1. Sodium citrate 3 g Formalin 1 mL Distilled water to 100 mL (Cheap and good) Or 2. Hayem’s fluid Mercuric chloride 0.5 g FIG. 9.6: WBC pipette Clinical Hematology 215 Sodium chloride 1.0 g Sodium sulphate 5.0 g Distilled water to 200 mL. (Needs to be made frequentl

 

2. Due to inappropriate erythropoietin increase in:

a. Benign/malignant tumors of:

• Kidney

• Liver

• CNS

• Uterus

• Ovary.

b. Renal disease (besides malignancies)

• Hydronephrosis

• Vascular impairment

• Cysts.

3. Associated with adrenocortical steroids or Androgens.

a. Adrenal hypercorticism (all types)

b. Virilizing tumors

c. Androgens used therapeutically (rarely corticoids).

4. Associated with chronic chemical exposure

a. Nitrites, sulfonamides, other substances producing methemoglobin and sulfhemoglobin.

b. Cobalt, shellac components, various alcohols.

5. Relative

a. Stress or spurious polycythemia

b. Dehydration: water deprivation, vomiting

c. Plasma loss: burns, enteropathy.

HEMATOCRIT/PACKED CELL VOLUME (PCV)

Definition

Hematocrit is the volume of red cells expressed as a

percentage of the volume of whole blood in the sample.

The venous hematocrit is almost same as that obtained

from a skin puncture. Dried heparin, EDTA or double

oxalate are satisfactory anticoagulants.

Methods

1. Using Wintrobe’s tube.

2. Using microhematocrit capillaries.

Wintrobe’s Tube

Fill the Wintrobe’s tube till the 100 mark on top with a

Pasteur pipette ensuring that there are no air-bubbles in

the blood column. Centrifuge this tube for 15 minutes

at 3500 rpm (or longer at lower speeds) until packing is

complete. After centrifuging, the blood is separated into 3

layers, a column of red blood cells at the bottom, a narrow

middle layer—buffy coat of WBCs and platelets and the

topmost fluid column of plasma. The percentage of the

height of the column of blood occupied by packed red cells

constitutes the hematocrit. Roughly, the hematocrit value

is three times the hemoglobin concentration.

Sources of Error

1. Inadequate mixing of blood.

2. Irregularity of the bore of the tube.

3. Incomplete packing.

Microhematocrit

This method is in common use in most well-equipped

laboratories. Capillary tubes coated with anticoagulant

can be filled with blood obtained from finger puncture or

from a venipuncture or with blood already anticoagulated.

One end of the filled capillary tube is sealed with sealing

wax (e.g. Plasticine) or the empty end is sealed with heat.

The sealed tube is centrifuged for 3 minutes in a special

Clinical Hematology 213

high-speed centrifuge. By reading the packed cell height

and the total height of the entire specimen, the hematocrit

can be determined. Special reading devices are available.

Values: If the red cells are of normal size (normocytic), and

the red cell count is 5 million, the hematocrit is about 45%.

Men—Range 42–52% Average = 47%

Women—Range 37–47% Average = 42%.

Interpretation

Causes of reduced hematocrit—causes of anemia. Causes

of raised hematocrit—causes of polycythemia.

If packed cell volume has been determined by

Wintrobe’s tube, one can obtain some more information.

Buffy coat: A buffy coat of thickness 1 mm approximately

corresponds to a total leukocyte count of about 10,000.

Absent or minimal buffy coat implies leukopenia, a

thickness more than 1 mm implies leukocytosis. In

addition, in sub-leukemic leukemia, a film can be made

from the buffy coat where a greater concentration of

WBCs will be available, and identification of atypical cells

would become easier and less time consuming. Another

advantage is for performing LE cell or phenomenon test,

for which also WBCs can be picked up from the buffy coat.

The platelets form a very thin layer above the white cells,

the coat is pinkish white but is of no use clinically, one has

to do platelet counts if necessary.

Plasma layer: The topmost layer of plasma can give

important clues by observing its color. Its normal color is

pale yellow or straw.

Yellow—jaundice

Pink—hemolysis

Creamy white—hyperlipidemia, especially chylomicrons

Brown—methemalbuminemia.

BLOOD CELL COUNTS

 White Blood Cell (WBC)

A white cell count (TLC) estimates the total number of

white cells in a cubic millimeter of blood. It is important in

the diagnosis of disease, especially when accompanied by

a differential white cell count.

The diluting fluid: WBC diluting fluid contains a weak acid

to lyse the RBCs and a stain for staining the nucleus of

WBCs, e.g. Turke’s fluid.

Glacial acetic acid 1.5 mL 1% aqueous solution of

gentian violet 1.0 mL distilled water 98.0 mL

(A pinch of thymol may be added to the diluting fluid to

prevent growth of moulds).

