一种同时测量血浆中残余血小板和红细胞的新型流式细胞术方法：质量控制的验证与应用

Jilma-Stohlawetz P, Marsik C, Horvath M, Siegmeth H, Höcker P, Jilma B

Department of Clinical Pharmacology, University of Vienna, Austria.

Transfusion. 2001 Jan;41(1):87-92. doi: 10.1046/j.1537-2995.2001.41010087.x.

BACKGROUND

Guidelines for the preparation of FFP in Austria and Germany require the quantification of residual RBCs in plasma. However, currently there is no validated method for enumeration of RBC counts as low as 1 x 10(9) per L.

STUDY DESIGN AND METHODS

A new flow cytometric method was developed for QC, to simultaneously determine if fresh unfrozen plasma contains residual platelets and RBCs. This method uses test tubes (TruCount, Becton Dickinson Immunocytometry Systems) that contain a known number of brightly fluorescent polystyrene beads. Plasma is pipetted into these tubes and mixed with FITC-labeled anti-CD41 and PE-conjugated anti-glycophorin A MoAbs. Acquisition can be performed on a flow cytometer after an incubation period of 15 minutes.

RESULTS

Individual standard curves for platelet counts revealed excellent correlation coefficients (r>0.998). Platelet counts were validated against simultaneous measurements by using a cell counter and a hemocytometer. While the flow cytometric method slightly overestimated platelet counts of >40 x 10(9) per L by 10 percent, its precision in the critical range was very good-that is, a deviation of platelets < or = 1 x 10(9) per L from target values, which was even better than microscopic enumeration. The limit of sensitivity was <0.5 x 10(9) per L of platelets. RBC counts were also validated against simultaneous measurements made with a second cell counter. Standard curves for RBC counts also revealed excellent correlation coefficients (r>0.997). The limit of sensitivity was <0.25 x 10(9) RBCs per L. Platelet counts in plasma obtained by plateletpheresis or plasmapheresis ranged from 3 to 60 x 10(9) per L. About 25 percent of all plasma samples had platelet counts greater than the allowed upper limit of 25 x 10(9) per L, while all plasma samples contained fewer RBCs than the upper limit of 6 x 10(9) per L.

CONCLUSION

This newly developed method provides a simple, quick, precise, and easily reproducible tool for simultaneous measurement of residual platelets and RBCs in fresh plasma.

背景

奥地利和德国关于新鲜冰冻血浆（FFP）制备的指南要求对血浆中残留红细胞进行定量。然而，目前尚无经过验证的方法来计数低至每升1×10⁹个的红细胞数量。

研究设计与方法

开发了一种新的流式细胞术方法用于质量控制，以同时测定新鲜未冷冻血浆中是否含有残留血小板和红细胞。该方法使用含有已知数量明亮荧光聚苯乙烯微球的试管（TruCount，Becton Dickinson免疫细胞计数系统）。将血浆移液到这些试管中，并与异硫氰酸荧光素（FITC）标记的抗CD41和藻红蛋白（PE）偶联的抗血型糖蛋白A单克隆抗体混合。孵育15分钟后，可在流式细胞仪上进行采集。

结果

血小板计数的个体标准曲线显示出极佳的相关系数（r>0.998）。通过使用血细胞计数器和血细胞计数板进行同步测量来验证血小板计数。虽然流式细胞术方法对每升大于40×10⁹个的血小板计数略有高估，高估了10%，但其在关键范围内的精密度非常好，即血小板每升与目标值的偏差≤1×10⁹个，甚至优于显微镜计数。灵敏度极限为每升血小板<0.5×10⁹个。红细胞计数也通过与第二台血细胞计数器的同步测量进行验证。红细胞计数的标准曲线也显示出极佳的相关系数（r>0.997）。灵敏度极限为每升<0.25×10⁹个红细胞。通过血小板单采术或血浆置换术获得的血浆中的血小板计数范围为每升3至60×10⁹个。所有血浆样本中约25%的血小板计数高于每升25×10⁹个的允许上限，而所有血浆样本中的红细胞数量均低于每升6×10⁹个的上限。