URINE ANALYSIS: KIDNEY ANATOMY Enzyme: sodium

PHYSIOLOGY AND FUNCTION balance)

• Juxtaglomerular complex- produce renin o I, 25-
• Cleaves plasma aangiotensinogen to dihydroxyvita
Excretion and osmoregulation in man
angiotensin I min D
Excretory organs • Converted to angiotensin II by (calcitriol)
angiotensin-converting enzyme (ACE) in the calcium
Accessory excretory organs lungs balance
main excretory organs • Stimulates the secretion of aldosterone by
ANATOMY OF THE KIDNEYS
the adrenal cortex- renin angiotensin-
aldosterone system  Bean-shaped, reddish-brown organs,
Lung skin liver
which are about 11cm long
liver • Responsible also to the final hydroxylation  Found on the posterior abdominal
of 25-hydroxycholecalciferol to form I, 25- wall either side of the vertebral
dihydroxycholecalcifero/ activated vitamin column between T12 and L3
Functions of kidneys D o Lateral margins- convex
o Medial margins- concave
• Primary function of the kidneys is to BASIC RENAL PHYSIOLOGY o Hilum- middle indention
regulate the extracellular fluid (plasma and (medial); where renal vessels
1. Clear metabolic waste products
tissue) environment in the body and the renal pelvis enter
from blood & excretion in urine
and exit the kidneys
2. Maintain the body’s water &
• Filter the blood and excrete waste
inorganic ion balance THREEE REGIONS OF THE KIDNEYS
and excess water as urine
3. Remove of foreign chemicals from
• Able to regulate: blood & excretion in urine Region Description
4. Others: Cortex The dark outer part of the kidney
• Blood plasma volume- also the  Gluconeogenesis; which contains the arcuate and
during prolonged interlobular arteries and veins and
blood pressure
fasting (liver) the cortical nephrons.
• Concentration of waste products  Secretion of Medulla Lies deep to the cortex and
contains the cone-shaped
–nitrogen containing wastes hormones
structures known as the renal
o Erythropoietin-
pyramids, which contain the vasa
• Electrolyte balance erythrocyte recta, nephron loops and
production collecting ducts.
• Ph control Pelvis Cup like projections called minor
o Renin- calyces then surround the tip of
Endocrine:
angiotensin each pyramid to collect urine.
Formation of renal erythropoeitic factor formation Several minor calyces then
control (BP & coverage to form a major calyx,
(REF) – needed to form erythropoietin
which in turn joins with other
major calyces to form the renal
 pelvis. Found in the central region  The walls of the glomerular capsule potassium ions and urea are
of the kidney, the renal pelvis is are thin, but only allow water and secreted into the PCT.
continuous with the ureter. small ions to pass through.  It contains as the loop of Henle.
 Filtrate (water and small molecules)
which is similar to blood plasma Reabsorption & Secretion
CROSS SECTION THROUGH A KIDNEY passes into the capsular space of
Loop of Henle
the glomerular capsule.
 A tubule with a long hairpin turn, its
Filtration
descending limb enters the medulla,
Glomerulus where it makes a 180 degree turn so
the its ascending limb enters the
 A tiny capillary network that lies cortex.
within a glomerular capsule.  Salts are reabsorbed from the loop
 The glomerulus receives blood at of Henle into the medulla of the
high pressure from a tiny branch of kidney ( making the medulla very
the renal artery, called the afferent salty compared to the filtrate).
arteriole  It ends in the cortex as the distal
 The filtered blood ( blood cells, convoluted tubule (DCT).
proteins and large molecules )
 Fibrous capsule
leaves the glomerulus via the Reabsorption
 Renal artery
efferent arteriole which goes on to
 Renal vein Distal convoluted tubule (DCT)
form a capillary plexus around the
 Renal pelvis
PCT, before draining into a tiny
 Renal sinus  A highly coiled tubule located in the
branch of the renal vein.
 Ureter cortex and surrounded by capillaries.
 Renal pyramids  Salts such as sodium are actively
Filtration
 Base of absorbed from the DCT under the
 Renal cortex Proximal convoluted tubule (PCT) control of a hormone called
 Minor calyces aldosterone.
 Major calyces  Originating from the glomerular  Hydrogen and potassium ions are
 Renal papilla capsule the PCT is a highly twisted actively secreted into the DCT to
and coiled tubule that descends regulate pH.
through the cortex  The rate of absorption and secretion
 It is the part of the nephron in the DCT are controlled by
responsible for most of the hormones.
COMPONENTS OF A NEPHRON AND RENAL reabsorption of the filtrate.  It empties into the collecting tubule
TUBULE  Water, glucose, amino acids and (CT).
salts are reabsorbed from the PCT
Glomerular (Bowman) capsule back into the blood. Active Secretion
 Drugs, toxins and solutes such as
 The start of the nephron. bicarbonate, hydrogen and
Collecting tubule (CT) URINE FORMATION  The cells of the tubule reabsorb vital
nutrients and water back into the
 They pass through the medulla Three important steps: blood, while retaining the waster
forming the pyramids of the kidneys. products that the body needs to
 Bicarbonate, potassium and 1. Glomerular filtration eliminate.
hydrogen ions, are secreted into the 2. Tubular reabsorption  The plexus formed by the efferent
CT to regulate pH. 3. Tubular secretion arteriole (from the glomerulus)
 Water and salts are absorbed from passes closely to the proximal
FILTRATION
the urea in the CT under the control convoluted tubule, allowing direct
of two hormones (one of them being  Filtration at the glomerulus is transfer into the blood.
anti-diuretic hormone that increases under pressure as the  In the loop of Henle the filtrate is
the CT permeability to water). afferent arteriole is so close further concentrated.
 Each CT opens into minor calyces at to the abdominal aorta.  Water is absorbed by osmosis, being
the apex of the renal pyramid.  The fluid that passes through transported down its concentration
 From here urine flows via funnel-like the wall of the glomerular gradient.
calyces into the pelvis of the kidney. capsule into the nephron is  The amount of the water reabsorbed
called glomerular filtrate and is controlled by an anti-diuretic
Reabsorption, Secretion & Transport is similar in composition to hormone secreted by the posterior
plasma. lobe of the pituitary gland.
MICROSCOPIC STRUCTURE OF THE NEPHRON
 Blood and protein cannot  The amount of salts reabsorbed is
 Glomerular capsule Blood Flow in pass into the filtrate but small controlled by aldosterone secreted
 Afferent arteriole Kidney waste molecules can. by the cortex of the suprarenal
 Artery glands.
Glomerular Filtration Rate  These hormones are increased or
 Vein Rental Artery
 Venule decreased according to the needs
 Volume of filtrate produced by both
 Descending limb of the body.
Afferent Arteriole kidneys per minute
 Ascending limb  1 15 mL/min- women ACTIVE SECRETION
 Peritubular capillaries  1 25 mL/min- men
 Renal medulla Glomerulus
 Average of 120 mL/min  During active secretion, wastes that
 Renal cortex  Equivalent to 7.5L filtrate produced were not initially filtered out of the
 Distal convoluted tubule Efferent Arteriole per hour or 180L per day or about 45 blood in the glomerular capsule such
 Efferent arteriole gallons as ammonia and certain drugs and
 Proximal convoluted tubule toxins are removed from the
Vasa Rectae
 Glomerulus capillaries into the distal convoluted
REABSORPTION tubule.
Venule
 The tubule of the nephron functions EVENTS IN THE PROXIMAL TUBULES
Rental artery to reabsorb most of the glomerular
filtrate.  Glomerular filtrate and plasma
Renal Vein
osmolality is the same (-300mOsm)
  Active transport of sodium across the o The walls of the ascending Released by the posterior pituitary
proximal tubule occur loop of Henle is not gland that acts on the collecting duct cells
o Maintained by the Na-K- permeable to water stimulating the formation of water channels
ATPase pump – sodium  Fluid becomes on the membrane
enters from the tubular lumen increasing diluted-
to renal cells via symport fluid the enters the Aquaporins- increases water
sodium transporter distal convoluted absorption
 The buildup of sodium within the tubule is hypotonic (-
renal cells of the PCT attract chloride 100mOsm) Diabetes insipidus
ions by passive transport  The deepest regions of the
o This establishes a solute medulla reaches as highas ~1200 Antidiuretic hormone deficiency
concentration gradient- mOsm
Polyuria with urine characteristically
allows water to move toward  In the descending loop of
hypotonic
the renal cells by osmosis henle- the salts are not actively
o About 85% of salt and water transported: impermeable to the
Urine is diluted: low specific gravity
are reabsorbed passive diffusion of salt
 Permeable to water – water Aldosterone
EVENTS IN THE LOOP OF HENLE
enters the blood by osmosis
 Thus, the concentration of the Control directly the levels of sodium in the
o Salt (NaCI) is actively
tubular fluid is increased, its volume blood
extruded from the tubular
decreased as it descends towards
lumen to the surrounding fluid
the tip of the loops Addison’s disease
 Sodium diffuses from
the filtrate into the Deficiency in aldosterone
cells of the ascending
loop of Henle Events in the distal tubules and the Hyponatremia- low sodium in the blood
accompanied by the collecting ducts
secondary active Hyperkalemia- high potassium in the blood-
transport of potassium Reabsorption of fluids and solutes – that only metabolic alkalosis
and chloride ( 1 Na to about 1% of the filtered volume is excreted
1 K and 2 CI) in the form of urine In primary aldosteronism or conn’s syndrome
o Sodium is actively
transported across the 1 to 2 litres of urine per day Results to exaggerated increase in the
basolateral membrane to levels of sodium in the blood
tissue fluid by Na-K-ATPase Approximately 99% of the filtrate is (hypernatremia)
pumps returned back to the circulation
o Chloride follows thesodium Accompanied by hypokalemia (low
The last stage of reabsorption is governed potassium in the blood – metabolic alkalosis
passively and potassium
by hormonal control (water and sodium)
diffuses back into the filtrate
URINE COMPOSITION
Antidiuretic hormone ADH OR VASOPRESSIN
Factors affecting composition:
 1. Nutritional status able to remove 2. Stable in urine during
2. State of metabolic processes filterable substance a possible 24-hour
3. Ability of the kidney to select from the blood collection period.
substances presented to it - Utilizes measurement 3. Consistency of
plasma level
General features:
 Tests for glomerular filtration 4. Availability to the
- Also known as the body
 97% water and 3% solid
clearance test 5. Availability of
 24-hour urine contains:
- General Equation: laboratory test for the
o 60-70 grams solid
GFR= concentration substance
o 35 grams organic salts
of substance in the
o 25 grams inorganic salts
urine multiplied by the
Organic salts: volume of urine per
2. TUBULAR REABSORPTION TESTS
minute divided by
3. TUBULAR SECRETION AND RENAL
a. Urea- 25 g/L (6 to 18 g/24h) plasma concentration
BLOOD FLOW TESTS
b. Creatinine- 1.5g/L (0.3 to 0.8 of same substance
1.1. Urea
g/24h) - Inulin or creatinine
- Earliest to be utilized
c. Uric acid- 0.6g/L (0.08 to 0.2 may be use
due to its presence in
g/24h) Indicator substance
all urine specimens
d. Ammonia- 0.6g/L should be:
1.2. Β2 microglobulin
e. Sugars, proteins, ketones, etc 1. Freely filtered
- Rapidly removed from
- Trace amounts 2. Neither
the plasma by
secreted nor
Inorganic salts: glomerular filtration.
reabsorbed
- More sensitive
a. Chloride (as NaCL)- major inorganic 3. Not
indicator of a
solid dissolved- 100 to 250 mEq/24h metabolized
decrease in GFR than
b. Sodium (as Na0)- 100 to 200mEq/24h by the kidney
creatinine clearance.
c. Potassium (as K20)- 50 to 70 mEq/24h 4. Easily
- Disadvantage: not
d. Magnesium oxalate analyzed in
reliable in patients
e. Calcium oxalate the urine &
who have history of
plasma
immunologic
Renal Function Tests:
disorders.
 Factors to consider in the
1. CLOMERULAR FILTRATION TESTS 1.3. Inulin
selection of a test substance:
 Clearance test - Most accurate
1. It is neither
- Standard test used to method
reabsorbed nor
measure filtering - Advantage:
secreted by the
capacity of the  Extremely
tubules.
Glomeruli. stable
- Measures the rate at
which the kidneys are
  Neither 2. Chromogens present in human - a proteinase inhibitor found in all
reabsorbed plasma react in the chemical nucleated red cells
nor secreted analysis. - Freely filtered by the glomerulus and
- Disadvantage: 3. Urinary creatinine will be broken reabsorbed in the proximal
Exogenous and toxic down by bacteria if specimen are convoluted tubules.
- Measured by kept at room temperature. - Correlates better with GFR than
immunoassays 4. Heavy meat diet will influence the creatinine in renal transplant
1.4. Creatinine results of tests. recipients.
- Waste product of 5. Not reliable indicator in patients - Measured by immunoassays.
muscle metabolism suffering from muscle wasting.
2. Tubular Reabsorption Tests (Concentration
that is normally found
Tests)
at a relatively
constant level in Formula: C= UV/P
-  The first function affected in renal
blood. Where: C- ml of plasma diseases.
- Method of choice cleared/min  Determine the ability of the tubules
U- mg/dL urine creatinine to reabsorb the essential salts and
V- urine volume in mL water that have been non-
Clearance test (vol/min) selectively filtered by the glomerulus.
P- mg/dL plasma creatinine
Urea clearance test (75ml/min) 2.1 Fishberg test
- To adjust clearance for body size:

Creatinine clearance test (70- C= (UV/P)x(1.73/A) - Patients were deprived of fluids for
24 hours prior to measuring the
157ml/min)
Where: 1.73- body surface specific gravity
Inulin or lothalamate clearance test area in average person (sq meter)
2.2 Mosenthal test
(120ml/min) A- actual body size in
sq meter - Compared the volume and specific
Std. clearance (ml/min)= U (mg/dl) X gravity of day and night urine
V (ml/min) X 1.73 By Cokcroft and Gault: samples to evaluate concentrating
ability.
Ccr= (140-age)( weight in kg)
P(mg/dl) A o Person with normal
72 x serum creatinine in
concentrating ability should
Glomerular Filtration Tests- Creatinine mg/dL have a specific gravity of
1.025 when deprived of fluid
Disadvantages over Inulin OR
for 16 hours.
1. Some is secreted by the tubules Ccr= 140-age; results multiplied by 2.3 Osmometry
(secretion increases as blood level 0.85 for Females Serum creatinine
increases.) NaCI- reference standard because it
1.5. Cystatin C is partially ionized and more
 representative of urine and plasma Ration of urine to serum mOsm: 1:1 completely removed from the
component. *after controlled fluid intake: plasma each time it comes in
3:1 contact with functional renal
Source of errors: tissues.
Interpretation:
- Lipemic serum Advantages:
- Lactic acid - Failure to respond to injected ADH
- Volatile substances ex. Ethanol indicates renal defect in 1. Non-toxic
- Failure to refrigerate in less than 20 concentrating ability. 2. High molecular weight and does not
minutes - Response to injected ADH is bind to plasma proteins allowing
associated with a defect in complete removal
Osmometry 3. PAH is secreted by the proximal
production.
concoluted tubule
i. Based on freezing point depression: 2.4 Free Water Clearance:
- The freezing point of a
solution by supercooling a - Performing the urine – to – serum Disadvantage: Exogenous
measured amount of sample osmolarity ratio using a timed urine
to approximately- 7°C. specimen. P-aminohippuric acid (PAH) test
ii. Vapor pressure osmometer - Tells how much water must be
- Uses microsamples (0.01 mL) Cosm (mL/min)= C (mg/dL PAH) X V
cleared each minute to produce a
- Actual measurement (mL/min Urine)
urine with the same osmolarity as the
performed in dew point- plasma. P (mg/dL PAH)
temperature at which water
favor condenses. 3.2 Titratable Acidity and Urinary
1. Compute the osmolar clearance
Ammonia
Cosm= Uosm XV
Clinical Significance:
where: V- urine volume Principle: Ability of the kidney to
Clinical use of osmolarity: Posm produce an acid urine dependent on
2. Subtract the osmolar clearance from the tubular secretion of hydrogen ions
1. Initial evaluation of renal the urine volume and production and secretion of
concentrating ability ammonia by the cells of the DCT.
CH20= V- Cosm
2. Monitoring of the course of renal
disease Clinical significance:
3. Monitoring fluid and electrolyte
therapy Tubular secreation and Rena Blood Flow 1. Normal person excrete
4. Establishing the differential diagnosis Tests approximately 7 mEq of acid per
of hypernatremia and hyponatremia day in the form of either
and polyuria. 3.1 p-aminohippuric acid (PAH) test titratable acid (H+) or
ammonium ion (NH4+).
Normal Osmolarity: Test most common test
- 2. Normal variation in urine acidity:
- Requirement: a. Alkaline tides- shortly after
Serum: between 274 and 300 mOsm
use substance that is arising and post-
Urine: between 50 and 1400 mOsm
 prandially (2pm and • The actual amount of urochrome ➢Bilirubin in urine – yellow foam appears
8pm) produced is dependent on the body’s when the specimen is shaken.
b. Lowest pH at night time. metabolic state (increased amounts
produced in thyroid conditions and fasting). ➢Normal urine – small amount of rapidly
Physical Examination of Urine
disappearing foam.
• Increased in urine that stands at room
Urine is normally 94% water and 6% solutes.
temperature. ➢Increase protein in urine – large amount of
Color
white foam.
• Constant rate excretion = Intensity of the
•Urine color varies from almost colorless to
yellow color in a fresh urine specimen can ➢Urobilin in urine – no yellow foam when
black (normal metabolic functions, physical
give a rough estimate of urine shaken.
activity, ingested materials, or pathologic
concentration.
conditions). Dark Yellow/Amber/Orange
✓Pale yellow Diluted urine
•The responsibility of the laboratory to • Biliverdin – Photo-oxidation of bilirubin
determine whether the color change is gives a yellow-green color urine.
✓Dark yellow Concentrated urine
normal or pathologic.
Normal Urine Color • Phenazopyridine or azo-gantrisin
Normal Urine Color compounds – yellow-orange urine
UROERYTHRIN & UROBILIN specimen, used to treat UTI.
Common description:
• Also present in the urine in much smaller ➢Interferes chemical tests.
✓Pale yellow
quantities.
➢Produces yellow foam when shaken
✓Yellow
• Contribute little to the color of normal, (mistaken for bilirubin).
fresh urine.
✓Dark yellow
Red/Pink/Brown
➢Uroerythrin – pink pigment, found in
Examine the specimen under a good light
refrigerated samples resulting in the • Most common cause of abnormal urine
source,
precipitation of amorphous urates. color.
Looking down through the container against
➢Urobilin – orange-brown color of urine • Presence of blood (color may range from
a white background.
(NOT FRESH), oxidation product of the pink to brown).
UROCHROME normal urinary constituent urobilinogen
• RBCs in an acidic urine for several hours
• Pigment that causes the yellow color of Abnormal Urine Color produces brown urine (oxidation of
urine. hemoglobin to methemoglobin).
Dark Yellow/Amber/Orange
• Is a product of endogenous metabolism Hemoglobin & myoglobin – produces red
(Constant rate). • Can be caused by the abnormal pigment urine and result in a positive chemical test
bilirubin. for blood.
In contrast: ➢Homogentisic acid (metabolite of
phenylalanine) – black urine from persons
➢RBCs – red and cloudy urine.
with alkaptonuria (inborn-error of
metabolism).
➢Hemoglobin & myoglobin – red and clear
urine. Blue/Green

