Physiology Lab

Blood Cell Counts and Hematocrit measurement

Goals
Understand physiology of blood formation
Understand meaning of hematocrit and related physiological aspects
Understand principle and procedure of blood cell counting methods
Principle
Include study of different kinds of anemia and their cause, characters, and treatment.
Background information
Hemoglobin (Hb)

Hemoglobin is a large complex protein made up of globins chains and heme found inside
the red cells. Heme contains iron which is the portion of the protein that actually binds to
oxygen.
Hematocrit (Hct)
Hematocrit is the percent of volume that is taken up by red blood cells. It is also called
the packed RBC volume because the measure is taken after centrifugation.
Red Blood cell count (RBC)
Red blood cell count determines the total number of red cells in a sample of blood.
Because the number of red blood cells is very large, it is practical to dilute a sample of
blood with an isotonic solution, count the number of red blood cells in a fraction of this
diluted blood, and then multiply by a correction factor.
White Blood Cell Count (WBC)
White blood cells are easily separated from red blood cells. Red blood cells are broken
and only nuclei are counted. This works quite well unless immature red cells that still
contain nuclei are present. These red cell nuclei are counted and the result is a falsely
elevated number. Corrected white cell counts have had the red cell eliminated from the
report.

Normal ranges
Tests
Hemoglobin (g/dl)

Hematocrit (%)
RBC count (/microlitter)
WBC count (/microlitter)

Class
Male
Female
Newborn
Children
Male
Female
Newborn
Children
Male
Female
Total

Normal range
14.0 18.0
12.0 -16.0
17.0 23.0
11.0 16.5
42 45
37 47
53 65
30 - 43
4.7 6.1 X 106
4.2 5.4 X 106
4.8 10.8 X 103

Hemocytometer

*9 large squares 1mm x 1mm

*Center square divided into 25 smaller
squares
0.2mm x 0.2mm = 0.04 mm2
*Depth of the counting chamber: 0.1
mm
*Volume of a small square:
0.04 mm2 x 0.1 mm = 0.004 mm3
= 0.004 (l)
*Volume of w square:
1 mm2 X 0.1 mm = 0.1 (l)

Materials
2

2% Acetic acid solution

Gowers solution (Na2SO4, Acetic Acid, and H2O)
Blood cell diluting pipettes
Hemocytometer
Heparinated capillary tubes
Clay (sealant)
Microhematocrit centrifuge
Unopette reservoir and capillaries
Methods
1. Blood collection
Collect blood on two heparinated capillary tubes.
Follow blood collection procedure to prevent coagulation of blood
Fill the capillary about 2/3 of it
Mix blood very well by up-and-down motion of capillary
2. WBC count
1) Take blood to 0.5 marked line of the white blood cell dilution pipette (one with
white ball)
2) Fill the pipette up to 11 marked line with 2% acetic acid solution. That would
make 20X dilution of the blood.
3) Mix the pipette by vigorously shaking for 2 minutes while holding both ends with
fingers
4) Discard three drops
15) Place the coverslip over the hemocytometer counting chamber and place a drop of
the cell suspension at the edge of the V shape of the chamber. Allow the
suspension to be drawn into the chamber by capillary action. Care should be taken
not to overfill or underfill the chamber. Fill the opposite chamber in the same
manner.
26) Place the chamber on the microscope stage.
37) The hemocytometer consists of nine 1 mm squares divided into smaller squares.
One of the 1 mm squares represents a volume of 0.1 mm3 or 0.1 l. By using the
10X objective, count the number of cells in a 1 mm square area (W on the
graphic). If there are fewer than 100 cells in a square mm, 2 or more 1-mm square
areas should be counted and the results averaged.
48) Use the same procedure to count the cells on the other side of the hemocytometer.
59) Calculate the concentration of the cells.
0
Dont forget to apply dilution factor of 20.
1
*When you count 4 of W area (1mm2): you counted 0.4 l, need to multiply 2.5
2
(to cover 1 l), and multiply 20 (to account dilution factor).
3
*When you count only one of W area (1mm2): you counted 0.1 l, need to
4
multiply 10 (to cover 1 l), and multiply 20 (to account dilution factor).
3. Red blood cell count
1) Take blood to 0.5 marked line of the red blood cell dilution pipette (one with red
ball).
2) Fill the pipette up to 101 marked line with Gowers solution. That would make
200X dilution of the blood.

