全血淋巴细胞流式细胞术精细免疫分型方法的建立

Establishment of a multiparametric flow cytometry assay for measuring the absolute and relative numbers of human lymphocyte subsets

目的建立双平台法(流式细胞仪和血细胞计数仪)检测人外周血淋巴细胞亚类绝对数及相对数的流式方法 ,分析更精细的淋巴细胞亚群数量,全面评估被测者免疫功能。方法取人EDTA抗凝外周血500μl,200μl用于血细胞计数仪测得淋巴细胞绝对数;300μl用于流式细胞分析,测得所需淋巴细胞亚群相对数。各淋巴细胞亚群相对数(百分率)×淋巴细胞绝对数,即得到各淋巴细胞亚群绝对数。此方法用同一标本的全血和PBMC进行分析,评价用微量全血进行精细免疫分型的可行性。结果利用双平台法,采用微量全血标本分析了辅助T细胞、细胞毒性T细胞和B细胞的14个细胞亚群,得到其绝对数和相对数。用本方法对同一标本的全血和PBMC进行检测,结果无统计学差异。结论成功建立使用微量全血检测精细淋巴细胞亚群的多色流式分析方法,较全面地评估被测者免疫功能,为免疫相关疾病早期诊断提供线索和依据,对临床治疗具有重要指导作用。

To establish a multiparametric flow cytometry （FCM） assay for measuring absolute and relative numbers of human lymphocyte subsets in lysed whole blood, 500 ~xl peripheral blood （PB） was collected in a vacutainer tube containing liquid ethylenediaminetetraacetic acid （EDTA） as an anticoagulant and processed within 2 hours from collection. The percentage values of studied subsets obtained by eytometric reading were converted into absolute values （cells/p^l） according to absolute number of lymphocyte count provided by the haematological analyser. To prove whole blood assay was reliable, whole blood and PBMC from the same samples were detected by this method. Data showed that there was no statistical difference between whole blood and PBMC samples in relative and absolute numbers of 14 lymphocyte subsets. In conclusion, this study has raised a method to quantitatively assess 14 lymphocyte subsets using lysed whole blood method, which appears to be an effective and rapid test for comprehensive assessment of immune function, thus provides clues and evidence for the diagnosis of immune diseases, and will plays an important role in clinical treatment.