Asbestos fibres burden in gallbladder: a case study
Alessandro Croce1, Silvana Capella2, Elena Belluso2, Federica Grosso3, Narciso Mariani4, Roberta Libener4, Caterina Rinaudo1
1 Department of Science and Technological Innovation, University of Piemonte Orientale, Alessandria, Italy; 2 Department of Earth Sciences and Interdepartmental Centre for Studies on
Asbestos and Other Toxic Particulates G. Scansetti, University of Torino, Torino, Italy; 3 Mesothelioma Unit – Oncology – SS Antonio e Biagio e Cesare Arrigo, General Hospital, Alessandria, Italy; 4 Pathology Unit – SS Antonio e Biagio e Cesare Arrigo, General Hospital, Alessandria, Italy.
Correspondence: Prof. Caterina Rinaudo, Department of Science and Technological Innovation, University of Piemonte Orientale, Viale Teresa Michel 11, 15121, Alessandria, Italy; e-mail:
caterina.rinaudo@uniupo.it.
Abstract
The methods conventionally used to determine the burden in asbestos fibres inhaled/incorporated in lung require chemical digestion of the biological matrix before counting of the inorganic fibrous phases under Scanning Electron Microscopy equipped with Energy Dispersive Spectroscopy (SEM/EDS).
In this work, we try to quantify the asbestos fibres in extra-pulmonary organs, in particular in gallbladder.
Although the standardized procedure needs almost 5·10-1 g of tissue, this amount of tissue results sometimes not available. We applied the procedure on about 9·10-4 g of gallbladder from a patient affected by Malignant Pleural Mesothelioma (MPM), therefore certainly exposed to asbestos, and with severe troubles in the bile-tract. The traditional procedure of digestion in NaClO and filtering of the obtained suspension was carried out. The filter was examined under SEM/EDS by two ways: 1. following the procedure standardized to assess the fibre burden in lung, therefore investigating only 2 mm2 of the filter (660 microscopic field); 2. analysing all the microscopic field in 1/4 of the filter (82 mm2). In parallel, histological sections, normally prepared for medical diagnosis, without digestion or manipulation of the sample are also studied under Variable Pressure SEM/EDS.
Keywords: asbestos fibres, microscopy, fibre quantification, histological sections, asbestos related
diseases.
Introduction
The relationships between asbestos fibres and severe diseases affecting the respiratory system (mesothelioma, asbestosis, pulmonary carcinoma) is now universally accepted (Westlake, 1965; Auerbach et al., 1980; Szendröi et al., 1983; Ehrlich et al.., 1985; Huang et al., 1988; Williams et al., 2001; Muller et al., 2001). Nevertheless other cancers, affecting larynx and ovary, have been recognized by IARC as related to asbestos (IARC, 2012), whereas the relationships between asbestos and gastrointestinal (GI) cancers are still debated by the scientific community (Constanza Camargo et al., 2011; IARC, 2012; Jacobsen et al., 2013). For this reason, the aim to define the asbestos role in the development of GI cancers is more and more at the basis of scientific works (Brandi et al., 2008, 2013; Boulanger et al., 2015; Di Ciaula & Gennaro, 2016; Di Ciaula, 2017). In our opinion, one way to face the problem is to set out number, type and localization of the asbestos fibres in the different human organs affected by neoplastic growth. During the time, when the presence of asbestos in the human body was under analysis, digestion of the biological matrix on almost 5·10-1 g of wet tissue sample was performed (Churg & Warnock, 1977; Churg, 1982; Karjalainen et al., 1996; Tossavainen, 1997; De Vuyst et al., 1998; Belluso et al., 2006). By this way, after digestion, identification and quantification of the fibrous mineral phases were performed under optical microscope (Roggli et al., 1986; Kobayashi et al., 1987; Pairon et al., 1994; de Ridder, 2016), scanning electron microscopy equipped with energy dispersive spectrometer (Roggli et al., 1986; Belluso et al., 2006; Roggli, 2006; Casali et al., 2015; de Ridder, 2016), or transmission electron microscopy (De Vuyst et al., 1983; Albin et al., 1990; Dodson et al., 1997). Nevertheless, during digestion the relationships between fibres and biological medium result definitely destroyed.
