British Journal of Industrial Medicine 1980;37:375-381

Scanning electron microscopy and x-raymicroanalysis of mineral deposits in lungs of apatient with pleural mesotheliomaE M OPHUS,* G MOWIt, K K OSEN, AND B GYLSETH

From the Institite of Occupational Health, Oslo, and the Department ofPathology, Rikshospitalet, Oslo,Norway

ABSTRACT Scanning electron microscopy of lung tissue, ashed at low temperature, and obtainedfrom an insulation worker who had died of pleural mesothelioma, showed the presence of numerousinorganic particles and fibres. A regional variation in fibre concentration in different tissue sampleswas found, and the size distribution of naked fibres and asbestos bodies was determined. By energydispersive x-ray microanalysis the fibres were identified mainly as amphibole asbestos. This methodalso showed the presence of particles containing titanium and of fragments of diatom shells. Despitea mean concentration of 33 x 106 fibres per gram of dry tissue no significant lung fibrosis was found.

The causal relation between asbestos exposure andpleural and peritoneal mesothelioma was establishedin 1960.1 Since then the increased incidence ofmesothelioma in individuals exposed to asbestos hasbeen widely documented.2 The relation to asbestosexposure may, however, be obscured by a longperiod between initial exposure and the appearanceof the tumour. The dose required for the initiation ofa mesothelioma is small, and may be acquired bynon-occupational exposure to asbestos.3A relation between the lung asbestos burden and

asbestos-related diseases has been suggested.4-6Identification and quantification of mineral particlesin the lungs requires the use of microanalyticaltechniques.7 For in situ identification of inorganicsubstances in lung tissue-for instance, in the studyof pneumoconiosis-scanning electron microscopycoupled with energy dispersive x-ray microanalysishas proved particularly useful.8 This instrumentationhas also been used for quantitative analysis ofmineral fibres in lung residues obtained by tissuedegradation methods. Gylseth et a19 reported thatlow temperature plasma ashing is the most con-venient and reliable technique for this purpose.

In the present study we have used these methods

*Present address: University of Trondheim, NorwegianInstitute of Technology, Division of Industrial Management,N 7034 Trondheim-NTH, Norway.

Received 2 July 1979Accepted 5 September 1979

to identify and quantify inorganic particles andfibres in different parts of lung tissue taken from aninsulation worker who died of pleural mesothelioma.

Case history

OCCUPATIONAL EXPOSUREThe patient was born in 1920. A complete occupa-tional history was obtained in 1976, when heparticipated in an epidemiological investigation.From 1935 to 1952 he was engaged in insulationwork in industry and shipyards. During this period(17 years), he was exposed to various insulationmaterials such as asbestos (mainly amphiboleminerals), man-made mineral fibres, cork, anddiatomaceous earth. No occupational exposure toasbestos was recorded after 1952. The lapsed timefrom first exposure until death in 1978 was 43 years.He started smoking when 15 years old and smoked10-15 cigarettes a day.

SYMPTOMS AND SIGNSIn 1976 he developed slight dyspnoea duringphysical activity. The lung function was normal:FVC 5000 ml and FEV1 3800 ml. Radiographicexamination showed a slight pleural reaction on theright side but no evidence of parenchymal changes(fig 1(a)). During summer 1977 he developedprogressive dyspnoea. In August 1977 a large pleuraleffusion was seen on the right side (fig 1(b)). Cyto-logical examination of fluid from the right pleura

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Ophus, Mowe, Osen, and Gylseth

showed malignant, probably epithelial, cells. Hebecame progressively ill until death in March 1978.

