameters in the evaluation of sperm
morphology include:
○ measurement of head, neck, and tail size
○ size of the acrosome
○ presence of vacuoles.
● performance of strict criteria evaluation requires the use of
a stage micrometer or morphometry
● at present, evaluation of sperm morphology using strict
criteria is NOT routinely performed in the clinical
laboratory but is recommended by the WHO

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 D.5) Sperm Vitality
● as estimated by assessing the membrane integrity of
the cells, may be determined routinely on all samples,
especially for samples with less than about 40%
progressively motile spermatozoa
● this test can provide a check on the motility
evaluation, since the percentage of dead cells should
not exceed the percentage of immotile spermatozoa
● it is clinically important to know whether immotile
spermatozoa are alive or dead
● decreased sperm viability
○ suspected when a specimen has a normal sperm
concentration with markedly decreased motility
● presence of a large proportion of vital but immotile
cells
Fig. Different morphological abnormalities of spermatozoa
○ indicative of structural defects in the flagellum
● epididymal pathology
○ high percentage of immotile and non-viable cells
Normal Values for Sperm Morphology
● sperm vitality should be assessed ASAP
Kruger’s Strict
● Normal forms ≥ 14% ○ after liquefaction of the semen sample
Criteria
○ preferably at 30 minutes, but in any case, within 1
Routine Criteria
● Normal forums ≥ 4% hour of ejaculation
(WHO)
○ to prevent deleterious effects of dehydration or of
changes in temperature on vitality.
● percentage of live spermatozoa is assessed by
identifying those with intact cell membrane, from dye
exclusion or by hypotonic swelling.
● normally, the percentage of viable cells should
exceed that of motile cells

Fig. Spermatozoon with double Fig. Spermatozoon with amorphous D.5a) Dye Exclusion Test
head, hematoxylin-eosin head, hematoxylin-eosin. ● based on the principle that damaged plasma
membranes, such as those found in dead cells, allow
entry of membrane-impermeant stains.
● uses Eosin- nigrosin
○ ⬆contrast between the background and the sperm
heads, which makes them easier to detect

Fig. Spermatozoon with double tail, Fig. Spermatozoon with bent neck
Procedure: Sperm Vitality
hematoxylin-eosin and spermatid, hematoxylin-eosin A) Dye Exclusion Test
1 ● mix the semen sample well
● Remove an aliquot of 50 ul semen and mix it with an
2 equal volume of eosin-nigrosin suspension
● Prepare 2 replicates
● for each suspension, make a smear on a glass slide
3
and allow it to dry in air
● examine each slide using the oil immersion objective
4
Fig. Immature spermatozoa, Fig. Nonviable spermatozoa (OIO)
hematoxylin-eosin demonstrated by the eosin- nigrosin
stain ● evaluate 200 spermatozoa in each replicate, in
5
order to achieve an acceptably low sampling error

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 ● tally the number of stained (dead) or unstained D.5b) Hypo-Osmotic Swelling Test
6
(vital) cells ● test presumes that spermatozoa with intact
● calculate the average and difference of the two membranes will swell within 5 minutes in a
7 hypo-osmotic medium
percentages of vital cells from the replicate slides
● all flagellar shapes are stabilized within 30 minutes
8 ● determine the acceptability of the difference ● hypo-osmotic medium mixture:
● IF the difference between the percentages is ○ (1) sodium citrate dihydrate
acceptable ○ (2) D- fructose in purified water
○ report the average percentage of vital
spermatozoa Procedure: Sperm Vitality
9
● IF it is too high B) Hypo-Osmotic Swelling Test
○ make 2 new preparations from two fresh
aliquots 1 ● mix the semen well.
○ repeat the assessment ● remove a 100 ul aliquot of semen and add to 1 ml
2 of swelling solution
● report the average percentage of vital
10 ● prepare 2 replicates
spermatozoa to the nearest whole number
● incubate at 37°C for 5 min 30 min
Live ● white heads 3 5 mins ● membrane swelling
Spermatozoa ● faint pink head 30 mins ● flagellar stabilization
Dead ● prepare replicate slides and examine at x400
● stained red or dark pink 4
Spermatozoa magnification.
● stain is limited to only a part of the neck ● evaluate 200 spermatozoa in each replicate, making
5
region a tally of unswollen (dead) and swollen (alive) cells.
“Leaky Neck ● rest of the head area is unstained ● calculate the percentages for each replicate
Membrane” ● NOT a sign of cell death and total 6 ● determine the percent average and difference of the
membrane disintegration two percentages of vital cells from the replicate slides
● assessed to be alive
7 ● determine the acceptability of the difference

● IF the difference between the percentages is

acceptable
○ report the average percentage of vital
spermatozoa
8
● IF it is too high
○ make 2 new preparations from two fresh
aliquots
○ repeat the assessment
● report the average percentage of vital
9
spermatozoa to the nearest whole number

Fig. Dye Exclusion Test

Fig. (a) shows no change in structure and images

(b-g) show the various types of tail changes

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● (a) Swollen spermatozoa are identified by changes in the
shape of the cell, as indicated by coiling of the tail.
● (b) Live cells are distinguished by evidence of swelling of
the sperm tail.

Lower Reference Limit

Dye Exclusion Test ● 58%

Hypo-Osmotic Swelling Test ● 58%

Acceptable Differences bet. 2 percentages

Acceptable Acceptable
Average % Average %
Difference Difference
0 1 66-76 9
1 2 77-83 8
2 3 84-88 7
3-4 4 89-92 6
5-7 5 93-95 5
8-11 6 96-97 4
12-16 7 98 3
17-23 8 99 2
24-34 9 100 1
35-65 10
*Acceptable differences between two percentages for a given average
determined from replicate counts of 200 spermatozoa (total 400 counted)

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Transcribed by: Abenes, Aliping, Guarin, Villanueva (2024). 20
 OTHER SEMINAL FLUID TESTS A.2) Citric Acid
● citric acid
A) CHEMICAL TESTS ○ major anion in semen
○ quantitated using spectrophotometric methods
A.1) Fructose ● ⬇CA levels indicate:
● fructose ○ dysfunction of the prostate gland
○ produced and secreted by the seminal vesicles ● normal semen total citric acid conc. = >52 μmol per
○ presence in semen reflects: ejaculate
■ secretory function: seminal vesicles
■ functional integrity: ejaculatory ducts & vas A.3) Acid Phosphatase
deferens ● acid phosphatase activity
● fructose level ○ useful marker to assess the secretory function of
○ most often determined when the sperm count the prostate gland
reveals azoospermia (i.e., no sperm) ● normal semen acid phosphatase = >200 units of
● normal semen fructose level = >13 μmol per ejaculate enzyme activity
● ⬇fructose levels & azoospermia may be ○ p-nitrophenyl phosphate (substrate)
encountered in cases of: ○ other body fluids contain insignificant amounts
○ retrograde ejaculation ● uniquely high concentration in semen
○ obstruction of the ejaculatory ducts ● prostatic acid phosphatase measurements are often
○ abnormalities of the seminal vesicles or vas used to determine whether semen is present in vaginal
deferens fluid specimens obtained from women following an
● several quantitative, spectrophotometric procedures are alleged rape or sexual assault
available for fructose determinations ● washings of the skin or stained clothing can reveal
significant levels of prostatic acid phosphatase, which
Test for Fructose positively identifies the presence of semen
● rapid and easy qualitative tube test based on
the development of an orange-red color in A.4) Other Biochemical Substances
the presence of fructose ● l- carnitine and α-glucosidase
● NO orange-red color = NO fructose ○ being evaluated as indicators of epididymal
● advantage:
function
Resorcinol ○ ease of performance
Test ○ rapid turnaround time ● specific lactate dehydrogenase isoenzymes of sperm
● disadvantage: are being examined for their clinical use in the
○ qualitative technique evaluation of male fertility
■ relies on the visual assessment of color
■ lacks sensitivity to decreased fructose
levels

NOTE:
● specimen should be tested within 2 hours or frozen to
prevent lysis of fructose

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 B) MICROSCOPIC EXAMINATION B.2) Sperm Vitality

B.1a) Sperm Motility: Hanging Drop Sperm Vitality Tests

● evaluates the speed and direction of sperm cells ● assess the ability of the sperm cells to
Results and Interpretation: Hanging Drop Revitalization become viable upon treatment with a
Test solution
0 ● no movement ● 5% glucose Ringer’s solution
● with movement ● uses 0.5% Yellow Aqueous Eosin
1
● no forward progression Bloom’s/Eosin Solution
● slow forward progression Test ○ live cells: unstained
2 ○ dead cells: stained pink-yellow
● noticeable lateral movement
● slow speed
3 B.3) Sperm Cell Count
● some lateral movement
● other fixatives/diluting fluids used:
4 ● rapid, straight-line motility ○ 1 % formalin
B.1b) Sperm Motility: Computer Assisted Sperm Analysis ○ 5 % sodium bicarbonate
(CASA) ○ 0.5 % chlorazene
● objective determination of kinematics of individual ○ 1% formalin in 3% trisodium citrate
sperm cells ○ 5 % sodium bicarbonate in 1% phenol and water
○ individual sperm cells:
■ velocity Sperm Function Tests
■ trajectory/direction of motion ● total absence of sperm cells
● sperm concentration and morphology ● results from:
○ bilaterally small underdeveloped
○ 200 motile sperm/spx: analyzed in the following
testes
velocity: Azoospermia
○ ejaculatory obstruction from the
■ smoothed path velocity previous traumatic or operative
■ track velocity procedures
■ straight line velocity ○ secondary to gonorrhea
■ amplitude of lateral head displacement
● few motile cells and low sperm
■ beat cross frequency
count
● factors affecting CASA
● resulting from:
○ sample preparation Oligospermia ○ bilateral or unilateral hypotrophic
○ frame rate testes
○ counting-chamber depth ○ possibly secondary to
○ sperm concentration hypothyroidism
■ high sperm concentration ● condition in which there is a low
● collisions w/ high frequency → errors → percentage of live
Necrospermia
dilute w/ seminal plasma ● high percentage of immotile or dead
spermatozoa in semen
Benefits and Disadvantages of ● condition characterized by inability of
Manual Sperm Analysis & CASA Astheno- vital sperm cells to move
Method Advantage Disadvantage zoospermia ○ slow moving sperm cells
○ “lazy” sperm cells
● takes relatively long
Manual time for analysis
● low acquisition cost
Analysis ● subjective counting
● only rough analysis
● fast analysis
● high acquisition costs
● highly reproducible
● regularly
Computer with same settings
maintenances
Assisted ● detailed analysis
necessary
Analysis ● high statistical power
● different settings may
(CASA) due to objective
dramatically change
analysis of numerous
results
sperms

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 C) POST-VASECTOMY SEMEN ANALYSIS Test for Anti-Sperm Abs
● normal sperm count post-vasectomy = 0 ● primarily to detect the presence of IgG
○ azoospermia within 12 weeks ● semen sample containing motile sperm is
● nonmotile sperm can be present for as long as 21 incubated with IgG anti-human globulin
months post vasectomy (AHG) and a suspension of latex particles
○ results from the release of nonviable residual or treated RBCs coated with IgG
Mixed
● bivalent AHG
sperm in the seminal vesicles & vas deferens Agglutination
○ binds simultaneously to both the
(abdominal portion) Reaction
antibody on the sperm and the antibody
● spx are routinely tested at monthly intervals (MAR) Test
on the latex particles or RBCs
○ beginning at 2 months post-vasectomy ○ (+) clumps of sperm and particles or
○ continuing until 2 consecutive monthly specimens cells (microscopic)
show no spermatozoa ● normal = < 10% of the motile sperm
attached to the particles
■ e.g. if vasectomy was conducted in March →
testing begins on May → continued every ● more specific
month until 2 consecutive months that the ● used to detect the presence of IgG, IgM,
specimen shows no sperm cell and IgA
● wet preparation ● demonstrates what area of the sperm
○ recommended testing for post-vasectomy (head, neckpiece, midpiece, or tail) the
autoantibodies are affecting
Test for Post-Vasectomy
Head- ● interfere with penetration into
● using phase microscopy for the presence
Directed the cervical mucosa or ovum
of motile and nonmotile sperm
Abs
Wet ● negative (-) wet preparation
Tail- ● affect movement through the
Preparation ○ followed by centrifugation of the specimen
Directed cervical mucosa
for 10 mins and examination of the
Immuno bead Abs
sediment
test
D) DETECTION OF ANTISPERM ANTIBODIES ● sperm are mixed with polyacrylamide
● under normal conditions, the blood-testes barrier beads known to be coated with either
separates sperm from the male immune system anti-IgG, anti-IgM, or anti-IgA.
● disrupted barrier may occur due to: ● microscopic examination of the sperm
○ surgery ○ shows the beads attached to sperm at
○ vasectomy reversal (vasovasostomy) particular areas
○ trauma ○ depending on the type of beads used,
○ infection the test could be reported as:
■ “IgM tail antibodies,”
● Ag on the sperm
■ “IgG head antibodies,”
○ produce an immune response that damages the
sperm → damaged sperm may cause the ● normal = presence of beads on < 20% of
production of antibodies in the female partner the sperm

(+) Abs in a E) MICROBIOLOGICAL ASSAY

● clumps of sperm are observed during a ● commonly performed when there is increased WBC
Male
routine semen analysis (>1 million/mL) in semen
patient
● suspected when there is a normal semen ○ indicating infection involving the prostate gland
analysis result yet there is continued ● routine aerobic and anaerobic cultures and tests for:
(+) infertility ○ Chlamydia trachomatis
Anti-sperm ● method to detect antisperm Ab ○ Mycoplasma hominis
Abs in a ○ mixing the semen with the female cervical
○ Ureaplasma urealyticum
Female px mucous or serum
○ (+) agglutination ○ Escherichia coli
○ (-) NO agglutination ■ most frequently performed
● the latter is implicated on the occurrence of sperm
agglutination and immobilization

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 F) SPERM FUNCTION TESTS

Sperm Function Tests

● sperms are incubated w/
Hamster Egg
species-non-specific hamster eggs
Penetration
● penetration is observed microscopically
Cervical
● observation of sperm’s ability to
Mucous
penetrate cervical mucous
Penetration
In Vitro
Acrosome ● evaluation of the acrosome to produce
Reaction enzymes essential for ovum penetration
(Acrosin)
● uses unfertilized oocytes obtained
through donation binding capacity of
Hemizona
sperm cells
assay
● assess possible success of invitro
fertilization (IVF) tests

G) MEDICO LEGAL TESTS

Medico Legal Tests

● detects for presence of choline (dark
Florence Test
brown, rhombic crystals) in semen
● for spermine in semen
Barbiero’s
○ yellow, leaf-shaped crystals
● substance is highly concentrated in
Acid
semen
phosphatase
● used for investigation of rape cases
● use of this instrumentation provides a
Fluorescence picture of the possible presence of
or UV semen that appears
procedures ○ brilliant, pale, lavender under
Wood’s lamp

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ANALYSIS OF URINE MLS 321

& OTHER BODILY FLUIDS 8438

LECTURE BS MLS 3 FINALS
M7: OTHER BODILY FLUIDS (UNITS 3 – 7)

UNIT 3: CERVICAL MUCUS, VAGINAL SECRETIONS, Clinical Significance

AND PREGNANCY TEST ● noticeable changes in the color, consistency, or quantity
of vaginal secretions may be linked to various
I. VAGINAL SECRETIONS conditions & infections such as sexually transmitted
diseases (STDs)
Vaginal Secretions ● STDs can be detected by performing several tests in
● composed of discharges coming from: the microbiology department & urinalysis department
○ uterus ● most common gynecologic complaints:
○ vagina ○ vaginal discharge
○ cervix ○ vaginal discomfort
○ vaginal odor
Endocervix ● three major causes:
● lined by a single folded layer of mucus-secreting ○ (1) bacterial vaginosis
columnar epithelium ○ (2) candidiasis
● endocervical glands ○ (3) trichomoniasis
○ secrete cervical mucus ● whereas the causative agent for each of these
■ an alkaline, clear, white to pale yellow viscous conditions is distinctly different, the clinical
fluid presentations can be nonspecific and similar
○ composition: ● NOTE:
■ (1) 90–95% water ○ when microscopic findings turn out to be negative
■ (2) mucin glycoproteins (heterogeneous (-) despite clinical findings → culture/ DNA
mixture) probe analysis may be performed
● cell-surface and gel-forming mucin
○ works as a barrier to prevent any foreign materials
from entering the cervix and uterus
○ prevents possible infection

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 SPECIMEN COLLECTION AND HANDLING Vaginal Secretion Collection
● appropriate collection and handling optimize the ● collected during a pelvic examination
recovery & detection of microorganisms, & other cellular ● speculum
elements ○ used to provide access to the vaginal fornices
○ non-lubricated
Specimen Collection Swabs ○ moistened only with warm water
● (1) polyester-tipped ● speculum lubricants
Recommended ○ e.g. Dacron ○ AVOIDED bec. it many contain antimicrobial
Sterile Swabs ● (2) swab on plastic shaft agents
● (3) sterile loop ● health care provider uses one or more swabs or a
Cotton Tipped collection loop to collect vaginal secretions
● toxic to Neisseria gonorrhoeae
Swab ● collected from the:
Wooden Shart ● toxic to Chlamydia trachomatis ○ (1) posterior vaginal fornix
○ or (2) vaginal pool
■ mucus and cells present in the posterior fornix
of the vagina when the female is in a supine
position

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 PHYSICAL EXAMINATION: VAGINAL SECRETIONS

1. Color
● indicator of infection

Pale Yellow ● normal

Creamy White ● candidiasis
Gray ● bacterial vaginosis
Yellow-Green ● trichomoniasis
● menstruation
Red
● cervical polyp

2. Consistency and Appearance

Vaginal Secretion Transport Clear ● normal

● specimen collected are transported to the laboratory Curd-like;
● candidiasis
ASAP requiring: Creamy
○ (1) labeled spx Thin,
● bacterial vaginosis
○ (2) in a biohazard bag Homogenous
○ (3) requisition slip Frothy ● trichomoniasis

Requisition Slip Must Contain Odor

● complete name
Standard Patient ● bacterial vaginosis
● age Foul, Fishy
Identification ● trichomoniasis
● sex
● patient’s menstrual status CHEMICAL EXAMINATION: VAGINAL SECRETIONS
● exposure to sexually transmitted
Patient History diseases 1. pH
● use of vaginal lubricants, creams, and ● normal pH of vaginal secretions = 3.8 - 4.5
douches ○ maintained by numerous lactobacilli and their
major metabolic end product, lactic acid
Specimen Processing ○ production of lactic acid and H2O
● upon receipt in the laboratory, the test must be ○ prevents overgrowth of
performed ASAP ■ G. vaginalis
● kept at room temp in case of delay ■ trichomoniasis
■ atrophic vaginitis
● since it adversely affects the
GENERALLY ● lactobacilli
○ recovery of N. gonorrhoeae
NOT for ○ known to produce hydrogen peroxide (H2O2) that
○ detection of trophozoite T. vaginalis
Refrigeration enhances the acidity of the vaginal canal
■ “flitting motility”
○ prevents the overgrowth of some indigenous
● detection of Chlamydia trachomatis microbe such as Gardnerella vaginalis
FOR
● detection of viruses
Refrigeration ⬇ or absence ● predisposing factor for the occurrence of
○ e.g., herpes simplex virus
(Exception) of H2O2 bacterial vaginosis
● prevents the overgrowth of normal flora
● pH is determined with the use commercial pH paper
before placing the swab into saline to differentiate
diagnosis of patient’s vaginitis
● indicative of:
○ bacterial vaginosis
>4.5 pH
○ trichomoniasis
○ atrophic vaginitis

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MICROSCOPIC EXAMINATION: VAGINAL SECRETIONS 3. Gram Staining
● used to distinguish and classify bacterial species into
1. Wet Mount Preparation ○ gram-positive bacteria
● immediate examination of specimen ○ gram-negative bacteria
● must performed ASAP for the detection of T. vaginalis
○ identification depends solely on observing motile
organisms

Procedure: Wet Mount Preparation

● directly placing the swab into a 0.5 to 1.0 mL of
1
sterile physiologic saline (0.9% NaCl)
2 ● agitated to release the secretions from the swab Fig. White blood cells. Notice the Fig. Normal red blood cells (×400).
multilobed nucleoli (×400).

