Добавил:
Sekretar
kiopkiopkiop18@yandex.ru
t.me/Prokururor I Вовсе не секретарь, но почту проверяю
Опубликованный материал нарушает ваши авторские права? Сообщите нам.
Вуз:
Предмет:
Файл:Ординатура / Хирургия / Библиотека им академика М.И. Перельмана / Книга_5543_Библиотеки_им_академика_М_И_Перельмана.pdf
308
Medical Laboratory Technology: Volume 1
• Microscope
• Freshly made sodium metabisulphite, 2% solution
Reagent
Sodium metabisulphite (2% w/v in water)
Sodium metabisulphite (Na2S2O50) 0.5 g
Distilled water (q.s.) 25 mL
Note Make a fresh solution before use. If sodium metabisulphite (preferred) is not available,
you can use sodium bisulphite.
Procedure (Figure 11.1)
1. Place a small drop of blood (20 µL) in the centre of the glass slide.
2. Add an equal drop of the sodium metabisulphite solution by means of a Pasteur
pipee.
3. If petroleum jelly is not available, keep the preparation in a moist chamber (Figure 11.1).
Note Place a wet lter paper at the boom of a Petri dish; place the slide supported
on two sticks on the lter paper; cover the Petri dish. Some authors believe that
sealing is not necessary when a reducing agent like sodium metabisulphite is used. A
wet chamber, however, will not be necessary if the cover slip is sealed by petroleum
jelly.
4. Mix carefully with the corner of the cover slip.
5. Cover with the cover slip, making sure that no air bubbles form under the cover slip.
Press down lightly on it to remove any excess blood, sodium metabisulphite mixture
and air bubbles.
6. Using the syringe and 19 gauge needles, carefully rim the cover slip with petroleum
jelly, completely sealing the mixture under the cover slip.
7. Wait for 15 min.
8. Examine the preparation under the high-dry objective (40x) of the microscope. Make
sure when the objective is changed to the high-dry, it does not touch the petroleum
jelly. Sickling is visible almost immediately in case of sickle cell disease (Hb S-S) and
will be obvious in sickle cell trait (Hb S-A) within 60 min. If the result is negative, reexamine after 2 h and after 24 h. The red cells remain round (unchanged) when the test
is negative. Slightly crenate red cells are also reported as negative. In case of positive
results, the red cells become sickle-shaped or banana-shaped often with spikes. In some
instances, the red cells may take the ‘holly-leaf' form, which is often found in the sickle
cell trait. It is important to examine several parts of the preparation as sickling can occur
more quickly in one part than in another. Do not mistake normal erythrocyte lying on
their side or crenated cells for sickle cells.
Additional information
• Sickle cells or the ‘holly-leaf’ shapes of the cell must have one or more pointed ends.
Elongated cells with rounded ends must not be confused with sickle cells.
• It is important to examine several parts of the preparation, as sickling might occur more
quickly in one part than in another.
• If the patient’s blood gives a positive result, examine his blood smear for the presence
of sickle cells, nucleated red blood cells (NRBC), target cells, poikilocytes and often
macrocytes.
False negative results might occur if reagents are outdated, concentration of Hb S is low
or the patient has moderate to severe anaemia. Whenever possible, electrophoresis of the
haemoglobin should be carried out to conrm a diagnosis of sickle cell disease. This can be
done in a reference laboratory.
Special Haematological Tests
309
Figure 11.1 Sickle cell test using sodium metabisulphite: (a and b) A drop of blood specimen,
obtained by nger stick, is placed on a slide, (c and d) A drop of freshly made sodium
metabisulphite solution is mixed with the blood specimen with the corner of a coverslip;
cover with the coverslip (no air bubble), (e) Place the slide in a moist chamber, (f)
Examine after IS min under high-dry objective (40×), (g) If the red cells are sickled,
report the test as positive
Solubility Test for Sickle Cell
This test can be conveniently carried out anywhere and is ideal for mass screening. It is
observed visually and requires no microscope. This test is more sensitive than the previous
sodium metabisulphite test.
Specimen
EDTA-anticoagulated venous blood, heparinized capillary blood or citrated blood can be
used. A fresh specimen is not necessary.
Principle
Haemoglobin S in the reduced state is less soluble than the normal Hb A. Dithionite in
phosphate buer reduces Hb S; thus it forms a turbid suspension of protein crystals. These
crystals prevent reading of lines on a paper card. Saponin is used to lyse the red cells.