Methods

NECROPSYA routine post-mortem examination of the visceraand the brain was performed. Tissue samples, in-cluding large slices of the lungs, were fixed byimmersion in buffered 10% formalin. Samples forhistological examination were embedded in paraffin.Besides the routine haematoxylin-azophloxine-saffron procedure, the lung sections were stained byperiodic acid-Schiff (PAS) and a modification ofMallory's method for connective tissue. Tumoursections were stained by several methods: (1) PASwith and without preincubation for one hour at37°C in 0-5 % malt diastase; (2) alcian blue with andwithout preincubation for 18 hours at 37°C in asolution of 1-25 mg testicular hyaluronidase in 50 ml0 - M acetate buffer, pH 5.610; and (3) Hale'scolloidal iron" with and without hyaluronidasedigestion, as described above. After removal of theparaffin wax small pieces of tumour tissue werehydrated, osmicated, dehydrated, and embedded inEpon. Semithin sections were stained with 0.1%toluidine blue in borax, pH 10, and examined bylight microscopy. Ultrathin sections were stainedwith uranyl acetate and lead citrate and examined inthe transmission electron microscope.

SCANNING ELECTRON MICROSCOPYThe concentration of fibres in the lungs was deter-mined by a scanning electron microscope (SEM)according to the method described by Gylseth et al.9Two lung slices (about 3 x 1 x I cm), including pleuraland parenchymal tissues, were divided into threepieces each by cuts parallel to the pleura. After dryingand low temperature plasma ashing followed by

Fig 1 Chest films taken (a) two years before death; (b) slight acid treatment, the ash residue was dispersed17 months later (six months before death). in water and collected on Nuclepore filters. The fibre

Fig 2 (a)-(f) are from a tumour metastasis in thigh muscles; (g) is from the lung; (e) is an electron micrograph; othersare light micrographs. (a) Tumour shows cell-lined slits and irregular groups ofpolygonal cells in an abundant connectivetissue stroma that also contains remnants of muscle fibres (arrowheads) and moderate amounts of leucocytes. (Paraffinsection, haematoxylin-azophloxine-saffron, x 85.) (b) Cell from lumen of a slit with a large, metachromatic vacuole (v).(c) Tumour cells with irregular nuclei (n) and numerous apical microvilli (arrowheads). ((b) and (c) Epon sections,toluidine blue, x 1200.) (d) Tumour cells with apical microvilli (arrowheads) are seen lining a slit. Two vacuolated cellsare embedded in underlying stroma. Vacuole (vl) of upper cell has a smooth wall and is filled with a PAS-negativesubstance. Lower vacuole (v2) appears empty with a ragged wall possibly provided with microvilli. (Paraffin section, PASwith diastase, x 1200.) (e) Electron micrograph from apical end ofa tumour cell with long and slender microvilli(arrowheads), (n) part of nucleus. ( x 25 000.) (f) Tumour cell vacuole (v) with a positively stained content. Note alsopositively reacting coarse granules (arrows) between cells. (Paraffin section, Hale's colloidal iron without hyaluronidase,x 1200.) (g) Alveolar wall with numerous capillaries (c). (Paraffin section, PAS, x 1200.)

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Scanning electron microscopy and x-ray microacnalysis 377

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38Ophus,Mowe, Osen, and Gylseth

density and size distribution was determined at4500 x in a JEOL JSM 35 SEM, operated at 25 kv inthe secondary electron mode. The mean fibre con-

centration in each of the six samples was given bythe arithmetic mean of three replicate counts. In eachcount the number of naked fibres and asbestosbodies in 200 "view fields" in the SEM was deter-mined by scanning intermittently across the filter.

X-RAY MICROANALYSISThe elemental composition of mineral particles andfibres, including naked fibres and asbestos bodies,was determined in the SEM by means of energy

dispersive x-ray microanalysis. The investigationwas carried out using a Princeton Gamma TechPGT-1000 spectrometer. The asbestos fibres were

identified by comparing the x-ray spectra with thoseobtained by analysis of standard UICC asbestossamples.

