Direct white blood cell counting assay

Summary

Principles and techniques of microscopic counting methods for palm-tip leukocyte counting.

Operation method

Direct white blood cell counting assay

Principle

With dilute acetic acid solution, the blood will be diluted and destroyed red blood cells, mixed and filled into the counting cell, in the microscope counting a certain volume of leukocyte count, after conversion to find the number of leukocytes per liter of blood, commonly used in the following two methods: l% hydrochloric acid; 2% acetic acid

Materials and Instruments

Blood
Dilute acetic acid solution Hydrochloric acid
Microscope Micropipette Counting plate Coverslip

Move

1. Take two small test tubes and add 0.38 ml of leukocyte diluent.

2. Use a micropipette to accurately aspirate 20 microliters of peripheral blood or 20 microliters of anticoagulated blood. 3. Wipe off the remaining blood outside the tip of the tube and insert the pipette into the bottom of the diluent. Inject gently, aspirate the supernatant to wash 3 times and shake well.

4. Wipe the counting plate and coverslip clean, coverslip on the counting pool and then use a micropipette to inhale the suspension, filling the thin gap between the clean counting pool and coverslip, rest for 2-3 minutes, and wait for the cells to sink. 5. Count the number of leukocytes in the four large squares in the four corners of the low-magnification microscope. 6.

5. Count the number of leukocytes in the four large squares at the corners using low magnification.

6. Calculate (number of leukocytes in four large squares/4) × 10 × 20 × ¹⁰⁶ = number of leukocytes/liter

Lab report format: ^{ΔΔΔ×109/L}

Reference value: Adult: (4-10) × ^109/L

Neonate: (15-20) ^×109/L

6 months-2 years old: (11-12) ^×109/L

Caveat

l. The diluent should be fixed with a Class A 1 mL pipette or a standard 0.38 mL sampler.

2. If the leukocyte count is too low (＜2×109/L), count the number of leukocytes in 8 large squares or recount the result in 40ul and divide by 2. If the leukocyte count is too high, increase the dilution factor and recount the number of leukocytes.

3. Leukocyte diluent can not destroy nucleated erythrocytes, therefore, in pathological cases, the peripheral blood appears to be a large number of nucleated erythrocytes should be subtracted from the WBC/liter = (pre-corrected nucleated cell/liter) × (100/100 + sorted count of nucleated cells in the 10O WBC).

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