Armed Forces of Liberia

Edward Binyah Kesselly Military Barracks

Margibi, Liberia

5 DECEMBER 2009

STANDARD OPERATING PROCEDURES

SUBJECT: BASIC LAB WORK/TESTS INSTRUCTIONS

Procedures for Routine Lab Work

Collecting, Staining and Reading Malaria Smears (Field’s Stain
Method)

Tick Film:

1. Clean the middle or ring finger using an alcohol swap.

2. Prick the finger using a sterile blood lancet. Squeeze gently to obtain a large drop
of blood. Collect the blood on a clean-grease free microscope slide.
3. Spread the blood using a piece of applicator stick evenly on the slide.
4. Label the slide with patient name or other identification number and place it on a
drier or leave to air dry.
5. When dry, dip the slide in Field’s stain A with smear facing downwards for 5
seconds.
6. Wash the slide in clean water gently so that smear does not wash off. Drain off
excess water.
7. Dip the slide into Field’s stain B for 3 seconds. Wash the slide in clean water and
drain off excess water.
8. Dry slide in a drying box or place slide in a vertical position to air dry.
When the thick film is completely dried, place a drop of immersion oil to an area
of the film which appears mauve colored (usually around the edges). Use the 100x
objective of the microscope to examine the stained slide.

Malaria parasites appear on the microscope as follows:

Chromatin (nucleus) of parasite----------------Dark red

Cytoplasm of parasites---------------------------Blue

Other Findings:
Reticulocytes-----------------------------------------Grey blue
Nuclei of neutrophils--------------------------------Dark purple

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 Granules of neutrophils-----------------------------Mauve purple
Granules of eusonophils----------------------------Red
Cytoplasm of mononuclear cells------------------Blue grey

Reporting Thick Film Malaria Smears:

Parasites:
1-10 per 100 high power fields---------------------------------1+
11-100 per 100 high power fields------------------------------2+
1-10 in every field-----------------------------------------------3+
More than 10 in every high power field-----------------------4+

Earl Taweh

Lab Tech
Examination of Urine (microscopy and Urine chemistry)

Urine is examined microscopically as a wet preparation to detect the following:

1 WBCs (white blood cells) in excess of 10 cells/micro liter of urine

2 Red cells
3 Casts
4 Yeast cells
5 Tricomonas vaginalis motile trophozoites
5 Schistosoma hematobium eggs
6 Bacteria (provided the urine is freshly voided)
.
To diagnose urinary schitosomiasis and to detect the above mentioned,
centrifugation of the urine is required to sediment red cells, wbcs, casts, yeast
cells, S. hematobium eggs, T. vaginalis motile trophozoites and bacteria.

Preparation and examination of urine

Chemistry
To do Urine chemistry, dip a urine multi-stick (dipstick) into the urine sample
prior to centrifugation and read result after two minutes. Compare the reagent
strip to the color chat on the dipsticks’ bottle and report abnormality (ies) if any,
for the following Chemicals (analytes):

Glucose Ketones
Proteins Ph
Specific gravity Nitrite
Bilirubin Urobilinogen
Blood Leucocytes

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 Centrifugation
1. Transfer about 10 ml of well mixed urine to a labeled conical tube.

2. Centrifuge at 500-1000 g for 5 mins. Discard the supernatant urine by completely

inverting the tube.

3. Remix the sediment by tapping the bottom of the tube. Transfer one drop of well
mixed sediment to a clean slide and cover with a cover glass.
Examination
4. Examine the preparation microscopically using the 10x and 40x objective with the
condenser iris closed sufficiently to give good contrast. Report the following:
-WBC (round with granules, 10-15 nanometers in diameter) report as:

-Few (up to10 wbcs/hpf (high power field)

- Moderate (11 to 40cells/HPF) Earl Taweh
- Many (more than 40cells/hpf) Lab Tech

