WEEK 2: SAFETY IN THE CLINICAL LABORATORY

➢ Airway filter to prevent circulation of contaminated air.

OUTLINE ➢ 99.97% filter even bacteria.
I Laboratory Safety ➢ 0.3 micrometer or larger organism will be filtered.
A Safety awareness for Clinical Laboratory Personnel
i CDC
II Biological Hazard • Universal Practices:
A Chain of infection o Wearing of gloves
B Prevention o Handwashing:
C CDC Guidelines for Hand Hygiene ▪ best way to break the chain of infection. Wash hands before
III Sharps Hazards and after handling specimen using antimicrobial soap.
IV Chemical Hazards o Laboratory coats
A. Chemical Spils
o Prohibited: eating, drinking, smoking, applying cosmetics, touching
B. NFPA Hazardous Materials Classification
C. Degree of Hazards contact lenses
V Radiation Hazards
VI Electrical Hazards • Vaccination against HBV:
VII Fire / Explosive Hazards o Requirements before you take internship.
A. RACE: When a fire is discovered o Blood borne infection from needle-prick
B. PASS: To operate a fire extinguisher o Must be 3 shots with same brand
C. Types of Fire and Fire Extinguisher
VIII Physical Hazards
IX Summary of Hazards (Tabulated) • Appropriate signs to identify hazards
X Handwashing Materials o Know hazard signs and symbol

BIOLOGICAL HAZARD
LABORATORY SAFETY • Anything known to be harmful to health is a biological hazards,
• Recognition of hazards standard precaution is needed
o We must be informed since we are constantly exposed with this
hazards. There’s different policies used in the laboratory. • SYMBOL:
o Be able to recognize diff hazards and know what are the o 3 circles connected with each other.
precautions needed to eliminate or minimize exposure. o Color: BLACK
o Background: yellow or orange
• Application of common sense (requirement)
o Some hazards are unique since we are processing specimen. CHAIN OF INFECTION
• Transmission of microorganisms
• Safety-focused attitude o Specimen in the laboratory
o When working in laboratory, focus and have presence of mind. o To make transmission possible you need
these three: SMS
• Good housekeeping in all laboratory work and storage areas.
o Know what you are doing and handling • Requires a continuous link between 3 elements
o 1. Source: SARS-COV 2
▪ Reservoir: Human, patient, or an inanimate object touched by
SAFETY AWARENESS FOR CLINICAL LABORATORY patients
PERSONNEL o 2. Method or Mode of Transmission: Airborne or Droplet
▪ Direct contact: the most common mode of transmission ex.
CDC: CENTERS FOR DISEASE CONTROL AND PREVENTION the unprotected host touches the patient specimen or
• Universal Precautions (1987): original name contaminated object (hand-to-hand).
o All substances with blood are infectious. ➢ Droplets: Travel with shorter distance only
▪ Indirect contact
• Standard Precautions (1996): expanded the content and change the
➢ Airborne: Respiratory infections, inhalation of aerosol
name.
particles circulating in the air, or particles attached in the
o All body fluids with blood or not are all infectious.
dust particle. (Bioterrorism agent transfers through air,
➢ Urine, stool, semen, cerebrospinal fluid etc.
faster transmission of disease). Travel with greater
.
distance, recirculate in the air.
• Blood and body fluid precautions should be consistently used for
➢ Vehicles: contaminated food and water, certain disease
all patients
can be transmitted through food and water this is why we
o Body fluids
can’t eat or drink in the laboratory
▪ Other samples from other sections in the laboratory
➢ ex. histopath like unfix tissue, organs.
➢ Vectors: animals, insect bites. This requires
▪ Blood slide in hematology
rodent/mosquito program to prevent them from
transmitting diseases, especially insect or animals from the
▪ Pus-filled wound: not source of sample directly, since dead
lab can cause outbreak outside the laboratory.
microorganism/cells here.
o 3. Susceptible Host: If exposed to source, host becomes then a
➢ Pus-filled tissue is used as a sample. Pus alone is not used
source.
as a sample
Chain Of infection: IREMES based kay Strasinger
• Unfixed tissues, organs or blood slides Table No. IREMES
o Precautions:
ACRO MEANING EXAMPLES
▪ Appropriate barriers (gloves, gowns or laboratory coats)
NYM
➢ Fluid specimen: counter-top glass to prevent splashing of
I Source SARS-COV 2
the sample. It’s a cover so that when accidental splashing
is encountered, it will not go directly to your eyes, mouth. R Reservoir Contaminated object, specimen
Can be the agent itself: human
▪ Appropriate engineering controls E Exit From reservoir
➢ HEPA Filter: High Efficiency Particulate Air Filter
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 Lesson 2 : SAFETY IN THE LABORATORY

Sneezing, coughing of the patient SHARP HAZARDS

Mouth, nose • MOSTLY ENCOUNTERED IN MICROSCOPY: Needles, broken glass
M Mode Droplet, airborne wares, coverslip (thin glass prone to injury)
of Transmission • Placed near workspace
E Entry • Closed when not in use
• Sealed when ¾ full, bawal punuin ¾ lang
S Susceptible
o Disposed in puncture-resistant, leak-proof container with the
Host biohazard symbol.
▪ Must be disposed properly always check your sharps
PREVENTION container.
• Personal Protective Equipment (PPE) ➢ It has a lead or the cap/cover, and the body of the container
➢ Always wear your complete PPE is a thick plastic.
➢ Eyes and face shield if possible ➢ Molds are visible if contaminated, not usable na (kapag
➢ PREVENTS THE RISK OF SPLASHING THAT MAY GO nabasa)
DIRECTLY TO YOUR MUCUS MEMBRANE, THIS
BECOMES A PORTAL OF ENTRY OF DISEASE NOW.

o Gloves –
o Fluid-resistant gowns
o Eye and face shields
o Countertop shields: FOR
FLUID SPECIMENS TO
AVOID ACCIDENTAL
SPLASHING. CHEMICAL HAZARD
• Chemical spills
• Proper handwashing o BEST FIRST AID: flush the area with large amounts of water for at
o HAND CONTACT is the primary method of infection transmission least 15 MINUTES continuous then seek medical attention.
o HAND WASHING is the BEST WAY to break the chain of infection. ➢ Reagent slips in microscopy
▪ ULTIMATE GOAL: PREVENT TRANSMISSION TO ➢ Make sure to handle them safely to prevent accidents.
PREVENT THE INFECTION. FOLLOW CDC GUIDELINES Every chemical we use should be presumed hazardous.
STRICLY.
o Board exam question:
• Disposal of biological wastes ▪ There is an accidental chemical spill in your skin, what’s the
o ALL biological waste, except urine , must be placed in appropriate first thing to do?
containers labelled with the _biohazard symbol ➢ Wash it with water for 15 minutes.
➢ Urine is also a biohazard
o Discard urine by pouring it into the laboratory sink, avoid splashing, ▪ A hot benedict reagent splash and have contact with your eye,
and then flush with water what should you do?
▪ Urine is disposed in the sink to prevent leakage in the yellow ➢ Wash with boric acid or wash with water and cry for help.
bag. Remember to disinfect the sink using 1:10 sodium ➢ Board Examiners: Choose the wash with boric acid in eye
hypochlorite is needed after disposing of the urine. area.
➢ It must be diluted since pure NaOCl is corrosive ➢ Based on MSDS: Stick on MSDS. Wash the eye area for
15 minutes.
o Disinfection of the sink using a 1:10 sodium hypochlorite and
should be performed daily. ➢ Context: Base on textbooks. Boric acid can be used as
neutralizers, and boric acid is a component found in eye
CDC GUIDELINES FOR HAND HYGIENE drops. (LEMAR Ans.) based on the MSDS in Benedict
• Wash hands with detergent soap or Antimicrobial soap: reagent, the first thing you should do is to wash the area
o When the hands are visibly soiled (contaminated hands) for 15 minutes. (Pioneer Ans.)
▪ Use liquid soap for less contamination.
➢ If your hands are contaminated and you touch the bar • In chemical bottles, find and follow NFPA hazard identification system
soap, disease, microorganism transmission is still possible (a diamond symbol) when handling chemicals.
causing contamination
o Before eating Table No.1 NATIONAL FIRE PROTECTION ASSOCIATION (NFPA)
o After using the restroom HAZARDOUS MATERIALS CLASSIFICATION
LSR Yellow White Blue Red
• NOT ALL TIMES YOU HAVE TO WASH YOUR HANDS WITH quadrant quadrant quadrant quadrant
WATER AND ANTIMICROBIAL SOAP: Reactivity/ Specific Health Flammability
o You may use alcohol or antiseptics Stability hazard hazards
▪ Disinfectant: used for inanimate objects
0 Stable OXY = Oxidizer Normal Will not burn
➢ Tables, equipment
material
▪ Antiseptic: used directly in the skin to clean our hands
1 unstable if ACID = Acid Slightly Above 200’F
heated hazardous
o Alcohol is an antiseptic and disinfectant solution.
2 violent ALK = Alkali Hazardous Below 200’F
chemical
• Alcohol-Based Hand rub
change
o Before having direct contact
3 shock and COR = Extreme Below 100’F
▪ Ex. before venipuncture, touching any equipment, typing
heat may Corrosive danger
results use sanitizers first.
deteriorate
➢ Never type with gloves on, remove gloves first, dispose it
and use alcohol 4 may = Use no Deadly Below 73 ‘F
deteriorate water
o Hands are not visibly soiled • **LS: LEVEL OF SEVERITY/REACTION
o After contact with inanimate objects
▪ e.g medical equipment • Reading of chemical bottles example:
o Right: Yellow, 1
▪ Unstable if heated, the particular chemical shall not be heated

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 Lesson 2 : SAFETY IN THE LABORATORY

o Up: RED Q1 • Chemistry, hematology, molecular biology: uses radioisotopes

▪ Flammable if expose to less than 200 F hazards • Pregnant women should be careful to prevent abnormality on babies
o Left: 1 when exposed to radiation
▪ Slightly hazardous
ELECTRICAL HAZARDS
• We cannot use machine if:
o If the are is wet: don’t turn on machine
o Frayed cord. Check cords durability/quality of life

• If electrical shock occurs, never touch the person or the equipment

involved
• Turn off the circuit breaker
• Unplug the equipment
• Move the equipment using a nonconductive glass or wood object

FIRE / EXPLOSIVE HAZARDS

• Volatile explosive, flammable metals: pure sodium shall never be mix
with water because it will explode, potassium, and etc.
• Flammable chemicals should be stored in safety cabinets & explosion
prof refrigerator in a remote area.
▪ Chemical stock room is separated in the laboratory for this
reason.
o Acids and alkaline chemicals shall be separated.

➢ RACE: WHEN A FIRE IS DISCOVERED

➢ PASS: HOW TO OPERATE A FIRE EXTINGUISHER
*CHEMICAL HAZARD IDENTIFICATION SYSTEM
RACE: Follow this when a fire is discovered
• The OSHA rule of January 1990 requires each facility to have a o Rescue
Chemical Hygiene Plan (CHP) that defines the safety policies and o Contain: close doors because the more oxygen enter, the more
procedures for all hazardous chemicals used in the laboratory. fire
o Extinguish if possible, if not evacuated.
• MSDS: Material Safety Data Sheet is now called SDS, Safety Data ▪ PASS: To operate a fire extinguisher
Sheet. o Pull the pin to unlock the extinguisher
o All of the important info regarding the chemical is written.
o Handling, storage, stability, reactivity, antidote or first aid measures
SECTION Type of hazard
1 Identification
2 Hazard Identification
3 Composition/Information on Ingredients
4 First-Aid Measures
5 Firefighting Measures
6 Accidental Release Measures
7 Handling and Storage
8 Exposure Controls/Personal Protection
9 Physical and Chemical Properties
10 Stability and Reactivity
11 Toxicologic Information
12 Ecologic Information (nonmandatory)
13 Disposal Considerations (nonmandatory)
14 Transport Information (nonmandatory)
15 Regulatory Information (nonmandatory)
16 Other information
Table No.2 Types of Fire and Fire Extinguisher
• DEGREE OF HAZARDS: NO SMS EX FIRE Type of Example Type of Extinguisher
o 0 = NO MINIMUM HAZARDS TYPE hazard materials
o 1 = SLIGHT HAZARD A Ordinary Paper, clothing, Water, dry chemical,
o 2 = MODERATE HAZARD combustibles wood loaded steam
o 3 = SERIOUS HAZARD B Flammable Gasoline, paints, Dry chemical, Carbon
o 4 = EXTREME HAZARD liquids oils dioxide, Halon foam
C Electrical Laboratory Dry chemical, Carbon
equipment equipment dioxide, Halon
D Flammable Na, K, Li Metal X, Sand, fought by
metals fire fighters only
E Arsenal fires Forest fire Allowed to burn out
** Rarely encountered in microscopy
K Cooking Cooking oil Liquid designed to
RADIOACTIVE HAZARDS media prevent splashing &
cool the fire
• When procedures using radioisotopes are performed

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 Lesson 2 : SAFETY IN THE LABORATORY

