RBC COUNT METHOD
The RBC count method comes under haemocytometry, which quantitatively measures the
number of RBCs in a blood sample. Haemocytometer or Neubauer’s chamber slide is a
manual method to count RBCs.
Nowadays, more accurate or automated devices have been developed like electrometric and
photometric counters that can count the cell constituents of the blood sample.
It is not possible to directly count the RBCs in a blood sample. Thus, it is necessary to dilute
the blood sample or blood specimen using one of the RBC diluting fluids (hayem’s or
formalin citrate diluting fluid).
In this context, we will discuss the requirements, preparations and procedure of the RBC
count through Neubauer’s chamber. You will also get to know the formula for calculating the
number of RBCs.
What is RBC?
RBCs stand for red blood cells. It is also called erythrocytes, which appears red-coloured due
to the coloured pigment (haem) and exists as a biconcave disc. RBCs possess a diameter of
7.5 to 8.7 μm and a thickness of 1.7 to 2.2 μm. It lacks a nucleus and has a life span of 120
days.
Requirements of Total RBC Count
Haemocytometer refers to the micro-slide through which the number of erythrocytes or RBCs
can be enumerated via two methods, namely microdilution and macrodilution. Following are
the requirements of the RBC count method:
Neubauer’s Chamber or Haemocytometer
It is a specialized thick glass slide used to count the eukaryotic cell suspension.
Haemocytometer has a size of 30 X 70 X 4 mm. Its central portion is ruled, where the cell
counting is performed. The counting grid has a size of 3 mm X 3 mm. One can estimate the
number of red blood cells using a haemocytometer after diluting the blood sample with RBC
diluent.
Nine large squares have an overall width of 3 mm.
Twenty-five medium squares in each large square have a width of 0.25 mm.
Sixteen small squares in each medium square have a width of 0.05 mm.
RBC Pipette
It is commonly used to dilute the blood sample with the RBC diluting fluid. It gives a dilution
of 1:100 and 1:200. The reading starts from 0.5 to the endpoint of 101. There is a red
bead within the RBC pipette, which mixes the RBC specimen with the diluting fluid.
There is a mouthpiece attached towards the end of the suction rubber tube. Through the
mouthpiece, the blood is sucked upto a point 0.5 and diluting fluid upto the endpoint 101.
The diagram below provides a well-labelled presentation of an RBC pipette.
RBC-Diluting Fluid
Hayem’s fluid and formalin citrate diluting fluids are generally used to dilute the RBC
specimen. Both function as an isotonic solution, which do not cause haemolysis and the
RBCs’ crenation. The composition of Hayem’s and formalin citrate diluting fluid is
mentioned below.
Glass Cover
It is a square-shaped coverslip, having a width of 20 mm. To count the number of eukaryotic
cells, you should keep the glass cover over the central portion or the ruled area of Neubauer’s
chamber. There is usually a 0.1 mm gap between the glass cover and the central area of the
haemocytometer.
Procedure
Sample preparation: It uses an RBC pipette to incorporate the blood specimen with the
diluent.
1. Take the blood sample upto a point (0.5). Then, wipe the RBC pipette’s tip using
blotting paper.
2. After that, suck RBC diluting fluid or diluent upto a mark 101.
3. Horizontally rotate the RBC pipette by using your palms.
Loading the sample over the haemocytometer slide:
1. Take a clean, grease-free haemocytometer slide and cover glass.
2. Place the cover glass on top of the haemocytometer’s lined region.
3. Before loading the RBC sample into the haemocytometer, discard 1-2 drops.
4. Then, you should carry the RBC pipette at an angle (45 degrees) and load a small
volume of RBC sample towards the edge of a cover glass.
5. Wait for 3-5 minutes in order to settle down the RBCs in the chamber.
6. Observe the prepared slide under the microscope to count the number of RBCs
manually.
RBC Count under Microscope
First, focus the rulings of the haemocytometer slide using a 10X objective lens. Using coarse
and fine adjustment knobs, focus on the five squares of the large central square to count the
number of red blood cells under the 40X objective.
A diagram below represents the pattern to count RBCs in all the five medium squares of a
large central square. As already discussed, each medium square possesses 16 small squares.
We need to manually count the number of RBCs in five medium squares via hand tally.
In each square, you need to count the red blood cells located within the square. The red lines
in the upper and right corners indicate the areas not to count RBCs, whereas green lines
indicate the areas to count the RBCs.
Calculation of RBCs
Total RBCs/µL = Number of RBCs counted X Dilution factor / Area X Depth
The number of red blood cells (N) =?
Dilution factor = 1:200 or 200
A large central square is subdivided into 25 medium squares or sub squares. So,
the area will be one sq. mm. The number of RBCs is enumerated in 5 squares out
of 25 squares. So, the area of 5 small squares will be 5/25 or 1/5 sq. mm.
Depth of the sample = 0.1 mm
Total RBCs = N X 200 / 1/5 X 0.1 = N X 200 X 50 = N X 10,000 cells/µL
Suppose, N or number of RBCs in the five squares is 486, then the equation will be
represented as:
Total RBCs = 486 X 10,000 = 48, 60, 000 cells/µL
Conclusion
We can conclude that the RBC or erythrocyte count method provides the concentration
of RBCs per µL of whole blood. Through a haemocytometer, RBCs are counted manually.
RBCs’ normal concentration in children, women and men range between 4.0-5.5
million/mcL, 4.2-5.4 million/mcL and 4.7-6.1 million/mcL, respectively.