Being our Hospital and our University placed in a region where asbestos are a very severe problem, and where not only respiratory diseases appear with higher percentages, during the last years our research group addressed attention to the identification of asbestos fibres in bile tract and in gallbladder to verify their presence in these parts of the human body. For this purpose, bile fluid and thin sections of gallbladder of patients, living in an asbestos-polluted area, and operated on for gallbladder stones have been examined using variable pressure scanning electron microscopy equipped with energy dispersive spectroscopy (VP-SEM/EDS). Also thin sections of the gallbladder from patients affected at the same time by Malignant Pleural Mesothelioma (MPM) and severe
troubles in the gallbladder were analysed. In all the studied cases, except in a patient too young to be exposed to asbestos, the technique allowed to identify, on the basis of their morphology and chemical composition, asbestos fibres in the examined tissues (Grosso et al., 2015, 2017). In our studies, we observed the asbestos fibres directly in thin histological sections without digestion of the biological matrix. This means that also the area of the tissue in which the fibres were incorporated could be determined (Grosso et al., 2017). Especially in the case of MPM patient, the analysis of gallbladder sections highlighted an important number of asbestos fibres. A further step appeared therefore the quantification of the asbestos fibres, but being the amount of tissue for the analyses very limited, we applied the traditional procedure on 9·10-4 g of tissue, available amount, of gallbladder included in paraffin. The filter obtained after digestion of the biological matrix was examined under SEM/EDS in two laboratories. A deep study of histological sections of gallbladder from the same patient was also carried out in order to locate the fibres in the biological medium where they were incorporated.
The aim of this study is on one hand to assess for different methods of quantification of asbestos fibres by SEM-EDS their scientific reliability and to compare cost/benefit ratios. On the other hand a way to obtain complementary information useful for a deep knowledge of the incorporation mechanism of the asbestos in the human body is described.
Materials and methods
In this work, following the definition of NIOSH, 2011, and Roggli, 2014, have been considered “fibres” all the particles showing length:thickness > 3:1 and “asbestos” all the fibres with chemical composition corresponding to a mineral phase regulated as “asbestos” (tremolite, actinolite, anthophyllite, amosite, crocidolite, chrysotile; D Lgs 277/91, Rinaudo et al., 2003, 2004; NIOSH, 2011; IARC, 2012; Roggli, 2014). In fact, the World Health Organization (WHO, 1986) and the US Occupational Safety and Health Administration (OSHA, 1992) definitions are referred to respirable fibres. The definition of asbestos fibres are respectively: crystal showing a length (l) >5 µm, diameter (d) <3 µm, and a ratio, length over diameter (l/ d) ≥ 3:1 (WHO 1986); crystal having l ≥ 5 µm, l/d ≥ 3:1, without limit for the diameter(OSHA 1992) . These kinds of dimensional criteria concern fibres biologically active in the alveolar region of the respiratory system, and therefore defined for the breathing process.
It has not been still defined if the reported values are sound when the fibres are incorporated through other mechanism than the breathing, ingestion as an example.
The fibre observations without digestion of the biological medium was performed directly on 5 µm histological thin sections, cut from cell blocks embedded in paraffin. Three thin sections for each patients were prepared on plastic support to avoid, as described in previous works (Croce et al., 2013; Grosso et al., 2015, 2017), interferences with the chemicals elements constituting the asbestos minerals.
Before VP-SEM/EDS analyses, the sections on the plastic holders were placed in an oven at 60°C overnight to eliminate the paraffin film. The sections were therefore examined under Variable Pressure Scanning Electron Microscopy (VP-SEM), ESEM Quanta 200 (FEI Company Hillsboro, OR, USA), equipped with energy dispersive spectroscopy (EDS, EDAX, Mahwah, NJ, USA) and a back-scattered electron detector (BSED). The back-scattered images (BSE), characterized by white/black contrasts produced by the inorganic phases plunged in the biological medium, allowed an easy detection of the asbestos fibres, as described in Grosso et al., 2015, 2017. The experimental conditions were: pressure of 90 Pa, working distance of 10 mm, and accelerating voltage of 20 kV. The EDS registered spectra were processed with GENESIS software version 3.6. To determine the real elemental composition of the inorganic phases observed, EDS microanalyses were carried out both on the inorganic phases and on the areas close to them, therefore acquiring several EDS spectra in different points of a fibre/bundle of fibres (analyses IN) and on the surrounding area (analyses OUT). The difference between the mean values obtained in the analyses acquired on the inorganic phase (analyses IN) and on the surrounding area (analyses OUT) was considered assessing the chemical composition of the observed fibre/bundle of fibres.
