Fluorescence flow cytometry methodology to exclude platelet aggregate interference when measuring feline CD4 and CD8 lymphocyte counts

Changes in individual feline lymphocyte subsets over the course of infection, immune-mediated disease, or treatment can be used clinically to monitor disease progression. However, interference by platelet aggregates is a common problem when measuring feline lymphocyte subtype counts using flow cytometry in whole blood specimens. In this study, buffer was used to lyse red blood cells so that lymphocytes could be isolated, and then a gate containing a highly purified population of lymphocytes was characterized and fixed using fluorescence flow cytometry analysis. After tagging platelets with anti-CD61AF647 antibody to reduce aggregate interference, lymphocyte subtypes were measured using simultaneous 3-color channels with fluorescent anti-CD markers. D61AF647 exclusion of platelet aggregates was used, CD4%, CD8%, CD8low% and CD4:CD8 counts increased significantly (all specimens, n = 66, P < 0.001; >20% CD61 in the fixed gate, n = 21, P < 0.01). The methodology showed robust stability and precision over 3 days (n = 10 specimens), yielding average day-to-day coefficients of variation (CVs) of 2.15%, 5.01%, 7.33%, 7.77% and 9.35% for white blood cell (WBC) counts, lymphocyte counts, CD4 lymphocyte counts, CD8 lymphocyte counts and CD4:CD8, respectively.