• Distinguishing between Hemoglobinuria Pathogenic causes:

and myoglobinuria may be possible by
examining the patients plasma. • Bacterial infection

➢Hemoglobinuria (in vivo breakdown of • Urinary tract infection (Pseudomonas spp.)

RBCs)– red plasma.
• Intestinal tract infections
➢Myoglobinuria (breakdown of skeletal
• Ingestion of breath deodorizers (Clorets)
muscle) – does not affect the color of the
green urine.
plasma and reddish-brown in urine.
• Medications (blue urine)
• Porphyrins – red (port wine) urine
specimens, resulting from the oxidation of • Methocarbamol (Robaxin)
porphobilinogen.
• Methylene blue
•Nonpathogenic causes (Red urine):
• Amitriptyline (Elavil)
➢Menstrual contamination
Clarity
➢Ingestion of highly pigmented foods
(beets, black-berries) • Refers to transparency or turbidity of a
urine specimen.
➢Medications (rifampin, phenolphthalein,
phenindione, phenothiazines) Brown/Black • By visually examining the mixed specimen
in a clear container (test tube) while holding
• May contain melanin or homogentisic it in front of a light source.
acid in a standing urine sample and have a
negative chemical test results for blood. • Common terminology used to report
clarity:
➢Melanin (oxidation product of
melanogen)– produced in excess with CLARITY TERM
malignant melanoma.
Milky May precipitate or be clotted
Preservatives Advantages Disadvantages Comments
Refrigeration Does not interfere with • Raise SG by hydrometer Prevent bacterial growth in 24 hours
chemical test • precipitates amorphous
phosphates and urates

Thymol Preserves Glucose and • interfere with acid precipitation

sediments well test for protein
• large amount interferes with O-
toluidine glucose test

Boric acid Preserves proteins and • large amount may precipitate • keeps ph about 8.0
sediments well crystals • bacteriostatic (18g/L)
Does not interfere with • interfere with drug and hormone - Can be used for culture transport
routine analyses other analyses
than ph

Formalin Excellent sediment • interfere with copper reduction test Can be used to rinse container for cell counts
preservative for glucose for preservation of cells and casts.
• causes clumping of sediments

Chloroform Interfere with sediment analyses May cause cellular changes

Toluene Does not interfere with Clings to pipettes and testing
routine test materials
Sodium fluoride •Prevents glycolysis Inhibit reagent strip tests for glucose, Will not interfere with hexokinase glucose test
•good preservation for blood and leukocytes
drug analyses

Phenol Does not interfere with Causes odor change Use 1 drop per ounce specimen
routine test
Commercial preservative •Convenient if May contain one or more of the Check product composition
tablet refrigeration not above preservatives
required
•Concentration is
controlled to minimize
interference

Urine C +S transport kit Urinalysis and culture Decreases ph Uses boric acid as preservative
can be run on serve
specimen
Urinalysis Quality Assurance C. Patient’s dietary and medicinal intake test.
PRE- ANALYTICAL FACTORS D. Method of collection • Urine is tested for glucose and ketones