• Check the label, preferred fire extinguisher material. HANDWASHING PROCEDURE

o A: use water • Follow the CDC Guideline
o ABC: Dry chemical used in buildings, may be use for Fire A,B,C.
o BC: Dry chemical, carbon dioxide or Halon foam • WET HANDS WITH WARM WATER.
o Electrical equipment fire = use halon o Before applying soap hands should be wet already.
o Arsenal: fire fighters only e.g forest fire for firefighters • APPLY ANTIMICROBIAL SOAP.
o K = never use water it’ll spread the fire, use foam extinguisher • RUB TO FORM LATHER, CREATE FRICTION, AND LOOSEN
DEBRIS.
o Each finger, up to wrist should be clean.(every corner of hand)
PHYSICAL HAZARDS • THOUROUGHLY CLEAN BETWEEN FINGERS, INCLUDING
• Things to avoid at the lab because there are occupationally THUMBS, UNDER FINGERNAILS AND RINGS, AND UP TO THE
acquired hazards and diseases. WRIST FOR ATLEAST15 secs (by CDC), 20 secs (by Strasinger).
o Eating in the laboratory • RINSE HANDS IN A DOWNWARD POSITION.
o Improper wearing of headcap, mask, not tying hair o Arm down to the fingertips to avoid contamination.
➢ If fingertips to arms, the germs from elbow will go back to
• General precautions: your hands.
o Avoid running in rooms and hallways • DRY WITH PAPER TOWEL.
o Watch for wet floors • TURN OFF FAUCETS WITH A CLEAN PAPER TOWEL TO PREVENT
o Avoid dangling jewelry RECONTAMINATION.
o Bend knees when lifting heavy objects
o Maintain clean, organized work area “Strength does not come from physical capacity. It comes from an
o Keep long hair pulled back indomitable will”
o Wear closed-toe shoes - Mahatma Gandh
Table No.3 Summary of the Safety Hazards
TYPE SOURCE POSSIBLE
INJURY
Biologic Infectious agents Bacterial, Fungal, Viral,
Parasitic infections
Sharps Needles, Lancets, Cuts, Punctures, or
Broken glass blood-borne pathogen
exposure
Chemical Preservatives and Exposure to toxic,
reagents carcinogenic, or caustic
agents
Radioactive Equipment and Radiation exposure
Radioisotopes
Electrical Ungrounded or wet Burns or Shock
equipment; frayed
cords
Fire / Explosive Open flames, organic Burns or
chemicals dismemberment
Physical Wet floors, heavy Falls, Sprains, or
boxes, patients Strains

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 WEEK 2.2: SPECIMEN COLLECTION AND HANDLING

OUTLINE
I Specimen Collection
A Urine Container
B Specimen Labeling
C Specimen Rejection
D Specimen Handling
E Specimen Integrity
i Analytes that increase
ii Analytes that decrease
iii Unaffected analytes
F Specimen Preservation

SPECIMEN COLLECTION
• Procedures in the laboratory must be followed thoroughly because
an error in collection produces an erroneous result.
• Collection of urine for lab analysis may be the first step in
determining diagnosis. Hence, minimize the risk of introducing error. • Storage container
• To obtain accurate result, the integrity of urine specimen must be o Used to collect timed specimen such as 12-24 hr. specimen
maintained. ▪ Note: There is no need for midstream clean catch for 24 hr.
o Ex. Change in urine composition because of improper storage specimen.
produces result that does not reflect the patients’ condition. o Can collect up to 3000ml
• Disposable, wide-mouthed, and flat-bottom containers with screw ▪ Normal volume of urine is 600-2000ml per each day.
caps are recommended ▪ It comes in different sizes such as 1500ml, 2000ml, 3000ml
o For routine urinalysis, it should be disposable and at least clean
container.
▪ Note: Not all the time we need to use sterile container.
➢ Ex. Routine urinalysis may use clean container instead
of sterile container. Unlike the ones used for culture and
sensitivity.
o Wide-mouthed – the mouth should be wider than the base.
o Flat bottom – to avoid spilling
o Screw caps - rather than snap on.
• Urine is considered as biohazardous substance. Hence, gloves
must be worn.
• Clear containers and at least 50 mL capacity
o However, it is filled with less than 50ml urine to have a room for
swirling the sample because urine containers cannot be • To distinguish sterile and non-sterile urine container:
inverted. o Sterile container has plastic bag
o For routine urinalysis, 10-15ml is already enough. But collecting o Non-sterile has no plastic bag
larger volume are encouraged for sufficiency in urine samples o Usually, it can also be found in the label if the container is sterile
when there is a need for retests. or not.
• Adhesive bags for pediatrics (pediatric wee bags) and large plastic
containers for 24-hour specimens SPECIMEN LABELING
o To quantify certain analytes/substances such as creatinine and
• If the requisition form has patient information, but the container has
urea in the sample. Literally, urine should be collected for 24 hrs.
no label, it should be rejected.
• Wear gloves when working with urine o Never do pre-labeling in the lab because it is prone to
mislabeling.
URINE CONTAINER o Usually, containers are labeled after collection. Including the
name, id number (if hospital based), date and time of collection.
• Sterile Disposable container o Sample scenarios:
o This is the preferred use. But it is not required for routine ▪ If a urine was collected at 6 am and was submitted at
urinalysis. 12noon, it should be rejected because the allowable time for
urine is only 2 hours.
▪ If a urine was collected at 7am and was refrigerated, it is
accepted because refrigerated urine can last up to 24 hrs.
▪ If a urine was collected at 6am and was submitted at 12noon
(the urine was preserved in an ice box), it is still accepted.
• Information on label:
o Patient’s name, ID number, date, time
o Additional information: age, location, physician
• PLACE LABEL ON CONTAINER, NOT LID
• Requisition form: Must accompany specimen
o Information must match label
o Time of receipt is stamped on requisition
o Other info: type of specimen, interfering meds
• Pediatric Wee Bag
• The information on requisition form and container must match. If not,
o Hypoallergenic
reject.
o The yellow is the adhesive. This will be adhered in the skin.
o 10-20ml of urine can be collected using pediatric wee bag.

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 Lesson 3: Specimen Collection and Handling

SPECIMEN HANDLING
• Changes in urine composition take place not only in vivo but also in
vitro

SPECIMEN INTEGRITY
• Test within 2 hours of collection
• Refrigerate if testing is delayed.
o Most common preservation method
• Most problems are caused by bacterial multiplication

ANALYTES THAT INCREASE:

• Color (normal color of urine is yellow, pale yellow, light yellow, dark
yellow) if it becomes dark brown, then it becomes aged/
unpreserved urine.
• Turbidity: Due to bacterial multiplication. Increases because of high
pH (alkaline).
• Sterile Container: Name, Date and Time, Gender (not often asked), o If the urine is clear, it does not guarantee that the patient is
date of birth. healthy.
• 24-hour urine container has a sticker that contains the starting date ▪ Ex. Diabetes mellitus, diabetes insipidus, they have polyuria
and time, also the end date and time. hence, it is already given that the urine produced have no
color
• pH: Increases because of ammonia. Main component of urine is
Urea. The urea converted to ammonia by urease producing bacteria.
o Old specimen produces ammonia like odor/ammoniacal
(mapanghi)

ANALYTES THAT DECREASE:

• Glucose: Because bacteria utilize glucose, hence it decreases.
• Ketones: Such as acetone. Ketones are increased if the fat
metabolism is fast. Since they are highly volatile, they can decrease
easily.
• Bilirubin: Sensitive to light/ artificial light. Hence, can be oxidized
easily.
• Urobilinogen: When oxidized, it becomes urobilin (Urobilin
provides dark/brownish color to the urine)
• Laboratory requisition form: Check the test that must be done. • Urobilinogen decreases, urobilin increases
• Doctor’s note can also be found here. • Cells: Decrease because in unpreserved urine, the pH is high
o Such as interfering medication and pre-diagnosis (alkaline). Thus, disintegrating the cells.
▪ Pre-diagnosis enables the MedTech to see the bigger
picture of the test result. UNAFFECTED ANALYTES
▪ Ex. The patient has diabetes mellitus according to the pre-
diagnosis. Hence, it is reasonable for her test result to have • Protein is unaffected in unpreserved urine sample. It is also not
4+ in reagent strip. normal to see protein in urine. Because this might indicate the
presence of proteinuria.
• Anything that leads to mislabeling must be rejected.
o Ex. If the patient’s name is Anna Marie Perez, but the label only • No significant amount of protein must be seen in a urine. Because
has Anna Perez, theoretically reject but practically accept. this indicates kidney problems.
• Interfering medication:
o Ex. If the patient intake ascorbic acid, the result may be affected. SPECIMEN PRESERVATION
Because ascorbic acid is a reducing agent, meanwhile, blood’s • Most routinely used method of preservation:
principle is oxidation. Hence, if the patient intake ascorbic acid, o Refrigeration (2⁰C to 8⁰C)
it will produce false negative result. o For transport: ice box, temperature must be 2⁰C to 10⁰C
• Note: Duplication and triplication of result will only be done if the ▪ High temperature stops bacteria metabolism.
initial result is abnormal. ▪ Refrigerators are bacteriostatic, meaning, they impede the
growth of bacteria.
SPECIMEN REJECTION ▪ Question: when the sample is placed inside the refrigerator,
will the bacterial growth completely stop?
• Unlabeled containers
▪ Answer: No. Metabolism is only minimized but does not
• Non-matching labels and requisitions
completely stop bacteria multiplication.
• Contaminated specimens o Decreases bacterial growth and metabolism
o feces, paper
• Contaminated containers • Urine preservatives: Theres no single urine preservative that is
o Containers with any contaminants must be rejected considered the best.
• Insufficient quantity • You must choose the preservative depending on what you want to
o Example scenario: 2-month-old baby only produced 5ml urine. test.
Will you accept it or reject? Note: 10-15 ml of urine is the o Ex. For glucose only, use thymol.
requirement.
▪ Yes. Because you cannot force the baby to produce more Table No.1 Urine Preservatives
than 5ml urine. Preservatives Advantages Disadvantages Additional
▪ There are some situations when you can accept even if Information
insufficient. This must be noted on the result (ex. Insufficient Refrigeration Does not Raises specific Prevents bacterial
urine volume submitted). interfere with gravity by growth 24 h3
• Delayed or improper transport chemical tests hydrometer
o Ice, refrigeration
Precipitates
▪ Ex. Not placed in an ice box
amorphous

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 Lesson 3: Specimen Collection and Handling

phosphates and abnormal cells seen in

urates Use to preserve the urine)
cells only
Thymol Preserves Interferes with
glucose and acid precipitation Formalin: All
sediments well test for protein form elements
Boric Acid Preserves May precipitate Keeps pH at about
proteins and crystals when 6.0
formed elements used in large
• Routine urinalysis: there’s rare chemical preservatives in US the
well amounts urine collected kit or the Urine container like ETS: it has needle heel
you just must insert the urine tube and there will be vacuum that will
Does not Is bacteriostatic suck the urine to prevent contamination. This is available in some
interfere with (not bactericidal) at part of Philippines however in Philippines it is not a practice to put
routine analyses 18 g/L; can use for chemical preservatives just refrigerate the urine. It should also be
other than pH culture transport* tested right away.
Interfere with drug
and hormone
analyses Urine Sets
Formalin Excellent Acts as a Rinse specimen
(formalde- sediment reducing agent, container with
hyde) preservative interfering with formalin to
chemical tests for preserve cells and
If you want to glucose, blood, casts
preserve formed leukocyte
elements, use
esterase, and
formalin.
copper reduction
Toluene Does not Floats on surface
interfere with of specimens and
routine tests clings to pipettes
and testing
materials
Sodium Prevents Inhibits reagent May use sodium
fluoride glycolysis strip tests for benzoate instead of Table No.2 Type of Urine Specimen
glucose, blood, fluoride for reagent Random/Occasional/Single • For routine and qualitative Urine Analysis
Remember: and leukocytes strip testing5 • Most common urine specimen submitted in the
Sodium fluoride Is a good
is not an anti- laboratory. Collect any time of the day and submit
preservative for it to the laboratory
coagulant. It is a
preservative. It drug analyses • Routine: as much as possible in a midstream-
inhibits enolase. clean catch to prevent chemical changes brought
by bacteria
Phenol Does not Causes an odor Use 1 drop per • It is a screening test for qualitative, not used for
interfere with change ounce of specimen quantitative like diabetes monitoring, detecting
specific analyst.
routine tests
First Morning Urine • Ideal specimen for routine UA and
Commercial Convenient May contain one Check tablet
Pregnancy Test (hCG)
preservative when or more of the composition to
tablets refrigeration not preservatives determine possible • Most Concentrated/Most Acidic
possible including sodium effects on desired • For evaluation of orthostatic proteinuria
Sometimes o Orthostatic proteinuria: Not clinically
Have controlled fluoride tests significant, urine has protein because of
mixture of concentration to
chemicals in your posture only.
minimize ▪ E.g nakatayo ka maghapon, tataas
tablet form for
the same interference yung blood pressure mo going to the
purpose, used to kidney. Some protein is filtered even
preserve certain though it shall not be filtered due to
substances in this.
urine. ➢ Random collection: Orthostatic
and True Proteinuria is both (+)
➢ First Morning: Only the TP is (+)
Urine Contains since nakatulog na, the posture
Collection collection cup, is changed.
Kits6 (Becton C&S
Dickinson, preservative tube o True proteinuria: Clinically significant, urine
Rutherford, or UA tube has protein, and it is abnormal. (+)
NJ)
Give a picture of your metabolism for the previous day.
Gray C&S Sample stable at Decreases pH; do Preservative is
tube room not use if urine is boric acid and may
Second Morning/Fasting • 2nd voided urine after fasting
temperature (RT) below minimum not be used for UA
Used for longer for 48 hr; fill line • For glucose determination
preservation • Metabolism that happened the night before
preserves collecting the sample.
(>24 hrs) of
bacteria • Fasting blood sugar and fasting urine samples.
urine
Yellow plain Use on Must refrigerate Round or conical 2 Hour-Postprandial • For diabetic screening and monitoring
UA tube automated within 2 hours bottom • Is the cell and insulin capable to metabolize
instruments glucose. Does it increase or decrease glucose?
Cherry Stable for 72 Bilirubin and Preservative is Glucose Tolerance • *Optional
red/yellow hours at RT; urobilinogen may sodium propionate; • Together with blood samples in glucose
top tube instrument- be decreased if conical bottom tolerance test
compatible specimen is • Like OGTT (Oral Glucose Tolerance Test)
Used for longer exposed to light • RARELY IN URINE: Check blood glucose level
preservation and left at RT of fasting blood sugar after 1 hr, 2 hrs, after
(>24 hrs) of collecting blood collect urine after 1 hr and 2 hrs
urine
to check the urine glucose level.
Saccomanno Preserves Used for cytology
Fixative cellular elements studies Fractional Specimen • At least 2 voided collections
• Series of blood and urine sample are
Very useful in (Cells only: RBC, collected at specific time intervals to
histopath Epithelial cells, compare the concentration of the sample.
abnormal cells, • To compare if there will be changes, you have to
Made up of bladder cells, cancer collect two different time. At least 2 voided
carbowax and cells, renal tubular collections then compare.
epithelial cells are
methanol Midstream clean catch • For routine UA and Bacterial culture

