The McGill Physiology Virtual Lab

Blood Laboratory

Blood cell indices > RBC count
  Among the most common hematological tests performed are those which determine the red blood cell count, the hematocrit, the hemoglobin content, ABO/Rh Blood Group typing, and the total and differential white blood cell count. Each one of these measurements may be carried out on a single drop of blood.
Review the topics in your textbook.
Theory

A convenient and inexpensive way to count blood cells is through the use of the hemocytometer. This instrument is a special microscope slide on which precise grids have been etched within a counting chamber designed to hold an exact volume of diluted blood sample. The blood cell determination involves counting cells in several squares of the grid and obtaining an average number. That number is multiplied by a factor that compensates for the amount of dilution. The final result expresses the number of red blood cells per cubic millimeter of the original blood sample.



The picture above shows a hemocytometer, on top of which a coverslip (thin square glass slide) was placed to delimit the area of each of the counting chambers, top and bottom. The groove (present on some hemocytometer models) on the edge of each counting chamber facilitates the positioning of the pipette tip and the loading of the diluted blood sample.
 

Procedure

The picture above shows a microcentrifuge tube (sometimes referred to as an eppendorf tube) containing the diluted blood sample in Gower's solution, a 20 ml adjustable pipette (or pipetman) and the hemocytometer.


In order to be able to effectively count cells, venous blood in EDTA (as anticoagulant) must be added to an isotonic solution (Gower's solution) which prevents lysis and crenation of erythrocytes. Gower's solution also contains a fixative which acts as a preservative and prevents agglutination of cells if counting cannot be done within an hour.

Preparation of the diluted blood sample:

Volume to volume dilution is one in which a unit volume of a solute is combined with an appropriate volume of a solvent liquid to achieve the desired concentration. The dilution factor is the total number of unit volumes in which the solute is dissolved in the solvent. This dilution factor method is based on ratios.
In this exercise, it is desirable to dilute blood 1:200; one "unit" of blood is to be diluted into 199 units of Gower's solution, for a dilution factor of 200. How much blood should you add to the Gower's solution to get a final volume of 1ml?

Loading the hemocytometer:

After adequate mixing, the diluted blood is introduced into the counting chamber where the cell count is performed microscopically.

Caution:



The 20 ml pipette tip is placed on the hemocytometer at the edge of the coverslip. The plunger of the adjustable pipette is slowly pressed so that the sample flows between the hemocytometer's raised shiny surface and the coverslip. Stop when the sample touches the three sides of the chamber (about 10 ml); do not overflow into the moat which surrounds the chambers.
Counting
Observe the grid of the hemocytometer below. Different areas are used for counting red blood cells and white blood cells. Red cells are counted in the areas indicated in red.
Consider the following: The central grid of 25 squares is 1mm x 1mm in area and 0.10 mm deep, and holds 0.1ml of liquid.

The dilution factor is 1:200.

Convert the number of red blood cells that are counted in 5 squares to the number of red blood cells/Ál. (N.B. 1 Ál (microliter) = 1 cubic mm ).

Since only 5 out of the 25 squares (1:5) are counted, you must multiply your count by 5.
Since you have diluted your sample by 200 (1:200), you must also multiply your count by 200 to arrive at the original concentration in the body.
You obtain a count which is contained in a volume of 0.1Ál (25 squares). So you must also multiply by 10 to express the count per cubic mm.

Click here to open a window which mimics what you might see looking through the 40x objective of a microscope. Only the center of the grid shown above is visible (where the 5 squares are coloured red). Try to verify this by observing the fineness of the grid as you move to the edges of the visible field. Count the red cells in the five squares and determine the red cell count as described above.

Important note: To avoid counting the same cells twice, cells that are touching the lines at the tops and left sides of the squares are counted, but cells that are touching the bottoms and right sides of the squares are not counted.
 
Female: 3.9-5.6 million/Ál
Male: 4.5-6.5 million/Ál

To continue with the next section, Hematocrit, click here