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298
Medical Laboratory Technology: Volume 1
• Saturated solution of brilliant cresyl blue
Brilliant cresyl blue 1.0 g
Trisodium citrate (Na3C6H4O72H2O) 0.4 g
Dissolve the dye and the trisodium citrate together in the sodium chloride solution. Filter
the solution into a staining bole. Label and write the date. Keep in the refrigerator. Filter
before use.
Caution Avoid contact with skin and eyes. Avoid formation of dust and aerosols. Provide
appropriate exhaust ventilation at places where dust is formed. Normal measures for
preventive re protection.
Storage Keep the container tightly closed in a dry and well-ventilated place.
Procedure (Figure 10.27)
1. Filter a lile of the cresyl blue solution into a test tube. In the boom of another tube
place two drops of ltered cresyl blue solution.
2. Collect a few drops of blood from the patient’s nger with Pasteur pipee or use
venous blood collected in EDTA dipotassium salt solution and mix well.
3. Add two drops 6f blood to the tube containing cresyl blue solution.
4. Mix by gently shaking the tube. Plug the tube with non-absorbent coon wool. Leave
for 15 min.
5. Take the tube and shake it gently. Remove one drop of the mixture. Place it on a slide
ready for spreading.
6. Make a thin smear of the mixture with the spreader. Leave the smear to air dry.
7. Examine the smear using the 100× oil-immersion obj ective. Look at the end of the
smear; there erythrocytes should be well separated from each other.
Note Erythrocytes stain pale blue.
8. Use of a hand tally counter (Figure 10.26) is recommended for the reticulocyte count.
Keep your eye xed on the microscopic eld and start counting the number of red cells.
As soon you see a reticulocyte, click on the hand tally counter. Then continue your
red cell count, changing the eld as you proceed. Remember to count the reticulocyte
along with the red cell. Reticulocytes are included in the red cell count.
Hand tally counter. The hand tally counter is used to count red cells during reticulocyte
count. Keep a separate count of reticulocytes while counting the red cells.
* Note: The percentage is of 100 red cells examined.v
Routine Haematological Tests
Number of reticulocytes counted1×000
9. Calculate the reticulocyte count as follows:
299
Reticulocyte count (%) =
Number of red cells examined
Reticulocyte count: (a, b and с) Freshly ltered new methylene blue or brilliant
cresyl blue stain is taken in a test tube, and (d and e) Mixed with equal amount of
anticoagulated whole blood, (f) Mix, (g) Wait for 15 min, (f-i) Prepare a smear of
stained blood cells, (k) Examine the smear under oil-immersion objective, (k) Look for
red cells with granules
75 100
Medical Laboratory Technology: Volume 1
Example Total number of red cells counted (reticulocytes are included in the RBC count) =
1500. Number of reticulocytes seen (covered in 15 elds) = 75.
Reticulocyte count (%) =
1500
5%
Reporting of results
Some haematologists prefer a report of the reticulocyte count as an absolute number of
reticulocytes in circulation per litre of blood, while others prefer them to be reported in terms
of the number fraction (%) relative to the number of red cells in circulation. Depending on
the practice in your laboratory or the specication of the requesting physician, make the
appropriate calculation.
Sources of error
1. Staining time should not be less than 10 min.
2. Mix the blood and the stain gently but thoroughly prior to making the smear. This
is important. The reticulocytes have a lower specic gravity than mature red cells
and therefore sele on top of the red cells in the mixture. Thus an unmixed or poorly
mixed blood specimen may not give the true picture.
3. Red cells showing highly refractile areas may be confused with reticulocytes. These
artifacts in the red cells are probably due to moisture in the air and poor drying of the
smear. Use a fresh specimen.
Additional information
• Supravital stains also stain several other red cell inclusions in addition to remnants of ri-
bosome and RNA in reticulocytes—Haemoglobin H bodies, Howell-Jolly bodies, Heinz
bodies and Pappenheimer bodies. While counting, examine these abnormal inclusions
and report if present.
• Haemoglobin H bodies appear as pale blue dots, variable in size. Unlike the reticulum
of reticulocytes, they occur in most of the erythrocytes. They are found in α-thalassemia
or haemoglobin H disease.
• Heinz bodies appear as blue granules, variable in size, lying to one side of the eryth-
rocyte near the cell membrane. They occur in glucose-6-phosphatase dehydrogenase
deciency following treatment with certain drugs.
