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Medical Laboratory Technology: Volume 1
4. Pour the stain gently into the staining trough, until all the slides are totally covered.
Stain for 30–45 min out of sunlight.
5. Pour clean water gently into the trough to remove the deposit on the surface of the
staining solution. Gently pour o the remaining stain and rinse again in clean water for
a few seconds, pour o the water.
Note In some laboratories with limited supply, the Giemsa stain is reused; however,
in such cases, the stain must be used on the same day.
6. Remove the slides with forceps and place in a slide rack to drain and dry. Keep the lm
side downwards but make sure that the lm does not touch the slide rack.
Rapid method
When urgent results are needed, the above method is slightly modied. It uses much more
stain than the regular method.
1. Allow the thick lm to dry thoroughly; fanning or briey exposing the slide to gentle
heat such as that from a microscope lamp. Care should be taken to avoid overheating
otherwise the thick lm will be heat-xed.
2. Prepare a 10% Giemsa solution in buered or diluted water with pH 7.2. If a small
quantity is being used, a few drops of stain per millilitres of buered water will give the
correct concentration of Giemsa solution. One slide requires about 3 mL of stain. Mix
the stain well with a glass rod.
3. Gently pour the stain on to the slides using a pipee. Stain for 5–10 min.
4. Gently ush the stain o the slides by adding drops of clean water.
Note Do not tip o the stain and then wash, as this leaves a deposit over the smears.
5. Place the slides in the slide rack to drain and dry, lm side downwards; make sure that
the lm does not touch the slide rack.
Field stain
Field staining has several advantages. It is quick and there is no dilution of staining as in
some other stains. It is recommended for a rapid detection of malarial parasites. The stain,
however, does not always stain the Schuner’s dots. It is recommended for thick smears.
Supplies and reagents
• Glass jars, slide racks
• Field stains A and B: See details in Chapter 10. Field stain has a long shelf-life (lter
every two days until the staining is weak). Washing does not require any buered water.
• Methanol
Procedure
The procedure of eld staining is described in Chapter 10 for the study of blood smear. A
general outline of the procedure is given here for convenience.
Thin lm
1. Fix the lm in methanol for 1 min.
2. Wash o the methanol with buered water. Do not x for extended period.
3. Using a pipee, cover the lm with diluted Field stain В (one volume of stain plus four
volumes of buered water).
4. Immediately add an equal volume of eld stain A solution and mix well by tilting the
slide.
5. Allow staining for 1 min.
6. Wash o the stain with clean water.
7. Place the slide upright in a slide rack to air-dry.
Thick lm
1. Dip the unxed lm into a jar containing Field stain A solution for 3 s.
2. Wash gently by dipping (once) into a jar of clean water for 5 s.
Special Haematological Tests
319
3. Dip the slide into a jar containing Field stain В solution for 3 s.
4. Wash the slide gently as in Step 2.
5. Finally, drain o the water and air-dry in a rack (smear side facing down). Do not blot
dry.
Evaluation of blood smears under microscope
Scan the blood smears (thick and thin) under the high-power objective (40x) and examine
closely under oil-immersion. The thick blood lm is used for reporting parasite density
(Figure 11.7). Count the number of parasites in each microscopic eld (l000×). This report
helps in the assessment of the clinical condition (high, >20; medium, 2 to 19; low, <2). In case
of high density the physician must be alerted. The density of parasite is also a characteristic
feature of the Plasmodium species.
Note Outlines of the red cells will not be seen in the thick smear after staining. The red cells
have lost the haemoglobin which is removed by the Field stain. The pink Schüner‘s dots can
still be seen around the parasite in the red cells (Figure 11.6). Cytoplasm of the trophozoites
(blue) remains after the staining and the leucocytes remain unchanged.
Figure 11.7 Thick smears are used for determining density of parasite: (a) High density with 20 or
more infected red cells in the microscopic eld (40×), (b) Medium density with 2–19
infected red cells, and (c) Low density with 1 or less infected red cells. The density of P.
falciparum is high, P. vivax is medium and P. malariae is low.
Interpretation
The various stages of the parasite seen in the peripheral blood smear are as follows—
trophozoites (immature and mature), schizont and gametocyte (Figure 10.17).
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Immature Trophozoites These are found in newly infected cells in the early phase of the
asexual life cycle. They are often identied as undivided nucleated cells with blue-coloured
cytoplasm or a ring of cytoplasm present within the red cells.
Mature Trophozoites These are enlarged trophozoites that have lost the ring structure and
have more compact cytoplasm which may be amoeboid in shapes.
