The effect of storage on immunophenotyping of sheep peripheral blood lymphocytes by flow cytometry

We have shown that when a whole blood method of cell staining is used for flow cytometric analysis of sheep lymphocytes (i.e. red cell lysis after addition of antibody), staining may be delayed for up to 48 h after blood collection without significant effect on expression of CD4, CD5, CD8 or B cell markers. If cells were stained immediately after blood collection, using the same whole blood method, and then fixed with paraformaldehyde, cell samples could be stored for 24 h without change in marker expression. However, by 7 days there was a significant decrease in the percentage of cells expressing CD8, T19 and B cell markers. Cryopreservation prior to staining was found to markedly affect the expression of all cell surface markers investigated. These results indicate that storage of sheep blood prior to flow cytometric analysis is feasible but may affect the results obtained. Thus is it important to standardise the handling of samples, especially when comparative studies are being undertaken.