Furumaki Hiroaki, Takeshita Akihiro, Ohto Hitoshi, Yamada Chiaki, Fujihara Harumi, Ishizuka Keiko, Shibata Hiroki, Shinba Takahito, Nemoto Naoki, Ino Kaede, Ozawa Akari, Watanabe Hiroko, Kawabata Kinuyo, Obata Yukako
Transfusion and Cell Therapy, Hamamatsu University School of Medicine, Hamamatsu, Japan.
Department of Blood Transfusion and Transplantation Immunology, Fukushima Medical University, Fukushima, Japan.
Vox Sang. 2021 Jul;116(6):725-734. doi: 10.1111/vox.13052. Epub 2020 Dec 13.
Anti-CD38 monoclonal antibodies, including daratumumab and isatuximab, often interfere with pretransfusion testing. Dithiothreitol (DTT) treatment of red blood cells (RBCs) negates this interference. However, the optimum DTT concentration and treatment time have not been well defined. Here, we quantified CD38 on RBCs before and after DTT treatment using a flow cytometric antibody binding assay (FABA) to specify the optimum conditions for CD38 inactivation.
For FABA, untreated or DTT-treated RBCs were incubated with fluorescein isothiocyanate-labelled anti-CD38 antibody, in the presence or absence of 100-fold or more excess of unlabelled anti-CD38 antibody, and then analysed by flow cytometry (FCM). Dissociation of CD38-positive and control histograms was determined from the D-value using the Kolmogorov-Smirnov test. The results from FABA were compared with those from conventional FCM, indirect antiglobulin test (IAT) and Western blotting.
The results from FABA were more consistent than those from conventional FCM. The D-value was found to be reliable in the analysis of difference between CD38 before and after DTT treatment. Our data showed that 0·0075 mol/l DTT for 30 min is sufficient to inactivate CD38 on RBCs. These results were stable and consistent with the findings from IAT.
Flow cytometric antibody binding assay is an objective way of evaluating the efficacy of DTT treatment for CD38 on RBCs. This approach allows the detection of a small number of cell surface antigens and will be useful for assessing the various chemical treatments to denature RBC antigens.
抗CD38单克隆抗体，包括达雷妥尤单抗和isatuximab，常常会干扰输血前检测。用二硫苏糖醇（DTT）处理红细胞（RBC）可消除这种干扰。然而，最佳的DTT浓度和处理时间尚未明确界定。在此，我们使用流式细胞术抗体结合测定法（FABA）对DTT处理前后红细胞上的CD38进行定量，以确定CD38失活的最佳条件。
对于FABA，将未处理或经DTT处理的红细胞与异硫氰酸荧光素标记的抗CD38抗体一起孵育，存在或不存在100倍或更多过量的未标记抗CD38抗体，然后通过流式细胞术（FCM）进行分析。使用柯尔莫哥洛夫-斯米尔诺夫检验从D值确定CD38阳性和对照直方图的解离情况。将FABA的结果与传统FCM、间接抗球蛋白试验（IAT）和蛋白质印迹法的结果进行比较。
FABA的结果比传统FCM的结果更一致。发现D值在分析DTT处理前后CD38的差异时是可靠的。我们的数据表明，0.0075 mol/l DTT处理30分钟足以使红细胞上的CD38失活。这些结果是稳定的，并且与IAT的结果一致。
流式细胞术抗体结合测定法是评估DTT处理红细胞上CD38效果的一种客观方法。这种方法能够检测少量细胞表面抗原，并且将有助于评估使红细胞抗原变性的各种化学处理方法。