After digestion
To perform fibre quantification after digestion of the biological matrix, five 15 µm thin sections of tissue from the same paraffin block were cut. Before digestion, the paraffin was eliminated from the sections by washing two times with xylol and two times with ethanol, each washing being carried out during 10 min. After drying, the tissue was weighted by means of an analytical balance KERN 770 (KERN & Sohn GmbH, Blingen, Germany), working with an instrumental error of d = 1·10-4 g. In the studied case, the weight of the sections resulted about 9·10-4 g. The chemical digestion was performed using 30 ml NaClO (Carlo Erba, 12.5%) at room temperature to eliminate the organic fraction. The inorganic material was then recovered by filtering the suspension on a mixed cellulose esters filter, porosity of 0.45 µm and diameter of 25 mm.
Before observations, the sample was made conductive by covering it with graphite film through a metallization process, using an auto carbon coater (JEOL JEC-530) and therefore divided in two parts: one was analysed under SEM/EDS, the other one was stored for future check needs.
The same prepared half-filter was examined in two laboratories following two different procedures: one time at the DISIT, University of Eastern Piedmont, under the VP-SEM/EDS described previously. In this case, only half of the sample was observed at 2000x magnification. On all the inorganic phase/aggregate, observed by means of BSE detector, detailed morphological characterization- determination of length, thickness, ratio length/thickness- were performed increasing magnification, then chemical analyses were performed.
The second procedure was carried out at the Department of Earth Sciences of the Turin University, using an Electron Microscope (JSM-IT300LV), equipped with EDS (Inca Energy 200 with INCA X-act SDD detector). The experimental conditions were: voltage 15 kV; working distance 10 mm; probe current about 1nÅ; the observations were performed at 2000x analysing 660 microscopic fields (MF), which correspond to 2 mm2. The MF were distributed along 6 parallel horizontal strips; the distance was defined to avoid overlap between the examined areas. The fibre dimensions have been determined under SEM and the chemical composition were determined by EDS spectra.
Results
Histological sections
We describe the results from observations under VP-SEM/EDS of the histological sections of gallbladder from a patient affected by MPM, therefore certainly exposed to asbestos, and suffering at the same time of secondary lymph node localization and necrotic inflammatory changes in the gallbladder.
Under VP-SEM/EDS, the asbestos fibres/bundles of fibres were detected by means of BSE images in different situations:
a) asbestos fibres appearing as agglomerates of very thin chrysotile fibrils as shown in Figures 1A and 1B;
b) bundles of chrysotile fibres, Fig. 1C, as revealed by EDS analyses, Fig. 1D, close or overlapped by phases of chemical composition not corresponding to asbestos and therefore appearing as aggregate of same or different inorganic phases;
c) fibrous phases showing chemical composition identifying “crocidolite”, Figures 1E and 1F; d) elongated crystals partially plunged in the biological matrix, with chemical composition corresponding to serpentine, Figures 1G and 1H. In these cases, we cannot surely determine the serpentine mineral phase that is chrysotile, i.e. an asbestos classified fibre or antigorite, i.e. a serpentine not classified as “asbestos”.
As it concerns the digested samples, at the University of Eastern Piedmont all the inorganic fibrous phases observed under VP-SEM/EDS on a quarter of the filter were investigated (corresponding to 82 mm2) and all the fibrous phases observed were characterized on their morphological and chemical aspects. At the University of Turin, only a portion of 2 mm2 of the filter was examined under SEM/EDS, as for fibre quantification in lung (Belluso et al., 2006). As specified before, in both laboratories only the asbestos fibres showing a ratio length: thickness > 3:1 have been considered for the counting (NIOSH, 2011; Roggli, 2014).
