Department of Pathology and Laboratory Medicine, University of California Los Angeles, Los Angeles, CA, USA.
Am J Transplant. 2013 Jul;13(7):1859-70. doi: 10.1111/ajt.12287. Epub 2013 Jun 13.
Solid phase multiplex-bead arrays for the detection and characterization of HLA antibodies provide increased sensitivity and specificity compared to conventional lymphocyte-based assays. Assay variability due to inconsistencies in commercial kits and differences in standard operating procedures (SOP) hamper comparison of results between laboratories. The Clinical Trials in Organ Transplantation Antibody Core Laboratories investigated sources of assay variation and determined if reproducibility improved through utilization of SOP, common reagents and normalization algorithms. Ten commercial kits from two manufacturers were assessed in each of seven laboratories using 20 HLA reference sera. Implementation of a standardized (vs. a nonstandardized) operating procedure greatly reduced MFI variation from 62% to 25%. Although laboratory agreements exceeded 90% (R(2) ), small systematic differences were observed suggesting center specific factors still contribute to variation. MFI varied according to manufacturer, kit, bead type and lot. ROC analyses showed excellent consistency in antibody assignments between manufacturers (AUC > 0.9) and suggested optimal cutoffs from 1000 to 1500 MFI. Global normalization further reduced MFI variation to levels near 20%. Standardization and normalization of solid phase HLA antibody tests will enable comparison of data across laboratories for clinical trials and diagnostic testing.
固相多重珠阵列用于检测和鉴定 HLA 抗体，与传统的基于淋巴细胞的检测方法相比，具有更高的灵敏度和特异性。由于商业试剂盒的不一致性和标准操作程序 (SOP) 的差异，检测的变异性妨碍了实验室之间结果的比较。器官移植抗体临床试验核心实验室研究了检测变异性的来源，并确定通过使用 SOP、共同试剂和归一化算法是否可以提高重现性。使用 20 个 HLA 参考血清，在七个实验室中的每个实验室中评估了来自两个制造商的十种商业试剂盒。实施标准化（而非非标准化）操作程序可将 MFI 变化从 62%大幅降低至 25%。尽管实验室间的一致性超过 90%（R(2)），但仍观察到一些小的系统差异，表明中心特定因素仍会导致变异性。MFI 因制造商、试剂盒、珠类型和批次而异。ROC 分析表明，制造商之间的抗体分配具有极好的一致性（AUC > 0.9），并建议使用 1000 至 1500 MFI 的最佳截止值。全局归一化进一步将 MFI 变化降低至接近 20%的水平。固相 HLA 抗体检测的标准化和归一化将使临床试验和诊断检测的实验室间数据比较成为可能。
