LYMPHOCYTE ANTIGENS: STABILITY OF EXPRESSION AMONG HEALTHY DOGS AND UPON STORAGE

Background: Reliable detection of fluorescence intensity (FI) by flow cytometry is fundamental. FI is dependent on instrument settings, and measurement should be done using controls with known FI, preferably microbeads or cell subsets with stable mean FI (MFI) within the population. Data about the stability of antigen expression in healthy dogs do not exist, both among different subjects and after sample storage. Objective: The aim was to evaluate MFI stability of the main lymphocyte antigens among different subjects and after storage (fresh vs 24h). The final aim was to detect antigens to be used as internal controls in flow analyses of FI. Methods: Fresh (n=14) and stored (n=20) EDTA peripheral blood samples from healthy dogs were submitted for flow MFI analysis of CD3, CD5, CD4, CD8, CD21, and cyCD79b using conjugated MAbs. Adding FlowCheck microbeads (Coulter) MFI was calculated as MFIratio (CD/beads). Stability among subjects was assessed as CV of MFIratio (fresh samples). For each CD, CV of MFIratios and mean CV of fluorescence CVs were considered. Comparison between MFIratio and CV of fresh vs stored samples was carried out (t-test). Results: Less variation between subjects was reported for CD79b, CD4 and CD21hi. Minor variation between lymphocytes of single subjects was reported for CD21hi and CD4. No CDs reported significant MFI differences between fresh and stored samples; a significant difference in CV was detected for CD5 and CD21. Conclusion: Considering that CD79 needs permeabilization and that a consistent CD21hi population is not constantly recognizable, CD4 is the best internal control for CD MFI evaluation.