Specimen should be well mixed and left on a rocker for at least 5 minutes before using. Check Unopettes for clarity and contents. If the Unopette chambers appear cloudy or the amount of reagent looks questionable, do not use.
With the reservoir on a flat surface, puncture the diaphragm of the reservoir using the protective shield of the capillary pipette.
a. Using a twist action, remove protective shield from the pipette assembly b. Holding the pipette and the tube of blood almost horizontally, touch the tip of the pipette to the blood. The pipette will fill by capillary action and will stop automatically when the blood reaches the end of the capillary bore in the neck of the pipette.
c. Wipe the excess blood from the outside of the capillary pipette. Be careful not to touch the tip of the capillary when wiping off excess blood.
d. Before entering the reservoir, it is necessary to force some air out of the reservoir. Do not expel any liquid and maintain pressure on reservoir.
e. Place an index finger over opening of overflow chamber and position pipette into reservoir neck.
f. Release pressure on reservoir and then remove finger. The negative pressure will draw blood into pipette.
g. Rinse the capillary pipette with the diluent by squeezing the reservoir gently two or three times. This forces diluent up into, but not out of, the overflow chamber and releases pressure each time to ensure the mixture returns to the reservoir.
h. Return protective shield over upper opening and gently invert several times to mix blood adequately.
i. Allow the Unopette to stand for 10 minutes to allow RBCs to hemolyze. Leukocyte counts should be performed within 3 hours.
4. Charge hematocytometer a. Mix the dilution by inversion and convert the Unopette to the dropper assembly.
b. Gently squeeze Unopette and discard first 3 or 4 drops. This allows proper mixing, with no excess diluent in the tip of the capillary.
c. Carefully charge hematocytometer with the diluted blood, gently squeezing the reservoir to release contents until chamber is properly filled. Be sure to charge both sides and not to overfill chambers.
5. Place the hematocytometer in the premoist-ened Petri dish and leave for 15 minutes. This allows the sample to settle evenly.
Cell Counts and Calculations
A WBC count is performed with a Neubauer hematocy-tometer.
1. Using the X10 microscope magnification, WBC are counted using all nine squares of the counting chamber. Count both sides of the chamber and average the count. Refer to diagram below.
2. When counting, the cells that touch the extreme lower and the extreme left lines are not included in the count.
3. Use the following formulas to calculate the WBC.
average No. of cells X depth factor (10) X dilution factor (100) Area
Example: side 1 = 85 cells Side 2 = 95 cells
90 cells average/all 9 squares counted 90 X 10 X 100
Adult: 4000 to 10,000 Newborn: 10,000 to 30,000 1. Platelet counts are performed with a Neubauer hematocytometer (Fig. 20.9).
a. Counting is done using X40 dry phase contrast objective. Platelets will have a faint halo. The middle square of the hemacy-tometer chamber is counted. It contains 25 small squares.
b. Count 5 of the 25 squares if the platelet count is > 100,000. Take the average of both sides. Refer to diagram below.
c. To calculate platelets, use the following formula
• If 5 squares of middle square counted
Multiply No. of platelets X 5000 = No. of platelets/mm3
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