It appears obvious that following the first methodology a higher number of fibres/bundles of fibres were noticed. Only in one case, aggregates of thin fibres, as that shown in Figure 1A, were observed: probably during filtering the fibres forming aggregates split up. The fibres may be observed in a pile of fibres as in Figure 2A or isolated as in Figure 2B, with chemical composition referable more frequently to chrysotile Figures 2C and 2D, less frequently to crocidolite (Fig. 2E and 2F) or asbestos tremolite (Fig. 2G and 2H).
The number of the observed fibres have to be standardized to the number of fibres per gram of wet weight tissue fg (Tww)-1, (Belluso et al., 2006), and multiplying by 0.7, coefficient, which correlates the fibre concentration in digested tissues from paraffin blocks to fibre concentration in wet tissues (Roggli et al., 1986, and Roggli & Sharma, 2014):
[(exposed area / observed area) * observed number of fibres*0.7] / weight of de-paraffinized tissue
In Table 1 the area of the studied filter, the number of observed asbestos fibres and their concentration are reported. From the exposed data, comparable results are obtained with the two procedural methodologies.
Discussion
The results described in two previous works (Grosso et al., 2015, 2017), proving the possibility for the asbestos fibres to reach gallbladder, not only are confirmed but also they were detected in high concentration. In fact, in the studied case, in gallbladder thin sections from a patient suffering at the same time of MPM, therefore certainly exposed to asbestos, and severe troubles in bile tract, the asbestos fibres were detected in the histological sections. After digestion of the biological matrix, performed on 9·10-4 g of gallbladder tissue from paraffin sections and after filtration of the obtained solution, the fibres detected were characterized under their morphological and chemical under SEM/EDS technique to be identified as “asbestos fibres”. Their concentration was proved to be about 3·105 fibres/cm3.
The quantification was obtained by two ways: in one laboratory a quarter of filter has been studied under VP-SEM/EDS and all the observed fibres characterized. In the second laboratory, only 2 mm2 of filter has been investigated under SEM/EDS. Comparing the results obtained with the two pathways, Table1, the fibre concentration appeared always of the same order of magnitude -105 fibres/cm3 or fibres/g of wet tissue.
From the described data, some considerations may be proposed:
the investigation of the histological sections, without digestion of the biological medium, let to locate the fibres in the tissues, to highlight them as single fibres or as bundle of fibres, to identify the asbestos in agglomerates of different inorganic phases; after digestion the fibres appeared mostly as single bundles of fibres, they are rarely observed in aggregates of different phases, probably consequent to a splitting of the different components of the aggregate during the digestion-filtration processes;
when digestion of the biological matrix is carried out, the characterization of all the observed fibres in a quarter of filter is certainly a more time consuming analysis, about 20 hours versus 2-3 hours, but a deeper and more accurate characterization of a higher number of incorporated fibres therefore a more precise data on the percentages of the different mineral phases observed may be obtained;
the methodology proposed for analysis on 5·10-1 g of lung may be applied also when limited amount of tissue is available. The procedure performed analysing only 2 mm2 at magnification 2000x is certainly fast and an useful tool to know fibre concentration, even if some limits in the determination of the characteristics- morphological, chemical- of all the incorporated fibres must be underlined, especially because a very limited amount of tissue is examined.
This work is certainly a preliminary study, other cases will be studied to corroborate the presented data and to obtain results more sound on the statistical point of view. Nevertheless, a conclusion may be proposed: the counting of the fibres in human tissues, not necessarily from respiratory system, may be obtained also when a limited amount of tissue is available. The amount in asbestos fibres can be obtained by analysing by SEM/EDS only 2 mm2 of filter surface, method which is cheaper and faster. Nevertheless, the poor number of fibres detected makes difficult a deep knowledge of the morphological and chemical-physical characteristics of the incorporated fibres.
Finally, the number of asbestos fibres which can reach the gallbladder appear important when exposure to asbestos is significant. In the studied case, exposure was proved by the development of MPM. It appears now important to perform studies allowing knowledge of the path used by
the fibres to reach the different organs and the different parts of the human body. For this goal the deep analysis of the histological sections, without digestion of the biological medium, and therefore preserving the relationships between asbestos and cellular medium appears very important.
