Introduction:
Natural killer (NK) cells are important players in the human immune response. Impaired NK function may lead to serious, life-threatening conditions. Defects may be consequences of genetic mutations or results of secondary factors such as infections, malignancies and autoimmune diseases. The cytotoxicity test is very useful, but its accessibility is limited to special immunological laboratories. Blood samples are often transported to remote centers, which takes time and requires special conditions. The aim of this study was to compare cytotoxicity assay results between samples preserved with three different anticoagulants to standardize the diagnostic procedure.

Material and methods:
Peripheral blood from healthy donors was taken with three anticoagulants: heparin, K2EDTA and citrate. Peripheral blood mononuclear cells (PBMC) were isolated and tested directly after blood drawing and after 24-hour storage. Cytotoxic abilities of NK cells were tested in 4 h co-culture with K562. NK cytotoxicity was measured by flow cytometry.

Results:
In most cases of analyzed healthy donors, cytotoxicity results were similar regardless of type of anticoagulant. However, the highest mean values were obtained in samples with citrate. There was a significant decrease in cytotoxicity after 24 hours of storage of the whole blood at ambient temperature. The mean drop in cytotoxicity results was substantial for all anticoagulants: 76% for heparin, 67% for citrate and 70% for EDTA.

Conclusions:
Results of spontaneous NK cytotoxicity seem to be affected by the anticoagulants used for blood protection. Commercial instant cytotoxicity testing and delayed analysis after blood storage gave the highest results in blood with sodium citrate.



中文翻译：

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血样保存及不同类型抗凝剂对NK细胞毒性试验结果的影响

简介：
自然杀伤（NK）细胞在人体免疫反应中发挥着重要作用。NK 功能受损可能会导致严重的危及生命的病症。缺陷可能是基因突变的结果，也可能是感染、恶性肿瘤和自身免疫性疾病等次要因素的结果。细胞毒性测试非常有用，但其可及性仅限于特殊的免疫学实验室。血液样本通常被运送到偏远的中心，这需要时间并且需要特殊的条件。本研究的目的是比较用三种不同抗凝剂保存的样品之间的细胞毒性测定结果，以标准化诊断程序。

材料和方法：
健康献血者的外周血采用三种抗凝剂：肝素、K2EDTA 和柠檬酸盐。抽血后和保存 24 小时后直接分离外周血单核细胞 (PBMC) 并进行检测。与 K562 共培养 4 小时，测试 NK 细胞的细胞毒能力。通过流式细胞术测量NK细胞毒性。

结果：
在大多数分析的健康供体案例中，无论抗凝剂类型如何，细胞毒性结果都是相似的。然而，在含有柠檬酸盐的样品中获得了最高平均值。全血在环境温度下保存24小时后，细胞毒性显着降低。所有抗凝剂的细胞毒性结果平均下降幅度很大：肝素下降 76%，柠檬酸盐下降 67%，EDTA 下降 70%。

结论：
自发 NK 细胞毒性的结果似乎受到用于血液保护的抗凝剂的影响。商业即时细胞毒性测试和血液储存后的延迟分析在含有柠檬酸钠的血液中给出了最高结果。