A. Random specimen F. 24- Hour (timed) specimen for quantitative

Changes in unpreserved specimens
chemical test
(Delayed urinalysis) • Most commonly received
•advantages: • 4- amino benzoic acid given to patient at
A Increased in:
1. Ease of collection start of collection period; concentration of
1. Bacteria 2. Lack of inconvenience to the patient excreted marker is measured to determine
2. pH- due to breakdown of urea to 3. Useful for routine screening (Glomerular completeness of collection.
ammonia by urease producing bacteria bleeding, albuminuria and cast seen at
anytime) G. Catheterized specimen
3. Turbidity- due to bacterial growth and
precipitation of amorphous material •disadvantage: produce erroneous results
• collected under sterile conditions
4. Nitrite- due to bacterial reduction of nitrite caused by dietary intake or physical
• commonly for bacterial infection, also
inactivity prior to collection.
routine
B. Decreased in:
B. First – morning specimen
H. Midstream clean- catch specimen
1. Glucose- due to glycolysis and bacterial • ideal for screening specimen
utilization • essential for preventing false- negative • Safer; less traumatic
2. Ketones- due to volatilization pregnancy test • for routine and bacterial culture
3. Bilirubin- from exposure to light • for evaluation of orthostatic proteinuria • advantages:
4. Urobilinogen- due to oxidation to urobilin • advantage: Concentrated specimen- • less contaminated by epithelial cells
assured of detecting chemicals and formed and bacteria
C. Disintegration of red blood cells,
elements • more representative of actual urine
especially in dilute alkaline urine
C. Fasting specimen (second morning) I. Suprapubic aspiration
D. Changes in color- due to oxidation or
reduction of metabolites • Second voided specimen after period of • collected by introduction of needle
fasting through the abdomen into the bladder
QUALITY ASSURANCE • for glucose (diabetic) monitoring • for bacterial culture; also cytologic
examination
PRE- ANALYTICAL FACTORS D. 2-Hour postprandial specimen
J. Three- glass collection
2. TYPES OF SPECIMEN • primarily for monitoring insulin therapy in
persons with diabetes mellitus • Determination prostatic infection
- Condition to obtain a specimen that is truly • quantitative cultures- on all specimens
representative of patient’s metabolic state: E. Glucose tolerance specimen
• microscopic examination- 1st and 3rd
• collected to correspond with blood specimens
A. Time of collection
sample drawn during glucose tolerance • control for bladder of kidney infection-
B. Length of collection period
2 specimen
nd
• Prostatic infection if: on a weekly basis III. QUALITY CONTROL
• 3rd specimen will have a WBC/hpf 1.2 bacterial count on a monthly basis • detect analytic or technical errors during
count and bacterial count 10 X that of the analysis and
first specimen Care of reagents strips:
• detect defects in the reagents or
• macrophages containing lipids may equipment to prevent reporting or incorrect
1. store with desiccant in an opaque, tightly
also be present patient results
closed container
2. Store in a cool place, but do not • may be INTERNAL or EXTERNAL quality
K. Pediatric specimen
refrigerate control
• use, soft clear plastic tags with adhesives 3. Do not expose to volatile fumes
Types of control system:
4. Do not use past the expiration date
L. Drug specimen collection 5. Use within 6 months after opening Two-level control
6. Do not use if discolored.
• observe chain of custody
• NORMAL control- constituents at
• volume: 30 to 45 mL
II. INSTRUMENT AND EQUIPMENT concentrations within non-disease reference
• urine temperature: taken 4 minutes from
range
the time collection – detect whether • Urinometer/refractometer • ABNORMAL control- constituents at
adulterated (
concentrations outside of the reference
temperature range 32.50 Celsius to 37.70 Calibration: Each shift using distilled water
range
Celsius) (1.000)and a known control
Also by 5% saline (1.022+/-0.001) Three-level control
ANALTICAL FACTORS Also by 9% sucrose (1. 034+/-0.001)
-may be: LOW, NORMAL, HIGH
1. reagent Refrigerator and water baths
2. instrumentation and equipment • when medically significant decisions are
3. quality control • Temperature taken daily and recorded made at each level
4. Preventive maintenance
Centrifuges Examples:
5. Access to procedure manual
6. Competency of personnel performing the • calibration performed every 3 onths • chek- stix- for reagent strips
tests • disinfected on a weekly basis • method of Hoeltge & Ersts – for
• centrifugation for 5 minutes at RCF 450 microscopic sediments
I. REAGENTS AND TEST PERFORMANCE gives optimum amount of sediment with
least chance of damaging the sediments OUT-OF-CONTROL PROCEDURE
• strips checked against known negative
and positive control solutions on each shift Microscope 1. Run control
and whenever a new bottle is opened. • record all routine and non-routine
• deionized water used for reagent maintenance performed IN control Out
preparation is controlled: • be kept clean at all times control
1.1 using ph and purify meter resistance
Proceed with testing Go • After the strip has been appropriately 2. Reduced costs (e.g., reagents, personnel)
step 2 wetted in a urine sample, chemical
reactions cause the reaction pads to 3. Enhanced test sensitivity and specificity
Chemical Examination of Urine change color.
4. Decreased the amount of urine required
REAGENT STRIPS • At the appropriate “read time,” results are for testing
determined by comparing the color of each
To ensure the integrity of reagent strips, their
Reagent strips enable rapid screening of reaction pad with the appropriate analyte
proper storage is essential, and the
urine on the color chart.
manufacturer’s directions must be followed.
specimens for:
✓pH Reducing agents such as ascorbic acid
✓Protein have the potential to adversely affect
several reagent strip test results, it is Care and Storage
✓Glucose
important that these and other potential • All reagents strips must be protested from
✓Ketones interferences are detected or eliminated. moisture, chemicals, heat, and light.
✓Blood • Any strips showing evidence of
✓Bilirubin deterioration, contamination, or improper
✓Urobilinogen storage should be discarded.
✓Nitrite • Tight-fitting lids, along with desiccants or
✓Leukocyte esterase drying agents within the product container,
help eliminate test pad deterioration due to
Routinely used for chemical analysis of moisture.
urine. • Fumes from volatile chemicals, acids, and
Single or Multiple (2 to 10) test pads
bases can adversely affect the test pads
Four commonly used brands of commercial
reagent and should be avoided.
Results are reported (depending on the test):
strips are: • All reagents strip containers protest the
✓Multistix (Siemens Healthcare Diagnostics ✓In concentration (milligrams per deciliter) reagent strips from ultraviolet rays and
Inc., Deerfield, IL) sunlight; however, the containers themselves
✓As small, moderate, or large must be protected on the label of the
✓Chemstrips (Roche Diagnostics, container
Indianapolis, IN) ✓Using the plus system (1+, 2+, 3+, 4+) • Reagents strips should be stored in their
original containers at temperatures below
✓vChem Strips (Iris Urinalysis-Beckman ✓As positive, negative, or normal
30°C (86°F); they are stable until the
Coulter Inc., Brea, CA) expiration date indicated on the label.
The specific gravity and the pH are
✓Aution Sticks (Arkray Inc., Kyoto, Japan) exceptions; these results are estimated in
their respective units. Quality Control Testing
• A commercial reagent strip or dipstick • Ensures that the reagent strips are
consists of reagent -impregnated test pads Reagent strips have significantly: functioning properly.
that are fixed to an inert plastic strip. 1. Reduced the time required for testing • Confirms the acceptable performance
and technique of the laboratorian using • At room temperature
them.
TECHNIQUE:
Negative and positive control levels must 1. Using uncentrifuged urine, mix specimen
be used to check the reactivity of well.
reagent strips. 2. Dip reagent strip briefly into urine to wet
Commercial or laboratory-prepared all reaction pads and start timing device.
materials can serve as acceptable 3. Remove excess urine by drawing edge of
negative/positive controls. • Commercial tablets tests (e.g., Ictotest, strip against rim of container or by blotting
Clinitest, Acetest [all from Siemens strip edge on absorbent paper.
4. At the appropriate times, read results of
Healthcare Diagnostics Inc., Deerfield, IL])
each reaction pad using the color chart on
An additional quality check on chemical • Must be handled and stored according to the
and microscopic examinations, as well as the inserts provided by the manufacturers. container.
on the laboratorian, involves: • Susceptible to deterioration from exposure 5. Discard strip into biohazard waste.
• Interlaboratory – aliquoting a well-mixed to light, heat, and moisture.
urine specimen from the daily workload and Reagent Strips
CHEMICAL TESTS
having a different laboratory analyze the
Urine specimen (room temperature) – it must
specimen.
• Sulfosalicylic acid (SSA) precipitation test – be tested within 2 hours after collection to
detect protein avoid erroneous results caused by changes
• Interlaboratory duplicate testing checks
• Hoesch test – detect porphobilinogen that can occur in unpreserved urine.
the entire urinalysis procedure and detects
(PBG) Urine specimen (Refrigerated) – it should be
innocuous changes when manual urinalyses allowed to warm up to room temperature
are performed, such as variations in the • Watson-Schwartz test – detect porphyria
before testing with reagent strips to avoid
speed of centrifugation and in centrifuge • Benedict’s test – detect glucose erroneous results.
brake usage. Urines that are highly pigmented due to
When new reagents are prepared, they medications can lead to false-positive
• Intralaboratory – aliquoting a well-mixed should be tested in parallel with current “in- reagent strip test results because the true
urine specimen from the daily workload and use” reagents to ensure equivalent color reaction is masked by the pigment
having a technologist on each shift analyze performance. present.
the specimen.
CHEMICAL TESTING TECHNIQUE Tablet and Chemical Tests
• Intralaboratory duplicate testing can
also be used to evaluate the technical Chemical and tablet tests are generally
Reagent Strips
competency of laboratorians. performed:
ROOM CONDITIONS:
TABLE AND CHEMICAL TESTS 1. To confirm results already obtained by
• Good lighting, preferably fluorescent; reagent strip testing;
Care and Storage avoid direct sunlight 2. As an alternative method for highly
pigmented urine;
URINE SPECIMEN:
3. More sensitive for the substance of interest
than the polyelectrolyte. As protons are released, the drugs (e.g., sulfonamides, streptomycin,
reagent strip test (e.g., Ictotest tablets); pH decreases and produces a color salicylate) and prevents stone formation
4. Specificity of the test differs from that of change of the bromthymol blue indicator from calcium oxalate, uric acid, and cystine
the reagent strip test (e.g., SSA test, from blue-green to yellow-green. crystals.
Clinitest). • Chemstrip and Multistix reagent strips only
• Polyelectrolyte used: <4.5 – suspect adulteration of urine
CHEMICAL TESTS specimen.
• Chemstrip: ethylene glycol-bis tetraacetic
>8.0 – indicates:
• Specific Gravity acid
• Presence of an iatrogenic alkaline
• pH • Multistix: polymethylvinyl ether/maleic substance (intravenous medication or
• Protein acid agent)
• Blood • Sensitivity • Improperly stored urine specimen
• Leukocyte Esterase • Chemstrip: 1.000–1.030 • Contamination with an alkaline chemical
• Nitrite • Multistix: 1.000–1.030 (preservative)
• Glucose • vChem: 1.000–1.035 4.5–6.9 (Acid urine) – associated with:
• Diet (high protein and cranberry ingestion) ,
• Ketones •Specificity
sleep,
• Bilirubin and Urobilinogen • Detects only ionic solutes; provides metabolic acidosis (e.g., ketoacidosis,
• Ascorbic Acid “estimate” in 0.005 increments starvation,
• Falsely low: severe diarrhea, uremia, poisons—ethylene
Specific Gravity Clinical Significance • Chemstrip: Glucose and urea >1 glycol,
g/dL methanol), respiratory acidosis (e.g.,
1.000 – same as pure water; suspect emphysema,
adulteration of urine specimen • Multistix: pH 6.5; add 0.005
chronic lung disease), urinary system
>1.040 – indicates presence of iatrogenic • Falsely high:
disorders:
substance (e.g., radiographic contrast • Protein approximately equal to 100– UTI with acid-producing bacteria
media, 500 mg/dL (Escherichia coli),
mannitol) • Ketoacidosis chronic renal failure, uremia, medications
1.010–1.025 – Indicates average used to
solute and water intake and excretion pH Clinical Significance induce: ammonium chloride, ascorbic acid,
1.001–1.009 – Dilute urine; associated with • Urine pH varies from 4.5 to 8.0. methionine, mandelic acid
increased water intake or water diuresis 7.0–7.9 (Alkaline urine) – associated with:
• Average individual excretes slightly acidic
(e.g., • Diet (vegetarian, citrus fruits, low
urine of pH 5.0 to 6.0.
diuretics, inadequate secretion/action of carbohydrate),
ADH*) • Alkaline tide – (during and after a meal)
metabolic alkalosis (e.g., vomiting, gastric
1.025–1.035 (1.040 maximum) – Concentrate urine produced is less acidic. lavage),
urine; associated with dehydration, fluid • Acid urine prevents stone formation by respiratory
restriction, profuse sweating, osmotic alkaline-precipitating solutes (e.g., calcium : UTI with urease
diuresis carbonate, calcium phosphate) and inhibits producing bacteria (Proteus sp., alkalosis
Physiologically Impossible the development of urinary tract infection. (e.g., hyperventilation),
urinary system disorders Pseudomonas sp.),
• Alkaline urine prevents the precipitation of
• Ionic solutes present in the urine cause renal tubular acidosis, medications used to
and enhances the excretion of various induce:
protons to be released from a
sodium bicarbonate, potassium citrate, • Temperature dependent (pH • Postrenal proteinuria
acetazolamide decreases with increasing temperature). • Prerenal or overflow proteinuria – an
• pH Test Papers increase in plasma low MW proteins leads to
• Do not add impurities to the urine. increased excretion in urine.
• Produce sharp color changes for • Proteins present:
comparison with a supplied color chart of •Normal proteins:
pH values. • Myoglobin
• Hemoglobin
Protein Clinical Significance • Acute phase reactants
• Normal urine – 150 mg (1 to 14 mg/dL) of • Abnormal proteins:
Physiologically Impossible
protein each day • Ig light chains (Bence Jones protein –
pH Reagent Strip Method • one-third albumin and two-thirds Multiple Myeloma)
globulins • An aliquot of the urine specimen
•Less than 0.1% (plasma albumin enters the would be heated, and if the urine
ultrafiltrate) coagulated at 40°C to 60°C and redissolved
• 95% to 99% (all filtered protein is at 100°C.
reabsorbed)
• <40,000 – readily pass through the Renal Proteinuria
glomerular filtration barriers • Glomerular proteinuria – GFB is defective,
• Principle: Double-indicator system allowing plasma proteins to enter
•>90,000 – unable to penetrate a healthy
• Indicators: methyl red and bromthymol
glomerular filtration barrier ultrafiltrate.
blue are used to give distinct color changes
from orange to green to blue (pH 5.0–9.0). • Proteins that originate from the urinary
• Proteins present:
• Sensitivity: tract:
• Selective – Increase in albumin and
• Chemstrip: 5.0–9.0, in 1.0-pH • Uromodulin (also known as Tamm-
increments moderate MW plasma proteins (Transferrin,
Horsfall protein)
• Multistix: 5.0–8.5, in 0.5-pH increments α1-Antitrypsin, α1-Acid glycoprotein)
• Urokinase (enzyme)
• vChem: 5.0–9.0, in 1.0-pH increments • Nonselective – Increase in all proteins,
• Specificity: pH; hydrogen ion • Secretory immunoglobulin A
including high MW plasma proteins
concentration •Causes: Glomerulonephritis, Diabetes
• No interferences known; unaffected by Protein Clinical Significance
mellitus
protein concentration. • Proteinuria – first indicator of renal disease
• Functional proteinuria – induce a mild
pH Other Methods (albumin).
glomerular or mixed pattern of proteinuria in
• pH Meter • Proteinuria can be classified into four
the absence of renal disease (Strenuous
• Alternative method categories:
exercise, fever, extreme cold exposure,
• Consists of a silver–silver chloride • Prerenal or overflow proteinuria
emotional distress, congestive heart failure,
indicator electrode with a pH-sensitive glass • Glomerular proteinuria – most common
and dehydration).
membrane connected by a salt bridge to a type of proteinuria encountered and is the
reference electrode (usually a calomel most serious. • Renal Proteinuria
electrode, Hg–Hg2Cl2). • Tubular proteinuria • Tubular proteinuria – defective tubular
reabsorption of protein • Presence of proteins other than
• Proteins present: Increase in the low MW • Reagent Strip Tests albumin
proteins (Plasma proteins – β2-microglobulin, • Highly colored substances that
retinol-binding protein, α2- microglobulin, or mask results, such as drugs
lysozyme) normally present in the (phenazopyridine, nitrofurantoin), beet
ultrafiltrate, including albumin. ingestion
• Causes: Acute/chronic pyelonephritis, • Principle: Protein error of indicators
Interstitial nephritis, Fanconi’s syndrome • Sensitive Albumin (Microalbumin) Tests
• When the pH is held constant by a
• SSA precipitation test used to detect total • Monitoring of urine for low-level albumin.
buffer (pH 3.0), indicator dyes release H+
protein. • Used for individuals with diabetes,
ions because of the protein present. Color
hypertension, or peripheral vascular disease.
change ranges from yellow to blue-green.
• Postrenal • Commercial reagent strip methods
• Indicator used: derivatives of
• Urine includes proteins produced by the available to screen urine for low-level
tetrabromophenol blue
urinary tract or the urine is contaminated increases in albumin include:
• Protein reagent strip results are reported as
with proteins during excretion. • OSOM ImmunoDip urine albumin test
concentrations in milligrams per deciliter
• Proteins present: (Sekisui Diagnostics
(mg/dL).
• Pus • Corporation, Framingham, MA)
• Menstrual and hemorrhoidal blood • Protein reagen (Roche Diagnostics)
Note that globulins, myoglobin, hemoglobin,
• Vaginal secretions immunoglobulin light chains (Bence Jones • Multistix PRO reagent strip test – A/C
• Prostatic secretions proteins), and mucoproteins are usually NOT ratio
DETECTED by the reagent strip test. • Clinitek Microalbumin reagent strip test
Protein Methods (Siemens Healthcare Diagnostics Inc.) – A/C
• Sulfosalicylic Acid Precipitation Test (SSA)– • Sensitivity: ratio
detects all proteins in urine—albumin and • Chemstrip: 6.0 mg/dL in 90% of urines
globulins. tested Determination of an A/C or protein-to-
• Not frequently performed today because • Multistix: 15–30 mg/dL creatinine ratio
reduces the numbers of false- positive and
it is nonspecific and time-consuming. • vChem: 20 mg/dL in 67% of urines
false-negative test results.
• False-positive SSA precipitation results (x- tested
ray contrast media and certain drugs) Specificity: More sensitive to albumin
Blood Clinical Significance
• False-positive results:
Differentiation of Hemoglobinuria and
• Highly buffered or alkaline urine (pH
Myoglobinuria
>9), such as alkaline drugs, improperly
preserved specimen, contamination with Parameter Hemoglobinuria Myoglobinuria
quaternary ammonium compounds Urine color Pink, red, brown Pink, red,
• Highly colored substances that brown
mask results, such as drugs Blood Positive Positive
(phenazopyridine), beet ingestion reagent
• False-negative results: strip test
Serum color Pink to red Pale yellow • Multistix: 0.02–0.06 mg/dL Hgb (6–20
(hemolysis) (normal) RBCs/μL*)
Serum • vChem: 0.02 mg/dL Hgb (5 RBCs/μL*) in
chemistry 67% of urines tested
tests Decreased to Normal
Haptoglobin absent • Specificity: Equally specific for hemoglobin
Normal Increased
and myoglobin Intact RBCs are lysed on
Myoglobin Increased Normal • Principle: Action of leukocyte esterases to
Free Increased, but Increased, reagent pad
cleave an ester and form an aromatic
hemoglobin <10 times upper but >10 times
• False-positive results: compound is followed by an azo coupling
Creatine reference limit upper
kinase (CK) reference limit • Menstrual contamination reaction of the aromatic amine formed with
• Hematuria – abnormal quantity of red • Peroxidases (e.g., microbial) a diazonium salt on the reagent pad. The
blood cells in the urine (Cloudy or smoky • Strong oxidizing agents (e.g., azo dye produced causes a color change
urine). hypochlorite in detergents) from beige to violet.
• Hemoglobinuria – indicatesthe urinary • False-negative or decreased results:
• Ascorbic acid: • Ester used:
presence of hemoglobin (Clear urine).
• Multistix (>9 mg/dL) • Chemstrip and vChem:
• Myoglobinuria – indicates the urinary
• vChem (>5 mg/dL) indoxylcarbonic acid ester
presence of myoglobin.
• Chemstrip NOT affected • Multistix: derivatized pyrrole amino acid
Blood Method • High specific gravity ester
• Captopril (Multistix)
• Formalin • Sensitivity:
• High nitrite (>10 mg/dL) • Chemstrip: approximately 10 WBCs/μL
• Multistix: approximately 5–15 WBCs per
Leukocyte Esterase Clinical Significance high-power field (⁓11–36 WBCs/μL)
Reagent Strip Test
• Normal urine – few white blood cells • vChem: approximately 15 WBCs/μL in
• Principle: Pseudoperoxidase activity of the (leukocytes) are present in urine (0 to 8 per 67% of urines tested
heme moiety high-power field).
• Specificity: Detects only granulocytic
• The chromogen reacts with a peroxide • Pathologic urine – Presence of leukocytes
in the presence of hemoglobin or myoglobin approximately 20 or more white blood cells
• False-positive results:
to become oxidized and produce a color per microliter.
• Highly colored substances that mask
change from yellow to green.
• Chemical detection of leukocyte esterase results, such as drugs (phenazopyridine),
• Chromogen used:
provides a means to determine the beet ingestion
tetramethylbenzidine
presence of white blood cells even when • Vaginal contamination of urine
• Sensitivity: they are no longer viable or visible. • Formalin
• Chemstrip: 0.02–0.03 mg/dL Hgb(5–
• Reagent Strip Test • False-negative results:
10RBCs/μL*)
• Lymphocytes are not detected
 • Increased glucose (>3 g/dL) or pad. The azo dye produced causes a color characterized by an inability to metabolize
protein (>500 mg/dL) change from white to pink. galactose to glucose.
• High specific gravity • Urine from children younger than 2
• Strong oxidizing agents (soaps, • Amine used: years should be tested with a copper
detergents) • Chemstrip: sulfanilamide reduction test to screen for reducing
• Drugs such as gentamicin, • Multistix and vChem: p-arsanilic acid substances that are formed as a result of
cephalosporins, tetracycline galactosemia.
• Aromatic compound:
• Diabetes mellitus is the most common
• Chemstrip and Multistix:
Nitrite Clinical Significance disease that causes hyperglycemia and
tetrahydrobenzoquinolinol
• Routine screening for urine nitrite provides glucosuria.
• vChem: N-(1-napthyl)-ethylenediamine
an important tool to identify urinary tract
infection. • Sensitivity: Glucose Method
• Chemstrip: 0.05 mg/dL nitrite ion in 90% of
• A urinary tract infection (UTI) can involve
urines tested
the bladder
• Multistix: 0.06 mg/dL nitrite ion
(cystitis), the renal pelvis and tubules
• vChem: 0.04 mg/dL nitrite ion in 67% of
(pyelonephritis), or both.
urines tested
• Nitrate-reducing bacteria (nitrate • Reagent Strip Tests
• Specificity:
reductase) are infecting the urinary tract
• False-positive results:
and adequate bladder retention time is • Principle: Double-sequential enzyme
• Highly colored substances that mask
allowed, these bacteria convert dietary reaction.
results, such as drugs (phenazopyridine),
nitrate to nitrite.
beet ingestion • Glucose oxidase on reagent pad
Nitrite Method • Improper storage with bacterial catalyzes the oxidation of glucose to form
proliferation hydrogen peroxide. The hydrogen peroxide
• False-negative results: formed in the first reaction oxidizes a
• Ascorbic acid (>25 mg/dL) chromogen on the reagent pad. The
interference second reaction is catalyzed by a
• Various factors that inhibit or prevent peroxidase provided on the pad. The color
nitrite formation despite bacteriuria change differs with the chromogen used.
• Chromogen used:
Glucose Clinical Significance • Chemstrip: tetramethylbenzidine
•Reagent Strip Test
• Multistix: potassium iodide
• Principle: Diazotization reaction of nitrite • Glucosuria – presence of glucose in urine.
• vChem: tolidine hydrochloride
with an aromatic amine to produce a • Glycosuria – non-glucose sugars (e.g.,
• Sensitivity:
diazonium salt is followed by an azo fructose, sucrose, lactose) or a combination
• Chemstrip: 40 mg/dL in 90% of urines
coupling reaction of this diazonium salt with of sugars are present.
tested
an aromatic compound on the reagent • Galactosemia is an inherited disorder
• Multistix: 75 to 125 mg/dL
 • vChem: 20 mg/dL in 67% of urines • Inability to utilize available
tested carbohydrates
• Diabetes mellitus
• Specificity: Specific for glucose • Insuficient carbohydrates consumption
• Affected by high specific gravity and • Starvation
low temperatures • Diet regimens
• False-positive results: • Alcoholism
• Strong oxidizing agents, such as Ketone Formation • Severe Exercise
bleach • Cold exercise
• Peroxide contaminants • Acute febrile illness in children
• False-negative results: • Loss of carbohydrates
• Ascorbic acid (>50 mg/dL) • Frequent vomiting (e.g., pregnancy,
• Improperly stored specimens (i.e., illness)
glycolysis) • Defective renal reabsorption (e.g., .,
Fanconi’s syndrome)
• Copper Reduction Tests (Benedict’s test)
• Digestive disturbance
• Reducing substances in urine that can
cause positive copper reduction test: Ketone Method
Limited carbohydrate availability, the liver
• Sugars (Glucose, Fructose, Galactose, must oxidize fatty acids as its main
Lactose, Maltose, except sucrose) metabolic substrate.
• Ascorbic Acid
• Cysteine • The terms ketones and ketone bodies
• Homogentisic acid identify three intermediate products of fatty
acid metabolism: • Reagent Strip Tests
• Acetoacetate – 20%
• β-hydroxybutyrate – 78% • Principle: Legal’s test—nitroprusside
• Acetone – 2% reaction.
• Acetoacetic acid in an alkaline
• Ketonuria – ketones are excreted in the medium reacts with nitroferricyanide to
urine, 70 mg/dL (the renal threshold level). produce a color change from beige to
• Acetone is also eliminated by the lungs– purple.
• A tablet version of Benedict’s test is the
breath of patients with ketonemia has a • Chemstrip and vChem reagent strips
Clinitest reagent tablet (Siemens Healthcare
distinctive acetonic or fruity odor. include glycine in the reaction pad, which
Diagnostics Inc.)
enables the detection of acetone
• A mixture of approximately 0.25 mL of Ketone Clinical Significance • Multistix strips do not
urine (5 drops) and 0.50 mL of water (10
• Urine ketone excretion is abour 20 • Sensitivity:
drops) is prepared in a test tube, and one
mg/day. • Chemstrip: 9.0 mg/dL acetoacetate
reagent tablet is added.
• Causes of Ketonuria: and 70 mg/dL acetone in 90% of urines
• Principle:
tested Bilirubin and Urobilinogen Formation Bilirubin and Urobilinogen Clinical Sig
• Multistix: 5.0 to 10 mg/dL acetoacetate Diagnostic Utility of Urine Bilirubin,
• vChem: 3.0 mg/dL ketone Urobilinogen, and Fecal Color
(acetoacetate) in 67% of urines tested
• Specificity: Does not detect β- Jaundi Conditions Urine Feca
hydroxybutyrate ce l
• False-positive results: Classifi Colo
cation r
• Compounds containing free
Prehep Hemolytic Bilirubi Nor
sulfhydryl groups, such as MESNA, captopril, • Sources of bilirubin: atic disorders n: mal
N-acetylcysteine •85% hemoglobin (Senescent RBC) (increa • Transfusion Negati
• Highly pigmented urines •Destroyed red blood cell sed reactions ve
• Atypical colors with •Heme-containing proteins (myoglobin or heme • Sickle cell Urobili
phenylketones and phthaleins degra disease nogen
cytochromes)
• Large quantities of levodopa dation) • Hereditary :↑
• Urobilin and stercobilin (orange- brown) – spherocytosis
metabolites color of feces. • Hemolytic
• False-negative results: • Urobilinogen (urine) – contribute to the disease of
• Improper storage, resulting in color of urine. newborn
volatilization and bacterial breakdown Ineffective
Bilirubin and Urobilinogen Clinical Sig erythropoiesis
Nitroprusside Tablet Test for Ketones • Thalassemia
(Acetest) • Pernicious
anemia
Hepati Hepatitis Bilirubi Nor
c Cirrhosis n: mal
(hepat Genetic Positiv
ocellul disorders e
ar Urobili
disord nogen
er) :
• Bilirubinuria – abnormally increased Norma
amounts of bilirubin in urine. l to ↑
•Tablet test for the detection of ketones in • Hyperbilirubinemia – abnormally Posthe Gallstones Bilirubi Pale,
urine is the Acetest (Siemens Healthcare increased amounts of bilirubin in the blood. patic Tumors n: chal
(obstru (carcinoma) Positiv ky,
Diagnostics Inc.). • Healthy individual – only trace amounts of
ction) Fibrosis e “ac
•Specimen type: urine, serum, plasma, or bilirubin (0.02 mg/dL) are excreted. Urobili holic
whole blood. • One drop of a specimen is • Jaundice – the yellowish pigmentation of nogen ”
placed directly on the tablet, and after the skin, sclera, tissues, and body fluids caused : ↓ to
appropriate timed interval, the tablet color by bilirubin. absent
is compared with a color chart provided. Bilirubin and Urobilinogen Clinical Sig
 •Sensitivity:
• Chemstrip: 0.5 mg/dL conjugated
bilirubin in 90% of urines tested
• Multistix: 0.4 to 0.8 mg/dL conjugated
bilirubin
• vChem: 1.8 mg/dL conjugated bilirubin 1. Urine (10 drops) is added to a special
in 67% of urines tested absorbent pad, and an Ictotest tablet is
placed atop the urine premoistened pad.
Bilirubin Methods • Specificity
2. Two drops of water are added to the
• Physical Examination • False-positive results tablet and allowed to flow onto the pad.
• Foam test – urines are agitated or • Drug-induced color changes,
shaken, the foam that forms has the such as phenazopyridine, indican-indoxyl 3. After 30 seconds the tablet is removed
characteristic yellow color that indicates the sulfate and the absorbent pad is observed for the
presence of bilirubin. • Large quantities of development of any purple or blue
chlorpromazine metabolites coloration, which indicates a positive test.
• Reagent Strip Tests • False-negative results
• Ascorbic acid (>25 mg/dL) Urobilinogen Methods
• High nitrite concentrations
• Normally present in urine in concentrations
• Improper storage or light
of 1 mg/dL or less or 0.5 to 2.5 mg/day.
exposure, which oxidizes or hydrolyzes
• Specimen of choice for quantifying or
bilirubin to nonreactive biliverdin and free
• Principle Azo coupling reaction of monitoring is a 2-hour collection after the
bilirubin
bilirubin with a diazonium salt in an acid midday meal (i.e., 2 PM to 4 PM) “alkaline
medium to form an azo dye. Color changes • Diazo Tablet Test for Bilirubin (Ictotest tide”.
from light tan to beige or light pink are Method) • Urobilinogen is labile in acid urine and
observed. • A tablet test for the detection of bilirubin in easily photo-oxidizes to urobilin.
• Diazonium salt used: urine is the Ictotest method (Siemens • Classic Ehrlich’s Reaction (Historical)
• Chemstrip: 2,6-dichlorobenzene Healthcare Diagnostics Inc.). • Used to qualitatively screen for
diazonium tetrafluoroborate • Concentrations of bilirubin as low as 0.05 urobilinogen.
• Multistix: 2,4-dichloroaniline to 0.1 mg/dL can be detected. • Based on the reaction of urobilinogen
diazonium salt • Greater sensitivity to bilirubin than is seen with p-dimethylamino- benzaldehyde
• vChem: 2,4- dichlorobenzene with reagent strip tests. (Ehrlich’s reagent) in an acid medium to
diazonium tetrafluoroborate produce a characteristic magenta or red
Ictotest method: chromophore.
Results are reported as negative,
small, moderate, or large, or
when the plus system is used, as
negative, 1+, 2+, or 3+.
 • The test is performed by mixing 1 part Drugs Blood* 9 mg/dL Reacts with
Ehrlich’s reagent with 10 parts urine in a tube • Sensitivity: H2O2 on
and observing the mixture for any pink color • Chemstrip: 0.4 mg/dL urobilinogen reagent pad
after a 5-minute incubation. • Multistix: 0.2 mg/dL urobilinogen Bilirubin 25 mg/dL Reacts with
diazonium
• vChem: 1.6 mg/dL urobilinogen in 67%
• Reagent Strip Tests salt on
of urines tested reagent pad
• Principle:
Nitrite 25 mg/dL Reacts with
• Chemstrip and vChem strips: azo • Specificity: The total absence of diazonium
coupling reaction of urobilinogen with a urobilinogen cannot be determined. salt
diazonium salt in an acid medium to form Reactivity increases with temperature, produced by
an azo dye. Color changes from light pink to optimum 22°C to 26°C first reaction
dark pink are observed. • False-positive results Glucose 50 mg/dL Reacts with
• Multistix strips: H2O2
produced by
• Any other Ehrlich’s reactive
first reaction
substance • Ascorbic acid is a strong reducing
• Atypical colors caused by substance because of its ene-diol group.
sulfonamides, p-aminobenzoic acid, p-
• Diazonium salt used: aminosalicylic acid • As a hydrogen donator, ascorbic acid
• Chemstrip: 4- • Substances that induce color readily oxidizes to dehydroascorbic acid, a
methoxybenzene-diazonium-fluoroborate mask results, such as drugs colorless compound.
• vChem: 3,2-dinitro-4-fluoro- (phenazopyridine), beet ingestion
4-diazonium-diphenylamine • Chemstrip and vChem strips: • Reagent strip tests that use hydrogen
tetrafluoroborate • Highly colored substances that peroxide or a diazonium salt are subject to
• Multistix strips: modified Ehrlich’s mask results, such as drugs ascorbic acid interference.
reaction. • False-negative results
• Urobilinogen present reacts • Formalin (>200 mg/dL), a urine
with Ehrlich’s reagent (p- preservative
dimethylaminobenzaldehyde) to form a red • Improper storage, resulting in
compound. Color changes from light oxidation to urobilin
orange-pink to dark pink are observed.
Ascorbic acid Mechanisms of Interference Ascorbic acid Methods
False-Negative or Decreased Reagent Strip • Reagent Strip Test
Results Due to Ascorbic Acid Interference
Ehrlich’s reactive substance:
TEST
Urobilinogen
Porphobilinogen Test Ascorbic Acid Mode of
Indican Affected Concentration Interference • Principle:
5-Hydroxyindoleacetic acid Needed • Ascorbic acid reduces a dye
impregnated in the reagent pad, causing a
color change from blue to orange.
• Dye used: Chem: 2,6-
dichlorophenolindophenol

