Aim – to estimate the packed cell volume of red blood cell
Relevance
Measurement of hematocrit (Hct) or packed cell volume (PCV) is the most accurate and
simplest of all tests in clinical hematology for detecting the presence and degree of anemia or
polycythemia. In comparison, hemoglobin estimation is less accurate, and RBC count far less
accurate.
Also, if Hb, RBC count, and PCV are determined at the same time, various absolute
corpuscular values (e.g., volume and Hb content of a single red cell) of a person can be
determined. These values help in the laboratory diagnosis of the type of anemia in a person.
APPARATUS AND MATERIALS
Sterile swabs,
Alcohol,
Syringe and needle
Container with anticoagulant (double oxalate or sequestrene)
Wintrobe tube (hematocrit tube)
Pasteur pipette
Centrifuge machine
PROCEDURES
1. Draw 5 ml of venous blood and transfer it to a container of anticoagulant. Rotate the bulb
between my palms.
i. To ensure proper mixing of cells and plasma (inaccurate results are likely if this
precaution is not taken).
ii. To oxygenate blood cells to remove CO2 (red cells are larger when CO2 is high,
in venous blood).
2. Fill the pasteur pipette with blood and take its nozzle to the bottom of the Wintrobe
tube. Expel the blood gently by pressing the rubber teat, and fill the tube from below
upwards while withdrawing the pipette but always keeping its tip below the level of blood.
Ensure that there is no air bubble trapped in the blood.
• Do not try to fill the tube from its top as blood will not flow down to its bottom
because of air present in the tube.
3. Bring the blood column exactly to the mark 10 (or the mark 0 on the other side of the
scale) at the top. There should not be any bubbles at the top of blood.
• lf less blood is available, note the level.
4. Close the mouth of the tube with its rubber cap and centrifuge it at 3000 rpm for 30
minutes (slower speed will not pack the red cells fully). Balance this tube with another
tube filled with water, or another sample of blood placed in the opposite tube holder.
5. At the end of 30 minutes, take the reading of upper level of packed red cells on the side of
the scale where zero is at the bottom. Replace the tube in the machine and centrifuge it again
for 15 minutes. Read the packed cell height again; it should be the same as before. If the
height is reduced, centrifuge it again for 5 minutes. To be reliable, at least 3 successive
readings, at intervals of 5 minutes, should be the same.
• Note that unnecessarily prolonged centrifugation may cause mechanical hemolysis of red
cells which must be avoided.
OBSERVATIONS AND RESULTS
Note that the blood has been separated into 3 layers:
i. A tall upper layer of clear plasma—amber or straw-colored. It should not be pink or red
which would indicate hemolysis of red cells in the sample or within the body (i.e., before
withdrawal of venous blood) in hemolytic diseases. If there is hemolysis, the test must be
repeated on a fresh sample.
ii. A greyish-white, thin layer (about 1 mm thick) the so-called “buffy layer”, consisting of
platelets above and leukocytes below it.
iii. A tall bottom layer of red cells which have been closely packed together. A greyish red
line separates red cell layer from the layer of leukocytes above it. This line is due to the
presence of reduced Hb in the red cells lying next to the leukocytes which reduce the
oxyHb of the cells. The line marks the upper limit of the red cell layer.
• The percentage of the volume of blood occupied by the red cells constitutes hematocrit
or packed cell volume, i.e., the percentage of whole blood that is red cells
Hematocrit (Hct) = Height of packed red cells (mm) × 100 / Height of packed RBCs and
plasma
For example, if the height of packed red cells is 45 mm, then
= 45 × 100/ 100 = 45 percent.
• It also means that out of 100 volumes (or parts) of blood 45 volumes (or parts) are red cells
and 55 volumes (or parts) are plasma. Thus, out of 1 liter of blood, 450 ml are red cells and
550 ml are plasma.
Normal values. The average value of PCV is 42% when the RBC count is 5 million/mm3
and their size and shape are normal.
Males: 44 percent (38–50 percent)
Females: 42 percent (36–45 percent)
The PCV for newborns is about 50 percent.
Venous blood hematocrit: The Hct of venous blood is slightly higher than that of arterial
blood, because as the pH changes from the arterial value of 7.41 to 7.37 in the venous blood,
the red cells gain a little water.
PRECAUTIONS
1. Use the recommended amount of EDTA as the anticoagulant. There should be no clotting
or hemolysis of the blood.
2. The test should be done within 6–8 hours of collection of the sample.
3. The Wintrobe tube should be filled from below upwards so that no air bubble is trapped in
the tube.
Results -