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 Lesson 3: Specimen Collection and Handling

• Non-invasive procedure, not sterile since it will

flow in the urethra. If you urinate the first portion
is discarded, in the middle you collect the urine.
o There’s still organism but not that much.
• Not applicable with kidney problems/failures
Catheterized urine • For bacterial culture
• Also, a sterile collection of urine.
• Used for patients with kidney stones, it requires a
catheter inserted in the urinary bladder.
o 2 types of catheters
▪ Urethral
➢ Passes from urethra to urinary
bladder.
▪ Ureteral
• Passes from ureter, much painful.
Suprapubic aspiration • Bladder urine for anaerobic bacterial
culture and urine cytology
• The most sterile collection because you correct
directly in the urinary bladder
o Urine is non-sterile if it flows at the urethra
because in urethra, we have normal flora
that contaminates the urine.
o It is normal to see bacteria in urine; not in
large amounts, that is a sign of UTI.
• Bacterial culture: Midstream, Catheterized, and
Suprapubic are all use for bacterial culture
Pediatric specimen • Using soft, clear plastic bag with adhesive
• Sterile specimen may be obtained by
catheterization or suprapubic aspiration
Three-glass technique • For prostatic infection
o First portion of voided urine
Used for men. To check if the o Middle portion of voided urine
patient has prostatitis. There’s pre- o Urine after prostatic massage
massage, midstream clean catch,
and post massage.
Massage: literal massage of Methods: Physician will insert fingers in the anus to
prostate glands for prostatic palpate the prostate gland.
secretion. This was done to check if This procedure must only be done by the doctor .
the prostate glands have bacteria or
pus.

Timed- Specimen • 24 Hour → Requires preservative

o Preservative used is formalin
Used for quantitative analysis. o For quantitation of creatinine and urea
• 12 Hour → For Addis count
o Counting of the cells and formed elements.
o Formalin is also used here.
• 4-Hour → For Nitrite determination
o 4 hrs. are the time requirement time for
nitrate to become nitrite.
o There are nitrate producing bacteria
wherein they reduced nitrate to nitrite.
o No need to use preservative.
• Afternoon → For Urobilinogen
determination
o Specific time: 2 -4 pm of urobilinogen.
o Urobilinogen is sensitive to acid.
o You cannot collect urobilinogen from first
morning sample.
o It must be collected during the afternoon
where the patient has alkaline tide.
▪ Alkaline tide – Increased of hydrogen
ions in the stomach after the
hydrochloric acid break down food in
the stomach.
o Hence, after eating, blood pH increases (it
becomes alkaline)
o Hence, you can only collect urobilinogen
during the afternoon.
Drug Specimen Collection • Chain of Custody (COC) →
Documentation of proper sample
Under Clinical Chemistry. identification from the time of collection to
the receipt of laboratory results.
If you want to preserve the urine, o Example of chain of custody:
you may add sodium flouride ▪ Witness collection: Specimen
collection from drug suspects. The
suspect will be assisted by the
MedTech to observe if the urine
specimen is collected properly.
• 30-45 mL/ 32.50C-37.70C
o Higher in volume for repeat testing and for
confirmation.
o Ex. In screening, the specimen was tested
positive. The sample will not be recollected,
instead, the sample will be sent to reference
laboratory (East Avenue Medical Center).
for confirmation.
• Requires blueing agent (dye)

Page | 4 | CAPORADO. MOLINA |

 WEEK 3: PHYSICAL AND CHEMICAL EXAMINATION OF URINE

OUTLINE Table No.1 Urine Clarity

I Physical Examination Clarity Term
A Volume Clear No visible particulates, transparent
B Color
Hazy Few particulates, print easily seen
i. urochrome
through urine
C Clarity
Cloudy Many particulates, print blurred
i. Turbidity
through urine
ii. Grading
Turbid Print cannot be seen through urine
D Odor
i. Associated disease Milky May precipitate or be clotted
E Specific Gravity
i. Urinometer o Clear: Nababasa yung nakasulat sa print
ii. Refractometer o Hazy: visible print, readable but with few partciles
iii. Corrections o Cloudy: Blur text, unreadable text
o Turbid: totally unreadable text, shadow is still there
II Chemical Examination
A Reagent strip test
o Milky: Lipiduria
i. 10 Parameters ▪ Presence of lipids in the urine. Specially if you problem in
ii. 4 Essential Parameters glomerulus, you will not be able to filter substances
iii. How to Use Reagent Strip anymore. So lipids accumulate in urine and the urine clots.
➢ Serum clots, with white/cloud like urine.

PHYSICAL EXAMINATION ODOR

• Normal: aromatic, fragrance smell
VOLUME • Ammoniacal: bacteria (UTI), or unpreserved
• ½ to ¾, wag pupunuin! • Mousy: hemophilia
• 50 ml: the capacity of the urine container • Maple syrup: Ketone
• Grape fruit: Ketone
COLOR
• Normal: yellow, pale yellow, dark-yellow, light yellow because of SPECIFIC GRAVITY
urochrome.

o Our body normally undergoes metabolism process. The higher 1. URINOMETER:

metabolism that you have the higher the pigment, the
o Check the number of solute concentration in the urine,
urochrome gives the yellow color.
o Direct determination for urine specific gravity.
▪ Fever: dark yellow, or yellow
o Determine the number of solute present in the urine.
▪ Starvation: dark yellow, or yellow
• How to use?
HOW TO KNOW THE COLOR? o 1. Transfer in cylinder and drop your urinometer let it touch the
o Transfer in test tube for at least ½ or ¾ of tubes, using a white tip, urine and spin it. Lulutang yan.
background check the color with spatial light. o 2. Check where the lower meniscus is. Start counting from the
bottom since di makikita dahil may meniscus. (Paatras)
o Urobilin: If unpreserved, aged, magiging dark. o

CLARITY • How to know the S.G?

• Using the same test tube check the clarity/turbidity. Check it using ▪ Check the grading (graduation) e.g 1.000 – 1.010
printed background. ➢ Sampung lines by 10
➢ Limang lines: by 2’s
• Unpreserved urine: o Start from the top, 1.000 to the bottom.
o Turbidity: increase, there’s bacterial growth
o Clarity: decrease, becomes turbid o Water S.G: 1.000

• Grading: o More solute in the urine: Urinometer floats

o Less solute in the urine: Urinometers sinks.

Page | 1 | MOLINA, CAPORADO |

 Lesson 3: Physical Examination, Chemical Examination of Urine

2. REFRACTOMETER ➢ It is still within 3 C. The base is (20, so 27 – 20 = 7 C).

Basically hindi pa lumalagpas sa 3, part pa rin siya ng
three). Kaya add 0.002 pa rin to.

REFRACTOMETER
• Refractometer needs 1 correction which is the solute concentration
ONLY.

RULES APPLICABLE FOR REFRACTOMETER

• Solute concentration
o In every 1 [Link] of Protein: subtract 0.003
o In every 1 g/dl of Glucose: subtract 0.004

• Principle: Refractive index, indirect method of determining specific PH

gravity • Use litmus paper, dip it in the urine sample.
• We are measuring the refractive index instead of the solute
concentration. (Kung saan nagkaroon ng boundary lines)
• Eyepiece: where you check the graduation
• Prism: The flat surface

• How to use?
o 1. Place 2 drops of urine samples here on the prism. Make sure
that it is cleaned before using. Wipe it.
o 2. Slowly close the daylight plate, and you’ll see that the urine
will spread all over the prism (No bubbles dapat)
o 3. Check at the eyepiece for graduation. Check for boundary
line, blue and white light intersection for specific gravity.
▪ If empty: totally blue color
▪ Estimate or approx. graduation.
o 4. Check at the UG, urine gravity at the right. CHEMICAL EXAMINATION

URINOMETER, REFRACTOMETER CORRECTION

URINOMETER CORRECTION
• Urinometer needs 2 correction which is the solute concentration and
the temperature calibration.

RULES APPLICABLE FOR URINOMETER

• Solute concentration
o In every 1 [Link] of Protein: subtract 0.003
o In every 1 g/dl of Glucose: subtract 0.004
REAGENT STRIP TEST
• Temperature: CALIBRATING TEMP: 20 C (CONSTANT)
• Not laborious like the biochemical testing done in biochemistry
o In every 3 C increase the CALIBRATING TEMP: add
• 2 types of reagent strip: Chemstrip by Rhoss Diagnostics Corp,
0.001
Multistix by Siemens
o In every 3 C decrease the CALIBRATING TEMP:
subtract 0.001 • It has 10 and 4 parameters

EXAMPLE 10 PARAMETERS:
• mas complete
• Specific Gravity/S.G: 1.040
• Glucose: 2 g/dl
4 ESSENTIAL PARAMETERS:
• Temperature: 26 C
• Glucose, Specific Gravity, pH, Protein (the most common but still
• Solution depends on the Manufacturer).
• Nowadays there’s the 11th parameter, which is the ascorbic acid it
(Solute causes false negative because it is an reducing agent.
Concentration) o If you positive in ascorbic acid, some parameters will be
negative.

(Calibrating T.) Table No.2 10 Parameters

10 parameters Time Principle Results

Glucose 30 secs Double Sequential + or -

➢ In this example since we have 2 g/dl of glucose we Enzymatic Reaction
subtracted 0.008 following the rule provided. (2 g/dl Bilirubin 40 secs Diazo Reaction Normal
glucose x 0.004 ). Next, the temperature is 26 C, so we
added 0.002 (0.002 increase since 3 C = 0.001). Now Ketone 45 secs Sodium Nitroprusside + or -
we have the corrected specific gravity using Reaction
refractometer which is 1.034. Specific 60 secs pKa change of Numerical
Gravity polyelectrolytes value
pH 60 secs Double indicator system Numerical
▪ What if the temperature is 27 C? value
Protein 60 secs Sorenseri’s Error of Indicator + or -

Page | 2 | MOLINA, CAPORADO |

 Lesson 3: Physical Examination, Chemical Examination of Urine

Blood 60 secs Pseudoperoxidase Activity of + or -

Hemoglobin
Urobilinogen 60 secs Ehrlich’s Reaction + or -

Nitrite 60 sec Greiss Reaction + or -

Leukocyte 120 secs Leukocyte Esterase + or -

HOW TO USE REAGENT STRIP?

1. Transfer the urine in the test tube at least ¾ so that the entire
pads will submerge in the sample.
2. Each pad is impregnated with chemicals. (Kung anong pad
ginamit na chemicals sa biochemical test same lang. Mas
konti lang yung urine sample na need and mas marami
maccheck na paramets)
3. Handle the pads correctly sa baba
4. Do not touch your pads (magkakaroon ng reactions
5. Touch it with urine with only 1 sec to prevent glitching off.
6. Remove the strips in the urine, blot the both side, back part of
the reagent strip in the tissue paper to remove the excess
urine.
▪ Urine can run off in the pads and cause erroneous results.
7. Use the same pads and bottle in comparing. Make sure that you
check it horizontal and not touching the bottle.
8. Grading is already written in the bottle. Except pH and S.G it is
written in numbers.
▪ Note: Urobilinogen will never be negative: It is only normal
or positive.
GRADING READING
1+ or + 1
2+ or ++ 2
+++ 3
+/- Trace (between the negative
and 1+)

