AUBF Lab Notes

1 ANALYSIS OF URINE AND OTHER BODY FLUIDS (LAB)
SAFETY IN THE • Vaccination against HBV
CLINICAL LABORATORY
• Appropriate signs to identify hazards
LABORATORY SAFETY
BIOLOGICAL HAZARD
• Recognition of hazards
board exam **ieexplain lang ang symbol ex. 3 dark
• Application of common sense boarded circle joined together, color black with yellow
• Safety-focused attitude background - Biohazard symbol
• Good housekeeping in all laboratory work and storage

areas CHAIN OF INFECTION
– Transmission of microorganisms
Safety Awareness for Clinical Laboratory Personnel – Requires a continuous link between:
CDC • HOST
Ø Universal Precautions (1987) • SOURCE
treat all blood as infectious • TRANSMISSION
Ø Standard Precautions (1996) 6 components of chain of infection (IREMES)

Blood and body fluid precautions should be consistently Infectious source/agent - virus
used for all patients
Reservoir - infected host
Portal of Exit - mouth, nose

• Potentially infectious materials:
Mode of Transmission - direct contact, airborne, droplets
– Body fluids
Portal of Entry - inhalation/ingestion
Unfixed tissues, organs or blood slides
Susceptible - elder, baby, immunocompromised,
Safety Awareness for Clinical Laboratory Personnel

Personal Protective Equipment (PPE)
• Precautions:
– Gloves
– Appropriate barriers (gloves, gowns or laboratory coats)
– Fluid-resistant gowns
– Appropriate engineering controls
– Eye and face shields
• Universal Practices:
– Countertop shields – for Clinical Chemistry
– Wearing of gloves
– Handwashing
• Proper handwashing
– Laboratory coats
– HAND CONTACT is the primary method of infection
– Prohibited: eating, drinking, smoking, applying transmission
cosmetics, touching contact lenses
– Handwashing is the BEST WAY to break the chain of Disposed in Puncture resistant container
infection.
• Disposal of biological wastes

CHEMICAL HAZARD
– ALL biological waste, except urine, must be placed in
Chemical spills
appropriate containers labelled with the Biohazard
Symbol BEST FIRST AID: flush the area with large amounts of
water for at least 15 minutes then seek medical attention.
– Discard urine by pouring it into the laboratory sink, avoid
splashing, and then flush with water *you can use boric acid to clean your eyes for 15 mins also
– Disinfection of the sink using a 1:10 dilution of Sodium you were born right** pnemonics
Hypochlorite and should be performed daily.
CDC GUIDELINES FOR HAND HYGIENE
• Wash hands with detergent soap or Antimicrobial soap:
– When the hands are visibly soiled

Degree of hazard
– Before eating
0 - no / minimal hazard
– After using the restroom
1 - slightly hazardous
• Alcohol-Based Hand rub
2 - moderately hazardous
– Before having direct contact
3 - serious hazard
– Hands are not visibly soiled
4 - extreme hazard
– After contact with inanimate objects
*board exam lab safety peyborit ng examiners
Alcohol Concentrations:
RADIOACTIVE HAZARDS
40 % (killing ability is low, for allergic px, to disinfect
working area/table) Ø When procedures using radioisotopes are performed
70 % (hand rub) Radioactive materials - damage to cell nucleus (DNA)
95%(easily evaporates) @risk of radioactive
70% di ganun kabilis at katagal mag evaporate that’s why 1. Pregnant women - abnormality in baby
killing time is enough
2. radioisotopes - radioactive iodine - to check activity of
• e.g medical equipment thyroid gland
SHARP HAZARDS esp. gamma rays
§ Placed near workspace
§ Closed when not in use UV Light - for disinfection
§ Sealed when ¾ full

ELECTRICAL HAZARDS Arsenal fires - forest fires
Ø If electrical shock occurs, never touch the person or the *Motor oil - type B
equipment involved
*never use h20 for cooking media such as oils and fats
Ø Turn off the circuit breaker because fire will just spread
Ø Unplug the equipment
machines should be grounded with 3- pronged plug to PHYSICAL HAZARDS

avoid electrical shock.
• General precautions:
Ø Move the equipment using a nonconductive glass or
– Avoid running in rooms and hallways
wood object
– Watch for wet floors
– Avoid dangling jewelry

FIRE / EXPLOSIVE HAZARDS
– Bend knees when lifting heavy objects
Ø Flammable chemicals should be stored in safety
cabinets & Explosion proof refrigerators in a remote area. – Maintain clean, organized work area
*wrap the burning person in wet blanket to smoothen the – Keep long hair pulled back
flame
– Wear closed-toe shoes
RACE
PASS
HANDWASHING PROCEDURE
• WET HANDS WITH WARM WATER.
• APPLY ANTIMICROBIAL SOAP.

dry chemical - most widely used fire extinguisher
• RUB TO FORM LATHER, CREATE FRICTION, AND LOOSEN
flammable liquids or inorganic chemicals - oil, paint, DEBRIS.
gasoline
• THOUROUGHLY CLEAN BETWEEN FINGERS, INCLUDING
electrical - halon is recommended THUMBS, UNDER FINGERNAILS AND RINGS, AND UP TO
THE WRIST FOR ATLEAST 15 SECONDS (Strasinger)
metals - for Mg, Na, Li, K use metal X
• RINSE HANDS IN A DOWNWARD POSITION (ELBOW-
Color of flames* board exams
FINGERTIPS) POSITION.
Na - yellow
• DRY WITH PAPER TOWEL.
Mg - blue
• TURN OFF FAUCETS WITH A CLEAN PAPER TOWEL TO
K - violet PREVENT RECONTAMINATION.
“Strength does not come from physical capacity. It comes ▪ Information on label:
from an indomitable will” – Mahatma Gandhi
▪Patient’s name, ID number, date, time
▪Additional information: age, location, physician

SPECIMEN COLLECTION PLACE LABEL ON CONTAINER, NOT LID
• Disposable, wide-mouthed, and flat-bottom ▪Requisition form: Must accompany specimen
containers with screw caps are recommended ▪Information must match label
• Clear containers and at least 50 mL capacity ▪Time of receipt is stamped on requisition
• Adhesive bags for pediatrics and large plastic ▪Other info: type of specimen, interfering meds
containers for 24-hour specimens
• Wear gloves when working with urine SPECIMEN REJECTION
▪Unlabeled containers
Urine Containers ▪Non-matching labels and requisitions
▪Contaminated specimens - feces, paper
▪Contaminated containers
▪Insufficient quantity
▪Delayed or improper transport
• Ice, refrigeration
1. Screw Top Lid
2. Pediatric Wee Bag

SPECIMEN HANDLING
3. Large Container
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
▪Most problems are caused by bacterial
Multiplication
SPECIMEN PRESERVATION
Refrigeration ( 2⁰C to 8⁰C)
Most routinely used method of preservation:
• Decreases bacterial growth and metabolism

SPECIMEN LABELING
Urine Preservatives table* postprandial specimen are compared with those of a

fasting specimen and corresponding blood glucose tests.
TYPES OF URINE SPX COLLECTION

5. Glucose Tolerance Specimens
• RANDOM
• FIRST MORNING • The urine is tested for glucose and ketones, and the
• FASTING (SECOND MORNING) results are reported along with the blood test results as
• 2-HOUR POSTPRANDIAL an aid to interpreting the patient’s ability to metabolize a
• GLUCOSE TOLERANCE measured amount of glucose and are correlated with the
• 24 HOUR (TIMED) renal threshold for glucose.
• CATHETERIZED • Collection of these specimens is an institutional option.
• MIDSTREAM CLEAN-CATCH
• SUPRAPUBIC ASPIRATION
• PROSTATITIS 6. 24-Hour (or Timed) Specimen
• PEDIATRIC
• DRUG SPECIMEN •Measuring the exact amount of a urine. A carefully
timed specimen must be used to produce accurate
1. Random Urine quantitative results
2. First Morning Urine • required when the concentration of the substance
• ideal screening specimen to be measured changes with diurnal variations and
• preventing false-negative pregnancy tests with daily activities such as exercise, meals, and
and for evaluating orthostatic proteinuria. body metabolism.
3. Fasting Specimen (Second Morning) 7. Catheterized Specimen
• A fasting specimen differs from a first morning • The most commonly requested test on a catheterized
specimen by being the second voided specimen after a specimen is a bacterial culture.
period of fasting. • If a routine urinalysis is also requested, the culture
• This specimen will not contain any metabolites from should be performed first to prevent contamination of
food the specimen.
ingested before the beginning of the fasting period. • A less frequently encountered type of catheterized
specimen measures functions in the individual kidneys.
• It is recommended for glucose monitoring. Specimens from the right and left kidneys are collected
separately by passing catheters through the ureters of
the respective kidneys.
4. 2-Hour Postprandial Specimen
• specimen is tested for glucose, and the results are

8. Midstream Clean-Catch Specimen
used primarily for monitoring insulin therapy in
• is less contaminated by epithelial cells and bacteria
persons with diabetes mellitus. and, therefore, is more representative of the actual
urine than the routinely voided specimen. Patients
• A more comprehensive evaluation of the patient’s
must be provided with appropriate cleansing materials,
status can be obtained if the results of the 2-hour a sterile container
• Strong bacterial agents, such as hexachlorophene or • most vulnerable part of a drug-testing program.
povidone-iodine, should not be used as cleansing
• The chain of custody (COC) is the process that provides
agents.
this documentation of proper sample identification from
• Mild antiseptic towelettes are recommended (Castile the time of collection to the receipt of laboratory results.
Soap Towelettes).
• that no tampering of the specimen occurred, such as
substitution, adulteration, or dilution
9. Suprapubic Aspiration
• aspiration provides a sample for bacterial culture that • 12. Drug Specimen Collection
is completely free of extraneous contamination.
witnessed or unwitnessed
• The specimen can also be used for cytologic
• Volume: 0-45ml of urine for drug testing
examination
• Temperature: must be taken 4mins within the time from
collection that will confirm the specimen will not
10. Prostatitis Specimen adulterated 32.5-37.7
Three-Glass Collection
• Quantitative cultures are performed on all specimens, Notes:

and the first and third specimens are examined
*reject spx only if less than 50 ml
microscopically.
• 2-4 pm urobilinogen
• In prostatic infection, the third specimen will have a
white blood cell/high-power field count and a bacterial • Nitrite – checked 4 hrs
count 10 times that of the first specimen. Macrophages • Addis count 4 hrs
containing lipids may also be present. • PPMT – mid stream clean catch
• Triple deionized h20 is not reactive
10. Prostatitis Specimen

PHYSICAL EXAMINATION OF URINE
Stamey-Mears four-glass localization method
URINE COLOR DETERMINATION
• Initial voided urine (VB1)
• Examine the specimen under a good light source
• midstream urine (VB2)
• Look down through the container against a white
• expressed prostatic secretions (EPS) background
• Post-prostatic massage urine specimen (VB3)
10. Prostatitis Specimen
Pre- and post-massage test (PPMT)
• A positive result is significant bacteriuria in the post-

massage specimen of greater than 10 times the pre-
massage count.
Color #4-8 – need to drink more h20

12. Drug Specimen Collection
Amber – urochrome, gives yellow or amber color of urine 1. Hormone
Darker – more concentrated, excretion of waste products 2. Baby pushing the urinary bladder
in little water ex. Fever
…….
Colorless/ Straw/ pale yellow – hydrated person
Phenazopyridine – mistaken as bilirubin

Urine Clarity
Presence of orange or yellow form or orange green
URINE VOLUME
Routine Urinalysis:
Volume required: 10-15 ml
Ex. 12 ml is still acceptable for routine urinalysis
Urine Clarity Determination

Drug Testing:
• Thoroughly mix the specimen
Volume required: 30-45 ml
• Examine the specimen while holding in front of a
light source
• View through a newspaper print or any printed
Polyuria
materials
2.5 L per day - Strasinger
2000 ml per 24 hrs – Herny’s

Milky – clouded/precipitated
Board exam* check for the unit used per day or 24 hrs
If the px have Kyluria
Diuresis
Odor
• Oliguria – px is dehydrated or may blockage sa

kidney tubules
400ml per day strasinger
<500 ml per 24 hrs henrys
• Anuria
<100 ml per 24 hrs
• Nocturia
>500 ml per night
Normal: Aromatic
Reasons:
*Maple Syrup – amoy leche flan
Rotting fish – have Trimethyl aminuria
Pungent – consumption of Asparagus
Specific Gravity
Checking of density of solutes on urine
Random Urine : 1.002 – 1.035 (most urine range from

1.015 – 1.030)
• Lower than 1.002 is not urine

• Higher than 1.040 use radiographic contrast media
URINOMETRY
-aka. Hydrometer, Direct method
When using the urinometer, an adequate amount of

urine is poured into a proper size container and the
urinometer is added with a spinning motion
The scale reading is taken at the bottom of the meniscus
Disadvantage:
• less accurate, not suggested by the CLSI, use a Computation:

large amount of urine A specimen containing 1 g/dL protein and 2 g/dL glucose has
• we are checking the amount of solutes but this a specific gravity reading of 1.040. Specimen Temp Reading:
is not longer performed in lab bcoz very time 23oC
consuming and it requires large
volume(disadvantage)
Calibrator: Potassium Sulfate (1.015)
May temp. correction

Calibration temp: 20 degree Celsius
• Every 3 degreee Celsius increase, add 0.001
3 degree Celcius decrease, subtract 0.001
Due to presence of glucose and protein also may temp.
• Corrected reading if there’s presence of protein
correct.
(subtract 0.003) and glucose (subtract 0.004)
Ex. 1.85 g/dL gluscose – round off to 2.00
1.3 g/dL – round off to 1.00
3 g/dL – no need to round off, just multiply to
0.004 glucose = 0.012
*Round off the temperature too
• Float shouldn’t touch the side of cylinder, read
below the meniscus
2. CLOSE THE DAYLIGHT PLATE GENTLY
3. SAMPLE MUST SPREAD ALL OVER THE PRISM SURFACE
make sure that there is sufficient urine that spread,

make sure that there are no bubbles
4. LOOK AT THE SCALE THROUGH THE EYEPIECE
5. READ SCALE WHERE THE BOUNDARY LINE