• Use of equal volumes of staining uid and blood specimen is not critical. In case of
low Hct value, use a larger proportion of blood and when the Hct is unusually high
(polycythaemia), use a smaller volume of blood. This variation in dilution helps
in the spreading of 100 to 150 red cells per microscopic eld and makes it easier to
count.
absolute platelet Count
Platelets are the smallest cells in circulating blood. They participate in the blood cloing
process. They originate in the bone marrow from megakaryocytes. Enumeration of platelets
is requested in the investigation of bleeding disorders. There are several problems in
counting platelets: they are small and readily disintegrate which makes them dicult to
distinguish from debris; they aach to each other due to their adhesive character which
adds further problems to their accurate counting and they may not be evenly distributed
in the blood. Three common ways of reporting the platelet count are by haemocytometry,
Routine Haematological Tests
by study of the blood smear (estimate only) and by automated counting. The manual
haemocytometric method will be described here, using the light microscope (Rees–Ecker
method). The procedure is similar to red cell counting, with a dierent diluent and
counting area. As platelets are small and easily oat away, they should be allowed to sele
down before counting.
Clinical significance
Increased platelet count (thrombocytosis) is found in polycythaemia vera, chronic myelogenous leukaemia, following splenectomy, and other clinical conditions. Decreased
platelet count (thrombocytopaenia) occurs in aplastic anaemia, acute leukemia, immune
thrombocytopenia, megaloblastic anaemia, hypersplenism and following cytotoxic chemo-
therapy and radiation treatment. Thrombocytopaenia is often associated with prolonged
bleeding and poor clot formation (described in greater detail in Chapter 13).
Normal value
Adult: 3 × 105 platelets/μL
Specimen
EDTA-anticoagulated blood is the recommended specimen for counting platelets. Capillary
blood can be used but venous blood generally gives more satisfactory results. Platelet counts
from capillary blood are generally lower than those from venous blood. The platelet count
must be completed within 2 h after blood collection. To obtain reproducible and representative
results, the blood specimen should be collected in a plastic syringe and immediately mixed
with an anticoagulant (EDTA).
Principle
The diluent prevents coagulation (citrate), xes platelets (formalin) and prevents them from
clumping. No aempt is made to lyse the red cells. Platelets are identied by their size, shape
and dark colour. The dye (brilliant cresyl blue) provides the background during cell counting.
This dye does not stain platelets, and it is not essential for the counting procedure.
Reagent
Diluting uid:
Trisodium citrate (0.106 M) 3.8 g (Na3C6H4O7 2 H2O)
Neutral formaldehyde (40%) 0.2 mL
Brilliant cresyl blue 0.1 g
Deionized water (q.s.) 100 mL
Dissolve ingredients in a 100-mL volumetric ask, make it up to volume (100 mL), lter,
centrifuge, and transfer to a tight-stopper bole and refrigerate. The diluting uid keeps indenitely but it is essential that it be made by using scrupulously clean glassware and with
fresh distilled water. Filter an aliquot of the diluting uid immediately before use.
Equipment
• Haemocytometer kit: The Neubauer chamber used for WBC and RBC counting can
be used for platelet counting. The entire central square is used for counting under
the high-power objective. The Spencer Brightline chamber, in which lines appear
white against a dark background, appears to have a denite advantage over other
types of counting chambers. The platelets are easier to see against the metal-coated
surface, of the Spencer Brightline chamber. As the chamber’s surface is smoother
Number of platelet counted Dilution
Medical Laboratory Technology: Volume 1
the platelet distribution is beer. However, this type of chamber is more dicult to
mount correctly.
• Sahli pipee (20 μL)
• Erlenmeyer ask (25-mL) or 10-mL test tubes
• Petri dish with lter paper (to prepare moist chamber; Figure 10.28)
Special note for cleaning of glassware
All glassware must be scrupulously clean. Clean with acid or hot detergent. Anything in
the pipee to which platelets could adhere must be removed. Clean the haemocytometer
thoroughly with mild detergent, make certain that the surface is free of all dirt and lint. The
use of 95% ethanol and a lint-free cloth is recommended for this purpose.
Procedure (Figure 10.28)
1. Transfer 3.98 mL of diluent (freshly ltered) into the Erlenmeyer ask or test tube.
2. Mix the blood specimen gently for about 2 min.
3. Add to the diluent 20 μL (0.02 mL) of anticoagulated blood with the help of a Sahli
pipee. Take care to wipe the outside of the pipee after removing the specimen.
Wash out contents of the pipee with the diluent at least 3 to 4 times. Empty the last
drop of liquid in the Sahli pipee and keep it aside as it will be used again for lling
the chamber.
