Popko Katarzyna, Paskudzka Monika, Pieśniewska Małgorzata, Dąbrowska Sylwia, Demkow Urszula, Stelmaszczyk-Emmel Anna
Department of Laboratory Diagnostics and Clinical Immunology of Developmental Age, Medical University of Warsaw, Poland.
Department of Laboratory Diagnostics, Medical University of Warsaw, Poland.
Cent Eur J Immunol. 2023;48(4):267-273. doi: 10.5114/ceji.2023.134360. Epub 2024 Jan 15.
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.
Peripheral blood from healthy donors was taken with three anticoagulants: heparin, KEDTA 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.
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.
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.
自然杀伤（NK）细胞是人体免疫反应中的重要参与者。NK功能受损可能导致严重的、危及生命的状况。缺陷可能是基因突变的后果，也可能是感染、恶性肿瘤和自身免疫性疾病等 secondary factors 的结果。细胞毒性试验非常有用，但只有特殊的免疫实验室才能进行。血样常常要送到偏远的中心，这既耗时又需要特殊条件。本研究的目的是比较用三种不同抗凝剂保存的样本之间的细胞毒性测定结果，以使诊断程序标准化。
用三种抗凝剂采集健康供体的外周血：肝素、KEDTA 和柠檬酸盐。分离外周血单个核细胞（PBMC），在采血后和储存24小时后直接进行检测。NK细胞的细胞毒性能力在与K562共培养4小时后进行检测。通过流式细胞术测量NK细胞毒性。
在大多数分析的健康供体案例中，无论抗凝剂类型如何，细胞毒性结果相似。然而，柠檬酸盐样本的平均细胞毒性值最高。室温下全血储存24小时后，细胞毒性显著下降。所有抗凝剂的细胞毒性结果平均下降幅度都很大：肝素为76%，柠檬酸盐为67%，EDTA为70%。
自发NK细胞毒性的结果似乎受用于血液保存的抗凝剂影响。商业即时细胞毒性检测和血液储存后的延迟分析在含柠檬酸钠的血液中得到的结果最高。
