【摘要】 目的研究液氮冻存对外周血单个核细胞(PBMCs)CD4+T和CD8+T百分比的影响,探索冻存PBMCs作为淋巴细胞免疫表型质控品的可行性。方法收集健康成人抗凝全血,用淋巴细胞分离液分离PBMCs后液氮冻存。定期复苏PBMCs检测CD4+T和CD8+T百分比,计算细胞存活率、回收率。并分析不同厂家试剂、复苏方法、固定时间等因素对CD4+T和CD8+T百分比的影响。结果液氮冻存0～12个月、12～24个月的PBMCsCD4+T和CD8+T百分比均保持在稳定水平。冻存12个月PBMCsCD4+T和CD8+T百分比的平均变异系数(CV)分别为14.5%和9.8%,细胞存活率为85.4%～94.3%,回收率33.3%～97.8%;冻存12～24个月PBMCs的CD4+T和CD8+T百分比的平均CV值分别为7.1%和6.5%,细胞存活率为88.3%～98.5%,回收率52.3%～101%。不同复苏方法和检测试剂的结果相对稳定,CD4+T和CD8+T百分比的平均CV值分别为8.1%和6.4%。复苏后的PBMCs经多聚甲醛固定可在48h内检测。结论液氮冻存的PBMCs有望作为淋巴细胞免疫表型测定的质控品。更多还原

【Abstract】 Objective To study the effect of liquid nitrogen cryopreservation on percentages of CD4+T and CD8+ T lymphocyte,and to explore the feasibility of cryopreserved PBMCs as a quality control panel for lymphocyte immonophenotypes.Methods Whole blood specimens from healthy adults were collected and PBMCs were isolated using standard ficoll-gradient centrifugation.After stored in liquid nitrogen,the PBMC samples were thawed at different intervals and then were assessed for percentages of CD4+ T and CD8+T lymphocyte,viability and recovery.Besides,the effects of thawing methods,lymphocyte immonophenotypes reagents,fixed time on percentages of CD4+ T and CD8+ T lymphocyte were evaluated.Results The percentage of CD4+ T and CD8+ T lymphocyte of PBMCs cryopreserved in liquid nitrogen from 0 to 12 monthes and from 12 to 24 monthes remained stable.The average CV of CD4+T and CD8+T percentages of PBMC cryopre-served at different intervals between 0 to 12 monthes were 14.5% and 9.8% respectively,with viabilities of 85.4%～ 94.3% and recoveries of 33.3%～97.8%.The average CV of CD4+T and CD8+T percentages of PBMCs cryopreserved at different intervals between 12 to 24 monthes were 7.1% and 6.5% respectively,with viabilities of 88.3%~98.5% and recoveries of 52.3%～101%.The results of using different thawing methods and lymphocyte immonophenotypes reagents remained stable.The average CV of CD4+T and CD8+T percentages were 8.1% and 6.4%.The thawed PBMCs could be detected correctly within 48 hours after fixed in polyformaldehyde.Conclusion Cryopreserved PBMCs could be used as a potential panel for quality control of lymphocyte immonophenotypes.更多还原