Department of Pathology, Dalhousie University, Halifax, NS, Canada.
Department of Pathology, Dalhousie University, Halifax, NS, Canada.
Hum Immunol. 2024 Sep;85(5):110857. doi: 10.1016/j.humimm.2024.110857. Epub 2024 Aug 2.
Compromised detection of HLA specific antibodies due to complement mediated interference (CMI) is a well-recognized limitation of the single antigen bead (SAB) assay. Serum treatment with EDTA prior to SAB assay testing is a common strategy used to prevent CMI, however, treatment of individual sera, especially in large clinical runs, can extend assay turnaround time and increase the risk of a sample mix-up. In this study, we describe a simplified EDTA treatment strategy that can be applied simultaneously to all sera in a testing run. This strategy effectively prevents CMI without added pipetting steps and the increased turnaround time associated with other strategies. In the novel bead treatment (BT) method, EDTA solution and SAB suspension are combined and added into the wells of a testing tray together, patient sera are then added to the SAB/EDTA mixture. This eliminates the separate EDTA pre-treatment step used in the standard procedure (ST). Parallel testing using the BT, ST, and no EDTA treatment strategies followed by antibody identification using the Rapid Optimized SAB (ROB) protocol was performed for 19 well characterized sera with known CMI at two different laboratories. Both BT and ST methods were equally effective in preventing CMI. In addition, excellent MFI correlation was observed for specificities not affected by CMI. Both negative and pooled positive control sera performed as expected using the BT method and the pooled positive control serum, specifically designed to exhibit CMI, served well as an EDTA treatment control for all sera. The modified BT protocol can be easily implemented for clinical testing and eliminates extra pipetting steps, reduces the likelihood of pipetting error, and decreases turnaround time, while effectively preventing CMI and ensuring accurate detection of HLA antibodies. This protocol was recently implemented in the Halifax HLA Laboratory and has been very positively received by the laboratory team.
由于补体介导的干扰 (CMI)，单抗原珠 (SAB) 检测法无法准确检测 HLA 特异性抗体，这是一个公认的局限性。在进行 SAB 检测之前，用 EDTA 处理血清是一种常用的策略，可以防止 CMI，但对个体血清进行处理，特别是在大量临床检测中，会延长检测周转时间并增加样本混淆的风险。在这项研究中，我们描述了一种简化的 EDTA 处理策略，可以同时应用于检测过程中的所有血清。该策略可以有效地防止 CMI，而无需增加移液步骤和与其他策略相关的周转时间增加。在新的珠处理 (BT) 方法中，EDTA 溶液和 SAB 悬浮液一起组合并添加到测试托盘的孔中，然后将患者血清添加到 SAB/EDTA 混合物中。这消除了标准程序 (ST) 中使用的单独 EDTA 预处理步骤。在两个不同的实验室中，使用 BT、ST 和无 EDTA 处理策略进行平行检测，然后使用 Rapid Optimized SAB (ROB) 方案进行抗体鉴定。对 19 份具有已知 CMI 的特征明确的血清进行了平行检测。BT 和 ST 方法在防止 CMI 方面同样有效。此外，对于不受 CMI 影响的特异性，观察到出色的 MFI 相关性。使用 BT 方法，阴性和混合阳性对照血清的性能均符合预期，并且专门设计为表现出 CMI 的混合阳性对照血清也可以作为所有血清的 EDTA 处理对照。该改良的 BT 方案可以轻松应用于临床检测，可以减少额外的移液步骤，降低移液错误的可能性，并缩短检测周转时间，同时有效地防止 CMI，并确保准确检测 HLA 抗体。该方案最近在哈利法克斯 HLA 实验室实施，并得到了实验室团队的高度认可。