4. Immediately mix the diluent with the specimen for at least 5 min.
5. Use the Sahli pipee to transfer a drop of the diluted specimen on each side of the
counting chamber.
Note Rinse the pipee with the diluent within the ask before transferring.
6. Place the mounted haemocytometer inside a moist chamber (Figure 10.28) and let it
stay undisturbed for 15 min. This permits platelets to sele and the moist chamber
prevents evaporation of uid from the haemocytometer chamber.
7. Place the haemocytometer on the stage of the microscope; focus the red cell counting
area under low magnication. Then move to the corner square of the red cell area and
carefully change to the high-dry objective.
8. Platelets are bluish and must be distinguished from debris. They are oval, round, or
comma-shaped, refractile bodies that vary in size normally from 1–5 μM.
9. Count platelets in the nely ruled central area (1 mm) of each side of the chamber.
Take the average counts of two sides.
Note In the improved Neubauer ruling there will be 25 small squares in the centre
and each of these squares has 16 smaller squares. The area covered by the 25 squares
is equivalent to 1 sq.mm.
10. Calculate the platelet count:
Platelet count/L=
Volu
mme of fluid
Volume of uid for the 1 sq.mm area = 1 × 0.1 = 0.1 μL (older expression, mm3)
Dilution = 200
Routine Haematological Tests
Platelet count: (a) Moist chamber used for settling down the moving platelets before
counting; the magnied area under low power covers the central 1 sq mm area, (b)
Platelets distributed in this square area are counted
Number of platelet counted 200
Medical Laboratory Technology: Volume 1
The above formula can thus be rewrien as:
Platelet count/L=
0.1
= Number of platelets counted × 2000
Sources of error and additional information
• Platelet count must be done within 2 h. Delay causes disintegration and clumping of
platelets.
• Debris and dust are the most important sources of error as they are easily mistak-
en for platelets. Keep the glassware scrupulously clean and lter the diluting uid
before use.
• Adjust blood dilution to 1:100 in case of thrombocytopaenia (lower count of platelets).
• An error of 15 to 25% is frequently observed in counting platelets.
Estimation of Platelet Count from Stained Blood Smear
Due to the variation of results in the platelet count by the direct method, extreme care must
be taken. A well-prepared blood smear can be used to check the results of direct counting.
Determine the ratio of platelets to red cells on a blood smear used for dierential count. If the
average number of platelets is 8 to 25 in 10 high-power elds, it is reported as adequate, and
if it is 0 to 5, it is reported as inadequate.
review Questions
1. What is the chemical nature of haemoglobin? Where is it located in the body? What
are its functions? How would you determine the concentration of haemoglobin in the
blood? Which pathological state is associated with a decrease of haemoglobin?
2. What is the clinical signicance of haematocrit determination? How would you report
haematocrit?
3. List the formed elements of blood and state their functions.
4. How would you report the following:
Total white cell count, Dierential count, Platelet count, and Red cell count?
5. Dene the following.
Microcytic Anaemia, Aplastic Anaemia, Leukemia, Leukocytosis, Leukemoid
Reaction, Target Cells, Auer Bodies, Howell-Jolly Bodies, Poikilocytosis, Anisocytosis,
Left Shift, Peiger Hüet Anomaly
6. What is the clinical signicance of dierential count? Arrange the following white
blood cells in the decreasing order of their distribution in dierential count:
lymphocytes, band neutrophils, segmented neutrophils, monocytes, basophils and
eosinophils.
7. What are the roles of diluting uids in (a) red cell count and (b) white cell count? Are
the white cells removed during the red cell counting?
8. What are reticulocytes? What is the clinical signicance of the reticulocyte count?
How would you stain reticulocytes? How does supravital staining dier from routine
staining of the blood smear?
9. List the common morphological abnormalities reported in red blood cells and white
blood cells. State their diagnostic signicance.
Routine Haematological Tests
10. Why is it necessary to let the haemocytometer stay in a moist chamber before obtaining
the platelet count?
11. The average number of platelets in 10 high power elds of a microscope was noted to
be 1%. What would you report as the platelet count—inadequate or adequate?
12. What is the principle of ow cytouorometry (FC)? How is it dierent from the old
automated blood cell counter?