Counting Chamber

The chamber normally used for cell counts is the improved

Neubauer’s chamber which has an area of 9 mm2

 and a

depth of 0.1 mm.

Other counting chambers can also be used including

the Burker’s chamber which has the same area and depth

as the improved Neubauer (Fig. 9.5) and the FuchsRosenthal ruled chamber which has the same area but a

double depth of 0.2 mm.

Methods

Using a WBC pipette (Fig. 9.6) of a hemacytometer, draw

well mixed venous blood or capillary blood and fill till the

0.5 mark. Clean the tip of the tube. Now draw WBC diluting

fluid till the 11 mark (or to 0.38 mL of diluting fluid add

0.02 mL of blood with a Hb pipette).

1. Mix the fluid and blood mixture gently, avoiding

bubbling.

2. Place the coverslip on the counting chamber at the

right place.

Shake the fluid-blood mixture and transfer the mixture

using a fine bore Pasteur pipette on to the counting chamber

(called charging the chamber), taking care that the mixture

FIG. 9.5: Platelet counting area count the cells in the 25 squares inside

the large center square circle

214 Concise Book of Medical Laboratory Technology: Methods and Interpretations does not overflow. If it does overflow, wash and dry the

chamber to be recharged again.

Allow the cells to settle to the bottom of the chamber

for 2 minutes. See that fluid does not get dried up (For

preventing this, take a petridish, place a wet filter paper

at its bottom, now place the charged chamber gently and

close off the dish for about 2 minutes).

For counting, clean the under part of the chamber if

it was left in the petridish and place it on the stage of the

microscope. Using 10X or low power objective, count

the WBCs uniformly in the four larger corner squares (as

indicated in the diagram). Cells present on the outermost

lines should be counted on one side and those present on

the line opposite should not be counted.

Calculate the number of cells per cubic millimeter of

blood as follows:

=

Cells counted × blood dilution × chamber depth

Area of chamber counted

= number of cells counted ×

20 × 10 (depth factor)

4

= number of cell counted × 50

(Dilution factor is 20 for there is no mixing of cells till

first 1 mark of the WBC pipette, hence 0.5 parts of blood

are present in 10 parts of the diluting fluid dilution factor,

then, is 10/0.5 = 20).

Falsely high counts occur due to:

1. Blood taken from an area where there was hemoconcentration.

2. Not wiping away the blood on the outside of tip of the

pipette.

3. Blood drawn above the mark in the pipette (happens

usually when the rubber mouthpiece is too short).

4. Diluting fluid not taken till the requisite mark.

5. Improper mixing.

6. Uneven distribution in the counting chamber.

7. Extraneous material present (yeast, dirt, etc.).

8. Errors in calculation.

Falsely low counts occur due to:

1. Dilution of the blood with tissue fluid due to edema

or squeezing.

2. Delay in counting (this does not affect RBCs as much

as WBCs, which are reduced by about 15% in 24 hours).

3. Blood not drawn up to the requisite mark.

4. Diluting fluid taken in excess of the requisite mark.

5. Saliva in the mouthpiece running into the upper end

of the pipette causing further dilution.

6. Improper mixing.

7. Uneven distribution in the counting chamber.

8. Uniform systematic counting not done.

9. Cells lost through hemolysis (in RBC count-for

instance).

10. Errors in calculation.

11. Clumping of cells or coagulation of the blood.

Causes of raised and reduced leukocyte counts would

be presented with differential leukocyte counts, since

raised or lowered total counts are usually accompanied by

abnormal differential counts.

Correcting the white cell count for nucleated red cells:

From peripheral smear, find out the number of nucleated

red cells per 100 WBCs counted.

Calculation

 Number of nucleated RBCs

100 + number of nucleated RBCs

×TLC

 = Nucleated RBC/cu mm

Corrected count = TLC–Nucleated RBC count.

Red Blood Cell (RBC)

Diluting Fluid

This should be isotonic so that RBCs are not hemolyzed.

Normal saline can be used but it may cause crenation of

the RBCs and allow rouleaux formation.

One can use:

1. Sodium citrate 3 g

 Formalin 1 mL

 Distilled water to 100 mL

 (Cheap and good) Or

2. Hayem’s fluid

 Mercuric chloride 0.5 g

FIG. 9.6: WBC pipette

Clinical Hematology 215

 Sodium chloride 1.0 g

 Sodium sulphate 5.0 g

 Distilled water to 200 mL.

(Needs to be made frequently and in hyperglobulinemia

one may set precipitation of protein so RBC clumping may

occur. Mercuric chloride acts as an antiseptic).

Method

Draw blood to the 0.5 mark in the RBC pipette (Fig. 9.7).