Differential Counting of Asbestos Using Phase Contrast and Fluorescence Microscopy

Our plain-English summary of Differential Counting of Asbestos Using Phase Contrast and Fluorescence Microscopy, by T. Nishimura, M. Alexandrov, T. Ishida, et al (The Annals of Occupational Hygiene, 2016).

The study

• Authors: T. Nishimura, M. Alexandrov, T. Ishida, R. Hirota, T. Ikeda, K. Sekiguchi, A. Kuroda
• Journal: The Annals of Occupational Hygiene
• Published: 2016;60(9):1104-1115
• Cited by: 3 articles, as counted by NIH iCite
• Read it: PubMed · Publisher

Abstract

Considering the increasing use of various asbestos substitutes, asbestos risk management in many industries may require accurate techniques for detecting and distinguishing asbestos from non-asbestos fibers. Using fluorescently labeled asbestos-binding proteins, we previously developed a novel method for detection and counting of asbestos fibers under fluorescence microscopy (FM). This method can provide speedy, on-site detection and identification of the asbestos fibers and has higher sensitivity than phase contrast microscopy (PCM). However, current asbestos exposure limits are derived from risk assessments based on epidemiological studies that were conducted using PCM fiber counts. Therefore, the sensitivity of asbestos testing should be maintained at PCM level to properly assess compliance with these limit values. Here, we developed and tested a novel application of FM as a differential counting method that complements PCM analysis and is fully compatible with the PCM-based epidemiological data. In the combined PCM-FM method, the fluorescent asbestos-binding probe is applied prior to filter clearing. The method makes it possible to easily switch between two microscopic techniques while analyzing the same fields of view: PCM is used for counting fibers, and FM for differentiating asbestos from non-asbestos fibers. Using airborne dust samples from demolition sites in Japan, we compared PCM-FM with scanning electron microscopy (SEM)-based differential counting method. Statistical analysis indicated a slight conservative bias of PCM-FM method, combined with relatively high variability across the full range of fiber concentrations in our sample set. Using correlative microscopy, we also evaluated the specificity of FM staining, which is a potential cause of variability between the two methods. The energy-dispersive X-ray analysis indicated that ~95% of fluorescently stained fibers in the demolition site samples were correctly identified as asbestos. While further research is needed to fully clarify the causes of variability between FM- and SEM-based differential counting, PCM-FM could be used for rapid and selective detection of asbestos fibers in field samples.

© The Author 2016. Published by Oxford University Press on behalf of the British Occupational Hygiene Society.

In plain English

This laboratory study tested a way of counting asbestos fibers in air samples that combines phase contrast microscopy (PCM) with fluorescence microscopy (FM). Fibers were counted under PCM. The same view was then checked for a fluorescent asbestos-binding stain, and only stained fibers were counted as asbestos. The authors compared this PCM-FM method with a scanning electron microscopy (SEM)-based method on 61 air samples from demolition sites across Japan. On average, PCM-FM gave asbestos concentrations 11.1% higher than PCM counts corrected using the asbestos fraction measured by SEM, but agreement varied widely (limits of agreement minus 43.5% to plus 65.7%). In 34 other demolition-site samples, 322 of 340 stained fibers (about 95%) were confirmed as asbestos. The other 18 included mineral wool, organic and ceramic fibers. In artificial samples made from insulation containing amosite, crocidolite or chrysotile, 17 of 701 fibers (2.4%) were not stained. The authors concluded the method may not be fully equivalent to electron microscopy-based counting. They noted that the two methods examine somewhat different fibers, and that missed asbestos fibers could not be measured directly in field samples.

We wrote this plain-English summary from the full article with the help of AI, and a second AI model checked each sentence against the article. It is not the authors' wording; the abstract and the article are their own account.