Schizonts (merozoites) These appear as individual nucleated cells often arranged in a circle
forming a rosee, or distributed throughout the red cell.
Gametocyte This is one compact and round (or elongated) gametocyte—male or female—
lling the entire red cell. This stage occurs when the multiplication of trophozoites and
infecting of new red cells has occurred for a while.
Pigment Some of the parasites have granules of pigment—yellow brown to black—in their
cytoplasm.
Morphology of Red Cells Infected red cells may look normal, deeply stained, enlarged,
oval-shaped, with jagged edges, or with pink dots (stippling) called
Schiiner’sdots.The
characteristic changes depend on the species of the infecting malarial parasite.
Identifying characteristics of Plasmodium species
Some of the characteristic features of P. falciparum and P. vivax are given in Table 11.1.
Table 11.1 Characteristic features of P. falciparum and P. vivax
Stage of development of
parasite P. falciparum Ρvivax
Immature trophozoites Cytoplasm thin hair like pale blue ring Not found frequently
Mature trophozoites Seldom seen in peripheral blood,
compact cytoplasm, dark pigment
Schizont Very rare in blood lms except in
serious conditions
Gametocyte Frequently found; sausage or crescent
shaped
Parasite density Very high density Medium density
Red cell morphology Size normal, colour normal, few red
dots
Malarial ring Frequently found, small 1/5-1/3 RBC
diameter; normal; multiple infection
of RBC more common than in other
species; Maurer’s clefts (under certain
staining conditions)
Parasite density Often high Medium density
Irregular or amoeboid outline with
strands of cytoplasm; Schuner’s dots
present
Quite frequently found; 12–18 large
compact red granules
Frequently found, oval or round
Size enlarged, pale coloured,
Schuner’s dots present
Frequently found, thick ring, round
to oval; occasionally Schuner’s
dots; occasionally mbriae; multiple
infection of RBC not uncommon
Parasite density
A report of the parasite density, as seen under the microscope, provides a clue regarding the
seriousness of the disease. In addition, it may also help in the identication of the species
(Table 11.1). A low density of parasites in adults in an endemic area may not be alarming.
Only thick lms are used for the reporting of the parasite population.
Special Haematological Tests
The parasite density is the number of parasites counted in each microscopic eld
(Figure 11.7). Two methods can be used to count malarial parasites—quantitative and
qualitative.
Quantitative report of parasite density is based on the number of parasites counted as
compared to the number of leucocytes.
Note These parasites, in trophozoite or gametocyte stages, were originally present in the
infected red cells; after staining, they appear as dots beside the white cells. You may use two
hand tally counters, one for the white cells and another for the parasites. If the white cell
count is known, the number of parasite per µL of blood can be calculated.
Example Ten parasites were counted against 200 white cells. If the white cell count is 8000/µL,
the parasite density will be reported as:
321
Number of parasites observed
Number of white cells counted
Or,
10
×
8000/mL = 400 parasites/mL
200
In case of P. falciparum, it is normal practice to count the number of gametocytes and the
asexual stage of the parasite separately. This helps in monitoring the response to antimalarial
drugs that are active against the schizont stage, and would not be expected to have any eects
on gametocytes.
In case of qualitative report, the reports are made, ranging from 1+ to 4+, indicating an
estimate of the parasite population.
1+ refers to 1–10 parasites per 100 thick-lm elds (only occasionally seen).
2+ is 11–100 per 100 thick-lm elds (or 1 per single thick-lm eld).
3+ is referred to 1–10 parasites per single thick-lm eld.
4+ refers to more than 10 parasites per single thick-lm eld.
Additional Information
• For proper identication and reliable parasite counting, use clean slides and well-made
and well-stained thick lms.
• Patients with very high parasite densities (4+) require urgent treatment. Therefore, if
you nd a high parasite density, state the result clearly in your report and notify the
patient’s physician.
• In your routine examination of the blood lm, in search of malaria infection, if there is
a positive report, specify the species of parasite found, the stage of development of the
parasite and the parasite density.
• A patient may harbour more than one species of malarial parasite.
• If the report is negative, report as no parasites found.
×
WBC count/mL = Parasites/mL
Rapid diagnostic test (RDT) for malaria
Peripheral blood smear examination has been the “gold standard” for the diagnosis of malaria.