-Bacteria (report only when urine is freshly voided): Usually seen as rods, but
sometimes seen as cocci or streptococci.
Red Cells: these are smaller and more refractile than wbcs and have no granules.
report as few, moderate or many per high power field.
Yeast cells: differentiated from red and white cells by their oval shape and sometimes
they usually show single budding. Report as few, moderate or many.
Trichomonas vaginalis: little larger than WBC and are motile. Report as T. vaginalis
trophozoites seen.
Epithelial cells: Larger than both red and white cells and are varied in shape. They
can be seen easily with the 10x objective. Report as few, moderate or many per low
power field.
Eggs of S. hematobium: Recognized by their large size (145x55 micro meters) and
spine on one end. Report as S. hematobium eggs seen.
Casts: contain solidified protein and are cylindrical in shape because they are formed
in the kidney tubules. The following casts are seen in urine:
- Hyaline casts: colorless and empty figures
- Waxy casts: thicker and denser than hyaline casts. They appear indented or
twisted and may be yellow in color
- Cellular casts: contain white or red cells. Red cell casts appear orange red
while white cell casts are yellow-brown in color
- Granular Casts: contain irregular sized granules originating from degenerate
cells and protein.

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EXAMINATION OF FEACES

Collection: Stool samples are collected in a dry and clean container (preferably plastic
container to avoid absorption of water when collected in a paper container).
Instruct the patient to collect the sample from the portion containing mucus or blood and
transfer to the lab as soon as possible.

Before preparation, describe the specimen as follows:

-color of the specimen (brown, yellow, green, etc)

-Consistency of the sample (formed, semi-formed, unformed or watery)
-Presence of blood, mucus or pus

Preparation (saline method): Put a drop of normal saline on a clean grease-free

microscope slide. Immerse a portion (about the size of the head of a match stick), usually
the mucus or bloody parts into the drop of saline and spread evenly. The preparation
should not be too thick. Place a cover glass on the wet prep and place the slide on the
stage of the microscope. Examine the slide using the 10x and the 40x objectives with the
iris diaphragm close sufficiently to give good contrast.

Report the following:

1. The presence of the eggs of parasitic pathogens, (Example, eggs of Ascaris

lumbricoides, Schistosoma spp., etc.
2. Trophozoites of parasites of medical importance (eg. Giardia lamblia, B. coli etc).
3. Larva of pathogenic parasites (eg. Syrongyloides strecoradis).
4. White Blood Cells, Red Blood Cells and Mucus threads.

If there are no eggs, larvae, and trophozoites in the sample, report as No parasites or
ova seen. Report the presence of wbcs and rdcs as few, moderate or many wbc/rbc
seen.

Note: To describe the appearance of eggs, trophozoites, larvae, WBCs, RBCs and mucus
in stool preparation, refer to a bench aid.

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 WIDAL TEST (FOR TYPHOID FEVER)
QUALITATIVE AND SEMI-QUANTITATIVE PROCEDURES
The qualitative procedure detects the presence of antibodies against typhoid causing
bacteria (salmonella typhi and paratyphi) in a patient’s serum. Where as in the semi-
quantitative procedure, the titer (level) of antibodies is measured in respect to the amount
of serum dispensed on the test card. The titer is taken from the last circle area that shows
agglutination.

QUALITATIVE PROCEDURE

1. Bring the reagents to room temperature.

2. Place 50 micro-liter or one drop of patient’s serum into separate circles of the
card.
3. Resuspend the antigen (contained in the reagent) gently.
4. Add one drop of the reagent to each circle next to the sample which is to be
tested.
5. Mix the sample and reagent using a stirrer and spread over the entire area
enclosed by the ring. Use a new stirrer for each sample.
6. Rotate the cards at 100 r. p. m. for 2 minutes.
7. Agglutination in any circle is indicative of a positive result.
8. Report as reactive if there is agglutination and non reactive if there is no
agglutination in any of the circled test areas.

SEMI-QUANTITATIVE PROCEDURE

1. Using a semi-automatic pipette, dispense the following quantities of undiluted

patient’s serum to 5 test circles:

Circle 1----------80 micro-liters

Circle 2----------40 ‘‘ ‘’
Circle 3----------20 ‘’ ‘’
Circle 4----------10 ‘’ ‘’
Circle 5----------5 ‘’ ‘’

2. Add one drop of the reagent to each circle.

3. Mix well using a stirrer.
4. Rotate the slide at 100 r.p.m. for 2 minutes.
5. Agglutination in any of the circles is indicative of the following results:

Circle 1-----------1:20
Circle 2-----------1:40
Circle 3-----------1:80
Circle 4-----------1:160
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 Circle 5-----------1:320
Earl Taweh
Note: A tenth-forth rise in antibody titration is clinically important. Lab Tech

Blood Collection for typhoid test

To obtain serum for the Widal test (typhoid test), aseptically collect 3-5ml of venous
blood in a plain test tube. Sit the blood until clot form. Centrifuge the clotted sample for
3-5 mins. The yellowish portion that sits atop the red cells is the serum. Collect the serum
using a plastic bulb pipette and proceed with the procedures for typhoid testing.