Conflict of interest
The authors declare no conflict of interest.
Acknowledgements
The work was supported by ASL AL (“Progetto globale di ricerca in tema di cure e di prevenzione per la diagnosi e la terapia del mesotelioma - CUP C68F14000270005”) and by CRT (“Amianto e colangiocarcinoma: una nuova malattia asbesto correlata?”). The authors wish to thank the Associazione Familiari Vittime dell’Amianto (AFeVA) for the encouragement for these studies.
References
Albin, M., Johansson, L., Pooley, F.D., Jakobsson, K., Attewell, R. & Mitha, R. (1990) Mineral fibres, fibrosis, and asbestos bodies in lung tissue from deceased asbestos cement workers. Br. J. Ind. Med., 47(11), 767-774.
Auerbach, O., Conston, A.S., Garfinkel, L., Parks, V.R., Kaslow, H.D. & Hammond, E.C. (1980) Presence of asbestos bodies in organs other than the lung. Chest 77(2), 133–137.
Bell, S. (2001) Measurement good practice Guide. A beginner’s guide to uncertainty of measurements. 11(2).
Belluso, E., Bellis, D., Fornero, E., Capella, S., Ferraris, G. & Coverlizza, S. (2006) Assessment of inorganic fibre burden in biological samples by Scanning Electron Microscopy – Energy Dispersive Spectroscopy. Microchim. Acta 155(1-2), 95-100.
Boulanger, M., Morlais, F., Bouvier, V., et al. (2015) Digestive cancers and occupational asbestos exposure: incidence study in a cohort of asbestos plant workers. Occup. Environ. Med. 72(11), 792-797.
Brandi, G., Di Girolamo, S., Belpoggi, F., Grazi, G., Ercolani, G. & Biasco, G. (2008) Asbestos exposure in patients affected by bile duct tumours. Eur. J. Oncol. 13(3), 171–179.
Brandi, G., Di Girolamo, S., Farioli, et al. (2013) Asbestos: a hidden player behind the cholangiocarcinoma increase? Findings from a case–control analysis. Cancer Causes Control 24(5), 911-918.
Casali, M., Carugno, M., Cattaneo, A., et al. (2015) Asbestos lung burden in necroscopic samples from the general population of Milan, Italy. Ann. Occup. Hyg. 59(7), 909–921.
Churg, A. (1982) Asbestos fibers and pleural plaques in a general autopsy population. Am. J. Pathol. 109(1), 88-96. Churg, A., & Warnock, M.L. (1977) Correlation of quantitative asbestos body counts and occupation in urban patients.” Arch. Pathol. Lab. Med. 101(12), 629-634
Constanza Camargo, M., Stayner, L.T., Straif, K., Reina, M., Al-Alem, U., Demers, P.A. & Landrigan, P.J. (2011) Occupational exposure to asbestos and ovarian cancer: a meta-analysis. Environ. Health Perspect. 119(9), 1211-1217.
Croce, A., Musa, M., Allegrina, M., Trivero, P. & Rinaudo, C. (2013) Environmental scanning electron microscopy technique to identify asbestos phases inside ferruginous bodies. Microsc. Microanal. 19(2), 420–424.
De Ridder, G.G., Kraynie, A., Pavlisko, E.N., Oury, T.D. & Roggli, V.L. (2016) Asbestos content of lung tissue in patients with malignant peritoneal mesothelioma: A study of 42 cases. Ultrastruct. Pathol. 40(3), 134-141.
De Vuyst, P., Karjalainen, A., Dumortier, P., et al. (1998) Guidelines for mineral fibre analyses in biological samples: report of the ERS Working Group. Eur. Respir. J. 11(6), 1416–1426.
De Vuyst, P., Mairesse, M., Gaudichet, A., Dumortier, P., Jedwab, J. & Yernault. J.C. (1983) Mineralogical analysis of bronchoalveolar lavage fluid as an aid to diagnosis of “imported” pleural asbestosis. Thorax 38(8), 628-629.
