Immunology

Experiment 7

Aim of the Experiment

Learn to count cells, observe and identify different bloodcells in a smear, quantify their proportions and count RBCs per μl (mm^3) using a haemocytometer.

Introduction

Red blood cells (RBCs) are the round shape, biconcave discs, present in the blood that helps in the transport of gases throughout the body. The biconcave shape helps the RBCs in rendering the red cells quite flexible so that they can easily pass through the capillaries. On an average, the size of the Red Blood Cells (RBCs) is 7.2 – 7.4 mm (microns). The mature RBCs are non-nucleated cells with an Iron-containing pigment known as Hemoglobin which helps in the transport of oxygen from the lungs to tissues and carbon dioxide from tissues back to the lungs for excretion. The Average lifespan of Red Blood Cells (RBCs) is 100 – 120 days.

Principle

Very large numbers of Red Blood Cells are present in the Blood Specimen. Practically, counting this the amount of Red cells directly under the microscope is highly impossible. So, the Red Blood cells are counted by using a special type of chamber, designed for the counting of blood cells in the specimen, known as Hemocytometer or Neubauer’s chamber.

For this, the blood specimen is diluted (usually in 1:200 ratio) with the help of RBC diluting fluid (commonly known as the Hayem’s Fluid) which preserve and fix the Red blood cells. The Hayem’s fluid is isotonic to the Red blood cells and does not cause any damage to it. The Normal Saline solutions can also be used for this but it causes the slight creation of red blood cells and allows rouleaux formation which may cause the errors in results. After diluting the specimen, the content is charged on the Hemocytometer / Neubauer’s chamber and the cells are counted in the areas specific for RBC count

Requirements

1. • Water sample

   • Blood sample

   • RBC diluting pipette with hose and bulb.

   • Plastic droppers

   • RBC diluent

   • 95% Alcohol for rinsing

   • 99% Methanol for fixation

   • Sterile lancet

   • Hemocytometer for RBC counting

   • Microscope

Calculation

The Neubauer’s Chamber has ruled the area of total 9 square mm and the depth is 0.1 mm as when the coverslip is placed on the surface of the counting chamber, the space between the bottom of the cover glass and the base of grooved area measures 0.1 mm in depth.

The central 1 square is highly ruled which is divided into 25 squares. Each square of the Central square is further subdivided into 16 small squares.

For RBC count the cells are counted in the 5 squares of the Central square as 4 Corner squares of the Central square (divided into 25 squares) and 1 central square of the Larger Central Square (divided into 25 squares).

Each square of the Central Square (divided into 25 squares) contains 16 small squares so the total no. of the area to be counted for RBC Count – 16 × 5 = 80 small squares.

Let’s consider it as ‘N’ no. of cells.

Now, the volume of the fluid inside the chamber is the product of Area and depth of the Hemocytometer / Neubauer’s chamber.

The central area is the 1 sq. mm which is divided into 25 parts so the area is 25 squares = 1 sq. mm

Out of these 25 squares, the RBCs are counted in 5 squares. So the Area of 5 small squares is 5/25 i.e. 1⁄5

The depth of the Hemocytometer is 0.1 mm as described above in a short description of Hemocytometer.

Now Apply the Following formula to get the Total Red Blood Cell Count –

Total RBC Count = N × Dilution / Area × Depth

N × 200 (or 100 as the dilution is made) / (1/5 × 0.1)