Granulocytes Affect Double-Strand Break Repair Assays in Primary Human Lymphocytes

Patients who develop therapy-related myelodysplasia/acute myeloid leukemia after autologous-hematopoietic stem cell (aHCT) transplant show lower expression levels of DNA repair genes in their pre-aHCT CD34+ cells. To investigate whether this leads to functional differences in DNA repair abilities measurable in patients, we adapted two plasmid-based host-cell reactivation assays for use in primary lymphocytes. Prior to applying these assays to patients who underwent aHCT, we wanted first to verify whether sample preparation affected repair measurements, as patient samples were simply depleted of erythrocytes (with hetastarch) prior to freezing, which is not the classical way to prepare lymphocytes prior to DNA repair experiments (with a density gradient). We show here that lymphocytes from healthy donors freshly prepared with hetastarch show systematically a higher level of double-strand break repair as compared to when prepared with a density gradient, but that most of this difference disappears after samples were frozen. Several observations points to granulocytes as the source for this effect of sample preparation on repair: 1) removal of granulocytes makes the effect disappear, 2) DSB repair measurements for the same individual correlate to the percentage of granulocytes in the sample and 3) nucleofection in presence of granulocytes increases the level of reactive oxygen species (ROS) in neighboring lymphocytes in a dose-dependent manner (R2 of 0.95). These results indicate that co-purified granulocytes, possibly through the release of ROS at time of transfection, can lead to an enhanced repair in lymphocytes that obfuscates any evaluation of inter individual differences in repair as measured by host-cell reactivation. As a result, hetastarch-prepared samples are likely unsuitable for the assessment of DSB repair in primary cells with that type of assay. Granulocyte contamination that exists after a density gradient preparation, although much more limited, could have similar effects, but might be circumvented by freezing cells prior to analysis.

自体造血干细胞移植（autologous-hematopoietic stem cell transplant, aHCT）后发生治疗相关骨髓增生异常综合征/急性髓系白血病的患者，其移植前CD34阳性细胞（CD34+ cells）中DNA修复基因的表达水平更低。为探究该现象是否会导致患者体内可检测到的DNA修复能力出现功能差异，我们优化了两种基于质粒的宿主细胞再激活实验（host-cell reactivation assay），使其可用于原代淋巴细胞（primary lymphocytes）检测。
在将上述实验应用于接受自体造血干细胞移植的患者之前，我们首先验证了样本制备方式是否会对修复检测结果产生影响：患者样本在冻存前仅通过羟乙基淀粉（hetastarch）去除了红细胞，这与DNA修复实验中经典的淋巴细胞制备方式（密度梯度离心法，density gradient）并不一致。
本研究结果显示，采用羟乙基淀粉法新鲜制备的健康供者淋巴细胞，其双链断裂修复（double-strand break repair, DSB repair）水平系统性高于采用密度梯度离心法制备的样本；但该差异在样本冻存后基本消失。多项观察结果表明粒细胞（granulocytes）是样本制备方式影响修复检测结果的来源：1）去除粒细胞后该差异消失；2）同一受试者的双链断裂修复检测结果与样本中粒细胞占比呈相关性；3）粒细胞存在时的核转染（nucleofection）会以剂量依赖性方式增加邻近淋巴细胞内的活性氧（reactive oxygen species, ROS）水平（决定系数R²为0.95）。
上述结果提示，共纯化得到的粒细胞可能通过转染过程中释放的活性氧，增强淋巴细胞的修复能力，从而混淆通过宿主细胞再激活实验评估的个体间修复能力差异。因此，采用羟乙基淀粉法制备的样本，并不适合通过该类实验评估原代细胞的双链断裂修复能力。尽管密度梯度离心法制备的样本中粒细胞污染程度更低，但仍可能产生类似影响，不过可通过在分析前冻存细胞的方式予以规避。