Temperature Affects Leukocyte Surface Antigen Expression

Factors that affect leukocyte immunophenotyping include sampling and processing temperature, the anticoagulant used, and the storage temperature of the blood sample.

Scientists at the University of Oulu, (Oulu, Finland), investigated the possible effects of anticoagulant, sample collection, storage and processing temperature, and sample storage time, on the expression of several leukocyte surface antigens. They collected blood from five male patients, aged 44-68, with severe sepsis requiring intensive care and five healthy staff members (two males and three females, aged 22-62). In this study, the patients and the healthy volunteers were combined into one study population.

Blood was collected into siliconized vacuum tubes containing acid citrate dextrose (ACD) or sodium heparin as an anticoagulant and stored either at 4 °C or at room temperature (RT). Aliquots of the blood samples were incubated with various antibodies at different temperatures and times and analyzed on a FACSCalibur flow cytometer (BD Bioscience; San Jose, CA, USA). The surface antigens of interest were neutrophilic cluster of differentiation (CD)11b and CD64, monocytic CD11b, CD14, CD40, CD64, CD80 and human leukocyte antigen, HLA-DR, and lymphocytic CD69 (separately in CD4+ and CD8+ T cells, B cells, and natural killer cells).

The fluorescence intensities were higher at RT than at 4 °C. During storage the intensities increased at RT, but at 4 °C there were only minor changes. The effects were similar with both anticoagulants studied. The authors concluded that the study shows in a quantitative way that storing the blood samples at RT may have pronounced effects on the intensities of several leukocyte surface antigens, something which is of interest in leukocyte activation studies, such as patients with sepsis. They recommended that flow cytometric analysis of leukocyte surface antigen expressions should be performed using 4 °C temperature throughout the process and within six hours. The study was published online March 15, 2011, in the Journal of Clinical Laboratory Analysis.

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