Optimization of airborne endotoxin exposure assessment: effects of filter type, transport conditions, extraction solutions, and storage of samples and extracts

Abstract

Endotoxin exposure occurs in homes and occupational environments and is known to cause adverse health effects. In order to compare results from different studies and establish standards, airborne endotoxin exposures should be assessed using standardized methods. Although the European Committee for Standardization (CEN) developed guidelines for endotoxin exposure assessment, these leave room for individual interpretation. The influence of methods of sampling, extraction, and analysis has never been investigated in a full experimental design. Thus, we sought to fully elucidate the importance of all facets of endotoxin assessment. Inhalable dust samples collected simultaneously were used to investigate the effects on and interactions with airborne endotoxin concentration in two working environments of filter type (glass fiber or Teflon), transport conditions (with/without desiccant), sample storage (-20 or 4 degrees C), extraction solution (pyrogen-free water [PFW] or PFW plus 0.05% Tween 20), extract storage (-20 or 4 degrees C), and assay solution (PFW or PFW plus 0.05% Tween 20). Four hundred samples were collected and randomly distributed over the 20 combinations of treatments. There were no differences found for transport conditions and storage temperature of extracts. Also, no interactions between study variables existed. Sampling on glass-fiber filters, storage of samples in the freezer, and extraction in PFW plus 0.05% Tween 20 resulted in 1.3-, 1.1-, and 2.1-fold-higher estimated endotoxin concentrations, respectively. Use of PFW plus 0.05% Tween 20 in the assay solution had an additive effect. Thus, this study investigated gaps in the CEN protocol and provides data with which to fully specify a protocol for standardization of endotoxin exposure assessment.

FIG. 1.

Schematic overview of the design of the experiment. Asterisks mark places from which the scheme follows the same route as is written out from the stage with the corresponding letter besides the design step. The number sign indicates that the influence of assay solution was investigated in part of the data (136 out of 386 samples). Numbers in parentheses indicate the number of samples that undergo that particular step of the scheme. Tw, Tween 20.

FIG. 2.

Pictures of the parallel sampler, which contains 10 sampling heads positioned in an annular chamber between the inner and outer cone. (a) Parallel sampler without outer cone; (b) parallel sampler with outer cone; (c) placement of sampling heads in parallel sampler; (d) parallel sampler with vacuum monometer and tube for connection with pump, but without outer cone attached.

FIG. 3.

The factor of influence and 95% confidence interval for the effect on endotoxin exposure levels in EU/m³ per combination of variables changed compared to the CEN protocol as a reference. T, Teflon filter; G, glass-fiber filter; nD, no desiccant during transport; D, desiccant during transport; R, refrigerator; F, freezer; WT, PFW with 0.05% Tween 20; W, PFW alone.