But microscopy is expensive, not easily available, and requires special training. On the other
hand, Rapid Diagnostic Tests or RDTs, based on immunochromatographic principles for the
detection of malaria antigens, developed in the past decade, have opened a new and exciting
avenue in malaria diagnosis. It is fast, accurate and requires minimal skill to perform. In
addition, RDTs do not require a laboratory, electricity, or any special equipment. The Rapid
Malaria Tests (RDTs) have been developed in dierent test formats like the dipstick, strip,
card, pad, well, or cassee. Antibodies are impregnated on nitrocellulose strip which is
322
placed in a plastic cassee or on a card. Cassees and cards tend to be more expensive than
using a test tube with lysed blood and buer in which the strip is dipped. But the cassees
and cards are simpler to use.
Microscopy and RDTs are both adequate to diagnose malaria in febrile patients.
Demonstration of the presence of malaria parasites is advised before the treatment with
antimalarial medicines, as diagnosis based solely on clinical symptoms is of poor accuracy and
leads to over diagnosis of malaria, waste of antimalarial medicines, an increased frequency of
adverse side-eects and increased drug pressure on resistant parasites.
Things to remember when using an RDT:
• Prior to perform the test, carefully read manufacturer’s instruction included in the kit
and the interpretation of results.
• Blood-safety precautions should be followed.
• The RDT should be discarded if the envelope is punctured or badly damaged.
• The test envelope should be opened only when it has reached ambient temperature and
the RDT should be used immediately after opening.
• The result should be read within the time specied by the manufacturer.
• An RDT cannot be re-used.
Use of a typical RDT malaria test kit (easy trust triline)
This kit is simple, easy to handle and aordable by poor laboratories.
Principle
Malaria rapid diagnostic tests (sometimes called “dipsticks”) detect specic antigens (proteins)
produced by malaria parasites (Plasmodium falciparum or Pf, P. vivax or Pv, Plasmodium ovale
or Po, and Plasmodium malaria or Pm). These antigens are present in the blood of infected or
recently infected people. The RDT signies their presence by a colour change on an absorbing
nitrocellulose strip. When in good condition, some RDTs can achieve sensitivity similar to
that commonly achieved by microscopy. Sensitivity, however, can vary between products
and density of parasites.
Supplies in the kit
• Instruction card
• Contents of device package:
▪ Alcohol swab
▪ Test card or strip placed in a separate package, inside the device package
▪ Diluent (buer and haemolysing agent) in a dropper bole
▪ Lancet to draw the blood sample
▪ Pipee to transfer blood
Storage of kit
• When stored at room temperature, the test kit components are stable until expiry date
printed on labels.
• The test kit should be kept away from direct sunlight, moisture and heat.
• Do not freeze the test kit.
Additional requirements
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Medical Laboratory Technology: Volume 1
Warning
• Read the instructions before starting the procedure.
• Do not use beyond the expiration date.
• Do not open the foil until you are ready to use it.
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323
Figure 11.8 Rapid malaria diagnostic test
Procedure
The test procedure varies between the test kits. In general following procedure seems to
describe the basic steps (Figure 11.8):
1. Take out the alcohol swab from its package and sterilize the nger.
2. Open the test card package and take out the test card.
3. Place the card on the work bench.
4. Make a routine skin puncture with the help of the lancet (EDTA anticoagulated blood
specimen from venepuncture can also be used).
5. Gently draw the blood (2 to 50 µL) with the help of a transfer pipee.
6. Place three drops of blood sample in the designated well.
7. Now take the diluent (buer mixed with haemolysing compound) in the dropper
bole and add 3 drops in the same well as the blood sample. The diluent will carry
the sample (antigen) through the absorbent strip to reach the antibody spots. Wait
for 5 min.
Note Some test kits require 15 min for the migration of the blood specimen. Follow the
instructions of the manufacturer of the kit.
8. After the waiting period of 5 min, closely observe the lines on the test strip or card.
As the blood sample is drawn up, the malarial antigens present in blood sample react
324
with the specic antibodies placed on the respective zones of test strip. The reaction
is recognized by the appearance of dark lines. Positive reaction between the antigen
and the antibody is visualized as dark bands by the presence of detectable marker like
colloidal gold. This is predeposited with the specic antibodies on the test strip by the
manufacturer.
9. Report the result after comparing the lines with the picture given in the test card for
dierent malarial parasites.
10. Change of colour on the control line is necessary to validate the test and its nonappearance, with or without colour changes on the test lines, invalidates the test. With
colour change only on the control line and without colour change on the other lines, the
test is interpreted as negative.