Page | 3 | MOLINA, CAPORADO |

 LESSON 7: MICROSCOPIC EXAMINATION OF URINE

▪ RCF: Relative Centrifugal Force

OUTLINE o Centrifuge for 5 minutes only. Do not over centrifuge sample since
I Pre-analytical Phase it may disrupt or destroy sample.
II Analytical Phase
A. Materials
o Strictly follow the standard: 5 minutes at 400 RCF or 1500 rpm
B. Slides & coverslip • Pour off the supernatant liquid. Sufficient urine remains in the tube to
C. Microscope suspend the sediment.
D. Procedure o Sediments that settle on the bottom will be our sample for testing.
III Role of Microscopic Examination of Urine o Excess urine: discard it leaving only sediments at the bottom of
IV What are the Sources of Errors in Handling Urine? the tube.
V. Preparation and Examination of Urine Sediments
o How: Decant the tube by fully inverting it.(discard the urine)
A. Specimen preparation
B. Specimen volume o Leave only a small amount: 0.5-1 ml urine
C. Centrifugation ▪ To resuspend the sediments so we can place it in the glass
D. Sediment preparation slide.
E. Volume of sediment examined • Shake the tube and transfer a drop on the slide. The drop should not
F. Examination of the sediment overrun the cover slip and there should be no air bubbles since both
G. Reporting of results tend to alter the results.
H. Correlation of results
o After that, mix or agitate the tube or sample.
o Transfer sample in the glass slide: aspirate using disposable
pipette or pasteur pipetter
• Microscopic Examination o Using Pasteur pipette: 1 drop
o Also called as sediment examination o Using Micropipette: 20 ul
o 3rd part of Routine Urinalysis o Place the coverslip. Make sure it is not overfilled or has excess.
o Main purpose: detect and identify insoluble materials present in o 1drop is enough already. Don’t overfill/ excess the urine drop:
urine ▪ Because there are some large formed elements that can be
▪ Ex: Cells casts, crystals lost when there is excess urine na lumabas sa cover slip.
o Serves as reflection of body’s state of metabolism ▪ Dapat sakto lang sa coverlip ang urine drop
• There is a developed protocol which is to increase the ▪ Ex: casts- nakikita sa edges or side ng coverslip kaya if
standardization of microscopic examination because: overfilled ang glass slide ng urine, these large elements will
o Very subjective part of urinalysis be lost. Lalabas sila sa coverslip.
o Depends on skill of medtech on how to identify certain elements • Examine under the microscope using LPO first the HPO.
o Aside form identification, you have to quantitate also insoluble o After placing coverslip, examine under microscope.
materials present in the urine o Examine:
▪ 10 LPF: Because some elements are too large, they can
PRE-ANALYTICAL PHASE occupy the entire HPO/HPF. Examine large elements in LPF.
• The site of the experiment should be sterilized. The medical ▪ 10 HPF
technologist should wear proper personal protective equipment before
the experiment. ROLE OF MICROSCOPIC EXAMINATION OF URINE
• Upon receiving the urine specimen in the laboratory, the receptionist • Discussed in lecture.
checks if the urine container is properly labeled then transfer it into a • Determine the correctness of diagnosis of a renal system infection and
test tube for centrifugation disease
o Basically, the role of microscopic examination is to confirm our
ANALYTICAL PHASE results form physical and chemical examination.
• Materials: o Kung ano nakikita sa previous test dapat ma confirm yun.
o Urine (freshly voided or random specimen, preferably first morning • Refrigerated urine preserve almost all cellular elements, crystals,
specimen) and cast, very well
▪ Any urine specimen: random is okay o If there is any delay in testing: preserve the specimen
▪ Preferred: first morning urine is better since it is most o Most common preservative: Refrigeration
concentrated. ➢ We can preserve most formed elements in the
➢ Want to see more elements, use 1st morning urine refrigerator.
o Slides and cover slips ➢ Disadvantage: promote precipitation of amorphous
▪ Frosted or non-frosted is okay materials.
▪ Preferred: Frosted (for label) ➢ Ex: Amorphous phosphate & Amorphous urates - interfere
o Microscope with microscopic examination of crystals
▪ Brightfield microscope is routinely used ➢ Solution for this : Pre-heat the sample at 37°C before
testing to dissolve amorphous crystals.
PROCEDURE • Fresh and concentrated urine: more accurate results
• Collect first the specimen. At least 10-15 ml urine specimen for routine o More accurate result: use fresh or 1st morning urine- more
microscopy/ urinalysis. concentrated urine
o Standard amount urine needed: 10-15 ml. • Hypotonic urine causes lysis of cells and casts
• Thoroughly mix your urine specimen in urine container. o Concentration of urine has effect as well.
• Pour 10 mL of the urine into a centrifuge tube or any test tube used ▪ Hypotonic: low number of solute/concentration in urine.
for that matter. ➢ If konti lang ang solute present in urine, the water will go
o Transfer directly to tube: either glass of disposable tube inside the RBC since mas higher ang concentration ng
o Disposable tube: conical tube is more preferred since formed solute sa RBC-> RBC will swell since water came in the
elements settle best in conical tube cell -> after swelling, it will lyse
o Glass tube: round end tube, still okay to use if it’s the only option. ➢ Hypotonic urine causes cell lysis.
o Transfer 10 ml. Mix it first by swirling since there are formed ▪ Hypertonic: high number of solute/concentration in urine
elements that settle at the bottom of container. ➢ Tendency is the water inside the RBC cell will go out to
• Centrifuge for 5 minutes at 1500 rpm. the urine since urine has higher concentration of solute
o Required: 1500 rpm or 400 RCF (double check centrifuge if in -> RBC cell will shrink.
RPM or RCF) ➢ Hypertonic urine causes shrinkage of cell.
▪ RPM: Revolutions/ Rotation per minute

Page 1 of 9 | CAPORADO, DELA CRUZ, MOLINA |

 Lesson #7: Microscopic Examination of Urine

• Proper collection avoid extra debris of the urethral meatus, vaginal • Blood (-)
secretions ➢ Microscopic examination
o Must be considered since it can cause misidentification and may • Bacteria and RBC is seen.
contaminate the sample. Especially if you’re not able to cleanse ▪ What can be the cause of these discrepancy: Ascorbic Acid
opening of meatus. ▪ Indicated sa form na nag overdose ng Ascorbic Acid si patient.
▪ It can cause confusion sometimes kasi pwede makakita ng ▪ Most of the time ascorbic acid can cause false negative result.
maraming bacteria (mistaken as UTI) but actually
contamination lang siya. ▪ After a week bumalik siya, and pagka-test normal lahat ng
• Proper use of subdued light in LPO before HPO: for accurate results result except for RBC. What is the cause of that?
o Why use decreased light/ subdued light: because there are ▪ Discrepancy: If may RBC sa urine dapat pati ibang parameter
some formed elements that has low refractive index (parang ay affected like protein. But normal si protein.
transparent). ▪ Cause: Menstrual contamination
o If increased ang light some elements with low refractive index will o That is how we correlate physical, chemical, and microscopic
not be seen by microscopist/ medtech. result.
o Make sure there is enough light but not too much light.
• Examine the urine specimen within 1 to 2 hours SPECIMEN PREPARATION
o If there is delay: refrigerate sample • Specimen should be examined while fresh/adequately preserved.
o If refrigeration is not available: use chemical preservatives like o For the specimen we receive in the laboratory, we need to check
Formalin. it properly.
o Formalin: especially used for cytology, preserves elements very o Check the label
well. o Check if the submitted sample is an old specimen
o Check if amount is sufficient for testing.
• Refrigeration may cause precipitation of amorphous urates and
WHAT ARE THE SOURCES OF ERRORS IN HANDLING phosphates.
URINE? o If sample is refrigerated, expect turbidity in increased due to
• Careless transfer of sediment (contamination) precipitation of amorphous urates & amorphous phosphates.
o Contamination: Not just because of collection but also equipment o Easy to remove this crystals: Pre-warm the sample for 37°C
we are using. As much as possible use disposable materials. before testing. Crystals will be dissolved.
o Ex: Pasteur pipette is better to use than glass pipette to avoid o Make sure specimen is fresh. Old specimen has high pH
contamination (alkaline). Alkaline sample has effect to cells- cells (RBC. WBC,
• Too much light (retractile bodies can not be seen) Casts) will disintegrate rapidly in alkaline urine
o Very low refractive index are not able to be seen with too much • Mix the specimen prior to decanting a portion into a centrifuge tube.
light. Decrease the brightness o Don’t forget to mix or swirl the container prior to transferring it in a
• Using the high power only tube since there are formed elements that settle at the bottom.
o Squamous Epithelial cell occupies entire field in HPO. This is a o Mix the specimen: It is done para maging accurate ang result and
problem with large elements, they can occupy the entire field, good representative ang sample.
especially in HPO.
▪ They have to be checked in LPO. SPECIMEN VOLUME
o Before going to HPO, scan first the specimen in LPO to look for • Standard amount of urine is about 10-15mL. (Average: 12 ml)
larger formed elements/ cells. • 12mL volume is frequently used.
• Specimen dries up only on long standing (false elements are seen) • If obtaining 12mL is not possible, volume of the specimen used should
o If you left the specimen in the glass slide for a long time, it can be noted.
promote/ cause precipitation of some crystals. Tendency it can o If less than 12ml: do we accept or reject?
mislead the result lalo pag nag dry. o Depending on the situation: if pediatric or geriatric patient or
o Dried specimen: nag mumukhang crystal (interfere with crystal depending on the case of patient (patient history)- we can still
identification) accept it.
• Dirty equipment o It depends only on the condition and who is the patient
o Routinely check if clean: Test tubes & centrifuge we are using ▪ If sample is insufficient (Ex: 6ml only): take note of that so
• Scratches on slides that physician can make some adjustments with the results.
o Madalas magkamali ang mga students. Mistaken as crystals. ▪ Pag konti masyado ang volume, physicians make
o What is seen are scratches in slide- it means mali ang focus. – adjustments.
Adjust further the fine adjustment knob until Squamous Epithelial
Cell is seen. CENTRIFUGATION
o Squamous Epithelial Cell: palatandaan naka focus na tayo, pag • Set correctly the centrifuge.
focused na si Squamous Epithelial Cell, tama ang focus ng • Speed and length of time should be consistent
microscope. • Centrifugation of specimen for 5 mins at 400 (RCF) or 1,500 rpm
o Pag puro scratches ang nakikita, further adjust the knob • RCF rather than RPM is used.
o Why we are using RCF rather than RCF: to correct differences in
PREPARATION AND EXAMINATION OF URINE SEDIMENTS diameter ng centrifuge head.
• Here is the step by step procedure of Microscopic examination: o Centrifuge head has different sizes. To correct differences- we
• Specimen preparation use Relative Centrifugal Force than Revolutions per Minute.
• Specimen volume o At the same time we have to makes sure that is the maximum
• Centrifugation speed.
• Sediment preparation o We can’t centrifuge it at a higher speed and longer time since
• Volume of sediment examined it disrupts the sediments/ destroy formed elements.
• Examination of the sediment
• Reporting of results • Formula :
• Correlation of results 𝑅𝐶𝐹 = 1.118 𝑋 10−5 × 𝑟𝑎𝑑𝑖𝑢𝑠 𝑖𝑛 𝑐𝑒𝑛𝑡𝑖𝑚𝑒𝑡𝑒𝑟𝑠 × 𝑅𝑃𝑀2
o Very important to correlate result especially the physical and • Use of braking mechanism can cause disruption of the sediment.
chemical examination. • All specimen must be centrifuged in capped tubes
o Why: Because the results from physical and chemical can be o Preferably during centrifugation, tubes are capped- urine is a
confirmed with microscopic examination. biohazardous specimen and produces aerosol.
▪ Also we are able to know if there is discrepancies in test result: o To prevent biohazardous aerosols all specimen are centrifuged in
o Example: capped tubes.
▪ In our exam, Patient x, if we check during her 1st visit in the o Capped tubes is not always followed in practice. If we don’t have
hospital, there is problem with urinalysis result: capped tubes, we can use parafilm as a seal and prevent aerosol
➢ Chemical examination droplets.
• Nitrite (-)

Page 2 of 9 | CAPORADO, DELA CRUZ, MOLINA |

 Lesson #7: Microscopic Examination of Urine

SEDIMENT PREPARATION CELLS

• After centrifugation, sediments settle at the bottom.
• Decant it to discard urine. Table No.2 Cells
Figure Characteristics
• Volume of 0.5-1mL are frequently used. RBC: most commonly
o Leave only 0.5-1 ml of the sample. encountered.
o Usually when we decant, may maiiwan na urine na mag-adhere
sa side or wall ng test tube. This is enough already. The RBC in urine shall be the
• To maintain a uniform sediment concentration factor, urine should be same shape in the blood.
aspirated off rather than poured off
• Sediment must be thoroughly resuspended by gentle agitation. Characteristics; Biconcave, non-
o After decanting, mix it. nucleated, very smooth
o There are formed elements that tend to settle at the bottom, the
deepest part of tube, while the smaller elements ay nasa ibabaw. Sometimes the characteristics of
To have equal distribution of elements, mix it. the urine affects the shape of the
o If not mixed we don’t have the representation of the sample. Mix blood cells.
it to have equal distribution of formed elements.
• Vigorous agitation should be avoided, as it may disrupt some cellular Shrinking: microcytic RBC due to
elements. hypertonic urine.
o Avoid vigorous agitation para hindi masira ung formed elements.
o Kailangan very gentle ang agitation: Tap the tip of the tube with
finger, it is enough to mix the sample. Ghost cell: RBC swelling due to
Hypotonic urine. Looks like a
VOLUME OF SEDIMENT EXAMINED shadow na lang, without Hgb part
• Glass slide we are using is the tempered size of glass slide. the intracellular component of
RBC is gone because it is already
• We can use the frosted or ordinary glass slide. What is available can
lysed.
be used.
• Conventional glass-slide method: 20µL (0.02ml)
• Gass cover slip: 22x22mm
o Only add 1 drop of sample, then carefully place the coverslip to
avoid any air bubbles.
o Kailangan walang air bubbles: it is a source of misidentification or
error.
• Overfilled : loss of heavier elements (casts)
o Avoid overfilling also to not lose the large elements like casts.
o Casts are seen at the edge of coverslip.
Comparing to other cells, RBC is
WBC
smaller

RBC: 0.2 – 0.3 per Hpf.

Abnormal: >0.2 – 0.3 per Hpf (1
Epithelial RBC Epithelial or two RBC is normal)

Transitional epithelial cell Larger

than WBC/RBC and Nucleus is
centrally located
EXAMINATION OF THE SEDIMENTS
RTE: nucleus is eccentric
• Examined both LPF and HPF minimum of 10 fields. >2 RTE per HPF: Tubular injury.
• LPF : Cast, squamous epithelial cells, mucus thread, abnormal It is the lining component of
crystals Renal tubules.
o Use to check large elements
o Small amount of this abnormal crystal in LPO is already clinically WBC
significant since they are not normally seen in the urine. If seen in
examination it must be reported in LPO. Glittering cell: Sometimes they
have glittering characteristics
• HPF : WBC , RBC , yeast cells, normal crystals, other types of due to swelling. Granules
epithelial cells (transitional, RTE) glitter/very shiny (clinical
o Transitional and RTE are not normally seen in urine, if RTE is significant)
encountered, the abnormal cells must also be reported in HPO.
o Other formed elements
WBC clump
Table No.1 Reporting
LPO HPO Neutrophils: The most
Cast average/lpf Rbc average/hpf commonly encountered WBC
Epithelial cells, crystal and other Wbc average/hpf because of infection, irritation.
elements are frequently reported
in semi-quantitative terms Eosinophil: Other WBC
encounter because of allergy.