• Sensitivity:
• vChem: 17 mg/dL in 67% of urines
tested

• Specificity:
• False-positive results
• Free sulfhydryl drugs (e.g., MESNA,
captopril, N-acetylcysteine)
RENAL DISEASES Glomerulonephritis

INTRODUCTION Result from immunologic disorders

• Immune complexes formed from

• Worldwide, chronic kidney disease has
immunologic reactions and increased serum
become a public health problem with
immunoglobulins-IgA deposited in the
increasing prevalence and high cost for
glomerular membrane
treatment and prevention.
• Complements neutrophils,
• It is projected that by 2030 there will be
lymphocytes, monocytes and cytokines are
about 2 million patients needing dialysis and
attracted to the area producing changes
transplantation. ACUTE POSTSTREPTOCOCCAL
and damage to the membranes.
• kidneys perform vital function in the GLOMERULONEPHRITIS
• Cellular infiltration or proliferation resulting
homeostatic processes of the human body
in thickening of the glomerular basement • Acute glomerulonephritis (AGN)
including:
membrane: and complement-mediated • A disease marked by a sudden onset of
• filtration of blood,
damage to the capillaries and basement symptoms consistent with damage to the
• maintenance of acid base,
membrane glomerular membrane
• water and electrolyte balance,
NONimmunologic causes: exposure to • Fever, EDEMA (most noticeably around
• regulation of hormone level’s and
chemicals and toxins that affect the tubules, the eyes) fatigue, hypertension, oliguria
• removal of waste products and
disruption of the electrical membrane (<400ml of urine/day), and hematuria
unwanted substance from plasma
changes as occurs in NEPHROTIC • Found in children and young adults
• Various hormones that have metabolic
SYNDROME syndrome following respiratory infections caused by
and physiological functions
• deposition of amyloid material from certain strains of group A streptococcus that
• erythropoietin
systemic disorders involving chronic contains M protein in the cell wall
• prostaglandins
inflammation acute-phase reactants • Forms an immune complexes with
• Several diseases:
• basement membrane thickening corresponding circulating antibodies and
• acute nephrotic syndrome
associated with diabetic nephelopathy are deposited on the glomerular membrane
• acute glomerulonephritis
• Refers to sterile, inflammatory process that inflammation
• can singly or in combination
effects the glomerulus and is associated • Success in management of
precipitate renal diseases
with the finding of blood, protein and cast in hypertension and electrolyte imbalance
urine and clearing on immune complexes in the
• Rapidly progressive glomerular nephritis blood will leave no permanent kidney
to chronic glomerulonephritis to the damage
nephrotic syndrome and eventually renal • Urinalysis: marked hematuria, proteinuria
failure and oliguria; accompanied by RBC cast,
dysmorphic RBC, hyaline and granular casts
and WBCs

GLOMERULAR DISORDERS • Blood Chemistry: BUN is elevated

•Immuno-serology: Positive for anti-group A Glomerular Disorders
streptococcal enzyme test
Goodpasture Syndrome Wegener Granulomatosis

 Morphologic changes in the  A granuloma-producing information

glomerulus resembling rapidly of the small blood vessels primarily of
progressive glomerulonephritis the kidney and respiratory system
 Cytotoxic autoantibody can appear  Antineutrophilic cytoplasmic
against glomerular and alveolar antibody (ANCA) in the serum
basement membranes after viral  Pulmonary symptoms – renal
respiratory infections involvement → hematuria,
 Attachment of this autoantibody to proteinuria, RBC casts and elevated
the basement membrane followed serum creatinine and BUN
Rapidly Progressive (Cresentic)  Testing: incubate serum with either
by complement activation,
Glomerulonephritis produces capillary destruction ethanol or formalin fixed with
→antiglomerular basement neutrophils under indirect
 More serious form of acute immunofixation – neutrophils are
membrane antibody in patient’s
glomerular disease- poorer fixed in ethanol the antibodies will
serum
prognosis, often terminating to renal form perinuclear pattern called p-
 Initial complaints: hemoptysis and
failure ANCA and when fixed with formalin
dyspnea followed by hematuria
 Immune systemic disorder → the pattern is granular throughout
 Urinalysis results include proteinuria,
Systemic Lupus Erythematosus (SLE) the cytoplasm called c-ANCA
hematuria and presence of RBC
o Damage by monophages to
casts
the capillary walls releases
 Progress to chronic
cells and plasma into
glomerulonephritis and end stage
Bowman’s space forming
renal failure is common
cresentric formations
containing macrophages,
fibroblasts and polymerized
fibrin causing permanent
damage to capillary tufts
o Initial laboratory findings:
similar to AGN but as it
progresses will show markedly
elevated protein and low
glomerular filtration rates
Glomerulonephritis
o May demonstrate fibrin
degradation products,
Henoch-Schonlein Purpura
cryoglobulins and deposition
of lgA immune complexes in  Primarily in children after upper
the glomerulus respiratory infections
  Initial symptoms include symptoms 2 different alteration: cellularity of
appearance of raised, red patches glomerulus and peripheral capillaries
on skin
 Respiratory and gastrointestinal Glomerulonephritis  Type 1- increased cellularity in the
symptoms – blood found in sputum subendothelial cells of the
and stools Membranous Glomerulonephritis mesangium (interstitial area of the
 Renal involvement is the most serious Bowman’s capsule)
complication that may range from Pronounces thickening of the glomerular o Progress to nephrotic
mild to heavy proteinuria and basement membrane resulting from the syndrome
hematuria with RBC casts deposition of lgG immune complexes  Type 2- extremely dense deposits in
 Complete recovery on 50% of cases the glomerular basement
Associated in SLE, Sjogren syndrome, membrane
secondary syphilis hepatitis B, gold and o Experience symptoms of
mercury treatments and malignancy chronic glomerulonephritis
 Mostly affected are children and has
Laboratory findings: microscopic hematuria poor prognosis
and elevated urine protein excretion  Laboratory findings: may vary but
with consistent hematuria,
proteinuria with decreased serum
complement levels
o Appears to be associated
with autoimmune disorders,
Henoch-Schonlein Purpura infections and malignancies
Pathogenesis • IgA-mediated
leukocytoclastic vasculitis
Clinical • Palpable purpura
manifestations • Arthritis/arthralgia
• Abdominal pain,
intussusceptions
• Renal disease similar to
IgA nephropathy
Laboratory • Normal platelet count
findings & coagulation studies
• Normal to ↑ creatinine
• Hematuria +/_ RBC
casts +/- proteinuria
Treatment • Supportive (hydration &
NSAIDs) for most patients
• Hospitalization &7 Membranoproliferative Glomerulonephritis
systemic glucocorticoids Chronic Glomerulonephritis (CG)
(MPGN)
in patients with severe
  Gradual worsening symptoms  Progression to chronic proteins by RTE), renal tubular
including fatigue, anemia, glomerulonephritis to end stage epithelial (RTE) cells; epithelial, fatty
hypertension, edema and oliguria renal disease develops gradually and waxy casts plus microscopic
 Laboratory findings: hematuria, that may take 20 long years of hematuria
proteinuria, glucosuria- of tubular asymptomatic cases
dysfunction; presence of many
variety casts (broad)
 Decreased GFR and increased in
BUN and serum creatinine levels and
electrolyte imbalance

Diagnostics of CG

1. Blood test: Hgb, RBC, WBC,

Platelets, ESR before and after
kidney biopsy
2. Chemstrips: hematuria,
proteinuria and leukocyturia
3. Protein/ creatinine ratio
4. Creatinine, BUN levels Nephrotic Syndrome
5. Clotting time
6. Allocated daily liquid intake and  Marked by massive proteinuria (>3.5
weight g/day)
7. Concentration of cyclosporine, o Low serum albumin- causing
tacrolimus in serum low oncotic pressure in the
capillaries; increases fluid loss
into interstitial spaces
accompanied by retention
Immunoglobulin A (IgA) Nephropathy Minimal Change Disease
of sodium = EDEMA
o Low immunoglobulin and
 Aka Berger disease  Aka lipid nephrosis- produces little
coagulation factors risk of
 IgA deposition on the glomerular change in the glomerulus except
infection and coagulation
membrane – most common cause some damage to podocytes and
disorders
of glomerulonephritis shield negativity increased
 High levels of lipids
 Findings: patient IgA serum level is protein filtration
 Pronounced edema
increased that result in mucosal  Associated with HLA-B12
 Acute onset: circulatory disruption
infection  Idiopathic- may be due to allergic
producing systemic shock that
 Frequently seen in children and reactions and recent immunization
decrease the pressure and flow of
young adults  Responds well to corticosteroids and
blood to the kidneys
 Others: macroscopic hematuria is prognosis is generally good
 Microscopy: oval flat bodies (due to
observed following or strenuous
absorption of lipid-containing Focal Segmental Glomerulosclerosis (FSGS)
exercise
  Only affects certain numbers and  To some extent filtration of large o Transport of filtered
areas of glomeruli amounts of hemoglobin and substances across tubular
 Immune deposits primarily IgM and myoglobin is also nephrotoxic membranes,
C3 o Disruption of cellular energy
Pathophysiology
 Seen also in association with abuse needed for transport
of heroin and analgesic o Or changes in the tubular
membrane permeability
Tubular Disorders

 Tubular function is disrupted as a

result of actual damage to tubules
and those in which a metabolic or
hereditary disorder affects the
intricate functions of the tubules

Acute Tubular Necrosis (ATN)

