2. Due to inappropriate erythropoietin increase in:
a. Benign/malignant tumors of:
b. Renal disease (besides malignancies)
3. Associated with adrenocortical steroids or Androgens.
a. Adrenal hypercorticism (all types)
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.
a. Stress or spurious polycythemia
b. Dehydration: water deprivation, vomiting
c. Plasma loss: burns, enteropathy.
HEMATOCRIT/PACKED CELL VOLUME (PCV)
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.
2. Using microhematocrit capillaries.
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.
1. Inadequate mixing of blood.
2. Irregularity of the bore of the tube.
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
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%.
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
Creamy white—hyperlipidemia, especially chylomicrons
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
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
The chamber normally used for cell counts is the improved
Neubauer’s chamber which has an area of 9 mm2
Other counting chambers can also be used including
the Burker’s chamber which has the same area and depth
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
2. Place the coverslip on the counting chamber at the
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
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
Cells counted × blood dilution × chamber depth
(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,
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
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.
6. Uneven distribution in the counting chamber.
7. Extraneous material present (yeast, dirt, etc.).
Falsely low counts occur due to:
1. Dilution of the blood with tissue fluid due to edema
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.
7. Uneven distribution in the counting chamber.
8. Uniform systematic counting not done.
9. Cells lost through hemolysis (in RBC count-for
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
Correcting the white cell count for nucleated red cells:
From peripheral smear, find out the number of nucleated
red cells per 100 WBCs counted.
100 + number of nucleated RBCs
Corrected count = TLC–Nucleated RBC count.
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.
(Needs to be made frequently and in hyperglobulinemia
one may set precipitation of protein so RBC clumping may
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