Problems with RDTs
False Positivity: False positive tests can occur with RDTs for many reasons. Circulating
antigen below detection level is one of them. Drug resistance or reaction with rheumatoid
factor can also occur. Cross reactivity with autoantibodies, such as rheumatoid factor (RF),
gives false positive result.
Sensitivity: Sensitivity of RDTs varies with parasite density. In cases of suspected severe
malaria or complex health emergencies, a positive result may be conrmatory but a negative
result may not rule out malaria. Further, a negativeRDTresultshouldalwaysbeconrmed
by microscopy. It should be emphasized that P. falciparum malaria, a potentially lethal disease,
must not be missed because of a false-negative dipstick test.
Interpretation of Results
Negative: When control line appears while other sites remain clean.
Invalid: The test is invalid if the “control” line (marked “C”) does not appear.
Positive: The test is positive when 2 or 3 lines appear in the test strip. Interpret results after
comparing with the results given on the instruction sheet.
Note Control line must appear in order to interpret the results.
Medical Laboratory Technology: Volume 1
Sleeping Sickness (Trypanosomiasis)
Sleeping sickness is caused by a protozoan that belongs to the genus Trypanosoma. The disease
is called Trypanosomiasis. It is prevalent in southern and western Africa. In Central and
South. America, Chagas disease is caused by T. cruzi. African trypanosomes (T. gambiense)
are transmied by tsetse ies (Glossina sp.) and humans are the main reservoir of infection.
Pigs, dogs and possibly other animal species can also harbour the parasite, but their role in
spreading the disease is secondary. Transmission occurs when tsetse ies ingest the blood of
infected humans or animals.
Clinical stages
African trypanosomiasis occurs in three phases—the acute phase, the parasitaemic phase and
the neurological phase. The parasite is transmied by the vector tsetse y. Two or three days
after the bite of an infected tsetse y, a chancre appears at the inoculation site, which disappears
within 2–3 weeks. From the site of the chancre, the trypanosomes invade the bloodstream,
giving rise to occasional episodes of intermient fever. The most common symptoms of the
rst or acute phase are headache, sleeplessness, pain in the joints and lymph nodes of the
posterior neck, swelling of eyelids and joints, weight loss and generalized intense itching,
especially in the region of the breast bone. Invasion of the central nervous system causes
irritability, paraesthesia, sleeplessness and eventually severe headaches and blurred vision,
as well as epileptic aacks, psychosis, drowsiness, mental lethargy and coma. Some species of
Special Haematological Tests
325
Trypanosome may be slow in their progress (T. gambierne) while others (T. rhodesiense) might
spread rapidly causing death in few months.
Laboratory diagnosis
Trypanosomes, the causal agent of sleeping sickness, are found in the lymph gland at an
early stage, particularly in case of T. gambiense infection. When the infection is about 4 months
old, the parasite disappears from the lymph gland and is found in the blood. Microscopic
examination of lymph node aspirate is helpful in the early diagnosis of sleeping sickness.
The lymphatics of the body drain into the lymph glands. Among various other functions
(production of “lymphocytes, transportation of lymphatics), lymph nodes appear to act as
lters keeping particulate maer, especially bacteria, from gaining entrance to the blood
stream.
Specimen
Lymph node aspiration is used in the diagnosis of trypanosomiasis at an early stage (within
two to three months following infection). The site for obtaining the lymph node aspirate must
be carefully chosen. Aected glands are swollen and are recognized among cervical glands
of the neck. The swollen glands have round lumps of 2–4 cm diameter and are rubbery and
slide under the skin, oering lile resistance to pressure. They become hard at later stages of
infection. The specimen must be immediately examined so that the agellar movement of the
parasite can be observed.
Principle
A wet mount made from the lymph node aspirate can reveal the presence of trypanosomes
at an early stage of infection. Flagellar movement among cellular elements identies the
parasite. A stained smear of the aspirate can also be examined.
Equipment and Supplies
• Microscope
• Microscopic slides and cover slips
• Blood drawing syringe, 5 or 10 mL. Both syringe and needle must be perfectly dry
• Needle (for subcutaneous injection) 25-gauge
• Tincture of iodine or thiomersal
• Ethanol (70%)
Reagents
• Saline (0.85% sodium chloride)
Dissolve 8.5 g of NaCl in 500 mL of distilled water in a 1000-mL volumetric ask and make
it to volume.
Procedure (see Figure 11.9)
1. Choose the site: Lymph nodes are found among the cervical glands of the neck.
Feel both the right and the left sides of the neck, from the base of the neck up to
the ears.
