Demonstration by Flow Cytometry of the Numbers of Residual White Blood Cells and Platelets in Filtered Red Blood Cell Concentrates and Plasma Preparations
Demonstration by Flow Cytometry of the Numbers of Residual White Blood Cells and Platelets in Filtered Red Blood Cell Concentrates and Plasma Preparations
复制标题
通过流式细胞术显示过滤红细胞浓缩液和血浆制剂中残留白细胞和血小板的数量
DOI：
10.1046/j.1423-0410.1997.7340220.x
复制
发表时间：
1997
期刊：
影响因子：
2.7
通讯作者：
W. Mayr
中科院分区：
文献类型：
--
作者：
J. Neumüller;D. Schwartz;W. Mayr
背景和目标：新一代聚酯过滤器可显著去除过滤的红细胞浓缩物（FRCC）和过滤的血浆制剂（FP）中的白色血细胞（WBC）和血小板（PLT）。本研究的目的是建立一种灵敏的流式细胞术方法来监测FRCC和FP中残留的WBC和PLT。材料与方法：我们使用50 μl抗CD 45（FITC标记）和CD 19（PE标记）的单克隆抗体（MAB）组合测定500 μl FP FRCC中的WBC数量。在红细胞裂解后，我们将特定数量的参考珠与剩余的WBC混合。与采集体积相关的残留WBC数量由采集的参比微珠定义。方法的检出限为3 WBC/μl。替代方法使用针对CD 45（FITC和PerCP标记的）和CD 14（PE标记的）或淋巴细胞亚群如CD 3（FITC标记的）和CD 19、CD 4、CD 8、CD 16和CD 56（PE标记的）的MAB与CD 45（PerCP标记的）组合。CD 45/CD 14染色的DL值为10 WBC/μl，CD 3+和CD 19+淋巴细胞测定的DL值为0.1 WBC/μl。对于FRCC或FP中的残留PLT，我们使用了抗CD 41的FITC标记MAB，并使用参比微珠确定采集体积。在前向光散射× 90°光散射信号点图中门控后，在绿色-荧光（FL 1）单直方图中显示PLT。PLT计数如WBC所述。DL值约为2 PLT/μl。结果如下：使用Pall WBF-1过滤器过滤可使WBC减少4 log，PLT减少3-4 log，导致细胞计数低于导致不良输血反应的临界限值。结论：与Nageotte血细胞计数器相比，流式细胞术技术提供了一种可重复的客观工具，用于计数血液制剂中的残留WBC和PLT。可获得白细胞和淋巴细胞亚群的绝对数量。
Background and objectives: New‐generation polyester filters provide significant depletion of white blood cells (WBC) and platelets (PLT) in filtered red blood cell concentrates (FRCC) and in filtered plasma preparations (FP). The aim of this study was to elaborate a sensitive flow cytometric method for monitoring residual WBC and PLT in FRCC and FP. Materials and methods: We determined the number of WBC in 500 μl FRCC of FP using 50 μl of a combination of monoclonal antibodies (MAB) against CD45 (FITC labeled) and CD19 (PE labeled). After lysis of red blood cells, we mixed a specific number of reference beads with the remaining WBC. The number of residual WBC related to the acquisition volume was defined by the acquired reference beads. Using this method, the detection limit (DL) was 3 WBC/μl. Alternative methods used MAB against CD45 (FITC and PerCP labeled) and CD14 (PE labeled) or lymphocyte subsets such as CD3 (FITC labeled) and CD19, CD4, CD8, CD16 and CD56 (PE labeled) in combination with CD45 (PerCP labeled). The DL values were 10 WBC/μl for the CD45/CD14 staining and 0.1 WBC/μl for the determination of both CD3+ and CD19+ lymphocytes. For residual PLT in FRCC or FP, we used an FITC‐conjugated MAB against CD41, with reference beads to determine the acquisition volume. PLT were demonstrated in a green‐fluorescence (FL1) single histogram after gating in the forward light scatter × 90° light scatter signal dot plot. PLT counting was as described for WBC. The DL value was about 2 PLT/μl. Results: Filtration with Pall WBF‐1 filters reduces WBC by 4 log and PLT by 3–4 log, resulting in cell counts which are below the critical limit for causing adverse transfusion reactions. Conclusions: Flow cytometry techniques provide a reproducible and objective tool for counting residual WBC and PLT in blood preparations compared with the Nageotte hemocytometer. Absolute numbers of leukocyte and lymphocyte subpopulations are obtainable.