Results

PATHOLOGICAL ANATOMICAL EXAMINATIONPost-mortem examination showed a malignanttumour in the visceral and parietal pleura bilaterally.In addition a massive, directly invasive growth oftumour was found in the mediastinum, the dia-phragm, the abdominal wall, and the retroperitonealtissue. These structures were all considerably en-larged and appeared like a continuous mass oftumour weighing about 5 kg. Distant metastaseswere found in the muscles of the thigh bilaterally,the myocardium, the intestinal submucosa, thevertebral column, the liver, and the kidneys. Themetastases, like the primary tumour, thus seemed togrow preferentially in mesenchymal tissues.On microscopy the tumour was found to have a

pure epithelial character being composed in part ofcuboidal or cylindrical cells lining slit-like cavities, inpart by more undifferentiated polygonal cells em-bedded in a dense connective tissue stroma (fig 2(a)).The apical end of the lining cells showed numerousslender microvilli, about 3-4 lum long (fig 2(c-e)).Round cells with a vacuolated cytoplasm were foundboth in the lumina of the slits and among the poly-gonal cells. Some of them had a typical signet ringshape with a large single vacuole that had either asmooth (vl in fig 2(d)) or a ragged (v2 in fig 2(d))circumference, the latter possibly being providedwith microvilli. While most vacuoles appearedempty or washed out, a few had a content that wasstained faintly by the alcian blue and distinctlypositive by the colloidal iron method (fig 2(f)).These reactions were not significantly affected by thehyaluronidase incubation. In toluidine blue sectionsthe filled vacuoles were metachromatic, assuming a

purple colour (fig 2(b)). All viable tumour cells werePAS-negative, but among them were found positivelystained cellular debris and scattered coarse granules,which were stained positively also by the colloidaliron method (fig 2(f), arrows). The PAS and colloidaliron staining of the granules disappeared afterdigestion with diastase and hyaluronidase, respec-tively. The tumour cells showed only scatteredmitoses and a moderate nuclear pleomorphy.

In the lungs the alveolar tissue was invaded bytumour cells only in the immediate vicinity of thetumour-containing pleura. There was no significantfibrosis or emphysema of the lungs as judged by thegross examination and the microscopy of routine,PAS, or Mallory stained sections. The bronchioli,alveolar ducts, and alveolar sacs seemed normal insize and, as illustrated in fig 2(g), the alveolar wallsappeared delicate with no discernible changes in thedensity of capillaries or collagen fibres. The peri-bronchial connective tissue nevertheless containedconsiderable amounts of anthracotic pigments,birefractive crystals, and asbestos bodies, also pres-ent in the enlarged, tumour-invaded mediastinallymph nodes.

FIBRE CONCENTRATIONTable 1 shows the fibre concentration in successiveregions of the two lung slices. The mean concentra-tion was 33 x 106 fibres/g of dry tissue with asignificantly larger concentration in the centralsample of slice 2. Ninety-two per cent of the fibresgave a positive asbestos (amphibole) identificationby the x-ray microanalysis. The true proportion ofasbestos fibres may have been higher because fibresless than 0-1 ,um in diameter could not be identified,due to the low signal-to-background ratio. Chrysotilewas not detected. Non-asbestos fibres were identifiedby x-ray microanalysis as other fibre-forming sili-cates. Few fibres were found in the thickened andcalcified pleura, indicating pleural fibre concentra-tions at least one order of magnitude less than thoseof the lung tissue.About 70% of the total number of fibres were

classified as asbestos bodies (fig 3(a)). The centralfibre could not be identified, due to the x-ray signalfrom the iron in the coating, in 17% of the bodies.In the remaining 83 % the coating was fragmented

Table I Regional distribution offibre concentrations inthe two lung slices

Distance from No offibreslg dry lung x 106 (range)pleura (cm)

Slice I Slice 2

0-1 22 (20-23) 24 (22-25)1-2 29 (27-31) 24 (23-26)2-3 32 (29-33) 66 (55-82)

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Sccnnin1g electroli microscopy and x-ray microanalysis

and showed the central core for identification. Onlyamphibole minerals (mainly crocidolite) were found.

Table 2 gives the relative proportion of asbestosbodies in different parts of the tissue. There were no

systematic differences related to the distance of thesample from the pleural surface.