INTERCEPTS IT
Video 6. WIPE THE SAMPLE FROM THE PRISM, CLEAN WITH
TISUUE AND WATER
Refractometer – Indirect Method

CALIBRATION:
Temperature corrections are not necessary but
there are still corrections for glucose and protein • DISTILLED WATER: 1.000
• Triple D. H20 – also 1.000
• 5% NaCl: 1.022 +/- 0.001
• 3% NaCl: 1.015
• 9% Sucrose: 1.034 +/- 0.001
TS – total solid meter
-Principle: Refractive Index/
light velocity air

light velocity in solution
Urine Specific Gravity (UG)
Can read 1.01 – 1.050
Ex. SG: 1.028
S.G Dilution
• For specimens with very high SG
• Multiply the decimal portion of SG by the dilution factor
Example:
Urine specimen diluted 1:4 has a reading of 1.014. What

is the actual SG reading?
0.014 x 4 = 1.056
radiographic contrast media used in MRI, small magnet

STEPS IN USING THE REFRACTOMETER solution SG: more than 1.040..45
1. PUT 1 OR 2 DROPS OF SAMPLE ON THE PRISM That’s why you have to dilute the spx
Ex. 1:4 • Clinitest Tablet
Dilution factor – reciprocal
Procedure
Chemical Examination of Urine • For Reagent Strips:
Collect first 10-15 ml for routine urinalysis

PRE-ANALYTICAL PHASE
• Completely immerse all reagent areas of the strip in
• The site of the experiment should be sterilized. The
fresh, well-mixed uncentrifuged urine* and remove
medical technologist should wear proper personal
immediately (because chemicals may leech
protective equipment before the experiment.
off/deteriorate from absorbent pad).
• Upon receiving the urine specimen in the laboratory,
*Preferably, transfer your urine in a 10-15 ml of test
the medical technologist checks if the urine container is
tube
properly labeled and records the information indicated.
• Tap edge of strip against the side of urine container to
remove excess urine. Hold the strip in a horizontal
ANALYTICAL PHASE position to prevent possible mixing of chemicals from
adjacent reagent area and/or soiling of hands with urine.
Materials:
- Avoid mixing of reagents in the test strip →cross
REAGENT STRIP
reactions = incorrect result
– simple and rapid, chemical impregnated absorbent - Blot it in a tissue paper to remove excess urine
pad
• Compare test areas closely with corresponding color
• Urine (freshly voided or random specimen(4.5-8.0 pH), charts on the bottle label at the times specified. Hold the
preferably first morning specimen(5.0-6.0 pH) – most strip closely to color blocks/chart and match carefully.
concentrated)
- Read from top to bottom
*8.5 pH of urine means improper preservation or - After 30 seconds you have to read it continuously,
presence of alkaline detergent in container because it can have false positive result if you read
it after 1 minute
• N-Multistix SG reagent strip *Glucose – fastest, first to read

Single – only 1 pad/analyte Principle: Glucose oxidase or double enzymatic reaction
Multi test strips – used in lab *For other reducing sugar, use clinitest
*Use Non-reagent strip testing procedures such as Procedure for Reagent Tablets (CLINITEST)
tablets or liquid chemicals for questionable results and
highly pigmented spx(ex. taking Phenazophyridine Clinitest Reagent Tablets
compounds) - Test for reducing sugars, semi-quantitative test for
urine sugar
Semi quantitative – you have to grade the intensity
REAGENT TABLETS of the color
As the color intensifies = increase amt. of analyte
- back up during interfering substances in reagent strip - Principle: Alkaline copper reduction test(Benedict’s
testing Test) – but in tablets, it doesn’t need heat but do
• Urine (freshly voided or random specimen) not use plastic tubes because it produces heat.
Benedict’s test – use OH lamp to heat rgt 10 – 4 parameters + Bilirubin, ketones, Blood,
- (-) – blue color Urobilinogen, nitrite, leukocyte esterase
- (+) – brick red
11 – 10 pararmeters + ascorbic acid
• Always use accompanying test tube and dropper.

Other equipment may give incorrect results. Principles:
• Holding dropper upright, put five drops of urine into a
clean, dry test tube, rinse dropper. Then using the same
dropper, add 10 drops of water.
• Drop one Clinitest reagent tablet. Watch test carefully

until boiling stops for another 15 seconds longer. (Do not
shake the tube during this period).
• Now shake the tube gently and compare the color of

contents with color chart. If during the test a bright
orange color appears (even for a moment) and then
changes to brownish color, more than 2% sugar is
present. When this occurs, do not compare with color
chart but record as “over 2%”. Failure to watch test and
note “orange flash” may result in comparing final color
with color chart, misleading results.
• For estimation of sugar concentration above 2%, dilute

Glucose –Glucose Oxidase or double enzymatic reaction
urine being tested with normal urine (negative with
Clinitest tablet) and repeat the test. Multiply the results Ketones –Sodium nitroprusside (nitroferricyanide)
obtained by dilution to give percentage of sugar. reaction
ascorbic acid –added because it cause false negative

result for GLUCOSE and BLOOD
POST-ANALYTICAL PHASE
Protein –Protein error of indicators
Rapid Testing – using reagent strip and tablet
Blood – Pseudoperoxidase activity of Hgb
Traditional – using biochemical testing, through
experiments SG – Pka change(dissociation constant) of a
polyelectrolyte in an alkaline medium
Ph – Double indicator system of methyl red(4-6pH) and

2 manufacturer:
bromthymol blue(6-9)
Multi stix – manufactured by Siemens Healthcare
Bilirubin – Diazo reaction
Diagnostics
Urobilinogen – Ehrlich’s reaction
Chem strip – manufactured by Roche Diagnostics
Nitrite – Greiss reaction
- Both vary in chemical reaction, *specificity(ex.
acetone – only detected by Chemstrip) and leukocyte esterase – esterase
sensitivity
*Ph seen in all parameter

Parameters:
*Blood – only seen in 10 and 11 parameters
4 – Glucose, protein, SG, Ph
3.Blot the edge of the strip on a disposable absorbent

pad.
Semiquantitative – 1+…4+
4.Wait the specified amount of time for the reaction to
- Giving mg/dL present in some test areas
occur.
Interfering substances – gives false positive and false
5.Compare the color reaction of the strip pads to the
negative
manufacturer’s color chart in good lighting.
- Tests are mostly oxidation, clean your test tube
properly (there should be no contamination of
detergent) esp. in Blood Blank (demerit ng 3 days pag
mali wash huhu)
Errors Caused by Improper Technique
- Oxidizers
- Reducing substance – ex. Ascorbic acid (false 1. Formed elements such as red and white blood cells sink
negative on blood, bilirubin,? leukocyte esterase, to the bottom of the specimen and will be undetected in
nitrite and glucose) an unmixed specimen.
Ascorbic acid 2. Allowing the strip to remain in the urine for an

- F(-) Reagent strip (double sequential enzymatic extended period may cause leaching of reagents from the
system - Glucose oxidase) pads.
- F(+) Reagent tablet(copper reduction test) - the urine cannot be used for microscopic exam, that’s
why it is preferred to transfer urine in another tube if
Before leaving, the medical technologist must do all of there are other additional tests
the following: 3. Excess urine remaining on the strip after its removal
• Returning of materials, slides and microscopes from the specimen can produce a run-over between
chemicals on adjacent pads, producing distortion of the
• Disposal of wastes and disinfection with liquid Lysol or colors.
10% sodium hypochlorite of the area.
4.The timing for reactions to take place varies between
• The PPE of each individual should be removed properly. tests and manufacturers, and ranges from an immediate
These cannot be exposed outside the laboratory reaction for pH to 120 seconds for leukocyte esterase.
premises
5. A good light source is essential for accurate
interpretation of color reactions.
Post Lab: 6. The strip must be held close to the color chart without
actually being placed on the chart. Do not contaminate
Reagent Strips
the color chart by the urine sample.
• provide a simple, rapid means for performing
7. Reagent strips and color charts from different
medically significant chemical analysis of urine
manufacturers are not interchangeable.
• consist of chemical- impregnated absorbent pads
8. Specimens that have been refrigerated must be
attached to a plastic strip
allowed to return to room temperature(thawed) before
to reagent strip testing, (Specific gravity can easily be
affected, refrigerated – high SG)
Reagent Strip Technique
1.Dip the reagent strip briefly into a well-mixed

uncentrifuged urine specimen at room temperature. * Care of the Reagent Strip
2.Remove excess urine by touching the edge of the strip 1. Store with Desiccant in an opaque, tightly closed
to the container as the strip is withdrawn. container.(to avoid light and air)
2. Store below 30°C (Room temperature) to prevent Lymphocytes – WBC that have no esterase
moisture; do not freeze.
Trichomonas - cause false pos in leukocyte esterase
- strips are temperature dependent
- high temp can deteriorate the chemicals
3. Do not expose to volatile fumes, if there’s

discoloration do not use it
4. Do not use past the expired reagent strip.
5. Do not use if chemical pads become discolored .
6. Remove strip immediately prior to use.
7. Do not touch the chemical pads, because it can have

possible reactions because of sweat *Color/Principle
- Machine reads the reagent strip

- Improve the reproducibility – precision
Quality Control of Reagent Strips
• Strips must be checked with both positive and negative

Reflectance Photometry – checks the reflection of light
controls a minimum of once every 24 hours. (before and read by reflector
after shift)
- Increase light reflection = decrease color intensity =
• Testing is also performed when: negative
- Principle: color intensity is proportional
– a new bottle of reagent strips is opened
concentration of solute
– Questionable results are obtained
Ex. Glucose and Proteins are 4+, check reaction by

Confirmatory Testing – using diff reagent or technologies
duplicating the test
to check for the reaction
• Distilled h20 is not recommended as a negative control
- because it can still have reaction on other chemicals

Non-reagent strip testing procedures:
- only used as calibrator
• Tablets
• Liquid chemicals
Double Sequential:
1. Glucose Oxidase
2. Peroxidase Clinitest – for reducing sugar except for sucrose
- use to test for ascorbic acid(oxidizer),

Diazonium salt – cephalosporin(reducer)
Ketones/ Legal’s test -Principle: Copper Reduction, same with Benedict’s test
Ehrlich – PDAB Reduction of CuSO4 → Cuprous Oxide = (+ Brick Red)
Protein error – measures the Hydrogen Cause False (+)
Pseudo peroxidase – adopting reaction of glucose • Uric Acid

• Ascorbic Acid - test for acetone(ketones) bcoz it have glycine
Hygroscopic – tending to absorb moisture from the air

that’s why it needs to be stored in tightly closed container.
False (-)
- not valid result if not absorbed w/in 30 secs, use
• Detergent (opposite kapag reagent strip) new tablet
1. Place a thick glass test tube in a rack; add 5 drops of
urine.
+ purple/violet) same also in reagent strip)
2. Add 10 drops of distilled water to the urine in the test
tube. • Bilirubin
• Ketone
3. Drop one Clinitest tablet into the test tube and observe
• Leukocyte esterase
the reaction until completion (cessation of boiling).
4. CAUTION: The reaction mixture gets very hot. Do not

touch the bottom area of the test tube. Use thick glass 1. Remove the Acetest tablet from the bottle and place
test tube only. on a clean, dry piece of white paper.
5. Wait 15 seconds after boiling has stopped and gently 2. Place 1 drop of urine on top of the tablet.
shake the contents of the tube.
3. Wait 30 seconds.
6. Compare the color of the mixture to the Clinitest color
chart and record the result in mg/dL or percent. Observe 4. Compare the tablet color with the manufacturer-
for the possibility of the “pass-through” phenomenon. If supplied color chart.
present, repeat the procedure using 2 drops of urine 5. Report as negative, small, moderate, or large.
instead of 5 drops.
Pass Through Phenomenon – from blue turns to brick Contains:

red then returns to blue, • Sodium nitroprusside - *
Occurs when > 2 g/dL sugar is present • Glycine
Blue → Green → Yellow → Brick red → Blue • Disodium phosphate/Na2HPO4
To prevent pass through, use 2 gtt of urine • Lactose – add to give better color differentiation
Then add 10 gtt of h20 to dilute the urine
Ictotest
CLINITEST TABLET - confirmatory test for bilirubin, manufactured by
The tablet contains: siemens
• CuSO4 – main reacting agent - color of mat is the one to be checked
• NaCO3 – to eliminate the interfering O2 from room air • Blue to purple color + result
• light pink/no color – result*
• Sodium Citrate buffer – responsible for heat production
1. Place 10 drops of urine onto one square of the
• NaOH – responsible for heat production too absorbent test mat.
2. Using forceps, remove one Ictotest reagent tablet,

Acetest – mostly manufactured by Siemens recap the bottle promptly, and place the tablet in the
center of the moistened area.
3. Place 1 drop of water on to the tablet and wait 5 Chemstrip – can detect both acetone and aceto acetic
seconds. acid
4. Place a second drop of water onto the tablet so that Acetone can only be detected using Glycine (70% of
ketones)
the water runs off the tablet onto the mat.
Glucose and blood – use tetramethyl
6. Observe the color of the mat around the tablet at the
end of 60 seconds.
pH, Bilirubin, Urobilinogen, LE, Ketones – more sensitive

ang Multi stix
Contains: PSSB
•P-nitrobenzene-diazonium-p-toluene sulfonate(P-NB D
P-TS)
•SSA – sulfosalicylic acid
•Sodium carbonate
•Boric acid
Chromogen used:
• Potassium Iodide – green to brown

