Liu Chang, Pang Sue, Phelan Donna, Brennan Daniel C, Mohanakumar Thalachallour
HLA Laboratory, Barnes-Jewish Hospital, St. Louis, MO.
Division of Nephrology, Washington University School of Medicine, St. Louis, MO.
Transplant Direct. 2017 Jul 11;3(8):e194. doi: 10.1097/TXD.0000000000000709. eCollection 2017 Aug.
Ethylenediaminetetraacetic acid (EDTA) pretreatment has been shown to overcome complement interference in the single-antigen bead (SAB) assay. However, a quantitative evaluation of its impact on the assay for preemptive application to diverse clinical samples is still lacking.
Serum samples from 95 renal transplant candidates were tested with and without EDTA-pretreatment in parallel. Changes in mean fluorescence intensity (MFI) values were analyzed to determine the impact of EDTA-pretreatment and the characteristics of complement interference.
MFI values from EDTA-treated and untreated sera showed good correlations (r = 0.99) and were linear after excluding outliers (slopes, 1; intercepts, -63.7 and -24.2 for class I and II, respectively). Using an assay cutoff of 2000 MFI, positive/negative assignments were concordant for 99% of the 9215 class I beads and 9025 class II beads tested. As defined by an MFI increment above 4000 after EDTA pretreatment, complement interference affected 172 class I beads in 12 samples (12.6%) and 60 class II beads in 7 samples (7.4%), and the findings were supported in 83% and 86% of these samples by dilution studies. In a case study, EDTA pretreatment prevented falsely low MFI values and facilitated the interpretation of titration curves. Finally, EDTA pretreatment reduced the coefficient of variance (CV) by 2.1% and 2.4% for class I and II beads respectively ( < 0.0001).
It is safe to preemptively treat all clinical samples with EDTA before SAB assay to prevent false negative results or falsely low MFI values. EDTA pretreatment has the added benefit of improved assay precision.
已证实乙二胺四乙酸（EDTA）预处理可克服单抗原珠（SAB）检测中的补体干扰。然而，对于其对检测的影响进行定量评估以抢先应用于各种临床样本仍缺乏相关研究。
对95例肾移植候选者的血清样本进行平行检测，一组进行EDTA预处理，另一组不进行预处理。分析平均荧光强度（MFI）值的变化，以确定EDTA预处理的影响及补体干扰的特征。
经EDTA处理和未处理的血清MFI值显示出良好的相关性（r = 0.99），排除异常值后呈线性关系（I类和II类的斜率均为1；截距分别为 -63.7和 -24.2）。使用2000 MFI的检测临界值，在检测的9215个I类珠和9025个II类珠中，99%的阳性/阴性判定结果一致。按照EDTA预处理后MFI增量高于4000来定义，补体干扰影响了12个样本中的172个I类珠（12.6%）和7个样本中的60个II类珠（7.4%），稀释研究在83%和86%的这些样本中支持了该结果。在一个案例研究中，EDTA预处理避免了MFI值过低的错误，并有助于滴定曲线的解读。最后，EDTA预处理使I类和II类珠的变异系数（CV）分别降低了2.1%和2.4%（P < 0.0001）。
在进行SAB检测前对所有临床样本进行EDTA预处理以防止假阴性结果或过低的MFI值是安全的。EDTA预处理还有提高检测精度的额外益处。