● alternative method:
○ done by placing a small drop of sterile physiologic
saline onto a microscopic slide → swab is directly
rolled for microscopic examination → brightfield or
phase-contrast microscopy

● overall distribution of the specimen

LPF (10x)
components is assessed Fig. Bacteria. A, Large rods Fig. single parabasal cell
characteristic of Lactobacilli, the surrounded
● quantified predominant bacteria in normal by numerous squamous epithelial
HPF (40x) ● includes the evaluation of the amount and vaginal secretions (×400) cells
type of epithelial cells present

Fig. Budding yeast cells Fig. Yeast cells showing mycelial

forms (×400).

2. 10% KOH Preparation & Amine Test

Fig. Clue cells (x400).s Fig. Parabasal cell surrounded by
● addition of 10% KOH directly into the drop of specimen epithelial cells (×400).
on the microscopic slide

● presence of fungal elements

● caused by trimethylamine
Fishy Odor ○ volatilization product of polyamines
○ results from the alkaline pH change when
KOH is added
Fig. Squamous epithelial cells Fig. Clump of squamous epithelial
● NOTE: cells (×400)
identifiable under low power
○ heat may be applied → allows KOH to dissolve the
epithelial cell and blood cells in the specimen
enhancing visibility of fungal elements

Transcribed by: Lustre, Aliping, Guarin, Villanueva (2024). 4

 Elements Typically Identified in Wet Mounts of Vaginal Secretions
Normal Remarks
Blood Cells
● any variation often associated with:
○ menstrual cycle
WBC ● few to moderate/hpf
○ ovulation
○ menses
● maybe present when collected around or during
RBC ● usually NOT present
menstruation
Microorganisms
● large
● non-motile
Lactobacillus ● 50% to 90% of the total microbe
● gram (+)
● produce lactic acid and H2O2
Other bacterial
morphotype
[Link] ● ⬇= lactobacilli compared to squamous epithelial cells
Peptostreptococcus ● remaining 10 % to 50 % signals microflora imbalance
Staphylococci ● ⬆= other bacterial types is abnormal
Prevotella spp.
Bacteroides spp.
● KOH distinguish yeast from RBC
● ⬆number (1+ or >) or presence of hyphae or
Yeast / Blastoconidia ● present occasionally
pseudophyphae is abnormal
○ e.g., in cases of candidiasis
● pear shaped unicellular bodies averaging 15 μm (5 – 30 μm)
Trichomonads ● none in length
● “flitting” or “jerky” motion
Epithelial Cell
● large, thin, flagstone-shaped elements with small nuclei and
abundant cytoplasm
● keratohyalin granulation
Squamous ● most dominant ○ due to cell aging
○ not to be mistaken for clue cells
● keratohyalin granules
○ larger & more refractile than bacteria.
● bacteria-laden cells with shaggy edges
● soft appearance with fine stippling due to bacteria
Clue Cell (–)
adherence
● indistinct cellular borders; nuclei may not be visible.
● cells beneath surface/luminal squamous epithelium of
vaginal mucosa
● diameter: 15 to 40 μm, oval to round with clear cytoplasmic
● none or few; may be increased
borders.
Parabasal Cells during menses or post-
● resemble transitional cells but with nucleus to cytoplasm
menopausal period
ratio of 1:1 to 1:2
● ⬆ in atrophic vaginitis and desquamative inflammatory
vaginitis
● basal layer of vaginal stratified epithelium
● size similar to white blood cells (10 – 16 μm).
Basal Cells ● none ● nucleus to cytoplasm ratio of 1:2.
● ⬆ in desquamative inflammatory vaginitis along with ⬆
WBC

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 II. CERVICAL MUCUS Dry / Tacky / Thick ● not fertile

Cervical Mucus (CM) Creamy / Sticky ● not fertile

● clear, gel-like to watery-like consistency Cloudy / Stretchy ● semi-fertile
● produced from the endocervix by the cervical glands
○ dependent on the level of estrogen throughout the Watery / Stretch (Egg
● most fertile
menstrual cycle White Consistency)
○ ectocervix generally non-glandular
● interaction of CM to spermatozoa
● work as lubricant during sexual intercourse
○ allows the spermatozoa to ascend into the uterine
● plays an important role in fertility studies
cavity
○ CM undergoes cyclic, physical, and chemical
○ NOTE: CM may also prevent sperm entry
changes in the different phases of the ovarian cycle
● CM in fertility studies
○ e.g., when ovulation approaches → CM becomes
○ examined at its peak of maximal sperm
thinner & watery (raw egg white consistency)
receptivity

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Maximal Sperm Receptivity Fern’s Test
● ovulating period ● must be performed during maximal sperm receptivity
● increased LH, accompanied by (ovulating)
○ marked⬇cervical mucus viscosity ○ allows entry of sperm cells
○ ⬆leukocyte alkaline phosphate activity ○ increased secretion of LH
○ lowest value of basal body temp ■ produced from the anterior pituitary gland
○ (changes in the fern test pattern ■ triggers ovulation
● “ferning”
FERTILITY STUDIES ○ arborization pattern of the electrolytes that
● must be performed during maximal sperm receptivity crystallized in the presence of protein
(ovulating) ○ observed in CM when spread thinly on a slide and
○ allows entry of sperm cells allowed to dry
○ increased secretion of LH ● fern test pattern
■ produced from the anterior pituitary gland ○ good indicator of the midcycle elevation of LH
■ triggers ovulation and of ovulation
■ better than spinnbarkeit
OVULATION TESTS ○ direct relationship to sperm receptivity
○ sensitive indicator of hormonal changes (estrogen)
Spinnbarkeit Stretchability Test
● evaluates the viscoelastic Procedure: Fern’s Test
properties of ovulatory ● obtain cervical mucus with a cotton swab introduced
1
cervical mucus into the cervical canal
○ ability of cervical mucus 2 ● smear mucus on a coverslip and allow to air dry
to be stretched into a
thread 3 ● observe crystallization pattern under LPF
● during the mid-cycle week, the cervical mucus is
tested daily for viscosity either by a viscometer or by Results and Interpretation: Fern’s Test
simple stretchability test ● linear ferning only
1+
○ minimal degree of estrogen effect
Procedure: Spinnbarkeit Stretchability Test ● arborization of leaves at 90° to each other
● expose the cervical surface using a non-lubricated 2+
1 ○ palm leaf appearance
speculum and clean the cervical surface
● moderate degree of arborization
● using a disposable 1 ml tuberculin syringe without a
2 3+ ○ palm leaf appearance involves angulation at 3 right
needle, aspirate the endocervical mucus
angles
● record physical properties:
○ amount ● maximal arborization
3 ○ color 4+ ○ palm leaves appearing at 4 right angles to each
○ clarity other
○ pH
● place 0.1 ml mucus on a slide and cover with a
4
coverslip
● pull the coverslip away from the slide and measure in
centimeter the spinnbarkeit
5 ○ measure: ability of the mucus to be drawn into a
thread, the length of which is measured before it
breaks

Results and Interpretation: Spinnbarkeit Test Fig. Single ferns are big and closely Fig. Empty spaces appear between
branched the ferns
● ovulating
≥ 9 cm ○ mucus may form a thread up to 9 cm & over
because of its watery consistenc

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 Sperm-Cervical Mucus Contact Test (SCMCT)
● evaluates the penetration & migration in cervical
mucus of non-agglutinated spermatozoa despite the
presence of auto agglutinins (antisperm Ab)
● spermatozoa in ejaculates with partial
autoagglutinins
○ change their progressive motility into quick,
Fig. Single ferns are little and Fig. No ferning is present; mucus shaking, and jerking movements as soon as they
incompletely formed with rare or no may appear in clusters and threads come in contact with cervical mucus
branching
○ unable to penetrate the mucoid barrier
○ same movement is seen when ejaculate is normal
CERVICAL MUCUS-SPERM CELL
but the mucus contains agglutinating antisperm
RELATIONSHIP TESTS
antibodies

Postcoital (Sims-Huhner) Test

Procedure: SCMCT
● sample aspirated 2-3 hours after intercourse
● beginning at 4 hours after coitus ● mix 1 drop of semen and 1 drop cervical mucus on
1
a slide
○ no. of spermatozoa in the mucus gradually
3 ● incubate for 15-20 mins
decreases
● abnormal postcoital test must be repeated several ● cover with coverslip and observe behavior of
2
spermatozoa
times to substantiate the diagnosis of
oligoasthenospermia or an abnormality of cervical
Results and Interpretation: SCMCT
mucus
● presence of significant antisperm antibodies
Procedure: Postcoital (Sims-Huhner Test) ● spermatozoa do not progress normally in the
● perform the test as close to the time of ovulation as cervical mucus
possible ○ a phenomenon depending on IgA in the
1 Jerking
● instruct the patient to abstain from sex for 2 days genital secretions
Motility
before test ● attachment of spermatozoa to the
● perform the test 2 ½ hour after intercourse glycoprotein of cervical mucus is responsible
● NOTE: for the jerking nonprogressive movement
2
○ if the test is not performed at the specified time, ●
record the interval between intercourse and test
● obtain mucus from the ectocervix and low
3 endocervix by means of a sterile tuberculin syringe
without needle
● place the mucus on slide, cover with coverslip, and
4
examine under 40x / HPF

Results and Interpretation: Sims-Huhner Test

● 25 or more progressively motile
Normal
spermatozoa/HPF
● 10 or more directional motility
Satisfactory
spermatozoa/HPF
● < 5 spermatozoa/HPF with “localized
motility”
● assoc. conditions:
○ oligoasthenozoospermia /
Abnormal
oligoasthenospermia
■ decreased sperm count with
decreased sperm motility
○ abnormality of cervical mucus

Transcribed by: Lustre, Aliping, Guarin, Villanueva (2024). 8

 TESTS FOR ANTISPERM ANTIBODIES Immobilization Method: Sperm Immobilization Test (SIT)
● useful tool for detecting sperm immobilizing antibodies
Agglutination Methods ● traditionally calculated by eye estimation using a
● tube-slide agglutination test (TSAT) microscope to determine the proportion of motile sperm,
● gelatin agglutination test (GAT) after the number of motile and immotile sperm were
● tray agglutination test (TAT) counted manually by a highly trained laboratory
embryologist
Antisperm Antibodies
● associated with both male and female subfertility Sperm-Cervical Mucus Methods
● may be present in semen or cervical mucus ● sperm-cervical mucus penetration test (SCMPT)
○ also present in blood = as IgM only ● sperm-cervical mucus contact test (SCMCT)
● aka spermatoagglutinating Ab
○ IgG, IgA, or IgM
● produced in response to the sensitization of the sperm
○ caused by testicular trauma, damage, surgery, resulting
to the blood vessel barrier
● main cause or risk factor = vasectomy
● can cause damage to spermatogenesis and for
infertility in the male
● specifically destroy the germinal cells of the testis
● sperm are foreign bodies for women and upon exposure,
are sometimes recognized as antigens, which can cause
an immunological reaction in the woman
○ husband has no sperm Ab → sperm comes into contact
with cervical mucus → sensitization occurs wherein the
female creates Ab against the sperm cells
○ inhibit the sperm enzyme hyaluronidase required for
Radiolabeled Antiglobulin Test
entry into the ovary
● modification of the radiolabeled Coombs Antiglobulin
test used to measure antiplatelet IgG antibodies
Tube-Slide Agglutination Test (TSAT) ● semen resuspended and incubated with radiolabeled
● Boettcher’s modification of the Franklin-Dukes rabbit anti-IgG
microscopic agglutination test ● plasma is also collected
● Baker’s buffer (pH 8.1)
● human serum Procedure:
Reagents ○ inactivated at 56 °C for 30 min ● two million donor spermatozoa, negative for antisperm
● semen suspension in Baker’s buffer 1 antibodies, are incubated with undiluted plasma from
○ 50 x 106 70% motile spermatozoa/mL each patient (male and female)
● place 10 drops of inactivated serum in a ● the specimen is then centrifuged, the sperm button
test tube and add 1 drop of semen 2 washed, and the spermatozoa resuspended and
suspension incubated with radiolabeled rabbit anti-IgG
○ incubate at 37°C. ● the spermatozoa are then washed, and the
Procedure at intervals of 1-4 hours, remove 1 drop
● sperm-associated radioactivity is determined in a
from the bottom of the test tube, place on a 3 γ-counter
slide, and cover with coverslip ○ expressed as the % of the total 125I anti-IgG
● examine microscopically and count the radioactivity incubated with each aliquot of sperm
number of agglutinates seen per 100 motile
non agglutinated spermatozoa Results and Interpretation: Radiolabeled Antiglobulin
Normal ● 1.15 ± 0.68

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 III. PLACENTAL SECRETIONS Urine Estriol
● produced by pregnant women as an indicator mainly
of fetal sources
● evaluates fetal age

● 2nd and 3rd trimester

Fetal
● from liver and adrenal glands
Maternal ● 1st trimester

● also excreted by nonpregnant women as

○ metabolic end product of ovarian enthrone
○ estradiol excretion
● detects pregnancy

Precursor of Estriol
● pregnant
Trophoblasts ● converted by the placental enzymes to
● cells forming the outer layer of a blastocyst, which estriol, and enters the maternal
provides nutrients to the embryo circulation
● develops into a large part of the placenta ○ results in a 1000-fold rise in
16-DHEA
● formed during the first stage of pregnancy and are the maternal serum estriol concentration
(α-dehydroepi–
first cells to differentiate from the fertilized egg androsterone) during an average pregnancy.
sulfate ○ can then be converted in the
PLACENTAL HORMONES maternal liver and excreted into the
Human Placental Lactogen urine as either
● produced by “fetoplacental unit” ■ glucuronic

■ sulfate derivatives
Production ● 6 weeks ● non pregnant
● 1st and 2nd trimester Estradiol ○ thus an increase in estriol does not
Peak or Rise
● detectable levels always indicate pregnancy
Plateau ● 3rd trimester
● rapidly (within minutes) falls after ● evaluate fetal placental unit
Decline
delivery
Decreased ● failure of the fetoplacental unit to grow or
● the plasma or urine levels can be used to monitor a Estriol function properly
growing trophoblast since Sudden 4
● fetal distress
mg/24-hr fall
Declining ● danger immediate abortion
Level ● miscarriage Increased
● normal fetal growth
Estriol
Estrogen
● pattern of production and decline parallels HPL Human Chorionic Gonadotropin (hCG)
● excreted as estriol in the urine ● “human placental trophoblast”
● evaluates the fetoplacental unit ○ predominantly produced by a very young
○ recommended to be performed at 3rd trimester (9-day-old) placental trophoblast
since estriol production is attributed to the maternal ○ smaller amounts are also produced in the pituitary
serum in the 1st trimester gland, liver and colon
● excreted out in urine basis of most tests for
● fetal distress ○ pregnancy
Declining ● fetal death ○ trophoblastic tumors in men and women
Level ○ fetal precursor substances are needed by ■ hydatidiform mole
the placenta to produce estrogen ■ choriocarcinoma germ cell tumors

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● a glycoprotein with galactose as the primary sugar and Extrauterine
● characterized by a low rate of increase,
composed of two subunits: (Ectopic)
without the typical doubling
Pregnancies
● nearly identical to the α subunit of LH and
FSH
α subunit ● checking serum levels is typically paired with ultrasound
● thus, cross-reacts with LH in certain bioassay
evaluation to improve sensitivity and specificity
systems to give false-positive results
○ since there is a large range of normal hCG levels
● determines the biologic and immunologic
β subunit and inconsistent rates of increase of this hormone
specificity of hCG
● primarily catabolized by the liver Pregnancy Test
○ although about 20% is excreted in the urine. ● not a direct test for pregnancy
○ beta subunit is degraded in kidneys to make a core ○ i.e. detection of a fetus
fragment which is measured by urine hCG tests ● a test to detect the presence of hCG in urine or blood
● measuring hCG levels can be useful
○ in identifying a normal pregnancy, pathologic ● detect pregnancy much earlier than using
Serum
pregnancy urine
○ following an aborted pregnancy ● best specimen is the first morning urine
○ in measuring hCG in a variety of cancers including because it is most concentrated
choriocarcinoma and extra-uterine malignancies Urine
● for optimal results, the SG should also be
Heat-Stable Serum Placental ALP ≥ 1.015
● an enzyme produced by the placenta that appears to Immunochromatographic Method
reflect placental function better than hCG ● used by most test kits
● largest increase occurs during the 2nd trimester ● measures urine hCG with a detection limit ranging
● eclamptic patients / eclampsia from 6 to 50 IU/L
○ prone to fetal distress and death ● urine applied to the kit is absorbed into a nitrocellulose
Increased ○ precedes fetal distress by 2-3 weeks bed
○ hypertension during pregnancy ○ hCG is concentrated into a narrow band as the
○ increased protein excretion urine migrates
Sudden ○ a labeled anti-hCG antibody in the kit binds to the
● placental failure
Decrease migrating hCG and passes through a zone having
Increased solid phase capture antibody to hCG
● normal fetal growth ○ the appearance of a colored line indicates a positive
Estriol
test
PREGNANCY: hCG False Positive False Negative
Serum
Levels of hCG
● can vary widely between women with normal ● hook effect
pregnancies ● ectopic production of hCG ○ hCG levels are too high
○ choriocarcinoma (≈ 500,000 mIU/mL)
● 1st trimester ○ germ cell tumors ○ prevents reaction due to
Exponential ○ doubling about every 24 hours during supersaturation
Rise the first 8 weeks Urine
● in serum and urine concentrations ● large amounts of blood, ● too dilute
Peak ● around 10 weeks of gestation protein (1 g/day) ● too soon testing after a
Decrease ● decrease levels until about the 16th week ● bacteria missed period
Constant ● 16th week until full term ● semen ● increased urine potassium
● drugs ● presence of
● nonviable pregnancy ○ aspirin ○ salicylates
○ hCG levels that plateau prior to 8 weeks ○ carbamazepine, ○ barbiturates
○ hCG fail to double antipsychotic drugs ● hook effect
○ whether intrauterine or extrauterine