Special Haematological
Tests
Chapter Outline
• Laboratory Diagnosis of Haemoglobinopathies
• Screening Test for Sickle Cell Anaemia
▪ Sickle cell preparation
▪ Solubility test for sickle cell
▪ Haemoglobin electrophoresis
• Laboratory Diagnosis of Blood Parasite Infection
▪ Malaria (Plasmodium)
▪ Sleeping sickness (Trypanosomiasis)
▪ Chagas disease
▪ Kala azar (Leishmaniasis)
• Review Questions
11
Anuradha Chakravarthy
Laboratory Diagnosis of HaemogLobinopatHies
Haemoglobinopathies are congenital disorders that originate from a globin chain defect in
the haemoglobin. In a normal adult, Hb A constitutes about 96% of total haemoglobin and
Hb A2 is less than 3.5%, while Hb F (foetal haemoglobin) is only 1%. In a new born, HbF
constitutes over 50% of the total haemoglobin. As the infant grows, the synthesis of Hb F
gradually decreases and is replaced by Hb A until only about 1% HbF remains by the end of
the rst year of life.
Haemoglobinopathies can be divided into two major categories—quantitative defects and
qualitative defects. Included in the quantitative defects are thalassemias where the normally
found haemoglobins Hb A, Hb A2 and Hb F are abnormally distributed.
A qualitative defect of haemoglobin synthesis is found in sickle cell anaemia where
abnormal haemoglobin (Hb S) is formed. In sickle cell anaemia, the homozygous condition
(Hb S-S), Hb A is in low concentration. In sickle cell trait, the heterozygous condition (Hb S-A)
both haemoglobins (Hb S and Hb A) will be found.
Other abnormal haemoglobins include Hb С, Hb D and Hb E. These may occur in
combination with each other, e.g., Hb S-C and others. As Haemoglobin С is relatively
insoluble, aected erythrocytes left in 3% sodium citrate solution for up to 12 h, may show
the typical at-sided intra-erythrocyte crystals in wet preparations. Identication of various
haemoglobins can only be done by electrophoresis.
Special Haematological Tests
307
screening test for sickLe ceLL anaemia
Sickle cell anaemia is a genetic disorder caused by the abnormal haemoglobin S. If inherited
from both parents (Hb S-S), it causes sickle cell anaemia, a serious disease. If inherited from
only one parent (Hb S-A), it causes sickle cell trait. Haemoglobin S occurs mainly in tropical
Africa but also in the Eastern Mediterranean region, among Americans of African origin, and
in some tribal populations in India.
Clinical Significance
Red cells with haemoglobin S-S (Hb S-S) are abnormal in shape, typically narrow,
crescent-shaped with defective membranes. Hb S is insoluble when the oxygen tension is
lowered and this makes the red cells susceptible to sickling. The sickled cells tend to clump
together and cannot freely ow through the circulation. Most of the clinical manifestations
of this disease are due to blockage of the blood supply in small blood vessels by these
red cell clumps. The blockage causes pain and these cells are easily haemolysed because
of their abnormal shapes. This results in a chronic haemolytic anaemia. The laboratory
diagnosis of sickle cell anaemia is based on three observations: (1) sickling of red cells
under deoxygenated conditions, (2) precipitation of haemoglobin S in a special medium
and (3) haemoglobin electrophoresis. The laer is a conrmatory test but requires a
considerable amount of the technician’s time. Other tests are relatively easy and are done
for screening. They cannot dierentiate between sickle cell anaemia (HB S-S) and sickle
cell trait (Hb S-A).
Red cells with homozygous abnormal haemoglobin (S-S, disease) or heterozygous
abnormal haemoglobin (S-Α, trait) look normal in the smear. The presence of sickle cells in
the peripheral blood smear is seen occasionally in patients with sickle cell anaemia. But these
normal looking red cells, with Hb S-S or S-A, will sickle under reduced oxygen supply. The
degree of sickling, however, depends on the concentration of Hb S in the cell. The following
screening tests may rst be performed and specimens with positive ndings may be subjected
to the conrmatory test of Hb electrophoresis.
Specimen
EDTA-anticoagulated venous blood, heparinized capillary blood or free-owing capillary
blood without anticoagulant obtained by skin puncture (nger, toe or heel). The laer is
placed directly on the slide.
Sickle Cell Preparation
This test is convenient but requires a microscope to observe sickling and it does not dierentiate
sickle cell trait from sickle cell disease.
Principle
Whole blood is mixed with sodium metabisulphite, a strong reducing agent that deoxygenates
haemoglobin. If cells contain Hb S, they become sickle shaped or half-moon shaped.
Equipment and supplies
• Dropper
• Pasteur pipee with ne tip or applicator sticks
• Petri dish
• Microscopic slides and cover slips
• Filter paper
• Syringe (5-mL) lled with petroleum jelly or nail polish
• Hypodermic needle, 19 gauge