What the study did and found

• Design: Laboratory methods-comparison study of an asbestos fiber counting technique (combined PCM-FM versus SEM-based differential counting), plus a check of stain specificity using correlative fluorescence and electron microscopy; no health outcomes were studied
• Who was studied: Airborne dust samples from demolition sites across Japan: 61 samples for the method comparison, and a different set of 34 samples for the staining-specificity check. Artificial samples were also prepared from three types of sprayed insulation. Collection dates are not given.
• Exposure: Airborne asbestos and non-asbestos fibers on sampling filters. Non-asbestos fibers found included mineral wool or rock wool, organic fibers and ceramic fibers. The artificial samples came from sprayed insulation containing 12.7% amosite, 49.3% crocidolite or 10.6% chrysotile.
• Main results:
  • In the 61 demolition-site samples, PCM-FM asbestos fiber concentrations were on average 11.1% higher than PCM counts corrected using the SEM asbestos fraction (PCM-SEM). The authors described this as a slight conservative bias.
  • The standard deviation of the relative differences between PCM-FM and PCM-SEM concentrations was 0.279 (27.9%), with limits of agreement of minus 43.5% to plus 65.7%. The variability between the methods was higher than the authors expected.
  • Comparing asbestos fiber ratios rather than concentrations, the mean ratio by PCM-FM was 0.05 higher than by SEM. The standard deviation was 0.17, and the limits of agreement were minus 0.28 to plus 0.38.
  • In 34 demolition-site samples (10 fibers per sample), chemical analysis by energy-dispersive X-ray (EDX) found that 322 of 340 fluorescently stained fibers (about 95%) were asbestos.
  • The 18 stained fibers that were not one of the 6 regulated kinds of asbestos were: 7 man-made mineral wool or rock wool, 1 probable man-made mineral fiber, 6 organic fibers, 2 ceramic fibers, 1 silicon-only fiber, and 1 unidentified fiber.
  • In artificial samples containing only asbestos fibers, 17 of 701 fibers (2.4%) were not stained. The unstained share was 3.4% for amosite, 2.1% for crocidolite and 1% for chrysotile.
• The authors' conclusion: The authors concluded that PCM-FM may not be fully equivalent to electron microscopy-based differential counting. They judged that the variability is only partly due to false positives and false negatives, and probably stems largely from differences in how fibers are seen under optical and electron microscopy. They also stated that the method could replace or supplement existing methods at demolition sites and in industries using fibrous materials.
• Limitations the authors state:
  • The false-negative rate (asbestos fibers left unstained) could not be measured directly in the field samples. It was estimated only from artificial samples.
  • SEM and PCM examine somewhat different subpopulations of fibers, because fibers differ in how visible they are under each method. This affects the comparison between the two methods.

Topics

The National Library of Medicine indexes this study under subjects our site covers: Chrysotile.

Medical terms in this study, explained in our Lexicon: phase contrast microscopy (PCM), Asbestos Exposure, Scanning Electron Microscopy (SEM).

Related studies

• Evaluation of the dark-medium objective lens in counting asbestos fibers by phase-contrast microscopy — E.G. Lee, J.H. Nelson, et al. (2015)
• Development of an automated asbestos counting software based on fluorescence microscopy — M. Alexandrov, E. Ichida, et al. (2015)
• Evaluation of sensitivity of fluorescence-based asbestos detection by correlative microscopy — T. Ishida, M. Alexandrov, et al. (2012)
• An inter-laboratory study to determine the effectiveness of procedures for discriminating amphibole asbestos fibers from amphibole cleavage fragments in fiber counting by phase-contrast microscopy — M. Harper, E.G. Lee, et al. (2012)
• Assessment of occupational exposure to asbestos fibers: Contribution of analytical transmission electron microscopy analysis and comparison with phase-contrast microscopy — C. Eypert-Blaison, A. Romero-Hariot, et al. (2018)
• Rapid on-site detection of airborne asbestos fibers and potentially hazardous nanomaterials using fluorescence microscopy-based biosensing — A. Kuroda, M. Alexandrov, et al. (2016)

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