CLINICAL

MICROSCOPY
 Laboratory Tests

1. ROUTINE URINALYSIS
 2. URINE MICRAL TEST
 3. PREGNANCY TEST
 4. L-FABP (L-TYPE FATTY ACID BINDING PROTEIN
 5. ROUTINE FECALYSIS
 6. FECAL OCCULT BLOOD
 7. CLOSTRIDIUM DIFFICILE
 8. FIT (FECAL IMMUNOCHEMICAL TEST)
 Laboratory Tests
9. SEMEN ANALYSIS
10. BODY FLUID ANALYSIS
a. CSF
b. PLEURAL FLUID
c. ASCITIC FLUID
d. SYNOVIAL FLUID
e. OTHER BODY FLUIDS
11. MICROFILARIA
 ROUTINE URINALYSIS

1. Pour 10-15 mL of a well-mixed urine specimen into a graduated disposable centrifuge

tube. Perform physical examination and reagent strip chemical evaluations. Centrifuge
at 450 g for 5 minutes.
2. Carefully remove and save the supernatant. The final volume used to re-suspend the
sediment may vary with the standardized system used but should remain constant
within any given laboratory. Use a disposable pipet, specialized tube, or pipet system
to concentrate the sediment.
3. Gently re-suspend the sediment in the remaining supernatant, and add one drop of
supravital stain if desired. Using an appropriate pipet, load/charge the examination
chamber of a standardized slide. Allow the urine to settle for 30-60 seconds.
4. Examine with low and high power objectives. Subdued light or phase-contrast
illumination will be required to detect sediment entities with a low refractive index.
The fine focus should be varied continuously while scanning.
 MICRAL TEST
 The MICRAL test is an immunospecific dipstick for detection of low
concentrations of albumin in urine (microalbuminuria).
 1. In a urine container, immerse for 5 seconds with the fluid level between the 2 black bars.
 2. After 1 minute, compare color development above the inscription micral.
 3. Manner of Reporting/unit of measurement used: mg/L
 PREGNANCY TEST
 TEST PROCEDURE
1. Collect the urine in a clear, dry and contaminant free plastic or glass container. The urine sample
can be taken at any time of a day. However, the first urine in the morning provides the highest
level of hCG concentrations.
 *Any sample collected and stored between +2o to +8oC should be used within 48 hours.
2. Remove the test from the packaging just before starting the test. Place the test on a flat surface.
3. Use the plastic dropper to collect the urine from the container.
4. Place 2-3 drops of urine in the sample cavity on the test kit.

EVALUATION OF THE RESULT
Results appear within 5 minutes of placing the urine sample on the test kit. Discard any results
 that appear after 10 minutes of testing.

 PREGNANCY TEST
NEGATIVE - the presence of a single line in the “C” result window only indicates a
NEGATIVE result.

POSITIVE - the presence of a line in the “C” result Window AND the “T” result window,
indicates POSITIVE result.

INVALID - If there are no lines in the result window after 5 minutes, the result is invalid.
The test must be re-done with a new test kit.
 L-FABP

 L-FABP is excreted to the urine in response to proteinuria observed by the progression of kidney
disease or ischemic and oxidative stresses. L-FABP can be detected in the early stage of the
progression of kidney disease, although most of biomarkers reflect the result of failure of
kidney function.
 SPECIMEN: Urine collected in a clean and dry sample container.
 PROCEDURE:
 1. Take 100 ul of urine sample (fill to the line of the dropper) and add to Pre-treatment
Micro Tube.
 2. Mix the sample with the Pre-treatment reagent slowly 10 times by using dropper to
avoid bubbling as much as possible.
 3. Take 100 ul of the mixed sample (fill to the line of the dropper). Place all the mixed
sample (100 ul) into L-FABP Test Cassette.
 4. Keep L-FABP Test Cassette flat and stable. After 15 minutes, read Test Line of L-FABP
Test Cassette. The L-FABP Concentration range of the sample is determined.
 L-FABP
 5. Do not read Test Line which has been left for more than 15 minutes following the addition of sample.
 TEST RESULT:

 When a line appears on the Control Line, read the color intensity of the Test Line and make an assessment based on the
three degrees of concentration.
 RESULT SCORE:
 If both Control Line and Test Line appear in the viewing area, the test indicates that L-FABP was detected in the
specimen.
 A. 0-12.5ng/ml (L-FABP < 12.5ng/ml). The burgundy color intensity of the Test Line is less than 12.5 of the color
block of the reference card.
 B. 12.5-100ng/ml (L-FABP > 12.5ng/ml and <100ng/ml): The burgundy color intensity of the Test Line is more than
12.5 of the color block of the reference card and less than 100.
 C. > 100ng/ml (L-FABP >100ng/ml): The burgundy color intensity of the Test Line is more than 100 of the color
block of the reference card.
 INVALID
 If there is no visible line on the Control Line area within 15 minutes, repeat the assay with a new test device.
L-FABP
 ROUTINE FECALYSIS/STOOL EXAM
 PATIENT PREPARATION: None
 SPECIMEN: Freshly collected stool specimen in a clean, clear, dry, wide mouth screw
 cap container.
 VOLUME: Thumb size (3-5 grams)
 COLLECTION AND TRANSPORT
 1. Collect fresh, diarrheal stool in a clean, dry bedpan or on a plastic, leak proof
container.
 2. Those portions of stool containing blood and mucus are especially significant
and should be transferred into the container.
 3. The patient should understand that the specimen should not be contaminated
with urine and toilet water that may contain chemicals.
 4. Label the specimen with patient’s full name, date and time of collection.
SPECIMEN STABILITY: Specimen must arrive at the laboratory within 1 hour of
collection.
METHODOLOGY: Direct Light Microscopy
 STOOL EXAMINATION/ROUTINE FECALYSIS
 PROCEDURE:
 1. Place 1 drop of NSS and small amount of stool onto slide, mix and read
 under the microscope.
2. Look for the presence of ova and parasite, amoeba (cyst and trophozoite)
 3. Type the result.
STOOL EXAM FOR OCCULT BLOOD
METHOD: HEMA SCREEN
The Fecal Occult Blood Test (FOBT) is a lab test used to check stool samples for hidden (occult)
blood. Occult blood in the stool may indicate colon cancer or polyps in the colon or rectum – though
not all cancers or polyps bleed.

 TEST INSTRUCTIONS:
 1. To develop specimen test area, lift flap.
 2. Place 2 drops of Hema Screen Developer over each smear.
 3. Apply 1 drop of developer onto control area.
 INTERPRETATION:
 BLUE COLOR should appear within 30 seconds if positive.
 NO CHANGE IN COLOR if negative.
 CLOSTRIDIUM DIFFICILE

This test is a rapid membrane enzyme immunoassay for the
simultaneous detection of Clostridium difficile glutamate
dehydrogenase antigen and toxins A and B in a single reaction well.
The test detects C. difficile antigen, glutamate dehydrogenase, as a
screen for the presence of C. difficile and confirms the presence of
toxigenic C. difficile by detecting toxins A and B in fecal specimens
from persons suspected of having C. difficile disease. The test is to be
used as an aid in the diagnosis of C. difficile disease. As with other C.
difficile tests, results should be considered in conjunction with the
patient history.
 CLOSTRIDIUM DIFFICILE
 PROCEDURE:
 1.) Add to a test tube:
 750 ul Diluent
 1 drop Conjugate
 25 ul (1ST graduation) of specimen
 Mix thoroughly.
 CLOSTRIDIUM DIFFICILE
 2.) Transfer 500 ul (highest graduation) from tube to small Sample well.
 Keep the cassette at room temperature and wait 15 minutes.

 3.) Add 300 ul Wash buffer to large Reaction Window. Allow to

 Completely absorb.
 CLOSTRIDIUM DIFFICILE
 4.) Add 2 drops Substrate to large Reaction Window. Keep the cassette at room
temperature and wait 10 minutes. Read results.


 INTERPRETATION:
 A positive result may be interpreted at any time during the 10 minute incubation. A
test cannot be ruled negative or invalid until 10 minutes has passed.

 If there are no dots ay C (control), the test is invalid.

