A clinical research consortium sponsored by NIAID
Bourcier K, Seyfert-Margolis V, Grant J, Liu Z, Wallace S, Wallace P
Immune Tolerance Network, University of California San Francisco, CA/Bethesda, MD Department of Flow Cytometry, Roswell Park Cancer Institute Buffalo, NY
Background: Recent innovations in flow cytometry methodologies have allowed clinical researchers to elucidate highly specific immune cell populations. The wide application of flow cytometry methods for definition of cell populations in various diseases and during treatment has resulted in ongoing efforts to standardize assay parameters. In this study we addressed how variation in blood tube collection and sample handling (blood shipping) can affect detection of various cell populations.
Methods: Two types of blood collection tubes were evaluated - tubes containing sodium heparin or K3EDTA/BCT as anticoagulants. Since in many clinical trials blood can be shipped to a central facility for standardized evaluations we also addressed the effect of shipping blood immediately after collection and processing within 24hr after collection. For determination of immune cell populations we used eight 5-color antibody panels to assess various populations of B cells, dendritic cells, T cells, NKT cells, and monocytes as well as activation markers such as CD62L, CD25, CD122, CD80, CD86.
Results: Blood collected in sodium heparin and K3EDTA/BCT tubes and shipped ambient had comparable expression of several B cell (memory, plasma, IgD+, IgM+) and T cell (CD8+, CD4+, naïve, memory, CD25+) populations. DC and NKT cell markers were unchanged regardless of tube type or shipment. CD62L expression decreased in K3EDTA/BCT tubes regardless of shipment (45%, sodium heparin; 5.5%, K3EDTA/BCT) (% = percent of gate). Lower expression of CD62L affected resolution of Tregs in K3EDTA/BCT tubes determined as CD62L+CD25high. CD69 expression was also lower in K3EDTA/BCT tubes (12%, sodium heparin; 1.5%, K3EDTA) and also decreased in the shipped sodium heparin tubes (~2%).
Conclusions: These preliminary results show the importance of standardizing blood tube collection protocols as well as post-collection handling as it might greatly affect expression of cell markers detected by flow cytometry and influence data interpretation.
