流式细胞术外周血样本预处理最佳试验条件探索

Exploration on optimal test conditions of peripheral blood sample pretreatment for flow cytometry

目的 探索流式细胞术基于外周血样本预处理抗体量、样本放置时间、样本固定与否的最佳试验条件。方法 采集3只正常大鼠EDTA抗凝全血进行白细胞及淋巴细胞计数,随机定义为样本A,B,C,各样本分为4份,分别用于检测各淋巴细胞亚群CD3^(+),CD3^(-)CD45RA^(+),CD3^(+)CD4^(+),CD3^(+)CD8^(+)的含量,每份样本抗体上样量都以标示量1μg的1/4,1/2,1,2倍孵育并标记。根据试验结果,1/4倍抗体完全满足检测所需,又采集3只正常大鼠血液样本,随机定义为样本D,E,F,处理同样本A,B,C,每份样本抗体上样量都以标示量1μg的1/4,1/8倍孵育并标记。将1/4抗体标示量处理好的样本D,E,F,即刻上机检测(固定前),之后将样本分成两部分:一部分分别于固定后15 min,24 h上机检测;另一部分2~8℃避光保存,分别保存0.5,1.5,3 h后上机检测。结果 (1)各样本淋巴细胞数处于47万~100万。样本A,B,C抗体使用量降至0.25μg时,各样本淋巴细胞亚群比例没有显著性下降。样本D,F抗体使用量在标示量的1/8时,CD3^(+),CD3^(-)CD45RA^(+),CD3^(+)CD8^(+)细胞的比例显著降低(P<0.05),样本E淋巴细胞亚群数量未发生显著性变化。(2)与固定前比较,固定15 min后样本D,E,F的CD3^(+),CD3^(+)CD4^(+),CD3^(+)CD8^(+)细胞比例在各血样样本中均有不同程度的下降(P<0.05)。(3)非固定样本D,E,F中CD3^(+),CD3^(+)CD4^(+),CD3^(+)CD8^(+),CD3^(-)CD45RA^(+)测定值的总体变化趋势为随保存时间延长逐渐下降,而其中样本D中CD3^(-)CD45RA^(+)测定值在保存3 h时未发生显著性变化。(4)与固定保存15 min比较,固定保存24 h后样本D、E中CD3^(+),CD3^(+)CD4^(+)细胞比例显著下降(P<0.05),样本F淋巴细胞亚群数量未发生显著性变化。结论 淋巴细胞数处于47万~100万时,0.25μg检测抗体完全满足检测所需。样本固定、固定后放置过长时间、样本不固定放置1.5 h以上都会对淋巴细胞亚群数量产生一定影响。

Objective To explore the optimal test conditions for flow cytometry based on the amount of antibody for peripheral sample pretreatment,sample placement time,and fixation or not.Methods White blood cells and lymphocytes were counted in the EDTA anticoagulant whole blood of three normal rats,which were randomly defined as A,B,and C.Each sample was divided into four parts to detect the contents of CD3^(+),CD3-CD45RA+,CD3^(+)CD4^(+),CD3^(+)CD8^(+)of each lymphocyte subgroup,respectively.The amount of loaded antibody per sample was incubated and labeled as 1/4,1/2,1,2 times the labeled amount(1μg).According to the test results,1/4 times the antibody fully met the requirements of detection,then three normal rat blood samples were collected,randomly defined as D,E,F,and treated the same as samples A,B,C,and the amount of loaded antibody in each sample was incubated and labeled as 1/4,1/8 times the labeled amount(1μg).Samples D,E and F with 1/4 antibody labeling amount were immediately detected on the machine(denoted as pre-fixation),and then the samples were divided into two parts.One part was fixed and tested at 15 min and 24 h after the fixation,respectively,while the other part was stored in dark at 2-8℃and tested after being stored for 0.5,1.5,3 h,respectively.Results①The number of lymphocytes in each sample ranged from 470000 to 1000000.The proportion of lymphocyte subsets in samples A,B and C did not decrease significantly when the antibody dosage was reduced to 0.25μg.When the amount of antibody was 1/8 of the labeled amount,the proportions of CD3^(+),CD3-CD45Ra+and CD3^(+)CD8^(+)cells were significantly decreased in samples D and F(P<0.05),and there was no significant change in the numbers of lymphocyte subsets in sample E.②Compared with those before fixation,the proportions of CD3^(+),CD3^(+)CD4^(+)and CD3^(+)CD8^(+)cells in samples D,E and F after fixation for 15 min were decreased in varying degrees(P<0.05).③The proportions of CD3^(+),CD3^(+)CD4^(+),CD3^(+)CD8^(+)and CD3-CD45RA+in unfixed samples D,E,F were gradually decreased with the extension of storage time.However,the value of CD3-CD45RA+in sample D showed no significant changes after 3 h storage.④After 24 h of fixed storage,the proportions of CD3^(+)and CD3^(+)CD4^(+)cells in samples D and E were significantly decreased compared to those after 15 min of fixed storage(P<0.05),and there was no significant changes in the number of lymphocyte subsets in sample F.Conclusion When the number of lymphocytes is 470000-1000000,0.25μg antibody is sufficient for detection.The number of lymphocyte subsets can be affected by fixed samples,prolonged sample placement after fixation,and unfixed placement of samples for more than 1.5 h.