A field method for cryopreservation of whole blood from a finger prick for later analysis with flow cytometry.

Abstract

OBJECTIVES\nFlow cytometry is a powerful tool for investigating immune function, allowing for the quantification of leukocytes by subtype. Yet it has not been used extensively for field work due to perishable reagents and the need for immediate analysis of samples. To make flow cytometry more accessible, we devise and evaluate a field protocol for freezing capillary blood.\n\n\nMATERIALS AND METHODS\nWe collected finger prick blood samples from 110 volunteers, age 18 to 42. Blood samples were analyzed immediately for 18 cell surface markers. Aliquots of whole blood were frozen in the vapor phase of a liquid nitrogen tank with 10% dimethyl sulfoxide in medium. Samples were analyzed on a Guava EasyCyte HT flow cytometer after 2, 4, or 14\u2009weeks.\n\n\nRESULTS\nMajor lymphocyte fractions in frozen samples were correlated with fresh values (T-cells: r = 0.82; Natural Killer [NK] cells: r = 0.64; CD4: r = 0.67; CD8: r = 0.82; Naïve CD4: r = 0.73, Naïve CD8: r = 0.71; B-cells: r = 0.73; all p\u2009<\u20090.001), and mean values were similar to those from fresh samples. However, correlations for smaller subsets of CD4 and B cells were generally poor. Some differences resulted from changes in non-specific binding for some antibody-conjugate pairs. Cryopreservation also resulted in a reduction in granulocytes more than lymphocytes.\n\n\nDISCUSSION\nOur results suggest that antibody/fluorochrome combinations should be validated before use on frozen samples, and that functional changes in cells may affect some cell markers. However, this simple freezing protocol utilizing finger pricks, whole blood, and a liquid nitrogen shipping tank is viable for obtaining samples for flow cytometry under field conditions.