 FECAL IMMUNOCHEMICAL TEST
(FIT)

 Test designed to detect human hemoglobin in fecal samples. The test is based on an
immunochemical method that improves specificity of detection of lower gastro-intestinal
disorders including colorectal cancers and adenomas without any dietary restrictions.
 1. Bring test cassettes and patient’s samples to room temperature prior to testing.
 2. Remove a test cassette from the sealed foil pouch and place it on a clean, level surface.
Label the test cassette with a patient or control identification. For best results, the assay
should be performed within one hour.
 3. Shake the specimen collection tube thoroughly to ensure proper mixing of fecal sample
with the specimens diluent buffer.
 4. Unscrew the white protection cap from the specimen collection tube. Using a piece of
tissue paper, break the tip of the collection tube using a twisting motion.
 5. Hold the specimen collection tube vertically and dispense 3 drops of solution into the
sample well (S) of the test cassette. Avoid trapping air bubbles in the sample well (S) and
do not add any solution to the result area.

 FECAL IMMUNOCHEMICAL TEST
(FIT)

6. Read the result after 5 minutes. Do not interpret results after more than 10 minutes.

RESULT INTERPRETATION:

Positive: Two coloured lines appear on the membrane. One line appears in the control line region (C)
and the other one in the test line region (T).
Negative: Only one coloured line appears in the control region (C).No coloured line appears in the test
line region (T).
Invalid: The control line (C) fails to appear.
 ROUTINE SEMEN ANALYSIS
 For the semen analysis result to be most valuable, proper collection of the specimen is essential.
The semen analysis is performed on a fresh specimen within 2 hours of collection. Before testing, a
period of 2 to 7 days of abstinence from ejaculation is recommended. Feelings of anxiety about
producing a specimen are common and should be discussed with the doctor or nurse.
 SEMEN COLLECTION METHODS
 The specimen is best collected by masturbation into a sterile container. This is most conveniently
performed in the facility provided at the laboratory however collection at home is acceptable
provided the sample is rapidly transported (within 1 hour) and kept at body temperature.
 Semen collected by interrupted intercourse is not favored as it risks the loss of sample, particularly
the first fraction of the ejaculate.
 Semen should never be collected into an ordinary condom, which contains substances that kill
sperm.
 If religious or personal practices prohibit masturbation, a special condom (SCD) can be used that
does not affect the sperm quality.
 ROUTINE SEMEN ANALYSIS
 Laboratories vary widely in their ability to provide high quality analyses. Semen analyses are best
performed in a specialized laboratory with extensive experience using the approved methods of the
World Health Organization. Analysis of the ejaculate includes the characteristics of the seminal fluid
(volume) and of the sperm themselves including the number of sperm (called sperm count or
concentration), their movement (motility) and shape (morphology).


 NORMAL VALUES OF SEMEN VARIABLES:



 The normal ranges for various parameters of semen quality are shown below:
 STANDARD TEST VOLUME: > 2.0 ml
 SPERM CONCENTRATION: >20 million sperm/ml
 SPERM MOTILITY: >50% with forward movement
 SPERM MORPHOLOGY: >15% normal forms
 WHITE BLOOD CELLS <1 million cells/ml
 ROUTINE SEMEN ANALYSIS
 A semen analysis, also called a sperm count measures the quantity and quality of semen or
sperm.
 1. Remind patient that abstinence for 3-5 days is observed.
 2. Note the time specimen was collected and received.
 3. Upon receipt of specimen, identify the color and volume.
 4. Under the microscope, check for the motility (motile, non-motile, sluggish).
 5. Note the liquefaction time.
 6. Make a smear and stain.
 7. Check the morphology of the specimen (double headed sperm, elongated head,
pyriform (pear-shaped) head, detached head, bent or coiled tail.
 SPERM COUNT FORMULA:
 Cells counted x 50,000/cc
 BODY FLUIDS ANALYSIS

 DIRECT METHOD – WBC COUNT

 1. Charge the fluid directly into the counting chamber.
 2. Count all cells in 9 large squares.

 COMPUTATION:
1 9
10 X 1
(DEPTH) (AREA)
= Cells counted x 100
9
 COMPUTATION OF BODY FLUIDS
 INDIRECT METHOD – WBC COUNT
 1. Using WBC pipette, suck the fluid up to 0.5 ml and WBC diluting fluid up to mark
11.
 2. Mix the blood and diluting fluid in the bulb of RBC pipette for 2-3 minutes by
rotating the pipette (horizontally) between your palms.
 3. Hold the pipette carefully and mix the solution again. Discard the first 1-2 drops
of fluid from the pipette.
 4. Charge into the counting chamber and counting of blood cells can be performed
in the 4 large squares by observing them under the microscope.