310
Medical Laboratory Technology: Volume 1
Reagent
Dithionite solution
Dibasic potassium phosphate:
Anhydrous K
HPO4 21.6 g
2
Monobasic potassium phosphate:
Anhydrous crystals, KH2PO4 16.9 g
Sodium dithionite, Na2S2O4 0.5 g
Saponin 0.1 g
Preparation of reagents
Place the K2HPO4 in a 100-mL volumetric ask. To facilitate preparation, rst dissolve the
K2HPO4 in about 60 mL of distilled water. Next, add the KH2PO4 and the dithionite; mix
until dissolved. It may be necessary to add a lile more water to dissolve crystals. Add the
remainder of the water up to the volumetric mark only after everything is dissolved. Finally,
add the saponin. Mix well. Date and refrigerate the bole. It is stable for one week.
Equipment
• Test tube rack with a background of lined reader scale made in the laboratory by taking
a white cardboard with parallel horizontal black lines, 2.5 cm apart.
• Test tube (10-mL)
• Sahli pipee (20-µL)
Procedure (Figure 11.2)
1. Pipee 2 mL of reagent in the test tube.
Note Bring the reagent to room temperature before dispensing. The test tubes may be
marked previously at 2 mL which avoids repeated pipeing.
Figure 11.2 Solubility test for the diagnosis of sickle cell anaemia: (a) Prepare a fresh solution of
sodium dithionite, (b) Take 2 mL of dithionite working solution in a test tube and 0.02
mL of whole blood, (c) Mix, (d) Hold at room temperature for 10 min, (e and f) Observe
turbidity; in case of a positive test, lines on the background cannot be seen
2. Add 20 µL of anticoagulated blood specimen. If a calibrated dropper is available, use
that. The volume of the specimen must correspond closely to 20 µL. It is a qualitative
test and hence accurate measurement of volume is not very critical.
Special Haematological Tests
3. Mix and wait for 10 min.
4. Place the tube in a test tube rack with a background of a lined reader scale.
5. Read the turbidity and note the results. A positive test is indicated by a very turbid
solution—the black lines on the lined reader scale cannot be seen through the solution.
6. Run positive and negative controls with each group of solubility tests.
Note Other haemoglobins (Hb С) may also cause turbidity, which is identied by
electrophoresis.
7. All specimens with a positive result must be subjected to Hb electrophoresis in order to
dierentiate between Hb S-S (sickle cell disease) and Hb S-A (sickle cell trait).
Additional information
• A false positive result might arise from conditions such as polycythaemia, too much
blood added to the reagent, hyperlipaemia and impure saponin. Always use the best
quality saponin.
• False negative results might arise from outdated reagents. It is often convenient to pre-
pare reagents individually (ve times concentrated) and later dilute before use. The
nal working solution should not be stored for more than one week. Always refrigerate
the phosphate buer.
311
Haemoglobin Electrophoresis
Electrophoresis is a method by which various proteins can be identied. When the mixture of
proteins is subjected to an electrical eld, the protein particles move with dierent velocities
towards the positive pole (anode). The rate of movement is dependent upon the nature of
protein and the number of charges the protein particle is carrying. In case of haemoglobin,
a conjugated protein, the normal and abnormal haemoglobins migrate towards the anode
(+ve pole) at dierent rates, which enable their identication and the laboratory diagnosis of
haemoglobinopathies. Haemoglobinopathies are congenital disorders that originate from a
defect in the globin chain (the protein component of the haemoglobin molecule).
As haemoglobin electrophoresis is generally performed at reference biochemistry
laboratories, these techniques are described for a more complete understanding of the process.
Specimen
EDTA-anticoagulated venous blood or heparinized capillary blood can be used. If samples
have to be transported, use of citrated blood is preferred.
Principle
Haemoglobin, obtained from haemolysed red cells, is placed on a supporting medium (cellulose
acetate gel or acrylamide) and allowed to migrate in an electrophoresis apparatus (Figure 11.3).
At the end of a specied period, abnormal haemoglobins are identied by comparison of their
migration with that of Hb A which is found in normal adults. Staining may not be necessary;
however, for beer clarity of the location, a protein stain can be used like Ponceau S.
Reagents
Tris buer
Tris (hydroxymethyl) amino methane 10.2 g
EDTA 0.6 g
Boric acid 3.2 g
Distilled water (q.s.) 1000 mL
Dissolve ingredients in a 1-L volumetric ask with about 800 mL of distilled water and then
dilute to the volume. Store the buer in a refrigerator.
