Interlaboratory evaluation of endotoxin analyses in agricultural dusts—comparison of LAL assay and mass spectrometry

Abstract

Endotoxin exposure is associated with wheeze and asthma morbidity, while early life exposure may reduce risk of allergy and asthma. Unfortunately, it is difficult to compare endotoxin results from different laboratories and environments. We undertook this study to determine if lipopolysaccharide (LPS) extraction efficiency could account for differences among laboratories. We generated and collected aerosols from chicken and swine barns, and corn processing. We randomly allocated side-by-side filter samples to five laboratories for Limulus assay of endotoxin. Lyophilized aliquots of filter extracts were analyzed for 3-hydroxy fatty acids (3-OHFAs) as a marker of LPS using gas chromatography-mass spectrometry. There were significant differences in endotoxin assay and GC-MS (LPS) results between laboratories for all dust types (p < 0.01). Patterns of differences between labs varied by dust type. Relationships between assay and GC/MS results also depended on dust type. The percentages of individual 3-OHFA chain lengths varied across labs (p < 0.0001) suggesting that each lab recovered a different fraction of the LPS available. The presence of large amounts of particle associated LPS and absence of a freezing thawing cycle were associated with lower correlations between LPS and bioactivity, consistent with an absence of Limulus response to cell-bound endotoxin. These data suggest that extraction methods affect endotoxin measurements. The LAL methods may be most suitable when comparing exposures within similar environments; GC-MS offers additional information helpful in optimizing sample treatment and extraction. GC-MS may be of use when comparing across heterogeneous environments and should be considered for inclusion in future studies of human health outcomes.

Effect of Extraction and Assay Media on Analysis of Airborne Endotoxin

“…Differences between laboratories in measured endotoxin concentrations of parallel samples have been reported (20,21), which poses a serious problem when an exposure limit has to be estimated or compliance with an exposure limit is required. It has been suggested that the harmonization of protocols can lead to more-comparable results (5,10).…”

Optimization of Airborne Endotoxin Exposure Assessment: Effects of Filter Type, Transport Conditions, Extraction Solutions, and Storage of Samples and Extracts

“…Several studies showed differences between laboratories when endotoxin samples were analyzed (3,17,28). One of these showed that the generally high variations between laboratories were reduced by using a common extraction protocol and endotoxin assay kit, although differences remained (3).…”

“…In most of the studies only one type of dust was investigated. Recent studies showed that variability between labs also depended on the source of dust that was analyzed (28,29), Thus, the environment sampled needs to be taken into account when effects of different procedures are investigated.…”

Optimization and Influence Mechanism of Sampling and Analysis of Airborne Endotoxin Based on Limulus Amebocyte Lysate Assay

“…Theoretically, three main endotoxin forms (LPS) were presented in the air: LPSs existed on the membrane of bacteria cells, LPSs associated with other biological or non-biological aerosol particles, and free LPS molecules (Duquenne et al, 2013). LPS molecules embedded in the cell membrane were not toxic and could not activate the enzymes that triggered the LAL reaction (Reynolds et al, 2005;Spaan et al, 2008). Ultrasonication not only broke cells to release LPS, but also separated LPS and fine particles.…”