Investigation of interleukin-1β release from cryopreserved blood stimulated with endotoxin

Abstract

The feasibility of an indigenously developed ELISA method to determine cytokine response to wide spectrum of pyrogenic stimuli utilizing fresh human whole blood is limited by the availability of healthy donors. The possibility of using cryopreservation of pooled human blood for detection of cytokine response to lipopolysaccharide is explored in this study. The effect of cryopreservation on blood parameters, cellular morphology and cytokine response were compared with that of the pooled fresh blood and cryopreserved blood from single and multiple donors. In vitro pyrogenic stimulation with 0.5 and 5 EU of LPS was monitored on fresh and cryopreserved pooled blood from single and multiple donors. The release of IL-1β was quantitated by Sandwich ELISA (1, 10, 25, 45 and 75 days) after storage. The results indicated that the cryopreserved blood displayed enhanced IL-1β release on stimulation with LPS, when compared to fresh blood. The maximum release of IL-1β level was observed at 2h when 5 EU of LPS was treated with pooled fresh blood which is similar to that of fresh blood. After 75 days storage of pooled cryopreserved blood the IL-1β release was maximum at 9 and 15 h when treated with 5 and 0.5 EU of LPS. Observations of the study suggest that cryopreserved pooled blood is an economically and experimentally viable alternative to fresh blood. This investigative study promises short term storage and regular supply of non-allergic, pathogen free human blood for the detection of interleukin-1β for the evaluation of in vitro pyrogenicity.