• Some form elements must be reported in average number. Nephritis diagnosis uses
• Some are reported in estimates. presence of WBC and bacteria.
o Rare, few, moderate, many If both are present, UTI yon.
o Too numerous to count (tntc)
If only WBC is present, and no
• Reporting is in semiquantitative terms bacteria is present it is an allergy
possible drug induced nephritis.

Page 3 of 9 | CAPORADO, DELA CRUZ, MOLINA |

 Lesson #7: Microscopic Examination of Urine

Inflammation of nephron RTE at phase contrast

because of certain medication microscope: It has singe lobe.

WBC has many lobe.

Eosinophil: granules are stained You may also use stain or dye to
using Hansel stain, check for the color and the
nuclear details

RTE variants

Oval fat body: RTE with fat

globules.
NEUTROPHILS: Nuclear details
is enhanced by adding acetic How do we know what type of
acid to remove the RBC. fat/cholesterol is this?
Triglyceride: Neutral fats, stained
with lipid stain Sudan four or oil-
red O (normal stain don’t work
with fats)
Cholesterol: never ma’stained
with lipid stain
Glitter cells: shiny yung
magkakadikit
Oval fat body: RTE with fat
globules.
Squamous epithelial cell: itlog-
like mistaken as dirt or RBC. The
cytoplasm is thin, large with folds.
It lines the urethra, vaginal lining
of female and lower urethra lining
of male. It is normally seen in
urine specially in the female.

In higher number, this will be 2 types of lipids:

clinically significant. 1. Triglyceride
2. Cholesterol
WBC Squamous epithelial cells
(SEC): very large Differentiation of lipids:
RBC 1. Staining: only works for
RTE: the nucleus is eccentric. Triglyceride ( Sudan four or oil-
TEC Has different shape red O)
2. Depolarizing microscope: only
Transitional epithelial cells works for Cholesterol (Maltese
SEC RTE
(TEC): Different shape. formation)
In lower light: mas kita ang formed
elements Calcium oxalate: at the upper
right Depolarizing microscope:

Cholesterol crystals: Under

Columnar RTE: RTE originated polarizing microscope, it
from PCT: Very large nucleus, formed the Maltese cross in
columnar pahaba the urine.

CAST
• Actually made up of protein, formed in the renal tubules and has the
same size of renal tubules,
Oval RTE: originated from DCT:
Eccentric nucleus, oval shape • Consist of Tamm Horsfall Protein (THP)
o produced by Renal Tubular epithelial cells. In the renal tubules if
there is any inflammation, infection, RTE will be triggered to
Hawig ng WBC so use phase produce this type of protein.
contrast microscope. o Also called as uromodulin.

• Sometimes we have different type of casts: columnar or curve

(naformed sa curve), cylindroid (with a tail).
Cuboidal RTE: originated from
• Pathological
collecting ducts
o Clinically significant
o Presence may indicate that the patient has kidney problems,
tubular injury

Table No.3 Casts

Page 4 of 9 | CAPORADO, DELA CRUZ, MOLINA |

 Lesson #7: Microscopic Examination of Urine

Figures Description
Coarsely granular cast:
Contrast microscope: use to parang nadurog.
see the cast easily.

Finely granular cast: further

Mahirap hanapin disintegration of coarsely
granular cast.
Hyaline cast at LPO: made up
of pure protein (THP)

See them on the edges of the

coverslip. Sometimes inside
the renal tubules this protein
matrix, are protein fibers that Mixed cellular cast: WBC,
can trap cells. Kaya from RBC combination. It has the
hyaline cast may cellular cast. matrix yung “supot na clear
Matrix surroundinig the cast”

Renal failure: kapag puro waxy

cast na lang nakikita mo.

Mixed cellular cast: WBC,

RBC combination. It has the
matrix

Hyaline cast at LPO

Granular cast: WBC that

disintegrates.

Cellular cast: hyaline cast with

trapped cells inside. (RBC
/WBC) WBC cast: lines/edge is very
smooth, it has matrix “supot”
Dipende kung anong natrap na
cell we’ll have RBC cast, WBC
cast. Somestimes, bacterial
cast.

Cells inside can be

destroyed/disintegrated. After
cellular cast it can be a granular
cast.
WBC clump: lines/edge is not
smooth.

RTE cast

RBC cast

Page 5 of 9 | CAPORADO, DELA CRUZ, MOLINA |

 Lesson #7: Microscopic Examination of Urine

Can be hyaline cast, cellular cast,

granular cast or waxy cast.
All type of cast can be broad cast
because It only becomes broadcast
when renal tubule widens.

Free RTE
RTE cast: mas pansinin yung
cast matrix. You have to be very Broadcast
careful in determining if it is a
RTE or WBC cast.

WBC: associated with

infection, may bacteria.

RTE: single-lobe nucleus, free

RTE. Waxy cast: Finely granular cast will disintegrate more turning into very
shiny appearance.
How to differentiate the two?
1. Use phase-contrast CRYSTALS
microscope
2. Nuclear details. Table No.5 Crystals
Figures Description

Amorphous Urate crystal

Amorphous urate color: Small, brown,
red, brick dust.

Sometimes, colorless same with

amorphous phosphate. Especially if
there’s no uroerythrin.

Fatty cast
Amorphous Urate

Parameter use to differentiate

amorphous urate from amorphous
phosphate; pH.

Fatty casts: highly refractile

kaya madali sila makita in Amorphous phosphate
phase-contract microscope.
Acidic pH = amorphous urate
Pinakamadaling makita na cast Alkaline pH = amorphous phosphate
dahil higly refractive ang fat
globules.

Amorphous crystals are not

pathological. They precipitate
because of the manner of preservation
(refrigeration)
If sample is refrigerated, there will be
presence of amorphous crystals.
Hence, can’t be differentiated unless
pH is checked.
Fatty cast: it has cholesterol
crystal. It will never be stained. Ammonium biurate. “Thorny apple”
shape.
Triglyceride: when stain it is
Cholesterol
crystals Orange
or red stained

Ammonium biurate. Seen in

ammoniacal specimen.
Broad cast- Because of its size
Usually causes of widening of
renal tubule due to renal failure
Also called as renal failure cast
because it is seen in patient
with renal failure

Page 6 of 9 | CAPORADO, DELA CRUZ, MOLINA |

 Lesson #7: Microscopic Examination of Urine

• Calcium carbonate. Can produce gas. • Cystine Crystal

Glacial acetic acid + bubble formation • Colorless unlike uric acid.
indicates the possibility of the
presence of calcium carbonate.
• Appearance: Sometimes in X form,
colorless and very small • Triple phosphate. Also called as
• Calcium oxalate crystal. Very common ammonium magnesium phosphate
in urinary sediment. • Very easy to see because it has a
• Appearance: Envelope shape, coffin lid shape.
colorless, very small.
• 2 Types: • Triple phosphate.
o Monohydrate- Dumbell shape
o Dihydrate – Envelope shape

• Calcium Oxalate
• Associated with: Renal calculi
(causing renal stone)
• Hardest among the different kidney • Triple phosphate.
stone. Hence, pinakamasakit na
stone.
• Food that is rich in oxalic acid such as
tomatoes, asparagus, and ascorbic
acid.
o Recommended vitamin C is 2000
mg each day or 4 tablets.
• Accidental ingestion of ethylene glycol • Triple phosphate.
(anti-freeze sa refrigerator)
o Doctor gives antidote, check for
Routine UA result.
▪ UA must have monohydrate of
calcium oxalate to see if
ethylene glycol is accidentally
ingested. • Calcium phosphate.
• Softdrinks. Because it is rich in oxalic • Plates are seen in the picture
acid.
o Coke float – rich in oxalic acid and
sundae is rich in calcium. Causing
formation of calcium oxalate.
• Calcium phosphate
• Rosette form. Very large, thin shape.
• Looks like a colorless platelet.
• Often mistaken as squamous epithelial • Calcium phosphate
cell. Especially if it’s shaped in plates. • Nail shaped calcium phosphate
because their shape differs.
• Cholesterol crystal
• Notched plate. “May punit sa side”
• Staircase pattern
• Oil droplets on the side.

• Cystine crystal. • Uric acid crystal

o Has Amino acid and usually seen • Most pleomorphic.
when there’s amino aciduria. o Lemon shape, hexagonal,
o When there’s amino aciduria, rosette
cystine increases. • Associated with gout or leukemia
• Hexagonal shape. especially if the patient is
undergoing chemotherapy.
o Cell destruction is highm =
• Cystine Crystal purine metabolism is
increased.
▪ Accumulation of uric acid
• Different shapes of Uric Acid
Crystals

• Cystine Crystal
• Looks like uric acid but uric acid is
yellow brown in color.

• Uric acid crystal in cuboidal form.

Page 7 of 9 | CAPORADO, DELA CRUZ, MOLINA |

 Lesson #7: Microscopic Examination of Urine

• Just a diaper fiber

• Radiographic contrast media • Often mistaken as cast
• To differentiate radiographic • Polarized, highly refractile.
contrast media to cholesterol o Casts can never be highly
crystal, check the specific gravity. refractile except for fatty
o Specific gravity is high = RCM cast.
• Resemble crystal. • Not a granular cast
• Trichomonas vaginalis • A glass fragment
• Usually mistaken as WBC • Often mistaken as crystal.
especially if the parasite is already Irregular shaped. Wala daw
dead. ganitong shape na crystal
o Dead T. vaginalis: It is • Spherical shape at the bottom
important to distinguish their arrow: Often mistaken as WBC
axostyle and flagella to but is actually a starcg granule
differentiate from WBC. o Starch granule: Sometimes
• Leukocyte esterase positive. from gloves.
• Rely in movement which is rapid o From coverslip: to prevent
jerky tumbling stacking, nilalagyan daw
ng starch.
• Yeast cells
▪ We are not using
• Budding or “Nanganganak”
coverslip in urinalysis.
• RBC is biconcave, (arrow)
o Already a ghost cell because
its color is faint. Meaning, it is
already lysed. REPORTING OF RESULT
• QUANTITATE an average of 10 representative fields.
• Amag o Quantitate 10 representative for LPO, HPO
• Mycelial form of yeast cell or fungi. o For miscellaneous elements, it depends on the protocol of the
If the infection is progressive, not laboratory.
just yeast cell but also mycelial • Do not quantitate budding yeast, mycelia elements, trichomonas, or
form can be seen in the patient. sperm, but do note their presence.
**table at the last page**

Table No.6 Urine Sediment Characteristic

Stain Action Function
• Mucus thread Sternheimer- Delineates structure Identifies WBCs,
• If there’s irritation in RTE. Tamm- Malbin and contrasting epithelial cells, and
horsfall protein is produced. colors of the nucleus casts
• Mucus thread has the same and cytoplasm
component which is Tamm-horsfall Toluidine blue Enhances nuclear Differentiates WBCs
protein. detail from RTE cells
o Protein material is produced 2% acetic acid Lyses RBCs and Distinguishes RBCs
not only in RTE but also in enhances nuclei of from WBCs, yeast, oil
other glands such as prostate WBCs droplets, and crystals
gland. Lipid stains: Oil Stain triglycerides Identify free fat
• Protein fibrils. Red O and and neutral fats droplets and lipid-
• Reported in LPO because it is very Sudan III orange-red; do not containing cells and
large. stain cholesterol casts
• Often mistaken as cotton fiber. Gram stain Differentiates gram- Identifies bacterial
• Very low refractive index unlike positive and casts
cotton fiber, can be seen in high gramnegative
brightness. bacteria
Hansel stain Methylene blue and Identifies urinary
• Spermatozoa eosin Y stains eosinophils
• Depends on the laboratory if eosinophilic granules
reporting is needed. Prussian blue Stains structures Identifies yellow-
• Sometimes, the absence or stain containing iron brown granules of
presence is the only reporting hemosiderin in cells
needed. and casts
• Present after sexual contact.
• Avoid bubbles during the • Sternheimer – Malbin: Most commonly used stain.
application of coverslip to avoid air o Pink cast
bubbles. o WBC is color blue
• Could interfere with the o A combination of crystal violet and safranin O.
identification of some formed o Board Exam question:
elements. ▪ Brand of Sternheimer Malbin: KOVASTAIN
• To differentiate: Add 2% acetic ➢ Color of the cast is blue
acid • Toluidine blue – to differentiate RTE and WBC by checking their
o Bubbles nuclear detail.
o RBC – nawala • Acetic acid – to remove RBC and differentiate it from the other sources
o Oil droplets/ yeast – can still be of error
seen • Lipid Stain – For triglyceride only. Cholesterol for polarizing
microscope
• Gram stain – For bacteria
• Hansel Stain – For eosinophil
• Prussian blue stain – If there’s presence of iron in the cells. Specially
RBC.
o Hemosiderin granules.