Note Pre-weighed commercial packages are also available.
312
Medical Laboratory Technology: Volume 1
Lysing reagent
• Stock solution of saponin, 1% w/v in water (use only certied grade of saponin)
Saponin 1 g
Distilled water (q.s.) 100 mL
Dissolve saponin in about 50 mL of water in a 100-mL volumetric ask, then make up to
the volume.
• Working solution of saponin, 0.1%
Dilute stock saponin 1:10 (10 mL made up to 100 mL in a volumetric ask). Add to
this 2.0 mL of 3% (w/v in water) potassium cyanide solution (Poison) and store in a
refrigerator.
Ponceau stain
Ponceau S 0.5 g
Trichloroacetic acid (TCA), 5% in water (q.s.) 100 mL
Note TCA is highly hygroscopic, so store it in a desiccator. Alternatively, prepare the
solution as soon as the bole is opened.
• Acetic acid, 5% v/v in water
• Sodium chloride solution, 0.85% w/v in water.
Equipment and supplies
• Electrophoresis apparatus with accessories: Sample applicator, sample holder, aligning
plate, electrophoresis chamber, power supply, bloing paper and disposable wicks.
• Appropriate containers to hold stain, buer solution, acetic acid and water.
• Cellulose acetate strips.
Procedure (see Figure 11.3)
1. Take 0.5 mL of the patient’s whole blood in a test tube. Wash the red cells with 0.8%
NaCl.
2. Place the tube in the freezer for at least 10 min. Then thaw the specimen in order to
obtain complete haemolysis.
3. Wet the cellulose acetate strip in buer solution. Immerse slowly in the pan containing
buer (about 40 mL); soak for 20 min; do not let air bubble form on the surface and do
not touch the dull side (acetate surface) with nger.
4. Set up the electrophoresis chamber with buer in the outer compartment and moisten
paper wicks to ensure buer contact. Use fresh buer.
5. Carefully blot the cellulose acetate strips between lter paper in order to remove excess
buer; place on the aligning base and secure each end. Apply 10 μL of haemolyşate by
means of an applicator.
6. Place the strip in the electrophoresis chamber. Put some weight on the strip to ensure
contact of cellulose acetate with buer. Put on the cover.
Note The dull side should face down and should be in contact with the buer.
7. Connect electrodes and rum on the power supply; adjust the voltage to 220 V and ran
the power for 2 h at 5 mA.
8. At the end of 2 h, tum o the power supply and remove strips gently and blot excess
buer.
9. Agitate the strip gently in a container with acetic acid solution (5%). Repeat the process
with fresh acetic acid solution (5%) until the strip retums to its white colour with the
red-stained spots of haemoglobin.
10. Air dry and identify the haemoglobins by comparing the migrating distances with the
known controls. Report haemoglobins in the order of decreasing concentrations as
estimated visually (Figure 11.3).
Special Haematological Tests
313
Figure 11.3 Electrophoresis apparatus
Laboratory Diagnosis of bLooD parasite infection
Blood parasites which are more prevalent in tropical countries can cause a number of ailments.
These blood parasites are either protozoa or nematodes. Only protozoan blood parasites
will be discussed in this section. Three of the common diseases caused by these protozoan
blood parasites are malaria (Plasmodium sp.), Kala azar (Leishmania sp.) and sleeping sickness
(Trypanosoma). These parasites are either non-agellates (e.g., Plasmodium) or agellates
(e.g., Leishmania, Trypanosoma). They are either found inside the red cells (e.g., Plasmodium)
or outside the red cells (e.g., Trypanosoma) or they may be in both places (e.g., Leishmania).
Filariasis is also caused by the parasitic infection of blood. The causal agents are a number of
nemathelminths—Wuchereria, Loa loa, Brugia and Mansonella. These will be discussed in
the Microbiology (Parasitology, Chapter 22) section of this book.
All the protozoan blood parasites need a carrier host (vector) usually an insect, through
which infection is transmied to humans. The human host always harbours the asexual phase
of the parasite’s life cycle, while the carrier host (denitive host) harbours the sexual phase.
Laboratory diagnosis of protozoan blood parasite infection primarily focusses on microscopic
examination of blood smear, a technique closely related to haematological investigation.
