フローサイトメトリーによるフィルター処理白血球除去血液製剤中の残存白血球数の確認

抄録

A dramatic improvement in the performance of leukocyte removal filters in recent years has made it difficult to count the few remaining leukocytes in the filtered platelet concentrates (PC) and red cell concentrates (RCC) using automated electronic cell counters or visual counts in hemocytometers. A cytospin method recently developed in our laboratory could detect residual leukocyte in filtered PC, but this method is time consuming and unsuitable for meauring leukocytes in filtered RCC. We have developed a simple flow cytometric method able to measure the small number of leukocytes remaining in both filtered PC and RCC. Our method uses the ORTHO CYTORON flow cytometer, as this instrument incorporates a sample delivery system which delivers a defined volume of sample to the flow cell. For testing, one part of pre- and post-filtered PC and RCC are added to 7.5 parts of propidium iodide solution containing RNase, sodium citrate and Triton X-100. Stained leukocytes are measured at a flow rate of 1μl/sec for 1min. The large numbers of leukocytes in the pre-filtered samples had first been measured in the hemocytometer and serially diluted with leukocyte-free PC or RCC prepared by repeated filtration. The number of leukocytes measured on the ORTHO CYTORON as a function of cell number expected from the hemocytometer count can be expressed by the regression lines: y=0.823x-0.454 (r=0.999) for RCC and y=0.801x+0.672 (r=0.999) for PC. After correction of measurements using these regression equations, these was a significant correlation between the measurement of leukocytes in PC and RCC by the ORTHO CYTORON and hemocytometer in the range of 10⁵-10⁷cells/ml (r=0.99). When RCC and PC had been filtered with several kinds of leukocyte removal filters, the automated electronic cell counter or hemocytometer frequently failed to detect leukocytes in the filtrate, whereas the flow cytometric method could detect leukocytes to concentrates as low as 1.77×10^-1cells/μl.