A new approach to deposit homogeneous samples of asbestos fibres for toxicological tests in vitro

Our plain-English summary of A new approach to deposit homogeneous samples of asbestos fibres for toxicological tests in vitro, by G. Della Ventura, A. Rabiee, A. Marcelli, et al (Frontiers in Chemistry, 2023).

The study

• Authors: G. Della Ventura, A. Rabiee, A. Marcelli, S. Macis, A. D'Arco, G. Iezzi, F. Radica, F. Lucci
• Journal: Frontiers in Chemistry
• Published: 2023;11:1116463
• Cited by: 2 articles, as counted by NIH iCite
• Read it: PubMed · Publisher · Free full text

Abstract

In this paper we describe the results obtained with a novel method to prepare depositions of asbestos fibres for toxicological tests in vitro. The technique is based on a micro-dispenser, working as an inkjet printer, able to deposit micro-sized droplets from a suspension of fibres in a liquid medium; we used here a highly evaporating liquid (ethanol) to reduce the experimental time, however other solvents could be used. Both the amount and spatial distribution of fibres on the substrate can be controlled by adjusting the parameters of the micro-dispenser such as deposition area, deposition time, uniformity and volume of the deposited liquid. Statistical analysis of images obtained by optical and scanning electron microscopy shows that this technique produces an extremely homogeneous distribution of fibers. Specifically, the number of deposited single fibres is maximized (up to 20 times), a feature that is essential when performing viability tests where agglomerated or untangled fibrous particles need to be avoided.

Copyright © 2023 Della Ventura, Rabiee, Marcelli, Macis, D’Arco, Iezzi, Radica and Lucci.

In plain English

This laboratory methods study compared two ways of spreading fibres onto silicon slides to prepare for cell-toxicity tests. Despite the title, the material was not regulated asbestos. It was a synthetic amphibole, Ni-richterite, which the authors called asbestiform because its needle-like crystals have the shape of asbestos amphiboles. The same suspension (5 milligrams of powder in 50 ml of ethanol) was deposited either with a conventional pipette or with a micro-dispenser that works like an inkjet printer. No cells were tested. In optical images, the pipette method gave a mean of 24 objects per image and the micro-dispenser 212, with similar surface coverage (4.4% versus 5.0%). Counting by hand under NIOSH Method 7400 rules found 92 versus 1557 fibres per square millimetre in optical images, and 65 versus 1916 in electron-microscope images, for the pipette and micro-dispenser respectively. The authors concluded that the micro-dispenser gave a much more even deposit with far more single fibres. They noted that ethanol is unsuitable for real cell experiments and that counting by hand may include operator error.

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 study comparing two ways of depositing fibres on a substrate (conventional pipette 'drop' method versus a 'micro-drop' dispenser), assessed by image analysis; no cells or animals were tested
• Who was studied: Fibre deposits on silicon substrates made from one ethanol suspension (5 mg powder in 50 ml ethanol). Optical microscope images: 52 for the drop method and 14 for the micro-drop method. Electron microscope (SEM) images: 26 for the drop method and 2 for the micro-drop method. Laboratories in Italy.
• Exposure: A synthetic amphibole powder, Ni-richterite, with needle-like crystals 5 to 15 µm long and less than 1 µm wide. It does not occur in nature and is not on the regulated asbestos list; the authors describe it as asbestiform, not asbestos.
• Main results:
  • In the representative optical images, the drop method gave an average surface coverage of 4.4% (range 1.1 to 10.4%) and a mean of 24 objects per image (5 to 37). The micro-drop method gave an average coverage of 5.0% (1.9 to 6.8%) and a mean of 212 objects per image (80 to 352).
  • Objects with a length-to-width ratio above 3 (fibrous particles) were about 15% of objects counted with the drop method (169 of 1144) and 31% with the micro-drop method (930 of 2964).
  • Small objects (area under 2 square micrometres) numbered 77 per square millimetre with the drop method and 1559 with the micro-drop method (about 20 times lower with the drop method). These were 25% and 52% of all counts, respectively.
  • With the drop method, about 60% of objects had a low length-to-width ratio (below 2.2). The authors interpreted this as heavily entangled, stubby objects.
  • Counting by hand under NIOSH rules in optical images found 92 fibres per square millimetre for the drop method and 1557 for the micro-drop method (about 18 times higher).
  • Counting by hand in electron microscope (SEM) images found 65 fibres per square millimetre for the drop method and 1916 for the micro-drop method (about 29 times higher).
  • In the micro-drop deposit seen by SEM, fibres 5 to 8 µm and 8 to 11 µm long were almost equal in number and together made up 70% of the fibres counted.
• The authors' conclusion: The authors concluded that, starting from the same suspension, the micro-drop technique gave a much more homogeneous distribution of particles and far more single fibres than the micropipette technique.
• Limitations the authors state:
  • Ethanol was used because it evaporates quickly, but it is not suitable for real experiments with cells because it may itself be toxic to cells.
  • Very small particles (under 2 µm long) were grouped into one class because they could not be reliably told apart from the background.
  • The image-analysis category of objects with a length-to-width ratio above 3 also includes objects that do not count as asbestos under the AIA rules.
  • Counting by hand may be affected by operator error, because the counter has to leave out entangled particles.
  • For the uneven drop deposit, the SEM count was likely underestimated because SEM covered a smaller area.
  • Automated image analysis is prone to error, especially with optical images because of their relatively low resolution.

Topics

Medical terms in this study, explained in our Lexicon: Deposition, Scanning Electron Microscopy (SEM).

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