How to remember: Povidone Iodine (color green
bottle, brown liquid)
*use pnemonics/palatandaan para matandaan
• Tetramethylbenzidine – yellow to green
1.030 – use refracto
1.040 – dilute specimen
*CPAP tips – stock knowledge pag nag exam tapos check

yung mga di na maalala yun ang reviewhin
*Multi stix – only Sodium nitroprusside, can’t detect Albumin – normal in urine except if may orthostatic
acetone, only aceto acetic acid proteinuria
BIOCHEMICAL • Add 3-5 drops of 5% acetic acid and heat again.
EXAMINATION OF URINE
• If albumin is present, the urine becomes cloudy; the
greater the cloudiness, the larger the amount of

albumin.
PRE-ANALYTICAL PHASE • Record the results according to the accompanying table
• The site of the experiment should be sterilized. The for heat tests.
• Upon receiving a urine sample, the medical
technologist checks if the sample is properly
labelled and subjects it to different qualitative tests
for protein and glucose.
Positive result: White Cloud ppt
Qualitative tests for Protein
Heat and acetic acid test Exton’s Test Or Sulfosalysilic Acid Test (SSA)
• Urine (freshly voided or random specimen, - to detect proteins other than albumin, such as Globulin
(not detected by reagent strip, (-) in RS and (+) in SSA)
preferably first morning specimen)
Proteinuria is caused by other types of proteins like
• Test tube holder
Globulin
• Alcohol lamp
• Urine (freshly voided or random)
• Test tubes
• Test tube holder
• 5% acetic acid
• Alcohol lamp
Acidification is needed to prevent formation of soluble
• Test tubes
acid and precipitation of carbonate and phosphate on
alkaline medium • Exton’s reagent - composed of Na2SO4, 3 % SSA and
H20
Macroglobulin positive in Exton?? Check in book ghorl

Procedure
PROCEDURE
Principle: proteins are denatured and coagulated on
heating to give a white cloud ppt • Principle: negative charge of SSA neutralize positive
charge of protein causing denaturation → ppt formation
Frederick eckers – white ppt bcoz of albumin
Cause False (+)
▪ Hematuria – bcoz of Hgb(contains Globin, a

• Fill a test tube 3/4 full of a clear or centrifuged urine.
protein)
• Gently heat the upper inch or so to boiling. Leave the ▪ radiographic contrast media
▪ sulfonamides
lower portion unheated for contrast. ▪ salycylates
▪ penicillins
▪ Highly conc. urine • Place a small quantity (3-5 ml) of pure nitric acid in a
▪ excess uric acid test
False (-) tube.
▪ highly diluted urine • Stratify a small amount of (2-3 ml) urine on the nitric
acid.
• Centrifuge urine sample. *Incline the tube when adding urine, so HNO3 will not be
disturb
• Pipette 3 mL of urine then add 3 mL 3% sulfosalicylic
acid. • A white ring(junction) at the point of contact indicates
the
• Invert to mix and let stand for 10 minutes then mix by
presence of albumin.
inverting twice.
• Record your results according to the accompanying
• Using ordinary room light, observe for the degree of
Table for ring tests.
precipitation and grade the results.
+ Result: White ring at the point of contact
Results for Heat Tests
Robert’s test
Robert’s rgt – combination of saturated MgSO4 and H20,
suitable for precipitation of proteins
Protein range/sensitivity
*Centrifuge urine if cloudy.
4+ is alarming, report it right away
• Place 1 mL of Robert`s reagent in 13 x 100 test tube.
• Stratify the reagent with 1 mL of urine.
• Record result.
White ring at the point of contact of the two fluids
indicates albumin. The larger the ring, the greater the
amount of albumin
Heller’s test or Hellers Nitric Acid Ring Test
Conc. HNO3 – causes denaturation and precipitation of

proteins
coagulum formed and report in gm%. Height of

coagulum is
the number of grams albumin in 1000 mL urine; obtain

the
amount of albumin in gm%, divide by 10
3. Read according to the plus system below and report in
gm%
*wait for at least 5 minutes for appearance of ring
Results:
+ No definite flocculation, cloud is distinguishable but not

granular
++ No definite flocculation, cloud is distinct and granular.

Quantitative tests for Protein Represents 0.04-0.1 gm% (height of coagulum) albumin
Kwilecki’s Modification of Esbach’s method +++ Marked flocculation with dense cloud. Represents
E. rgt – contains 1 g of Picric acid and 2 g of Citric Acid 0.2-2.3 gm% albumin
Procedure: ++++ Heavy thick precipitate almost to boiling solid.
1. Fill the esbach’s tube to mark “U” with urine. Represents 0.5 gm% albumin or higher, 3 gm% albumin
2. Add 10 drops of 10% FeCl3 (ferric chloride solution) boils solid.
3. Mix. - powdery, almost to boiling solid
4. Fill the tube to the mark “R” with Esbach’s reagent
5. Place in water bath at 72’C for 5 minutes. Qualitative tests for Sugar
1. BENEDICT’S TEST
– commonly used, color BLUE rgt
Kingsbury Clark Method Glucose reduces Cu3+ to Cu2+ (Copper reduction
test)
- use 3% SSA
in hot alkaline
• Procedure: - CuSO4, NaCO3, Na Citrate or Citric Acid,
NaOH(strong alkaline)
1. Pipet 2.5 mL of centrifuged urine into a test tube
graduated at 10 mL and add 3% sulfosalicylic acid to the Na Citrate – prevent the precipitation of copper?
10 mL mark. - aside from reducing sugar can also detect

2. Immediately after heating, read the height of the metabolites such as:
▪ gluconic acid
▪ Homogentisic acid
▪ uric acid
▪ Creatinine
▪ drugs such as ascorbic acid(reducer)
*all cause False (+) result
False (-) Detergent(oxidizer)
Sensitivity: 200 mg/dL (less sensitive than rgt strip test)
Trace – may green opacity/solution with no or green ppt/
1+ if green opacity w/ yellow ppt or yellow green ppt • Procedure:

2+ if green to yellow solution w/ yellow ppt
1. Mix equal parts of Fehling`s solution A and B in a test
3+ if muddy orange color w/ yellow ppt
tube.
*yellow ppt – presence of sugar
4+ orange to brick red ppt 2. Dilute this mixture 2 or 3 times the amount of water
and boil for a few seconds
*if too much heat, solution can splash
3. Continue boiling, adding urine drop by drop until
*pass through phenomenon only in clinitest tablets
about 5 mL has been added.
4. Refer to the accompanying table for interpretation of

PROCEDURE:
results.
• Place 5 ml of Benedict’s solution in a test tube
• Add 8-10 drops of urine and mix
• Place the test in boiling water for 2-3 minutes
• Let stand until cool.
3. NYLANDER'S METHOD
- to detect presence of sugar
0.5 mL N Rgt – combination of Rochelle’s Salt(potassium

sodium tartrate tetrahydrate ) + Bismuth Subnitrate
+ NaOH
+ KOH
2. FEHLING’S TEST PROCEDURE:

Fehling’s Rgt – *CuSO4 is still the main rgt 1. Place 5 ml of urine in a test tube
- contains KOH and Sodium potassium tartrate in place 2. Add 0.5 ml of Nylander’s reagent
of NaCO3 and Na Citrate in Benedicts test
3. Heat for 3-5 minutes
- not used anymore bcoz less sensitive and less specific
4. Allow to stand for few minutes (let it cool)
- cause Caramelization – when we use highly alkaline
solution
Before leaving, the medical technologist must do all of

the
following:
•Returning of materials, slides and microscopes
•Disposal of wastes and disinfection with liquid Lysol or
•The PPE of each individual should be removed properly.

These cannot be exposed outside the laboratory
RESULT premises
• Black indicates presence of sugar
• Brown indicates trace amount of sugar
• No change of color: negative (colorless)

BIOCHEMICAL
*If solution turns black after cooling, reaction due to EXAMINATION OF URINE
substances other than sugar
(Test for other sugars)
*monitor the color while heating

4. MOORE HELLER’S METHOD medical technologist should wear proper personal
- using 10% KOH only
• Upon receiving a urine sample, the medical technologist
• Procedure:
checks if the sample is properly labelled and subjects it to
1. Pipette 2 mL of urine into 13 x 100 test tube. different qualitative tests for fructose, lactose, and
pentose.
2. Add 1 mL of 10% KOH.
3. Boil the upper part of the mixture for 2-3 minutes.

FRUCTOSES
1. SELIWANOFF’S TEST
*If phosphates are present in large amount, filter
• Principle: when heated w/ conc. HCL, fructose forms
the preparation and examine the filtrate. Oxymethyl furfurol(due to Hydrolysis of fructose) that
gives RED color in combination with rescorcinol
- Can also use to check presence of fructose in semen esp.

RESULT AND INTERPRETATIONS(Qualitative)
in low motility
1 % or less Canary yellow
For fructose only not for other sugar?
1 - 2 % Wine yellow
PROCEDURE:
2 - 3 % Cherry yellow
1. 1.5 ml of urine in test tube
3 - 4 % Rum yellow
2. Add 2 ml of Seliwanoff reagent(conc. HCL and
>4 % Dark brown or black color rescorcinol) - gives the red color)
3. Heat
POST ANALYTICAL PHASE

- use Lead Acetate and Ammonia H20? as main

reacting agent
PROCEDURE:
1. 10 ml of urine, add 3 g lead acetate powder then shake
2. Filter off the precipitate and heat the filtrate for
few minutes (yellowish-brown appears)
3. Add ammonium hydroxide and continue heating
RESULT: 4. Let it cool upon heating
•Positive result - red and precipitates a dark sediment
•Negative - no change of color (colorless)
2. BORCHARDT’S TEST
– uses rescorcinol and 25% of HCL, same procedure
with Seliwanoff’s
Red color – presence of fructose
PROCEDURE:
RESULTS:
1. Take 5 ml of urine in a tube and add 5 mL of 25% HCl.
• red color with red precipitate: presence of lactose
2. Add a few crystals of resorcinol and boil for not more
than half a minute. A red color represents fructose. • Red color with yellow precipitate: glucose
• Negative: milky appearance with flocculation
PENTOSES
Ex. deoxy ribose and ribose
1. BIAL ORCINOL METHOD
- HCL and 10% FeCl3
RESULT: PROCEDURE:
•positive result - red and precipitates a dark sediment 1. Boil 5 ml Bial’s Orcinol reagent in a test tube
•Negative - no change of color (colorless) 2. Remove from the flame and add urine drop by drop
until no more than 1 ml urine has been used
LACTOSES
- esp. in lactose intolerance
1. RUBNER’S METHOD
BIOCHEMICAL EXAMINATION OF
URINE (Special Urinary Test)

• Upon receiving a urine sample, the medical technologist

checks if the sample is properly labelled and subjects it to
different qualitative tests for ketones, bilirubin, blood,
RESULTS:
indican, electrolyes such as calcium and chloride.
•Positive for pentose turns green immediately after
addition of urine
KETONES
•Negative –no reaction (yellow)
- significant for Px with diabetes and diabetic ketoacidosis
- increase activity of lipolysis could lead to = ketosis or

ketonuria
3 Ketone bodies:
the
▪ B-hydroxybutyric acid – most numerous, cannot
following:
determine by usual tests unlike the other
•Returning of materials, slides and microscopes ketones.
▪ Acetoacetic acid
•Disposal of wastes and disinfection with liquid Lysol or ▪ Acetone
•The PPE of each individual should be removed properly. 1. Legal’s Test (Acetone)
premises Principle: In reaction with the –NO group, sodium
nitroprusside, acetone and acetoacetic acid form
isonitroacetone which remains trapped in the complex
Quiz: anion
Reflectance photometry - decrease reflection = high (+) Acetone: purple or violet red colored complex
concentration (+) Acetoacetic acid: red
Procedure:
1.To few mL of urine, add enough NaOH or KOH solution
to render it slightly alkaline.
2. Add a 2-3 gtt/few drops of 10% sodium nitroprusside

solution(from red → yellow)
3. Add a few drops of conc. acetic acid to side of test tube

(yellow → violet)
4. Observe the result and record as positive or negative. PROCEDURE:
- Check the line/junction of the 2 fluid to form a Carmine To a 5 ml of urine in a test tube, add drop by drop
or purple ring
10% ferric chloride until no further precipitation occurs
(+) Bordeau red
(-) rusty color or no reaction
2. Gunning’s Test
Test only for acetone,
Principle: Reaction of acetone bodies and alcoholic iodine

→ form iodoform crystals *w/ the addition of Sulfuric acid with ether and ferric
chloride, the test will be more specific and the color turns
(+) Yellow ppt
to violet red ?
Procedure:
BILE PIGMENT
1. Pour 5 mL of urine into a test tube
To detects bile leakage – excrete bilirubin in feces or
2. Add 5 drops of strong ammonia urine
3. Add Lugol’s solution(alcoholic iodine) enough to 1. Smith’s Test

produce a black cloud which does not disappear
Principle: oxidation of bile pigments/bilirubin with acid,
immediately.
bilirubin(yellow) → biliverdin(green)
4. Let stand for a few minutes.
Procedure:
5. Take a small quantity of the urine sediment and
1. Take 5 mL of urine in a test tube.
examine microscopically. Formation of Iodoform crystals
appear as yellowish six-pointed stars or six-sided 2. Overlay the urine with the tincture of alcoholic iodine
plates(under microscope)
3. Observe the result and report as positive or negative
6. Report as positive or negative.
3. Gerhardt’s Test
For the presence of acetoacetic acid/di acetic acid
only
- non-specific test bcos ferric chloride can also use for
amino acid products and ketones that can result to
interferences(false pos or neg)
Principle: Urine is acidified w/ Sulfuric acid with ether and

ferric chloride (oxidize the acetoacetic acid)
(+) emerald green ring at the point of contact bet. the 1. Place 20 drops of urine.
urine and iodine tincture
2. Add 1 drop of potassium dichromate as an indicator.
(-) yellow in color
3. Finally, add only 16-20 drop of 2.9 % silver nitrate
solution until permanent distinct red brown is produced.
2. Harrison’s Spot Test >20 gtt of HgNO4 and 12 gtt in Potassium Dichromate
indicates Hyperchloremia
Principle: oxidation of bile pigments/bilirubin with using
FeCl3 and TCA The number of drops required to produce reddish
brown color expresses the amt of chloride present in the
Procedure:
urine in gm/L.
1. Take 5 mL of urine and 5mL of 10% barium chloride
*Increase Na = inc Cl
solution in a test tube. Mix and stand for a few minutes.
2. Filter. Spread the filter paper bearing the precipitate on
another piece of dry filter paper.
3. Place 3 drops of Fouchet`s reagent on the

precipitate.
F. rgt - (combination of 0.9% FeCl3 and 25%
Trichloroacetic acid)
4. Observe the result and report as positive or negative.
(+) Blue to green color ppt BLOOD
(-) yellow, no change in color 1. Benzidine Test
For the determination of blood(RBC or Hgb) in urine
Also for hidden/occult blood
Principle: Pseudo peroxidase activity of Hgb

decomposes H2O2, it will release O2 which in turn oxidize
the benzidine to give the GREEN or BLUE color(presence
of blood)
Procedure:
1. Prepare about 3 mL saturated solution of benzidine in

