Multiple myeloma • Megaloblastic anemia. 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

 

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.

RBC count

=

No. of cells counted × Dilution factor × Depth factor

Area counted

Where Dilution is 1 in 200, Depth is 1/10 mm

Area counted is

80

400 =

1

5 mm2

 Number counted × 200 × 10

1/5

 = Number counted × 10,000

Interpretation

RBC counts are low in anemia and high in polycythemia,

the causes of these have already been discussed.

Platelets

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

water).

Method

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

be used instead

¾ Charge the chamber with the help of the pipette

employed

¾ Using 40X objective with reduced condenser aperture,

count the platelets in the same squares as indicated for

RBC counting

¾ Calculate as (if RBC pipette used)

Cells counted × blood dilution × chamber depth factor

Area of chamber counted

= N × 200 × 5 × 10

= N × 10000

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.

Diluting Fluid

Consists of:

Trisodium citrate 3.8 g

Neutral formaldehyde 0.2 mL

Brilliant cresyl blue 0.1 g

FIG. 9.7: RBC pipette Deionized water 100 mL

216 Concise Book of Medical Laboratory Technology: Methods and Interpretations Dissolve the ingredients in 100 mL volumetric flask,

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

fluid immediately before use.

All glassware must be scrupulously clean. Dirt or dust

particles can resemble and may be counted as platelets.

Procedure

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

tube 3–4 times

¾ Immediately mix the diluent with the specimen for at

least 5 minutes or so

¾ Employ the Sahli’s pipette for charging either side of the

chamber

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

microns.

¾ Count the platelets in the finely ruled center area

(1 mm2

) 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

25 squares is equal to 1 mm2

).

Platelet count/mL or cu mm =

 Number of platelets counted × dilution

Volume of fluid

Where,

volume of fluid for the 1 sq mm area = 1 × 0.1 = 0.1 mL (cu mm)

Dilution = 200

So platelet count/cu mm =

 Number of platelets counted × 200

0.1

 = Number of platelets counted × 2000

Rough Estimation of Platelet Count from Stained

thin Smear

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.

Causes of Thrombocytopenia

1. Causes of platelet production failure: Selective

megakaryocyte depression:

• Drugs

• Chemicals

• Viral infections.

 Part of general bone marrow failure:

• Aplastic anemia

• Leukemia

• Myelosclerosis

• Marrow infiltration, e.g. in carcinoma, lymphoma

• Multiple myeloma

• Megaloblastic anemia.

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

• Splenomegaly.

4. Dilutional loss

• Massive transfusion of old blood to bleeding

patients.

Raised Platelet Count (Thrombocytosis)

Can occur as a part of generalized myeloproliferative

disorder, e.g. CML or following acute hemorrhage.

ERYTHROCYTE INDICES

These can be calculated from:

a. Hematocrit,

b. Hemoglobin concentration, and

c. Red cell counts.

The Mean Cell Volume (MCV)

 MCV =

Packed cell volume

Red cell count per liter × 1015 fl

 Normal Values

 Adults 76–96 fl.

 Infants, full term cord blood average 106 fl.

Clinical Hematology 217

 Children, 1 year 76–87 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)

 MCH =

Hemoglobin in g/L

Red cell count per mL pg.

 Normal MCH in adults is from 27 to 32 pg.

 MCH is reduced in hypochromic anemias.

The Mean Cell Hemoglobin Concentration (MCHC)

 MCHC =

Hb in g%

PCV × 100 = 31–35 g%

 This too, is low in hypochromic anemias.

COMPLETE BLOOD COUNT (CBC)

Normal values

Contd.. Contd..

Contd...

Normal range, unit SI units

Hematocrit (HCT)

Adult females 37–47% 0.37–0.47 L/L

Pregnant

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

Children

Newborn 42–68% 0.42–0.68 L/L

3 months 29–54% 0.29–0.54 L/L

1 year 29–41% 0.29–0.41 L/L

3 years 31–44% 0.31–0.44 L/L

10 years 34–45% 0.34–0.45 L/L

Hemoglobin (HGB)

Adult females 12–16 g/dL 7.4–9.9 mmol/L

Pregnant

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

Normal range, unit SI units

Panic low level <5 g/dL <3.1 mmol/L

Panic high level >18 g/dL >11.2 mmol/L

Children

Newborn

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

>18 g/dL >11.2 mmol/L

Red Blood Cell (RBC) Count

Adult females 4.0–5.5 million/µL 4.0–5.5 × 1012/L

Pregnant

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

Children

Newborn

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..

Contd... Contd...

Normal range, unit SI units

2–8 weeks 33% 20.48 mmol/L

3–5 months 34% 21.10 mmol/L

6–11 months 33% 20.48 mmol/L

3 years 35% 21.72 mmol/L

1–2 years 32% 19.86 mmol/L

4–15 years 34% 21.10 mmol/L

White Blood Cell (WBC) Count

Adult females 4500–11,000/mL 4.5–11.0 × 109

/L

Pregnant

Trimester 1 6600–14,100/mL 6.6–14.1 × 109

/L

Trimester 2 6900–17,100/mL 6.9–17.1 × 109

/L

Trimester 3 5900–14,700/mL 5.9–14.7 × 109

/L

Postpartum 9700–25,700/mL 9.7–25.7 × 109

/L

Adult males 4500–11,000/mL 4.5–11.0 × 109

/L

Children

Newborn 9000–30,000/mL 9.0–30.0 × 109

/L

3 months 5700–18,000/mL 5.7–18.0 × 109

/L

1 year 6000–17,500/mL 6.0–17.5 × 109

/L

3 years 5700–16,300/mL 5.7–16.3 × 109

/L

10 years 4500–13,500/mL 4.5–13.5 × 109

/L

White Blood Cells Differential

Granulocytes

Segmented

Neutrophil (Segs) 54–62% 0.54–0.62

Adults 3800/µL or mm3 3800 × 106/L

Children

Birth 8400/µL or mm3 8400 × 106

/L

12 hours 12,100/µL or mm3 12,100 × 106

/L

24 hours 8870/µL or mm3 8870 × 106

/L

1 week 4100/µL or mm3 4100 × 106

/L

2 weeks 3320/µL or mm3 3320 × 106

/L

1–2 months 2750/µL or mm3 2750 × 106

/L

4 months 2730/µL or mm3 2730 × 106

/L

6 months 2710/µL or mm3 2710 × 106

/L

8 months 2680/µL or mm3 2680 × 106

/L

10 months 2600/µL or mm3 2600 × 106

/L

12 months 2680/µL or mm3 2680 × 106

/L

2 years 2660/µL or mm3 2660 × 106

/L

4 years 3040/µL or mm3 3040 × 106

/L

6 years 3600/µL or mm3 3600 × 106

/L

Normal range, unit SI units

Mean Cell Volume (MCV)

Adults 82–98 µ3 82–98 fl

Children