Transcribed by: Lustre, Aliping, Guarin, Villanueva (2024). 11

 Other Pregnancy Test Methods OTHER USES OF hCG
Biological Assays
● involves the use of test animals where interpretation of Gestational Trophoblastic Disease (GTD)
result is based on the biological changes observed in ● detection of hCG is also useful in the evaluation of
these test animals trophoblastic disease, including
Biological Change Animal Used Method
○ complete and partial hydatidiform mole
● male grass frog ○ post-molar tumor
○ Rana pipiens ○ gestational choriocarcinoma
○ Rana clamitans ○ testicular choriocarcinoma
Spermiation ● male toad ● Galli-Mainini Test
○ placental site trophoblastic disease
○ Bufo bufo
○ Bufo ● all of these entities produce hCG, varying levels of
americanus which are reported on commercial assays
● female toad
○ Xeneous laevis ● Hogben’s Test ● highly suggestive of a complete
Ovulation
● South African ● Friedman’s Test hydatidiform mole in early
clawed frog pregnancy
Total hCG
● Ascheim-Zondek Test >100,000 mUI/mL ● although many normal pregnancies
● immature female
Hyperemia ● Frank- Berman Test
white mouse
● Kupperman Test
may reach this level at their peak
● mature virgin ● Friedman’s Test around 8 to 11 weeks of gestation
Corpora
Hemorrhagica female rabbits ● Hoffman’s Test
Diagnosis of Ectopic Pregnancy
Radioimmunoassays
● hCG fails to double
● based on competitive binding of hCG (present in test ● hCG usually remains < 6,500 mIU/mL
serum) and a radiolabeled hCG on the binding sites of
hCG antibody
● bound radiolabeled hCG is related to the Evaluation of Abortion
concentration of hCG in the specimen
Radio Receptor Assays 94% ● hCG levels <1000mIU/mL in a patient
Predictive of with vaginal bleeding
● based on the
Abnormal
○ ability of radiolabelled hCG to bind to specific ● progesterone level of <5 mg/mL
Pregnancy
receptors prepared from the cell membranes of
gonadal tissues Spontaneous ● beta-hCG is non-detectable within 9-35
■ from rat or pig testes Abortion days
■ corpora lutea from ovaries of pregnant cows
○ inhibition of this binding by unlabelled hCG present
Diagnosis and Evaluation of Down Syndrome
in the biological samples
● beta-HCG is increased as it is measured in maternal
Immunometric Assay
serum during the 2nd trimester
● a solid phase immunoassay that makes use of labelled
antibody rather than labeled antigens
● the Ag-Ab binding is directly proportional to the amount of Surveillance of Ovarian Germ Cell Tumors
antigen present ● choriocarcinoma
● the monoclonal antibody attached to the solid state is ● endodermal sinus tumor
directed towards an epitope of the alpha subunit ● embryonal carcinoma
● the second monoclonal antibody labeled with a ● dysgerminoma
radioisotope or an enzyme (alkaline phosphatase) is
directed at an epitope on the beta subunit
Evaluation of Testicular Tumors
● indicative with hCG levels > 5,000 IU

Ectopic hCG Production

● in non-trophoblastic tumors
○ such as in breast, ovarian, pancreatic, cervical,
gastric and hepatic carcinoma
● in ascites with malignant tumors

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 UNIT 4: GASTRIC FLUID, DUODENAL CONTENTS, Composition of Gastric Fluid
SPUTUM AND SWEAT Cells Substance Secreted
Mucous Neck ● mucusprotects linings
GASTRIC FLUID
Cells ● bicarbonate
● gastric acid (HCl)
Composition of Gastric Fluid
○ primary contributor of gastric
● HCl, electrolytes
acidity
● mucus Parietal Cells
● intrinsic factors
○ a mixture of mucoprotein & mucopolysaccharides
○ Ca2+
that increases the viscosity
○ vitamin B12 absorption
● non-digestive enzymes
○ LDH, AST, ALP, ALT and ribonuclease ● histamine
● digestive enzymes ○ stimulates parietal cells for
○ pepsin, gastricsin, renin and lipase Enterochromaffin- secretion of HCl
like Cells ○ most important positive regulation
mechanism of the secretion of
● major digestive enzyme of gastric secretion
gastric acid in the stomach
by the chief cells (peptic cells) and is
Pepsin ● pepsin(ogen)
activated by gastric acid at an optimal pH of
1.6 to 2.4 Zymogen Chief ○ pepsin = acted upon by HCl
Cells ○ pepsin = degrades proteins
● has a proteolytic activity at pH 3.2 and has a
Gastricsin ● gastric lipase
higher concentration than pepsin
● somatostatin
● has weak proteolytic activity while gastric D Cells
○ inhibits acid
Renin lipase is important in the digestion of dietary
fat at an optimum pH of 4.5 ● gastrin
G Cells ○ stimulates parietal cells to produce
HCl
● other components include
○ small amounts of serum albumin,
○ gamma globulin
○ lactic acid
○ butyric and other organic acid

Production of Gastric Fluid

● tightly regulated by positive and negative feedback
mechanisms
● nerves that influence the secretion significantly in the Reasons for Gastric Fluid Analysis
digestive tract
Evaluate Ability ● especially patients diagnosed
○ endings of the vagus nerve
To Secrete pernicious anemia and stomach ulcer
■ lays a role on stimulation of gastric production
Gastric Acid gastric acid in the stomach
○ intramural nervous plexus
● nerve endings in the stomach Measure Amount ● peptic ulcer, stomach cancer,
○ secrete two stimulatory neurotransmitters Acid Produced duodenal cancer
■ acetylcholine ● indicated by hypochlorhydria in the
■ gastrin-releasing peptide presence of elevated gastrin
○ action directed on parietal cells ● tumors in the pancreas and
○ mediated through the secretion of gastrin from G Diagnosis Of duodenum
cells and histamine from enterochromaffin-like cells. Zollinger-Ellison gastrinoma
●
Syndrome ○ tumor that hypersecretes gastrin
● triad of symptoms
○ non B-cell tumor / duodenal tumor
○ duodenal ulceration
○ increased production of gastrin

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 ● removal of one of the vagus nerve → PHYSICAL EXAMINATION: GASTRIC FLUID
decreased gastrin production → ● a thin, colorless, acidic liquid
decreased acidity ● specific gravity of 1.001 to 1.010
Evaluate
● usually performed on px with cases
Completeness of
with overly produced gastrin or Physical Examination: Normal Gastric Fluid
Vagotomy
gastrinoma px Volume ● 50 – 60 mL (fasting)
● patient responds to an acid stimulation Color ● colorless
test → vagotomy not successful
Odor ● odorless; slightly sour
Differential
Specific Gravity ● 1.001 – 1.010
Diagnosis of
● gastric ulcer lower gastrin production pH ● 1.6 – 1.9
Gastric Ulcer
than duodenal ulcer
from Duodenal
Ulcer Volume

Decreased
SPECIMEN COLLECTION: GASTRIC FLUID ● gastric hypermotility
Volume
● 8 – 12 hrs fasting
● hypomotility
○ do not swallow too much saliva = may neutralize Increased
● pyloric obstruction
gastric fluid Volume
● Zollinger-Ellison
● sitting position for best recovery
○ or lie on left side with head elevated ≈ 45º
Color
Gastric Lavage
● increased bile
● intubation using sterile Levin tube through the Yellowish-Green
○ indicates obstruction in biliary tree
○ mouth = orogastric
Reddish or
○ nose = nasogastric
Coffee-Ground ● presence of blood
● toxicants are removed by flushing saline into the
Appearance
stomach → suction of gastric contents
● suction to aspirate fluid
● basal and maximal secretion Odor
○ basal = absence of stimulus
Fecal ● suggests intestinal obstruction
○ maximal = stimulus given to px to maximize
Rancid ● increased lactic acid content
secretion of gastric fluid
Foul ● suggests necrotic lesion

pH

● contamination with large amount of

saliva during collection
Neutral pH
● gastric cancer
● marked chronic gastritis
● increased gastric acid
Decreased pH
○ common in cancers

Transcribed by: Lustre, Aliping, Guarin, Villanueva (2024). 14

 MICROSCOPIC EXAMINATION: GASTRIC FLUID ● to evaluate completeness of vagotomy
● normal secretions ● to determine if px requires vagotomy
○ yeasts ● hypoglycemia produced by insulin
○ starch granules ○ causes increased gastric secretion by
○ normal bacterial flora of mouth and throat acting through the vagus nerves
○ small amounts of mucous ● insulin is used as a test to see whether
vagotomy would be of value and to
Microscopic Examination: Gastric Fluid determine whether vagotomy has been
Pathologic completed
Stain
Findings
Procedure
Fats ● Sudan III or IV = red
● insulin infused to px → insulin induces
Yeasts ● Sudan III or IV = yellow hypoglycemia → cause stimulation of
Hollander’s
● Sudan III or IV vagus nerve to stimulate production of
Test for
Starch ○ lilac-purple gastrin
Insulin
○ black-blue ● insulin is injected intravenously
● Gram’s stain ● FBS obtained
○ large non motile lactic acid bacilli ○ every 30 mins for 2 hours
○ appearing in chains ● gastric aspiration
Boas Oppler ● iodine = brown ○ every 15 mins for 2 hours
Bacilli ● indicates stasis with fermentation in ● at least one of these samples should
the absence of free HCL demonstrate a blood sugar < 40 mg/dL
○ especially with achlorhydria seen in Intact Vagus ● hypoglycemia,
cancer of the stomach Nerve ● increased gastrin
● Gram’s stain, they appear as Complete Vagus ● no hypoglycemia
○ large coccoid in tetrads Nerve Removal ● increased gastrin
Sarcinae ● iodine = brown
● associated with stasis and high
● titration
stomach acidity
○ to determine amount of acid produced
○ titrant = 0.1N NaOH
OTHER LABORATORY TESTS: GASTRIC FLUID ● sum of two titrations determines total
acidity of the specimen
Tests for Gastric Fluid Gastric
Acidity Indicators
● tubeless gastric analysis
Determination Topfer’s ● red to yellow at pH 3.5
○ no intubation
Reagent ● measures HCl
Procedure ● colorless to faint pink
Phenolph–
● ion-exchange resin (amberlite cation) at pH 8.4
thalein
mixed with blue dye azure A ● measures organic acid
○ given orally to px after caffeine
Diagnex Blue: ● lactic acid is normally absent
stimulation which stimulates acid
Tubeless ○ may be seen in small amounts,
secretion
Gastric ● indicates fermentation of CHO from
● presence of free HCl + ion exchange
Analysis foods like sour milk
resin with blue dye → blue dye detaches
Lactic Acid
from exchange resin → excreted in Tests
Determination
urine (blue) ● Modified Uffelman’s
● dye remains attached to ● Strauss
No Free
the resin and does NOT ● Kelling’s
Acid in the
appear in the urine within ● Simon modification of Kelling’s
Stomach
two hours

Transcribed by: Lustre, Aliping, Guarin, Villanueva (2024). 15

 ● blood is normally absent PHYSICAL EXAMINATION: DUODENAL CONTENTS
○ but small amounts may be observed
due to trauma during collection. Physical Examination: Normal Duodenal Fluid
● large amounts are observed in Volume ● 7–20 dL / 24 hours
○ peptic ulcer Color and Clarity ● clear and colorless
○ gastric cancer
Determination ○ esophageal or stomach varices ● active state = thin, watery secretion
of Blood ● basal state = thick, more viscid
○ chronic passive congestion Viscosity
○ due to low sialic acid and high
○ prolonged vomiting
fructose content
○ blood dyscrasia
Specific Gravity ● 1.007 – 1.042
Brown
● formed due to HCl pH ● 7.0 – 8.7
Hematin
degrading blood
Crystals
Viscosity

DUODENAL CONTENTS Increased ● associated with a decrease in

● duodenum function primarily for chemical digestion Viscosity pancreatic enzymes
○ receives secretions from the liver and pancreas
○ mucosa contains large numbers of goblet cells and
Brunner's glands MICROSCOPIC EXAMINATION: DUODENAL CONTENTS
■ secrete a watery fluid that is rich in mucus and
bicarbonate ions Pancreatic Juice Reveals the Following
● bile-stained columnar epithelial cells
Reasons for Duodenal Fluid Analysis
● bile-stained pus cell
● recommended as a standard practice for direct ○ indicates bacterial infection when increased
assessment of ● cholesterin and calcium bilirubinate crystals
○ pancreatic insufficiency ● parasites
○ intestinal maldigestion ○ amoebas
● measurement of pancreatic enzymes ○ flagellates
○ hookworm
○ e.g. lipase, amylase and chymotrypsin ○ Strongyloides
○ Ascaris
● quantitation of bile salts that facilitate intestinal
absorption Interpretation of Findings
Bile-Stained Cells and
● stasis
Crystals
Occasional Cholesterin or ● normal
Ca Bilirubinate Crystals ● increased = cholecystitis

SPECIMEN COLLECTION
● Levine tube
● lying on right side
● Meltzer-Lyon method
○ Mg sulfate is administered to patient to drain the
bile from biliary tract then duodenal juice is
aspirated

Transcribed by: Lustre, Aliping, Guarin, Villanueva (2024). 16

 Tests for Duodenal Contents Starch Tolerance Test
● stimulation of pancreatic function ● patient receives 100 g starch
○ measurement of pancreatic secretion ● two-day test of measuring amylolytic
after intravenous injection of activity of the pancreatic juice
secretin and pancreozymin Radioactive Iodine (I-131) Triolein Test
● serum amylase and lipase are also
● I-131 triolein
measured at the same time
○ fat consisting of oleic acid groups
○ since levels may rise in the presence of
esterified with 1 mole of glyceryl
pancreatic duct obstruction
● in the process of absorption of fat there is
Post-Pancreozymin (10-min period) Test for no appreciable loss of iodine
Volume 16 – 72 mL Malabsorption ○ thus, the amount of I-131 in the blood
or feces can be accepted as evidence
Bicarbonate Con. 8 – 24 mEq/L
of absorption or lack of it
Amylase 28 – 182/ IU/L at 37ºC
● absence of
Poor Absorption
Post-Secretin (60-min period) pancreatic enzymes
Secretin & of I-131 triolein
● mucosal inadequacy
Pancreozymin Volume 99 – 337 mL
Stimulation Maximum ● detects problem in pancreas through
Test 69 – 126 mEq/L
Bicarbonate Con. synthesis of trypsin

Interpretation ● negative control

Increased ● pancreatic Tube 1 ● boil & cool to deactivate trypsin
Post-secretin stimulation occurred and other digestive enzymes
Same volume ● problem in the ● positive control
Tube 2
Post-secretin stimulation ● add small amount of trypsin
● reliable index of ● patient’s sube
Reduction in Tube 3
pancreatic ● enhance alkalinity
Bicarbonate
dysfunction ● add duodenal secretion to all tubes
Above Normal ● indicates ductal Trypsin ● add gelatin to all tubes
Serum Enzymes obstruction ○ acts a substrate
● indicates ductal ○ trypsin degrades gelatin
Absence of Bile ● incubate overnight at 37°C
obstruction

● based on the principle that excess acid Dissolved

● trypsin is present
liberates CO2 from bicarbonate Gelatin
○ equivalent amount of H+ is measured to ● trypsin deficiency
Titration of determine bicarbonate concentration ● problem in pancreatic
Intact
Bicarbonate ● reagents: synthesis of trypsin
Gelatin
in Duodenal ○ hydrochloric acid, 0.05 N ○ common in chronic
Aspirate ○ sodium hydroxide, 0.01 N pancreatitis

Reduction in ● indicative of ● duodenal secretion + starch (substrate) =

Bicarbonate pancreatic damage starch is hydrolyzed
Amylase
● investigation of pancreatic function Starch Not
● absent amylase
Hydrolyzed
Glucose Tolerance Test
● patient receives 100 g glucose
Tolerance
● performed for detecting any destruction
Test
of the islets by either
○ chronic pancreatitis
○ pancreatic tumor

Transcribed by: Lustre, Aliping, Guarin, Villanueva (2024). 17

 SWEAT ● 50 mg collected sweat → transferred to a capillary for
● sweat analysis also is rarely done conductivity measurement using the same apparatus
● an emerging non-invasive technique used to provide furnished in iontophoresis
insights to genetic abnormality
Sweat Chloride Concentration
● common sweat diagnostic tests include testing for
● 5 – 35 mEq/L (mmol/L)
○ cystic fibrosis Normal
● average of 25 mEq/L
○ illicit drugs (sometimes)
Adults: 50-60 mEq/L ● suggestive of fibrocystic diseases in
Cystic Fibrosis (Mucoviscidiosis) Children: >60 mEq/L the absence of adrenal insufficiency
● congenital autosomal recessive disorder ● patients with malnutrition
● associated with dysfunction of the exocrine glands ● hyperhidrotic ectodermal dysplasia
● nephrogenic diabetes insipidus
○ pancreas ○ lacrimal glands ● renal insufficiency
○ salivary glands ○ sweat glands > 60 mEq/L
● glucose-6-phosphate deficiency
○ peritracheal and ○ mucosal glands of ● hypothyroidism
peribronchiolar the small bowel ● mucopolysaccharidosis
glands ○ bile ducts ● fucosidosis
● characterized by
○ signs of chronic pulmonary disease
○ pancreatic deficiency NOTE:
○ abnormally high levels of electrolytes in sweat ● tends to be slightly lower than those
Sodium
of chloride in patients with cystic
Concentration
SPECIMEN COLLECTION fibrosis, relative to sweat concentration
False-Negative ● patient with cystic fibrosis in the
Pilocarpine Iontophoresis Sweat Test presence of hypoproteinemia edema
● sweating is induced in a local area by the action of the ● cyclic fluctuation of sweat
drug pilocarpine, which is introduced into the skin by electrolytes in about half of a group of
means of an electric current (iontophoresis) premenopausal adult women
○ two electrodes are placed on the opposite sides of Premenopausal
● reaching a peak chloride con.
an arm or leg Adult Women
○ slightly under 65 mEq/L
(+) Electrode ● moistened with a pilocarpine ○ most commonly 5- 10 days prior to
(–) Electrode ● moistened with Na nitrate sol’n the onset of menses.