Although malarial parasites (Plasmodium) are easily recognized from the examination of
blood smears, for the diagnosis of sleeping sickness (Trypanosoma), microscopic study of
lymph node aspirate (early stage) and spinal uid (late stage) may be necessary. As the
extracellular trypanosomes are only a few in blood circulation during the middle stage of
314
the disease, concentration of blood is required prior to its microscopic examination. These
techniques will be discussed below. Laboratory diagnosis of kala-azar is based on the
microscopic examination of biopsy material (e.g., bone marrow), which is reviewed in the
histology section (Chapter 37) and by the chemical screening (aldehyde test) of serum, which
will be discussed here.
Medical Laboratory Technology: Volume 1
Malaria (Plasmodium)
Malaria is common in tropical countries and is often caused by two species of Plasmodium—P.
vivax and P. falciparum. The two other species of Plasmodium—P. malariae and P. ovale are
relatively rare. It is transmied to humans through the inoculation of Plasmodium sporozoites
by the female Anopheles mosquitoes or by blood transfusions. The sporozoites travel
through the blood to the liver, where they transform into large tissue schizonts containing
considerable numbers of merozoites (tissue schizogony). These begin to rupture after 5–20
days, (depending on the species) and the released merozoites invade circulating erythrocytes.
The replication cycle is repeated at regular intervals. Plasmodium malariae and P. ovale are
common in some parts of Africa.
Clinical significance
The rst clinical symptoms of a malarial infection are low-grade fever, headache, muscle
aches and malaise. These symptoms are often misinterpreted as being the result of a viral
infection.
Following a history and physical examination, microscopic study of the blood smear should
be performed to make the diagnosis. Two blood lms are made, thick and thin, and stained
with Field and Giemsa stains. They may also be detected by immunologic procedure, known
as dipstick test that is described in the Chapter 24. In case of a mixed infection, a patient can
harbour more than one species. Although it is often dicult to identify the species in malarial
infections, such aempts should be made, and if it is not possible, report the presence of
malarial parasite with the comment ‘species unknown’.
Specimen
Anticoagulated venous blood or non-anticoagulated capillary blood from skin puncture can
be used. The most suitable time for collection of blood is at the height of an episode of fever,
when parasites are most numerous in the blood. Blood specimens should always be collected
before antimalarial drugs are administered.
Microscopic examination of blood smear
Malaria is caused by a parasite in the blood. The parasites (Plasmodium) are very small
and can be seen only under a microscope with high magnication. Thus the most reliable
method for diagnosing malaria is microscopic examination of a patient’s stained blood lm.
This requires skilled technician. Before the parasites can be seen, however, a blood lm
must be made, dried, stained and examined under the microscope. When the microscopist
sees stained parasites, the diagnosis of malaria is conrmed. Microscopists can identify the
stages and species of malaria parasite and the density of the infection. Few real changes in
the microscopy of malaria parasites have occurred since 1991, but much has changed in the
way malaria is diagnosed and treated. Where microscope is not available, Rapid Diagnostic
Tests (RDT) can be used for quick diagnosis. One should, however keep in mind that the
RDTs have less accuracy and false positives and can also be falsely negative in the rst 24 h.
Principle
Two blood smears are made—thin and thick (Figure 11.4). The thick lm helps in the detection
of the parasite while the thin lm allows microscopic examination of erythrocytes under
Special Haematological Tests
Figure 11.4 Preparation of blood lm for blood parasite examination: (a) Take a drop of blood on
a microscope slide, (b) Prepare a routine thin smear with a spreader slide, (c) Also
prepare a thick smear, with a bigger drop of blood and spread the drop with the corner
of another microscope slide by a circular motion
315
high power in order to identify species. The thin blood smear is stained with Giemsa stain and
Field stain. Giemsa stain is in a water medium, which removes the red cells by haemolysis
(Figure 11.5). It, however, leaves behind imprints of the parasite as pink Schüner’sdotsand
other structures (Figures 11.5 and 11.6). The thin lm allows you to study the morphology of
the red cells. The morphological abnormalities of red cells can identify the stage of the parasite
in the life cycle as well as characteristics of certain species of Plasmodium. The Leishman stain
is not used as much as the xative interferes in the development of Schüner’s dots.
Equipment and supplies
• Microscope
• Clean grease-free microscopic slide, spreader slide
Note Clean the slide rst with detergent, rinse with water, nally clean with wet gauze
or coon wool containing 95% ethanol. A clean slide gives beer thin blood smear.