FIBRE SIZE DISTRIBUTION

While the fibre concentrations were higher in thedeeper than in peripheral parts of the lung (table 1),the length distribution was relatively constant,although fibre lengths ranged from 1 to 50,um. In allsamples nearly 700% of the naked fibres were lessthan 5 ,um in length. The diameters of the fibresnever exceeded 2 ,um, and 950% of the fibres were

less than I ,tm in diameter.The length distribution of the central fibres in the

asbestos bodies differed from that of the nakedfibres (fig 4(a)). About 900% of the central fibresexceeded 5ttm in length compared with 30°/ for thenaked ones. The diameter distribution of the coated

fibres showed only a small bias towards increased

values as compared with the naked ones (fig 4(b)).About 2-0 x 106 naked fibres and 1.5 x 106 coatedfibres longer than 10 jm were present per gram ofdry lung tissue.

OTHER PARTICLES

Fragments of diatom shells (kieselguhr) that were

often found in the lung tissue were identified by

morphology (fig 3(b)) and the pure silicon Kat ay

spectrum. Cases of diatomaceous earth pneumo-

_Vww coniosis have been described.12 On average, 1-4 x

106 shell fragments were found per gram of dry lung. ~~tissue. The distribution of diatom shells resembled

F_ 2 0 1 ~~~~~~~~~~~~~~~~~501

(ll.20-

0

Fig 3 Lung tissue incinerated and collected on a F l3 3< <5 5< -10 10<-.20 20<-<50Nuclepore filter. (a) Asbestos body ( x 2600). (b) Diatomfragment ( x 15 000). 50- ( Naked fibres O-O

Central fibres in40- asbestos bodies

Table 2 Regional distribution of asbestos bodies in the ./30- /

two lung slices. Concentrations are expressed in 20percentage of total number offibres within each sample ,/ \*

I 46, ---5-.01 01--03 03-SG505<-s07 07<-s09 09<

Fibre diameter (Aum)

Fig 4 Size distributions of 1459 nakedfibres and 114central fibres in asbestos bodies. (a) Length. (b)Diameter.

Distance fro1m Percentage asbestos bodiespleura (cnm)

Slic e 1 Slice 2

0-1 76 9 61-2 11-5 6 52-3 6-3 2 8

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380

that of asbestos bodies and showed no preferentialsite for deposition in the lung.

Further investigation of the low temperature ashedpleural and parenchymal tissues showed variousparticles containing silicon or titanium. The latterpresumably consisted of titanium dioxide pigments,which may behave as a mild irritant in the pulmonaryinterstitium."3 14

Discussion

According to the necropsy findings the patient diedfrom a malignant mesothelioma present in bothpleurae, but possibly originating on the right side andspreading to several other locations either by directinvasion or metastasis. The thickened, tumour-invaded pleurae evidently must have affected boththe respiratory space and the respiratory movements,but in agreement with the radiographic findings twoyears before death we found no discernible fibrosis oremphysema of the lungs.

Despite the epithelial character of the tumour, theexistence of a metastatic carcinoma or a bronchiolo-alveolar cell carcinoma could be ruled out by theelectron microscopical appearance of the tumourcells, particularly their long and slender microvilli.According to Wang15 these microvilli constitute ahighly characteristic feature of malignant meso-theliomas. Similar microvilli are found only in theadenomatoid tumours of the genital tracts, which arealso supposed to be of a mesothelial origin.16Microvilli of carcinomas, including pulmonarytumours, are, as a rule, shorter, broader, and moreirregularly club-shaped.'7 In the present case theabsence of any other possible primary tumour alsostrongly favoured the diagnosis of a mesothelioma.Although these findings leave no doubt that the

present tumour was a pure epithelial mesothelioma,we have not been able to prove the presence ofhyaluronic acid, which is a characteristic feature ofsuch tumours.10 Tumour cell vacuoles with villous orsmooth walls containing alcian blue material labileto hyaluronidase have been described by Wang15 intwo cases of diffuse mesotheliomas. In our materialthe cell vacuoles appeared very much like thoseillustrated by Wang,15 except that most of themseemed empty or washed out. A few vacuoles mayhave contained acid mucopolysaccharides as judgedby their metachromasia, positive staining by alcianblue and colloidal iron, and negative PAS-staining.18In our hands, however, these staining reactions werenot significantly changed by preincubation withhyaluronidase. The discrepancy may possibly bedescribed to a prolonged formalin fixation that mayhave removed the water-soluble hyaluronic acid,leaving only remnants of a less soluble and more