● a current of about 1.5 milliampere is passed between

Chloride Electrode Method
the electrodes
● measures directly the concentration of chloride ions
● some of the pilocarpine is carried into the skin, where it
in solution together with the required electric
stimulates the eccrine sweat glands
measuring instrument (expanded-scale pH meter)
○ sweat is collected on a filter paper pad or gauze
● sweat production is induced by
● amount of sweat collected is eluted from the pad with
○ iontophoresis
water and the solution analyzed for sodium and
○ applying a heating block (42 °C) to the skin for a
chloride concentration
short time
■ interscapular region is use for infants
TEST FOR CHLORIDE IN SWEAT
● when proper sweating has been induced, the chloride
electrode is placed on the moist skin and the reading
Cystic Fibrosis Analyzer using Conductivity Bridge
taken on the meter
● estimates the chloride content of the sweat by
measuring the electrical conductivity produced ● Cotlove chloridometer
Other Tests
● sweating is induced by pilocarpine iontophoresis and ● Microtitration with Mercuric Nitrate
for Chloride
sweat is collected in a small plastic cup taped over (Schales and Schales method)
the stimulated site

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 SPUTUM Volume
● an important aid in the diagnosis of pneumonia and
other infectious diseases involving the pulmonary Diminished ● acute bronchial irritation
system Secretion ○ acute bronchopneumonia
● goblet cells = produces mucus ● chronic irritation
Increased
○ bronchiectasis
Secretion
Composition ○ chronic bronchitis
● water ● pulmonary adenomatosis
● solutes ● alveolar cell carcinoma of the lung
○ electrolytes Copious Volume ● bronchiectasis
○ lipids ● abscesses and cavities (tuberculosis)
○ proteins that open into bronchi
○ enzymes
○ hormones Color
● bronchial epithelial cells and WBC
○ hallmark for sputum identification Greenish Gray ● indicates an inflammatory process
○ absence = saliva Purulent ● indicates increased WBC
Cheesy ● indicates cavitary tuberculosis
SPECIMEN COLLECTION ● acute heart failure
● expectoration Blood-tinged
● acute lung failure
○ an early morning specimen is “coughed up from
Rusty ● chronic passive congestion of lungs
deep down in the chest”
Purulent and ● infection with pyogenic (pneumococcus)
● bronchoalveolar lavage
Rusty organisms
○ should contain only lower respiratory tract
secretions
○ followed by washing Viscosity
● heated aerosol technique Watery ● saliva
○ performed to induce sputum release if patient is
Frothy
non-productive ● indicates pulmonary edema
(maybe blood tinged)
● swab from the posterior pharynx while attempting
● Klebsiella pneumoniae infections
to induce a cough Tenacious
● asthma
○ may be successful it is not always certain that such
Opaque and Foul
a specimen comes from the lungs and not from the ● indicates increased amount of pus
Smelling
nasopharynx
Caseous ● observed in cases of tuberculosis
PHYSICAL EXAMINATION
Odor
Physical Examination: Sputum
● suppurative, cavitary and necrotizing
● depends on the number of active Foul or Fecal
lesions
Volume mucus-secreting cells present in
the bronchial mucosa at a given time Specific Gravity
Color and Clarity ● colorless
Viscosity ● mucoid Purulent ● 1.015 – 1.060

Specific Gravity ● 1.004 – 1.008 (mucoid) Serous ● > 1.037

pH ● 6.5 – 7.0

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 Macroscopic Elements: Sputum Cell Types
Cheesy Masses ● fragments of necrotic lung tissues
● plugs from bronchi or bronchioles Bronchial Epithelial Cells
● consists of cellular detritus, fat, and ● these are sloughed-off bronchial cells
Dittrich’s Plugs bacteria ● reflects a pathologic state of the bronchi and lungs
● most are columnar and ciliated
● seen in chronic lung disease
● appears as degenerated bizarre-shaped cells showing
(bronchiectasis) “myelin degeneration”
● spirally twisted mucoid strands
enclosing Hematologic Cells
Curschmann’s ○ epithelial cells,
● chronic bronchitis, differential count
Spirals leukocytes (chiefly eosinophils)
○ Monocyte: 1-2%
○ sometimes Charcot-Leyden
○ PMN: up to 75%
crystals
○ Histiocytes: 5-20%
● consists of fibrin and mucus ○ Bronchial epithelial cells: 5-15%
● usually grayish white
● seen in asthma, chronic
● may be reddish brown because of Eosinophils
bronchitis and aspergillosis
blood pigment
Bronchial Cast ● common in sputum
● frequently so tangled that they Lymphocytes
● increased number reflects
cannot be recognized until they are & Monocytes
inflammatory process
floated in water over black
● often reflect patient’s
background resistance to antibiotics
● mixture of calcium carbonate and ● hallmark for sputum identity
Pneumoliths phosphate ● may contain
(Broncholiths, ○ dust and carbon particles
Lung Stone) ● seen in chronic lung disease
(tuberculosis) Macrophages ■ e.g., pneumoconiosis
○ fat droplets in lipid
● Echinococcus granulosus
pneumonia
○ hydatid sand in sputum ○ hemosiderin in chronic
● Toxocara canis heart failure
Parasite
○ visceral larva migrans ■ heart failure cells
● Paragonimus westermani Leukocytes
● Ascaris lumbricoides
Pneumoconiosis
● rounded masses (fungal balls) of ● characterized by the presence of large
opportunistic fungi such as masses number of phagocytic leukocytes
Mycetomas of Aspergillus (aspergilloma) containing various dust particles such as:
● seen in patients on long term
antibiotic or steroid therapy silicosis silica or sand
anthracosis coal
MICROSCOPIC EXAMINATION siderosis iron
calicosis marble or lime
Stained Preparation bagassosis silica-impregnated fibers
● Wright-Giemsa stain for hematologic cells asbestosis asbestos
● Gram stain for bacteria
● India ink or Gomori methenamine silver stain for fungi Asbestos Bodies
● Acid fast stain for acid fast organisms ● highly refractive bodies with a central
● Papanicolaou stain for cytologic examination needlelike crystal (greenish tinge) around
which is deposited with a yellowish
homogenous substance
● often have clubbed ends and show
segmentation
● (+) prussian blue reaction for iron

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 ● small number are normal but large
Erythrocytes numbers indicate hemorrhage or severe
infections (viral and bacterial)
● hexagonal, slender crystals with pointed
Charcot-Leyden
Crystals
ends
● believed to be derived from eosinophils
Elastic
● highly refractive and curlylike springs
Fibers

CHEMICAL EXAMINATION

Neuraminic Acid Assay

● glands in the bronchial tree produce acid
glycoproteins (form bulk of sputum glycoprotein) that
consists of several amino acid (serine, glycine, proline),
sialic acid and neuraminic acid
○ serves as the basis for the viscosity of sputum

● neuraminic acid is high and so is the

Asthma
viscosity
● has no neuraminic acid activity
Saliva
○ thus low viscosity

a-Antitrpsin
● a serum protein that is usually decreased in cases of
○ pulmonary emphysema in adulthood
○ liver disease in childhood
○ e.g., neonatal hepatitis, cirrhosis, hepatocellular
carcinoma

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 UNIT 5: SEROUS FLUIDS Permeability of Parietal Membranes
● the permeability of the capillaries in the parietal
SEROUS FLUIDS membrane
● a yellowish fluid enclosed within cavities ● regulates the rate of ultrafiltrate formation and its
○ pleural protein composition
○ pericardial ○ must be well regulated to only form only controlled
○ peritoneal amount of fluid
● each of these cavities is lined by two serous ● increased permeability of endothelium → allows more
membranes which are covered by mesothelial cells proteins into the cavities → accumulation of protein
Parietal enhances movement of fluid into cavity → effusion
● covers directly the cavity wall
Membrane
Hydrostatic Pressure [HP]
Visceral
● covers directly the organ within the cavity ● hydrostatic pressure in the capillaries forces the
Membrane
plasma ultrafiltrate to form in the cavity → initiate
serous fluid formation
● ↑ HP = ↑ ultrafiltration = effusion
Oncotic Pressure [OP] (Colloid Osmotic Pressure)
● generated by trapped proteins in the parietal membrane
○ opposes ultrafiltration
○ initiates fluid absorption back into the capillaries
● regulates or counteracts the increased in hydrostatic
pressure
○ OP only increases when HP increases
■ this mechanism produces a continuous
exchange of serous fluids and maintain the
normal volume of fluid between the membranes
FORMATION OF SEROUS FLUIDS
○ increased OP → excessive counteraction on HP →
● considered as an ultrafiltrate of plasma
no ultrafiltration → no fluid formed
○ composition similar to that of serum/plasma
● decrease OP → increased HP → more fluid being
● serves as a lubricant between the membranes
formed into the cavities → effusion
○ allows free movement of the enclosed organs
● produced and maintained through Lymphatic Pressure
○ plasma ultrafiltration in the parietal membrane ● absorption of fluid by the lymphatic system
○ absorption by the lymphatic system through the ● prevents the buildup of fluid in the cavities
visceral membrane ● if obstructed or damaged → interrupts reabsorptive
● protein process → fluid buildup in the cavities → effusion
○ rate limiting component differentiating plasma and
serous fluid SEROUS EFFUSIONS

FACTORS AFFECTING FORMATION Effusion

● an increase in fluid between the membranes due to a
disruption of the mechanisms of serous fluid formation
and reabsorption causes
● generally categorized into either transudates or
exudates
○ classifying them accordingly provides an initial
diagnostic step for any additional testing

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 TRANSUDATE EXUDATE
● testing is usually not necessary ● testing is performed for diagnosis
● formed as a result of non-inflammatory processes ● results from inflammatory process
that disrupts the balance in the regulation of fluid ○ infections: bacterial, tuberculous, viral, fungal
filtration and reabsorption ○ tumors/neoplasms
○ thus, low protein content ■ lung cancer for pleural exudat
● occurs in conditions that ■ hepatic cancer for peritoneal exudate
○ increase the hydrostatic pressure ○ systemic disorders: rheumatoid arthritis, SLE
■ congestive heart failure ○ gastrointestinal diseases
■ hepatic cirrhosis ○ trauma
■ shock ● such conditions increase the permeability of the
○ decrease oncotic pressure capillary endothelium in the parietal membrane
■ hypoproteinemia in nephrotic syndrome ○ proteins can easily pass through the membrane →
● effusion due to water crossing the membrane draws in more fluid → high protein content
● may also occur in a defect or decreased absorption of
the lymphatic system
○ lymphoma
○ metastasis
○ trauma
○ surgery

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 SPECIMEN COLLECTION AND PROCESSING Collection Tubes and Volume Required
Chemical Examination
SPECIMEN COLLECTION
● Plain Tube
Glucose ● 3 – 5 mL
Paracentesis ● Sodium Fluoride
● collection of serous fluid through percutaneous Protein
puncture of a body cavity for the aspiration of fluid LDH
● performed a licensed physician
Amylase ● Plain Tube ● 5 – 10 mL
○ should note the color and clarity of the fluid right
Triglyceride
after serous fluid collection, then record this
information on the test request form at the bedside Cholesterol
pH ● Heparin ● 1 – 3 mL
● aspiration on the chest wall through
Thoracentesis the pleural cavity to obtain pleural Microscopic Examination
fluid Cell & Differential ● EDTA
Pericardiocentesis ● to collect pericardial fluid ● 5 – 8 mL
Counting ● Heparin
● abdominal paracentesis Serous Fluids for ● Sodium Heparin ● 25 – 50 m
Peritoneocentesis ● collection of peritoneal fluids Cytology Tests
○ ascites; “ascitic fluid” (employing
Papanicolaou ● EDTA or Plain Tube ● 10 – 20 mL
Blood Collection staining and cell
● collected shortly before or after the paracentesis block may require)
procedure to enable comparison studies of the Microbiological Examination
chemical composition of the effusion with that of Gram Staining ● Sterile Container
the patient’s plasma ● SPS ● 10 – 20 mL
Bacterial Culture
● to compare the concentration of the biochemical ● Sodium Heparin
substances detected in order to determine the serous ● Sterile Container
fluid-plasma ratio Acid Fast Staining ● Sodium Heparin ● 15 – 50 mL
● enable classification of the effusion to either ● SPS
○ transudate or exudate
○ chylous or pseudochylous NOTE: Syringe
● syringe acceptable for glucose tests since it uses plain
Collection Tubes and Specimen Volume test tube
● the same type of specimen collection tube should be ● plain syringe for pH testing not acceptable
used for both the fluid specimen and the blood ○ syringe must be moistened with sodium heparin
collection for chemical analysis and maintained anaerobically at refrigerator
● at least 100mL of serous fluids should be collected, temp. to prevent false increase
transported and tested as soon as possible

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 SPECIMEN HANDLING Color
Temperature Pale to Light
● normal
● maintained at room temperature and transported Yellow
without delay ● presence of blood
● delayed transportation Red ○ traumatic collection
○ cellular degradation ○ hemorrhagic effusions
○ bacterial proliferation ● peritoneal fluid involving
○ chemical degradation Greenish ○ increased bile
○ pancreatic damage
■ e.g. decreased in glucose due to glycolysis
Brown ● peritoneal fluid involving liver abscess
● refrigeration at 4° C to 8°C
Black ● pleural fluid involving Aspergillosis
○ should NOT be used since it adversely affects the
viability of microorganisms NOTE: Bloody Serous Fluid
○ exception: samples intended for cytology ● due to traumatic collection or hemorrhagic effusions
examination
Traumatic Hemorrhagic
Clot Formation ● blood present at the hub
● normally, no blood or fibrinogen is present upon puncture but amount
● active bleeding from a ruptured blood vessel may occur of blood decreases as
● if amount of blood does
aspiration continues
and a hemorrhagic effusion is produced after a not decrease in
● most common cause
traumatic puncture procedure continuous aspiration
● hemothorax = bloody
● proper container to be used for specimen collection another
pleural fluid due to a
should be considered to prevent clot formation that traumatic puncture in the
entraps microorganism pleural cavity
● serous fluid Hct is 50% ● serous fluid Hct is 50%
SPECIMEN ANALYSIS: SEROUS FLUID greater than the blood lower than the blood

PHYSICAL EXAMINATION Parameter Chylous Pseudochylous

● the reference values are not available because in ● thoracic duct
healthy individuals, the fluid volume in these cavities is leakage
small and the fluid is not normally collected ● obstruction of ● chronic disease
lymphatic system ○ TB
● clarity and color are usually assessed by collecting Cause
○ exudative ○ RA
physician pleural effusion ○ vascular disease
Clarity ● hepatic cirrhosis
● vein thrombosis
● most frequently observed physical characteristics and
Onset ● sudden ● gradual
may vary accordingly
● milky
● cloudy and milky are usually associated with chyle, and ● milky white
Appearance ● greenish metallic
should be differentiated if either: ● bloody yellow
sheen
○ chylous
Chylomicrons ● present ● absent
○ pseudochylous
Cholesterol
● absent ● present
Clear ● normal Crystals
● increased WBC Triglycerides ● >110 mg/dL ● <110 mg/dL
○ usually due to inflammatory process ● acidic
Cloudy pH ● alkaline ○ due to crystals
○ e.g. tuberculosis (pleural fluid)
● increased fats, lipids, or chyle ● alkaline
● more opaque than cloudy Odor ● odorless ● odorless to foul
Milky ● increased chyle Reaction to
○ due to lymphatic obstruction or damage Sudan III ● strong ● weak
[stains triglycerides]
Shows Clearing
and Decrease in
Volume with Ether ● yes ● no
and HCl

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Transcribed by: Lustre, Aliping, Guarin, Villanueva (2024). 26
 CHEMICAL EXAMINATION ● glucose levels of <60 mg/dL
● performed both on serous fluid and blood to ● difference between the serum and serous
○ differentiate if transudate or exudate fluid is >30 mg/dL
○ establish diagnosis Exudative ○ rheumatoid arthritis
● specific diagnosis based on laboratory findings from ○ bacterial infections
serous fluids is limited to: ○ tuberculosis
○ malignant neoplasm
● when malignant cells are recovered
Malignancy Bacterial ● decreased glucose levels
and identified
● when characteristic lupus Infections ● increased lactate and pyruvate
SLE erythematosus cells are found during
the microscopic examination Amylase
Infectious ● when microorganisms are identified by ● diagnosis of pancreatitis & gastrointestinal damage
Disease gram stain or culture ○ increased amylase particularly in pleural and
peritoneal fluid
pH
● more significant in pleural fluids ● pancreatitis
Amylase Level of
○ an abnormally low pH associated with ● esophageal rupture (salivary
Serous Fluid is 1.5
parapneumonic effusions that require aggressive amylase)
–2.0x Higher Than
treatment ● gastroduodenal perforation
the Serum Value
■ esophageal rupture ● metastatic disease.
■ involve the parietal and visceral membranes,
producing pus in the pleural cavity Triglyceride
● to differentiate if chylous or pseudochylous
● placement of drainage tubes for
pH < 7.30 resolution of the effusion while pleural >110 mg/dL ● chylous
effusion with despite antibiotic therapy
<50 mg/dL ● pseudochylous
pH > 7.30 ● usually completely resolve following
antibiotic treatment alone ● requires performing lipoprotein
electrophoresis to identify the
Between
NOTE: presence of chylomicrons
50 – 110 mg/dL
● pericardial and peritoneal fluid pH measurements ○ (+) chylous effusion
currently have no clearly established clinical value ○ (–) pseudochylous