Associated with cystinosis and Hartnup
 Decreased blood flow that causes a
disease
lack of oxygen (ISCHEMIA) or
presence of toxic substances in the Hereditary and Metabolic Tubular Disorders
Alport Syndrome
urinary filtration damage to the
RTE cells  Disorders affecting tubular function
 Inherited disorder of collagen
may be caused by systemic
production affecting glomerular
Causes: conditions that affect or override the
basement membrane
tubular reabsorptive maximum for
 Shock (cardiac failures, sepsis  Sex-linked or autosomal genetic
particular substances normally
involving toxigenic bacteria, disorder- male inheriting the
reabsorbed by the tubules
anaphylaxis, massive hemorrhage autosomal gene
 Or by failure to inherit a gene or
and contact with high-voltage  Curing respiratory infection, males
genes required for tubular
electricity), younger than 6 years may exhibit
reabsorption
 Trauma (crushing injuries), macroscopic hematuria and
 Surgical procedures Fanconi Syndrome continue to exhibit microscopic
 Exposure to nephrotoxic agents- hematuria
o Aminoglycoside antibiotics,  Generalized failure of tubular  May develop hearing and visual
o Fungal agents amphotericin reabsorption in the proximal abnormalities
B, convoluted tubule  Glomerular basement membrane
o Cyclosporine  Substances noticeably affected are has a lamellated appearance with
o Radiographic dye, glucose, amino acids, phosphorus, areas of thinning
o Organic solvents (ethylene sodium, potassium, bicarbonate and o No antibodies present
glycol, heavy metals water
o Toxic mushrooms  The tubular dysfunction include:
  Currently most common end-stage  Polycystic kidney disease and
renal disease sickle cell anemia
 Glomerular membrane damage is
Renal Glycosuria
not only due to glomerular
thickening but also increased
 Affects only the reabsorption of
proliferation of mesangial cells and
glucose
increased deposition of cellular and
 Inherited as an autosomal recessive
noncellular material within the
trait
Prognosis ranges mild to persistent glomerular matrix → accumulation
hematuria and renal insufficiency in later life of solid substances around the Characteristics:
to the nephrotic syndrome and end-stage capillary tufts
renal disease o Poorly controlled glucose  Decreased in the number
level glucose transporters
o The vascular structure of the  Affinity of the transporter of
glomerulus develops to glucose is decrease
sclerosis o Urine glucose is increased
 Microalbumin is a biomarker of onset while normal blood
of DN glucose concentrations
Uromodulin-Associated Kidney Disease  Modification of diet and control BP
Interstitial Disorders
can decrease progression of renal
 Is a glycoprotein and is the only disease  Closely related to tubules → referred
protein produced by the kidney – to as tubulointerstitial disease
Nephrogenic Diabetes Insipidus (DI)
proximal and distal convoluted  Most cases involve infections and
tubules → primary protein found in  Disruption of Antidiuretic hormone inflammatory conditions
normal urine (ADH) produced  Urinary tract infection (UTI)
 It is primarily an inherited disorder  Excessive amount of urine are  Involving the lower urinary tract
caused by an autosomal mutation in excreted (urethra and bladder)
the gene that produces the o Inability of the renal tubule to  Involving the upper urinary tract
uromodulin – replaced by the respond to ADH → (renal pelvis, tubules and interstitium
abnormal form → damage the nephrogenic DI  Mostly observed in infection of the
tubular cells o Failure of the hypothalamus bladder (cystitis) –more serious upper
o Renal monitoring and to produce ADH → UTI left untreated
eventually renal nephrogenic DI  Affects more women and children –
transplantation s/s urinary frequency and burning
 Mutation of cause an increase in Characteristics:  Microscopy: presence of numerous
serum uric acid (gout) in teenagers WBCs and bacteria (fresh sample);
before detectable renal disease  Sex-linked recessive gene
accompanied by mild proteinuria
 Or acquired medications
and hematuria and increased in pH
Diabetic Nephropathy including lithium and
amphotericin B Acute Pyelonephritis
  Infection of the upper urinary tract o Pencilin, methicillin, o Burns
(tubules and interstitium) ampicillin, cephalosporins, o Surgery
 General term: pyelonephritis- acute sulfonamides, NSAID’s o Septicemia
or chronic thiazide diuretics  Renal
 Acute- ascending movement of  use of steroids and supportive renal o Acute glomerulonephritis
bacteria from lower UTI into the renal dialysis to lessen inflammation and o Acute tubular necrosis
tubules and instertitium which get rid metabolites o Acute pyelonephritis
interfere with the downward flow of o Acute intersitial nephritis
urine (ureters to bladder) or Renal Failure  Postrenal
incomplete emptying of the bladder o Renal calculi
 Exist in both acute and chronic forms
 s/s: urinary frequency, burning o Tumors
 Progression to end-stage renal
sensation on urination and lower
disease is characterized by: Renal Lithiasis
back pain
o Marked decrease in GFR (<25
 Obstructions- renal calculi,
mL/min);  Renal calculi (kidney stone) – in the
pregnancy and reflux of urine
o Steady increased in serum calyces, pelvis of the kidney ureters
(bladder back to ureters)
BUN and creatinine values and bladder
vesicoureteral reflux
(azotemia);  Lithotripsy – procedure using high-
Chronic Pyelonephritis o Electrolyte imbalance; energy shock waves to break stone
o Lack of renal concentrating located in the upper urinary tract
 Serious and may cause permanent ability → isosthenuric urine; into pieces
damage to renal tubules – o Proteinuria,  Pathogenesis similar to urinary
progression to chronic renal failure o Renal glucosuria and crystal formation: pH, chemical
 Congenital urinary structural defects o Abundance in granular, concentration and urinary stasis
producing reflux nephropathy are waxy and broad cast
most common Substances that cause kidney stone:

Acute Interstitial Nephritis (AIN)  75% of kidney stone are of calcium

oxalate (metabolism or diet) or
 Marked by inflammation of the renal calcium phosphate origin
intersitium followed by inflammation o Magnesium ammonium
of the renal tubules phosphate (struvite) found in
 Initial s/s: fever and skin rashes infection of urea splitting
 Acute renal failure (ARF)- sudden loss
 Rapid onset s/s: oliguria edema, bacteria (7.0 pH)
of renal function and frequently
decreased renal concentrating o Uric acid (high purine in diet)
reversible
ability and GFR – acidic pH and
 Primarily associated with allergic Causes: o Cystine ( hereditary disorders
reaction to medications that binds to in metabolism)
the interstitial protein  Prerenal
 s/s develop after 2 weeks o Decreased blood pressure/
administration cardiac output
o Hemorrhage
 OVERFLOW METABOLIC RENAL
INHERITED
Phenylketonuria Infinite Hartnu
tyrisinemia p
diseas
e
Tyrosinemia Melanuria Cystin
uria
Alkaptonuria Indicanuria
Maple syrup urine 5-
disease hydroxyindole
acetic acid
Organic Porphyria
acidemias
Cystinosis
Porphyria
Metabolic Disorders Mucopolysaccha
ridoses TYROSINEMIA
Disruption of enzyme function can be Galactosemia
Lesch-Nyhan  Defective Process: Amino Acid
caused by failure to inherit the gene to Transport- Excretion of Tyrosine And
disease
produce a particular enzyme, this is referred Trytophan Catabolites In Urine
to inborn error of metabolism (IEM).  Defective Enzyme:
 Tyrosine Aminotransferase (Ii);
Abnormal metabolic constituents or  4-hydroxy-phenylpyruvate Oxidase
conditions detected in the routine urinalysis Phenylalanine and Tyrosine Metabolic (Iii);
Pathways  Fumarylacetoacetate Hydrolase (I)
Color Odor Crystal
Homogentistic Phenylketonuria Cysteine ALKAPTONURIA
acid
Melanin Maple syrup Leucine  DEFECTIVE PROCESS: TYROSINE
urine disease DEGRADATION
Indican Isovaleric Tyrosine  DEFECTIVE ENZYME:
academia HOMOGENTISATE-1, 2-DIOXYGENASE
Porphyrin Cystinuria Lesch-  Original “inborn error of metabolism
Cystinosis Nyhan  Darkening of urine at long standing
Homocystinuria disease
 OCHRONOSIS – generalized
pigmentation of tissues and arthritis
Major disorders of protein and carbohydrate like degeneration
metabolism associated with abnormal
urinary constituents, classified by functional PHENYLKETONURIA
defect
  DEFECTIVE PROCESS: CONVERSION 450nm; excitation  Decrease in activity of ASA
OF PHENYLALANINE TO TYROSINE wavelength at 360nm synthetase →Citrullinemia
 DEFECTIVE ENZYME: PHENYLALANINE  Confirmed diagnosis is based on • Citruline is elevated in MS/MS
HYDROXYLASE (Phenylalanine-4- findings increased plasma and • Argininosuccinic aciduria –
mono-oxygenase) urinary levels of the three branched- milder elevations
 An autosomal recessive trait – urine chain amino acids and their • Citrullinemia – dramatic
has a musty odor ketoacids with LEUCINE (highest: elevations
 Compounds seen in both urine and above 4mg/dL) • Orthinine and arginine are seen
blood:  Prenatal diagnosis: measuring increased I older infants
 Phenypyruvic acid – primary decarboxylase enzyme
ISOVALERIC ACIDEMIA
catabolite concentration in cells cultured from
 Phenyllactic acid –product of amniotic fluid
 DEFECTIVE PROCESS: DEGRATIVE
deaminzation
PATHWAY OF LEUCINE
 Phenylacetylglutamine – produced
CYSTINURIA  DEFICIENCY ENZYME : ISOVAREYL-
from oxidation of phenylpyruvic acid
CoA DEHYDROGENASE
MAPLE SYRUP URINE DISEASE  DEFECTIVE PROCESS: AMINO ACID  “Sweaty feet” odor urine
TRANSPORT SYSTEM RATHER THAN • Elevtion of glycine conjugate:
 Defective process: Metabolism of METABOLIC ENZYME DEFICIENCY isovaleric acid and isovarylglycine
three essential branched-  20-30 fold increase in the urinary
LESCH-NYHAN DISEASE
chainamino acids (Leucine, excretion of cystine as a result of
Isoleucine and Valine) genetic defect in the renal
 Purine disorders
 Defective enzymes: Branched chain resorptive mechanism
 A genenetic associated with 3 major
alpha-Keto Acid Decarboxylase  Other amino aids ecreted: ornithine,
clinical elements: overproduction of
Complex lysine and ariginine
uric acid, neurologic disability, and
 Burnt sugar odor of urine, breath and • Cystine being relatively insoluble
behavioral problems
skin insoluble and once accumulated will
• rare inherited disorder (inherited as
 Screening: Modified Guthrie test – tend to precipitate in the kidney
an x-linked recessive disorder)
metabolic inhibitor of B. subtilis which tubules forming calculi
caused by mutations in the gene
is 4-azaleucine is impregnated in the  Remedy: high fluid intake and
encoding hypoxanthine-guanine
medium alkaliizing the urine: penicillin
phosphoribosyltransferase (HPRT)
o POS (+) for MSUD = bacterial  Diagnosis: cyanide-nitroprusside
 Overproduction of uric acid is
growth (red-purple color)
associated with hyperuricemia
 Microfluorometric assay using
 If left untreated, it can produce
leucine dehydrogenase
nephrolithiasis with renal failure,
o Filter paper specimen is
ARGININOSUCCINIC ACIDURIA AND gouty arthritis, and solid
treated with methanol and
CITRULLINEMIA subcuntaneous deposits known as
acetone to denature
tophi
hemoglobin  DEFECT PROCESS: UREA CYCLE  Neurologic disability is dominated by
o The NADH fluorescence  DEFECTIVE ENZYME: dystonia but may include
produced is measured at ARGINOSUCCINIC ACID LYASE
 choreoathetosis, ballismus, spaticity, enzymes of the heme  Both have: skeletal structure is
or hyperreflexia biosynthetic pathway or from abnormal and severe mental
increased activity of the retardation; without treatment both
PORPHYRIN DISORDERS
erythroid form of the first enzyme are fatal during children.
in the pathway, Aminolevulinic  Sanfilippo syndrome (abnormality is
acid (ALA) synthase-2 (ALAS 2) only mental therapy are the most
 Acute porphyrias – manifest promising treatment for these
attacks of abdominal, mental, disorders
and neurologic symptoms  Screening test: acid-albumin and
• They are typically triggered by cetytrimethylammonium bromide
drugs, gormonal activity in young (CTAB) turbidity tests and the
women, and other exogenous metachromatic staining spot test →
factors POSITIVE white tubidity forms when
 Cutaneous porphyias – tend to mucopolysaccharides are found in
cause continuous or intermittent the urine sample; graded 0-4 after:
symptoms involving cutaneous • 30 minutes with acid-albumin
photosensitivity • 5minutes with CTAB
 Urine discoloration ( red or
CARBOHYDRATES DISORDERS
reddish brown) may occur in the
symptomatic phase of all
 Galactosuria – dificiency in any 3
porphyrias excepterythropoietic
enzymes:
 Porphyrias are rare disorders in protophyria (EPP) and ALAD-
• Galactose-1-phosphate uridyl
which hemoglobin is abnormally deficiency porphyria
transferase (GALT) – fatal
metabolized due to genetic or • Red or port wine color
symptoms in infants
acquired deficiencies of  Discoloration results from
• Galactokinase- deficiency will
enzymes of the heme oxidation of the porphyrinogens,
cause cataract in adults
biosynthetic pathway the porphyrin precursor
• UDP-galactose 4-epimerase –
• these deficiencies allow heme porphobilinogen (PBG), or both
mild symptoms
precursors to accumulate • Sometimes color develops after
 Lactosuria – seen during
causing toxicity the urine has stood in air or light
pregnancy and lactation
 Porphyrias are define by the for minutes to hours, allowing
 Fructosuria – parenteral feeding
specific enzyme deficiency time for non-enzymatic
and pentosuria with ingestion of
• Two major clinical oxidation.
large amounts of fruits.
manifestation occur:
MUCOPOLYSACCHARIDE DISORDERS
• neurovisceral abnormalities
(the acute porphyrias)  Hurler syndrome
• cutaneous photosensitivity CEREBROSPINAL SEMINAL, SYNOVIAL AND
(mucopolysaccharides accumulate
(the cutaneous porphyrias) SEROUS FLUIDS
in the cornea of the eye) and Hunter
 Porphyrias reslts from a syndrome (inherited as sex-linked CSF COMPOSITION
deficiency of any of the last 7 recessive and rarely seen in females • CSF is the third major fluid of the body
 • Adults volume 90-150 ml • Evaluate for intracranial hemorrhage
• Neonatal volume 10-60 ml • Diagnose malignancies, leukemia
• Investigate central venous system
CSF PRODUCTION disorder0
• Produced at the Choroid plexus of the 4
ventricles by modified Ependymal cells SPECIMEN COLLECTION AND HANDLING
• At rate of 20 ml/hr (adults)
• 150 ml/day is produced
• CSF flows through the Subarachnoid • Normal – Crystal clear, colorless
space • Descriptive Terms – hazy, cloudy, turbid,
• Where a volume of 90 – 150 ml is milky, bloody, xanthrochromic
maintained (adults) • Often are quantitated – slight, moderate,
• Reabsorbed at the Arachnoid  Routinely collected via lumbar marked, or grossly.
villus/granulation puncture between 3rd and 4th, or 4th • Unclear specimens may contain
• That is eventually reabsorbed into the and 5th lumbar verterbrae under increased lipids, proteins, cells or bacteria.
blood sterile conditions Use precautions.
 Intracranial pressure measurement • Clots indicate traumatic tap
BLOOD BRAIN BARRIER taken before fluid is withdrawn. • Milky – increased lipids
• Occurs due to tight fitting endothelial cells
• Oily – contaminated with x-ray media
that prevent filtration of larger molecules.
• Controls/restricts/filters blood components
• Restricts entry of large molecules, cells,
etc.
• Therefore CSF composition is unlike blood’s
CSF is not an ultrafiltrate

CSF FUNCTIONS  Tube 1 – chemestries and serology

• Supplies nutrients to nervous tissues  Tube 2 - microbiology cultures
• Removes metabolic wastes  Tube 3 – hematology
• Protects/cushions against trauma  Testing considered STAT
 Specimen potentially infectious
FOUR MAJOR CATEGORIES OF DISEASE  If immediate processing is not • Xanthrochromic – Yellowing discoloration
• Meningeal infections possible of supernatant (may be pinkish, or orange).
• Subarachnoid hemorrhage • tube 1 ( chem-sero) frozen • Most commonly due to presence of
• CNS malignancy • Tube 2 ( micro) room temperature ‘old’ blood.
• Demyelinating disease • Tube 3 ( hema ) refrigerated • Other causes include increased
CSF APPEARANCE bilirubin, carotene, proteins, and melanoma
INDICATIONS FOR ANALYSIS
• To confirm diagnosis of meningitis
 • Cerebral hemorrhage
• Even distribution of blood in the
numbered tubes
• Clot formation possible
• Xanthrochromatic supernatant
• RBCs must have been in CSF @ 2+hours
• D- dimer, fibrin degradation product
from hemorrhage site
• Clots - indicates increased fibrinogen and • Microscopic presence of
usually due to traumatic tap, but may erythrophages, or siderophges, Hemosiderin
indicate damage to blood-brain barrier. granules
(See below)
CSF PROCEDURES  Formula for calculations- results in #
• Pellicle formation in refrigerated specimen
cells/µL
associated with tubercular meningitis.
o Count and record cells from
• Pellicle formation – picture at right
both sides of the chamber.
(pellicle in Left tube, right is normal)
o Average the two sides
• Milky – increased lipids
o Multiply by dilution factor (if
• Oily – contaminated with x-ray media
no dilution is made. This
TRAUMATIC COLLECTION VS CEREBRAL number is 1)
 All specimens chould be examined
HEMORRHAGE o Divide by number of squares
microscopically- hematology
counted X volume of each
 Stat priority, RBC lyse in 1 hour, WBC
square
in 2 hrs. Refrigerate if not able to
 Large square, such as
process immediately.
#1-9 below have
 Electronics counters generally
volume of 0.1
unsuable. Manula count
 Small squares- in
 No dilution usually required (use
center #5 have
saline needed)
volume of 0.004
 Standard Neubauer hemacytometer
counting chamber EXPECTED RESULTS

