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OBJECTIVE To validate a flow-cytometry-based method for the measurement of lymphocyte subgroups in cynomolgus monkeys and Sprague Dawley rats,respectively.METHODS 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 variability.Besides,intra-animal precision was investigated in monkeys.RESULTS 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 d.Samples after fixation were stable while stored in the refrigerator(+ 4℃) for 24 h.For 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 h.However,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 d.In 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 Sprague Dawley rats.
OBJECTIVE To validate a flow-cytometry-based method for the measurement of lymphocyte subgroups in cynomolgus monkeys and Sprague Dawley rats, respectively. METHODS 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 cells (CD3 + CD4 +) and T cytotoxic cells (CD3 + CD8 + For Sprague Dawley rats, other two panels of fluorescent antibodies, respectively 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 variability. Besides, intra-animal precision was investigated in monkeys .RESULTS 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 ° C for 1 d.Samples after fixation were stable while stored in the refrigerator (+ 4 ° C) for 24 h. For 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% Blood samples were stable for CD3 + / CD4 + and CD3 + / CD8 + while stored in the refrigerator (+ 4 ° C) for 24 h.However, rat blood samples were unstable for CD3 / 161a when kept at room temperature for 6h and unstable for CD3 / 45RA when kept at 4 ° C for 1 d. 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 cynom olgus monkeys and Sprague Dawley rats.