Total Protein and Lactate Dehydrogenase (LDH) Tumor Markers (Serological Test)
● to differentiate if transudate or exudate ● proteins used to evaluate pleural and peritoneal
effusions from patients who
● TP ratio <0.5 ○ have a previous history of tumor
Transudate
● LD ratio <0.6 ○ are suspected of having a tissue-producing tumor
● TP ratio >0.5
Exudate ● for identifying the
● LD ratio >0.6 ● colorectal
CEA primary source of
cancer
tumors producing ascitic
exudates
● source is from the
Glucose ● ovarian cancer ovaries, fallopian
● to differentiate if transudate or exudate CA 125 ● gastrointestinal tubes, or endometrium
● not significant for diagnosis of DM cancer
○ (+) CA 125
● Benedict’s Qualitative Test
○ (–) CEA
○ qualitative method most commonly used for
detecting glucose in these fluids CA 153 ● breast cancer
CYFRA 21-1 ● lung cancer

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Adenosine Deaminase (ADA) Total Cell Counts: Serous Fluids
● to identify effusion is due to Pericardial ● WBC count >1000 cells/uL ● pericarditis
○ tubercular effusion in pericardial fluids
○ tubercular peritonitis from peritoneal fluid ● WBC count >500/uL
● bacterial
Peritoneal ○ with predominance of
peritonitis
Tubercular neutrophil
● pericardial fluids > 40 U/L
Effusion ● neoplasms or
Tubercular Pleural ● RBC count >10,000/uL trauma of the
● peritoneal fluid > 36 U/L
Peritonitis pleura

BUN to Creatinine Ratio NOTE: Diluting Fluids

● used to evaluate peritoneal fluids ● used when counting cells for bloody effusion
● commonly used
Increased ● ruptured or punctured urinary ○ hypotonic saline
BUN: Creatinine bladder during a paracentesis ○ saline with saponin
● acetic acid diluents
Alkaline Phosphatase (ALP) ○ must NOT be used because they cause cells to
● highly diagnostic of intestinal perforation clumps, causing counting to be difficult
● for obstructive type of liver disease
WBC Count Computation
Bilirubin
WBC Population Clarity
● measured when leakage of bile into the peritoneum
Low Clear
is suspected after trauma or surgery
● a test for total bilirubin is acceptable since bile contains
primarily conjugated bilirubin

Rivalta’s Test Moderate Hazy to Cloudy

● to determine presence of serosa mucin
● involves dropping a specimen into a
solution containing 100 mL water and a
drop of acetic acid
○ normal body fluid does not produce
any turbidity, . High Milky
● figure presented shows a heavy cloud
that slowly floats down to the bottom of
the tube (drop-or jelly-fish-like), *total cell count is averaged

indicating that the fluid is exudate

Differential Count
Transudate ● slight clouding ● routinely performed on serous fluids, preferably on
Exudate ● heavy precipitate ○ Wright stained
○ cytocentrifuged specimen
MICROSCOPIC EXAMINATION ○ slides prepared from the sediment of centrifuged
specimen
Total Cell Count ● fluid smear
● usually performed to differentiate transudates from ○ prepared using similar procedure used in preparing
exudates blood smear and stained with Wright’s stain
Transudate ● WBC count of >1,000/uL
Exudate ● RBC count >100,000/uL

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 Differential Count: Serous Fluid
● often not performed because a variety of
conditions can produce the same cell
differential
Pericardial
● high percentage of neutrophils
○ bacterial endocarditis
○ WBC count >1000/uL
● neutrophils predominate (>25%) in most
exudates
○ bacterial infection
Peritoneal ● lymphocytes predominate
○ tuberculosis
○ neoplasms
Malignant Cells
○ lymphatic obstruction,
● neutrophils predominate in about 90% of ● characterized by
○ nuclear and cytoplasmic irregularities
effusions caused by
○ hyperchromic nucleoli
○ pneumonia ○ cellular clumps with cytoplasmic molding (community
○ pancreatitis borders)
○ pulmonary infarction ○ abnormal nuclear to cytoplasmic ratio
Pleural ● lymphocytes predominate in 90% of ● malignant effusions mostly contains large,
effusions caused by ○ irregular adenocarcinoma cells
○ tuberculosis ○ small or oatcell carcinoma cells resembling large
○ viral infections lymphocytes
○ malignancies ○ clumps of metastatic breast cancer cells
○ autoimmune diseases (RA and SLE) ● present in
○ primary adenocarcinoma
Other Cells ○ small cell carcinoma
Eosinophils ● peritoneal fluid
● increased eosinophils (>10 %) observed in cases of ○ malignant cells of ovarian, prostatic and colonic origin
○ trauma with presence of air or blood in the cavity are frequently seen
○ allergic reactions
○ parasitic infections and SLE
Mesothelial Cells
● lining cells of the serous membrane
● in pleural fluids, normal and reactive forms of
mesothelial cells have no significance
○ but they may be increased in pneumonia and
malignancy
● tuberculosis
○ decreased mesothelial cells
○ increased plasma cells

● often appear as singles

● small or large round cells with abundant
Normal blue cytoplasm
● single round nuclei with uniform dark purple
cytoplasm
● often appear in cluster
● have varying amounts of cytoplasm
Reactive ● eccentric nuclei, prominent nucleoli
● multinucleated
○ closely resembling malignant cells

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Cytologic Examination MICROBIOLOGICAL EXAMINATION: SEROUS FLUID
● a test specific in the diagnosis of primary and ● acid-fast stains, adenosine deaminase, and cultures
metastatic neoplasm may also be requested for tuberculosis
○ requires large volumes for fluids: 10 – 200 mL ● molecular testing using PCR is a more sensitive
○ fluid is concentrated to increase yield of cells and specific method for the diagnosis of bacterial
○ a cell block and cytospin smears can be prepared infections, including M. tuberculosis
● for detection of psammoma bodies in peritoneal fluid
Staining Techniques
● group of cells made up of concentric
● in smear preparations, concentrated or
striations of collagen-like materials
cytocentrifuged specimen is recommended for
● indicative of cancer in peritoneal fluid
immediate identification of microorganisms
○ ovarian cancer
● sensitivity to the stain depends on the
○ thyroid cancer
○ appropriate collection, processing, and handling of
Psammoma the fluid specimen
Bodies ○ technical competence of the microscopist reading
the smear

Staining Techniques
Stain Gram’s Stain Acid-Fast Stain
● positive in 30% to ● positive in 10% to
Effusion 50% of bacterial 30% of tubercular
effusion effusions
Pericardial ● bacterial endocarditis ● tubercular effusion
Fluid
Peritoneal ● bacterial peritonitis ● tubercular peritonitis
Fluid

Culture
● aerobic and anaerobic culture
○ sensitivity of positive cultures depends on the origin
of the fluid and the organism present
○ inoculation of fluid into blood culture bottles
immediately at the bedside increases the recovery
of anaerobic organisms

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 ● 80% of all bacterial effusions Lysozyme Test
Culture ● 50% – 70% peritoneal tuberculosis cases ● for the diagnosis of tuberculous effusion
Positive ● 50% of all pericardial effusion cases ○ screening procedure for cases of TB
● 30% of all pleural tubercular effusion cases ● transformation of monocytes to
macrophages and epithelial cells
Tuberculosis
● increase in lysosomal enzymes
including lysosome

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 UNIT 6: AMNIOTIC FLUID 12 weeks ● 25-50ml
37 weeks ● 1200ml
AMNIOTIC FLUID (AF)
● 800- 1200ml
● liquid medium that bathes a fetus throughout its Peak
● decreases just before the delivery
gestation
● product of fetal metabolism, with its components
FORMATION OF AMNIOTIC FLUID
providing information on the fetus’ condition and
● contributors
development
○ maternal plasma
● found in the amnion
○ fetal metabolism (e.g. fetal urination)
● formed from the metabolism of fetal cells & fetal urine

● a sac or membrane composed of a Trimester Major Contributor Average Volume

Amnion single layer of cuboidal epithelial cells ● 25 – 50 mL
1st ● maternal plasma
● surrounds the fetus Trimester ● ≈ 35 mL
● fetal metabolism
2nd ○ possible minimal
amniotic fluid from ●2
Trimester
lungs, GIT and
umbilical cord
● 800 – 1200 mL
3rd ○ gradually
● fetal metabolism
Trimester decreases after
delivery

As the Gestation Progresses

● water and solutes exchange between the fetus and
1st Trimester ● week 1 to week 12 of pregnancy the amniotic fluid through:
2nd Trimester ● week 13 to week 27 of pregnancy ○ fetal urination
3rd Trimester ● week 28 to week 40 of pregnancy ○ intestinal absorption following fetal swallowing of
amniotic fluid
○ capillary exchange in the pulmonary system alveoli,
Primary Functions of the Amniotic Fluid
as the alveoli of the fetal lungs are bathed with
1. exchange of water and chemicals bet. the fetus and
amniotic fluid.
maternal circulation
○ electrolytes
Later Stages of Pregnancy
○ nitrogenous compounds
● fetal swallowing & urination
○ proteins
○ play a significant role in the amniotic fluid volume
○ enzymes
and composition
○ lipids and hormones
● similar exchange occurs between amniotic fluid and
2. permit proper fetal lung development
maternal plasma
3. stabilization of temperature to protect the fetus from
○ maternal plasma removes metabolic waste
extreme temperature changes
products and replenishes them with water,
4. allows fetal movement;
nutrients, and electrolytes
5. serves as a protective cushion for the fetus
● removes water and electrolyte from the
Amniotic Fluid Volume amniotic fluid and replaces them
● regulated by: Fetal
through urination with metabolic
○ (a) production from fetal urine and lung fluid Swallowing
by-products such as urea, creatinine,
○ (b) absorption from fetal swallowing and uric acid
○ (c) intramembranous flow.
■ absorption of amniotic fluid water and solutes
into the fetal blood

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 AMNIOTIC FLUID REGULATION Final Chemical Composition of Amniotic Fluid
● begins once fetal amniotic fluid begins ● amniotic fluid contains different components and may
vary once fetal production begins
Fetal Swallowing of Amniotic Fluid
● fetus must begin swallowing amniotic fluid ● ultimate source of amniotic fluid water and
Placenta
● continuous cycle solutes
● used for the assessment of the fetus’
Intramembranous Flow condition and development
● ⬆ conc. once fetal production begins:
● absorption of amniotic fluid in the placenta through the
○ creatinine
blood vessels of the fetus ○ urea
○ uric acid
Transmembranous Flow Biochemical ● ⬇ conc. once fetal production begins:
● absorption of amniotic fluid through the amnion Substances ○ glucose
○ protein
Transcutaneous Flow ● unaffected substances:
○ electrolytes
● absorption through skin of fetus
○ enzymes
Variations in Volume of AF ○ hormones
○ metabolic end products
● > 1200 mL AF
● (1) alpha-fetoprotein
● associated with ⬇ fetal swallowing Biochemical
● (2) acetylcholinesterase
● detected through ultrasound Markers
○ amniotic fluid index ● detect neural tube defects

Conditions: Maternal Urine v.s. Amniotic Fluid

○ fetal distress ● clear distinction should be made so as to determine
○ neural tube defect (NTD) whether the fluid collected is amniotic fluid or whether it
■ open spina bifida = underdeveloped is urine aspirated from the bladder
Poly- spinal chord
hydramnios ■ anencephaly = failure of skull to close Differentiation Maternal Urine Amniotic Fluid
properly ● > 4 mg/dL
○ rapidly growing fetal edema Creatinine ● as high as 10 ● ≤ 4 mg/dL
○ hydrops fetalis mg/dL
● > 30 mg/dL
Secondary Conditions:
Urea ● as high as 300 ● ≤ 30 mg/dL
○ fetal structural anomalies
mg/dL
○ cardiac arrhythmias (fetal heart failure)
Glucose ● essentially (–) ● ≤ 46 mg/dL
○ congenital infections
○ chromosomal abnormalities. Protein ● essentially (–) ● 2 – 8 g/L
● < 800 mL AF Fern’s Test ● negative ● positive
● associated with ⬆fetal swallowing

Conditions:
○ urinary tract deformities
○ membrane leakage
Oligo-
○ donor twin transfusion syndrome
hydramnios
○ congenital malformation
○ premature rupture of the amniotic
membrane
○ umbilical cord compression
■ decelerated heart rate

■ fetal death

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 SPECIMEN PROCESSING: AMNIOTIC FLUID Methods of Amniocentesis
● AF is reflective of fetal development ● much preferred
Transabdominal ● sterile syringe inserted through
Fetal Gestation ● allow early antenatal diagnosis of
Amniocentesis abdomen
(15 – 18 weeks) genetic and congenital disorders
● recommended due to lesser risk
● assess fetal pulmonary maturity
32 – 42 weeks
later in pregnancy ● insert tubing through vaginal canal
Vaginal ● has higher risk for infection
Other Diagnosis Assessed in AF: Amniocentesis ● more prone to contamination with
● (1) estimate the degree of fetal distress caused by vaginal secretions
● affect fetal lung maturity tests
isoimmunization or infection
● (2) detection of metabolic disorders & chromosomal
Reasons to Perform Amniocentesis
abnormalities
● early diagnosis of genetic and congenital disorders
○ karyotyping
● assessment of fetal lung maturity
○ FISH (Fluorescence in Situ Hybridization),
● assess fetal distress
○ SKY (Spectral Karyotyping) and DNA;
● diagnose metabolic disorders
Blood Tests for Maternal Serum alpha-Fetoprotein (AFP)
Pregnancy Stage and Conditions Detected
Triple ● maternal AFP
Screening ● HCG ● detect neural tube defect (NTD)
Test ● unconjugated estriol ● mother’s age is 35 or more at delivery
● family history of chromosomal
● maternal AFP
Quadruple abnormalities
● HCG
Screening ○ e.g., Down syndrome
● unconjugated estriol
Test ● allow early antenatal diagnosis of
● inhibin A
genetic and congenital disorders
*HCG- human chorionic gonadotropin
○ parent with known chromosomal
abnormality
SPECIMEN COLLECTION: AMNIOCENTESIS
○ previous child with chromosomal
Amniocentesis abnormality
● collection is performed through amniocentesis 15 – 18 weeks ○ parent is a carrier of a metabolic
● performed after 14 weeks gestation disorder
● one must consider the purpose of doing the procedure ● history of genetic diseases
and when it is done ○ sickle cell
○ Tay-Sachs disease
10-20 mL ● normal collection volume
○ Hemophilia
30 mL ● maximum volume that can be collected ○ muscular dystrophy
● discarded ○ sickle cell anemia
● contaminated with: ○ Huntington’s disease
2-3 mL ○ maternal blood ○ cystic fibrosis
○ tissue fluid ● elevated maternal alpha fetoprotein
○ cells ● history of three or more miscarriages
● upon collection, it is placed in an amber-colored ● specifically for assessment of genetic
16th week
container or aluminum foil defect/ chromosome analysis
○ protect the specimen from sunlight that can cause Near End of ● specifically for assessment of uterine
bilirubin photo-oxidation to biliverdin 2nd trimester growth restriction
● NOTE:
○ glass containers should be avoided because
cells will adhere to it, affecting the test result
○ advised to discard 2-3 mL to eliminate the
contaminated portion

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 ● primarily used to assess the pulmonary PHYSICAL EXAMINATION: AMNIOTIC FLUID
and overall health status of the fetus
● indicated to evaluate: COLOR
○ fetal lung maturity (FLM)
Colorless to
○ fetal distress ● normal AF color
20 – 42 weeks Pale Yellow
■ cases of hemolytic disease of the

newborn caused by Rh blood type ● an indication of traumatic tap,

incompatibility abdominal trauma, or intra-amniotic
○ possible toxemia, diabetes mellitus and hemorrhage
infection during pregnancy Kleihauer – Betke test
● blood (+) specimen should be
Blood-Streaked
centrifuged immediately to remove
SPECIMEN HANDLING (Red / Red
any intact red blood cells before
● process ASAP Tinge)
hemolysis occurs
● hemolysis causes the formation of
For Fetal Lung Maturity Test (FLM)
oxyhemoglobin which can interfere
● specimen must be placed on ice for delivery to the
with several chemical analyses
laboratory
●
● refrigerated before testing within 72 hrs
Yellow or ● related to bilirubin in cases of hemolytic
● transporting the specimen at room temperature is
Amber colored disease of the newborn (HDN)
acceptable since the specimen is stable for 16-24 hrs
● specimen may or may not be centrifuged ● (+) biliverdin
● (+) meconium
● 500-1000 x g, no longer than 5 mins ○ gelatinous or mucus-like material that
● low centrifugation speed is applied forms in the fetal intestine due to
L/S ratio
● with the recommended filtration method to swallowed amniotic fluid and fetal
prevent loss of phospholipids intestinal secretions
● 1500-2000 x g for 5-15 min Dark Green ○ infants excrete meconium as their
Spectral
● to maximally clear the supernatant of first bowel movement after birth
Analysis
turbidity
● NOTE:
For Cytogenetic Study ○ fetal distress can cause premature
● maintained at room temperature or body release of meconium into the
temperature to prolong the life of cells needed for amniotic fluid
analysis Dark
● fetal death
● centrifugation = 140 x g Red-Brown

For Chemical Assay Kleihauer-Betke Test

● need to separate cellular elements from debris ASAP to ● determines the source of blood (maternal or fetal)
prevent distortion of chemical constituents by cellular ● upon elution with an alkaline buffer, fetal cells would
metabolism and disintegration. demonstrate resistance due to presence of Hgb F
● centrifugation = 500 to 1000 x g, 5 mins ○ microscopically, they appear as large, red and
● bilirubin quantitation refractile cells
● glucose determination ● acid elution testing
○ adult Hgb = sensitive = ghost cells
○ fetal Hgb = resistant = bright pink

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 ● demonstrate resistance due to presence of CHEMICAL EXAMINATION: AMNIOTIC FLUID
Hgb F I. Test for Fetal Age
Fetal Cells
● microscopically, appear as large, red and A. Creatinine Determination
refractile cells II. Test for Fetal Distress
A. Pigment Analysis
Maternal ● appear uncolored ghosts or dull red
1. Hemoglobin Analysis
Cells ● contains poorly stained intermediate cells 2. Bilirubin Analysis
B. Alpha-Fetoprotein
C. Acetylcholinesterase Level
● alternate methods
D. Bacterial Count
○ immunofluorescence staining of Hgb F III. Test for Fetal Lung Maturity (FLM)
○ electrophoresis or isofocusing of Hgb F A. Lecithin-Sphingomyelin Ratio
○ flow cytometry B. Phosphatidylglycerol (PG) Determination
1. Amniostat-FLM
○ MCV examination
2. Thin Layer Chromatography
■ fetal cells = 140 fL C. Foam Stability Index
■ adult cells usually = < 100 fL D. Microviscosity: Polarization Assay
E. Lamellar Body (LB) Count

