electron microscope characteristics of inhaled chrysotile ... · department of mineral...

Brit. J. industr. Med., 29, 146-153

Electron microscope characteristics ofinhaled chrysotile asbestos fibre

F. D. POOLEYDepartment of Mineral Exploitation, University College, Cardiff

Pooley, F. D. (1972). Brit. J. industr. Med., 29, 146-153. Electron microscope characteristicsof inhaled chrysotile asbestos fibre. Specimens from 300 lungs have been examined underthe electron microscope to determine the morphology and diffraction characteristics of anychrysotile asbestos they contained. In 120 cases, material was prepared by alkali digestionand the residual dust was examined. In all cases standard 6-micron histological slides werepartially ashed before the residue was transferred to the electron microscope grids. Of the 300specimens examined, 20 came from men with prolonged industrial exposure to chrysotile, 87from cases of mesothelioma, and the remainder from control groups drawn from rural andindustrial populations.

Chrysotile fibres were readily identified by the characteristic polycrystalline diffractionpattern. The hollow appearance of the single fibres and their shape and arrangement also helpin the identification. Specimens from men without industrial exposure contained either singleshort fibres or aggregates scattered throughout the lungs. In specimens from industriallyexposed men, fibres were very numerous and found as strands of single fibres mainly groupedtogether in discrete locations. Ferruginous bodies were found rarely and only on straightfibre bundles of over several micrometers in length.

Chrysotile asbestos which comprises over 90% of thetotal asbestos mineral used by our industrial societyis a very unique polymorph of the mineral serpentine.It is composed of single, very fine fibres which havebeen shown by Maser, Rice, and Klug (1960) andYada (1967) to be less than 30nm (300A) in diameter.When seen in hand specimens or under the opticalmicroscope, however, only strands or aggregates ofthese fine fibres are observed.

Chrysotile has the ability to form a very largevariety of particle shapes and sizes because of thevery fine nature of its single fibre structure. Becauseof this large variation in particle morphology, it ispossible to define only the size of the single chryso-tile fibril. It is almost impossible to define particlesof larger size than the single fibril by any singleparameter as strands composed of large numbers ofsingle fibrils often branch and bush at their ends andintertwine with other strands to become irregularaggregates (Fig. 1).

The degree of aggregation obtained in any particu-lar chrysotile dust sample is governed by themechanical treatment the sample has received.Vorwald, Durkan, and Pratt (1951) found that ball-milled chrysotile had a great tendency to form smallspherules which prevented the efficient dispersion ofthe material as fibres in their animal inhalationstudies. The ability of chrysotile fibres to form suchaggregates thus implies that it is impossible tomanufacture a chrysotile dust with any specificdimensions and that the characteristics of chrysotiledust clouds will vary with the method of productionof the particles. Speil and Leineweber (1969) showthat the degree of length disintegration of fibrebundles depends not only upon the severity of themechanical action imposed on the sample, but alsoupon the brittleness or harshness of the mineral.They also found that the fibrous nature, together withthe actual structure, of the mineral can be completely

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Electron microscope characteristics of inhaled chrysotile asbestos fibre 147

FIG. 1. A sample of chrysotile dust paiticles (x 2000).

destroyed when intensive grinding of a chrysotilesample is performed.

Because of the heterogeneous nature of chrysotiledust particles, a number of questions can be raisedconcerning the influence of the particle form on boththe aerodynamic behaviour of the dust when air-borne, and also the form in which it may be a dangerto health.

Because of lack of a more specific method ofidentification, light microscopists have been un-

willing to identify fibres of less than 3:1 aspect ratioas chrysotile. However, Timbrell, Pooley, andWagner (1970) observed that chrysotile seen in lungtissue with the electron microscope bears littleresemblance to the same material viewed by opticalmeans. A similar result by Lynch and Ayer (1968)obtained from the examination of airborne chryso-tile dust particles showed that the electron micro-scope was essential for the study of the majority ofchrysotile dust particles which could not be resolvedin the optical microscope.Some doubt has been expressed about the preva-

lence of asbestos in the lungs of the general populationof the industrial countries of the western world whoare not occupationally exposed to asbestos. Gross,deTreville, and Haller (1969) found that they couldnot identify chrysotile in the core of 28 ferruginousbodies extracted from the lungs of urban dwellers notoccupationally exposed to asbestos in America.Langer (1970), however, states that the analysis of a

number of ferruginous bodies gave results which were

consistent with magnesium-leached chrysotile.Chrysotile has been shown to be a very commonconstituent of the dust contained in the lungs of a

rural population group in Great Britain by Pooley,Oldham, Um, and Wagner (1970) and also of a citypopulation group in America by Langer, Selikoff, andSastre (1971). As chrysotile appears to be rarelydetected in the cores of ferruginous bodies foundin the lung and yet appears to be a common con-stituent of environmentally exposed lung tissue, itseems that greater effort should be made toestablish the presence and characteristics of thismineral in lung tissue of those people likely to havebeen exposed to dusts containing it.

Chrysotile is the only asbestos mineral which canat present be positively identified as particles lessthan 1 micron in size. This is partly because of itsunique morphology but also because the mineralgives very characteristic electron diffraction patternswhen subjected to diffraction analysis in the electronmicroscope. Therefore, once it is detected in lungtissue, there is no difficulty whatsoever in establishingits identity with the aid of the electron microscope.

This paper reports the characteristics of chrysotilefibre found in lung tissue of people occupationallyexposed to chrysotile asbestos and also in the tissueof random samples of the general population. Theresults were obtained from a study of material drawnfrom a wide range of environments, including thosewho lived in rural, urban, and industrial areas, andalso from persons previously employed in theproduction and processing of chrysotile asbestos.No attempt will be made to report the incidence ofchrysotile obtained from the cases examined as thisinformation is to be published separately. Approxi-mately 300 cases have been examined and these breakdown into the groups listed in the Table.