0
1
2
3
4
5
6
7

3) Mix the pipette by vigorously shaking for 30 times while holding both ends with
Fingers.
4) Discard three drops.
65) Place the coverslip over the hemocytometer counting chamber and place a drop of
the cell suspension at the edge of the V shape of the chamber. Allow the
suspension to be drawn into the chamber by capillary action. Care should be taken
not to overfill or underfill the chamber. Fill the opposite chamber in the same
manner.
76) Place the chamber on the microscope stage.
87) Find 0.04mm square (R area on the graphic) on the center area of the chamber.
One of the 0.04 mm squares represents a volume of 0.004 mm3 or 0.004 l. By
using the 10X or 40X objective, count the number of cells in the 0.04 mm square
area (5 different areas as indicated on the graphic). If there are fewer than 100
cells in a square mm, 2 or more 1-mm square areas should be counted and the
results averaged.
98) Use the same procedure to count the cells on the other side of the hemocytometer.
109) Calculate the concentration of the cells.
Dont forget to apply dilution factor of 200
*When you count 5 of R area (0.4 mm2): you counted 0.2 l, need to multiply 5
(to cover 1 l), and multiply 200 (to account dilution factor)

* Tip on cell counting when cells are on the line.

When cells are on the line, count cells on
left and top, but not right and bottom.
Then, be consistent throughout counting
process.

4. Alternative way of blood dilution

(1) Puncture the Unopette reservoir with the capillary pipette shield
(2) Remove the cover from the capillary tube and draw a sample into the tube by
capillary action. The blood should flow into the capillary tube and fill the entire
tube.
(3) Squeeze the reservoir chamber, insert the tip of the capillary into the chamber, and
release the chamber, drawing blood from the capillary into the reservoir fluid.
(4) Withdraw the capillary tube from the reservoir turning it around and placing it
back on the reservoir so it forms a small dropper bottle. Gently swirl the reservoir
to mix the contents, and invert and discard three to five drops of fluid into a

cotton swab to clear the capillary tube. The diluent consists of blood and Hayems
solution.
(5) Place the coveslip on the hemacytometer and fill the chamber with the capillary
tube. Do not force excess liquid under the hemacytometer glass.
(6) Let the suspension sit one to two minutes for an even dispersion of the cells.
*** Calculation
WBC: (Counts from four corners) X 2.5 X20(dilution factor)
***20l blood in 380l
RBC: (Counts from five sectors) X 40 (dilution factor)
***10l blood in 380l
5. Hematocrit measurement
1) Fill a heparinated capillary tube about with blood.
2) Seal the unused end of the tube by holding it near the tip and pushing it into a tray
of sealing clay at a 90-degree angle with a twisting motion. A plug of clay will
then seal the tube.
3) Place the tube in one of the grooved of a microhematocrit centrifuge with the
sealed end facing the perimeter of the centrifuge tray. Then, close the top.
4) Centrifuge for 4minutes.
5) Remove the tube from the centrifuge. You may notice fractions of plasma, a thin,
lightly colored buffy coat, and red blood cells.
6) Lay the tube on a white paper background, and measure the total length of the
sample from the clay-RBC border to the end of the plasma. Then, measure the
RBC portion only. Report in a form of % of red blood cell fraction.

Unopette Procedure
Unopette(r) for RBC count.

Puncturing the diaphragm of diluent with the capillary

pipette shield.

Drawing blood into the Unopette capillary tube. (A) From a finger puncture; (B)
From a venous blood sample.

Preparing reservoir to receive blood from

the capillary tube.

Mixing blood sample and diluent.

Loading the counting chamber.