TESTS FOR FETAL AGE

Creatinine Determination
● serum method: Jaffe’s reaction
● e.g. 2 mg/dL at 37 weeks = fetal growth not fully
achieved

1.5 – 2.0 ● normal level

mg/dL ● fetal age < 36 weeks
> 2.0
● fetal age > 36 weeks
mg/dL

TESTS FOR FETAL DISTRESS (QUANTITATION TESTS)

Pigment Analysis
● color variation association

Hemoglobin Analysis
CLARITY ● gestational age increase = Hgb increase
● amniotic fluid would usually have a slight to moderate ○ too much elevation of Hgb = intraamniotic hemorrhage
turbidity.
● turbid sample is due to ⬆ number of: Bilirubin Analysis
○ vernix caseosa (major contributor) ● normal bilirubin level = 10-30 mg/dL
■ cheese like white substance coating fetus for ○ low and undetectable
fetal development ● ⬆ bilirubin level
○ fetal cells ○ due to maternal antibodies crossing the placenta and
○ hair destroying the fetal cells
● to remove particulate matter causing the turbidity,
perform centrifugation and filtration Spectrophotometric Analysis
● measurement of amniotic fluid bilirubin
● normally, the OD is highest at 365 nm and decreases
linearly to 550 nm
● (+) bilirubin = 450 nm (max absorbance)

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 ● NOTE:
Optical Density ○ oxyhemoglobin (410 nm)
■ interference in bilirubin detection
365 nm ● increased
550 nm ● declines
450 nm ● increased bilirubin
410 nm ● increased oxyHb

Liley Graph
● used as a basis of:
○ risk of hemolysis
○ frequency of amniocentesis
○ whether fetal transfusion is needed

Alpha-Fetoprotein (AFP) Test

● major protein produced by the fetal liver during early
gestation (before 18th week)
○ also, from fetal intestine and yolk sac
○ normally low during pregnancy
● maximal production observed at 12th - 15th week then
eventually declines
● also found in the maternal serum due to
○ combining fetal-maternal circulation
○ AF diffusion
○ excretion of fetal urine
● both AF and maternal serum are collected for
comparison

⬆AFP ● family history of previous neural tube

Maternal defects
● ‘low risk zone’ Serum ● history of multiple pregnancies
○ absence or mild hemolysis ⬆AFP ● fetal neutral tube defects
Zone 1 ○ amniocentesis: every 3 weeks Amniotic ○ anencephaly
○ no fetal transfusion needed Fluid ○ spina bifida
○ mother may proceed to full term delivery
● ‘low intermediate risk zone’ ● unit of MoM (multiple of median)
○ moderate hemolysis ○ concentration of AFP is measured
○ amniocentesis: every 2 weeks
○ no fetal transfusion needed ● normal
Zone 2 < 2 MoM ● reference level for a given week of
● ‘high intermediate risk zone’
○ moderate hemolysis gestation
○ amniocentesis: once a week ● abnormal
> 2 MoM
○ fetal transfusion is needed ● value is 2x than the median value
● ‘high risk zone’
○ severe hemolysis Acetylcholinesterase Level (AChE)
Zone 3 ○ amniocentesis: more than once a week ● confirmatory test for fetal neutral tube defect (NTD)
○ fetal transfusion is needed after an elevated amniotic fluid AFP level
○ cesarean section ● more specific for NTD
● cholinesterase
○ component of nerve tissue

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⬆AChE ● neural tube defect (NTD) L/S Ratio
False ⬆ ● bloody specimen >2 ● fetal lung immaturity
AChE ● naturally, blood contains AChE <2 ● FLM

Bacterial Count ● L/S ratio is a better predictor of the maturity of fetal

● bacterial infection during pregnancy can affect lungs than of immaturity
development of the fetus ○ some infants with an L/S ratio > 2.0 still develops
RDS, despite having a mature L/S ratio
> 50 ul of AF ● (+) bacterial infection
○ in contrast, some infants with an L/S ratio between
1.5 and 2.0 do not develop RDS and are falsely
TESTS FOR FETAL LUNG MATURITY
identified by the L/S ratio as having an “immature”
● performed to detect respiratory distress syndrome
pulmonary system
(RDS)
○ most frequent complication of early delivery
Disadvantages and Interferences of L/S Ratio
○ cause of mortality in the premature infant
○ usually result from insufficient production of lung Blood ● falsely lowers the ratio
surfactant and structural immaturity of fetal lungs Meconium ● falsely increases the ratio

● produce and secrete about 90% of

Type II Phosphatidyl Glycerol (PG) Determination
phospholipids and 10% proteins in the
Pneumocytes ● used in place of the L/S ratio to detect respiratory
form of lamellar bodies
distress.
Lamellar ● release surface-active compounds known
Bodies as surfactants ● phospholipid that enhances the spread of
● reduce the alveoli's surface tension surfactants across the alveolar surface
○ prevent them from collapsing during Phosphatidyl ● 35th week = increase production
Surfactants expiration -glycerol ○ parallel to lecithin
○ reduce the amount of pressure needed ● delayed production
to reopen the alveoli during inspiration ○ occurs with diabetic mothers

● NOTE: Amniostat-FLM
○ Fetal Lung Maturity (FLM) Tests are only ● based on the principle of an immunologic agglutination
performed at 32 weeks of gestation ○ anti-PG agglutinates PG
● results are based on the agglutinate size and the degree
of background clearance
Lecithin-Sphingomyelin Ratio
● L/S ratio is considered as the reference method for
● FLM
FLM tests Positive (+) ● clinically valuable, indicating pulmonary
● utilizes thin layer chromatography (TLC), which was maturity
later on replaced by phosphatidylglycerol immunoassay, ● fetal lung immaturity
fluorescence polarization, and lamellar body density Negative (-) ● provides no useful information due to high
procedures number of false negative produced

● primary component of surfactants ● more rapid for the assessment of FLM

● early weeks = low production ● NOT affected by specimen contamination with blood and
● 35th week meconium
Lecithin ● CANNOT be used until late pregnancy (>35 week’s
○ noticeable increase in its production
gestation)
occurs for the stabilization of fetal
lung alveoli
Thin Layer Chromatography
● 26th week = increase production
● more labor-intensive test as compared to the
● 35th week = decrease production
Sphingomyelin Amniostat-FLM
● serves as a control based on lecithin ● reference method of respiratory distress syndrome
increase (RDS)

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Foam Stability Index (FSI) Test/ Shake’s / Foam’s Test Low ● dye bound to surfactant
● mechanical screening test for lung surface Polarization ● longer fluorescence lifetime
lipids/surfactants (e.g., phospholipid) based on their High ● dye bound to albumin
physical or functional characteristics observed in Polarization ● decreased fluorescence lifetime
amniotic fluid
● good correlation with L/S ratio and PG test ● surfactant/albumin ratio expressed in milligrams
surfactant to grams albumin
Procedure: Foam Stability Index Test
● shaking vigorously equal parts of amniotic fluid and ● (+) FLM
1 > 55 mg surfactant
95% ethanol for 15 secs ● correlates well with a L/s ratio of
per gram albumin
2 ● stand for 15 mins 2.0
< 39 mg surfactant
● immature
per gram albumin
Results and Interpretation: Foam Stability Index Test
40-54 mg surfactant ● cannot be declared “mature” or
● continuous line of bubbles around the per gram albumin “immature” indicators
outside edge
● contamination of lipids
Positive ○ indicates sufficient amounts of Falsely Decreased
● unfiltered sample
(+) phospholipids available to reduce the
surface tension of the fluid even in the
NOTE:
presence of alcohol (antifoaming agent)
● polarization assay requires at least 1 ml of amniotic
fluid
Procedure: FSI Modification
● fluid should be filtered prior to examination
● mixing equal volumes of amniotic fluid with differing ○ prevent sedimentation of the lipids and reporting a
volumes of ethanol
falsely decreased result
1 ○ concentration of ethanol in each tube represents
the possible index values ● specimens contaminated with blood, meconium, maternal
■ ethanol concentration = 0.43 - 0.55 urine and bilirubin should NOT be used
2 ● followed by vigorous shaking
Lamellar Body (LB) Counting
● identify the highest concentration of ethanol with a
● surfactants responsible for FLM are stored in the
stable foam present
3 ○ stable foam is one in which the bubbles remain form of structures termed lamellar bodies
around the entire meniscus of the tube 15 minutes ● secretion begins at 20-24th weeks gestation and by
after shaking the 3rd trimester
● normal LB count = 50,000 to 200,000/uL
Results and Interpretation: FSI Modification ● number of lamellar bodies correlates with the amount of
phospholipid present in the fetal lungs
> 0.48 ● (+) FLM
○ FLM = ⬆ lamellar body = ⬆phospholipids & L/S
Falsely ● (+) blood and meconium ratio
High Index
Procedure: Lamellar Body Counting
Microviscosity: Polarization Assay
1 ● AF is usually centrifuged at 2000x g for 10 mins
● based on the measurement of the decrease in
microviscosity of amniotic fluid due to the presence ● optical density (OD) is measured at 650nm
of phospholipids 2 ○ eliminates optical interference from hemoglobin
○ (+) phospholipids = ⬇microviscosity of AF but not meconium
● employed by the Abbot TDx analyzer with the
TDx/TDXFLx FLM II assay system Results and Interpretation: Lamellar Body Counting
● quantitative measurement of the ratio of surfactant to
● (+) FLM
albumin in amniotic fluid for assessment of FLM
● correlates:
● polarization of the fluorescent dye that was 0.150
○ L/S ratio = > 2
combined with the surfactant and albumin is
○ (+) phosphatidylglycerol
measured

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● alternative method: Automated Platelet Channels
○ lamellar body counts can also be obtained with the
use of automated platelet channels of hematology
analyzers

Blood ● falsely increase or decrease counts

Meconium ● falsely increases count

Results and Interpretation:

LB Count - Automated Platelet Channels
● (+) FLM
≥ 32,000 or
● correlates:
more
○ L/S ratio = > 2
particles/ul
○ (+) phosphatidylglycerol

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 CYTOLOGIC STUDIES: AMNIOTIC FLUID Results and Interpretation: Nile Blue Stain
● cytologic studies are performed for:
○ fetal sex chromatin determination > 20 orange ● (+) FLM
staining cells ● pregnancy over 36 weeks
○ enzymatic studies
○ cell culture (simultaneous for 5-10 days)
○ karyotyping (for fetal cell)
● nile blue stain
○ commonly employed stain in cytologic study of AF
○ two-color stain

● keratinized
● superficial anucleated cells
Orange
● ⬆ rapidly during the last 4-5 weeks of
Stained Cells
pregnancy
●
● non-keratinized
Blue Stained
● anucleated squamous cells
Cells
● deeper parabasal-like cells

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 UNIT 7: SYNOVIAL FLUID FORMATION OF SYNOVIAL FLUID
● synovial fluid = plasma ultrafiltrate + secretion of
SYNOVIAL FLUID synoviocytes
● ultrafiltration of plasma across the synovial
Synovial Joint membrane
● strength of joints is provided by bones which are ○ non selective = almost all components can pass
capped by a layer of subchondral bone and then by a through = almost the same composition with
thin layer of cartilage plasma
○ excludes high MW substances (proteins)
● made from building blocks called
Cartilage ● secretions by synoviocytes
proteoglycans
○ can synthesize collagenase, hyaluronic acid,
Proteoglycans ● manufactured by chondrocytes proteins
● major cell type that are very good in ● chemical constituents are similar to plasma (glucose,
Chondrocytes
retaining water uric acid)
● total protein & immunoglobulin conc.
● strong healthy cartilage ○ only 1/4 to 1/2 of that of plasma
○ high-water content ● healthy synovial fluid = very few cells
○ provides a virtually frictionless surface for the bones
● cells within the synovial membrane
to glides against
Synoviocytes ● manufacture hyaluronic acid and
many other essential molecules
● makes synovial fluid very thick and
slippery
Hyaluronic Acid ● helps the synovial fluid contribute to
the frictionless movement (lubricant)
● lessen the shock of joint compression

SPECIMEN COLLECTION, PROCESSING

AND ANALYSIS

Synovial Fluid Reference Intervals

Parameters Normal Value
Physical Examination
Volume ● 0.1-3.5 mL
Synovial Fluid (SyF)
Color ● colorless to pale yellow
● bathing the ends of the two articulating bones
Clarity ● clear
(diarthroses or synovial joints) & lined by a synovial
membrane Viscosity ● able to form a string 4-6 cm long
● thick fluid that is similar in composition to plasma with Spontaneous Clot
● no
the exemption that it contains high amount of Formation
hyaluronic acid Microscopic Examination
● provides ALL the nutrients needed to nourish the WBC Count ● <200 cells/ul
cartilage enclosing it RBC Count ● <2000 cells/ul
Monocyte &
● 60%
Importance for Synovial Fluid Macrophage
● provides strength to articular cartilage Lymphocytes ● 30%
○ reduces friction between moving joints Neutrophils ● 10%
● reduces joint compression Crystals ● none
● for diagnosis of joint disorders

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 Chemical Examination “Dry Tap”
Glucose: ● a small yield of synovial fluid
Plasma-Synovial Fluid ● <10 mg/dl ● fluid may be in the needle bore
Difference ● collected when effusion is not present
Total protein ● 1-3 g/dl
Lactate ● 9-33 mg/dl SPECIMEN HANDLING AND TRANSPORT
Hyaluronate ● 0.3-0.4 g/dl 1 Chemistry or Immunological
2 Microscopic Studies
3 Microbiology

Sodium Heparin
● best anticoagulant for synovial fluid
● 25 units per milliliter of synovial fluid or liquid EDTA
● anticoagulants prevent clotting if fibrinogen is present
but do not form crystals

Oxalate & Powdered EDTA

● must be avoided
● produce crystalline structures
● resemble monosodium urate crystals

Synovial Fluid Analysis and Specimen Requirements

Analysis/
Test Required Volume
Examination
● color
Physical ● clarity ● 1 mL
● viscosity
● total cell count ● 2 - 5 ml
● differential count ○ sodium heparin
Microscopic ● crystal identification ○ liquid EDTA
● cytologic studies ● 5 - 50 ml
e.g., malignant cells ○ sodium heparin
● 1 - 3 ml
● glucose
○ NaF or red top
● total protein
Chemical ● uric acid
● 1 to 3 ml
● lactate
SPECIMEN COLLECTION: ARTHOCENTESIS ○ red top
● triglyceride
● cholesterol
Arthrocentesis ● 3 - 10 ml
● collection of SyF by the physician using sterile needles ○ sodium heparin
and syringes. Microbiology ● culture
○ SPS
● px should be fasting for a minimum of 4 to a ○ red top
maximum of 8 hours
○ allow the chemical constituents of the plasma and ● NOTE:
the synovium to equilibrate ○ there is NO upper limit to the amount of fluid that
● blood sample can be submitted
○ can also be collected just to ensure the differences ○ large volumes of SyF =⬆ the recovery of cellular
between the blood glucose serum and the synovial elements & microbial organisms
fluid

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 Insufficient Sample Tube Order Results and Interpretation: Mucin Clot Test/ Ropes Test
Amount Tube Test Good ● solid clot
● culture Fair ● soft clot
0.5 - 1mL ● sterile
● gram stain
Low ● friable
● plain or lavender ● WBC cell count
0.5 - 1 mL Poor ● no clot
top ● differential count
0.5 - 1 mL ● gray top ● glucose
● protein
● plain or lavender
1 - 2 mL ● special
top
chemistries
● crystal
0.5 mL ● green top
identification

Transport
● transport ASAP at room temp
● biohazard container
Delayed Sample
● cells in the fluid can alter the chemical composition
● detection of microbes can be jeopardized
● cellular elements such as red cells and white cells can
start to lyse
● specimen should NOT be refrigerated Metachromatic Stain
○ adversely affect the viability of any microorganisms
present Procedure: Metachromatic Stain
○ enhance crystal precipitation ● a few drops of synovial fluid are placed onto a filter
1
paper
TESTS FOR DIFFERENTIATION OF SYNOVIAL FLUID 2 ● 0.2% toluidine blue is added
FROM OTHER BODY FLUIDS
Mucin Clot Test/ Ropes Test
Results and Interpretation: Metachromatic Stain
● provides a semiquantitative index of the degree of
Positive (+) ● drops of fluid will stain blue
polymerization of hyaluronic acid in SyF
False (+) ● heparinized sample was used
Procedure: Mucin Clot Test/ Ropes Test
● mix about 1 part of the supernatant from centrifuged
1 synovial fluid with 4 parts of 2 % to 5% glacial acetic
acid
● acid causes precipitation or clumping of synovial
2
fluid mucin
3 ● after gently mixing, the clumped mucin is observed
● NOTE:
○ test should NOT be performed with samples
collected in EDTA but only in heparin or with no
anti-coagulant

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 PHYSICAL EXAMINATION Clarity
● done by the physician right after collection and must be
recorded Clear ● normal

Color ● encountered with the presence of cellular

elements:
Colorless to ○ RBC
● normal
Pale Yellow ○ WBC
● inflammatory and noninflammatory joint ○ synoviocytes
Deep Yellow
disorder ○ synovial fat droplets
● trauma during collection ○ fibrin
● any disorders that disrupt the synovial
fluid membrane: Turbid Other Cellular Elements
Red or Brown ○ joint fracture rice ● white, free-floating particles
○ tumor bodies ● resemble polished grains of rice
○ hemorrhagic arthritis ● made up of collagen covered by
○ traumatic arthritis fibrinous tissue
Greenish or ● septic arthritis ochro- ● pepper-like particles from
notic broken cartilage
Purulent ● tuberculous arthritis
shards
● tuberculous arthritis
Milky Fluid
● SLE Milky ● presence of crystals

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Viscosity Plasma: Synovial Fluid Glucose Difference
● synovial fluid has a viscosity higher than water and is
similar to an egg white consistency. < 10 mg/dL ● normal fasting patient

● non-fasting patient
Procedure: String Test
< 20 mg/dL ● non-inflammatory JD
1 ● expelling the fluid from the syringe one drop at a time ● hemorrhagic JD
20 mg/dL ● inflammatory JD
● observe the formation of a "string" at least 4 to 6
2 ● septic JD
centimeters long before breaking > 40 mg/dL
● range of 20-1006