NEUBAUER HEACYTOMETER / COUNTING  Normally 0 RBCs/uL regardless of age

CHAMBER  WBCs
o Adult- up to 5 mononuclear
WBCs/ uL
o Newborn- up to 30
mononuclear WBCs/ uL
 o Children (1-4)- up to 20
mononuclear / uL
o Children (5+) – up to 10 CSF SLIDE DIFFERENTIAL
mononuclear / uL
o Increased numbers =
Pleocytosis
 Normal differential values
o Aduts: 70% lymps, 30%
WBC COUNT monos.
o Children / newborns:
 3% acetic acid can be used to lyse monocyte
RBC  Types of cells
 Methylene blue staining will improve o Neutrophils – occasionally
visibility  Wright stained smear of (with normal count)
concentrated sediment. o Macrophages – increase
Correction of WBC count from traumatic
 Cytocentrifuge- places cells on filter/ following CVA
tap contamination.
membrane. Increases number of o Ependymal cells, and normal
 Uses ratio of WBCs to RBCs in blood cells to evaluate, however, risk of cell lining cells can also be seen.
and compares it to same ratio distortion from the centrifugation
(WBC/RBC) in CSF process.
 If patient’s peripheral cell counts are o Use of albumin reduces cell
normal, can subtract 1 WBC for distortion
reach 700 RBCs counted in CSF.  Count and differentiate 100
 Great chance for considerable error nucleated cells.
makes this of little value.  Any cell found in peripheral blood
may be seen in CSF, other nucleated  Entire smear should be evaluated for
QUALITY CONTROL cells and malignant cells can also be o Abnormal cells
found. o Inclusions within cells
 CSF controls o Clusters
 Entire smear should be evaluated for
 Check techniques o Presence of intracellular
o Abnormal cells, inclusions
 Check of reagents organisms
within cells, Clusters,
 Check of centrifuges
Presence of intracellular
CSF CELLULAR ELEMENTS
Decontaminate all counting chambers organisms
in bleach water for @15 minutes. Rinse in
water and cleaned again with alcohol.
 Lymphocytes and monocytes /
macrophages  Eosinophils  Suspicious / unclassified or malignant
o often associated with cells are reported as “other” or
parasitic/ fungal infections, “unclassified” AND are sent to
allergic reactions including pathology. (as seen below)
reation to shunts and other  Cytology- send unstained slide to
foreign objects. cytology / pathology
 1986 CAP CM10 CSF- blasts
(appearance similar to peripheral
blood, always consult with
L-lymphocytes and macrophages hematology specialist / pathologist)
(see below right)

CELLULAR INCLUSIONS
 Ependymal cells
o Normal cell, unique to CSF  Erythrophage
o Line the ventricles, produce  Siderophage
CSF fluid  Hematoidin crystals (see below)
o Large cell with distinct round
/ oval nucleus, sometimes
found in sheets
Mono / macro, segmenters and
CSF CELLULAR ELEMENTS
lymphocytes

CSF CELLULAR ELEMENTS

1991 CAP 30 CSF hematoidin crystal /

bilirubin crystal
  Decreased levels not significant disease and
 Increased levels effectiveness of
o Damaged B-B (as in treatment
meningitis or hemorrhage) o IgG levels (both serum and
o Production of CSF)
immunoglobulins within CNS  IgG index= CSF IgG
(MS) mg/dL)/ serum IgG
 Degeneration of neural tissue (g/dL)
 Dye-binding methods – preferred CSF
1991 CAP 30 CSF hematoidin crystal / o Alkaline biuret albumin (mg/dL) /
bilirubin crystal o Coomassie brilliant blue- a serum albumin (g/dL)
blue color produced is o Albumin (both serum and
proportional to the amount CSF)
of protein present (Beers o IgG synthesis rate
Law)
CEREBROSPINAL FLUID (CSF)- GLUCOSE
CEREBROSPINAL FLUID – MS PANEL
 Selective transported across blood-
brain barrier
ASCP 6 macrophage, lymphocyte,  Normal values: 60-70% of blood
siderophage glucose
 STAT procedure, gylcosis reduces
CHEMISTRY level quickly
 Procedure performed as for blood
 Blood- brain barrier causes selective
specimen
filtration
 Decreased levels seen in bacterial &
 Abnormal values  Multiple Sclerosis fungal meningitis
o From altered permeability o Diagnosis is difficult- no one o Hypoglycemia
o Increased production specific test o Brain tumors
o Increased metabolism o CSF Protein electrophoresis o Leukemias
 Looking for o Damage to CNS
CEREBROSPINAL FLUID (CSF) – PROTEIN
oligoclonal bands
 Normal 15-45 mg/dL o Myelin Basic Protein OTHER TESTS
o Albumin fraction. If lgG- from  Abnormal protein that
 CSF Lactate
damaged B-B, or CNS indicates
o Normal values= 11-22 mg/dL
produced? Can demyelination of
o Increase as result of hypoxia
electrophoresis to evaluate neuron axons
 Bacterial meningitis.
oligoclonal / malignant  Measurement used to
Head injury
bands. monitor course of
  CSF Glutamine + gram Pellicle + india ink  Neisseria meningitidis
o Normal 8-18 mg/dL stains formati with  Streptococcus
o Increased levels associated on Cryptococc pneumoniae
with increases in ammonia us o Staphylococcus auteus (it a
neoformans
(toxin) shunt is present)
+bacteri +immunolog
 CSF Enzymes al ical test for o Immunocompromised
o Lactate dehydrogenase (LDH antigen C.  Cryptococcus
or LD) tests neoformans neoformans
 5 isoenzyme types;  Candida albicans,
LD1& LD2 are in brain coccidioides or any
tissue CEREBROSPINAL FLUID (CSF) - opportunistic
o Creatine kinase (CPK or CK) MICROBIOLOGY organism
 Isoenzyme CK#/ CK-
 Gram stain- extremely important for INDIA-INK / NIGROSIN PREPARATION
BB from brain tissue
early diagnosis of bacterial
 Following cardiac
meningitis
arrest, patients with
o Even when well performed,
CSF levels <17 mg/dL
10% false negatives occur
have favorable
o Use of cytospin to
outcome.
concentrate specimen  Negative stain to view the
DIFFERENTIAL DIAGNOSIS OF MENINGITIS BY increases sensitivity encapsulated Crytococcus
LABORATORY RESULTS  Cultures- Aerobic & Anaerobic. neoformans (often AIDs/
Culture blood at same time immunocompromised complication)
Bacterial Viral Tubercu Fungal  Instead of stain, can also use dark
lar field microscopy for same effect.
Increase Increas Increas Increased
 These direct procedures have @ 25-
d WBC ed ed WBC WBC count
50% sensitivity
count WBC count
count o Prefer latex agglutination
Neutrop Lymphs Lymps & Lymps & tests, better results
hils Monos Monos  Organisms
Marked Mod. ↑ Mod- Mod- SEROLOGY
o Newborns
↑ protein protein Marked Marked 1
1 protein  [Link] & groups B Strep  VDRL (Veneral Disease Research
protein o Children Laboratory)
Marked ↔ ↓ Normal to ↓  Streptococcus o For detection of neurosyphilis
↓ normal glucose glucose pneumoniae o On CSF test low sensitivity, but
glucose glucose  Hemophilus influenza great specificity
Lactate Lactate Lactate Lactate > 25  Neisseria meningitidis o FTA-Abs also used on CSF,
> 35 normal > 25 mg/dL
o Adults more sensitive, but must
mg/dL mg/dL
 prevent blood Composition of Semen:
contamination.
 Spermatozoa 5%
SEMEN ANALYSIS  Seminal Fluid 60-70%
 Prostate Fluid 20-30%
SEMEN  Bulbourethral Glands 5%  A sample may also be collected
during intercourse in a special type
SEMEN ANALYSIS of condom known as a collection
condom
 A semen analysis evaluates certain  Collection condoms are made from
characteristics of a male’s semen silicone or polyurethane, as latex is
and the sperm contained in the somewhat harmful to sperm.
semen.  Many men prefer collection
condoms to masturbation, and some
WHY IS IT DONE?
religions prohibit masturbation
A semen analysis is done to determine entirely.
Semen Production: whether:
COITUS INTERRUPTUS
 Seminiferous tubues:  A man has a reproductive problem
o spermatogenesis  With this technique, the man
that is causing infertility.
 Epididymis: removes his penis from his partner
 A vasectomy has been successful.
o Sperm Maturation near the end of intercourse and
 The reversal of a vasectomy has
 Ductus Deferens: ejaculate into a cup.
been successful.
o Propel sperm to ejaculatory  Withdrawal or pull-out method
ducts COLLECTION METHODS
PERCUTANEOUS EPIDIDYMAL SPERM
 Seminal Vesicles:
 Masturbation ASPIRATION (PESA)
o Provide nutrients for sperm
 Collection Condom
and fluid  If a blockage in the vas deferens is
 Coitus Interruptus
 Prostate Gland: suspected to impede fertility, semen
 PESA
o Provide enzymes and can be taken directly from the
 TESE
proteins for coagulation and epididymis.
liquefaction MASTURBATION
 Bulbourethral Glands:
o Add alkaline mucus to  The most common way to collect a
neutralize prostatic acid and semen sample is through
vaginal acidity masturbation, directing the sample
into a clean cup.
SEMEN Percutaneous epididymal sperm aspiration
COLLECTION CONDOMS (PESA) is an efficient and simple method of
retrieving sperm. Additionally, sperm  Normal
collected through PESA can be o Gray-white color, translucent,
cryopreserved and stored for potential characteristic musty odor
future use.  Abnormal
o Increased white turbidity
TESTICULAR EPIDIDYMAL SPERM EXTRACTION indicates the presence of
(TESE) WBCs
o Red coloration of semen is
associated with the presence
of RBCs
o Yellow coloration; urine
contamination, prolonged
 Sperm Concentration
abstinence & medication
o Uses Neubauer chamber (5
 The process of removing a small
SPERM VOLUME WBC squares)
portion of tissue from the testicle
o Uses 1:20 dilution
under local anesthesia and
 Normal o Sodium bicarbonate and
extracting the few viable sperm cells
o 2-6 milliliters (mL) per formalin are the traditional
present in that tissue
ejaculation diluting fluids
 The testicular sperm extraction
 Abnormal o Good results can also be
process is recommended to men
o An abnormally low or high achieved using saline and
who are unable to produce sperm
semen volume is present, distilled water
by ejaculation as a result of primary
which may sometimes cause o Sperm counted 1,000,000=
testicular failure, congenital absence
fertility problems. sperm/mL
of the vas deferens or non-
 Sperm Count
reconstructed vasectom LIQUEFACTION TIME
o Sperm concentration volume
PARAMETERS IN SEMEN ANALYSIS  Normal of specimen= sperm/
o 30-60 minutes after collection ejaculate
 Appearance
 Abnormal
 Volume
o An abnormally long
 Viscosity
liquefaction time is present,
 pH
which may indicate an
 Sperm Concentration
infection.
 Sperm Count
 Sperm Motility SPERM COUNT/ CONCENTRATION
 Sperm Morphology
 Liquefaction Time
SPERM COUNT
APPEARANCE
 HYDROGEN ION CONCENTRATION (pH) o More than 30% of the sperm
have normal shape and
 Normal structure.
o 7.2-8.0 (alkaline)  Abnormal
 Abnormal o Sperm can be abnormal in
o Increased pH indicates several ways.
reproductive tract infection o Abnormal sperm may be
o Decreased pH is associated unable to move normally or
with increased prostatic fluid to penetrate an egg.

 Some abnormal sperm are

usually found in every normal
semen sample.
 But a high percentage of
abnormal sperm may make it
more difficult for a man to
SPERM MORPHOLOGY father a child.

 Normal
o 40 million spermatozoa per
ejaculate or more
o 0 sperm per milliliter if the
man has had a vasectomy SPERM MOVEMENT (MOTILITY)
 Abnormal
o A very low sperm count is  Normal
present, which may indicate o More than 60% of the sperm
infertility. show normal forward
o But a low sperm count does movment.
not always mean that a man  Abnormal
cannot father a child. o Sperm must be able to move
o Men with sperm counts forward (or “swim”) through
below 1 million have cervical mucus to reach an
 Normal
fathered children. egg.
 o A high percentage of sperm  Sperm Viability • Quantitative Test
that cannot swim properly  Seminal Fluid Fructose
may impair a man’s ability to  Antisperm Antibodies  Positive – equal or greater than 13
a father a child.  Microbial and Chemical Testing µmol ejaculate
 Postvasectomy Semen Analysis  Spectrophotometric method
Gradation of Sperm Motility – WHO 2010  Sperm Function Tests
ANTISPERM ANTIBODIES
 Grade 4 (A): Sperm with progressive
motility. These are the strongest and  It can be present in both men
SPERM VIABILITY
swim fast in and women
straight line.  Viability is evaluated by mixing the  They may be detected in semen,
 Grade 3 (B): Sperm with non-linear specimen with an eosin-nigrosin cervical mucosa or serum
forward motility. These also move stain, and counting the number of  Normally, the blood-testes barrier
forward but end to dead cells in 100 soerm. separates sperm from the male
travel in a curved or  Live cells – bluish white color immune system.
crooked motion.  Dead cells – red  When this barrier is disrupted, the
 Grade 2 (C): These have non- antigens on the sperm produce
progressive motility because they do Seminal fluid fructose an immune response that
not move forward despite damages the sperm.
 Low sperm concentration maybe • Vasovasectomy, trauma,
they move their tails.
cause by lack of the support infection
 Grade 1 (D): These re immotile and
medium produced in the seminal  The damages sperm may cause
fail to move at all.
vesicles the production of antibodies in
 Low to absent fructose level in the the female partner
semen  Mixed agglutination reaction test
 Resorcinol test • A screening procedure used
• Procedure: primarily to detect the presence
•Reagent – 50 mg resorcinol in 33 ml of IgG ANTIBODIES
concentrated HCL diluted to 100 ml • The sperm sample containing
with water. motile sperm is incubated with
 Mix 1 ml semen with 9 ml IgG antihuman globulin and
reagent suspension of latex particles or
 Boil treated RBCs coated with IgG.
 Observe for orange – red • The bivalent AHG binds
color simultaneously both the antibody
on the sperm and the antibody
on the latex particles or RBCs,
• Qualitative Test forming microscopically visible
ADDITONAL TESTING clumps of sperm and particles or
 Positive- orange – red color
cells.
 • Less than 10% of the motile INDICATES INFECTION WITHIN THE  MUCH LESS INVOLVED
sperm attached to the particles REPRODUCTIVE SYSYTEM, FREQUENTLY PROCEDURE COMPARED TO
is considered normal THE PROSTATE. FERTILITY ANALYSIS
 Routine aerobic and anaerobic  THE ONLY CONCERN IS THE
cultures as tests for chlamydia PRESENCE OR ABSENCE OF
trachomatis, mycoplasma hominis SPERMATOZOA
and ureaplasma. Urealyticum are  SPECIEMEN ARE ROURINELY
most frequently performed. TESTED AT MONTHLY
 CHEMICAL TESTS: INTERVALS, BEGINNING AT 2
o NEUTRAL ALPHA- MONTHS POSTVASECTOMY
 Immunobead test
GLUCOSIDASE AND CONTINUING UNTIL 2
o A more specific procedure
o 20 Mµ/ CONSECUTIVE MONTHLY
used to detect the presence
EJACULATE SPECIMENS SHOW NO
of IgG, IgM and IgA
o Decreased SPERMATOZOA
antibodies
values
o Demonstrates what area of APERM FUNCTION TESTS
suggests a
the sperm the autoantibodies
disorder in
are affecting
epididymis
o Head-directed abs affect
movement through cervical •zinc
mucosa
o In the immunobead test, o > 2.4 µmol/ ejaculatr
sperm are ,mixed with o Decreased values
polyacrylamide beads indicates lack of prostatic
known tobe coated with fluid
either anti-IgG, anti-IgM or
•Citic acid
anti-IgA
o Microscopic examination of o > 52 µmol / ejaculate
the sperm shows the beads o Decreased values indicates
attached to sperm at lack of prostatic fluid
particular areas. o Hamster eggpenetration
o The presence of beads on •acid phosphatase • Sperm are incubated with species-
less than 20% of the sperm is nonspecific hamster eggs and
considered normal. o >200 units/ejaculate penetration is observed
o Decreased values indicates microscopically
MICROBIAL AND CHEMICAL TESTING lack of prostatic fluid o Cervical mucus penetration
• Observation of sperm penetration
 THE PRESENCE OF MORE THAN 1 POSTVASECTOMY SEMEN ANALYSIS ability of partner’s midcycle cervical
MILLION LEUKOCYTES PER MILLILITER mucus
 • Hyaluronic acid CLARITY culture
• Mucopolysaccharides INCLUSION Crystal Uric
• Limited amount of plasma protein S Acid
• Glucose and uric acid levels equivalent VISCOSITY Cytology Lactic
to plasma acid
CLOTTING LDH
SPERM FUNCTION TESTS FUNCTION MUCIN Rheuma
• Supplies nutrients CLOT toid
o HYPO-OSMOTIC SWELLING • Lubrication of joint Factor
• sperm exposed to low sodium
concentration are evacuated to Reasons for analysis: CLASSIFICATION OF SYNOVIAL FLUID
membrane integrity and sperm • Infection
viability  Normal
• Hemorrhage
In Vitro Acrosome reaction  Non-Inflammatory
o • Degenerative disorders (arthritis)
• Evaluation of the acrosome to o Degenerative joint diseases
• Inflammatory disease (SLE)
produce enzymes essential for ovum  Inflammatory
penetration COLLECTION o Immunologic disorder (ie.
• Arthrocentesis Lupus, RA, gout crystals,
SEROUS FLUIDS – PERITONEAL FLUID etc…)
 Septic
o 1992 cap cm41 Peritoneal fluid.
o Microbial infections
Segmenter, macro, yeat
 Hemorrhagic
o Traumatic injury, tumors,
hemophilia, anticoagulant
overdose, etc…
• Tubes
o
• Heparin – chemistries, immunological
test
• Sterile tube – culturing and crystal
evaluation SYNOVIAL FLUID – LABORATORY
• EDTA - hematology PROCEDURES