CHLORIDE glacial acetic acid(to acidify urine, already contained in
solution) in a test tube.
1. Fantus Test
2. Add an equal volume of 3% hydrogen peroxide, mix
Principle: Silver nitrate reacts with chloride in the urine thoroughly.
forming silver chloride ppt
3. Should the solution turn green or blue, it must be
(+) brick red color of solution (no ppt) discarded.
*If only few chloride, Excess Silver nitrate reacts to 4. Add about 1 mL of urine and shake.
potassium dichromate(serves as an indicator only)
(+) green or blue color indicates the presence of occult
that’s why red ppt is formed
blood.
Procedure:
Sulkowitch rgt – contains Oxalic acid, Ammonium

Oxalate, Glacial Acetic Acid, D. H2O
Procedure:
1. Pipette 3 mL of urine specimen. (Centrifuge urine if not

clear).
2. Add equal amount of sulkowitch reagent.
3. Mix and allow to stand for few minutes.
4. Observe and record result obtained.
(+) Turbidity or Precipitation

2. Guiac Test
Procedure:
1. To a small amount of powdered guiac in a test tube,
add 2 mL of 95% alcohol and mix → Guiac’s solution
2. Mix 5 mL of hydrogen peroxide with 5 mL of the guiac
solution prepared above.
3. Make the urine strongly acidic with acetic

acid(nakahiwalay sa solution)
4. Apply the ring and contact test.
(+) Blue ring at the junction of solution RESULT INTERPRETATIONS:
CALCIUM /100 ml of urine?
1. Sulkowitch Test INDICAN

- Semi Quantitative Test Formed due to degradation of certain amino acids
- Checks the Calcium level of body excreted in urine (Tryptophan) then converted to Indole(by the Gut
- To check the digestion or absorption of Calcium bacterium in the intestine) → Indican(consist of 3-
hydroxy indole/indoxyl)
Principle: Urine mix with the buffered oxalate solution or
Sulkowitch rgt and the intensity of the ppt will have the Indole converted to Indican(happens in liver)
turbidity approximately proportional to the Calcium Blue Diaper Syndrome – presence of Indican in the urine
concentration. of infant
Qualitative Tests: 4. A 0.5% KMnO4 may be used in place of calcium

1. Obermayer’s Test hypochlorite if preferred. Five drops of KMnO4 are used
with 10mL of urine.
- Efficient screening tool for presence of indicans esp. in
adults for Dysbiosis(a disorder, bacterial imbalance of Blue or chloroform droplets that sinks to the bottom if
normal flora in the gut or intestine) indicant is present
Principle: when subjected to acid hydrolysis, urine (+) Blue color indicates indican
indican liberates the indoxyl and then oxidize by
obermayer’s rgt

Procedure:
the
1. To 5 mL urine in a test tube, add 5 mL Obermayer’s
following:
reagent(composed of indoxyl sulfate and FeCl3 solution
in conc. HCl) •Returning of materials, slides and microscopes
2. Mix and add chloroform to settle. A blue coloration of •Disposal of wastes and disinfection with liquid Lysol or
the chloroform indicates the presence of indican. 10% sodium hypochlorite of the area.
•The PPE of each individual should be removed properly.

premises
*This tests aren’t used now but for the sake of BOARD
EXAM HAHAHAHAH
*Reaction/Principle and positive result are important!
(+) Blue discoloration (Dark blue means higher # of

indicans in the urine) MICROSCOPIC EXAMINATION
(-) clear or yellow(no change in color) OF URINE
2. Jaffe’s Method • The site of the experiment should be sterilized. The

- to check for indicanuria medical technologist should wear proper personal
Procedure:
• Upon receiving the urine specimen in the laboratory,
1. To a small amount of urine in a test tube, add an equal
the receptionist checks if the urine container is properly
volume of HCl
labeled then transfer it into a test tube for
2. Mix and add 10-15 drops of chloroform. centrifugation.
3. Add drop by drop a strong fresh solution of calcium

hypochlorite shaking after the addition of each drop →
ANALYTICAL PHASE
blue or purple chloroform droplets that sink to the
bottom if indicant is present Materials:
• Urine (freshly voided or random specimen, preferably • Refrigerated urine preserve almost all cellular
first morning specimen) elements, crystals, and cast, very well
• Slides and cover slips *Formalin can also be used in urine
• Microscope • Fresh and concentrated urine: more accurate

results
• Hypotonic urine causes lysis of cells and casts

ANALYTICAL PHASE
Hypotonic – decrease solute → low specific gravity
Receiving area – spx should be sufficient, reject or
accept spx - causing swelling and lysis of cell
Internship: prone to demerit • Proper collection avoid extra debris of the urethral
meatus, vaginal secretions
Routine urinalysis – 10-15 ml of urine
*clean the opening of urethra
• Proper use of subdued light in LPO before HPO: for

Procedure:
accurate results
1. Thoroughly mix your urine specimen.
• Examine the urine specimen within 1 to 2
2. Pour 10 mL of the urine into a centrifuge tube or any hours(after 2 hrs most of the elements will be
test tube used for that matter. lysed/disintegrated)
*Conical tube are preferred, bcos most of the

sediments will settle at the bottom, it will provide
What are the sources of errors in handling urine?
adequate volume
• Careless transfer of sediment (contamination)
3. Centrifuge for 5 minutes at 1500 rpm.
- Use cleaned pipettes
4. Pour off the supernatant liquid. Sufficient urine
remains in the tube to suspend the sediment. • Too much light (retractile bodies cannot be seen)
*Should be fully decanted (what’s left: 0.5 to 1 ml (500 • Using the high power only
uL to 1000 uL) of urine and conc. formed elements)
*some elements can only be seen in LPF bcos they are
5. Shake the tube and transfer a drop on the slide. The big
drop should not overrun the cover slip and there
Ex. Squamous Epithelial Cells
should be no air bubbles since both tend to alter the
results. Renal Tubular Epithelial (RTE) Cells – viewed in HPF,
pathologic, smaller
*drop usually 20 um in slide
Transitional Epithelial Cells
*should be no bubbles(became artifacts and mistaken
as cells), should not be overflowing and covered by • Specimen dries up only on long standing (false
the cover slip elements are seen) or prolong refrigeration
6. Examine under the microscope using LPO first • Dirty equipment – glass tubes, washed properly to
before the HPO. avoid detergent contamination
• Scratches on slides – can look like crystals in the

microscope
Role of Microscopic Examination of Urine
• Determine the correctness of diagnosis of a renal

system infection and disease
Preparation and examination of urine sediments Tachometer or strobe light – to calibrate

centrifuge
• Specimen preparation –
• Formula:
• Specimen volume
• Centrifugation
• Sediment preparation
• Volume of sediment examined
• Use of braking mechanism can cause
• Examination of the sediment disruption of the sediment.
• Reporting of results • All specimen must be centrifuged in capped
• Correlation of results tubes to avoid biohazardous aerosols
Specimen preparation Sediment preparation

• Specimen should be examined while • Volume of 0.5-1mL(decant) are frequently
fresh/adequately preserved. Test w/in 2 hrs used.
Hyaline casts will easily be disintegrated in dilute • To maintain a uniform sediment
alkaline urine* concentration factor, urine should be
aspirated off rather than poured off
• Refrigeration may cause precipitation of *Pasteur pipettes can be used if there’s no
amorphous urates(pink sand) and phosphates. automatic pipette
- should be warmed in 37’C before centrifuge to • Sediment must be thoroughly resuspended by
dissolve the crystals gentle agitation/mixing
- using commercial system pipette or by
• Mix the specimen prior to decanting a portion repeatedly tapping of tube w/ finger
into a centrifuge tube. • Vigorous agitation should be avoided, as it may
disrupt some cellular elements.
- concentrated ang nasa bottom
Volume of sediment examined

Specimen Volume
• Conventional glass-slide method: transfer 20µL
• Standard amount of urine is about 10-15mL.
(0.02ml)
• 12mL volume is frequently used - multi strip
• Glass cover slip: 22x22mm
is easily immersed in this volume
• Overfilled will cause loss of heavier elements
• If obtaining 12mL is not possible, volume of
(casts) – seen on the edge of glass cover slip,
the specimen used should be noted in result
hindi makikita
or report form
Ex. UTI pediatric px(3-4 ml) – can be
accepted but should be noted! Corrected by
physician and also by medtech Examination of the sediments
Ex. 6ml urine centrifuged x 2 = 12 ml • Examined both LPF and HPF minimum of 10
fields.
• LPF: Cast, Epithelial cells, mucous threads,
Centrifugation abnormal crystals
• Centrifugation of specimen for 5 mins at 400 • HPF: WBC , RBC , yeast cells, there are also E.
(RCF) or 1500 Rpm cells
• RCF (relative centrifugal force) rather than
RPM (rotation per minute) is used.
Reporting:
LPF
• Cast average or range/lpf

Ex. 20/lpf in cast
• Epithelial cells, crystals and other elements are
frequently reported in semi-quantitative terms.
Epithelial cells reported in: occasional, few,
moderate, many(4+), too numerous to
count(TNTC)
HPF
• Rbc average/hpf
• Wbc average/hpf
• RTE average/hpf
• TC semiquantitative (rare, few, moderate,
many)
RBC
– smooth, non-nucleated discoid shape, smaller

Correlation of Results:
compared to WBC
- Microcytic and Crenated/shrink(hypertonic) RBC are

usually seen in urine
*Ghost cell(hypotonic) - swell or hemolyzed
NV:0-2 or 0-3/ HPF
Hematuria – presence of RBC
- help you to analyze the formed elements
Turbid – very high amt of formed elements
Crystals – very important to check the pH(basic and

acidic) Dysmorphic RBC due to Glomerular membrane damage
- RBC will force/push themselves in glomerular
Ex. Urates – acidic, Phosphates - alkaline membrane
*Glomerulus -filters the blood
Cells
WBC – nucleated, multilobed
Budding yeast – can be mistaken as RBC
WBC – highly refractile, granulated, w/ lobes
RTE cells– most clinically significant epithelial cells,

usually came from the nephrons, either rectangular,
polyhedral, cuboidal or columnar in shape
- “eccentric nucleus”/nasa side
2 variations of RTE:
1. Oval fat bodies – RTE w/ fat globules, present in

lipiduria or in nephrotic syndrome
Epithelial cells – largest cell, pyknotic(small and dark - viewed with Oil red O and Sudan 3(neutral stain
nucleus), very visible most commonly used)
2. Bubble cells – looks like oval fat, RTE w/ non-lipid
filled vacuoles, butas butas na RTE, seen in Renal
tubular necrosis
*Malaki na bilog(right) – Urothelial or Transitional cells –

usually seen in the bladder, “centrally located” nucleus,
polyhedral or caudate(may buntot) in shape
To differentiate: Check Nucleus:

• Squamous – pyknotics/small and dark nucleus
• Transitional – center
Air bubbles or oil droplets – no formed elements
• RTE - eccentric
- prevent using cotton swab when cleaning the slide
bcos cotton contains oils
WBC
– larger than RBC
Pyuria – presence of WBC in urine
RBC – smaller compared to WBC Normal: 0-5 or 0-8 per HPF
In Hypotonic sol:
RBC can become ghost cells
WBC can become Glitter cells(Brownian movement) and
misidentified as parasites(Trichomonas vaginalis)
Neutrophil – most commonly found in urine, Granulated

and multi lobed(3-4)
To differentiate if UTI or Drug induced interstitial
nephritis RTE cells (Under HPO)
- use Hansel’s stain “mukhang centrally located” so check the nucleus - may
- presence of nitrite(chemical exam) – screening test for granules and striations on side
UTI
Also WBC and RBC around
Eosinophils
N: <1%
>1 means clinical significant, seen in Drug induced
interstitial nephritis(allergy with certain medications
such as corticosteroids), Basophils also increase
RBC, crenated RBC(hypertonic urine)
Eosinophils using Hansel stain(bind in granules)
Intact RBC
Ghost cells – they lost the Hgb content, but there’s still
membrane, seen in hypotonic urine(low specific gravity
in physical exam.)
WBC treated with 2% acetic acid(to lyse the RBC)

Transitional EC in caudate shape

– checked under HPF, reported as semiquantitative
(rare, few, moderate, many)
Squamous Epithelial cells – largest cell, reference cells
for focusing microscope, check in LPF
- have a pyknotic, thick nucleus, irregular shape of
cytoplasm, can fold and mistaken as hyaline cast
WBC or glitter cells bcos of hypotonic sol (right)
Kova – brand of Sternheimer malbin(composed of

crystal violet and safranin O)
*Color of hyaline cast in Kova stain – PINK (board exam)
Eosin stain or Hansel stain – composed of eosin Y and

Squamous EC, WBC and RBC methylene blue, stains eosinophilic granules and identify
it
Squamous EC, RTE, Transitional EC in caudate shape

(left to right)
Clump of Squamous EC (under LPF)

