标本处理对淋巴细胞免疫表型检测的影响

Study on the sample preparations affecting flow cytometric immunophenotyping of blood lymphocytes

目的研究标本处理对流式细胞术(FCM)检测全血淋巴细胞免疫表型(CD3、CD4、CD8、CD16+56)的影响。方法分别将①提取淋巴细胞法、手工溶血法及仪器法(Q-prep免疫标本处理仪);②血液采集后室温放置1.5h、6h与7h;③染色、固定后于4℃放置1h、24h、48h与72h;④仪器法处理后洗涤和不洗涤等方法处理的全血标本用FCM检测免疫表型。结果使用淋巴细胞法、手工溶血法及仪器法,在血液采集后1.5h与6h,固定后1h、24h、48h,仪器法处理后洗涤与不洗涤等条件下分别比较FCM分析结果,差别无显著性意义(P>0.05)。血液采集后放置1.5h与7h、6h与7h,染色、固定后1h与72h、24h与72h、48h与72h分别比较FCM测定结果,CD3+、CD3+CD8+、CD3+CD16+56+百分率差别有显著性意义或极显著性意义(P<0.05或P<0.01),CD3+CD4+百分率差别无显著性意义(P>0.05)。结论FCM检测全血淋巴细胞免疫表型结果受多种标本处理因素的影响。标本采集后应尽快进行免疫荧光染色和固定,最迟不超过6h;染色、固定后标本于4℃放置48h,对结果影响不大;仪器法处理后的标本可不必洗涤。

Objective To study the sample preparations affecting flow cytometric immunophenotyping of blood lymphocytes.Methods The whole blood were prepared by separating lymphocytes or by hemolyzing or by Q-prep immunology workstation;1.5h、6h and7h in room temperature after sample collection;1h、24h、48h and 72h in 4℃ after fixing;washing or not washing after Q-prep immunology workstation preparation.FCM was adopted to detect the immunophenotype of lymphocytes.Results There was no difference of lymphocyte immunophenotyping among the methods of separating lymphocytes、blood hemolyzing and Q-prep immunology workstation.And the placing period of 1.5h and 6h,laying period of 1h、24h and 48h and washing after Q-prep preparing didn't affect the results(P>0.05).The percentage of CD3+、CD3+CD8+ and CD3+CD16+56+ were different significantly in groups of 1.5h、6h and 7h after sample collection.There were obvious difference in groups of 1h or 24h or 48h and 72h after fixing(P<0.05 or P<0.01),but the percentage of CD3+CD4+had not been affected(P>0.05).Conclusions Many factors may affect flow cytometric lymphocyte immunophenotyping.Prepare samples with Q-prep immunology workstation is a simple、speedy and reliable method.Samples should be dyed and fixed as soon as possible(no more than 6h).Samples can be placed 48 hours after dying and fixing with no difference of the results.Washing is not necessary for Q-prep preparing.