Wipe tip clean and draw diluting fluid to the 101 mark.
Shake for 3 minutes. Charge the chamber. Count the RBCs
using 40X objective in the 80 smallest squares as indicated
in the diagram of the chamber.
No. of cells counted × Dilution factor × Depth factor
Where Dilution is 1 in 200, Depth is 1/10 mm
RBC counts are low in anemia and high in polycythemia,
the causes of these have already been discussed.
Preferably, use venous blood for platelet counts. Finger
prick may cause clumping of platelets. In small children,
however, this clumping can be prevented by thinly
smearing Vaseline over the area to be punctured (make
sure that there has been no clotting of blood).
The blood is diluted in 1% ammonium oxalate stored
refrigerated at 4°C which hemolyzes the RBCs (prepared by
dissolving 1 g of ammonium oxalate in 100 mL of distilled
¾ Fill blood and diluent (in this case 1% ammonium
oxalate) as described for the RBC count and using the
RBC pipette. If platelet count is low, a WBC pipette can
¾ Charge the chamber with the help of the pipette
¾ Using 40X objective with reduced condenser aperture,
count the platelets in the same squares as indicated for
¾ Calculate as (if RBC pipette used)
Cells counted × blood dilution × chamber depth factor
However, if a WBC pipette is employed, the appropriate
formula and method should be used. Platelet counts are
made in the small 5 RBC squares only.
Platelet count = N × 20 × 5 × 10 or N × 1000
Normal platelet counts = 1.5–3.5 lakhs/cu mm.
Rees-Ecker Method for Platelet Count
Various components of the diluting fluid used have
various functions, e.g. citrate prevents coagulation while
formalin fixes the platelets and prevents their clumping
together. Here, no attempt is made to lyse RBCs. Platelets
are identified by their size, shape and dark color. Brilliant
cresyl blue (the dye used) provides the background during
cell counting. This dye does not stain the platelets and,
therefore, is not essential for the counting procedure.
FIG. 9.7: RBC pipette Deionized water 100 mL
filter, centrifuge, transfer to a well-stoppered bottle and
keep at 2–8°C (refrigerate). This fluid if not contaminated
will stay good indefinitely. Filter aliquot of the diluting
All glassware must be scrupulously clean. Dirt or dust
particles can resemble and may be counted as platelets.
¾ Take 3.98 mL of diluent (freshly filtered) into a test tube
¾ Add to the diluent 0.02 mL (20 μL) of well-mixed
anticoagulated blood. With the help of a Sahli pipette,
wipe out the outer tip of the pipette before dilution.
Wash out the contents in the pipette into the diluent
¾ Immediately mix the diluent with the specimen for at
¾ Employ the Sahli’s pipette for charging either side of the
¾ Keep the charged hemocytometer inside a moist
chamber (can be a petri dish with a moistened or wet
filter paper — on which the chamber can be kept). Let
stay for about 15 minutes. This permits the platelets
to settle down, and the moistened chamber does not
allow evaporation of the fluid
¾ Place the hemocytometer on the stage of the
microscope, focus the RBC counting area under low
magnification. Now move to the corner square of the
red cell area carefully to high dry objective
¾ 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 to 5
¾ Count the platelets in the finely ruled center area
) of each side of the chamber. Take the average
counts of two sides. (In the new improved Neubauer
ruling, there are 25 small squares and each of these
contain 16 smallest squares. The area covered by the
Number of platelets counted × dilution
volume of fluid for the 1 sq mm area = 1 × 0.1 = 0.1 mL (cu mm)
Number of platelets counted × 200
= Number of platelets counted × 2000
Rough Estimation of Platelet Count from Stained
A well-prepared peripheral blood smear can be used to
check the results of direct counting. Determine the ratio
of platelets to red cells on a thin blood smear used for
differential leukocyte count. If the average number of
platelets is 8 to 25 in 10 fields, it is reported to be adequate,
and if it is 0 to 5, it is reported as inadequate.
1. Causes of platelet production failure: Selective
Part of general bone marrow failure:
• Marrow infiltration, e.g. in carcinoma, lymphoma
2. Increased destruction of platelets
• Acute or chronic ITP (idiopathic thrombocytopenic purpura)
• Secondary immune thrombocytopenia (postinfection, SLE, CLL, and lymphomas).
3. Abnormal distribution of platelets
• Massive transfusion of old blood to bleeding
Raised Platelet Count (Thrombocytosis)
Can occur as a part of generalized myeloproliferative
disorder, e.g. CML or following acute hemorrhage.
b. Hemoglobin concentration, and
Red cell count per liter × 1015 fl
Infants, full term cord blood average 106 fl.
Children 10–12 years 76–93 fl.
MCV is reduced in microcytic anemias
MCV is raised in macrocytic anemias
The Mean Cell Hemoglobin (MCH)
Normal MCH in adults is from 27 to 32 pg.
MCH is reduced in hypochromic anemias.
The Mean Cell Hemoglobin Concentration (MCHC)
This too, is low in hypochromic anemias.