Ophus, Mowe, Osen, and Gylseth

hyaluronidase-stable substance.With respect to the composition of the PAS- and

colloidal iron-positive granules found between thetumour cells, their loss of PAS-staining after diastasedigestion speaks in favour of glycogen. It is doubtful,however, whether this can explain their lability tohyaluronidase.According to the occupational history and the

burden of fibre in the lung, this man must have hadconsiderable exposure to asbestos. The mean con-centration of fibres, 33 x 106/g of dry lung tissue, issignificantly higher than in people with low orunknown occupational exposure (Mowe et al at theVth International Conference on Pneumoconiosis inCaracas, 29 October-3 November 1978). Similarfibre concentrations have been reported by Ashcroftand Heppleston4 in patients with mesothelioma withmild asbestosis. According to them a significantcorrelation exists between the lung fibre concentra-tion and the degree of pulmonary fibrosis, in mildand moderate grades of asbestosis. Despite therelatively high fibre concentration in the present caseno pulmonary fibrosis was found. The lapsed timefrom the initial asbestos exposure to the appearanceof the tumour (42 years) corresponds with theaverage period reported in other cases of asbestos-induced mesothelioma.2The size of the asbestos fibres may be of importance

for the development of mesothelial tumours.Experimental studies by Stanton et a1'9 showed thatthe carcinogenicity probably depended on fibredimensions. According to them, fibres longer than8 Ktm and thinner than 1-5 /um yield the highestprobability of pleural sarcomas. In future studies ofpleural mesotheliomas both the concentration and thesize distribution of coated and naked fibres in thelung tissue should be determined.

This study indicates that there may be regionalvariations in the fibre concentration within the samesegment of the lung. Possibly, greater variations maybe found between different lung segments or lobes.The existence of intra- and inter-lobular variations infibre concentration should be borne in mind whendesigning comparative studies of asbestos fibre levelsin populations with various occupational exposuresor with various asbestos-related diseases.6 For suchinvestigations, whole slices of both lungs should beused for systematic sampling. Care should also betaken in comparing quantitative data from studieswhere different techniques of sample preparation orcounting procedures have been applied.9The evaluation of the lung asbestos burden is

further complicated by the tendency of asbestosfibres, particularly chrysotile, to be cleared from thetissue in vivo.20 In our patient many amphiboleasbestos fibres were found, in agreement with the

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Scanning electron microscopy and x-ray microanalysis

occupational history. Yet the presence of chrysotilecannot be excluded, because fibres (fibrils) too thinto be identified by the x-ray microanalysis mayrepresent chrysotile degraded in vivo or duringsample preparation. The predominance of amphibolecores in asbestos bodies is in accordance with thefindings of Churg and Warnock.21The proportion of coated fibres (7.1 %) in this

patient has been found in other cases with a highexposure to asbestos (Mowe et al at the Caracasconference, 1978). Botham and Holt22 found thatmature asbestos bodies have a beaded appearancethat develops with time and finally leads to frag-mentation of the body. Despite the fact that ourpatient had not been occupationally exposed toasbestos fibres during the last 20 years of life, nearly17% of the asbestos bodies were covered with anunbroken iron/protein sheet, and 80% had only thebare ends protruding from the cover. This observa-tion suggests the presence of newly formed, im-mature bodies in the lungs, and may indicate thatasbestos fibres undergo coating and fragmentationseveral times.The presence of several other minerals in the lungs

is consistent with the occupational history of thepatient, but the significance of these agents in thepathogenesis of the mesothelioma is uncertain.