WHITE BLOOD CELL (WBC) TOTAL COUNT

The WBC total count is used to investigate infections and unexplained fever and to
monitor treatments which can cause leucopenia (reduced white cells).

Sample Collection: only EDTA anticoagulated blood or capillary blood should be used
for counting white cells. EDTA anticoagulated blood is obtained by collecting venous
blood in a tube containing EDTA anticoagulant or finger-prick blood in a capillary tube
containing EDTA anticoagulant. Capillary blood is a finger-stick free flowing sample.

Test Method:
1. Measure 0.38 ml of WBC diluting fluid and dispense it into a small test tube.
2. Add 20 micro-liters (0.02 ml) of EDTA anticoagulated venous blood or free-
flowing capillary blood and mix. The volume of blood should be correct.
3. Assemble the Counting Chamber: Make sure the central grid areas of the chamber
and the special cover glass are completely clean and dry. Slide the cover glass
into position over the grid areas and press down on each side.
4. Re-mix the diluted sample. Using a plastic bulb pipette, fill one of the grids of the
chamber with the sample, taking care not to overfill the area.

Important: The chamber must be refilled if the sample overfills beyond the grid or
an air bubble forms in the grid area.

5. Leave the chamber undisturbed for two minutes to allow time for white cells to
settle.
6. Dry the underside of the chamber and place it on the stage of the microscope.
Using the 10x objective of the microscope with the condenser close sufficiently to
give good contrast, focus the rulings of the chamber and the white cells. Focus the
white cells until they appear as small black dots.
7. Count the cells in the four large corner squares of the chamber. Include in the
count the cells lying on the lines of two sides of each large square.
8. Report the number of white cells/cmm by multiplying the total number of cells
counted in four large corner squares by 50.

Example: If you counted 79 cells in all four squares, your report will be
4,582 cells/cmm, if you multiply 79 by 50.
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 SICKLE CELL SLIDE TEST
In the absence of the HBS solubility filtration test, the sickle cell slide test is useful in
detecting sickle cells in patients who have either sickle cell disease or sickle cell trait.

Materials and reagents:

- Freshly made 20 g/l (2% w/v) sodium metabisulphite or sodium dithionite solution
- A triple beam balance or an electric digital balance
- distilled water or deionized water
- Microscope slides and cover glasses
- Microscope
- Test tubes
- Pipettes
-Applicator sticks/stirrer
-Lancets, needles and syringes

Test Procedure

1. Weigh 0.1g of the chemical (sodium metabisulphite) and transfer to a test tube
capable of holding 15 ml of water.
2. Add 5 ml of distilled or deionized water, stopper, and mix until the chemical is
fully dissolved. The chemical can only be used on the day it was suspended(with
in 8 hrs)
3. Deliver one drop of patient’s capillary blood or well mix venous blood on a slide
and add an equal volume of freshly made reagent, mix and cover with a cover
glass. Exclude any air bubbles.
4. Place the slide in a plastic box or Petri dish with damp piece of blotting paper or
tissue at the bottom to prevent drying of the preparation. Close the container and
leave at room temperature.
5. After 10-20 minutes, examine the preparation microscopically for sickle cells.
Focus the cells first with the 10x objective and examine for sickling using the 40x
objective. Examine several fields. Sickling usually takes place in one part of the
preparation than the other.
6. Sickle cells usually appear crescent shape with pointed ends or holly leaf shape.
Report as “sickle cell test positive” when crescent shape cells are seen, or ‘sickle
cell test negative” when cells appear rounded or oval shape.

Syphilis Ultra Rapid Test

Immunochromatography(IC) Method

The syphilis ultra rapid test strip method detects the presence of Treponema pallidum
(TP) antibodies in a patient’s specimen (serum, plasma, or whole blood).