Adult females 37–47% 0.37–0.47 L/L
Trimester 1 35–46% 0.35–0.46 L/L
Trimester 2 30–42% 0.30–0.42 L/L
Trimester 3 34–44% 0.34–0.44 L/L
Postpartum 34–44% 0.34–0.44 L/L
Adult males 40–54% 0.40–0.54 L/L
Adult females 12–16 g/dL 7.4–9.9 mmol/L
Trimester 1 11.4–15.0 g/dL 7.1–9.3 mmol/L
Trimester 2 10.0–14.3 g/dL 6.2–8.9 mmol/L
Trimester 3 10.2–14.4 g/dL 6.3–8.9 mmol/L
Postpartum 10.4–15.0 g/dL 6.4–9.3 mmol/L
Adult males 14.0–18.0 g/dL 8.7–11.2 mmol/L
Panic low level <5 g/dL <3.1 mmol/L
Panic high level >18 g/dL >11.2 mmol/L
Day 1 15.5–24.5 g/dL 9.6-15.2 mmol/L
Days 2–3 19.0 g/dL 11.8 mmol/L
Days 4–8 14.3–22.3 g/dL 8.9–13.8 mmol/L
Days 9–13 16.5 g/dL 10.2 mmol/L
2–8 weeks 10.7–17.3 g/dL 6.6–10.7 mmol/L
3–5 months 9.9–15.5 g/dL 6.1–9.6 mmol/L
6–11 months 11.8 g/dL 7.3 mmol/L
1–2 years 9.0–14.6 g/dL 5.6–9.0 mmol/L
3–9 years 9.4–15.5 g/dL 5.8–9.6 mmol/L
10 years 10.7–15.5 g/dL 6.6–9.6 mmol/L
11–15 years 13.4 g/dL 8.3 mmol/L
Panic levels <5g/dL <3.1 mmol/L
Adult females 4.0–5.5 million/µL 4.0–5.5 × 1012/L
Trimester 1 4.0–5.0 million/µL 4.0–5.0 × 1012/L
Trimester 2 3.2–4.5 million/µL 3.2–4.5 × 1012/L
Trimester 3 3.0–4.9 million/µL 3.0–4.9 × 1012/L
Postpartum 3.2–5.0 million/µL 3.2–5.0 × 1012/L
Adult males 4.5–6.2 million/µL 4.5–6.2 × 1012/L
Day l 4.1–6.1 million/µL 4.1–6.1 × 1012/L
Days 2–8 5.1 million/µL 5.1 × 1012/L
Days 9–13 5.0 million/µL 5.0 × 1012/L
2–8 weeks 3.8–5.6 million/µL 3.8–5.6 × 1012/L
3–5 months 3.8–5.2 million/µL 3.8–5.2 × 1012/L
6–11 months 4.6 million/µL 4.6 × 1012/L
1–2 years 3.6-5.5 million/µL 3.6–5.5 × 1012/L
3 years 4.5 million/µL 4.5 × 1012/L
4 years 4.0–5.2 million/µL 4.0–5.2 × 1012/L
5 years 4.6 million/µL 4.6 × 1012/L
6–10 years 4.7 million/µL 4.7 × 1012/L
11–15 years 4.8 million/µL 4.8 × 1012/L
218 Concise Book of Medical Laboratory Technology: Methods and Interpretations Contd.. Contd..
Adult females 4500–11,000/mL 4.5–11.0 × 109
Trimester 1 6600–14,100/mL 6.6–14.1 × 109
Trimester 2 6900–17,100/mL 6.9–17.1 × 109
Trimester 3 5900–14,700/mL 5.9–14.7 × 109
Postpartum 9700–25,700/mL 9.7–25.7 × 109
Adult males 4500–11,000/mL 4.5–11.0 × 109
Newborn 9000–30,000/mL 9.0–30.0 × 109
3 months 5700–18,000/mL 5.7–18.0 × 109
1 year 6000–17,500/mL 6.0–17.5 × 109
3 years 5700–16,300/mL 5.7–16.3 × 109
10 years 4500–13,500/mL 4.5–13.5 × 109
White Blood Cells Differential
Neutrophil (Segs) 54–62% 0.54–0.62
Adults 3800/µL or mm3 3800 × 106/L
Birth 8400/µL or mm3 8400 × 106
12 hours 12,100/µL or mm3 12,100 × 106
24 hours 8870/µL or mm3 8870 × 106
1 week 4100/µL or mm3 4100 × 106
2 weeks 3320/µL or mm3 3320 × 106
1–2 months 2750/µL or mm3 2750 × 106
4 months 2730/µL or mm3 2730 × 106
6 months 2710/µL or mm3 2710 × 106
8 months 2680/µL or mm3 2680 × 106
10 months 2600/µL or mm3 2600 × 106
12 months 2680/µL or mm3 2680 × 106
2 years 2660/µL or mm3 2660 × 106
4 years 3040/µL or mm3 3040 × 106
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