Manual WHITE 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.
Determining Chamber Factors [Derivation of Formula]
Hand White Counts
1. Area for 1 WBC square x Depth = Volume of 1 WBC square
example: 1mm2 x 0.1 mm = 0.1mm3
2. Volume of 1 WBC square x # of WBC squares = Volume of squares counted
example: 0.1mm3 x 4 = 0.4mm3
3. volume desired = chamber Factor
volume counted
example: 1 = 2.5
0.4
4. Chamber Factor x Dilution Factor = WBC Factor
example: 2.5 x 20 = 50
5. Number of WBC's counted in
4 WBC squares x WBC Factor = number WBC
mm3 of blood
example: 100 x 50 = 5,000
[ندعوك للتسجيل في المنتدى أو التعريف بنفسك لمعاينة هذه الصورة]WBC Diluting Fluids
1. Must hemolyze RBC's so that WBC's are not obscured
2. 2% Acetic Acid
or
3. 1% (0.1N) HCl
UNOPETTE Procedure : WBC Counts
ALL MANUAL COUNTS MUST BE DONE IN DUPLICATE!!
1. Using the pipet shield of a 20 microliter capillary pipet, open the diaphragm of the dilution vial.
2. Blood from fingerstick or venipuncture is allowed to fill the capillary of the UNOPETTE by capillary action. Filling of the capillary will stop when it is full.
3. Wipe excess blood carefully from the outside of the pipet.
4. Using one finger to cover the base of the pipet, insert the pipet tip first into the diluent reservoir while simultaneously squeezing the container.
5. Release the reservoir and the end of the pipet simultaneously. this will draw the blood sample into the diluent. Blood is rinsed from the pipet by squeezing and releasing the diluent container several times.
6. Cover the opening at the base of the pipet with a finger and invert the container several times to mix thoroughly.
7. The base of the pipet can be reinserted into the vial for transportation and may be labelled on the outside.
8. Allow to stand for 10 minutes for complete hemolysis of red cells to occur. The pipet serves as the dispensing dropper for loading the hemacytometer.
9. Discard the first three drops of mixture from the capillary prior to loading the hemacytometer.
10. Load the hemacytometer as described previously and count the cells. Counts from each side of the chamber should match within 25%. Average the values for the duplicate determinations when reporting results.
11. The dilution ratio for the WBC count is 1:100.
CALCULATIONS:
# of Cells Counted X Dilution Factor X Depth Factor = WBC per mm3
# of Square Millimeters Counted
Example: If 45 WBC's were counted in 9 WBC squares.
45 X 100 X 10 = 5000 mm3
9
The UNOPETTE manufacturer recommends the following calculation, to avoid having to divide by 9 in the above formula:
Add 10% of the number of cells counted to the count. This is the equivalent of counting an additional square millimeter. Then the depth factor and the # of squares counted cancel one another out. You then have, using the above example:
49.5 X 100 X 10 = 49.5 X 100, or 4,950 mm3
10
Interpretation of Results
A normal white count is 5,000-10,000/mm3. An increased white count is referred to as leukocytosis and can be seen in many pathological and sometimes non-pathological conditions. Conditions associated with a leukocytosis include:
1. bacterial infections
2. leukemia
3. pregnancy
4. newborn
A decreased white count is referred to as a leukopenia and can be seen in:
1. viral infections
2. Lupus erythematosus
3. overwhelming bacterial infections
4. pernicious anemia
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. chipped pipette tips
2. obscure markings on pipettes
3. non-optically plane cover glasses
4. dirty glassware
5. inaccurate rulings on chamber
(2) Errors caused by personal technique:
1. not thoroughly mixing blood
2. inadequate shaking
3. failure to discard first 4 drops
4. not loading chamber properly (overfilling, trapped air bubbles)
5. counting cells inaccurately (skipping cells, counting cells twice, counting on wrong borders)
6. calculation error
7. clerical error
(3) Inherent errors in hemocytometry include:
1. "field errors" - relates to the random distribution of cells on the counting chamber
2. 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)
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موضوع: Manual WHITE Cell Counts 
