Method validation of lymphocyte subgroup phenotyping analysis in cynomolgus monkeys and Sprague-Dawley rats using flow cytometry
作者单位：Center for Drug Safety Evaluation and ResearchShanghai Institute of Materia MedicaChinese Academy of Sciences
会议日期：2013年
学科分类：10[医学]
摘 要：OBJECTIVE To validate a flow-cytometry-based method for the measurement of lymphocyte subgroups in cynomolgus monkeys and Sprague Dawley rats,*** For cynomolgus monkeys,two panels of fluorescent antibodies,namely CD3-FITC/CD4-PerCP/CD8-PE and CD3-PE-Cy5.5/CD16-PE/CD20-FITC were used to analyze the proprotion of T cells including T helper cell(CD3~+CD4~+) and T cytotoxic cells(CD3~+CD8~+),NK cells(CD3~- CD16~+) and B cells(CD3~- CD20~+).For Sprague Dawley rats,other two panels of fluorescent antibodies,namely CD3~- FITC/CD4-PE/CD8a-PerCP and CD3-FITC/CD45RA-PE-Cy5.5/CD161a-PE were used to analyze the proprotion of T cells,NK cells(CD3~-CD161~+) and B cells(CD3~-CD45RA~+).Blood samples from 10 monkeys and 10 rats were analyzed for intra-assay precision,sample stability and inter-subject ***,intra-animal precision was investigated in *** For cynomolgus monkeys,intra-assay CV and intra-animal CV for all parameters were within 15%.The positive binding signals of all isotype controls were less than 1%.Furthermore,monkey blood samples were stable for all parameters when kept at room temperature for 6 h or at 4℃for 1 *** after fixation were stable while stored in the refrigerator(+4℃) for 24 *** rats,intra-assay CVs for all parameters were within 15%.The positive binding signals of isotype controls of CD3~+/CD4~+ and CD3~+/CD8~+ were less than 1%.Furthermore,rat blood samples were stable for CD3~+/CD4~+ and CD3~+/CD8~+ while stored in the refrigerator(+4℃) for 24 ***,rat blood samples were unstable for CD3/161a when kept at room temperature for 6h and unstable for CD3/45RA when kept at 4℃for 1 *** latter two cases,the positive binding signals of the isotype controls were around 2%.CONCLUSION All the parameters tested fulfilled the criteria of acceptance with a few minor exceptions related to stability data from rat blood samples. It is suitable for the measurement of lymphocyte subgroups in cynomolgus monkeys and S