Practical hematology Assist.Prof. Dr.Sajeda Al-Chalabi

RBC count HEMOCYTOMETER Hemo: blood Cyto: cell Meter: measurement/counter Thus, it is an instrument used to count the blood cells.

It includes: Neubauer’s slide Cover slip RBC pipette WBC pipette

NEUBAUER’S SLIDE It is the name given to a thick glass slide NEUBAUER’S SLIDE It is the name given to a thick glass slide. In the centre of the slide, there is an H- shaped groove. On the two sides of the central horizontal bar, there are scales for counting the blood cells. The depth of the scales is 1/10mm or 0.1mm.

Neubauer Hemocytometer

Hemocytometer Chamber

The central square is subdivided into twenty five smaller squares and each of these smaller squares is further subdivided into sixteen smallest squares. These are meant for platelet and RBC counting.

the RBCs are counted in five small squares, four of corners and one of center. (total of 80 smallest squares)

Each of the smaller squares is further subdivided into sixteen smallest squares. Length of one smallest square = 1/5 x 1/4 = 1/20mm Width of one smallest square = 1/20mm Depth of one smallest square = 1/10mm Volume of one smallest square = 1/20 x 1/20 x 1/10 = 1/4000mm³

Cell Counts by Hemocytometer

Procedure For RBC counting Blood is filled till mark 0.5 and Hayem’s fluid is then filled till mark 101.Both are thoroughly mixed and then few drops are discarded which contain just the diluting fluid in the stem. Thus, 1 portion out of 101 is discarded. So, 0.5 part of blood is in 100 parts of fluid or, 1 part of blood is mixed in 200 parts of fluid Thus, dilution factor for RBC counting is 200. You can count blood cells with as little as a drop of blood. Because the cell density is very high, you have to dilute .

Procedure Before starting ensure that both the hemocytometer and its coverslip are clean by removing any dust particles with lens paper.  make sure to first place the coverslip over the counting surface before loading the cell suspension. Then place the pipette tip with your sample into one of the V-shaped wells, as in Figure, and gently expel the sample. The area under the coverslip fills by capillary action. Enough liquid should be introduced so that the mirrored surface is just covered,,

Procedure The loaded hemocytometer is then placed on the microscope stage and the counting grid is brought into focus at low power. Allow the sample to settle for a couple of minutes and avoid moving the coverslip as it might introduce air bubbles and make counting difficult.

Hayem's solution Hayem's solution contains: sodium chloride 0.5%, sodium sulphate 2.5% and mercuric chloride 0.25%. The sodium chloride to prevent hemolyses, the sodium sulphate discourages clumping of the erythrocytes and the mercuric chloride is a preservation.

FOCUSING 4X to see the general formation of slide. 10X for WBC counting 40X for RBC counting

procedure

Counting Rule Do not count cells touching Bottom line Right line Count cells touching the upper& left lines This is to avoid double counting.

Calculation Number of cells/mm³= counted cells in 80 small squares × diluting factor × volume correction factor. =N × 200 ×50=N × 10000

Normal Results Normal RBC ranges are: Male: 4.7 to 6.1 million cells /mm³ Female: 4.2 to 5.4 million cells/ mm³ What Abnormal Results Mean Higher than normal numbers of RBCs(polycythemia) Your RBC count will increase for several weeks when you are in a higher altitude. Lower-than-normal numbers of RBCs may be due to Anemia

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