Decreto Legislativo del Governo n° 277 del 15/08/1991. (1991) Attuazione delle direttive n. 80/1107/CEE, n. 82/605/CEE, n. 83/477/CEE, n. 86/188/CEE e n. 88/642/CEE, in materia di protezione dei lavoratori contro i rischi derivanti da esposizione ad agenti chimici, fisici e biologici durante il lavoro, a norma dell'art. 7 legge 30 luglio 1990, n. 212.
Di Ciaula, A. (2017) Asbestos ingestion and gastrointestinal cancer: a possible underestimated hazard. Expert Rev. Gastroenterol. Hepatol. 11(5), 419-425.
Di Ciaula, A. & Gennaro, V. (2016) Possible health risks from asbestos in drinking water. Epidemiol. Prev. 40(6), 472-475.
Dodson, R.F., O'sullivan, M., Corn, C.J., McLarty, J.W. & Hammar, S.P. (1997) Analysis of asbestos fiber burden in lung tissue from mesothelioma patients. Ultrastruct. Pathol. 21(4), 321-336.
Ehrlich, A., Rohl, A.N. & Holstein, E.C. (1985) Asbestos bodies in carcinoma of colon in an insulation worker with asbestosis. JAMA 254(20), 2932-2933.
International Agency for Research on Cancer (IARC). (2012) Asbestos (chrysotile, amosite, crocidolite, tremolite, actinolite, and anthophyllite). IARC Monographs on the Evaluation of Carcinogenic Risks to Humans (ed. by IARC), vol. 100C, pp. 219–309. Lyon, France.
Grosso, F., Randi, L., Croce, A., et al. (2015) Asbestos fibers in the gallbladder of patients affected by benign biliary tract diseases. Eur. J. Gastroenterol. Hepatol. 27(7), 860–864.
Grosso, F., Croce, A., Trincheri, N.F., Mariani, N., Libener, R., Degiovanni, D. & Rinaudo, C. (2017) Asbestos fibres detected by scanning electron microscopy in the gallbladder of patients with malignant pleural Mesothelioma (MPM). J. Microsc. 266(1), 48-54.
Huang, J., Sakai, K., Iwata, M. & Ono, Y. (1988) Asbestos fibers in human pulmonary and extrapulmonary tissues. Am. J. Ind. Med. 14(3), 331-339.
Jacobsen, M., Foda, A., Ronk, C. & McKinley, M.A. (2013) Chrysotile asbestos exposure and ovarian cancer: is there an association? Am. J. Epidemiol. 177(11suppl.), S123.
Karjalainen, A., Nurminen, M., Vanhala, E., Vainio, H. & Anttila, S. (1996) Pulmonary asbestos bodies and asbestos fibers as indicators of exposure. Scand. J. Work Environ. Health 22(1), 34–38.
Kobayashi, H., Watanabe, H. & Ohnishi, Y. (1987) A quantitative study on the distribution of asbestos bodies in extrapulmonary organs. Acta Pathol. Jpn. 37(3), 375-383.
Muller, A.M., Neumann, V., Muller, K.M. & Fischer, M. (2001) Association of asbestos fibers and colon cancer: an electron-microscopic study of mineral fiber concentrations in colon tissue of asbestos exposed patients with and without colon cancer. Z. Gastroenterol. 39(12), 993-1000.
NIOSH. Department of Health and Human Services. Centers for Disease Control and Prevention. National Institute for Occupational Safety and Health. (2011) Asbestos fibers and other elongated mineral particles: state of the science and roadmap for research. Current Intelligence Bulletin 62.
OSHA. Occupational Safety and Health Administration, (1992) Occupational exposure to asbestos, tremolite, anthophyllite and actinolite. [Docket No. H-033-d], 29 CFR Parts 1910 and 1926: Federal Register, v. 57, no. 110. Pairon, J.C., Orlowski, E., Iwatsubo, Y., et al. (1994) Pleural mesothelioma and exposure to asbestos: evaluation from work histories and analysis of asbestos bodies in bronchoalveolar lavage fluid or lung tissue in 131 patients. Occup. Environ. Med. 51(4), 244-249.
