Lambrecht Bernd, Spengler Hans-Peter, Nauwelaers Frans, Bauerfeind Ursula, Mohr Harald, Müller Thomas H
Blood Centers of the German Red Cross Chapters of NSTOB, Institute Springe, Springe, Germany.
Transfusion. 2009 Jun;49(6):1195-204. doi: 10.1111/j.1537-2995.2008.02079.x. Epub 2009 Feb 6.
A fully automated single-tube assay with tubes (BD TruCOUNT, BD Biosciences) for absolute counting of residual cells in freshly prepared plasma by flow cytometry was developed (BD Plasma Count).
The nucleic acid dye thiazole orange stains white blood cells (WBCs). The monoclonal antibodies anti-CD41a-peridinin chlorophyll protein-Cy5.5 and anti-glycophorin A-fluorescein isothiocyanate label platelets (PLTs) and red blood cells (RBCs), respectively. No fixation, permeabilization, or washing steps were required. Validation was done according to guidelines of the International Conference on Harmonization and the National Committee for Clinical Laboratory Standards. Cell-free plasma was spiked with each cell type for accuracy, reproducibility, and linearity measurements.
Results showed no carryover or drift under automated sample acquisition conditions. Nonspecific background was fewer than 0.3 cells per microL for residual WBCs (rWBCs), fewer than 2.7 cells per microL for rRBCs, and fewer than 85 cells per microL for rPLTs. Determinations of rWBC and rPLT counts were linear with a coefficient of variation of less than 12 percent for the imprecision. Owing to cross-linking of the anti-glycophorin A antibody, linearity and precision for rRBCs diverged up to 21 percent at a count of 6000 rRBCs per microL. In a 2-year period, five operators investigated 2666 quality control (QC) samples of fresh-frozen plasma on 108 working days. Maximum cell numbers found were 196 for rWBCs, 3960 for rRBCs, and 28,952 for rPLTs per microL. In 31 cases (1.2%) rWBCs were out of specification. No outlier was observed for rRBCs and rPLTs. Residual RBC cell numbers determined were always within the acceptable concentration range of the assay.
These data demonstrate that the single-tube test is suitable for routine QC assessment of the cellular contaminants of therapeutic plasma according to the European recommendations.
开发了一种用于通过流式细胞术绝对计数新鲜制备血浆中残留细胞的全自动单管检测法（BD TruCOUNT，BD生物科学公司）（BD血浆计数法）。
核酸染料噻唑橙可对白细胞（WBC）进行染色。单克隆抗体抗-CD41a-多甲藻叶绿素蛋白-Cy5.5和抗血型糖蛋白A-异硫氰酸荧光素分别标记血小板（PLT）和红细胞（RBC）。无需固定、通透或洗涤步骤。根据国际协调会议和美国国家临床实验室标准委员会的指南进行验证。向无细胞血浆中加入每种细胞类型，以进行准确性、重现性和线性测量。
结果表明，在自动样本采集条件下无残留或漂移现象。残留白细胞（rWBC）的非特异性背景低于每微升0.3个细胞，残留红细胞（rRBC）低于每微升2.7个细胞，残留血小板（rPLT）低于每微升85个细胞。rWBC和rPLT计数的测定呈线性，不精密度的变异系数小于12%。由于抗血型糖蛋白A抗体的交联，在每微升6000个rRBC时，rRBC的线性和精密度差异高达21%。在两年时间里，五名操作人员在108个工作日内对2666份新鲜冷冻血浆的质量控制（QC）样本进行了检测。每微升中发现的最大细胞数为：rWBC为196个，rRBC为3960个，rPLT为28952个。在31例（1.2%）中，rWBC超出规格。未观察到rRBC和rPLT的异常值。所测定残留RBC细胞数始终在该检测方法可接受的浓度范围内。
这些数据表明，根据欧洲建议，该单管检测法适用于治疗性血浆细胞污染物的常规QC评估。
