Introduction To Hemocytometry

 

 

Aim: Study of Hemocytometry and use of it.

HEMOCYTOMETER

A hemocytometer is a procedure of counting the number of cells in a sample of blood; the red cells , the white cells and the platelets being counted separately. It is assumed that the cells are homogenously mixed(suspended) in the plasma in all regions of the body. However, even under physiological conditions, there are slight differences(e.g. higher red cells count in venous and capillary blood than in arterial blood) which, through minor, are accentuated by muscular exercise, changes in posture, meals etc. Nevertheless, important clinical information can be obtained of cell counts are done carefully on a venous blood sample.

Principle 

Since the number of blood cells is very high, it is difficult to count then even under the microscope. This difficulty is partly overcome by diluting the blood to a known degree with suitable diluting fluid and then counting them.

The sample of blood is diluted in a special prepared and is then pleased in a capillary space of known capacity(volume) between a specially ruled glass slide( counting chamber) and a coverslip. The cells spread out in a single layer which makes their counting easy. knowing the dilution employed, the number of cells in undiluted blood can then easily be calculated.

Units for reporting

The result of cell counting is usually expressed as " number of cells per cubic millimeter" ( c mm ; mm3;μl) of blood, e.g. RBC count = 5 million/ cmm.

STEPS IN HEMOCYTOMETRY:

The whole process of cell counting involves the following steps:

1.   Keeping all the equipment's ready.
2.   Getting a sample of blood.
3.   Pipetting, i.e. filling th pipette with blood and diluting it.
4.   Charging, i.e. filling the counting chamber with diluted.
5.   Counting the cells and reporting the results.

HEMOCYTOMETER

The Hemocytometer consists of the following:

1. Counting Chamber: It is a thick glass slide with two identical ruled areas separated by empty space and having two elevated ridges on their both sides. Either of the ruled areas is used for counting the cells. There are different types of counting chamber viz. Old neubauer counting chamber, improved neubauer chamber, burker counting chamber and fuch's rosenthal counting chamber.

Neubauer Counting Chamber Here, the counting square is of 9 sq.mm area (3mm x 3mm). The four corner squares of area 1 sq.mm. each (1mm x 1mm) are used for while blood cell count. In this the triple lines which dividing the central large square are very much closer to each other. The central ruled area is divided into 25 large squares of 0.04mm²  each. These squares are subdivided to form 16 smaller squares each with an area of 1/400 of 1 sq.mm( 0.0025mm² ). The four corners and one middle sub-squares are used for RBC count. The depth of improved neubauer chamber is same that is 0.1mm. This counting chamber is used to enumerate blood cells, sperm, eggs, pollen, fungal spores, large bacteria, etc. or non-living particles visible in light microscope.

      

                

2. Coverslips: A special cover glass is used which has a very smooth, flattened surface and even thickness. Different  thicknesses are: 0.3mm 0.4mm(most common), 0.5mm. two sizes are common: 16 x 22 sq.mm and 22 x 23 sq.mm.

3. Diluting Pipette: It is a glass pipette with rubber sucking arrangement. Diluting pipettes are different for RBCs and WBCs. These pipettes are sometime called as "cell pipette" or blood pipette. The third pipette, which students will be using is the hemoglobin pipette, which does not have a bulb. Parts of diluting fluid are:

•      The stem: The long narrow stem has a capillary bore and well-grounded conlical tip. It is divided into 10 equal parts (graduations) but has only two numbers etched on it -0.5 in the middle of the stem, and 1.0 at the junction of stem and bulb.
•      The bulb: the stem widens into bulb which contains a free-rolling bead -- red in RBC pipette and white in WBC pipette. The bead helps in mixing  the blood and the diluent and also helps in quick identification at a glance. Just beyond the bulb, the number 101 is etched on the RBC, and 11 on the WBC pipette.
• Rubber tube and mouthpiece: the bulb narrows again into a short to which a long, narrow, soft rubber tube bearing a mouthpiece( red in RBCs and White in WBCs).