RTE - very unique to kidney, formed in Distal CT and

Left up: Columnar shaped RTE with Oval fat bodies collecting duct, Cylindrical or Curve
Lower right: seen in Phase Contrast Microscope Tam Horsfall protein – major constituent of cast,
produce by RTE(found in DCT and CT)
3 shapes of RTE (depending on which renal tubule they
came from) Cylindroid - Cast w/ tail
Columnar: from proximal convoluted tubules
Oval: from distal convoluted tubules Cylindruria – presence of Cast or Cylindroid in urine
Cuboidal: from collecting duct
Oval Fat Bodies
Hyaline cast under LPF(hindi na kita)
oval fat body(checked in average per HPF)
Amorphous urates(crystals) - dust-like, in acidic urine
stained using sudan 3
Hyaline cast
in polarizing microscope showing Maltese cross

formation(bcos of presence of cholesterol) - also seen in
starch crystals
Cast
Hyaline Cast under phase contrast(to observe the and presence of pre-RBC(for identification)
structure)
*Cellular cast became Coarsely granular cast
Mixed Cellular Cast and cocobacilli and bacilli in

Cast w/ Hgb pigment
background
- seen in px with Glomerulonephritis(can also see WBC,
RBC, mixed cellular cast) and Pyelonephritis(infection in
kidney, can see WBC and bacterial cast, RTE cast)
To check if cast or just clump of cells:

1. Check the Cast matrix - shadow around or supot ng
cell
Different types of Cast
Mixed cellular cast – contains either RBC and mostly

WBC
Cast w/ hypochromic and dysmorphic pre-RBC around
Mixed cellular cast
Kova stained Cast under Phase contrast microscope
Disintegrating WBC cast(should have WBC on the side)

*If Disintegrating RBC cast – may RBC sa side
Coarsely granular cast - disintegrating RBC cast

RTE cell cast
Kova stained WBC cast

Covas stained
Clump of WBC/ WBC cast RTE cell cast w/ bilirubin stain(to confirm if true RTE
No cast matrix cast)
RTE cell cast
RTE cell cast that contains fat globules
RTE cell cast (sternheimer malbin stained) and several

free floating RTE cells
Artifact: glass fragment(yung color green)

WBC cast
- Assc. w/ Pyelonephritis
*Acute interstitial nephritis can also have WBC
cast(neutrophils), due to irritation in nephrons or renal
tubules, RTE produce Tamm Horsfall protein(catch the
bacteria and cells)
Compact RBC cast – contain biconcave shaped cells

Oval fat bodies in hyaline cast stained w/ Sudan 3
Orange red color in Sudan – Triglycerides
Fatty cast
- assc. w/ nephrotic syndrome(Albuminuria and
Lipiduria) or in Toxic Tubular necrosis
Coarsely granular cast
- proteins aggregate
Fatty casts Coarsely granular cast with squamous cells and some
Lower: under phase contrast mucous threads
Right: granular cast turning into waxy cast
Fatty cast loaded with fats

Cholesterol Crystal – yung arrow
Hyaline cast and RBCs

Hyaline cast that contain purely tamm Horsfall

Broad cast and Broad casts w/ cracks
CRYSTALS
curved hyaline cast w/ calcium oxalate
Monohydrate – mostly dumbbell shape
Hyaline cast(left) di kita

Waxy cast(right) - medyo shiny
Triple phosphate – “fern leaf appearance” (board

exam)
Waxy cast
Broad cast w/ cracks turning into waxy cast(seen in
renal failure)
Ammonium biurate – thorny apple, radial

concentric striations, formed during ammoniacal
fermentation
Amorphous urate - brick red color
Seen in ph 5.7-7.0
Small, yellow brown like sand, appears in clumps,

assemble in granular cast, but no matrix
Ammonium biurate w/out spikes
- yellow to golden brown color
Left: Amorphous phosphates – sand or chalk like,

colorless
- Found in alkaline, dissolve in acetic acid but not

by heat Bilirubin crystals
– golden yellow color
Monosodium urate, needle like, seen in px with

Gout, pencil like prism, reported as “urate crystal”
Calcium carbonate crystals – produce gas after

addition of acetic acid
- looks like an airplane

CaPO4 or MgPO4 aka Appatite - colorless plates,

flat, rectangular shape, often in rosette form
- confused w/ Sulfonamide crystals esp. if neutral

Calcium oxalate crystal(either di or monohydrate) pH of urine, cannot be dissolved in dilute acetic
acid
▪ Dihydrate or Weddelites – common,
envelope or octahedron shape - CaPO4 – dissolved in D. acetic acid
▪ Monohydrate or Whewellites –
uncommon, dumbbell and ovoid
rectangle shape
- Soluble in HCl
Right: In polarizing micro:
Monohydrate – blue
RBC - dark pigments Cholesterol crystal – w/ notched plate

appearance, assc. w/ Lipiduria, soluble in
Chloroform
-“staircase pattern”
- seen together w/ oval fat bodies and fatty cast
- resembles the Radiographic contrast media,

check the specific gravity of urine to
identify(1.040), highly dense
URIC ACID
Cystine crystals - hexagonal in shape
– to confirm: + in Cyanide nitroprusside test

Uric acid
- soluble to ammonia and D. HCl
- 4 sided, rombic or diamond in shape
– assc. in increase purine metabolism esp. in Px

w/ Leukemia undergoing chemotherapy
Seen in Orange diaper syndrome in Leisch Nyhan

Syndrome
Increased in Gout
Triple phosphate crystals – aka Ammonium Uric acid have many forms: 4 sided, rosette
magnesium phosphate, soluble in dilute acetic formed, wet stone, lemon shape, cube
acid
- aka Struvite, colorless modified prism w/

oblique ends, “fern leak like appearance”, coffin
lid like
- presence mean possible to have stasis of urine in

kidney, form kidney stone
Uric acid in cube form

Looks like sugar
Leucine crystals Squamous epithelial cells w/ Gardnerella

– have concentric striations, mas dark ang nucleus vaginalis(bacteria) or “Clue cells”
compared to transitional and RTE
- highly refractile, usually yellow or brown circle,
concentric striations or scallop like striations
- mistaken as fat globules
- nipple like
Transitional epithelial cells
RTEs – cuboidal in shape
Tyrosine crystals
- cross at various angles, appear in black center or
needle in cluster
X-ray contrast media

– check the SG(1.040, high)
- soluble in 1% NaOH
Dihydrate calcium oxalate w/ rod shape bacilli
Sperm and some bacteria(streptococci)

Trichomonas vaginalis Reported as: positive or negative or semiquantitative
- yellow: anterior flagella
Red: axostyle
- movement mistaken as Ghost cell RBC(Brownian
movement) because of darting movement
Air bubble and RBCs

Add 2% acetic acid → nag-lyse means RBC
Budding yeast and Ghost cell RBC
Diaper fiber –
very detailed structure
mistaken as hyaline cast(low fractive index and not
Yeast w/ mycelial forms usually in diabetic px bcos of
detailed)
increase sugar in urine
Starch granules
Mucous thread - looks like RBC
–contain tamm Horsfall as its major constituent - show maltese cross appearance in polarize microscope
- very long strand
- mistaken as clot fibers of underwear Arrow: glass fragments
Hemosiderin – protein, storage of iron
Hyaline Cast – unang stage ng Cast
Sudan 3 – specific stain for Triglycerides
Glitter cells/WBC in sternheimer malbin is blue color*

transitional and Casts – ave range/lpf
Crystal – hpf but semi quantitative*
Reporting of result
• QUANTITATE an average of 10 representative fields.

• Do not quantitate budding yeast, mycelia elements,
trichomonas or sperm, but do not avoid ? their
presence.
Post Analytical Phase
• Before leaving, the medical technologist must

do all of the following:
• Returning of materials, slides and microscopeso
• Disposal of wastes and disinfection with liquid
Lysol or 10% sodium hypochlorite of the area.
Nomarski – differential interference microscope
• The PPE of each individual should be removed
Hoffman – modulation microscope properly. These cannot be exposed outside the
laboratory premises.
Urinary Screening Test for 1. Place 1 mL of urine in a tube
Metabolic Disorders
2. Slowly add five drops of 10% ferric chloride
3. Observe color for a permanent blue-green color

- defect on how the body breaks down
food/metabolism
- usually a genetic disorder
- usually done in newborn screening, series of simple

test to diagnose metabolic disorder(6 diseases)
G6pd – popular in Ph
Urine organic acid, amino acid – Aminoaciduria
Urine spot test
Pre-Analytical Phase
The site of the experiment should be sterilized. The

protective equipment before the experiment. *Permanent – di na nawawala
*Transient – after prolong standing, nagbabago na
ang color/bumabalik sa dati
• Upon receiving the urine specimen in the laboratory, Acdg Henry’s: Confirmatory Test for phenylketonuria:
the receptionist checks if the urine container is Ion exchange High Performance Liquid
properly labelled and proceed to screening test Chromatography
1. Ferric Chloride Tube Test 2. Nitroso-Naphthol Test for Tyrosine
Most commonly used screening test for most inborn - screening test for Tyrosinuria – presence of tyrosine,
error of metabolism rancid butter odor of urine
▪ Alkaptonuria - automatic na may Tyrosenemia

▪ Tyrosyluria
Tyrosiluria?
▪ Argentaffinoma
▪ Melanuria 1. Place five drops of urine in a tube.
▪ Indicanuria
2. Add 1 mL of 2.63N nitric acid.
▪ Phenylketonuria
3. Add one drop of 21.5% sodium nitrite.
aside from this test, can also used Phenistix – rgt pad
used for the presence of Phenyl alanine in the urine of 4. Add 0.1 mL 1-nitroso-2-napthol. * take note the amt!
newborns(4-6 weeks)
5. Mix.
- also a screening test for phenylketonuria
6. Wait 5 minutes.
- should change color from Gray to gray green (waiting
time: 30 sec) 7. Observe for an orange-red color, indicating tyrosine
metabolites
Mga naunang test
Branched chain amino acid disorder:
1. 2,4-Dinitrophenylhydrazine (DNPH) Test for MSUD
MSUD – branch chain amino acid disorder
3 amino acid that increase in blood and urine:
Isoleucine, Leucine and Valine

(+) orange-red color
Renal type – problem in reabsorption

Confirmatory Test Acdg Henry
Amino acid is 40% freely filtered in glomerulus and
▪ Chromatography/HPLC should be reabsorbed back to the circulation, if not it
▪ Quantitative Assay in serum – detect the will appear in urine
tyrosine in the blood
1. Place 1 mL of urine in a tube.

3. Homogentisic Acid Test
2. Add 10 drops of 0.2% 2,4-DNPH in 2N HCl.
- detect Alkaptonuria
3. Wait 10 minutes
- Homogentisic Acid urine - Alkaline lover
4. Observe for yellow turbidity or precipitate.
- used for faster detection
- Alkalinization of fresh urine
▪ 10% NH4OH – gives the characteristic alkaline

for urine → (+) Black color
▪ Or fresh urine after prolong standing @RT →
alkaline urine → (+) black color
1. Place 4 mL of 3% silver nitrate in a tube.
2. Add 0.5 mL of urine
3. Mix Confirmatory acdg Henry’s : Amino Acid

chromatography
4. Add 10% NH4OH by drops.
5. Observe for black color.

2. Cyanide-Nitroprusside Test for Cystine
- for Cystine disorder
Cystinuria – sulfur odor, Renal type, defective tubular

reabsorption of Cystine, Ornithine, Lysine and
Arginine
- Cystine is the least soluble in urine kaya

present that’s why it forms Cystine crystals – piatos like
Phenyl – Alanine Tyrosine disorder ^ *Cystinosis – defective gene for Cystine metabolism
Aka Brands Modification of Legal’s Nitroprusside – - impair metabolism in Mucopolysaccharides or

same rgt w/ Legals test Glycosaminoglycans
Mucopolysaccharides – combination of
Polysaccharide and Protein, seen in connective Tx
Different types of Mucopolysaccharidosis
2. Add 2 mL sodium cyanide.
▪ Herner – accumulation of mucopolysaccharide
3. Wait 10 minutes.
in the cornea of the eye
4. Add five drops 5% sodium nitroprusside. ▪ Hunter
▪ Sanfilippo disorder – mental retardation
5. Observe for red-purple color. abnormality
*all can be screened using CTAB
2. Add 1 mL 5% CTAB in citrate buffer.
3. Read turbidity in 5 minutes
+ White turbidity
3. Silver Nitroprusside Test for Homocystine
- instead of cyanide, we use silver nitroprusside
- detection of Homocystine/Homocystinuria
2. Add two drops concentrated NH4OH. Aside from CTAB, Other screening Tests:
3. Add 0.5 mL 5% silver nitrate. ▪ Acid Albumin (+ white turbidity)

▪ Mucopolysaccharide Paper Test (+ Blue color)
4. Wait 10 minutes.
5. Add five drops sodium nitroprusside.