Page 8 of 9 | CAPORADO, DELA CRUZ, MOLINA |

 Lesson #7: Microscopic Examination of Urine

Fat globules examination under

Table No.7 Expected Staining Reactions of Sediment Constituents unstained in a polarized light
Elements in Usual Comments pink matrix indicates double
Urinary Sediment Distinguishing refraction
Color of
Stained
Elements Red cell inclusion Pink to orange- Intact cells can be
RBCs Neutral—pink to casts red seen in matrix
Identifies
purple WBCs,
Acid—pink Blood Orange-red No intact cells
epithelial cells, (hemoglobin) casts
(unstained)
Alkaline—purple\
and casts
Nuclei Cytoplasm Bacteria Motile: do not Motile organisms
WBCs (dark- Purple Purple stain are not impaired
staining cells) granules Nonmotile: stain
purple
Glitter cells Colorless or light Pale blue Some glitter cells
(SternheimerMalbin blue or gray exhibit brownian Motility is
positive cells) movement Trichomonas Light blue-green unimpaired in
Light shade vaginalis fresh specimens
Renal tubular Dark shade of of blue- when
epithelial cells bluepurple purple recommended
volumes of stain
Bladder tubular Blue-purple Light purple are used;
epithelial cells immobile
organisms also
Squamous Dark shade of Light purple identifiable
epithelial cells orangepurple or blue
Inclusions and Mucus Pale pink or pale
Matrix blue
Background Pale pink or pale
Hyaline casts Pale pink or pale Very uniform
purple
purple color; slightly
darker than *Be familiarize*
mucous threads
• Staining – different staining reaction
Coarse granular Dark purple • Sometimes, nucleus and cytoplasm have a different color.
inclusion casts granules in o Nucleus – nucleic acid (acidic)
purple matrix
▪ Stain should be alkaline
Finely granular Fine dark purple o Cytoplasm – Alkaline
inclusion casts granules in pale ▪ Stain used should be acidic
pink or pale
purple matrix POST ANALYTICAL PHASE
• Before leaving, the medical technologist must do all of the following:
Waxy casts Pale pink or pale Darker than
purple hyaline casts, but o Returning of materials, slides and microscopeso
of a pale even o Disposal of wastes and disinfection with liquid Lysol or 10%
color; distinct sodium hypochlorite of the area.
broken ends o The PPE of each individual should be removed properly. These
cannot be exposed outside the laboratory premises.
Rare; presence is
Fat inclusion casts confirmed if

Table No.8 Reporting of result

QUANTITATED None Rare Few Moderate Many
Epithelial cells LPF 0 0-5 5-20 20-100 >100
Normal Crystals HPF 0 0-2 2-5 5-20 >20
Bacteria HPF 0 0-10 10-50 50-200 >200
Mucus thread LPF 0 0-1 1-3 3-10 >10
Casts LPF 0 # RANGES: 0-2 , 2-5, 5-10 , >10
Rbc HPF 0 #RANGES: 0-2, 2-5, 5-10, 10-25, 25-50, 50-100, >100
Wbc HPF 0 #RANGES: 0-2, 2-5, 5-10, 10-25, 25-50, 50-100, >100

*Memorize this table*

Page 9 of 9 | CAPORADO, DELA CRUZ, MOLINA |

 LESSON 7: MICROSCOPIC EXAMINATION OF URINE (Face -to-Face)

OUTLINE CASTS: _______/ HPF

I Procedure
II Patient Information
III Physical Examination CRYSTALS:
IV Chemical Examination Normal: _______/ HPF
V. Microscopic Examination
A. Quantitation of Formed Elements per Field Abnormal _______/ LPF
B. Reporting of Result in Estimate Number
C. Reporting of Result in Average Number
OTHERS:

Mucus Thread _______/ LPF

PROCEDURE • To check for the cast, look at the edge of the coverslip.

• Thoroughly mix your urine specimen. Table 2. Reporting of Result in Estimate Number
• Pour 10 ml of urine in the test tube. REPORTING OF RESULT – Estimate Number
• Centrifuge for 5 minutes at 1,500 RPM. Formed Field Rare Few Moderate Many
• Pour off the supernatant liquid. Sufficient urine remains in the tube to Element
resuspend the sediment. Epithelial
LPF 0-5 5-20 20-100 >100
• Shake the tube after transfer and drop on the slide. Cells
• Examine under the microscope using LPO & HPO (10 fields). Mucus
LPF 0-1 1-3 3-10 >10
• Transfer ¾ of urine to test utbe Swirl first Thread
• Centrifuge for 5 mins. 400 RCF Normal
HPF 0-2 2-5 5-20 >20
o Label the tube with group number and section, no need to put Crystals
parafilm Bacteria HPF 0-10 10-50 50-200 >200
• After 5 mins., decant the tube
o Isang diretsong tapon lang hanggang sa may matira na 1ml or
.5ml Table 3. Reporting of Result in Average Number
• Mix the sediment left in the tube REPORTING OF RESULT – Average Number
• Add a drop of urine sample in the glass slide using a pipette, then Formed Field Range
cover with a coverslip. Elements
• Examine under the microscope 0-2
o 10 fields per HPO 2-5
Cast LPF
o 10 fields per LPO 5-10
▪ List the number every field then after looking at 10 field, you >10
can total the average. 0-2
o Reading should be done within 10-15 mins. to prevent drying of 2-5
the specimen 5-10
o When reading reached 30 mins, the slide will already dry causing RBC HPF 10-25
lots of crystal in microscopic examination 25-50
50-100
PATIENT INFORMATION >100
• Patient’s name 0-2
• Date & Time of Collection 2-5
• Age/ Gender 5-10
WBC HPF 10-25
PHYSICAL EXAMINATION 25-50
• Color 50-100
• Clarity >100

CHEMICAL EXAMINATION
• Glucose
• Bilirubin
• Ketone
• Specific Gravity
• pH
• Protein
• Blood
• Urobilinogen
• Nitrite
• Leukocyte

MICROSCOPIC EXAMINATION
Table 1. Quantitation of Formed Elements per Field
Formed Elements Quantitation per Field
CELLS:
RBC _______/ HPF
WBC _______/ HPF
Bacteria _______/ HPF
Epithelial cell _______/ LPF

Page 1 of 1 | SURNAME |
 ACTIVITY #4&5 : SEMEN ANALYSIS, FOBT

• Stain: Eosin and Nigrosin

OUTLINE o Separate stain that will be combine.
I Seminalysis o Technique:
A Physical Examination
B Sperm motility ▪ In a test tube, place one drop of sample and Eosin
C Sperm morphology (orange)stain. Next place 4 drops of Nigrossin (purple).
D Sperm viability ▪ From this combination, place one drop of solution in the slide.
E Sperm concentration ▪ Pull-away/Pull-apart method: One drop next pagpatungin
F Sperm count yung dalawang slide and then pull-apart.
▪ No need to fix just dry before examining in the OIO. Use
II FOBT
caedar wood oil.
A Physical Examination
B Procedures and Results ▪ Make sure that you’re sample is already focused in HPO
before you proceed with OIO.
➢ Tip: Mas maraming oil, mas malinaw siya.
SEMINALYSIS • Count 100 sperm, in the red background you’ll see unstained sperm,
like a shadow. As long as they have the energy they will not take up
I. PHYSICAL EXAMINATION the stain kapag patay lang stained.
• Color: o
o Increased flavine causes color yellow.
▪ If it is yellowish ask the patient how long did he abstained. • Normal:
• Volume: 2.5 ml o Alive (Bluish-White): 75%
o 2-5 ml more than is caused by prolonged abstinence. 2-3 days o Dead (Red / Pink)
abstinence to see enough sperm cells.
o If not abstain, low count of sperm and increase volume is expected • What will be the reading if you observe light pink? Dead. As long as
o If longer than normal abstinence, volume increase and no. of it take stain/color it is dead.
viable sperm will be affected.
• Viscosity: Check viscosity before liquefaction. Use applicatot stick
• In 100 sperm, count it regardless if it is dead or alive. For example,
and if it form a string it is still clotted or still not gel-like, di pa pwede
56 dead, 44 alive. The sperm viability is 44% viable.
iprocesses. Need watery muna, wala ng string na naf’form.
• 21 sperm, 14 alive, 7 dead. The sperm viability is 66%
o 0 - watery
o 4 - gel-like
• pH: slightly alkaline because of Bulbourethral glands. It neutralize the
[Link] MORPHOLOGY
acidity of vagina during ovulation. • Procedure
o use litmus paper. Either acidic or alkaline. o Prepare a thin smear of seminal fluid
• Odor: not determined anymore. Clorox-like, musty o Dry and fix by heat.
o Stain the smear by gram’s method
o Examine under OIO and count 200 cells, counting both the normal
and abnormal forms
II. MICROSCOPIC EXAMINATION o Report Normal and Abnormal forms in percentage
A. SPERM MOTILITY • Procedure:
• Procedure o First you have to make your smear
o Place one drop of seminal fluid on a slide and cover with coverslip o Pull-apart method
o Examine 20 HPO and report the number of motile and non-motile o Heat Fix
sperms in percentage. o Instead of Gram stain use Crystal violet (just 1 dip) and let water
• Purpose: You’ll be able to know if the sperm can penetrate the (di malakas pressure) flow to fingers not directly in the slide to
cervical mucus for fertilization. prevent wash out. Maalis lang konti stain, too much stain di
• In 20 HPO you just to estimate the amount of motile and non-motile makikita maayos.
sperms. Normally more than 30%. o Count 100 cells, look for normal form. Check the head and tail.
Use percentage.
• Normal: >50% motile (within 1 hr) o Head: normal size not pinhead or giant head, amorphous head.
• Quality: >2.0 (atleast 2) o Tail: not coiled, double tail, if no tail present it is spermatids that
cause infertility to males.
• Grading
o 0 = no motility • Normal:
o 1+ = has motility but has no forward progression (at place lang) o >30% normal forms
o 2+ = has forward progression, lateral movement (diretso or o <50% Abnormal forms
paliko)
o 3+ = straight and paliko konti, some lateral movement D. SPERM CONCENTRATION
o 4+ = straight and rapid swimming • Procedure
B. SPERM VIABILITY o Aspirate sperm sample up to 0.5 mark
• Procedure o Aspirate diluting fluid up to 11 mark.
o Prepare a thin smear of seminal fluid o Mix for atleast 1 minute
o Heat fix using alcohol lamp o Discard the first few drops (2-3 drops)
o Cover and read under OIO (count 100 sperms) o Charge your sample in the Neubauer counting chamber
• Purpose: Determine if the sperm has enough energy.
• 2 types of sperm • Count cells like in the Hematology using wbc pipette pipette 0.5 cc.
o Gymnosperm: sperm that forms female child (slower, low Then add diluting fluid at 11 mark.
mortality) • Mix 1-2 minutes
o Angiosperm: sperm that forms male child (Faster, high mortality) • Discard few drops dahil diluting fluid lang yung nasa sterm ng WBC
▪ Deep penetration, girl nasa taas. pipette. Kapag yun yung binilang konting sperm lang present there.
• OB-GYN will teach you position na kung anong want niyong gender Use the solution in the bulb where sperm is present.
of child. • Double check the counting chamber if may lines, charge na if meron.
• Look for secondary squares, we have 9. 4 WBC and 1 RBC square.

Page 1 of 2 | CAPORADO, DELA CRUZ, MOLINA |

 Lesson 4 : Fecalysis, Semenalysis

• Count in the 2 WBC square only Table No.1 Normal values

• Tertiaty of wbc = 16 Sperm Values
• Tertiary of rbc = 25 Motility >50% motile (within 1 hr)
• Quarternary of rbc = 16 Quality >2.0
Viability 75% Alive (Bluish-White)
• 2 wbc square (shortcut) Morphology >30% normal forms
o Number of cells x 100,000 mL Concentration 20-160 M/mL

• Example Count >40 M/ejaculate

o 40 x 100,000 mL
o 4,000,000 mL

• 1 wbc only Table No.2 Lenses

o Use the long method Test Lenses
Motility HPO
Sperm concentration: No. of cells counted x dilution factor x 1,000 Viability OIO
No of square counted (area) x 0.1 Morphology OIO

Sperm concentration: 20 x 20 x 1,000

1 x 0.1

Sperm concentration: 4,000,000 mL

FORMULA (LONG METHOD)

Sperm concentration: No. of cells counted x dilution factor x 1,000

No of square counted (area) x 0.1

Convert to uL to ml by multiplying it to 1,0000

Normal
o 20-160 M/mL

E. SPERM COUNT
• Multiple sperm concentration to the volume of the sample
• Determine the exact volume: transfer to the graduated cylinder.
• The unit is per ejaculate

FORMULA
Sperm count = sperm concentration x Volume

• Normal
o >40 M/ejaculate

FOBT
I. PHYSICAL EXAMINATION
• Color: brown
• Consistency: formed

• It’s different per manufacturer for the protocol. Check the insert.
Usually may paper na regarding the test and procedure.
• Front: Place the sample here together with the information of the
patient.
o When adding sample just apply small size in the middle of the
stool to prevent false positive kapag sa size.
o During the defecation merong blood masasama sa slide, dapat sa
middle kukuha ng feces lalo if constipated ka.
• Small amount only and put color developer
o Component of color developer:
▪ H2o2 and chromogen (Guaiac chromogen)
• Back window: Check the result here

II. FOBT
• Result
o Positive: bluish discoloration
▪ The guaiac is oxidized that caused the discoloration.
▪ Dapat sa control din apply the color developer. If there is no
changes in the color deteriorated na ang color developer

o Negative: no bluish discoloration

Page 2 of 2 | CAPORADO, DELA CRUZ, MOLINA |

 LESSON 14: SEMEN ANALYSIS

OUTLINE Table No.2 Macroscopic Examination

I Pre-analytical Phase
A. Specimen Collection
Macroscopic Examination
II Analytical Phase Normal 2 to 5 ml
A. Physical Examination
Volume Increased in Increased abstinence
B. Macroscopic Examination
1. Sperm Motility Decreased in Infertility, incomplete collection
2. Sperm Viability Normal Pour in droplets
3. Sperm Morphology (highly viscous)
4. Cell count Viscosity Increased viscosity Decreased sperm mobility
5. Sperm Concentration
C. Chemical Examination
Reporting: • 0- Water
1. Seminal Fructose Screening Test • 4- Gel-like
Normal 7.2- 8.0
7.3- 8.3
pH Increased pH Infection
PRE-ANALYTICAL PHASE
• Upon receiving the semen specimen in the laboratory, the receptionist Decreased pH Increased prostatic fluid
checks if the container is properly labeled and records the information
indicated.
• The site of the experiment should be sterilized. The medical
technologist should wear proper personal protective equipment before Table No.3 Materials
the experiment.
SPERM SPERM SPERM
CELL COUNT
SPECIMEN COLLECTION MOTILITY VIABILITY MORPHOLOGY
• Abstinence of 2-3 days for fertility testing, 2-3 samples must be Spermatic Spermatic Spermatic fluid Spermatic Fluid
examined at 2 weeks interval with a 2 abnormal samples considered fluid fluid
significant Slides Eosin- Alcohol lamp WBC pipette
Nigrossin-
stain
METHOD OF COLLECTION:
Cover slips Cover slips Slides Neubauer
• collect the entire ejaculate Slides Cover slips Counting
o Masturbation Crystal Violet Chamber
o Coitus interruptus Gram’s Iodine Cover slips
o Condom Method Acetone-Alcohol WBC diluting
▪ use non-lubricant containing rubber or polyurethane condom
fluid
o Vaginal vault aspiration
Safranin
• Time of collection- preferably in the morning , brought to the lab
within 30 minutes and examined within 1 hour. SPERM MOTILITY
• During transport- maintained by body temperature • Within one hour of collection, check the percentage of motility and
• Take note of the time of specimen collection, specimen receipt and repeat after four hours.
liquefaction • Place one drop of seminal fluid on a slide and cover with a cover slip.
• Analysis should be done after liquefaction (usually 30- 60 minutes) • Examine under HPO and report the number of motile and non-motile
• Specimen awaiting analysis should be kept at 37°C sperms in percentage.