LABORATORY RESULTS

MACROSC MICROSC CHEMIC OTHER

SYNOVIAL FLUID OPIC OPIC AL
VOLUME Cell Protein Aerobi
COMPOSITION AND FORMATION counts c
• Secreted by cells of synovial membrane culture
• Very viscous, clear ultra filtrate of plasma COLOR Differentia Glucose Anaer
• Contains AND l obic  Hematology
 • Physical properties tight ropey mass • Monocytes 48%, macrophages
• Color and Clarity - light yellow/ • Poor - appears 10%
straw and clear friable or fails to form • and synovial lining cells 4%
• Abnormal colors/ clarity as  Synovial lining cells
for other fluids • (look somewhat similar to
• Bloody mesothelial cells)
• Hemorthrosis
• Traumatic tap
• White/ opaque with turbidity
• Indicates pus cells or
debris  Hematology
• Xanthrochromia term not used!

 LE CELLS

• Cell counts
• 0 RBCs / ul
• <200 WBC/ ul
• Must let hemacytometer sit
longer to allow cells to settle before
counting.
• If dilution needed must use
 Physical properties saline  TART CELLS
• Viscosity If you use diluent with an acid, such
• Screening – ‘String Test’ as Unopettes, the sample will clot.
drop from pipette  Cell differential - Wright’s stain
• Evaluates
viscosity
• Normal - @ 5+
cm long before breaking
 OTHER CELLS
• Rope’s test for mucin
clot
• measures
degree of hyaluronate • Cells of peripheral circulation
• Neutrophil 7%
polymerization
• Lymphocytes 24%
• Good/ normal -
 • Chemistries Color Stra Straw Yello Varia Red
• Total protein NV = 1.07 – 2.13 G/DL w / w ble
• Increases seen in inflammatory yello
conditions and following joint hemorrhage w
Clarit Cle Clear Clou Clou Varia
• Glucose – similar to current blood level
y ar dy dy ble
 MALIGNANT CELLS • Decreased in inflammation or sepsis Visco Hig High Low Varia Low
 ORGANISMS • Lactase – assist in differentiation of sity h ble
septic and inflammatory arthritis Cell <20 200- 2,000 >100. Same
• Uric acid – increased in gouty arthritis. coun 0 2,000 - 000 as
• If gout is suspected, but no crystals, t/ul 75,00 bld.
0
may need uric acid level
%PM <25 <25% >50% >75% Same
• Microbiology Ns % as
bld.
• Gram stain, acid fast stain and cultures
Gra Ne Nega Nega >50% Negat
SYNOVIAL FLUID – LABORATORY PROCEDURE • Certain organisms associated with age m gati tive tive Pos ive
groups stain/ ve
• Children 16-50 – H. influenza cultu
• Adults 16-50 – staph. Strep. re
Pneumoniae, strep pyogenes, Neisseria Cryst Ne Nega Frequ Neg Negat
als gati tive ently ative ive
gonorrhea
ve
• Adults > 50 – staph. Aureus
Asso Dege Immu Non- Traum
ciate nerat nolog gono a,
• Serology
d e ic cocc hemo
• Serum results more reliable, so not often cond joint disor al or philia,
done for diagnosis of RA or Le ition disea ders- gono antic
• Autoantibodies se LE, ccal oagul
Microscopic exam for crystals
• Complement levels RA, septi ant
• Use regular and polarized light Gout, c overd
• QC- no commercial controls available,
• Crystals may be intra-cellular or extra- pseu arthri ose,
use serum controls if appropriate
cellular do tis etc.
• Monosodium urate - gout arthreritis gout
SYNOVIAL FLUID CLASSIFICATION
• Calcium pyrophosphate –
pseudogout Classification of Synovial Fluid by Test Results
REVIEW OF KEY POINTS
• Cholesterol – nonspecific; chronic Nor Non- Infla Septi Hemo
mal infla mmat c rrhagi • Synovial fluid analysis
inflammatory
mmat ory c • plasma ultra filtrate secreted by
• Apatite – calficic artheritis (mineral
ory synovial fluid
change in cartilage)
Volu < > 3.5 > 3.5 > 3.5 > 3.5 • hyaluronic acid and
• Corticosteroid – drug injections me 3.5 (ml) (ml) (ml) (ml) mucopolysaccharides make it viscous
(ml) • lubricates and nourishes joints
 • Infection, hemorrhage, degenerative 1989 cap cm 23 Synovial fluid, segd, and
and inflammatory diseases are reasons for macrophages
analysis
• Collection is by arthrocentesis
• EDTA (hematology), heparinized
(chemistries and serology) and sterile
(cultures and crystals) are collected
• Straw yellow, clear and viscous are
normal characteristics
• 0 RBC and <200 WBC/ul are normal Synovial fluid with acute inflammation and
• Cell counts requiring dilution must be monosodium urate crystals. (Wright-Giemsa
made with saline. stain and polarized light).
• Any peripheral circulating cell can be
Lupus erythematosus cell – far right side @3
seen as synovial lining cells in normal
oclock
patients.
• Abnormalities are classified as non-
inflammatory, inflammatory, septic or
hemorrhagic

1993 cap cm 21 SYNOVIAL FLUID.

Synovial fluid with acute inflammation and
Segmented and leukophage
monosodium urate crystals. The needle-
Lupus erythematosus (LE) cells shaped crystals demonstrate negative
birefringence, because they are yellow
when aligned with the compensator filter
and blue when perpendicular to the filter
(wright-giemsa stain and
polarized/compensated light).

SYNOVIAL FLUID – LABORATORY

MICROSCOPIC ANALYSIS: CRYSTALA-URIC PROCEDURES
ACID
 Synovial fluid with acute inflammation and • Parietal – lines cavity wall
calcium pyrophosphate dehydrate crystals • Visceral – covers organs contained
within
(wright-Giemsa stain and polarized light). • Serous fluids fill the space between

• “ULTRA FILTRATE” of the plasma

• Closely resembles the plasma (as
LEFT – needle shaped monosodium urate opposed to CSF)
crystals seen in patient with gouty arthritis
Appearance Possible reasons
Right 1987 CAP CM 18B synovial fluid. Pale yellow and Normal
Monosodium urate crystals clear
White, turbid WBCs/infection
Bloody RBCs/hemorrhage
Milky Chyle – lymph and
emulsified fats
Viscous Increased hyaluronic
Synovial fluid with acute inflammation and acid/ malignant
calcium pyrophosphate dehydrate crystals. mesothelialoma
The rhomboidal intracellular crystal (center)
demonstrates positive birefringence, • Produced by hydrostatic and
because it is blue when aligned with the oncotic(protein) pressure in the capillaries
compensator filter (wright-Giemsa stain and lining the membranes
polarized/compensated light) • Normally produced at a constant rate.
• Production ( parietal membrane)
SEROUS FLUIDS • Re-absorption (visceral membrane)
1989 CAP CM 24 synovial fluid. Calcium
pyrophosphate - polarized • Small amount of fluid that lies between the •Production and re-absorption are
membranes lining the body cavities influenced by:
MICROSCOPIC ANALYSIS: CRYSTALS OTHERS
(parietal) and those covering the organs • Changes in osmotic and hydrostatic
within the cavities (visceral) pressure in the blood
• acts as lubricant, • Concentration of chemical constituents
• provide nutrients, in the plasma
• remove wastes • Permeability of blood vessels and the
membranes
• Body cavities
•Types of serous fluids
• Pericardial – heart
• Pleural – lungs • Types of serous fluids
• Peritoneal – abdominal • Pericardial fluid – around heart
• Membrane • Pleural fluid (thoracic fluid) – lung cavity
• Lined with mesothelial cells
 • Peritoneal (ascetic fluid) – abdominal  Though of as an inflammatory  Pleural fluid
cavity process - Normal appearance: clear,
 Exudate examples: pale yellow
• Reasons for analysis - Pleural exudate – - Turbidity: white blood cells
• INFECTIONS carcinoma, pneumonia, and microorganisms
• Hemorrhages trauma - Blood: traumatic injury,
• Malignancies, - Pericardial exudate – malignancy, traumatic tap
• and other disorders. infection, cardiovascular - Milky: chylous or
disease (CV) trauma, pseudochylous material
Specimen collection and handling cancer - Neutrophils: bacterial
• Needle aspiration infection
Differentiation between transudates and
• Paracentesis - Lymphocytes: tuberculosis,
exudates malignancy
• Thoracentesis
• Pericardiocentesis - Normal glucose: parallels
CHARACTERISTIC/TES TRANSUDA EXUDAT
serum glucose
T TE E
• Lavage (i.e. Peritoneal lavage) - Low glucose: tuberculosis,
Color Pale Any
• Ringer’s lactate/saline is infused into rheumatoid- inflammation,
yellow abnorm
abdomen then retrived for analysis al color malignancy
• Specimen sometimes called ascites Clarity Clear Bloody - Low ph: tuberculosis,
fluid. cloudy, malignancy, esophageal
purulent rupture
SEROUS FLUIDS - COMPOSITION AND , turbid - Elevated amylase:
FORMATION Specific gravity <1.015 >1.015 pancreatitis
• EFFUSION Glucose Equal to Over 30  Pericardial fluid
serum mg less - Normal appearnce: clear,
• an increase in the serous fluid due to
than pale yellow
some disruption in production and re- serum - Milky: lymphatic drainage
absorption processes. level - Turbidity: infection,
• Classification of cause of an effusion is Protein <3.0 g/dl >
malignancy
aided by determining if the fluid is a Fluid/serum protein <0.5 >
- Blood: tuberculosis, tumor,
“transudate” or an “exudate” . ratio
Fibrinogen/spontane No Possible cardiac puncture
ous clotting - Neutrophils: bacterial
• Exudates
Fluid/serum amylase <2.0 >2.0 endocarditis
 Term to classify the effusion that is Fluid/serum bilirubin <0.6 >0.6 - Low glucose: bacterial
result of a problem with the ratio infection, malignancy
membrane themselves. Lactate <60% of >60% of  Peritoneal fluid
dehydrogenase serum serum - Normal appearance: clear,
 Produced by conditions that directly
Fluid/serum LD ratio <0.6 >0.6 pale yellow
involve the membrane of the
Cell counts (total) <300/l >1000/l - Tuebidity: peritonitis, cirrhosis
particular cavity, ex. Infections,
SUMMARY OF SEROUS FLUID TESTING - Blood: trauma
inflammations, and malignancies
 - Neutrophils: peritonitis • both fluid and current
- Low glucose: tubercular serum level to make
peritonitis, malignancy comparison: fluid protein/
- Elevated amylase: serum protein
pancreatitis, gastrointestinal - LDH enzymes
perforation • both fluid and current
- Elevated alkaline serum level to make  Unique to serous fluids, originate from
phosphatase: intestinal comparison: fluid LDH/ serum lining of peritoneal, pleural, and
perforation LDH pericardial cavities
- Elevated urea or creatinine: - Cultures  Large round cell with abundant blue
ruptured bladder - Serology – rarely done on cytoplasm and purple nucleus which
serous fluids as blood testing may be eccentric
is adequate  Cell sometimes described as having
SEROUS FLUIDS
- Cytology/pathology – if a “ fried egg” apperance – usually
malignancy is suspected are single or may be in sheets
 SPECIMEN COLLECTION AND
HANDLING  Nucleus round to oval and has a
SEROUS FLUIDS
- EDTA tube for cell count and smooth outline, takes up @1/3 – ½ of
differential  Hemotology/ gross examination the space
- Heparin tube for chemistries, - COLOR  Smooth spherical neceoi may be
serology, microbiology and • NV= yellow and clear seen.
cytology (other terms as for CSF are
- Since procedure not sometimes used, EXCEPT
performed unless an effusion ‘xanthrochromic’
exists, large amount of fluid - CELL COUNT
often collected. • same as CSF
- Blood specimen usually
collected at same time and Reactive mesothelial cells
comparisons of test results  Pleomorphic
made.  If ‘ reactive’ may appear in clusters,
have prominent nucleoli and be
SEROUS FLUIDS – TESTING OVERVIEW
multinucleated
 Variety od tests used aid in  Nucleus still distinct and around with
determining the cause of the uniform staining chracteristics.
effusion  A cluster of reactive mesos may
- DIFFERENTIAL resemble malignant cell clusters, but
- Apperance • any cell in peripheral
- Evaluation of clotting ability the mesos display “cell windows”
blood,
whether or not it will form • mesothelial cells,
clot, etc. • malignant cells SEROUS FLUIDS
- Cell counts
- Protein level MESOTHELIAL CELLS
 - Increased nuclei/ cytoplasm
ratio.

