食蟹猴外周血淋巴细胞亚群的流式法分析验证和正常参考值测定

Method validation and determination of normal reference values of peripheral blood lymphocytes phenotyping measure-ment in cynomoigus monkeys using flow cytometry

目的：建立并验证流式细胞法检测食蟹猴外周血淋巴细胞亚群（辅助性 T 细胞、细胞毒性 T 细胞、B细胞、NK 细胞）的方法，建立正常参考值范围，比较性别和年龄差异。方法 EDTA‐K2抗凝管采集食蟹猴血样，采用两组三色荧光抗体组合 CD3‐FITC／CD4‐PerCP／CD8‐PE 、CD3‐PE‐CYTM 5．5／CD16‐PE／CD20‐FITC 进行细胞分类检测。测定批内差异和批间差异以考察方法精密度；并分别对血样室温放置6 h ，4℃放置24 h ，检测处理后样本4℃放置24h 的样品稳定性进行考察。进一步扩大检测样本数量，确立不同性别、年龄段的正常参考值范围，并比较性别和年龄差异。结果精密度检测各指标批内变异度为2．51％～12．51％，批间变异度为1．32％～8．63％。血样经不同处理和放置不同时间后的测定结果与新鲜采集血样后的检测结果偏差均小于10％。确立了1．5～2岁和3～5岁两个年龄段，雌雄食蟹猴淋巴细胞亚群的正常参考值。比较发现，3～5岁与1．5～2岁相比，外周血 NK 细胞（CD3－ CD16＋）亚群比例升高；B 淋巴细胞（CD3－ CD20＋）亚群有年龄和性别差异。结论食蟹猴外周血淋巴细胞亚群检测的血液样本在室温放置6 h ，4℃放置24 h 内检测，或处理后4℃放置24 h 内检测，结果稳定性良好。不同年龄段和性别食蟹猴的淋巴细胞亚群正常值范围具有差异，提示选择食蟹猴进行相关研究评价时应予以注意。

Objective To establish and validate the method of peripheral blood lymphocytes phenotyping test in cynomolgus monkeys （including helper T lymphocyte ,cytotoxic T lymphocyte ,B lymphocyte and NK cell） using flow cytometry and determine reference value of the general peripheral blood lymphocytes phenotyping and compare the differences between ages and sexes .Methods Collect blood samples in EDTA‐K2 anticoagulant tube from cyno‐molgus monkeys and select two groups of fluorescent antibodies CD3‐FITC/CD4‐PerCP/CD8‐PE and CD3‐PE‐CYTM 5 .5/CD16‐PE/CD20‐FITC for cell sorting test to validate the precision （intra‐assay and inter‐assay） and sample sta‐bility （blood samples at room temperature for 6 h ;4 ℃ ,24 h ;stained samples 4 ℃ ,24 h） and expand the number of samples ,detection of different gender ,age normal reference range segment .Results Parameters CV were 2 .51% -12 .51% for intra‐assay and 1 .32% - 8 .63% for inter‐assay .Results deviations for blood samples kept at different conditions and time lengths were less than 10% .Normal reference value of 1 .5 - 2 and 3 - 5 years old cynomolgus monkeys were determined .Compared with the values of 1 .5 - 2 years old cynomolgus monkeys ,CD3 - CD16 ＋ NK cell ratio increased in 3 - 5 year old animals ,age and gender differences were found in CD3 - CD20 ＋ B cell .Conclusion Measuring results of Cynomolgus peripheral blood samples kept at room temperature for 6h and 24 h （4 ℃ ） and the stained samples within 24 h （4 ℃ ） are stable .Sex and age differences are found of some lymphocytes subgroups in cynomolgus monkeys and this call for attention when cynomolgus monkeys are chosen for studies .