A new method to measure air-borne pyrogens based on human whole blood cytokine response

Abstract

Air-borne microorganisms, as well as their fragments and components, are increasingly recognized to be associated with pulmonary diseases, e.g. organic dust toxic syndrome, humidifier lung, building-related illness, "Monday sickness."We have previously described and validated a new method for the detection of pyrogenic (fever-inducing) microbial contaminations in injectable drugs, based on the inflammatory reaction of human blood to pyrogens. We have now adapted this test to evaluate the total inflammatory capacity of air samples. Air was drawn onto PTFE membrane filters, which were incubated with human whole blood from healthy volunteers inside the collection device. Cytokine release was measured by ELISA.The test detects endotoxins and non-endotoxins, such as fungal spores, Gram-positive bacteria and their lipoteichoic acid moiety and pyrogenic dust particles with high sensitivity, thus reflecting the total inflammatory capacity of a sample. When air from different surroundings such as working environments and animal housing was assayed, the method yielded reproducible data which correlated with other parameters of microbial burden tested. We further developed a standard material for quantification and showed that this assay can be performed with cryopreserved as well as fresh blood.The method offers a test to measure the integral inflammatory capacity of air-borne microbial contaminations relevant to humans. It could thus be employed to assess air quality in different living and work environments.

The human whole blood pyrogen test – lessons learned in twenty years

“…), incriminated samples and > 80 products, direct comparisons to rabbit testing; not disturbed by adjuvants Variants: commercial kit available 1 , medical devices/solid materials (Hasiwa et al, 2007), airborne pyrogens (Kindinger et al, 2005), adsorb/wash-extraction/enrichment (Daneshian et al, 2006(Daneshian et al, , 2008, cell therapies (Montag et al, 2007), one-plate assay (Poole et al, 2003) 1 http://bit.ly/1xB1WmS…”

“…In contrast to the LAL, the whole blood test reflects the potency of different LPS species in the rabbit (Fennrich et al, 1999a). Since a cell suspension is used, blood as a reagent can be brought into contact with any material, including medical devices (Hasiwa et al, 2007;Mazzotti et al, 2007) or filters loaded with specimens from air samples (Kindinger et al, 2005). In contrast, cell lines in the validation study showed drifts and shifts in their responsiveness to pyrogens, letting MonoMac-6 cells fail the prevalidation and the two THP-1 tests fail the validation phase.…”

“…-The possibility to measure on surfaces allows determining adsorbed (filtered) samples without prior dissolution (which is very difficult for lipophilic LPS and LTA) directly on these surfaces. This enables complete measurement of airborne pyrogens (Kindinger et al, 2005), the enrichment of pyrogens for high-sensitivity measurements, e.g., for dialysis solutions (Daneshian et al, 2008), or the isolation of pyrogens from interfering, e.g., cytotoxic, samples (Daneshian et al, 2006). -Inter-individual responses can be tested using different blood donors, which seem to play a role for some non-endotoxin pyrogens.…”

Evidence for the detection of non-endotoxin pyrogens by the whole blood monocyte activation test

“…In contrast to the lAl/Bet, the whole blood MAt reflects the potency of different LPS species in the rabbit (Fennrich et al, 1999a). Since a cell suspension is used, blood as a reagent can be brought in contact with any material, including medical devices (Hasiwa et al, 2007;Mazzotti et al, 2007) or filters loaded with specimens from air samples (Kindinger et al, 2005). In contrast, cell lines in the validation study have shown drifts and shifts in their responsiveness to pyrogens, letting MonoMac-6 cells fail the prevalidation and the two tHP-1 tests fail the validation phase.…”

Development, validation and applications of the monocyte activation test for pyrogens based on human whole blood

“…the higher pyrogen retrieval by impingement when compared to filtration, possibly due to the incomplete eluation of the sample from the filters, was demonstrated by Zucker et al (2000). A new approach of measuring the integral inflammatory activity in air samples in different environments by WBt was reported by Kindinger et al, 2005. A defined amount of air is drawn through a filter in a sealable plastic monitor.…”