Hematology Laboratory: The Hemacytometer

CLS 312

Observation & Discussion of the Hemacytometer for Cell Counting

Specific Laboratory Objectives: Upon completion of this laboratory the student will be able to: 1. Given a sample of anticoagulated blood, make the proper dilution of blood to prepare for cell counting. 2. Properly load and count cells in the hemacytometer. 3. Make the necessary calculations to determine the number of cells per cubic milliliter of whole blood.

Manual Cell Counts Calculating the Dilution Factor In order to perform a manual cell count, the blood specimen must be diluted with the appropriate dilution fluid. A dilution factor must be used in the final cell count calculation to compensate for the dilution of the blood. In order to determine the dilution factor it is necessary to review the blood diluting pipet.

In performing a red blood cell count the blood is normally drawn to the 0.5 mark and the dilution fluid is drawn to the 101 mark. The mixing chamber holds 100 volumes of fluid (blood and diluting fluid). Therefore, 0.5 volumes of blood and 99.5 volumes of diluting fluid results in a dilution of 0.5 volumes of blood in 100 volumes of total fluid, written as 0.5:100 or: 0.5 100 The standard expression of this dilution is 1 or 1:200. Therefore the normal dilution factor for 200 a red cell count is 200.

Consider a situation in which the dilution factor for red counts varies from the normal. For example if blood was drawn to the 0.4 mark in a red blood cell pipet instead of the normal 0.5 mark what would the dilution factor be? 0.4 volumes of blood diluted in 100 volumes of total fluid results in a dilution of: expressed in standard form 1:250 or: Thus, the dilution factor is 250. Determining Chamber Factors Hand Red Counts 1. Area of 1 RBC square x Depth = volume of 1 RBC square example: 0.04mm 2 x 0.1 mm = 0.004mm 3 2. Volume of 1 RBC square x # of RBC squares counted = volume of all squares counted example: 0.004 mm3 x 5 = 0.02mm 3 3. volume desired volume counted example: = Chamber Factor 1 . 250 .4 - or, 100

1 = 50 0.02

4. Chamber Factor x Dilution Factor = RBC Factor example: 50 x 200 = 10,000 5. Number of RBC's counted in 5 squares x RBC Factor = example: 500 x 10,000 = 5,000,000 RBC/mm 3 of blood number RBC mm3 of blood

Diluting Fluids RBC Diluting Fluids 1. Isotonic, so that cells are not hemolyzed or crenated. 2. Must fix the cells to avoid agglutination 3. Hayems solution: (HgCl2), NaCl, Na2SO4 H2O 4. Gowers solution: Na2SO4, acetic acid (HAc) and H2O

Sources of Error Errors in hemocytometry most frequently arise as a result of: 1. apparatus 2. personal technique 3. inherent error (1) Errors caused by apparatus: 1. 2. 3. 4. 5. chipped pipette tips obscure markings on pipettes non-optically plane cover glasses dirty glassware inaccurate rulings on chamber

(2) Errors caused by personal technique: 1. 2. 3. 4. 5. 6. 7. not thoroughly mixing blood inadequate shaking failure to discard first 4 drops from pipet before filling counting chamber not loading chamber properly (overfilling, trapped air bubbles) counting cells inaccurately (skipping cells, counting cells twice, counting wrong borders) calculation error clerical error

(3) Inherent errors in hemocytometry include: 1. 2. "field errors" - relates to the random distribution of cells on the counting chamber statistical error - occurs when total number of cells is too low to give statistical confidence in result (this error is reduced when larger numbers of cells are counted)

140004950.doc Monday, April 15, 2013