Effects of blood sample storage time, temperature, anti-coagulants and blood stabiliser on lymphocyte phenotyping

Medical diagnostic laboratories have come under further scrutiny to ensure quality standards of their service and external quality assurance (EQA) programs involving multiple laboratories have been used to gauge this quality based on a consensus. However, because of the geographical distances within a country or internationally, cell surface marker expressions may change due to time delays and transport temperatures. Attention was given to this issue some decades ago and hence requires a re-evaluation in consideration of updated methods, reagents and instruments for flow cytometry and phenotyping. We have undertaken an extensive study to examine the effects of various conditions on blood storage akin to that experienced by patient samples as well as EQA programs, examining expression of lymphocyte surface markers, CD3, CD4, CD8, CD2, CD19, CD20, CD16/56 and HLA-DR. Assessment of lithium-heparin anticoagulated whole blood showed an increase in percentage of CD3⁺ and CD8⁺ T cells and a decrease in CD16/56⁺ NK cells after storage at room temperature (RT) for 24 and/or 48 h. In comparison, storage at 4°C led to a decrease in percentage of CD4⁺ and increase in percentage of CD8⁺ cells. The low temperature also caused an increase in percentage of B cells (CD19⁺, CD20⁺). While storage at RT did not alter levels of HLA-DR⁺ CD3⁺ T cells, there was a significant increase in percentage of these cells after 48 h. Changes were also seen at both temperatures when EDTA was used as an anti-coagulant. Assessment of blood treated with a stabiliser, normally used in the EQA samples (Streck Cell Preservative), reduced the range of lymphocyte subsets affected, with only CD2⁺ and CD20⁺ cells being significantly different at both temperatures, We conclude that 24-48 h storage/transport can affect the percentage of CD3⁺, CD4⁺ T cells, CD8⁺ T cells, B cells, NK cells and HLADR⁺ T cells which can be minimised by using the blood stabiliser as per EQA programs and we emphasise the need to adopt this in the processing of patients' blood samples.