Tissue Typing Lab, Transfusion Medicine, Mayo Clinic, Rochester, MN, USA.
Transfusion Lab, University of Nebraska Medical Center, Omaha, NE, USA.
Vox Sang. 2021 Jan;116(1):116-122. doi: 10.1111/vox.12989. Epub 2020 Aug 14.
Daratumumab (DARA), a human IgG1 monoclonal antibody targeting CD38, is used to treat refractory multiple myeloma patients. CD38 is expressed on many cell types (RBCs, granulocytes, lymphocytes, etc.), and thus, DARA can interfere with serological tests. Information regarding how DARA affects anti-granulocyte antibody (AGA) testing and optimal neutralization of DARA will help laboratories perform accurate testing.
Screening of AGA was performed by the granulocyte agglutination test (GAT) and the flow cytometric granulocyte immunofluorescence test (Flow-GIFT). Samples were tested from patients on DARA (n = 7), non-transfused blood donors (healthy controls, n = 7) and AGA reactive samples (positive controls, n = 5). Two neutralization experiments, CD38 removal with DTT and DARA epitope blockage with mouse anti-CD38, were evaluated.
Positive reactivity of human IgG binding was observed in 5/7 DARA cases when tested by Flow-GIFT; however, all 7 cases had negative GAT agglutination results. Further studies by Flow-GIFT revealed DARA concentrations >0·63 μg/ml bound to granulocytes. DARA binding was negated by DTT though a reduced Flow-GIFT sensitivity was observed in positive control samples due to increased background detection of human IgG. Mouse anti-CD38 neutralized the detection of human IgG observed in DARA-treated patient serum without effecting controls.
We established that DARA can interfere with AGA testing, leading to false positive Flow-GIFT results without causing GAT agglutination. DTT treatment increased background binding of secondary antibodies causing a decrease in Flow-GIFT sensitivity. In comparison, blockage of the DARA binding epitope using mouse anti-CD38 antibody was effective in neutralizing DARA interference while maintaining Flow-GIFT sensitivity.
达雷妥尤单抗（DARA）是一种靶向 CD38 的人源 IgG1 单克隆抗体，用于治疗难治性多发性骨髓瘤患者。CD38 表达于许多细胞类型（红细胞、粒细胞、淋巴细胞等），因此 DARA 可能会干扰血清学检测。关于 DARA 如何影响抗粒细胞抗体（AGA）检测以及如何最佳中和 DARA 的信息，将有助于实验室进行准确的检测。
通过粒细胞凝集试验（GAT）和流式细胞术粒细胞免疫荧光试验（Flow-GIFT）进行 AGA 筛查。检测了接受 DARA 治疗的患者（n=7）、未输血的献血者（健康对照，n=7）和 AGA 反应性样本（阳性对照，n=5）的样本。评估了两种中和实验，即用 DTT 去除 CD38 和用鼠抗 CD38 阻断 DARA 表位。
在 Flow-GIFT 检测中，5/7 例 DARA 病例观察到与人 IgG 结合的阳性反应；然而，所有 7 例均出现 GAT 凝集阴性结果。进一步的 Flow-GIFT 研究显示，浓度＞0.63μg/ml 的 DARA 与粒细胞结合。DTT 处理可消除 DARA 结合，但阳性对照样本的 Flow-GIFT 敏感性降低，原因是人类 IgG 的背景检测增加。鼠抗 CD38 中和了 DARA 处理患者血清中观察到的人 IgG 检测，而对对照无影响。
我们发现 DARA 可干扰 AGA 检测，导致 Flow-GIFT 结果出现假阳性，而不引起 GAT 凝集。DTT 处理增加了二级抗体的背景结合，导致 Flow-GIFT 敏感性降低。相比之下，使用鼠抗 CD38 抗体阻断 DARA 结合表位可有效中和 DARA 干扰，同时保持 Flow-GIFT 敏感性。
