Kelliher Abigail S, Parent David W, Anderson David C, Dorn Michelle E, Hahn Jessica L, Eapen Sara, Preffer Frederic I
Department of Pathology, Massachusetts General Hospital, Boston, Massachusetts 02114, USA.
Cytometry B Clin Cytom. 2005 Jul;66(1):40-5. doi: 10.1002/cyto.b.20055.
The effective and accurate diagnosis of hematologic malignancies relies on flow cytometric immunophenotyping. Selected combinations of monoclonal antibodies (mAbs) arranged in multicolor panels allow for the accurate definition of normal and abnormal hematologic cell populations. The most time-consuming and crucial step in the staining process involves dispensing combinations of multiple mAbs into their appropriate staining tubes. This step is prone to error, requires concentration and accuracy, and is dependent on technologist experience. The Becton Dickinson BioScience (BD) FACS Sample Prep Assistant (SPA) is touted as a breakthrough in automated in vitro diagnostic sample preparation. The SPA is designed to automate BD MultiTESk and BD TriTest lyse/no-wash assays. However, because most cases in our laboratory require tedious application of unique four-color mAb cocktails for leukemia and lymphoma testing, we wondered whether the SPA would be helpful in accurately dispensing these mixtures.
The mAb panels were prepared by the SPA in two separate timed runs and on separate days. Eleven specimens (nine from patients and two from normal volunteers) were split and stained with four-color cocktails created by the SPA or manually. The percentage of positive (%P) cells and mean fluorescent intensity for each mAb pair were determined. These values were plotted against each other and correlation values were calculated. To quantitate timesaving in the laboratory, two technologists prepared individually the same mAb panels and were timed.
The correlation between the two methods was high; r(2) was 0.988 for 158 %P antigen pairs; no bias between the manual and robotic methods was detected with the Wilcoxon rank test. Bland-Altman analysis indicated no obvious relation between the difference and the mean of %P cells, suggesting that the SPA successfully dispensed antibodies for leukemia/lymphoma panels. The two methods may be interchangeable, although the limited sample size prohibits this conclusion from Bland-Altman statistics alone. In addition, one possible error was detected in the SPA-prepared panels. The SPA averaged 65 min/run, the experienced technologist 12.95 min/run, and the inexperienced technologist 54.9 min/run.
SPA dispensing time was twice the average manual dispensing time; however, SPA use was completely automated and freed the technologist to perform other tasks. SPA use permitted preemptive preparation of mAb panels and thus streamlined processing; however, the cost of the assay and the amount of reagent waste increased. It is certain that software modifications by BD could decrease the SPA reagent dispense time and decrease the cost associated with reagent waste when the SPA is used in this novel fashion.
血液系统恶性肿瘤的有效且准确诊断依赖于流式细胞术免疫表型分析。排列在多色组合中的单克隆抗体（mAb）的特定组合可准确界定正常和异常血液细胞群体。染色过程中最耗时且关键的步骤是将多种mAb组合分配到合适的染色管中。此步骤容易出错，需要专注和精准，且依赖技术人员的经验。贝克曼库尔特生物科学公司（BD）的FACS样本制备助手（SPA）被誉为体外诊断样本自动化制备方面的一项突破。SPA旨在实现BD MultiTESk和BD TriTest溶血/免洗检测的自动化。然而，由于我们实验室的大多数病例在白血病和淋巴瘤检测中需要繁琐地应用独特的四色mAb组合，我们想知道SPA在准确分配这些混合物方面是否会有所帮助。
SPA在两个不同的定时运行且在不同日期制备mAb组合。将11个样本（9个来自患者，2个来自正常志愿者）分开，用SPA制备的或手动制备的四色组合进行染色。测定每个mAb对的阳性细胞百分比（%P）和平均荧光强度。将这些值相互绘制并计算相关值。为了量化实验室中的时间节省，两名技术人员分别制备相同的mAb组合并计时。
两种方法之间的相关性很高；158个%P抗原对的r²为0.988；通过Wilcoxon秩和检验未检测到手动和机器人方法之间的偏差。Bland - Altman分析表明%P细胞差异与均值之间无明显关系，这表明SPA成功为白血病/淋巴瘤组合分配了抗体。尽管样本量有限，仅根据Bland - Altman统计无法得出此结论，但这两种方法可能具有互换性。此外，在SPA制备的组合中检测到一个可能的错误。SPA平均每次运行65分钟，经验丰富的技术人员每次运行12.95分钟，经验不足的技术人员每次运行54.9分钟。
SPA的分配时间是平均手动分配时间的两倍；然而，使用SPA完全自动化，使技术人员能够腾出时间执行其他任务。使用SPA允许预先制备mAb组合，从而简化了流程；然而，检测成本和试剂浪费量增加。可以肯定的是，当以这种新颖方式使用SPA时，BD对软件进行修改可以减少SPA试剂分配时间并降低与试剂浪费相关的成本。