COMPUTATION:
 1 1 4
 10 X 20 X 1
 (DEPTH) (DILUTION) (AREA)
 CELLS COUNTED x 50
 COMPUTATION OF BODY FLUIDS
 DIRECT METHOD – RBC COUNT
 1. Charge the fluid directly into the counting chamber.
 2. Count all cells in 9 large squares.
 COMPUTATION:
 1 9
 10 X 1
 (DEPTH) (AREA)

 = Cells Counted x 10
 9
 COMPUTATION OF BODY FLUIDS
 INDIRECT METHOD – RBC COUNT
 1. Using RBC pipette, suck the fluid up to 0.5 ml and RBC diluting fluid
up to mark 101.
 2. Mix the blood and diluting fluid in the bulb of RBC pipette for 2-3 minutes
by rotating the pipette (horizontally) between your palms.
 3. Hold the pipette carefully and mix the solution again. Discard the first
1-2 drops of fluid from the pipette.
 4. Charge into the counting chamber and counting of blood cells can be
performed either in the central large square or in the
corner squares by observing them under the microscope.
 COMPUTATION OF BODY FLUIDS
 COMPUTATION:
 The formula for RBCs count is = number of cells
counted (N) × Dilution factor / Area x Depth
 Where,
 Number of Red blood cells (N)= ?
 Dilution factor = 200
 Area of 5 small squares =5/25 i.e. 1/5sq. mm in length
 COMPUTATION OF BODY FLUIDS

 Depth of the chamber = 1/10 mm = 0.1 mm

 Total RBCs/mm3 = N X 200 / (1/5 X 0.1) = N X 200 X 50
 = N X 10,000 million/mm3
 For example, suppose, N or number of RBCs in the five squares
is 500, then the total RBCs will be:
 = 500 x 10, 000 = 5,000,000 (5.0 million)
 = 5,000 x 10^9/L
COMPUTATION OF BODY FLUIDS
 MICROFILARIA
 SPECIMEN COLLECTION:
 Timing:
 Whenever possible, specimens should be collected before treatment is initiated.
When malaria and babesiosis are suspected, blood smears should be obtained and
examined without delay. Since the parasitemia may fluctuate, multiple smears might
be needed. These can be taken at 8 to 12 hour intervals for 2 to 3 days.
 Type of Sample:
 Venous blood samples provide sufficient material for performing a variety of
diagnostic tests, including concentration procedures (filariasis, trypanosomiasis).
However, in some parasitic diseases (e.g. for diagnosis of malaria in particular),
anticoagulants in the venous blood specimen can interfere with parasite morphology
and staining characteristics; this problem can be further compounded by excessive
delays prior to making the smears. In such cases, capillary blood samples are
preferrable.
 MICROFILARIA
 Capillary blood obtained by fingerstick:
 1. Label pre-cleaned slides (preferably frosted-end) with the
patient’s name (or other identifier) and date
and time of collection.
 2. Clean the site well with alcohol; allow to dry.
 3. Prick the side of the pulp of the 3rd or 4th finger (alternate
sites include ear lobe, or in infant’s large toe or
heel).
 4. Wipe away the first drop of blood with clean gauze.
 5. Prepare at least 2 thick smears and 2 thin smears.
 MICROFILARIA
 Venous blood obtained by venipuncture:
 1. Label collection tubes and pre-cleaned slides (preferably
frosted-end) with the patient’s name (or other
identifier) and date and time of collection.
 2. Clean the site well with alcohol; allow to dry.
 3. Collect the venous blood in a vacuum tube containing
anticoagulant (preferably EDTA); alternatively,
collect the blood in a syringe and transfer it to a
tube with anticoagulant; mix
well.
 4. Prepare at least 2 thick smears and 2 thin smears as soon as
possible after collection.
 MICROFILARIA
 Wet Smear:
 1. Prepare a wet mount by placing a drop of capillary
blood on a clean glass slide, adding a
drop of NSS and a coverslip.
 2. Look for live microfilaria under LPO and HPO.

 Manner of Reporting:
 POSITIVE or NEGATIVE for Microfilaria
 Prepared By:
CRISTINA M. LABRADOR, RMT