淋巴细胞免疫表型质控品的初步研究

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PBMCs CD4^＋T和CD8^＋T百分比均保持在稳定水平。冻存12个月PBMCs CD4^＋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。

Preliminary Study on a Potential Panel Used for Quality Control of Lymphocyte Immonophenotypes

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 ficollgradient 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 cryopreserved 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.