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Materials:
Test strips
Disposable specimen droppers
Buffer
Test cards
Lancets
Alcohol Cotton swabs

Test Method:
Allow test strips and buffer to reach room temperature prior to testing.
1. Remove test strip from the sealed foil pouch and use it as soon as possible. Best
results will be obtained if the assay is performed within one hour.
2. Peal off the tape from the test card and stick the test strip in the middle of the test
card with arrows pointing downwards.
3. For serum or plasma specimen: Hold the dropper vertically and transfer 2 drops
of serum or plasma onto the specimen pad of the test strip. Then add one drop of
buffer. For Venipunctured Whole Blood specimens: Hold the dropper vertically
and transfer 2 drops of whole blood onto the specimen pad of the test strip and
add one drop of buffer. For Fingerstick Whole Blood specimens: Allow 2
hanging drops of fingerstick whole blood to fall onto the specimen pad of the test
strip, then add one drop of buffer. Set the timer for 10 minutes. Read results after
10 mins (up to 30 mins). The result should not be read after 30 mins.

Interpretation of result:
Positive: Two distinct red lines appear on the test strip (one in the test (T) region
and the other in the control(C) region).
Negative: One red line appears in the control(C) region and no red line in the test
region.

Determine HIV 1/2 Test Method

The above test method is an Immunochromatographic (IC) test method for the qualitative
detection of antibodies to HIV 1and HIV 2. In case you don’t have the above mentioned
kit, follow the manufacturer’s test manual provided in the test kit available. The test
procedure requires Whole blood, Plasma or Serum.

Materials:
Lancets
EDTA capillary tubes
EDTA test tube
Pipettes
Syringes and needles
Cotton swabs

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Test Procedure
1. Remove the desire test strips from the 10-tests card and peal off the protective foil
cover from each test strip. Label the test strip with patient’s name or other
identification numbers.
2. For serum or plasma samples: Apply 50 micro-liters(2drops) of serum/plasma
to the sample pad (marked by the arrow symbol).
3. For whole blood (venipuncture) Samples: Apply 50 micro-liters (2 drops) of
Sample to the sample pad, wait 1 min,
add one drop of chase buffer.
4 For whole blood (fingerstick) samples: Apply 50 micro-liters (2 drops) of the
sample to the sample pad. Wait until
sample is absorbed into the sample pad.
Add one drop of chase buffer to the sample
pad. Wait a minimum of 15 mins (up to 1 hr)
and read results

Interpretation of results

Positive: Two red bars appear in both the control window (labeled ‘C’) and the
patient window (labeled ‘patient’) of the strip. Any visible red color in the patient
window should be read as positive.

Negative: One red bar appears in the control window of the strip (labeled ‘C’) and no
red bar appears in the patient window of the strip (labeled ‘patient’).

Determine HBsAg Test method

The above test method is a visual read, qualitative immunoassay for the detection of
Hepatitis B surface Antigen. Hepatitis B, as you may know, is a virus that causes liver
damage in some patients. Detecting the infection at an early stage is very important in the
treatment process.

The procedure for the above test method, using the Determine HBsAg test kit, is the same
for the Determine HIV 1 and HIV 2.Therefore, follow exactly, the outlined procedure for
HIV 1 and HIV 2 on the previous page.

Blood Collection
Many lab tests use Whole Blood, Plasma or Serum as specimen to carry out assays.
Therefore, the way you collect and handle blood is very important. For example, if you
handle blood in such a way that you hemolise (break the cells) the blood, the results may
come up to be wrong (falsely high or low). On the other hand, if you place blood samples
in the wrong containers, the results will also be altered. In any case, always use the
appropriate containers for blood samples and handle blood with ultimate care to avoid

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hemolisis. Temperature should also be given concern (blood samples and other body
materials should always be kept away from high temperature (temperatures around 2-25
degree Celsius are accepted).

Whole Blood: is blood that is not separated in any way. It could be venipunctured
whole blood or fingerstick whole blood. Venipuncture whole blood is blood taken from
the veins where as fingerstick whole blood is blood obtained from a clean-punctured
finger.

Plasma: refers to the yellow-brown fluid portion of the blood. Plasma can be obtained
when you place blood (freshly extracted) in a container (test tube) containing
anticoagulant. Anticoagulants are agents (chemicals) that allow blood to stand on
laboratory desk without clotting. After centrifuging the tube containing blood and
anticoagulant, the yellow portion that sits atop the sediment is the plasma.

Serum: Looks exactly like plasma. It is called serum because it is obtained from clotted
blood placed in a plain tube. The difference between plasma and serum is that plasma
contains fibrinogen and serum doesn’t. Some lab tests may require serum while other
tests will need plasma as specimen for assays. This is why you must know what is plasma
and what is serum.

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