PHYSICAL EXAMINATION • Sperm Motility Grading

• Materials:
o Spermatic fluid
o Specimen container
o Graduated cylinder
o Litmus paper
• Steps:
o Check for color and odor. Normal color for spermatic fluid is gray-
white, pearl white while odor is musty or chlorox like. o Performed undiluted, examine microscopically (20 hpf)
o Using litmus paper, check for pH. o Normal Values
o Note for the liquefaction time. Normal liquefaction time is 30-60 ▪ > 50 % motile (within 1 hour)
mins. ▪ Quality= ≥2.0
o Transfer specimen in graduated cylinder for specimen volume.

MACROSCOPIC EXAMINATION
Table No.1 Table No.4 Sperm Motility Grading
Appearance Grade WHO Criteria
Normal color • Gray-white, translucent 4.0 a Rapid, straight-line motility
• Pearly white, colorless to 3.0 b Slower speed, some lateral movement
creamy white 2.0 b Slow forward progression, noticeable lateral
Odor • Musty or bleach/ chlorox 1.0 c No forward progression
odor 0 d No movement
Increased white turbidity • Infection/ increased WBC
Red Coloration • Increased RBC
Yellow Coloration • Increased contamination,
urine contamination or
medication

Page 1 of 3 | SURNAME |
 Lesson # 14 : Semen Analysis

• Normal Values
o Examine at least 200 spermatozoa under OIF
o Routine criteria= > 30% normal forms or <50% abnormal forms
o Kruger’s strict criteria= >14% normal forms or < 70% abnormal
▪ Measure the head, neck, and tail using a micrometer

• Stains
o Wright’s stain
o Giemsa stain
o Hematoxylin
o Crystal violet
o Papanicolau’s stain

SPERM VIABILITY

CELL COUNT
• Using a WBC pipette, draw semen to the 0.6 mark
• Draw WBC diluting fluid to the 11.0 mark
• Shake for two minutes.
• Modified Bloom’s Test • Discard the first few drops and charge in 2 large squares ( similar to
o Prepare a thin smear of seminal fluid. WBC count)
o Heat fix using alcohol lamp.
o Add a drop of Eosin- Nigrossin stain.
o Cover and read under OIO.

SPERM CONCENTRATION
• Normal Value: 20-160 million/mL
o Reagent: Eosin and Nigrosin
▪ Count 100 sperms METHODS
➢ Living sperm:
unstained, • Improved Neubauer Counting Chamber
bluish white (75%) o Dilution- 1:20
➢ Dead sperm: red o Diluents: to immobilize the sperm
▪ Cold water
▪ 5% NaHCO3 in 1% phenol
SPERM MORPHOLOGY ▪ 1% Formali
▪ 1% formalin in trisodium citrate
▪ 5% Sodium bicarbonate
▪ 0.5% chlorozene

• Makler Counting Chamber

o For undiluted sample
o Uses heat to immobilize the sperm cells

• Prepare thin smears of seminal fluid.

• Dry and fix by heat
• Stain the smears by gram’s method.
• Examine under OIO and count 200 cells, counting both the normal and LONG METHOD FOR SPERM COUNT CONCENTRATION
abnormal forms. Also, report any epithelial cells, testicular cells, COMPUTATION
RBCs, WBCs and crystals seen.
(STANDARD NEUBAUER CALCULATION FORMULA)
• Report Normal and Abnormal forms in percentage
#𝑐𝑒𝑙𝑙𝑠 𝑐𝑜𝑢𝑛𝑡𝑒𝑑 𝑥 𝑑𝑖𝑙𝑢𝑡𝑖𝑜𝑛
Sperm concentration =
# 𝑠𝑞𝑢𝑎𝑟𝑒𝑠 𝑐𝑜𝑢𝑛𝑡𝑒𝑑 𝑥 0.1

Page 2 of 3 | CAPORADO, DELA CRUZ, MOLINA |

 Lesson # 14 : Semen Analysis

• Examples
o 1 sperm counted, 2 WBC squares Table No.5 Composition of Semen
o 1 sperm counted, 5 RBC squares
COMPOSITION OF SEMEN
5% Spermatozoa • Seminiferous tubules (Testes)
SHORTCUT METHOD FOR SPERM COUNT o Spermatogenesis
CONCENTRATION COMPUTATION o Sertoli cells = serve as nurse cells for
developing sperm cells
• 2 WBC squares
• Epididymis
o # of sperm counted x 100,000= sperm in M/mL
o Sperm maturation
(Sperm become motile)
• 5 RBC squares
o # of cells counted x 1,000,000= sperm in M/mL
60-70% Seminal • Seminal vesicles
Fluid o Provide nutrients for sperm and slightly
alkaline fluid
o Rich in fructose for sperm motility
20-30% • Acidic Fluid
Prostatic Fluid • Contains ACP, Zinc, Citric acid and other
enzymes
• For coagulation and liquefaction
5% Bulbourethral • Thick alkaline mucus
gland • Neutralize acidity from prostatic secretions
(Cowper’s gland ) and vagina

NORMAL VALUES FOR SEMEN ANALYSIS

COMPOSITION OF SEMEN

CHEMICAL EXAMINATION Volume 2-5 mL

• Materials: Viscosity Pour in droplets
o Spermatic fluid
o Fructose reagent pH 7.2- 8.0
▪ (50 mg resorcinol in 33 ml. conc. Sperm concentration ≥ 20 million/ mL
HCl diluted in 100 ml with water)
o Test tube Sperm count ≥ 40 million/ mL
o Test tube holder Motility ≥ 50 within 1 hour
o Alcohol lamp
Quality ≥ 2.0 or a, b, c
Morphology • >30% normal forms (routine
criteria)
• > 14% normal forms (strict
SEMINAL FRUCTOSE SCREENING TEST criteria)
• Prepare the Fructose reagent Round cells < 1 million / mL
o (50 mg resorcinol in 33 ml. conc. HCl diluted in 100 ml with water
reagent)
• Mix 1 ml semen with 9 ml fructose reagent.
• Boil
• Observe for orange-red color

• Seminal Fluid Fructose

o Normal Value: ≥13 umol/ejaculate
o Screening test
▪ Resorcinol test= (+) orange-red color

Table No.5 Chemical Testing

CHEMICAL TESTING
ANALYTE NORMAL VALUE DECREASED
VALUE INDICATES
Fructose ≥ 13 umol/ejaculate Decrease seminal
fluid
Neutral α- ≥ 20 mU/ejaculate Epididymis disorder
glucosidase
Zinc ≥ 2.4 umol/ejaculate Decrease prostatic
fluid
Citric Acid ≥52 umol/ejaculate
Acid phosphatase ≥ 200
Units/ejaculate

Page 3 of 3 | CAPORADO, DELA CRUZ, MOLINA |

 LESSON 6: FECAL ANALYSIS

o Applicator sticks
OUTLINE o Slides and cover slips
I Pre-Analytical Phase o Pea sized stool
A Materials
B Precaution Before Collection • Split fat procedure
C Specimen Collection o 36% acetic acid
D Specimen Preservation o Sudan III
II Physical Examination o Applicator sticks
III Chemical Examination o Slides and cover slips
A Fecal Occult Blood o Pea sized stool
B Acid Steatocrit
IV Microscopic Examination
A Methylene Blue Procedure for Fecal Leukocytes PRECAUTION BEFORE COLLECTION
B Muscle Fiber Procedure • Before collection, instruct the patients with these things, check the
C Neutral Fat Stain Procedure request form from the physician to see if the patient has any
D Split Fat Stain Procedure interference medication that may cause changes in chemical and
V Post-Analytical Phase physical composition. Confirm with patient if there is any.
• Interference medication = affects the physical, chemical, microscopic
components of the feces.
PRE-ANALYTICAL PHASE
• Upon receiving the fecal specimen in the laboratory, the receptionist • Patient should avoid the following things for at least 48 hours before
checks if the container is properly labeled and records the information collection of stool:
indicated. o Mineral oils, bismuth, non absorbable anti diarrheal drugs,
• The site of the experiment should be sterilized. The medical antimalarial drugs, antibiotics, etc
technologist should wear proper personal protective equipment before o Patient should not have barium before stool examination
the experiment. o Avoid iron containing drugs, meat, fish etc for at least 48 hours
before stool for occult blood
• Fecal analysis
o Use to assess the GIT if there is bleeding, malabsoprtion, FOR FOBT:
maladigestion or infection.
o Composition of the feces: pure water 60-80% water, ¼ is solid the • Instruct patient to avoid red meat = 3 days
bacteria, food stuff residues, intestinal secretion, fats, and other • Avoid aspirin = 7 days
soluble substances.

MATERIALS SPECIMEN COLLECTION

• I. CONTAINER – clean, dry, non-
PHYSICAL EXAMINATION breakable container, leakproof, screw-
capped multiple-day collection: large
• Sterile container containers
• Applicator stick o A clean container will do as long as
• Pea sized stool its leak proof. Avoid leakage
specially if the stool sample is
CHEMICAL EXAMINATION watery. (check consistency if
• Fecal occult blood test watery, formed)
o Guaiac solution o Not necessarily sterile.
o Hydrogen peroxide
o Glacial acetic acid
o Serologic pipettes • II. TYPE AND AMOUNT COLLECTED
o Filter paper pea-sized: FOBT, WBCs, qualitative fecal fat 2- to 3-day fecal
o Pasteur pipettes collection: quantitative tests
o Applicator sticks
o Pea sized stool SPECIMEN PRESERVATION
• Refrigeration
• Acid steatocrit procedure • Freezing in dry ice
o 5N perchloric acid • Formalin (2%, 5%, 10%)
o Deionized water • Alcohol
o Capillary tubes • 20% glycerin in saline (Cumming Method)
o Pea sized stool • Methiolate – Iodine Formaldehyde (MIF) Solution
o Vortex mixer • Polyvinyl alcohol (PVA) Fixative

MICROSCOPIC EXAMINATION • If there’s any delay store it properly

• Methylene blue stain procedure for fecal leukocytes o Most common is refrigeration: the best preservation
o Loeffler methylene blue o Freeze: use dry ice
o Applicator stick o 10% formalin = all purpose fixative
o Slides and cover slips ▪ the best chemical preservation it does not kill or destroy
o Pea sized stool organisms.

• Muscle fiber procedure o MIF = (merthiolate-iodine as stain) and (formaldehyde as fixative)

o 10% eosin in alcohol ▪ It can’t adequately preserve the morphology of trophozoites
o Applicator sticks ▪ Not for trophozoites
o Slides and cover slips
o Pea sized stool o PVA – good preservatives for trophozoites and cyst, very low
• Neutral Fat Stain Procedure viscosity, it can’t be use for the preservation of helminths eggs and
o Sudan III in 95% alcohol larva.

Page 1 of 5 | CAPORADO, DELA CRUZ, MOLINA |

 Lesson # : Unit Title

• Form stool = 1 hr tested na agad dapat

• Watery = mas mabilis kasi possible mawala yung mga organism which
is pathological, within 30 mins test the sample.

PHYSICAL EXAMINATION
• Pass out stool in a bedpan lined with newspaper. Avoid admixture of
urine, fibers, dirt, gauze threads and tissue papers.
• By means of an applicator stick, get a pea-size or marble-size of stool
from the mid-portion. If a portion is mucoid or bloody, collect from that
portion.
• In case of liquid stool, collect 10ml or 1/3 full of an eight-ounce
container.
• Examine the specimen immediately.
• Note the color, odor and consistency.

