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Enhanced flow cytometric method for counting very low numbers of white cells in platelet products
1Research and Development, COBE BCT, Inc., Lakewood, Colorado 80215, USA. dumont-larry@cobe.com
Background:
The increased demand for leukocyte-reduced platelet products has prompted actions both by blood suppliers and device manufacturers to develop effective quality assurance methods and improved devices for leukocyte-reduced platelet preparation. White cell (WBC) counting methods capable of counting WBCs substantially below 1,000 WBC/mL enhance these activities. The purpose of this study was to enhance and validate a method capable of counting 5-10 WBC/mL in platelets.
Methods:
We added marker chicken red blood cells (cRBC) at a known concentration to an aliquot of platelet concentrate containing propidium iodide stain solution (PI). A modified BD FACScan was used to analyze this mixture. The ratio of cRBC and WBC events observed was used with the cRBC concentration to calculate the WBC concentration. Validation was performed by analyzing standard curves prepared with leukocyte-depleted platelet diluent. Methods of preparing leukocyte-reduced platelets by apheresis and filtration were evaluated using this method.
Results:
The method was linear from 5,000 WBC/mL to 10 WBC/mL (r2 = 0.983). Residual WBCs in the platelet diluent hampered validation below 10 WBC/mL. Repeatability (CV) was 44% at 8 WBC/mL and 6.5% at 1,000 WBC/mL. The FACScan required frequent mixing of the sample and flushing with cleaning agents during acquisition.
Conclusions:
This method has good linearity and reproducibility from 10-5,000 WBC/mL for platelets. It is suited for research and development work, and may be a useful adjunct method for quality assurance in the blood center.