Guthrie Bacterial Inhibition Test
6. Observe for red-purple color
- another test for Phenylketonuria
- Phenylalanine – detected in blood of newborns by

using disc by Guthrie(who have a child that have PKU)
Specimen: Blood spots
Agar Base: Phenylalanine free agar base (Demain’s

medium) + Bacillus subtilis + inhibitor(to prevent the
growth of B. subtilis)
4. Cetyltrimethylammonium Bromide (CTAB) Test for ▪ If px have Phenylketonuria, presence of

Mucopolysaccharides (or Glycosaminoglycans) phenylalanine will counteract the action of
inhibitor → bacteria will grow (+)
▪ If no growth (-) - stones accumulate as kidney stones/Renal lithiasis/

Nephrolithiasis
- Measure the diameter of colony in blood disc
- hard solid mass of material that forms in the kidney
based on the materials present in urine (Ex. Ca)
1. A small drop of blood is taken from the heel of a
newborn and applied to a card.
• Kidney stones or calculi develop as a result of various
2. Punch-out of the dried disc was incubated on a petri
metabolic disorders which affect the fate of calcium
dish plated with bacteria (Bacillus subtilis) in the
and other mineral elements in the body.
presence of a growth inhibitor, B-2-thienyl-alanine
Ex. Coke Float
Ice cream – contain dairy/milk → Ca
Cola contains Oxalic acid → Oxalates
= Calcium Oxalate
• Shape of renal stones may be formed in the kidney

depending on the area:
▪ Urinary bladder - form smooth stones

▪ Ureter
▪ Urethra
▪ Renal pelvis - mostly forms here
High levels of Phe in the blood sample overcome the urine stasis → accumulation of crystals →
inhibition, and allow the bacteria to grow blockage of urine → form kidney stones
▪ Renal pyramids – form stag horns
Fluorometric Method can also be used (pinakamasakit, kasi tusok tusok)
Conditions Favoring the formation of Renal Calculi
1. pH – imbalance in urine
▪ Alkaline urine – forms Calcium stones
▪ Acid urine– forms uric and Cystine stone
2. Chemical Concentration
Diagnosed Px will be:
3. Urinary stasis
Phe restricted diet for 10 yrs to lessen the mental
defect of Px Hyperparathyroidism – increase Ca if there’s
urine stasis
*Increase Phenylalanine in foods like: – fish, eggs,
nuts Microscopic Hematuria - present in Primary
Urinalysis finding for Px with kidney stones
Diet: should be: Low protein bread + supplements
Renal calculi
PHOSPHATE - caused by super saturation of urine with

calcium phosphate
- 5-10%
Rich in calcium: Milk and dairy products
STRUVITE or Triple phosphate
- seen in presence of Urea splitting bacteria
▪ Proteus
5 commonly encountered Renal Calculi Formation: ▪ Pseudomonas
▪ Klebsiella
▪ Staphylococcus
CALCIUM OXALATE – present in 80% of Px with renal

stones, most common type Calcium oxalate Stone:
▪ Beer – high oxalate, comes in wheat - Basta may calcium oxalate = super hard stone, dark
▪ Spinach color w/ rough surface
▪ Ice cream, soft drinks etc.
URIC ACID
Uric acid – present in acidic urine, soluble in alkaline
- present 5-10% in Px
- present in Gout Px (painful joints→aspirate Toffy –

mukhang white ice cream/icing)
High Purine Foods: Red meat, red wine, beans,

munggo, legumes, taho etc. → cause uric acid stones Uric Acid stone: – moderately hard, yellowish to
brownish red color
CYSTINE CALCULI - Seen in Hereditary disorders of

cystine metabolism
10-15%, caused by cystine crystal formation when

urine stasis happens, acidic urine
Rich in cystine: meat, milk, cheese, egg

Methods for Analysis of CalculI
- to check for presence of the actual

calculi(microscopic)
•Optical Crystallography
•Radiograph Diffraction
Cystine calculi: smooth surface, yellow brown, very •Infrared spectroscopy

greasy, resemble as “old soap” (board exam!) • Electron Beam analysis
- least common calculi usually 1-2% only •Mass spectroscopy
- looks like “Himalayan salt”
Optical – actual
Lithotripsy
- medical procedure, non invasive(no need for

surgery, - Px is sleeping but painful)
• Extracorporeal Shockwave lithotripsy (ESWL)
• Non-invasive management
Ca phosphate: pale or chalk-like, very thin and • Medical procedure used to treat certain types of
friable(easily crumbled) kidney stones and stones in the other organs such as
gallblader or liver.
- main composition is Calcium
• Uses of high energy shock waves to break up stones
in the kidney.
- apply shock wave in pelvic part → crush the stones

inside the kidney → stones pass through in urine
Surgical/Invasive procedure:
Triple phosphate:
- commonly seen in dogs, rare in human
- branching or staghorn calculi (mukhang luya),

accompanied in urinary infection of urea splitting Percutaneous Nephrolithotomy – insert apparatus w/
bacteria like Proteus Vulgaris(OX2) camera and crusher

Post Analytical
the following:
MATERIALS
• Disposal of wastes and disinfection with liquid
Lysol or 10% sodium hypochlorite of the area. 1. PHYSICAL EXAMINATION/Macroscopic
• The PPE of each individual should be removed
- physical characteristic
properly. These cannot be exposed outside the
laboratory premises. • Pea sized stool
Ref of this lesson: Henry’s book - to check if sufficient/not (small amt)
------------------------------------------- *If test for fecal fat component, quantitate it(collect a 3

day/72 hr stool)
Henry’s Urinalysis part – helpful in Board Exam!
• Sterile container
•Applicator stick
EXAM:
Do you reject 1.040 urine?

2. CHEMICAL EXAMINATION
Common fecal bacteria, Gardnerella vaginalis?
Fecal occult blood test
- presence of blood
Quiz:
• Guaiac solution
Calculi common in women – struvite?
• Hydrogen peroxide
• Glacial acetic acid

FECAL ANALYSIS • Pea sized stool
- magkasama sa Clinical microscopy and parasitology • Serologic pipettes
- To assess gastro intestinal bleeding, liver and biliary • Filter paper

disorders, malabsorption syndromes, infection • Pasteur pipettes
- components of feces(60-80% volume of H20, 20- • Applicator sticks
40% volume of solid components such as
bacteria(non-pathogenic), intestinal secretions, food
residues, fat droplets and other soluble substances Acid Steatocrit Procedure
- aprroximately 100-200 grams feces are excreted – for presence of fats or steatorrhea (Increase fat in stool
per day/24 hrs *take note the time >6g/day)
• 5N perchloric acid
PRE-ANALYTICAL PHASE • Deionized water
• Upon receiving the fecal specimen in the laboratory, the • Capillary tubes
receptionist checks if the container is properly labeled • Pea sized stool
and records the information indicated.
• Vortex mixer • Slides and cover slips
• Pea sized stool
3. MICROSCOPIC EXAMINATION
- can be stained or non-stained (only NSS) PRECAUTION BEFORE COLLECTION
Methylene Blue Stain Procedure for fecal leukocytes Patient should avoid the following things for at least 48
hours before collection of stool:
• Loeffler methylene blue
Instruct px to restrict from this food:
• Applicator stick
• Mineral oils, bismuth(change color of stool), non-
• Slides and cover slips
absorbable anti diarrheal drugs, antimalarial drugs,
• Pea sized stool antibiotics(esp. for culture and sensitivity) etc.
*Antiparasitic drugs – it either interrupt or kill the

parasite
Muscle Fiber Procedure
• Patient should not have barium before stool
- Microscopically rectangular or cylindrical fibers with examination
cross striations
• Avoid iron containing drugs, meat(contains blood and
Creatorrhea - increase meat fibers pseufoperoxidase), fish etc for atleast 48 hours before
• 10% eosin in alcohol stool for Occult Blood
• Applicator sticks
• Slides and cover slips SPECIMEN COLLECTION
• Pea sized stool Collect at least 3 grams of stool (pea sized)
Leucine – for muscle fiber?? ▪ If watery: at least ¼ of container, examined w/in

30 mins
▪ If formed: examined w/in 1 hr.
Neutral Fat Stain Procedure Preserve if delayed – Refrigeration
– stains triglycerides or neutral fat
• Sudan III in 95% alcohol *Check the requisition slip of doctor

• Applicator sticks *Use a fresh sample and don’t forget the label!
• Slides and cover slips *Board exam: Which of the ff. is the appropriate
• Pea sized stool container for stool exam for parasites?
Correct answer from Henry’s: Waxed cardboard
Split Fat Procedure
– check free fatty acids I. CONTAINER – clean, dry, non-breakable container,
• 36% acetic acid leakproof, screw-capped
• Sudan III If multiple-day collection: large containers
• Applicator sticks
II. TYPE AND AMOUNT COLLECTED
pea-sized: FOBT, WBCs, qualitative fecal fat
2 to 3 day fecal collection: quantitative tests(Ex. Van de

kamer titration, should be 3 days fecal collection, not
routinely done only in special lab)
SPECIMEN PRESERVATION
• Refrigeration – mostly used
• Freezing in dry ice – for send out spx

Black – useful to know if upper(total black bcos oxidized)
or lower(red kasi fresh pa)
Fixatives: Charcoal – used in poisoning
• Formalin (2%, 5%, 10%) *If mix black and red – means contaminated or ingestion
of something like iron(black)
• Alcohol
• 20% glycerin in saline (Cumming Method)

Red
• Methiolate Iodine Formaldehyde (MIF) Solution – fixed
and stain the organism(parasitic) Rifampin – all body fluids will become red(semen, spinal
fluid etc.)
*Iodine – toxic for protozoan trophozoites
• Polyvinyl alcohol (PVA) Fixative – good preservative for

the morphology of protozoan trophozoites and cysts Pale/Yellow
Due to Bile duct obstruction – not enough urobilin(gives

the stool its brown color)
PHYSICAL EXAMINATION
Color, volume(for quantitative test), consistency, odor,

adult helminths/parasites esp. for coma px Green
1. Pass out stool in a bedpan lined with newspaper. Avoid Vegetables – not digested esp. if rich in fiber
mixture of urine, fibers, dirt, gauze threads and tissue
Ex. Corn – plastic like coating that cannot be digested
papers.
2. By means of an applicator stick, get a pea-size or

marble-size of stool from the mid-portion. If a portion is Mucoid in feces – ex. in pneumonia px, so check also the
mucoid or bloody, collect from that portion. gastric fluid for respiratory infection, no effect in color
3. In case of liquid stool, collect 10ml or 1/3 full of an
eight ounce container.
Lactobacillus casei – normal flora, good for digestion
4. Examine the specimen immediately.
5. Note the color, odor and consistency.

*eating too much fiber can cause
dehydration/constipation
Butter like Colitis is a chronic digestive disease characterized

by inflammation of the inner lining of the colon
*Board exam – Frothy is due to pancreatic disorder
Ribbon-like / flattened - peristaltic activity is not active
▪ Intestinal Constriction
MACROSCOPIC STOOL CHARACTERISTICS
▪ obstruction in lower colon
Color / Appearance & Clinical Significance ▪ spastic colitis
▪ syphilis
Light to dark Brown
Normal (Urobilin/ Stercobilin) Rice watery
▪ Cholera
Black
Upper Gastrointestinal Bleeding Pea soup
Charcoal
▪ Typhoid
Iron therapy
Bismuth (antacids) Scybalous (Goat droppings)
Red ▪ Constipation
Lower Gastrointestinal Bleeding ▪ spastic colitis
Beets and food coloring ▪ decrease fluid intake
Rifampin
Pale yellow, white, gray Small stool due to cancer, ulcer, tumors
Bile duct obstruction large caliber(large stool, massive enlargement of
Barium sulfate intestine – Hirsch sprung disease)
Green
Biliverdin
Oral antibiotics
Green vegetables
Butter-like Cystic fibrosis
MACROSCOPIC STOOL CHARACTERISTICS
Color / Appearance & Clinical Significance
Bulky/ Frothy
Bristol stool chart^
▪ Bile Duct Obstruction
▪ Pancreatic Disorders
▪ Steatorrhea
Mucus, Blood-streaked mucus
▪ Colitis
▪ Dysentery - Shigella dysenteriae
▪ Malignancy(colon cancer)
▪ Constipation
2. Make the stool specimen acidic using glacial acetic acid

(test using litmus paper).
3. Apply a small portion of stool in the filter paper.
4. Add 2-3 drops of the prepared gum guaiac and

hydrogen peroxide solution. A (+) blue color will appear
in the filter paper in the presence of blood. (checking the
pseudo peroxidase activity of blood → H2O2 oxidize the
guiac → oxidized guiac → forms blue discoloration
❖ Fecal occult blood test (BENZIDINE TEST)
- Most sensitive test among the 3 but highly toxic
1. 4 gm benzidine in 100 ml of glacial acetic acid

(reagent)
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

Score:
4. Add several drops of H2O2 (pseudoperoxidase act)
1 – very hard/constipate/dehydrated
5. Blue colour indicates positive reaction
Optimal – 3 and 4(most optimal)
7 – watery, possible diarrhea, hydrated, inc. secretion of

❖ Fecal occult blood test (ORTHOTOLIDINE
h20
TEST)
1. Smear the stool on a filter paper with an applicator

Indole and Skatole – gives the normal odor of the stool
2. Pipette a few drops of the reagent on to the filter
from Intestinal bacterial formation and putrefaction?,
bcos of this anaerobic bacteria – Bacteroides fragilis Paper (200mg Orthotolidine Barium Peroxide + 5 ml
(normal flora), that’s why we produce gas Glacial Acetic Acid)
Ph: Neutral or slightly acidic or alkaline, Varies from 6.9 3. After 30 sec examine for a blue colour
to 7.2 pH (but not acid due to carbohydrate fermentation
or alkaline due to increase protein fermentation, bcos of 4. Blue green colour within 30 sec means positive test
increase ammonia and crea**)
FECAL OCCULT BLOOD TEST - FOBT

CHEMICAL EXAMINATION Occult = hidden
3 reagents: •Screening test for colorectal cancer/ GIT bleeding
❖ Fecal occult blood test (GUAIAC TEST) •Pathologically Significant = >2.5 mL blood/ 150 g of
- Most preferred test, non toxic stool
- to screen px suspected for colorectal cancer •Sample = center portion of the stool
1. Mix 5 ml of 3% hydrogen peroxide with 5 ml of Guaiac Principle: Pseudo peroxidase activity of Hemoglobin
Solution(consist of 5ml of guiac to 5 ml of 95% ethanol).
Pseudo peroxidase Chromogens
1. Benzidine- most sensitive
2. Guaiac- preferred
3. O-toluidine
False (+) FOBT Dietary Pseudo peroxidases:
•Red Meat
•Melon, broccoli, cauliflower, horseradish
•Aspirin & other anti-inflammatory drugs(NSAIDS) =

(avoid for 7 days to avoid GIT irritations)
False (-) FOBT Reducing agents: CHEMICAL EXAMINATION