• Coplin jars or beakers to hold Field stain
• Draining rack
• Applicator sticks (or capillary tubes) and grease pencil
Procedure
A. Preparation of blood smear
1. With the patient’s left hand palm upwards, select the third or fourth nger (the big toe
can be used in infants. The thumb should never be used for adults or children). Use
coon wool lightly soaked in ethanol to clean the nger—using rm strokes to remove
dirt and grease from the ball of the nger. Dry the nger with a clean piece of coon
wool or lint.
2. With a sterile lancet, puncture the ball of the nger, using a quick rolling action. By
applying gentle pressure to the nger, express the rst drop of blood and wipe it away
with dry coon wool. Make sure that no strands of coon wool remain on the nger.
3. Working quickly and handling clean slides only by edges, collect the blood as described
in the following steps.
316
Medical Laboratory Technology: Volume 1
4. Apply gentle pressure to the nger and collect a single small drop of blood on to the slide
for making a thin lm as has been described in the section describing the dierential
count.
5. Apply further pressure to express more blood and collect two or three larger drops, on
a dierent slide for making thick lm. Wipe the remaining blood away with coon wool
and apply pressure to stop bleeding.
6. Thin lm: Using a spreader slide make a thin blood lm in the same way as you have
done in dierential count (Chapter 10). Keep the spreader slides at 45° angle or less
(30°–35°) while spreading the blood lm (Figure 11.4b).
7. Thick lm: Always handle slides by the edges, or by a corner to make the thick lm.
Using a second slide or a cover slip, join the larger drops of blood and spread them
to make an even, thick smear over an area of about 1 cm. It is important to have an
even thickness (Figure 11.4c). Smears that are too thick or too thin will not stain well.
It must be translucent (not opaque) when wet. Allow the smear to air-dry for about
30 min. Use of an electric fan expedites the process. Overnight drying is recommended.
Note Allow the thick lm to dry in a at, level position protected from ies, dust
and extreme heat. Label the dry lms with a grease pencil and include the date. Some
prefer to have both thick and thin smears on the same slide (Figure 11.5) and use
Giemsa stain.
8. Proceed to staining.
Figure 11.5 Examination of blood smears for blood parasites: (a) Thick and thin smears of blood are
used for the examination of blood parasites, (b) Field or Giemsa stain can reveal the
presence of ghost red cells (infected) with Schuner’s dots (b) which is characteristic of
P. vivax
B. Staining
Thin blood lms are usually stained by Giemsa stain (Figure 11.5), while thick blood lms are
stained with the Field stain (Figure 11.6).
Giemsa stain
Principle
During the staining of the blood lm, haemoglobin present in erythrocytes dissolves and is
removed by the water in the staining solution. All that remain are parasites and leucocytes,
which can be seen under the microscope.
Special Haematological Tests
Figure 11.6 Staining of blood lm for the microscopic examination of malarial parasites: (α-d) Transfer
a drop of whole blood to a clean slide with the help of capillary tube, (e) Prepare a
thin and a thick smear; the thick smear should barely allow seeing through, (f-i) Stain
with eld stain with stain A and stain B, (j) Dry on a rack and then examine under oilimmersion objective, (k-r) Presence of malarial rings and Schüner‘s dots are diagnostic
of malarial infection. Comparison of ring forms trophozoites (m-o) and gametocyte stages
(p-r) of Plasmodium falciparum (m, p), P. malariae (n, q) and P. vivax (o, r), respectively.
317
Equipment and supplies
• Microscope
• Measuring cylinder, beakers (50- and 250-mL)
• Staining troughs, glass rods, wash bole, slide forceps, slide racks, timer
Reagent
Giemsa stain, methanol in drop bole and buered water (pH 7.2); these are described in
Chapter 10.
Procedure
Thin smear
1. Fix the thin blood lm by adding three drops of methanol or by dipping into a container
with methanol for a few seconds. With prolonged xation it may be dicult to detect
Schüner‘s dots and Maurer’s clefts*. To permit dehaemoglobinization, the thicklm
should not be xed. Therefore, avoid exposure of the thick lm to methanol or its
vapour.
2. Put the slides back to back in a staining rack.
3. Prepare 3% Giemsa solution to buered or distilled water, pH 7.2, in sucient quantity
to ll the number of staining troughs being used. Mix the stain well.
*Maurer clefts are synonemous to Maurer dots. These are nely granular precipitates or irregular cytoplasmic
particles that usually occur diusely in red blood cells infected with the trophozoites of Plasmodium falciparum,
occasionally those of P. malariae; rarely observed in P. falciparum blood smears because its trophozoites seldom are
seen in peripheral blood.