2. Ask the patient to sit down and disinfect with tincture of iodine (or use thiomersal).
3. Wash o the iodine with absolute alcohol.
4. Take the lymphatic gland between thumb and index nger of the left hand and hold it
steady.
5. Introduce a sterilized needle at a right angle into the centre of the gland. Caution Do
not puncture a vein or artery.
6. Gently knead the gland with the left hand and revolve the needle in both directions
inside the gland with the right hand. This should not last for more than 90 s. Normally,
the piston is not necessary to pull the aspirate, but in case of a dry gland apply gentle
suction.
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Figure 11.9 Microscopic examination of lymph node aspirate for the diagnosis of trypanosomiasis:
(a-d) Lymph node aspiration,(e and f) Preparation of wet mount, (g) Shape and size of
Trypanosoma gambiense compared against red blood cells
7. Withdraw the needle and apply iodine.
Note Do not allow the iodine to touch the needle.
8. Aach the needle to the syringe and push out the contents on three slides with a drop
of saline on each (Figure 11.9e). In case of the third slide, draw a drop of saline into the
syringe and washout the contents onto the slide.
9. Place the cover slip (Figure 11.9f) and examine under low-power objective (10×). Switch
to high power (40x) once you have located the parasite in order to examine it in greater
detail. Close the condenser iris diaphragm suciently to give a sharp image. Wait until
the convection currents stop. It is impossible to see the movement of trypanosomes
among moving cells.
10. Look for motile agellar trypanosomes, which are two to three times larger than red
blood cells (20 µm lengthwise). The trypanosomes move among the cellular elements
following a zigzag course (Figure 11.9g).
Special Haematological Tests
327
Examination of blood films for trypanosoma Brucei Gambiense
In African trypanosomiasis, trypanosomes appear in the blood at intervals for a period of
a few days, mainly during the rst three months of disease and especially during bouts of
fever. Thick and thin lms of blood are made on separate slides and stained with Giemsa
or Field stains. The method is described in the laboratory diagnosis of malarial parasites
(see above). The blood parasite, when present, may be identied under the microscope on a
stained preparation.
Microscopicexaminationofvenousbloodconcentratedbycentrifugation
Blood collected by venepuncture, as described in Chapter 5, is anticoagulated with citrate,
subjected to centrifugation in order to concentrate parasites and then examined under a
microscope.
Equipment, supplies and reagents
• Microscope
• Centrifuge
• Microscopic slides, cover slips, conical centrifuge tubes, Pasteur pipee
• Trisodium citrate, 3.2% solution (anticoagulant). Mix 3.2 g of trisodium citrate salt in a
100-mL volumetric ask, dissolve in about 50 mL of water and then make it to volume
(100 mL). Keep the solution refrigerated. Label with date.
Procedure
1. Take 1 mL of the anticoagulant solution in a conical-shaped centrifuge tube (#1).
2. Add to this 9 mL of blood collected from venepuncture.
3. Mix and centrifuge (3000 G for 3 min).
4. Draw o the supernatant plasma and leucocytes above the level of erythrocytes into a
second centrifuge tube (#2).
5. Centrifuge second centrifuge tube (3000 G for 3 min).
6. Draw o the supernatant uid from centrifuge tube # 2 but save the deposit. Place the
supernatant uid of tube # 2 in another centrifuge tube (#3) and centrifuge this last tube
for 10 min at 3000 G.
7. Examine deposits of tubes #2 and #3 between a slide and a cover slip under a microscope.
The trypanosomes appears in the deposit from tube #3 and occasionally in the deposit of
tube #2.
Alternative microhaematocrit method
If a microhaematocrit centrifuge is available, take the citrated blood in a plain capillary
tube, seal it and then subject it to centrifugation. When examined under microscope, motile
trypanosomes, if present, can be found in the plasma just above the layer of leucocytes. First
use the 10× objective with reduced condenser aperture to detect any movement. Then switch
to 40× objective to see trypanosomes more clearly.
Immunologic card test for trypanosomiasis (card agglutination test)
A drop of capillary blood is taken on a strip of lter paper, along with the skin puncture
for making blood smear. The dried drop of blood is used in the immunological testing of
trypanosomiasis (card agglutination test). The method is described in immunology (Chapter
24 of Volume II).
Chagas Disease
Chagas disease is caused by another species of Trypanosoma—T. cruzi. It primarily aects
children and is characterized by intermient or continuous high fever. About 50% of children
manifest unilateral swelling of the eyelids. On other areas of the face or body, cutaneous
lesions (chagomas) that resemble furuncles occur near the inoculation site. There may be