•Ascorbic acid (Vitamin C) – reducer, even just ❖ Acid Steatocrit Procedure
>250mg/day and iron therapy (contains vit. C)
- rapid test to estimate the amt of fat excretion esp in
- oxidized guiac pediatric px
- can lead to false negative it not checked for blood after - screen especially pediatric px for steatorrhea(presence
30 sec – 1 min of fat in stool)
- Rapid Gravimetric Method – use centrifuge to make

layers of fats, water and solid components
- rapid and inexpensive, like in microhematocrit
1. 0.5g of feces from a spot collection is diluted 1 to 4 with

deionized water.
2. Vortex for 2 minutes to homogenize the specimen.
3. A volume of 5N perchloric acid equal to 20% of the

homogenate volume is added and the mixture is then
^Place stool at the back then on the other side, apply the
color developer (guiac solution) then wait for 30 sec – 1 vortexed for 30 seconds. Confirm the pH to be <1.
min for color change 4. Place the acid-homogenate mixture in 75 uL plain
hematocrit capillary tube. Seal the end with wax.
*check also the pos and neg control
*Video – FOBT: 5. The capillary tube is centrifuged horizontally at 13000

rpm for 15 minutes in a microhematocrit centrifuge. This
separates fat as an upper layer overlying a solid fecal fat.
6. The length of the fat and solid layers are measured ▪ 10-20% equivocal
using a magnifying lens. ▪ >20% abnormal (steatorrhea)
7. Calculate the acid steatocrit in percent.
8. Calculate the fecal fat in grams per 24 hours. MICROSCOPIC EXAMINATION
- Titration of fecal fat, 2-7 g? ❖ Methylene Blue Procedure for fecal leukocytes
Normal: < 3
Invasive condition: >3 per HPF (infection or diarrhea,

consistency is watery)
▪ Diarrhea w/ WBC – Campylobacter, Yersinia, E. coli,

Salmonella and Shigella (CYESS)
▪ Diarrhea w/out WBC – from toxin producing
bacteria ex. S. aureus, V. cholerae and viruses such
as Rotavirus, parasites
^After 15 mins (normal – middle – abnormal) Charcot Leyden – disintegrated neutrophils
• top most: fatty layer (quantitate in percent, Procedure:
derived from the volume of fatty layer divided by
volume of solid layer x100) 1. Place mucus or a drop of liquid stool on a stool. (wet
• 2nd – liquid preparation)
• Last layer: non fatty solid layer ex. food debris 2. Add two drops Loffler methylene blue.
• SL -sealant
3. Mix with a wooden applicator stick.
Acid Steatocrit Procedure 4. Allow to stand 2-3 minutes.
• SPECIMEN: Single stool 5. Examine for neutrophils under HPF
• 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. Fecal Leukocytes Determination
• No laxatives should be given. 1. Wet Preparation = Stool + Loeffler’s Methylene Blue
*Laxatives can remove all the waste in intestine including 2. Dried Preparation = Stool + Wright’s/ Gram stain – no
fat longer used
• Stool for steatocrit testing is stable for seven days under 3. Lactofferin Latex Agglutination Test
refrigeration. If longer term storage is necessary, it Lactoferrin = secondary granules of neutrophils
should be frozen.
(+) invasive bacterial pathogen
• Specimen must not contain foreign material such as
toilet paper.
❖ Muscle Fiber Procedure
Reference Intervals Creatorrhea -
Units: expressed as percentages (%) 1. Emulsify a small amount of stool in two drops of 10%
eosin
Age: >6 months:
in alcohol.
▪ <10% normal
2. Coverslip and let stand for 3 minutes.
3. Examine under HPF for 5 minutes.
4. Count the number of undigested fibers.
❖ Split fat stain procedure
- check the # and size of the fat globules
1. Mix emulsified stool with one drop of 36% acetic acid.
2. Add two drops saturated Sudan III.
3. Mix and coverslip.

Abnormal: > 10 undigested striated muscle fiber 4. Heat gently almost to boiling.
Due to biliary obstruction, gastrocolic fistulas, pancreatic 5. Examine under high power.
insufficiency (cystic fibrosis)
6. Count and measure the orange droplets per high
▪ Undigested - may 2 striations in both direction power field.
▪ Partially digested – one direction striation
▪ Completely digested - no striations
Include red meat in the diet, spx is check w/in 24 hrs*
❖ Neutral Fat Stain Procedure
- checks only the # of fat
- increase triglycerides mean problem in

digestion/maldigestion (malabsorption syndrome)
Procedure:
1. Homogenize 1 part stool with 2 parts water.
2. Mix emulsified stool with one drop 95% ethyl alcohol

on slide.
3. Add two drops saturated Sudan III in 95% ethanol.
*can also use sudan IV and oil red

Fecal Fat Determination
4. Mix and coverslip.
Qualitative Test:
5. Examine under high power field.
1. Neutral Fat Stain (Triglycerides) Suspension + 95%
6. Count orange droplets per high power field. ETOH + Sudan III Orange Droplets (Neutral Fats or
Triglycerides) ≥ 60 droplets/HPF = Steatorrhea
2. Split Fat Stain
Emulsified stool + 36% Acetic acid + Sudan III
Orange Droplets (Fatty Acids)
•Normal = 100 droplets (< 4 um)
•Slightly increased= 100 droplets (1-8 um)
•Increased = 100 droplets (6-75 um)

^Parasites
Quantitative Test:
Van De Kamer Titration the
•Gold Standard for fecal fats following:
•For definitive diagnosis of steatorrhea • Returning of materials, slides and microscopes
•Sample = 3 day stool • Disposal of wastes and disinfection with liquid Lysol or
10%
•Titration with NaOH
sodium hypochlorite of the area.
Normal value = 1-6 g fats/day

Quiz:
Steatorrhea= > 6 g fats/day
High protein diets increased fecal pH/alkaline
Quali: for screening Feces in blood
Quanti: for confirmatory Blood in stool - hemorrhoids or diverticulitis

10% formalin – all purpose fixatives
Black stool – Melena
Bright red stool – Hematochezia
0.1-0.2 mg per day

*REMEMBER CONVERSIONS!
Semen Analysis ▪ Decrease sperm count bcos most sperm are on the
first part of entire ejaculate
Seminal fluid – vehicle for spermatozoa * last part has the enzymes from Seminal Vesicle,
Prostate Gland and bulbourethral gland
Aka Sperm Count test ▪ increase pH bcos of bulbourethral gland/calper’s
▪ to analyze the health and viability of man’s gland – secrete alkaline fluid
sperm cells ▪ Clotted - will not be liquified
▪ to check for male infertility/sterility → no If last portion is missing:
sperm/movement of sperm
▪ Need also for Vasectomy Semen Analysis – the ▪ Inc sperm count
process where vas deferens is cut, needs to be ▪ Dec pH - acidic
successful → there is still fluid excreted bcos ▪ will not clot bcos no enzyme
there is still seminal vesicle and prostrate but no (responsible for clotting)
sperm cells can be seen Method of Collection:
▪ also used for forensic analysis for alleged rape
cases 1. Masturbation – self collection, most
appropriate/preferred, lesser contamination
- there is also isolated in gastric fluid(rare cases)
2. Coitus interruptus/ withdrawal method
3. Condom Method *use non-lubricant containing

PRE-ANALYTICAL PHASE rubber or polyurethane condom/silastic condom
• Upon receiving the semen specimen in the laboratory, 4. Vaginal vault aspiration – collect seminal fluid inside
the receptionist checks if the container is properly the vaginal wall, can become contaminated
labeled and records the information indicated.
*collection can also be done inside the lab in some
• The site of the experiment should be sterilized. The special laboratories
Specimen Collection
Time of collection: preferably in the morning, brought

to the lab within 30 minutes and examined within 1
Specimen Collection hour *normal liquefaction time for seminal fluid: 30
Abstinence for 2-3 days, just enough for fertility testing. mins to 1 hr bcos of enzyme
Then 2-3 samples must be collected and examined at 2
weeks interval with an abnormal samples considered
significant If sperm sample results to >2 cm length of spring in
Spring Test means viscous sample → perform
▪ <2 days – lesser sperm cells are isolated Liquefaction Induction
▪ >5 days – increase volume, decrease
viability/dead cells and motility
Liquefaction Induction
Method of Collection: collect the entire ejaculate If sperm sample is not liquefying w/in 2 hrs (highly
viscous) → use proteolytic enzyme such as alpha
Total Ejaculate: 5ml *bladder should be empty to chymotrypsin or bromelain added in Dulbeco’s
prevent contamination of urine Phosphate Buffer Saline (equal volume) →
If first part of ejaculate is missing/ not collected liquefaction is induced in sample
properly, it will result to:
Transport: Should be maintained in body ▪ Gray-white, translucent

temperature(placed in the armpit of male Px/ center of ▪ Pearly white, colorless to creamy white
bra if female Px will carry the sample)
Odor:
Take note: time of specimen collection, specimen
▪ Musty or bleach/ Chlorox odor
receipt and liquefaction
▪ Putrid odor – may infection
- Analysis should be done after liquefaction (usually 30-
60 minutes) ▪ Red Coloration Increased RBC/blood, have
damage in the way of sperm excretion or testis
- Specimen awaiting analysis should be kept at 37 C, ▪ Yellow Coloration - Increased contamination of
Never placed in refrigerator urine contamination or medication
Normal: no WBC
PHYSICAL EXAMINATION ▪ Increased white turbidity infection/ increased
Check the volume, ph, color and odor WBC
bcos of infection
Materials:
▪ Spermatic fluid Volume

▪ Specimen container
▪ Graduated cylinder Normal 2 to 5 mL (if >10 ml – abnormal)
▪ Litmus paper ▪ Increased volume in increased abstinence
▪ Decreased volume in infertility, incomplete
collection
1. Check for color and odor. Normal color for spermatic Viscosity
fluid is gray-white or pearl-white while odor is musty or
Chlorox-like(bcos of acidic prostate fluid from prostate Normal: Pour in droplets (highly viscous)
gland) ▪ Increased viscosity(>2cm in string test) -
2. Using litmus paper, check for pH(Normal: slightly decreased sperm motility
alkaline – to neutralize the acidity of vagina) Reporting:
Pre-cum – fluid in bulbourethral gland, highly alkaline, 0 - watery
Purpose: to lubricate the vagina and to neutralize the
acidity of vagina during intercourse 4 - gel-like
3. Note for the liquefaction time. Normal liquefaction
time is 30-60 mins. pH
*If more than 2 hrs → induction of liquefaction Normal 7.2 to 8.0(strasinger) or 7.3 to 8.3 (henry’s)
*depends on literature
4. Transfer specimen in graduated cylinder for specimen
▪ Increased pH - Infection
volume. Spx volume: 2-5 ml ▪ Decreased pH - Increased prostatic fluid
Macroscopic Examination SG
Appearance Normal: 1.033 or near
Color:
MICROSCOPIC EXAMINATION
MATERIALS:
*Normal: Grade should be 2,3 and 4 or A and B only
1. Sperm Motility - responsible for penetration of the

CASA – Computer Assisted Semen Analysis
ovary
- Within 1 hr of collection, check the percentage of - provide for both sperm velocity and trajectory, speed
motility and repeat after 4 hrs. and way/movement is determined, also morphology
and motility
- Place one drop of seminal fluid on a slide and cover
with a cover slip. Video: Sperm Motility
- Examine under HPO and report the number of motile

and non-motile sperms in percentage. 2. Sperm Viability
– not routine
- Prepare a thin smear of seminal fluid.

WHO Criteria for Sperm motility reading
- Heat fix using alcohol lamp.
- Add a drop of Eosin - Nigrosin stain.
- Cover and read under OIO.
Modified Bloom’s Test
1 drops of semen, 1 drop of 5% eosin stain and 4 drops

of Nigrosin stain
Diluting fluid – for sperm count
Reagent: Eosin and Nigrosin
*Sperm cells have mitochondria and produce ATP which

Sperm Motility Grading absorb stain
Performed undiluted, examine microscopically (20 hpf) Count 100 sperms
Normal Values: ▪ Living sperm: unstained / bluish white (75%)
▪ Dead sperm: red / pink / light pink
≥ 50% motile (within 1 hour)
Quality = ≥ 2.0 or 2 grade

Video: Eosin Nigrosin Stain
normal and abnormal forms. Also, report any epithelial

cells,
testicular cells, RBCs, WBCs and crystals seen.
- Report Normal and Abnormal forms in percentage
* cannot use eosin and nigrosin stain
Collection → Staining → Smear → Air dye →

microscope Normal Values
- Examine at least 200 spermatozoa under OIF
2 criteria:
▪ Routine criteria - >30% normal forms or <50%

abnormal forms
▪ Kruger’s strict criteria - >14% normal forms or <70%
abnormal forms
Measure the size of head, neck, and tail using a

micrometer
Stains
▪ Wright’s stain
▪ Giemsa stain
▪ Hematoxylin
▪ Crystal violet
▪ Papanicolaou’s stain - preferred
3. Sperm Morphology
Oval shape head – approx. 5 um long and 3 um wide
- Routine
check the acrosomal cap or shape of the head - Acrosomal cap – contains enzyme necessary for ovum
(important in penetration to ovum) and tail part penetration, check the shape
- Prepare thin smears of seminal fluid. Tail: measure from the neck up to the last part, 45um
- Dry and fix by heat *remember defects
- Stain the smears by Gram’s Method (or Pap Stain) Spermatid – detached head, no tail, immature sperm
cell, mistaken as WBC(stain w/ methylene blue for
- Examine under OIO and count 200 cells, counting both
confirmation)
the
4. Cell Count Long Method for Sperm Count Concentration

Use diluting fluid to immobilized the sperm bcos u Computation
cannot count them while moving
(Standard Neubauer Calculation Formula)
*can also use Sodium bicarbonate, 1% formalin or 3%
trisodium citrate as diluting fluid
- Using a WBC pipette, draw semen to the 0.6 mark
- Draw WBC diluting fluid to the 11.0 mark Examples:

- Shake for two minutes. ▪ 1 sperm counted, 2 WBC squares
▪ 1 sperm counted, 5 RBC squares
- Discard the 3-5 drops and charge in 2 large squares (
similar to WBC count)
Normal cell count: ≥ 40 million/ml
Sperm Concentration – performed before sperm count

100 → Per uL only → multiply to 1000 = 100,000 sperm
Normal Value: ≥ 20-160 million/mL
per ml
*compute like in hematology!
Shortcut Method for Sperm Count Concentration

