A hematopoietic chimerism assay is the laboratory test for monitoring engraftment and quantifying the proportions of donor and recipient cells after hematopoietic stem cell transplantation recipients. Flow cytometry is the reference method for determining the purity of CD3⁺ cells on the chimerism of selected CD3⁺ cells. In the present study, we developed a single‐step procedure that combines the CD3⁺ purity assay (using the PCR‐based Non‐T Genomic Detection Kit from Accumol, Calgary, Canada) and the qPCR chimerism monitoring assay (the QTRACE qPCR assay from Jeta Molecular, Utrecht, the Netherlands). First, for the CD3⁺ purity assay, we used a PCR‐friendly protocol by changing the composition of the ready‐to‐use reaction tubes (buffer and taq polymerase) and obtained a satisfactory calibration plot (R² = 0.8924) with a DNA reference scale of 2 ng/μl. Next, 29 samples (before and after CD3 positive selection) were analyzed, the mean cell purity was, respectively, 19.6% ± 6.45 and 98.9% ± 1.07 in the flow cytometry assay; 26.8% ± 7.63 and 98.5% ± 1.79 in the PCR‐based non‐T genomic detection assay. Our results showed that the CD3⁺ purity assay using a qPCR kit is a robust alternative to the flow cytometry assay and is associated with time savings when combined with a qPCR chimerism assay.

中文翻译：

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造血干细胞移植后分选的CD3 +细胞纯度和嵌合性的一步测定

造血嵌合试验是一种实验室测试，用于监测植入和造血干细胞移植受者后供体和受体细胞的比例。流式细胞术是用于确定CD3的纯度参考方法⁺所选CD3的嵌合细胞⁺细胞。在本研究中，我们开发了结合CD3 ⁺纯度测定法（使用来自加拿大卡尔加里Accumol的基于PCR的Non-T基因组检测试剂盒）和qPCR嵌合体监测测定法（得自QTRACE qPCR测定法）的单步骤程序Jeta Molecular，荷兰乌得勒支）。首先，对于CD3 ⁺纯度测定，我们通过改变现成的反应管（缓冲液和taq聚合酶）的组成，使用了PCR友好的方案，并获得了令人满意的校准图（R ² = 0.8924），DNA参考标度为2 ng /微升 接下来，分析了29个样品（CD3阳性选择之前和之后），在流式细胞仪检测中，平均细胞纯度分别为19.6％±6.45和98.9％±1.07。在基于PCR的非T基因组检测分析中，分别为26.8％±7.63和98.5％±1.79。我们的结果表明，使用qPCR试剂盒进行CD3 ⁺纯度测定是流式细胞仪测定的可靠替代方法，与qPCR嵌合测定结合使用时可节省时间。