Rinaudo, C., Belluso, E. & Gastaldi, D. (2004) Assessment of the use of Raman spectroscopy for the determination of amphibole asbestos. Mineral. Mag. 68(3), 455-465.
Rinaudo, C., Gastaldi, D. & Belluso, E. (2003) Characterization of chrysotile, antigorite and lizardite by FT-Raman spectroscopy. Can. Mineral. 41(4), 883-890.
Roggli, V.L. (2006) The role of analytical SEM in the determination of causation in malignant mesothelioma. Ultrastruct. Pathol. 30(1-2), 31-35.
Roggli, V.L. (2014) Appendix: tissue digestion techniques. Pathology of asbestos related disease. (ed. by T.D. Oury, T.A. Sporn, & V.L. Roggli), Third edition, pp. 343-344. Berlin, Germany: Springer.
Roggli, V.L., Pratt, P.C. & Brody, A.R. (1986) Asbestos content of lung tissue in asbestos associated diseases: a study of 110 cases. Br. J. Ind. Med. 43(1), 18-28.
Roggli, V.L. & Sharma, A. (2014) Analysis of tissue mineral fiber content. Pathology of asbestos related disease. (ed. by T.D. Oury, T.A. Sporn, & V.L. Roggli), Third edition, p. 256. Berlin, Germany: Springer.
Schneider, C.A., Rasband W.S. & Eliceiri, K.W. (2012) NIH Image to ImageJ: 25 years of image analysis. Nat. Methods 9(7), 671-675.
Szendröi, M., Németh, L. & Vajta G. (1983) Asbestos bodies in a bile duct cancer after occupational exposure. Environ. Res. 30(2), 270–280.
Tossavainen, A. (1997) Asbestos, asbestosis and cancer: the Helsinki criteria for diagnosis and attribution. Scand. J. Work Environ. Health 23(4), 311-316.
Westlake, G.E. (1965) Penetration of colonic mucosa by asbestos particles. An electron microscopic study in rats fed asbestos dust. Lab. Invest. 14(11), 2029-2033.
Williams, M.G., Dodson, R.F., Dickson, E.W. & Fraire A.E. (2001) An assessment of asbestos body formation in extrapulmonary sites: liver and spleen. Toxicol. Ind. Health 17(1), 1–6.
Table 1
Method Analysed area(mm2)
Number of observed asbestos fibres Asbestos fibres/gwt University of Eastern Piedmont method 82 62 2.9·105 University of Turin method 2 3 3.3·105
Table 1: Amount of asbestos fibres on the digested sample from gallbladder of MPM case, obtained by the two different methodologies described in Material and Methods (gwt = gram of wet tissue).
Figure Captions
Figure 1: (A) BSE image of a bundle of very thin fibrils; (B) EDS spectrum recorded on the bundle of Figure 1A, allowing identification of “chrysotile”; (C) BSE image of a bundle of fibres appearing close to phases with chemical composition not corresponding to asbestos; (D) EDS analyses carried out on the bundle, arrowed in figure, identifying “chrysotile”; (E) BSE image of a fibrous phase; (F) EDS spectrum recorded on the fibre shown in Figure 1E, the chemical composition indicates “crocidolite”; (G) BSE image of an elongated crystal, partially plunged in the biological matrix; (H) EDS spectrum of the crystal in Figure 1G, the chemical composition identifies serpentine, but the morphology does not allows to distinguish it as “chrysotile” or “antigorite”.
Figure 2: (A) BSE image of a pile of fibres observed in the digested sample; (B) BSE image of an isolated fibre; (C) EDS spectrum obtained from the fibres in figure 2A, allowing to identify the phase as “chrysotile”, the iron detected may be in part ascribed to white particles showing iron composition; (D) EDS spectrum recorded on the fibre of Figure 2B: also in this case the mineral phase is “chrysotile”; (E) BSE image of another fibre in the digested sample; (F) EDS spectrum of the fibre in Figure 2E identifying “crocidolite”; (G) BSE image of a fibre in the digested sample; (H) EDS spectrum obtained on the fibre in Figure 2G: the relative intensities of the peaks allow to identify the crystal as “tremolite”.