Results and Interpretation: String Test

Total Protein
Normal ● 4-6 cm of string ● not routinely performed
Viscosity
● used as an indicator of hemorrhagic & inflammatory
● string breaks earlier or forms discrete JD increased in:
Low water-like droplets ○ rheumatoid arthritis
Viscosity ● seen in inflammatory conditions of the ○ crystal synovitis
joint ○ septic arthritis

● normal TP concentration
< 3g/dL
● ⅓ of plasma level

Uric Acid
● indicator of gout
● along with the presence of monosodium urate
crystals when observed microscopically

Lactate
● test does not require equilibrium and comparison with
plasma lactate levels

● anaerobic glycolysis in the synovium

⬆ lactate ● > 250 mg/dl
levels ○ rheumatoid
Clot Formation
○ septic arthritis
● (+) fibrinogen = clot
● assoc. conditions: ⬇lactate
● gonococcal arthritis
○ disorders that can damage the synovial membrane levels
○ traumatic arthrocentesis with blood contamination
Enzymes
CHEMICAL EXAMINATION
● rheumatoid arthritis
● in the presence of a joint disease or disorder (JD),
⬆ LDH ● gout
○ some analytes maintain the same concentration as
● infectious arthritis
in blood plasma (e.g., uric acid)
⬆ACP ● rheumatoid arthritis (not specific)
○ some analytes also differ depending on the type of
the disease

Glucose
● most frequently requested test for synovial fluid
● specimen is ideally placed in a sodium fluoride tube
● assayed within 1hr of collection to minimize
glycolysis

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 MICROSCOPIC EXAMINATION ● monocytes or macrophages
● normal value = 60%
Clear Sample ● undiluted sample
● assoc. conditions:
Turbid / Bloody ● diluted ○ ⬆ in rheumatoid arthritis
Differential
Sample ● NSS or any appropriate diluent
Count ● lymphocyte
● hyaluronidase enzyme ● normal value = 30%
○ used as a buffer to reduce the
● neutrophil
viscosity in order to count the cells
● normal value = 30%
efficiently
● samples are incubated with Differential Count
hyaluronidase prior to slide Monocytes &
Thick / Very preparation ● normal value = 60%
Macrophage
Viscous Sample ● centrifuged (cytocentrifuge) to
● normal value = 30%
concentrate the fluid's cells at the
Lymphocyte ● assoc. conditions:
bottom of a test tube
○ ⬆ in rheumatoid arthritis
● placed on a slide, treated with special
stain (if needed) ● normal value = 10%
● evaluation of the different kinds of cells ● ≥80%
present is performed Neutrophil ○ bacterial arthritis
○ rheumatoid arthritis
○ gout
● acetic acid
○ CANNOT be used as a diluent ● not usually found in SyF
○ causes hyaluronate to form a mucin clot and ● >2%
cells to clump ○ rheumatoid disease
● temperature and pH changes ○ tubercular arthritis
○ affect crystal formation and solubility Eosinophil ○ parasitic infestations
○ normal temp. = room temp. ○ Lyme disease
● delayed processing result in: ○ metastatic carcinoma
○ WBC lysis ○ following arthrography
○ phagocytosis of crystals by WBC ○ radiation therapy

Crystal Identification
Total Cell Count
● very important for diagnosis of joint disorders
● diluting fluid used
● crystals often result from:
○ a. NSS with methylene blue
○ metabolic disorder
○ b. 0.3% saline
○ decreased renal excretion of crystallizing chemicals
○ c. 1% saponin in saline
○ degeneration of cartilage/bone
○ d. 0.1N HCL
○ injection of medications (corticosteroids) into a joint
● normal value = < 2000/uL ● polarized light microscopy
RBC Count ● assoc. conditions: ○ recognize the presence of birefringent crystals
○ ⬆ in hemorrhagic effusions ○ distinguish the types that are present
● birefringent crystals
● most frequently performed
○ appear as bright objects against a black
● normal value = < 200/uL
background
● assoc. conditions:
● presence of artifacts
○ > 2000/uL
○ artifacts found in SyF
WBC Count ■ bacterial arthritis
■ anticoagulant crystals starch
■ acute gouty arthritis
■ cartilage and prosthesis fragments
■ rheumatoid arthritis
■ collagen fibers
○ > 100,000/uL
■ fibrin and dust particles)
■ severe infection
○ may also show birefringence; hence, differentiation
with crystals is necessary

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 Synovial Fluid Crystals
Compensated
Crystals Characteristics Clinical Significance
Polarized Light
● fine, ● gouty arthritis
Monosodium Urate ● needle-like (-) birefringence
● pointed ends
● pseudogout or chondrocalcinosis
○ assoc. with the calcification of
articular cartilages
■ degenerative arthritis
Calcium ● rhombic
(+) birefringence ■ arthritides
Pyrophosphate ● square rods
● metabolic diseases
○ hypothyroidism
○ hyperparathyroidism
○ diabetes mellitus
● chronic inflammatory conditions
● flat
○ e.g., rheumatoid arthritis
● notched rhombic plates
Cholesterol (-) birefringence ○ nonspecific & frequently occur
● needle-like (rare
● chronic effusions found in other body
encountered)
cavities
● no clinical significance
● flat (+) and (-)
Corticosteroid ● only indicates an intra-articular injection
● variable shaped plates birefringence
of corticosteroid
Calcium Oxalate ● envelope shapes (-) birefringence ● observed among px having dialysis
● osteoarthritis
Hydroxyapatite ● tiny ● calcific deposition
no birefringence
(Calcium Phosphate) ● needle like crystals ○ collectively termed apatite assoc.
with arthropathies

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Other Synovial Fluid Cells

Cells and Inclusions Seen in Synovial Fluid

Cell inclusion Description Significance
● bacterial sepsis
Neutrophil ● polymorphonuclear leukocyte
● crystal-induced inflammation
Lymphocyte ● mononuclear leukocyte ● non-septic inflammation
● large mononuclear leukocyte ● normally found but may increase in viral
Macrophage (Monocyte)
○ may be vacuolated infections
● normally found and may indicate
● similar to macrophage, but may be
Synovial lining cell disruption of synovium during
multinucleated, resembling a mesothelial cell
arthrocentesis
● neutrophil containing characteristic ingested
LE cell ● lupus erythematosus
“round body”
● vacuolated macrophage with ingested
Reiter cell ● reiter syndrome/ reactive arthritis
neutrophils
● neutrophil with small, dark cytoplasmic granules
● rheumatoid arthritis immunologic
RA cell (ragocyte) containing immune complexes (rheumatoid
inflammation
factor)
Cartilage Cells ● large, multinucleated cells ● osteoarthritis
● macroscopically resemble polished rice; ● tuberculosis
Rice
microscopically show collagen and fibrin ● septic and rheumatoid arthritis
● traumatic injury
Fat Droplets ● retractile intracellular and extracellular globules
● chronic inflammation
Hemosiderin ● inclusions within clusters of synovial cells ● pigmented villonodular synovitis
● pepper like particles from pigmented cartilage ● ochronotic arthropathy, a consequence
Ochronotic Shards
that has eroded and broken loose into the fluid of alkaptonuria and ochronosis

Fig. Unstained wet prep of MSU Fig. Wright's-stained neutrophils Fig. Extracellular MSU crystals under Fig. MSU crystals under
crystals . Notice the characteristic containing CPPD crystals (×1000). compensated polarized light. Notice compensated polarized light. The
yellow-brown of the urate crystals. the change in color with crystal yellow crystal is aligned with the
alignment slow vibration

Fig. Strongly birefringent MSU Fig. sWeakly birefringent CPPD

crystals under polarized light crystals under polarized light Fig. CPPD crystals under compensated polarized light. The blue crystal is
by numerous squamous epithelial aligned with the slow vibration
cells

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 SEROLOGIC TEST MICROBIOLOGIC TEST
● association of the immune system to the inflammation
● allows for the direct observation of
process
Gram Stain bacteria or fungi under a microscope
● serologic testing plays an important role in the
● normal = no microbes present
diagnosis of joint disorders
● arthritis ● performed to determine what type of
○ frequent complication of Lyme disease (caused by microbes are present
Borrelia burgdorferi) ● (+) bacteria
○ demonstration of antibodies to the causative agent ○ susceptibility testing against certain
Culture and
in the serum can confirm the cause of arthritis antibiotics can be performed to guide
Susceptibility
● most of the test are performed using serum antimicrobial therapy
Testing
● extent of inflammation ● NOTE:
○ determined through measurement of the ○ whether or not the gram stain is
concentration of fibrinogen & C-reactive protein positive, ALL synovial fluids should
be cultured
● test for the presence of mycobacteria
● help diagnose tuberculosis
Acid Fast ● molecular test methods
Staining ○ are more sensitive and specific than
traditional cultures and may also be
performed

CLASSIFICATION OF JOINT DISORDERS

● analysis of synovial fluid plays a major role in the
diagnosis of joint diseases
● when infective arthritis and crystal-induced synovitis are
suspected, examination of the synovial fluid may
indicate a definitive diagnosis

Classification, Laboratory Findings and Pathologic Significance of Joint Disorders

Group II Group II
Group I Group IV
Inflammatory Inflammatory Group III Septic
Non-inflnflammatory Hemorrhagic
(Immunologic) (Crystal induced)
Volume > 3.5 mL
Color yellow yellow white, gray yellow white yellow green red brown
cloudy, opaque,
Appearance clear - hazy cloudy cloudy, purulent cloudy
milky
Clot fair poor
Viscosity high low
WBC count
<3000 2,000 - 100,000 10,000 - >100,000 > 5000
cells/ul
Neutrophils <25% >50% <70% >75% >25%
Culture and
(-) (-) (-) (+) (-)
gram stain
● osteoarthritis ● rheumatoid arthritis ● crystal synovitis ● bacterial infection ● trauma
● osteochondritis ● reactive arthritis (gout pseudogout) ● fungal infection ● blood disease (e.g.
● osteochondromatosis ● SLE ● mycobacterial hemophilia, SC
Assoc. ● traumatic arthritis ● scleroderma infection disease)
Diseases ● neuroarthropathy ● polymyositis ● tumor
● SLE ● ankylosing ● joint prosthesis
● early stages of spondylitis ● anticoagulant
rheumatoid arthritis ● lyme arthritis overdose

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Transcribed by: Lustre, Aliping, Guarin, Villanueva (2024). 52
ANALYSIS OF URINE MLS 321L

& OTHER BODILY FLUIDS 8438L

LABORATORY BS MLS 3 FINALS
M7U8: FECAL ANALYSIS

UNIT 08: FECAL ANALYSIS

FECES
● body’s waste products
● ¼ composition
○ undigested foodstuffs ○ gastrointestinal
○ sloughed intestinal secretions
epithelium ○ bile pigments
○ intestinal bacteria ○ electrolytes
● ¾ composition
○ water
● normal fecal volume excreted = 100-200g/day

PHYSIOLOGY AND FORMATION OF FECES

● fecal formation happens in the small intestines and
large intestines
● digestive enzymes secreted by the
pancreas into the small intestines
○ trypsin
Small ○ chymotrypsin
Intestines ○ amino peptidase ● fermentation of carbohydrates
(SI) ○ lipase ○ by intestinal bacteria in the large intestines
● bile salts ○ produces odorous intestinal gas/flatus = 400-700
○ provided by the liver also helps in the ml/day
digestion of fat ○ e.g., oligosaccharides
● limited absorption of: ■ resistant to digestion
Large CHO that ferments in the LI
○ water (2700-3000 mL) ■
Intestines
○ sodium ● intestinal gas, when incorporated in feces
(LI)
○ chloride ○ result in foamy and floating stool
○ usually normal
● excessive gas production also occurs in
lactose-intolerant individuals
○ intestinal bacteria metabolize the lactose from
consumed milk or lactose-containing substances

Fig. Fecal Formation

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 SPECIMEN COLLECTION, PROCESSING AND A) MACROSCOPIC EXAMINATION
ANALYSIS
● testing is not very common Normal Physical Characteristics of Feces
● provides diagnostic information for the early detection Color ● brown
of: Consistency ● semi-formed or formed
○ gastrointestinal bleeding Form ● cylindrical
○ liver and biliary duct disorder
○ maldigestion/malabsorption syndromes A1. Color
○ inflammation
○ causes diarrhea and steatorrhea Stool Colors
● caused by:
SPECIMEN COLLECTION AND HANDLING ○ stercobilin
● important to note that individuals have limited control in Orange-Brown
○ mesobilin
the timing of fecal excretion ○ urobilin
● collection is highly undesirable Clay-colored ● post-hepatic obstruction
● it is best to have verbal and written instructions or Gray ● ingestion of too much barium sulfate
provided to the patient along with an appropriate Pale Yellow or ● acholic stools
specimen container White ○ lacking bile
● specimen containers ● blood from the lower GI tract
○ may vary depending on the amount of specimen to ● consumption of:
○ beets
be collected
Red ○ food dyes
○ generally, specimen containers must be: ○ drugs
■ clean ■ BSP dye
■ non-breakable ■ rifampin
■ sealable ● blood from the upper GI tract
■ leak-proof ● consumption of:
● type and amount collected varies with test to be ○ charcoal
Black
performed ○ iron therapy
○ bismuth (medications or
● test that may be performed: suppositories)
Small Amount ○ occult blood ● excess consumption of green vegetables
of Randomly
○ WBC Green ● production of biliverdin during antibiotic
Collected Spx
○ qualitative fecal fat therapy
● test that may be performed:
3-Day Fecal ○ quantitative tests for the daily A2. Consistency
Collection
excretion of any substance
● requirement needed for: Stool Consistency
Dietary ○ occult blood Semi-Formed ● normal consistency
Restrictions ● aka scybalum
○ quantitative fecal fat Hard
● usual indication of constipation
Contamination ● must be avoided
w/ Urine, Toilet ● adversely affect detection of parasites ● ⬆ fecal water content
Tissue, or ● normal
Toilet Water and most chemical testing Soft ○ if related to intake of laxatives
● pathologic
● occur in cases of flatulence ○ involves GIT disorders
Spx w/ ● containers must be covered in tissue
Accumulated Watery ● correlated to diarrhea or steatorrhea
before opening
Gas
○ prevent splashing of fecal matter

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A3. Form

Stool Forms
● associated to:
Narrow, ○ bowel obstruction
Ribbon-Like ○ strictures
■ intestinal narrowing
Small, Round ● constipation
● associated to:
○ steatorrhea
Bulky/ Frothy
○ bile duct obstruction
○ pancreatic disorder

A4. Other Characteristics

Other Stool Characteristics

Foamy,
● ⬆ gas production
Floating
Greasy,
● steatorrhea
Spongy
● not normally present in feces
● associated to:
○ constipation
○ tumor in the colon
Mucus ○ gastrointestinal disorders
(translucent, ■ colitis
gelatinous) ■ intestinal tuberculosis
■ ulcerative diverticulitis
■ bacillary dysentery B) MICROSCOPIC EXAMINATION
■ neoplasms ● performed to detect the presence of:
■ rectal inflammation
○ (1) leukocytes
● associated to: ■ associated with microbial diarrhea and
○ dysentery
Blood-Streaked undigested muscle fibers
○ colitis
Mucus ○ (2) fats
○ malignancy
○ constipation ■ associated with steatorrhea
B1. Fecal Leukocyte
● primarily neutrophil or pus-exudates containing
WBC
● NOT normally present in feces, even in cases of:
○ amoebic colitis
○ viral gastroenteritis
○ bacterial diarrhea caused by toxin production
■ S. aureus and Vibrio spp.
● (+) fecal leukocyte indicates that intestinal wall is
infected or inflamed due to:
○ ulcerative colitis
○ bacillary dysentery
■ invasive bacterial pathogens:
■ Salmonella, Campylobacter, Yersinia, and
enteroinvasive E. coli
○ ulcerative diverticulitis
○ intestinal tuberculosis
○ abscesses or fistula

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(-) Fecal ● S. aureus B2a. Fecal Fat Tests: Qualitative Tests
Leukocyte ● Vibrio spp. ● plays an important role in screening for suspected
● Salmonella cases of steatorrhea
(+) Fecal ● monitoring patients undergoing treatment for
● Campylobacter
Leukocytes
● Yersinia malabsorption disorders
● enteroinvasive E. coli ● px preparation
○ normal dietary intake of fat
■ avoid contamination of spx with mineral oils or
cream that can produce false-positive results

Procedure:Qualitative Tests
1st Slide
● feces is placed on a glass slide with several drops of
1
95% ethanol
2 ● stain is added
● coverslip is applied and observed microscopically for
Fig. Fecal Leukocytes 3
presence of fat globule

B1a. Fecal Leukocyte Test: Smear Preparation (Wet or

Stained) Results and Interpretation: Qualitative Tests (1st Slide)
● may be stained with: Normal ● 60 globules of neutral fat/HPF
○ Wright’s Feces
○ Gram stain Steatorrhea ● > globules of neutral fat/HPF
○ Methylene Blue
Procedure:Qualitative Tests
Procedure: Smear Preparation (Wet or Stained) 2nd Slide
1 ● placing mucus or a drop of liquid stool on a slide ● aliquot of fecal sample is acidified with acetic acid
2 ● add 2 drops Loffler methylene blue and heated
1
○ it will provide estimation of total fecal fat content:
3 ● mix with a wooden applicator stick ■ neutral fats + fatty acids + fatty acid salts (soap)
4 ● examine for neutrophils under HPO
Results and Interpretation: Qualitative Tests (2nd Slide)
B1b. Fecal Leukocyte Test: Lactoferrin Latex Agglutination
Test ● contain <100 globules/HPF
● prefers a refrigerated and frozen specimen Normal Feces ● should NOT exceed 4 μm in diameter
● lactoferrin (about half of RBC)
○ component of granulocyte secondary granules ● increased numbers of globules with
Steatorrhea
extremely large size (40 to 80 μm)
Results and Interpretation: Lactoferrin Latex
Agglutination Test ● 1st slide: normal amount of fecal neutral
Intestinal fats
● presence of lactoferrin Malabsorption
(+) ● 2nd slide: increased
● indicative of invasive bacterial pathogen
Intestinal ● ⬆ neutral fats on both slides
Maldigestion
B2. Fecal Fat
● spx from suspected cases of steatorrhea can be
screened microscopically for the presence of excess
fecal fat
● quantitative determinations are used especially for
quantitation of:
○ unstained phospholipids & cholesterol esters