Computation
▪ 2 WBC squares
# of sperm counted x 100,000 = sperm in M/mL
▪ 5 RBC squares
# of cells counted x 1,000,000 = sperm in M/mL
Methods:
1. Improved Neubauer Counting Chamber
Dilution - 1:20
Diluents: to immobilize the sperm
▪ Cold water – most preferred

▪ 0.5% chlorozene
▪ 1% Formalin
▪ 1% formalin in trisodium citrate
▪ 5% Sodium bicarbonate
▪ 5% NaHCO3 in 1% phenol
CHEMICAL EXAMINATION
Materials:
2. Makler Counting Chamber
▪ Spermatic fluid
- For undiluted sample/ no need for dilution
▪ Fructose reagent (50 mg resorcinol in 33 ml
- Uses heat to immobilize the sperm cells conc. HCl diluted in 100 ml with water)
▪ Test tube
▪ Test tube holder ▪ Seminiferous tubules (Testes)

▪ Alcohol lamp
- Spermatogenesis
- Sertoli cells = serve as nurse cells for developing

Seminal vesicle – release fructose - energy source for sperm cells
sperm cell
▪ Epididymis
Decrease fructose – motility is also decreased →
- Sperm maturation (Sperm become motile)
perform the fructose test to confirm, can also be due to
androgen deficiency
60-70% Seminal Fluid

Seminal Fructose Screening Test – used only for • Seminal vesicles
decreased motility
- Provide nutrients for sperm and slightly
1. Prepare the Fructose reagent (50 mg resorcinol in 33 alkaline fluid
ml. conc. HCl diluted in 100 ml with water reagent).
- Rich in fructose for sperm motility
2. Mix 1 ml semen with 9 ml fructose reagent.
- most fluid comes here
3. Boil
4. Observe for orange-red color

20-30%. Prostatic Fluid
*Seliwanoff’s test is also a screening for fructose
Acidic Fluid
Contains ACP, Zinc, Citric acid and other enzymes

Seminal Fluid Fructose
For coagulation and liquefaction
Normal Value: ≥13 umol/ejaculate through
spectrophotometric method
Screening test 5% Bulbourethral gland (Cowper’s gland)
Resorcinol test = (+) orange-red color Thick alkaline mucus
Neutralize acidity from prostatic secretions and vagina
Citric Acid and acid phosphatase(ACP) – very important

for the liquefaction or clotting of the seminal fluid
Composition of Semen
5% Spermatozoa
protective equipment before the experiment
• Upon receiving the sputum in the laboratory, the
receptionist checks if the sample is properly labeled and
the amount of sample is adequate then records the
information indicated.
Materials:
• Sputum
• Specimen container
Microscopic
Make a smear
the following:
Stain
• Returning of materials, slides and microscopes
• Disposal of wastes and disinfection with liquid Lysol or • Sputum
10% sodium hypochlorite of the area. • Applicator stick
• Inoculating loop
• Glass slide and cover slips
*Nagkakaroon lang ng problem sa penetration kapag • Alcohol lamp
abnormal ang head but no effect on baby • Cedarwood oil
▪ Androsperm – deep penetration, most probably boy • Ziehl – Neelsen acid fast staining solution:
ang baby, increase motility but increase death • Carbol fuschin – primary stain
▪ Genosperm – shallow penetration, most probably • 3% Acid alcohol - decolorizer
girl ang baby, decrease motility increase • Methylene blue – counter stain
survival/viability
*kapag nag ka pre-cum, hindi panibagong abstinence

- hindi nakakabuntis, only for neutralization of vagina’s
acidity and lubrication during intercourse
Auto anti-sperm antibodies – cause infertility, detected

using serologic testsaq Sputum Collection
First morning - Most ideal, highly concentrated, not
contaminated
SPUTUM ANALYSIS • 24 hour - Volume measurement
• Throat swab - ideal for children, infant, pediatric
Components of Sputum: patient
Plasma • Sputum induction - Non-cooperative patients
Electrolyte – using nebulizer, use 5-10% Saline solution to induce
Water production of sputum for px who can’t
Normal flora(bacteria) Or put salt in warm water tapos mag-tataklob para mag-
Mucin excrete ng sputum
• Tracheal Aspiration- Debilitated patient
Collected in a dry, wide mouthed with 25 ml capacity

and leak proof container
– dilate the airways, for very weak patient, invasive,
insert a tube in the nostril to trachea to collect and
aspirate sputum
*check for food debris

3 repeated deep inhalations then exhalation followed
by a forced cough → 3-5 ml or 1 spoonful of sputum
should be collected
Deep cough in the chest If px can’t cough take several deep breath and hold
Usually collected at home, instruct the Px for correct breath momentarily to induce the cough → sputum
labelling
In lab, there should be an area where air is not flowing Super sticky phlegm – cannot release → need for
to prevent aerosol esp. if px have M. tuberculosis induction to reduce the viscosity of sample
Observe if sputum or saliva: check the macroscopic

Sputum Container characteristic of sample
1. Volume capacity of 50 mL(suggested by DOH) – much ▪ Sputum: Clear, watery, mucoid
better Check for presence of PMN and monocytes or
2. Made of transparent or translucent material - lung macrophage/dust cells
preferred ▪ Saliva: clear and very watery
3. Wide-mouthed (at least 35mm in diameter)
4. Screw-capped
5. Unbreakable and leak-proof
6. Clean and sterile (preferably)
Sterile – for culture and sensitivity
7. Single-use, combustible material Purulent or Mucoid – good quality spx
8. With walls that can be easily labeled ^usually collected in Tuberculosis Px
Gene expert machine – detect bacteria in 30 mins to 1

hr instead of making a culture (take days)
Bloody sputum – shouldn’t be examined bcos mucoid

materials still needs to be visible
Saliva – w/ presence of bubbles, used in CC2 for alcohol

level and secretor status(chew a paraffin wax) in IMHM Decreased in:
• Early pneumonia – when the bacteria/virus multiplies
*Sputum – not a normal body fluid, it’s always assc. with in the lungs, they interfere with the mucus production
a dse • Bronchial asthma - there’s a blockage
• Acute Bronchitis – inflammation or infection
*Video Increased in:

By suctioning • Bronchiectasis
• Lung abscess
• Edema
• Gangrene
• TB
• Lung Cancer
• Severe infections
Normally, Lung produce 20-30ml mucus per day for

functioning of cilia of lungs, serve as lubricant →
It is called sputum when excess amt is produced and
Storage: needs to be excreted
• Store in a cool (2-10°C), dry place away from sunlight
until ready for transport to the laboratory to avoid Macroscopic Examination
liquefaction Color: Colorless and translucent or whitish to faint
• Refrigeration also reduces the growth of contaminants yellow
in the specimen *Colorless indicates that sample is just a mucus, normal
– bacteriostatic which stop the multiplication of
organism Odor: Odorless
Transfer your specimen in ice pack or in cooler during • Foul/Putrid: Cavitary TB, Lung abscess, Gangrene
transportation • Fruity: P. aeruginosa
*Should transport w/in 3 hrs?
• Do not use any chemical preservative for storage Consistency: Watery
• Mucoid: asthma/bronchitis
Physical Examination • Serous/frothy: lung edema
• Examine the quantity, consistency, color and odor
directly from the container or preferably in a petri dish. pH: 6.5 to 7.0 (slightly acidic)
*Odor – no longer advised especially for TB patients bcos - bcos of carbonic acid when we exhale and bcos of
of aerosols bacteria (normal flora) in air passage
lactic acid – increase bcos of bacteria
• Record for minute macroscopic masses:
Macroscopic masses – large mass that could be seen in
specimen ex. aggregates
o Cheesy masses
o Bronchial casts
o Dittrich’s plugs
o Lung stones
Macroscopic Examination (Volume)

The necessary amount of sputum for most tests is 5 ml
P. aeruginosa – produce color bcos of pyoverdin and

pyocyanin
Rusty red – Board exam: anchovy sauce (bagoong isda)

color
*shuta iniiba kulay sa board exam - old blood
*not all red color are caused by TB, it can also be due to
excessive damage in throat(non-pathological) to make
sure, we perform DSSM…
Direct Sputum Smear Microscopy (DSSM)
- a routine method for TB dots → coiling → check for
Acid fast bacilli
Black – black lungs bcos of deposition of chemicals from

cigarettes
*Vape fluid – only lesser chemicals than cigarettes
*Use Nicotine patch instead for smoking withdrawal
White, yellow or gray – indicates puss and epithelial

cells, Tuberculosis px
Bronchial casts made up of fibrin and protein
*Paragonimus westermani and TB – can be
materials, can also have lymphocytes – branch tree-
differentiated microscopically but not macroscopically
like, follows the shape of bronchi
- it can be released bcos of deep cough
- Pulled up using a scope or camera
Macroscopic Examination
- Px have difficulty in breathing, seen usually in Lobar
Specific Gravity
pneumonia
• Mucoid- 1.004 to 1.008
• Purulent- 1.015 to 1.060
• Serous - 1.037 or higher
Features
• Cheesy Masses
• Dittrich Plug
• Curschmann’s spiral
• Pneumoliths / Broncholiths (Lungstones) Cheesy masses
• Casts/ Bronchial Casts - microscopically, but can also seen in macro –
• Parasites Curschmann’s spiral bcos of accumulation
• Mycetomas - rounded masses of fungal debris
Dittrich plug – very offensive odor, like a stool
*Lung migrating parasites of larva to mature – Ascaris,
Strongyloides Lungstone – esp. in fungal infections or histoplasmosis
• Select a portion of the sputum using an applicator stick
• Spread it on the glass slide
• Fix the specimen into the slide by passing 2-3 times
over the flame
• Stain the smear with Ziehl-Neelsen stain
• Observe under OIO • Pigmented Cells - endothelial leukocytes taking up

DSSM – coiling pigmented granules
o Carbon-Laden or Dust Cells- contains black

granules; due to Anthracosis
o Heart Failure Cells/ Siderocytes - hemosiderin
Laden(w/ iron deposits/siderocytes)
- Macrophage in the alveolar spaces
1. Sample
2. Smear
3. Airdry
4. Heat fix
5. Stain – Ziehl Neelsen using Carbol fuschin (primary
stain) color red → apply heat before boiling to stain
properly
6. Rinse with water → absorb by bacilli
7. Decolorize – acid alcohol (15-20 sec)
8. Wash
9. Methylene blue – counter stain
Cursch → CLC crystals → Creola bodies
Acdg DOH^ These 3 are always present in bronchial asthma^
Microscopic Examination 3. Myelin Globules - no significance, mistaken as

Blastomyces bcos they have same structure
• Crystals
4. Yeast- budding forms seen in antibiotic treatment
• Charcot-Leyden Crystals - most significant, seen in
asthma(cause by allergic reaction) 5. Fungi and Molds
Mistaken as Creola Bodies (cluster of columnar cells) 6. Parasites – strongyloides (pic)
*CLC is made up of degenerate eosinophils (increase

during allergy together with basophil)
• Hematoidin
• Cholesterol
• Fatty Acids

the following:
• Returning of materials, slides and microscopes
• Disposal of wastes and disinfection with liquid Lysol or

……
Micral test
Hemorrhoids – not occult blood

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1 ANALYSIS OF URINE AND OTHER BODY FLUIDS (LAB)

SAFETY IN THE • Vaccination against HBV

• Good housekeeping in all laboratory work and storage

Ø Standard Precautions (1996) 6 components of chain of infection (IREMES)

Portal of Exit - mouth, nose

Safety Awareness for Clinical Laboratory Personnel

• Disposal of biological wastes

CDC GUIDELINES FOR HAND HYGIENE

• Wash hands with detergent soap or Antimicrobial soap:

– When the hands are visibly soiled

70 % (hand rub) Radioactive materials - damage to cell nucleus (DNA)

95%(easily evaporates) @risk of radioactive

SHARP HAZARDS esp. gamma rays

§ Placed near workspace

§ Closed when not in use UV Light - for disinfection

§ Sealed when ¾ full

ELECTRICAL HAZARDS Arsenal fires - forest fires

Ø Unplug the equipment

machines should be grounded with 3- pronged plug to PHYSICAL HAZARDS

– Avoid dangling jewelry

• WET HANDS WITH WARM WATER.

• APPLY ANTIMICROBIAL SOAP.

▪Additional information: age, location, physician

• Disposable, wide-mouthed, and flat-bottom ▪Requisition form: Must accompany specimen

• Clear containers and at least 50 mL capacity ▪Time of receipt is stamped on requisition

containers for 24-hour specimens

• Wear gloves when working with urine SPECIMEN REJECTION

Urine Containers ▪Non-matching labels and requisitions

▪Contaminated specimens - feces, paper

▪Delayed or improper transport

2. Pediatric Wee Bag

▪Test within 2 hours of collection

▪Refrigerate if testing is delayed

▪Most problems are caused by bacterial

Refrigeration ( 2⁰C to 8⁰C)

Most routinely used method of preservation:

• Decreases bacterial growth and metabolism

Urine Preservatives table* postprandial specimen are compared with those of a

TYPES OF URINE SPX COLLECTION

timed specimen must be used to produce accurate

1. Random Urine quantitative results

2. First Morning Urine • required when the concentration of the substance

• ideal screening specimen to be measured changes with diurnal variations and

and for evaluating orthostatic proteinuria. body metabolism.

3. Fasting Specimen (Second Morning) 7. Catheterized Specimen

• specimen is tested for glucose, and the results are

• Quantitative cultures are performed on all specimens, Notes:

10. Prostatitis Specimen

• Post-prostatic massage urine specimen (VB3)

10. Prostatitis Specimen

Pre- and post-massage test (PPMT)

• A positive result is significant bacteriuria in the post-

Color #4-8 – need to drink more h20

Amber – urochrome, gives yellow or amber color of urine 1. Hormone

Phenazopyridine – mistaken as bilirubin

Volume required: 10-15 ml

Ex. 12 ml is still acceptable for routine urinalysis

Urine Clarity Determination

2000 ml per 24 hrs – Herny’s

• Oliguria – px is dehydrated or may blockage sa

*Maple Syrup – amoy leche flan

Rotting fish – have Trimethyl aminuria

Pungent – consumption of Asparagus