References

Wagner JC, Sleggs CA, Marchand P. Diffuse pleuralmesothelioma and asbestos exposure in the NorthWestern Cape Province. Br J Ind Med 1960;17:260-71.

2 Selikoff IJ, Lee DHK. Asbestos and disease. New York:Academic Press, 1978:241-306.

3Newhouse ML, Thompson H. Mesothelioma of pleura andperitoneum following exposure to asbestos in theLondon area. Br JInd Med 1965;22:261-9.

l Ashcroft T, Heppleston AG. The optical and electronmicroscopic determination of pulmonary asbestos fibreconcentration and its relation to the human pathologicalreaction. J Clin Pathol 1973 ;26:224-34.

5 Whitwell F, Scott J, Grimshaw M. Relationship betweenoccupations and asbestos-fibre content of the lung inpatients with pleural mesothelioma, lung cancer, and

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other diseases. Tlhorax 1977 ;32 :377-86.6 Bignon J, Sebastien P, Fondimare A, et al. Etude quantita-

tive et qualitative des fibres d'amiante dans l'appareilrespiratoire humain. Paris: Institut de RechercheUniversitaire sur l'Environnement, 1978.

7Ruttner JR, Spycher MA, Sticher H. The detection ofetiologic agents in interstitial pulmonary fibrosis. HiumanPathol 1973;4:497-512.

Funahashi A, Siegesmund KA, Dragen RF, Pintar K.Energy dispersive x-ray analysis in the study of pneumo-coniosis. Br J Ind Med 1977;34:95- 101.

9 Gylseth B, Ophus EM, Mowe G. Determination of in-organic fiber density in human lung tissue by scanningelectron microscopy after low temperature ashing.ScandJ Work, Environ Health 1979;5:151-7.

10 Wagner JC, Munday DE, Harington JS. Histochemicaldemonstration of hyaluronic acid in pleural mesothelio-mas. J Pathol Bacteriol 1962;84:73-8.

11 Culling CFA. Handbook of histopathological techniques(including museum techniquies). 2nd ed. London:Butterworth, 1963:233.

12 Dutra FR. Diatomaceous earth pneumoconiosis. ArchEnviron Health 1965;11:613-9.

13 Elo R, Maatta K, Uksila E, Arstila AU. Pulmonarydeposits of titanium dioxide in man. Arch Pathol 1972;94:417-24.

1' Maatta K, Arstila AU. Pulmonary deposits of titaniumdioxide in cytologic and lung biopsy specimens. LabInvest 1975;33:342-6.

15 Wang N-S. Electron microscopy in the diagnosis of pleuralmesotheliomas. Cancer 1973 ;31:1046-54.

16 Salazar H, Kanbour A, Burgess F. Ultrastructure andobservations on the histogenesis of mesotheliomas''adenomatoid tumors" of the female genital tract.Cancer 1972;29:141-52.

1 Kuhn C. Fine structure of bronchiolo-alveolar cell car-cinoma. Cancer 1972;30:1107-18.

18 Pearse AGE. Histoche,nistrl'. Theoretical and applied. 3rded. Edinburgh and London: Churchill Livingstone,1972:1030-2.

'9 Stanton MF, Layard M, Tegeris A, Miller E, May M,Kent E. Carcinogenicity of fibrous glass: pleural res-ponse in the rat in relation to fiber dimension. J NatlCancer Inst 1977;58:587-603.

20 Le Bouffant L. Investigation and analysis of asbestosfibers and accompanying minerals in biologicalmaterials. Environ Health Perspec t 1974 ;9 :149-53.

21Churg A, Warnock ML. Correlation of quantitativeasbestos body counlts and occupation in urban patients.Arch Pathol Lab Med 1977;101:629-34.

22 Botham SK, Holt PF. Development of asbestos bodies onamosite, chrysotile, and crocidolite fibres in guinea-piglungs. J Pathol 1971 ;105:159-67.

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