• Always send suspicious cells to

cytology/ pathology

SEROUS FLUID MALIGNANT CELLS

 Macrophage engulfed Candidia - Acsp 7, Case 1 peritoneal fluid,

species in a pleural fluid, mesothelial malignancy
cells.
- Ascp 12 Case 4, 30 year old with
back pain and inability to work.
SEROUS FLUIDS Pleural effusion fluid – malignant
tumor on spinal cord
 Malignant cells
- A frequent concern in any
serous fluid due to possibility
of cancer of any organ and/ -
or metastasis of CA from one - Ascp 9 Case 2 pleural fluid 42 year
location to another old, breast cancer
- Cells have irregular size,
shape, and staining -
characteristics of nucleus
and cytoplasm. Usually
deeply basophilic, molded
or balled up clusters of cells
with little distinction from one - Sheets of atypical cells with irregular
cell to the next. May be nuclear contours, nuclear
vacuolated. hyperchromasia, basophilic
cytoplasm, and jagged outline of
- cell borders.
SEROUS FLUIDS - Ascp 10 Case 3, ascitic fluid, 62 year - Squamous cell carcinoma (x400, Diff-
old admitted for GI bleeding Quik staining)
 Malignant cells
• Characteristics
- Irregular shape
- Uneven chromatin
distribution
- Prominent large irregular
nucleoli
- Community borders
 • suspicion of perforated intestine
- ph and ammonia

 MICROBIOLOGY
- Gram stain and acid fast
- Cultures – aerobic and anaerobic
 SEROUS FLUIDS
- 1987 CAP CM21 Pericardial
fluid, intracellular bacteria

SEROUS FLUID-LE CELLS

 Seen in patients with Systemic Lupus

Erythmatosis (SLE) a systemic disease
in which an autoantibody attacks
the patients organs and body
systems
 LE cells is a neutrophil that has
engulfed a homogeneous mass of
purple staining nuclear material

- 1992 CAP CM41 Peritoneal

fluid, Segmenter, Macro,
Yeast

SEROUS FLUIDS
 CHEMISTRY
- Total protein, and ratio to serum
protein
- LDH and ratio to serum LDH
- Glucose
- Amylase anf lipase – pancreatic
disorders
- Bilitubin – peritoneal fluid
•suspicion of perforated GI or gall
bladder
- Alkaline phosphatase – peritoneal
fluid
Microscopic Examination of Urine, - 20 µL (0.02) covered by 22 x 22  * Addis count
mm glass cover slp - use to quantitate
Automation in Urinalysis and Testing 6. Examination of the sediment: formed elements in the urine
- Observation of a minimum of 10 microscopic analysis to monitor
fields under both low (10x) and the course of diagnosed case
MICROSCOPIC EXAMINATION high (40x) power of renal disease
* First examined under low (10x)  - Uses a hemacytometer
•Purpose: power: to count the number of red
- to detect casts and white blood cells, casts,
 To detect and identify insoluble
- to ascertain general and epithelial cells presents in
materials present in the urine
composition a 12-hour specimen
• Preparation and Examination: 7. Reporting of the microscopic - Normal values:
examination: 0 to 500,000 red
1. Examined while fresh or  Casts are reported as blood cells
adequately preserved: average number per10 0 to 1,800,000 white
 RBC’s, WBCs, and hyaline casts – low power fields blood cells
disintegrate rapidly in alkaline  RBCs and WBCs are 0 to 5,000 hyaline
urine. reported as average casts
 Refrigeration may cause number per 10 high power  Commercial systems:
precipitation of amorphous fields  KOVA (ICL Scientific, Fountain
urates and phosphates and other  Semiquantitative (rare, Valley, CA)
non-pathologic crystals. few, moderate or  Fisherbrand Urisystem (Fisher
 Warming specimen at 370c may abundant; or 1+,2+, 3+ or Scientific, Pittsburgh, PA)
dissolve crystals. 4+)  Count-10 System (V-Tech, Inc.,
2. Standard volume: usually 10 to 15 - Epithelial cells Palm Desert, CA)
ml is centrifuged - Crystals
3. Speed of the centrifuge and the Urine Sediments
- Other elements
length of time the specimen is
centrifuged should be consistent • Phase contrast microscope
- time= 5 minutes - for identification and differentiation of
- Centrifugal force = 450 RCF formed elements and unstained cellular
4. Uniform volume should remain in components
the tube after decantation
• Polarized Microscopic 1. Red Blood Cells
- 0.5 to 1.0 ml.
- for fats and lipids 1. Normal:
5. Volume of sediment placed on
• Smooth, non-
the microscope slide;
• Standardization of urine specimen: nucleated, biconcave
 disks Ghost cells
• Average diameter of 7 - Usually seen in dilute urine
µm
• Urine value: (Hyposthenutic/hypotonic
• 0 to 2 RBCs/hpf
(Henry)
• O to 3 RBCs/hpf
(Stransinger)
2. White Blood Cells/ Leukocytes
- Large than RBCs
Isosmorphic - Average diameter of 12 um
- Normal value:
- Intact red cells <5 WBCs/hpf
- Seen in Non-Glomerular hematuria
ex. Ureter calculi

Urine Sediments – Red Blood Cells

Glitter cells/ Sterheimer – Malbin cells

Dysmorphic
- Sparkling appearance due to
- With cellular protrusions or
Brownian movement
fragmentations
Seen in hypotonic/dilute urine
Created or Irregular shaped - Seen in Glomerular hematuria
ex. Glomerulonephritis URINE SEDIMENTS – White Blood Cells
- Usually seen in concentrated urine
Lyzed WBCs
(Hypersthenuric/hypertonic)
- Seen in hypotonic / dilute or alkaline
urine

Possible sorce of errors:

  Originate from the
renal pelvis, ureters,
urinary bladder
and/ or urethra
 Round or
polygonal; less
 From the lining of commonly pear-
Transitional// Renal tubular cells
vagina and female shaped, tailed, or
- granular, larger and polyhedral
urethra and lower spindle-shaped.
portion of the male Smaller and
urethra smoother in outline
 Thin, flat cells, than squamous
usually with an cells, but larger
angular or irregular than WBC.
outline and a small
round nucleus
Red Blood cells  Represent normal
sloughing
- Non – granular, smaller

3. Epithelial Cells
2. Derived from linings of the
genitourinary system. Transitional cells
• Squamous epithelial cells  Increase in number;
- largest cell in the urine “Clue cells” with abnormal
• Transitional (Urothelial) morphology seen
epithelial cells  Covered with coccobacilli in:
• Renal Tubular cells (RTW)  Seen in infection with Gardnerella - Malignancy
- most significant of the vaginalis - Viral infection
epithelial cells 3. Renal Tubular Cells (RTE)
2. Transitional cells
Urine sediments – Epithelial cells

1. Squamous cells
Collecting duct renal a e
tubular cells l a
 Varyin g c
g size r t
and a i
shape f o
 Large t n
granul r - V
ar e i
rectan j r
gular e a
cells c l
to t i
cuboi i n
dal or o f
colum n e
nar - P c
cells y t
not e i
much l o
larger o n
than n
WBCs e
 Increa p
se h
numb r
ers i
mayb t
e seen i
in s “Bubble Cells”
TUBUL - T
 With large non –
AR o
lipid vacuoles
NECR x
 Injured cell in which
OSIS: i
the endoplasmic
- R c
reticulum has
e
n r
 dilated prior to cell 2. Increased ionic
death concentration – salt
concentration
3. Stasis or obstruction –
reduced urine outflow
4. Increased plasma proteins
entering the tubules

Types of Casts:
Hyaline cast
i. Matrix
“Oval Fat Bodies”  Contains almost entirely of Tamm
1. Hyaline
– Horsfall proteins
 Containing lipids 2. Waxy
 Most frequently observed
 Seen in: nephrotic syndrome ii. Inclusion
 Translucent and homogenous
Trauma 1. Granular
 Associated with lipiduria 2. Fatty Hyaline casts
3. Others – hemosiderin,
crystals, melanin  Icreased in:
4. Casts
iii. Pigmented Physiologic Pathologic cause
 Formed primarily in
1. Hemoglobin response
the distal
2. Hemosiderin 1. Strenuo 1. Renal disease
convoluted tubules
3. Myoglobin us - Acute
and collecting duct
4. Bilirubin exercis glomerulo
 Tamm – Horsfall e nephritis
protein – major iv. Cellular
1. Red blood cells 2. Dehydr - Pyelonep
constituent; forms ation hritis
the matrix of the 2. White blood cells
3. Heat - Chronic
casts 3. Epithelial
exposu renal
 Cylinduria – 4. Mixed cellular re disease
presence of casts in v. Miscellaneous casts/cats – like 4. Emotio 2. Congestive
the urine structure nal heart failure
1. Bacteria stress
Urine Sediments – Casts 2. Mucus threads 5. Diuretic
treatm
Conditions that increase cast formation: ent

1. Acidic ph
  Textured appearance which
ranges from fine to coarse in
character
 Granular originate from:
1. Lysosomes
2. Cellular remnants
Red blood cell cast
3. Proteins
 Appear with:
Waxy cast 1. Glomerular disaes
2. Tubular disease
 Reflect final phase of dissolution 3. Tubolointerstitial disease
of the fine granular and hyaline 4. Renal allograd-ft refection
casts 5. Pyenonephritis Epithelial cell cast
 Smooth consistency, more 6. Viral infections
refractile, commonly have 7. Chronic lead poisoning
squared off ends 8. Stress and strenuous
 Broader than hyaline exercise
 Clinical significance:
Fatty Cast
 Indicate localized nephron
obstruction and oliguria White blood cell cast
 Chronic renal disease
Urine sediments – Casts
“Renal failure cells”
 Pseudocasts
 Unusually broader
 Seen in end stage renal disease

Granular Casts  Identified by the presence of

refractile lipid droplets
 Seen in:
1. Heavy proteinuria Amorphous urates
2. Nephrotic syndrome  May form cylindrical shapes as
the sediment settles under the
Cellular Cast cover glass
 Highly refractile, and lack well
defined borders

5. Crystals
  Formed by the precipitation of 2. Lesch – Nyhan syndrome 1. Severe chronic renal disease
urine salts subjected to changes 3. Gout 2. Ethylene glycol or
in ph, temperature, or methoxyflurance toxicity
concentration. 3. Crohn’s disease
4. Genetically susceptible dose of
Normal urinary crystals
ascorbic acid
In acidic ph In alkaline ph
1. Uric acid 1. Amorphous
2. Amorphous phosphates
urates 2. Calcium
3. Calcium phosphates Amorphous urates
oxalate 3. Triple
phosphates  Yellow – brown mass of small
4. Ammonium rounded particles. White – colorless, fine or lacy
biurate  Soluble in alkali and heat precipitate
 Convert to uric acid crystals Amorphous phosphates
with acidification with acetic  Soluble in dilute acetic acid
Urine sedimentals – Normal Crystals
acid  Precipitate upon prolonged
Uric acid – most common standing at room temperature or
in refrigeration

 Yellow to reddish - brown;

 In variety of shapes - Colorless 3 – side prisms, plates or
rhombic plates, rosettes, Small, colorless, octahedron,
needles
wedges and needles that resembles an envelope;
CALCIUM PHOSHATES
 Birefringence with dumbbell shape and avoid
 SOLUBLE IN DILUTE ACETIC ACID
polarized light may occur
 OFTEN SEEN IN INFECTED URINE OF
• Dissolves in alkali ALKALINE PH
Calcium oxalate
• SEEN IN:
 Soluble in dilute HCL
1. Leukemia
Oxaluria:
 Urine sedimentals – Abnormal Crystals

Cystine

Yellow – oily appearing sphere with

radial and concentric striations
Yellow brown color; appear as
Colorless, refractile, hexagonal plates
spheres with radial or concentric  Soluble in hot alkali or acid
striations and irregular projections  Soluble in ammonia, dilute HCL,  Seen in severe liver disease,
of thorns – thorny apples water at ph<2 or >8 inherited metabolic disease
 Confirmed by cyanide –
Ammonium Biurate nitroprusside reaction
Tyrosine
 Soluble in acetic acid with heat
Cholesterol
at 60˚C

Urine sedimentals –Crystals

Abnormal urine crystals

 usually seen in acid ph

1. Cysteine
2. Cholesterol Maybe colorless or yellow; forms fine
3. Leucine Rectangular plate with a notch in one needles that maybe arranged in
4. Tyrosine or more corners sheaves or clumps
5. Bilirubin
6. Sulfonamides- Maybe seen  Soluble in chloroform  Soluble in alkali and dilute HCL
in neutral ph  Associated with lipiduria  Seen in severe liver disease,
7. Radiographic dyes Nephrotic syndrome severe leukemia, typhoid fever,
8. Ampicillin  Seen in conjuction with oval fat and small pox
9. Others bodies
Bilirubin
Mercaptopurine Primidone
Leucine
 6. Other Sediments

Reddish brown; amporphous

Bacteria
needles, rhombic plates, or cubes spermatozoa
 Soluble in Acetic acid, HCL, Contaminants
NaOH, Ether Chloroform
 Seen in severe liver disease,
hemolytic disease

Sulfonamides

Yeast

Talc

Yellow brown sheaves of wheat with

central bindings; striated sheaves with
eccentric bindings: rosettes; Parasite Starch
arrowheads; petals; needles; and
around forms with radial striations

 Soluble in acetone
 Seen in patients on sulfonamide
therapy
 1. detection of
Stain Action Function
malignanciee
Supravital Contracting Identitfies:
stain colors for WBCs, 2. Provide more definitive
Sternheim nucleus and Epithelial information about renal
er – Malbin cytoplasm cells, Casts tubular changes
stain
- Suitable specimens:
Pollen Grains 0.5% Enhances Differentiate
Toluidine nuclear s: 1. Voided or
blue detail WBCs, catheterized
Yeast, oil 2. Bladder washing or
droplets and brushing
crystals 3. Washing and
2% Acetic Enhances Distinguishes brushing of ureter or renal pelvis
Cotton Fibers acid WBCs nuclei :
EXAMINATION TECHNIQUES RBCs from - Fixative: 50% Ethyl alcohol
WBCs,
yeast, oil - Satin: Papanicolau stain
1. Sediment Stains
droplets
 Increase overall Lipid Stain TGC Identifies 3. Microscopy
visibility by oil red O and neutral free fat
changing refractive Sudan III fat droplets and Techniques Function
index lipid Bright – field Routine analysis
containing microscopy
sediments Phase contrast For elements of
Gram stain Differentiate Identifies microscopy low refractive
d bacteria bacterial index indices
casts Hyaline casts,
Hansel Stain Urinary mixed cellular
stain eosinophilic eosinophils casts, mucous
granules threads, and
Prussian Stain iron Hemosiderin trichomonas
blue in cells and Polarizing Identify cholesterol
casts microscopy in oval fat bodies,
fatty casts, and
crystals
2. Cytopathologic Interference 3- dimensional
examination microscopy image
 Purpose:
AUTOMATION IN URINALYSIS  Microscopy: Flow 4. Electron beam
cytometry analysis
4. AUTOMATED URINALYSIS - Employs 5. Mass spectroscopy
Goal: o Impedance
1. Improve reproducibility 2. URINARY SCREENING FOR
detection
and color discrimination METABOLIC DISEASES
o Light scattering
2. Increased productivity o Fluorescence
3. Standardization for
reporting Flow Cytometry

Automation

i. Phenylalanine – tyrosine
disorders

URINALYSIS
 Automated macroscopic/ SPECIAL URINE TESTING
chemical analysis
*Spectrophotometry 1. INVESTIGATE STONE FORMERS
 Automated microscopic  Urine Examinations
urine analysis 1. Routine – crystal identification
*Flow cytometric studies 2. 24 – hour urine collection – Na,
 Spectrophotometry – Ca, P, UA, Oxalate, Ccr
reflectance photometry 3. Urine ph determination
- Light reflection from the  Serum chemistry – Ca, P,
test pads decreases in UA and electrolytes
proportion to the intensity
Investigate Stone Formers
of color produced by the
concentration of test  Stone analysis
substances 1. Optical
- Light is reflected to a crystallography
photodetector and an 2. X-ray diffraction
analog/ digital converter 3. Infrared
spectroscopy
Disease cooments Methods of
testing
Phenylket Absence of 1. Reagent
onuria↑ phenylalanin strip: bacteria
-
e PHENISTIX
ii. Branched Chain Amino malabsorpt
hydroxylase 2. High
Acid Disorders ion
↓ indoacetic Performanc
acid in urine e LIQUID syndromes
Disease comments Methods of - hartnup
5- Chromatog testing
hydroxylindol raphy disease
Maple HYPERVALI 1. - inherited
eacetic acid 3. Guthrie – syrup NEMIA, dinitrophenylh disorders
in urine for blood urine ISOVALERIC ydrazine test
Alkaptonu Deficiency of 1. Ferric 5– 5 Nitrosonapht
disease ACADEMIA 2. gas or thin hydroxyind hydroxyind hol test
ria homogentesi chloride (MSUD) Urinary layer
c acid test ole acetic ole acetic
“SWEAT excretion chromatograp acid acid
↑ oxidase 2. Silver FEET of leucine, hy
Homogentesi nitrate test normaly - seen in
ODOR” isoleucine, Nuclear daily argentaffin
c acid of 3. “MAPLE valine and magnetic
urine Confirmato excretion: 2 tumors
SYRUP, their resonance to 8 mg
ry tests: CARAME correspond Spectroscopy.
Paper or LIZED ing keto
thin – Layer SUGAR, acids iv. Cysteine Disorders
chromatog OR
raphy CURRY” disease commen Methods of
URINE ts testing
Capillary ODOR Cystinuria Cysteine 1. cyanide –
electropho Cystinosis in the nitroprusside
resis urine
Tyrosiluria Urinary 1. iii. Tryptophan Disorders Homocystinur 1. 1. Cyanide
succinylacet Nitrosonap ia –
one and hthol test disease comments Methods of nitroprusside
succinylacet 2. Milon’s testing (+) red
oacetate tests Indicanuria Amounts of 1. oxidation purple
tyrosine and 3. tryptophan when 2. Silver –
phenolic acid Chromatog are exposed to nitroprusside
raphy for converted air test
confirmatio “blue to indole: (+) only for
n diaper Intestinal 2. Ferric homocytine
Melanuria Production of 1. sodium syndrome” disorders: chloride 3.
melanin; also nitroprussid - electrophore
protein, e obstruction sis
thyroxine, (Nitroferric - abnormal
epinephrine, yanide)
and tyrosine 2. Ferric
sulfate chloride
 v. Other metabolic Disorders a
m
disease comment Methods of m
s testing o
Porphyrin Abnormal Ehrlich’s ni
disorders ity in the reaction u
basic m
pathway Ultraviolet light o
for heme from wood’s x
sysnthesis lamp al
Mucopoly Abnormal Cetytrimethyla at
saccharid ity mmonium e,
e involving bromide gl
disorders the (CTAB) turbidity a
glycosam test ci
inoglycan al
s: Acid – albumin a
Hunter’s tests c
syndrome et
Hurler’s Metachromatic ic
syndrome staining spot a
Sanfilippo test ci
’s d
calciuria Increase Sulkowitch test - (+
urinary – qualitative )
calcium measurement tu
- R rb
e id
a it
g y
e
nt
:
o
x
al
ic
a
ci
d,