Standardization of whole blood assay for determination of pyrogenic activity in organic dust samples

To characterize bioaerosol exposure at workplaces standardized methods are necessary. Activity of endotoxin, one component of organic dust, can be quantified with the Limulus-Amoebocyte Lysat test (LAL test). Further information with respect to pyrogenic activity of the organic dust can be achieved by measuring cytokine release of human blood after stimulation with the dust or its extract (whole blood assay). The aim of our study was the standardization of the whole blood assay (WBA) while using cryo-preserved human blood (Qualis Laboratorium) and to compare the outcome of the different cytokines determined by incubation of the blood cells with extracts from dust samples collected at various workplaces. Cytokine release (IL-1 beta, IL-6, IL-8, TNF-alpha, MCP-1) was measured by ELISA after stimulation of fresh blood from ten donors as well as cryo-preserved human blood. In both cases blood was stimulated with E. coli endotoxin as well as with 30 dust filter extracts from various workplaces. All dust filter extracts were investigated in the WBA using cryo-preserved blood as well as with LAL test. E. coli endotoxin stimulated the release of IL-1 beta, IL-6, IL-8, TNF-alpha and MCP-1 in a dose-dependent manner in fresh as well as cryo-preserved human whole blood. 200 pg/ml E. coli endotoxin induced maximal cytokine release in cryo-preserved blood (mean value for IL-1 beta 2509+/-418 pg/ml; n=13 experiments) whereas fresh blood of single donors reached a maximum release when stimulated with 50 ng/ml endotoxin (mean value of ten donors 1125+/-553 pg/ml IL-1beta). Using cryo-preserved blood the coefficient of variation (CV) regarding the interassay variability was below 21% for all cytokines measured. Regarding 26 dust sample extracts correlation coefficient r2 for LAL test and WBA was between 0.90 and 0.93 (Pearson) for IL-1 beta, IL-6, IL-8 and TNF-alpha whereas correlation for MCP-1 was lower (r(2)=0.59). Two dust sample extracts which showed similar reactivity patterns in LAL test as well as in WBA with respect to IL-1 beta, IL-6, IL-8 and TNF-alpha could be differentiated by measuring MCP-1. In conclusion, cryo-preserved blood pools are suitable to standardize WBA. Combination of different outcome variables like IL-1 beta and MCP-1 improve the characterization from the inflammatory potency of workplace related dust samples.