Ekong T, Kupek E, Hill A, Clark C, Davies A, Pinching A
Department of Immunology, St Mary's Hospital Medical School, London, UK.
J Immunol Methods. 1993 Sep 15;164(2):263-73. doi: 10.1016/0022-1759(93)90319-3.
The typing of lymphocyte subsets may be influenced by a variety of technical influences including the duration and temperature of sample storage and the method used for staining samples. We have extended a previous study examining the effect of storage conditions on the baseline values of a number of lymphocyte subsets. EDTA-anticoagulated samples from 13 HIV-1-positive and 15 healthy laboratory controls were analyzed for a number of lymphocyte subsets (CD3+, CD4+/CD3+, and CD8+/CD3+ T cells and CD19+ B cells) (whole blood lysis method, Becton-Dickinson FACScan flow cytometer and reagents) at 0, 24, 48, 72, and 96 h after storage at 4 degrees C, 17 degrees C or 21 degrees C. During storage at both 4 degrees C and 21 degrees C, there were significant changes in baseline values of the majority of lymphocyte subsets and some of these were related to the HIV status of the donor. The optimum temperature for storage in our system appeared to be around 17 degrees C in both our study groups. We have also used propidium iodide in order to discriminate between viable and non-viable cells during flow cytometry of lymphocytes from eight HIV-1-positive and five control subjects. The results show that for both HIV-positive and control samples stored at 4 degrees C, and for control subjects at 21 degrees C, the changes in baseline values of lymphocyte subsets observed were not due to selective loss of particular subsets arising from cell death during storage. However, there was substantial loss of cells from all three subsets in HIV-positive subjects during storage at 21 degrees C, with loss of CD8+ and CD3+ T cells being more significant than loss of CD4+ T cells.
淋巴细胞亚群的分型可能受到多种技术因素的影响，包括样本储存的持续时间和温度以及用于样本染色的方法。我们扩展了之前一项研究，该研究考察了储存条件对多个淋巴细胞亚群基线值的影响。对13名HIV-1阳性患者和15名健康实验室对照的乙二胺四乙酸（EDTA）抗凝样本，在4℃、17℃或21℃储存0、24、48、72和96小时后，分析多个淋巴细胞亚群（CD3 +、CD4 + /CD3 +、CD8 + /CD3 + T细胞和CD19 + B细胞）（全血裂解方法、BD FACScan流式细胞仪及试剂）。在4℃和21℃储存期间，大多数淋巴细胞亚群的基线值有显著变化，其中一些与供体的HIV状态有关。在我们的两个研究组中，我们系统中储存的最佳温度似乎都在17℃左右。我们还使用碘化丙啶，以便在对8名HIV-1阳性患者和5名对照受试者的淋巴细胞进行流式细胞术检测时区分活细胞和死细胞。结果表明，对于储存在4℃的HIV阳性样本和对照样本，以及对于储存在21℃的对照受试者，观察到的淋巴细胞亚群基线值变化并非由于储存期间细胞死亡导致特定亚群的选择性丢失。然而，在21℃储存期间，HIV阳性受试者的所有三个亚群都有大量细胞丢失，CD8 +和CD3 + T细胞的丢失比CD4 + T细胞的丢失更显著。
