标本放置时间及年龄对CD4^+CD25^+调节性T细胞的影响

Influence of age and samples' preparation time on the assay of the CD4^+CD25^+ regulatory T cells

目的探讨标本放置时间及年龄对外周血CD4+CD25+调节性T细胞的影响,明确标本可放置的时间界限,建立不同年龄段的正常参考值范围。方法以171例不同年龄健康人的外周血为标本,采用双色直接荧光素标记法和多参数流式细胞术检测和分析。结果10份标本均分别在室温下放置0、1、2、3、4、5和6h检测时,CD4+CD25+Treg及CD4+CD25highT细胞分别为(12.00±1.12)%、(11.56±1.19)%、(10.72±1.29)%、(10.29±1.57)%、(8.11±1.28)%、(7.97±1.07)%、(7.70±0.93)%及(1.41±0.26)%、(1.45±0.26)%、(1.32±0.26)%、(1.27±0.28)%、(1.09±0.24)%、(0.95±0.18)%、(0.91±0.21)%。4~6岁组(n=34)、7~14岁组(n=24)、20~59岁组(n=95)、60岁以上组(n=18)健康人群CD4+CD25+Treg参考值分别是(6.55±1.28)%、(9.41±2.08)%、(11.67±2.14)%和(9.73±2.79)%,CD4+CD25high分别是(0.92±0.30)%、(1.21±0.45)%、(1.44±0.52)%及(1.51±0.55)%。结论外周血CD4+CD25+Treg和CD4+CD25highT细胞的检测结果随标本放置时间延长呈逐渐降低趋势,因此为真实反映体内CD4+CD25+Treg水平,标本放置时间应限制在3h以内。不同年龄段健康人群外周血中的CD4+CD25+Treg和CD4+CD25highT数值有明显差异,在探讨各种免疫相关性疾病与CD4+CD25+Treg之间的关系时,应该以与其年龄段相匹配的正常参考值为依据。

Objective To explore the influence of age and samples＇ preparation time on assay of human peripheral blood CD4^＋ CD25^ ＋ regulatory T ceils and establish a normal reference range. Methods At various preparation times, peripheral blood samples with fluoresce-labeled monoclonal antibody from 171 healthy individuals from the age of 3-year-6-month to 77-year were detected by using multiparameter flow cytometry. Results At the time points of 0, 1, 2, 3, 4, 5, and 6 h, the proportion OfCD4^＋ CD25^＋ of peripheral bloed CD4＋ lymphocyteswas （12.00±1.12）%, （11.56±1.19）%, （10.72±1.29）%, （10.29±1.57）%, （8.11±1.28）%, （7.97±1.07）%, and （7.70±0.93）% respectively, while the CD4^＋ CD25^high was （1.41 ±0.26）%, （1.45±0.26）%, （1.32±0.26）%, （1.27±0.28）%, （1.09±0.24）%, （0.95±0.18）%, and （0.91 ±0.21）%. The reference ranges of CD4^＋ CD25^＋ T ceils in the groups of 4- 6, 7- 14, 20- 59 and elder than 60 was （6.55 ± 1.28）%, （9.41± 2.08）%, （11.67 ± 2.14）% and （9.73 ± 2.79）% respectively, while CD4^＋ CD25^high was （0.92±0.30）%, （1.21±0.45）%, （1.44±0.52）%, and（1.51±0.55）%.Conclusion For acquiring aecurate resultsof CD4^＋ CD25^＋ Treg ceils, the peripheral blood samples should not be placed longer than 3 hours at room temperature before detection. The age of patients should be taken into consideration when we do some researches on these cells involved in immunity diseases.