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B2b. Fecal Fat Tests: Quantitative Tests ● rapid procedure for fecal fat that
● definitive test for steatorrhea requires less stool handling
○ confirms abnormal amounts of dietary lipids ○ requires 48-72-hr stool collection to
○ does NOT identify the cause of the increased exclude day-to-day variability
● does NOT require reagents after
excretion Near-Infrared
homogenization of the sample
● px preparation Reflectance
● result is based on the measurement and
○ 2 days before spx collection Spectroscopy
computed processing of signal data from
(NIRA)
■ px are provided with special dietary (100g/day reflectance of fecal surface
fat) instructions to ensure adequate fat and ○ scanned with infrared light
caloric intake ■ 1400 nm & 2600 nm wavelength

○ px are not allowed to skip any meals or take any ● results are calculated from calibration
derived from known sample
medications
○ spx should be at least 3-day sample and must be
refrigerated to prevent bacterial multiplication

Procedure: Quantitative Tests

● px collects ALL feces excreted for 3 (min) to 6 days in
1
a large, pre-weighed collection container
● sample is weighed and homogenized using a
2
mechanical shaker
● fecal fat is reported as
3 ○ grams of fat excreted per day;
○ or the coefficient of fat retention per 24 hours Fig. Several orange-red neutral fat globules present in a fecal
suspension stained with Sudan III

Results and Interpretation: Quantitative Test

Meat Fibers
Normal ● 2-7 g/day
● undigested foodstuffs
Feces ● coefficient of fat retention = at least 95%
○ meat
Steatorrhea ● < 95% ○ vegetable fibers
● characterized by rectangular and cross-striations
% Fat Retention Formula ● identification and qualitative assessment for meat fibers
is included while doing qualitative fecal fat
examination
● helpful in the diagnosis and monitoring of patients with
pancreatic insufficiency such as:
Methods Used for Fecal Fat
○ cystic fibrosis
● gold standard for fecal fat
Titrimetric quantitation ○ biliary obstruction
Technique ● unable to recover medium-chain fatty ○ gastrocolic fistulas
(Van de Kamer acids completely ● px preparation
Titration) ● measures only approx. 80% of the total ○ include red meat in diet prior to specimen
fecal lipid content collection
● rapid test to estimate the amount of fat ○ specimen should be examined within 24 hours
excretion (extracts all fecal lipids
present)
● similar to microhematocrit test
Acid Steatocrit
● more convenient than a 72-hour stool
(Gravimetric
collection
Method)
● reliable tool to monitor a px’s
response to therapy
● screen for steatorrhea in pediatric
populations

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 Procedure: Evaluating Meat Fibers
● used by applying few drops of a fecal suspension to a
1 slide and stain it with a solution of eosin in 10%
alcohol (enhance muscle fiber striation)
2 ● use coverslip and let stand for 3 minutes
3 ● examine entire slide for exactly 5 minutes
● count red-stained undigested fibers with well-
preserved striations
○ running both vertically and horizontally
4 ● partially digested fibers
○ striations in one direction and digested fibers have
no striations
● IF there is >10, it is reported increased

Results and Interpretation: Meat Fibers

● ⬆ amount of fecal meat fibers
(+) ○ often correlates with impaired digestion
Creatorrhea
and rapid intestinal absorption

Fig. Meat fibers present in fecal Fig. Note striations on meat fiber C) CHEMICAL EXAMINATION
emulsion specimen using brightfield present in a fecal emulsion
microscopy examination (×400) specimen (×1000).
C1. Fecal Blood
● presence of blood in stool is often due to:
○ bleeding in the GI tract from the mouth (bleeding
gums) → anus (hemorrhoids)
● early symptom of possible colorectal cancer
○ annual screening is recommended by American
Cancer Society on all individuals older than 50
years old
● clinical significance related to fecal blood:
○ esophageal varices
○ ulcers
○ inflammatory conditions
○ various drugs that irritate the intestinal mucosa
■ aspirin
■ iron supplements
● most of the time, fecal blood is considered occult
(hidden)
● large amount of blood
○ it can be macroscopically apparent
● ‘melena’
○ excretion of dark or black, pitchy stools resulting
from the presence of large amounts of blood
○ 50-100 mL/day

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● normal blood excretion = 2.5 mL/day or 2 mg ● defective guaiac or peroxidase developer
● hemoglobin/g of stool = 2.5 mL/150g of stool ● storage of specimen or prepared slides
● ⬆ fecal blood beyond 6 days
○ requires further investigation to discover its source ● medications
○ salicylates (aspirin)
● GI tract bleeding is intermittent
○ iron supplement
○ blood is frequently hidden within the stool ■ false positive (F+)
○ requires several stool specimens ○ ascorbic acid
■ false negative (F-)
Bright Red Blood ● lower GI tract bleeding ● avoid eating: the ff for 3 days prior to
Dark or Tarry ● upper GI tract bleeding specimen collection:
○ red meat
○ horseradish
C1a. Fecal Blood Test: Occult Blood Slide Test
○ melons
○ raw broccoli
Fecal Blood Test: Occult Blood Slide Test
○ cauliflower
● based on the pseudoperoxidase activity ○ radishes
of hemoglobin reacting with hydrogen ○ turnips
peroxide to oxidize a colorless compound ● to prevent the presence of dietary
to a colored compound Patient pseudoperoxidase in the stool
Preparation
● aspirin and other NSAIDs
Indicators ○ should NOT be taken for 7 days prior
● most sensitive but to specimen collection
benzidine carcinogenic ○ prevent possible GIT irritation
● no longer used ● as much as possible intake of vitamin C
ortho- ● second sensitive and iron supplements containing vitamin C
toluidine
should be avoided for 3 days prior to
● indicator of choice
collection
● reduces false positive
Principle ○ AA is a strong reducing agent
results
■ may interfere with peroxidase activity
● cause of F+
○ dietary factors
○ myoglobin & C1b. Fecal Blood Test: HemoQuant test
hemoglobin ● based on the chemical conversion of nonfluorescent
guaiac heme → intensely fluorescent porphyrins
■ meat and fish
○ peroxidases ● enables detection and quantitation of total fetal
■ bacteria
hemoglobin
■ ingested fruits &
○ portion converted to porphyrin in the intestine
vegetable ○ portion remaining as hemoglobin
● gFOBT ● measures only:
○ heme
○ heme-derived porphyrins
● NOT affected by those substances that interfere with
● (+) = blue color
qualitative test
Interpretation

● intensity = amount of enzymatic activity

● improper specimen collection
● application of too much or not enough
Sources of feces
Error ● use of chemically contaminated specimen
● specimen with menstruation or hemorrhoid
blood

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C1c. Fecal Blood Test: Hemoccult ICT Results and Interpretation: Meat Fibers
● immunochemical fecal occult blood test (iFOBT)
(+) maternal ● change in the color (yellow-brown)
● specific for globin portion of human hemoglobin origin
● uses anti-human hemoglobin antibodies
(+) fetal ● pink color remains the hemoglobin
● advantages it offers include: hemoglobin
○ a. NO dietary or drug restriction
○ b. more sensitive to lower GI bleeding C3. Fecal Carbohydrates
■ indicator of colon cancer or other ● determination of fecal carbohydrates are commonly
gastrointestinal disease performed to detect:
■ does not detect bleeding from other sources ○ (1) insufficiency of enzymes for disaccharide
■ decrease chance of F+ result due to upper GI metabolism as seen in lactose intolerance;
bleeding ○ (2) intestinal inability to reabsorb carbohydrates
○ c. used for patients taking aspirin and other as seen in celiac disease
anti-inflammatory drugs ● lactose intolerance
○ characterized by:
C1d. Fecal Blood Test: Radiometric assays ■ bloating
● uses chromium-51 ■ flatulence
● detect and localize gastrointestinal bleeding. ■ explosive diarrhea
● aliquot of the patient’s RBC is bound with radioactive ○ due to intestinal bacteria actively fermenting CHO
chromium and injected back into the patient present in the intestinal lumen
● comparisons are made between: ○ results to:
○ (1) amount of radioactivity recovered in the feces ■ ⬆production of large amount of gas
■ resulting from intestinal bleeding ■ ⬇ pH (5.0 to 6.0) compared to normal pH (>
○ (2) radioactivity remaining in the blood 7.0)

C2. Fetal Hemoglobin Fecal Carbohydrates Tests

● qualitative test that is based on the alkaline resistance
● acidic feces
of fetal hemoglobin (Hgb F) pH
○ carbohydrate fermentation
● serves to differentiate the source of fecal blood in the Determination
● alkaline feces
(uses pH meter)
neonate ○ protein fermentation
○ if it is coming from his own gastrointestinal tract or ● uses 1:3 dilution of the supernatant
from maternal blood. CHO from diarrheal stool to which it
● specimen needed: Determination: identifies reducing sugars only
○ should be fresh, bloody fecal specimen or a soiled, CLINITEST ● carbohydrate intolerance
Tablet Test ○ considered if carbohydrate level is
bloody diaper >250 mg/dL or 0.5 g/dL
● black, tarry stool
Chromato- ● used for screening of lactose
○ NOT accepted since hemoglobin was degraded graphic Specific deficiency through histochemical
to hematin already Chemical Test examination of the intestinal epithelium
● ingestion of a measured amount of
Procedure: Fetal Hemoglobin appropriate intestinal
● when performing the procedure, a suspension of the disaccharidase
1 feces with water (to release hemoglobin) is made and ● IF the px has adequate and
centrifuged appropriate disaccharidase
Tolerance Test ○ disaccharide is hydrolyzed to its
● 5 mL aliquot of the resultant supernatant is transferred
using Specific corresponding monosaccharides →
into 2 tubes wherein one tube will be used as a
2 Sugar absorbed into the patient’s blood
reference to evaluate color changes in the second
(Sucrose, ● adequate enzyme activity
tube.
Lactose) ○ increase in blood glucose >30
● to the second tube, 1 mL sodium hydroxide (0.25 mg/dL above the glucose fasting
mol/L) is added and a change in the original pink glucose level
3
color changes to yellow or brown in 2 minutes is ● enzyme deficiency
observed ○ < 20 mg/dL above fasting glucose
level

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 ● used to differentiate CHO ● most frequently used to measure
malabsorption & CHO maldigestion chymotrypsin
using xylose
● xylose ● more resistant to intestinal
○ pentose that does not depend on degradation
liver or pancreatic function for ● more sensitive indicator of
digestion Spectro-
photometric less severe case of
○ readily absorbed in the small
intestine Method Advantages pancreatic insufficiency
● NOT present at significant levels in the ● stable in feces for up to 10
blood, NOT metabolized by the body days at room temp
Xylose and readily passes through the ● can be measured by
Absorption Test glomerular filtration barrier for gelatin hydrolysis test
excretion in urine
● procedure involves patient’s
ingestion of a dose of xylose ● immunologic assay that uses monoclonal
followed by the collection of: antibodies against human pancreatic
○ 2-hour blood sample elastase-1
○ 5-hour urine specimen ● differentiate pancreatic from
● normal conc. of xylose in blood and non-pancreatic causes of steatorrhea
urine ● elastase-1
○ at least 16% to 24% of the ○ present in high concentrations in
ELISA
ingested dose of xylose is normally pancreatic secretions (account for
excreted by adults about 6% of all secreted pancreatic
enzymes)
○ strongly resistant to degradation
C4. Fecal Enzymes ○ pancreas specific
● enzymes produced by the pancreas into the GIT are ○ ⬇ elastase-1
very essential for the digestion of dietary proteins, ■ indicate steatorrhea of pancreatic origin
carbohydrates and fats
● decrease in production of these enzymes CONDITIONS ASSOCIATED WITH FECAL PHYSIOLOGY
(pancreatic insufficiency) AND FORMATION
○ associated with disorders:
■ cystic fibrosis A) DIARRHEA
■ chronic pancreatitis ● increase in the volume (200 g/day), liquidity, and
frequency of bowel movement
Fecal Enzyme Tests
● characterized as watery with odor as normal or
● stool is emulsified in water → x-ray paper
unremarkable
is exposed to it
● (+) trypsin ● classified based on the duration of illness, severity, stool
○ clear area is formed characteristics and mechanism of action
○ trypsin digest gelatin in the paper ● duration
● test is considered insensitive ○ acute < 4 weeks
○ detects only severe cases of ○ chronic which persists > 4 weeks
pancreatic insufficiency ● categorized as secretory, osmotic and intestinal
● due to: hypermotility diarrhea.
Gelatin Test False ● proteolytic activity of
Positive (+) bacterial enzymes in old Classification Of Diarrhea
specimen ● (1) acute
● more common Duration of ○ < 4 weeks
● due to: Illness ● (2) chronic
False ○ intestinal degradation of ○ > 4 weeks
Negative (-) trypsin
● (1) secretory
○ presence of trypsin Mechanism ● (2) osmotic
inhibitor in the feces ● (3) intestinal hypermotility diarrhea

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 Common Causes of Osmotic Diarrhea
● characterized by inability of the
intestines to convert foodstuffs into
readily absorbable substances
● occurs in most hepatic and pancreatic
disorders

● ⬇bile salts
○ fat emulsification
● micelle formation
● assoc. conditions:
○ hepatocellular disease
(severe)
A1. Secretory Diarrhea
Hepatic ○ bile duct obstruction
● ⬆solute secretion by the intestine → causing an Disorder ○ biliary cirrhosis
⬆fluid to be presented to the large intestine → ○ bile acid deconjugation
exceeding intestine’s absorptive capacity caused by stasis
Maldigestion
■ strictures
Common Causes of Secretory Diarrhea ■ blind loop syndrome
● stimulate intestinal secretions rich in ■ diabetes visceral
electrolytes neuropathy
● enterotoxin-producing organisms
Enterotoxin- ○ Vibrio cholerae ○ Clostridium ● deficiency in pancreatic
producing ○ Salmonella ○ Staphylococcus digestive enzymes
organisms ○ lactase
○ Shigella ○ Protozoa
○ Escherichia coli ○ Cryptosporidium ○ lipase
Pancreatic assoc. conditions:
●
● viral gastroenteritis Disorder ○ chronic pancreatitis
● ulcerative colitis ○ cystic fibrosis
Mucosal ● drugs ○ pancreatic cancer
damage ● Crohn disease ○ Zollinger-Ellison
● lymphocytic colitis syndrome
● diverticulitis ○ ileac resection
● caffeine
Drugs or ● characterized by normal digestion but
● prostaglandin
hormones inadequate absorption of processed
● vasoactive intestinal peptide
foods
Collagen
● assoc. conditions:
Vascular ● hyperthyroidism
○ parasitic infections (giardiasis)
Disease & ● Zollinger-Ellison syndrome
○ mucosal disease
Endocrine ● vipoma
Malabsorption ○ lymphatic destruction
Disorders
■ lymphoma
■ Whipple’s disease
A2. Osmotic Diarrhea ○ abetalipoproteinemia
● ⬆ingestion of osmotically active solutes that remain ○ disaccharidase deficiency
in the lumen ○ surgical procedures altering the
● cause the secretion of: mucosal structure
○ water ● antacids
Laxative
○ electrolytes ● sorbitol
Action of
● tetracycline
■ sodium, bicarbonate & potassium Some Drugs
● lincomycin
● exceeds the absorptive capacity of the large intestine,
thus causing diarrhea

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Differentiation of Secretory and Osmotic Diarrhea B) ALTERED MOTILITY
● osmotic and secretory diarrhea are differentiated in
terms of: Hypermotility ● motility is enhanced
○ fecal electrolytes Constipation ● motility is slow
○ fecal osmolality
○ stool pH Irritable Bowel Syndrome (IBS)
● fecal osmotic gap is calculated using fecal sodium ● functional disorder in which the nerves and muscles of
and potassium levels with this formula: the bowel are extra sensitive
● causes:
○ cramping
○ bloating
Differentiation of Secretory and Osmotic Diarrhea ○ flatus
● osmotic gap ○ diarrhea
○ > 50 mOsm/kg ○ constipation
● pH ● IBS is triggered by food, chemicals, emotional stress,
○ < 5.6 and exercise
■ indicates malabsorption of
carbohydrates →osmotic diarrhea
Rapid Gastric Emptying (RGE) Dumping Syndrome
● other common fecal tests:
Osmotic ● hypermotility of the stomach and the shortened gastric
○ microscopic fecal fats
Diarrhea emptying half-time causes the small intestine to fill too
○ muscle fiber detection
quickly with undigested food from the stomach
○ qualitative & quantitative fecal test
● hallmark of early dumping syndrome (EDS)
○ trypsin screening
● caused by disturbances in the gastric reservoir or in the
○ muscle fiber detection
transporting system
○ CLINITEST
● alterations in the motor functions of the stomach
○ D-xylose tolerance test
○ results in the accumulation of large amounts of
● osmotic gap
osmotically active solids and liquids to be
○ < 50 mOsm/kg
transported into the small intestine
● other common fecal tests:
Secretory ● associated conditions:
○ stool cultures
Diarrhea ○ gastrectomy
○ ova and parasite examination
○ gastric bypass surgery
○ rotavirus immunoassay
○ post- vagotomy status
○ fecal leukocyte
○ Zollinger-Ellison syndrome
○ duodenal ulcer disease
C3. Intestinal Hypermotility ○ diabetes mellitus
● results to diarrhea when there is rapid absorption of ● gastric emptying is controlled by:
digested food in the intestines ○ fundic tone
● stimulated by: ○ duodenal feedback
○ intestinal distention ○ GI hormones
○ chemicals, nerves, & hormonal changes
Gastric Emptying Half-time
Common Causes of Intestinal Hypermotility ● 35 - 100 mins
Secretory & Osmotic ● increased fluid lumen fluid cause Normal
● may vary with age and gender
Diarrhea intestinal distention
RGE ● < 35 mins
Parasympathetic
● enhancement of GI motility
Nerve Activity
Laxatives ● castor oil
Cardiovascular ● digitalis
Drugs ● quinidine

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 C) STEATORRHEA

Fats
● likewise observed in stool
● product from the digestion of lipids
● normal fat excretion = <6g/day (measured as fatty
acids)
● source of fat:
○ diet
○ gastrointestinal secretions
○ bacterial by-products of metabolism
○ sloughed intestinal epithelium

Steatorrhea
● fecal fat excretion = > 6g/day
● characterized by:
○ pale
○ greasy
○ bulky
○ spongy, or pasty with extremely foul odor
○ fluidity
■ may float or be foamy (presence of large
amounts of gas)
● detection of this is useful for the diagnosis of
pancreatic insufficiency & small bowel disorders
that cause malabsorption and fat maldigestion

Transcribed by: Lustre, Aliping, Guarin, Villanueva (2024). 12