• Check the physical characteristics/ Observable characteristics: BRISTOL STOOL CHART

Consistency, color, odor (not included) • Base on stool consistency
• Most optimal: Type 4
• Check the volume • Optimal: 3 and 4
o Watery = collect at least 10 ml to perform all of the test in cases
of diarrhea.
CHEMICAL EXAMINATION
Table No.1 Macroscopic Stool Characteristics
Color/Appearance Clinical Significance FECAL OCCULT BLOOD TEST (GUAIAC TEST)
Light to dark Brown Normal (Urobilin/ Stercobilin) • Most preferred FOBT is Guiaic because it is cheaper and safer than
Black Upper Gastrointestinal Bleeding benzidine, ortholidine
• presence of blood Iron therapy • Screens colorectal cancer
o Oxidized: upper GIT Charcoal
bleeding (melena) Bismuth (antacids) • 1. Mix 5 ml of hydrogen peroxide with 5 ml of guaiac solution.
• 2. Make the stool specimen acidic using glacial acetic acid (test using
Red Lower Gastrointestinal Bleeding litmus paper).
• presence of blood Beets and food coloring • 3. Apply a small portion of stool in the filter paper.
o Unoxidized/Fresh: reddish Rifampin • 4. Add 2-3 drops of the prepared gum guaiac and hydrogen peroxide
or hematochezia solution.
• 5. A blue color will appear in the filter paper in the presence of blood.
Pale yellow, white, gray Bile duct obstruction
Barium sulfate
• From the liver, andun yung PROCEDURE:
gallbladder then we have the • Prepare the guaiac solution and h202
bile duct going to the intestine. • Preparing guaiac solution: 1g Guaiac powder is mix with 5 ml
• If there is blockage in the bile 95% ethanol
duct, the bile together with the • In a filter paper, acidify the stool specimen. Drop a glacial
bilirubin, urobilinogen (will not acetic acid until it acidify.
be oxidized in the intestine) will • The emulsify stool specimen will be use to make a smear
not be able to pass the bile • Add hydrogen peroxide and a drop from the 10 ml guaiac
duct. solution in the smear.
o Blockage of bile duct: • Observe the color and grade.
unoxidized urobilinogen =
pale color of stool
Table No.2 Results/Grading for Guaiac Test
Green Biliverdin RESULTS GRADING
Oral antibiotics Faint blue green in 1 min trace amount of blood
Green vegetables Light blue (slowly, gradual) 1+
Butter-like Cystic fibrosis Clear blue (rapidly) 2+
Bulky/ Frothy Bile Duct Obstruction Deep blue (almost immediately, 3+
• BDO: bile salts is important for Pancreatic Disorders after 2-3 secs)
cholesterol degradation. Bile Steatorrhea Deep blue (rapidly/right away after 4+
salts can’t pass through drop)
• Lipase from pancreas. In
pancrease problems, fats FECAL OCCULT BLOOD TEST (BENZIDINE TEST)
contents of stool increases. • 1. 4 gm benzidine in 100 ml of glacial acetic acid
o If wala both magiging bulky
• 2. Emulsify pea sized bit of feces in 5 ml of water.
• 3. Mix 1 ml emulsion and 1 ml of reagent in test tube
Mucus, Blood-streaked mucus Colitis
• 4. Add several drops of H2O2
Dysentery
• 5. Blue colour indicates positive reaction
Malignancy
Constipation
Ribbon-like / flattened Intestinal Constriction, spastic
colitis,
obstruction in lower colon,
syphillis
Rice watery Cholera Infection
Pea soup Typhoid Fever Infection
Scybalous (Goat droppings) Constipation, spastic colitis,
decrease fluid intake

Page 2 of 5 | CAPORADO, DELA CRUZ, MOLINA |

 Lesson # : Unit Title

Chromogens 1. Benzidine- most sensitive

2. Guaiac- preferred
3. O-toluidine
False (+) FOBT Dietary Pseudo peroxidases
o Red Meat
o Melon, broccoli, cauliflower, horseradish
(pseudo peroxidase food)
o Aspirin & other antiinflammatory drugs=
(avoid for 7 days)
False (-) FOBT Reducing agents:
o Ascorbic acid (Vitamin C) and iron therapy
Restrictions • Red Meat-free diet for 3 days
• Bleeding with >2.5ml/1500g stool it is
considered as pathologically significant if
• Tubes there is bleeding.
o Tube 1: GAC + Benzidine (Mixed together) • Food causing pseudo peroxidase
o Tube 2: Emulsified stool
o Tube 3: 1 ml from Tube 1 and 1 ml from Tube 2 • Amino acids: reducing agents will interfere the
test since the test is oxidation.
PROCEDURE:
1. Complete the tubes. No need to perform in the filter paper.
2. Add H2O2.
3. Observe color change
4. Grade

Table No.3 Results/Grading for Benzidine/Ortholidine Test

RESULTS GRADING
Faint blue after 1 min trace amount of blood
Blue green (gradual/slowly green) 1+
Blue green (rapidly green) 2+
Blue (almost rapid blue or dark 3+
blue) • Nowadays, MedTechs don’t perform manual Guaiac Test since there
Blue or dark blue (immediately 4+ is already test cards available with color developer, chromogen, h202
which is easier to use.

FECAL OCCULT BLOOD TEST (ORTHOTOLIDINE TEST) ACID STEATOCRIT PROCEDURE

• Intermediate sensitive: in between of guaiac and benzidine test. • An alternative test if Van de Kamer titration can’t be performed.
• The problem here is that it is expensive • Rapid test to estimate the amount of fats
• Advantage: Single stool collection, pediatric yung mahihirap kuhaan
ng samples
• Procedure: • Screening test of steatorrhea
o 1. Smear the stool on a filter paper with an applicator • Semi=quantitative/check the percentage of fats
o 2. Pipette a few drops of the reagent on to the filter paper • Gravimetric procedure; relies on the weight or mass of the fat’s liquid
o (orthotolidine barium peroxide 200 mg+ glacial acetic acid 5 ml) and solid layer
o 3. After 30 sec examine for a blue colour • NV: <10%
o 4. Blue green colour within 30 sec means positive test • Steatorrhea:; >10% either maldigestion or malabsorption of fats.
• Procedure
o 0.5g of feces from a spot collection is diluted 1 to 4 with deionized
PROCEDURE: water.
• Make a smear o Vortex for 2 minutes to homogenize the specimen.
• Place a reagent: drop Ortholidine and Gac directly in the smeared o A volume of 5 N perchloric acid equal to 20% of the homogenate
filter paper. volumE is added and the mixture is then vortexed for 30 seconds.
• Check for color Confirm the pH to be <1.
• Use the results/grading of Benzidine o Place the acid-homogenate mixture in 75 microliter plain
hematocrit capillary tube. Seal the end with wax.
o The capillary tube is centrifuged horizontally at 13000 rpm for 15
Table No.4 Fecal Occult Blood Test FOBT minutes in a microhematocrit centrifuge. This separates fat as an
•Occult= hidden upper layer overlying a solid fecal fat.
•Screening test for colorectal cancer o The length of the fat and solid layers is measured using a
•Significant= >2.5 mL blood/ 150 g of stool magnifying lens.
•Sample= center portion of the stool o Calculate the acid steatocrit in percent.
Principle Pseudo peroxidase activity of Hemoglobin o Calculate the fecal fat in grams per 24 hours.

• H202 of hemoglobin
oxygen will be H2O2 + Guiac ---------------------------à Oxidized
donated to the Guiac (blue) + H2O
guaic
• the results will Pseudoperoxidase
be oxidized
guaiac (blue
chromogen)
and h2O. (loss
of 1 Oxygen
from the
h202).

Page 3 of 5 | CAPORADO, DELA CRUZ, MOLINA |

 Lesson # : Unit Title

PROCUDURE:
• Dilute the stool sample with deionized water. 1:4 dilution
• Ensure it is properly mix, use the vortex.
• Acidify the sample using perchloric acid (very hazardous/corrosive
susunog ng balat). Too acidic, acidify pH dapat maging <1.
• Non-heparinized capillary tube: blue (no anticoagulant)
• Centrifuge.
o Fatty layer: Small layer on the top
▪ To determine the fat, get the %
➢ %: get the size of fat and solid layer FECAL LEUKOCYTES
➢ Formula: volume of fat layer over the layer of the solid
• Determination
layer x 100 = %
o Wet Preparation = Stool + Loeffler’s Methylene Blue
➢ >10%: quantify it using Van de Kamer titration, to find
o More preferred: since it can stain the granules, the bilobed
the exact amount in grams.
nucleus is more visible.
o Intermediate layer: water, liquid
o Solid layer: non-fatty solid layer
o Dried Preparation= Stool + Wright’s/ Gram stain
▪ Make a smear of feces then stain using the Wright or gram
o Lactofferin latex agglutination test
▪ Lactoferrin= secondary granules of neutrophils
▪ (+) invasive bacterial pathogen
ACID STEATOCRIT ➢ , if theres a lot mas maraming WBC
➢ Greater/Strong agglutination = ↑ lactoferrin = ↑ WBC =
• SPECIMEN: Single stool Invasive condition
• It is preferable that patients have been on a diet containing 70-100g
fat per day for at least 3 days prior to and during the collection period.
MUSCLE FIBER PROCEDURE
• No laxatives should be given.
• Stain: 10% eosin stains directly the muscle fiber
• Stool for steatocrit testing is stable for seven days under refrigeration.
• Purpose: Use to determine if there is any maldigestion problems
If longer term storage is necessary, it should be frozen.
• Specimen must not contain foreign material such
• Emulsify a small amount of stool in two drops of 10% (alcoholic eosin)
as toilet paper.
eosin in alcohol.
• Coverslip and let stand for 3 minutes.
REFERENCE INTERVALS
• Examine under high power field for 5 minutes.
• Units: expressed as percentages (%) • Count the number of undigested fibers.
• Age >6 months: o Check striations
o <10% normal ▪ Both direction Striation: fully undigested
o 10-20% equivocal ▪ No striation: fully digested
o >20% abnormal (steatorrhea) ▪ Unidirected partially undigested

MICROSCOPIC EXAMINATION ABNORMAL

METHYLENE BLUE PROCEDURE FOR FECAL • -> 10 undigested muscle fiber
LEUKOCYTES • UNDIGESTED STRIATED MUSCLE FIBER : biliary obstruction,
• Purpose: Used to determine if there is any invasive procedure or gastrocolic fistulas, pancreatic insufficiency (cystic fibrosis)
condition / infection.
• Is it Normal to see few fecal leukocytes?
o Yes, unless there are in significant number if there’s >3
Neutrophils per hpf
• Invasive condition: >3 Neutrophils per hpf
• Wet preparation using Methylene blue
• Place it on glass slide
• Place a coverslip and examine under HPO

Procedure
• Place mucus or a drop of liquid stool on a stool.
• Add two drops Loffler methylene blue.
• Mix with a wooden applicator stick.
• Allow to stand 2-3 minutes. NEUTRAL FAT STAIN PROCEDURE
• Examine for neutrophils under high power. • Qualitative fecal analysis
• Malabsorption problems

PROCEDURE • Procedure
• Invasive condition: >3 Neutrophils per hpf o Homogenize one part stool with two parts water.
• Wet preparation using Methylene blue o Mix emulsified stool with one drop 95% ethyl alcohol on slide.
• Place it on glass slide o Add two drops saturated Sudan III in 95% ethanol.
• Mix Methylene Blue and specimen o Mix and coverslip.
• Place a coverslip and examine under HPO o Examine under high power field.
o Count orange droplets per high power field.

Page 4 of 5 | CAPORADO, DELA CRUZ, MOLINA |

 Lesson # : Unit Title

Table No.3 Fecal Fat Determination

Qualitative Test
PROCEDURE: 1. Neutral Fat Stain 2. Split Fast Stain
• Homogenize the sample: make the stool in watery form, 1:2 (Triglycerides)
parts, 1 for stool, 2 for water Emulsified stool + 36% Acetic
• Drop 95% ethyl alcohol • Suspension + 95% ETOH + acid + Sudan III
• Add Sudan III stain, examine under the microscope Sudan III Orange Droplets (Fatty acids)
• The orange globules is triglycerides, count this different • Orange Droplets (Neutral Fats • Normal = 100 droplets (<4
globules. or Triglycerides) um)
• If you see more than or >60 orange droplets per hpf then you • ≥ 60 droplets/hpf = Steatorrhea • Slightly increased = 100
have steatorrhea. Here, we are not after the size, look for the droplets (1-8 um)
presence. • Increased = 100 droplets (7-
75 um)
Quantitative Test
3-day stool,
quantify the amount of fats
Van de Kamer Titration • Sample= 3 day stool
• Gold Standard for fecal fats • Normal value= 1-6 g fats/day
• For definitive diagnosis of • Steatorrhea=> 6 g fats/day or
steatorrhea 18 – 21 g
• Titration with NaOH

• If you are positive for the 2 qualitative test, it is a must to perform the
quantitative test.

SPLIT FAT STAIN PROCEDURE

• Check Fatty acids, Identify if you have malabsorption
o Natunaw ang trigylecirides naging fatty acids but di mo maabsorb

• Procedure
o Mix emulsified stool with one drop of 36% acetic acid.
o Add two drops saturated Sudan III.
o Mix and coverslip.
o Heat gently almost to boiling.
o Examine under high power.
o Count and measure the orange droplets per high power field.

POST-ANALYTICAL PHASE
• Before leaving, the medical technologist must do all of the following:
PROCEDURE: • Returning of materials, slides and microscopes
• Emulsify the stool directly with 1 drop acetic acid • Disposal of wastes and disinfection with liquid Lysol or 10% sodium
• Stain hypochlorite of the area.
• No need to use alcohol, instead boil it. (mabaho, kumakapit sa
damit)
• In glass slide, padaanin sa alcohol lamp until magka-usok then
stop. Automatic you’ll see fats, not for the number but obeserve
the size.
• Examine in the microscope.
• Count the orange droplets per hpf
o Count 100 and check the size, if there’s 6-7um, (large)
most of them, then it is steatorrhea. You have lots of fatty
acids but it is not absorbed.

Page 5 of 5 | CAPORADO, DELA CRUZ, MOLINA |