TABLE

Source Description Number ofcases

Dorchester, General population 48England groupLiverpool, General population 40England groupMalmo, Pleural and peritoneal 33Sweden mesotheliomaMalmo, General population 32Sweden controlsLondon, Retrospective general 45England population groupCanada Mesothelioma cases 16Holland Mesothelioma cases 28Holland Mesothelioma controls 13Great Mesothelioma cases 18BritainGreat Industrial exposure 20Britain and groupSouthAfrica


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148 F. D. Pooley

Methods and materialsThe lung material examined was received as standard6-micron histological sections in paraffin wax, but in alarge number of cases larger portions of formalin-fixedtissue were available from which digested tissue extractswere prepared. The digestion technique employed wasthat formulated by Gold (1967), in which diced tissue isdissolved away in a 40% solution of potassium hydroxideat a temperature between 60 and 1000.

Both forms of specimens were piepared for electronmicroscope examination as follows:(a) The digested lung residues were transferred directly toelectron microscope grids covered with a carbon supportfilm by allowing a drop of the residue suspension to dryon the carbon film. Where suspensions also containedlarge quantities of carbon or organic material, portionswere first dried and then ashed at low temperatures, e.g.,400 to 450°C, and then resuspended in distilled water forexamination. During this whole process the tissue residuestreated this way were retained in 15-ml glass centrifugetubes and then resuspended by shaking the tubes by hand.It is known that heating causes chrysotile to become brittleand that any agitation of a heated sample may causedisintegration of particles; care was therefore taken inthe interpretation of the results from such preparations.These numbered only 20 in approximately 120 digestedresidues examined.(b) Six-micron histological sections were examined fromall of the cases listed. They were prepared for an examina-tion of their mineral content by a combination of ashingand transference of the ashed section onto electron micro-scope grids. The sections on their glass slides were firstwashed in xylene and alcohol to remove the majority ofthe embedding paraffin wax. Sections were then ashed atbetween 450°C and 500°C on their glass slides in a smallmuffle furnace. The slides were held in the furnace for asufficient time to reduce the tissue to a state such thatparticles of all sizes could be resolved from the tissueremains. Complete ashing destroys all the tissue. Removalof the sections before total ashing was obtained ensuredthat the outlines of the histological preparation werepreserved, while the ashed residue itself still retained somemechanical integrity which was extremely useful inobtaining a successful transference of the partly ashedsection onto the electron microscope support grid. Anashing time of 15 minutes at the stated temperatures wasfound to be sufficient to give the type of specimenrequired.The tissue ash plus mineral was removed from the glass

slide by an extraction replica process, using a thin film ofpolyvinyl alcohol as a carrier for the ashed section. Thepolyvinyl alcohol used was a high viscosity preparationof the polymer and was applied to the ashed section as a10% solution by weight in distilled water. A thin film ofthe polyvinyl alcohol solution was allowed to dry over theashed section on the glass slide and was then removed,lifting the adhering ash from the slide. This film was theninverted and placed in a standard vacuum coating unit anda layer of carbon was evaporated onto it, thus coating theadhering tissue ash. The preparation was then placed withthe plastic film facing downwards on a warm distilledwater bath to remove the plastic coating, leaving thetissue ash adhering to the carbon film. Random portionsof this film were picked up on electron microscope

_ _ _ _ _ Gloss slide plusashed section residue

2PVA film applied tocover ashed section

3 PVA film removed fromglass slide with adheringashed section

4Carbon film applied toadhering ashed section

5PVA film removed toleave final specimen

m glass slide m PVA filmashed section carbon film

FIG. 2. Schematic presentation of the preparation ofashed thin sections for electron microscope examination.

support grids for examination. A schematic presentationof the preparation procedure is given (Fig. 2).Both the techniques employed have the advantage that

they produce specimens for examination which arerepresentative of the whole mineral content of the lung.There is no bias in the selection of particles as in the caseof micromanipulation, for example. The digested lungresidues have one distinct advantage, that the mineral isconcentrated and so is suitable for specimens where theasbestos fibre content is very low. The ashed thin sectionshave the advantage that the mineral is located in the finalspecimen as it was in the tissue, so that, together with theoutline of the ashed tissue, a great deal of informationabout the location and deposition of any fibrous materialcan be obtained.

ResultsIt was found from examination of over 300 lungspecimens that chrysotile is an easy fibre type todetect with an electron microscope because of thedistinctive fine fibre structure of the mineral. Asthe particles of chrysotile were usually composedof numerous single fibres, no difficulty was encount-ered in obtaining good polycrystalline electrondiffraction patterns for positive identification of themineral. Figures 3 and 4 are two examples of thediffraction patterns obtained from chrysotile particlesby means of selected area electron diffraction.

Selected area electron diffraction patterns obtainedfrom chrysotile dust particles vary in character, de-pending partly on the size of the dust particle fromwhich the diffraction pattern is obtained, and also onthe size of the diffraction aperture employed whenrecording the pattern. The larger the diffraction aper-ture used, the larger the area of the particle or speci-men which is selected and thus contributes to thepattern. As the size of chrysotile particles increases


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FIG. 3. FIG. 4.

FIGS 3 and 4. Characteristic selected area electron diffraction patterns obtain.d from chrysotile dust particles.

the number of single fibrils from which they are com-posed also increases. Diffraction patterns obtainedfrom particles of chrysotile larger than a single fibrilare produced from a number of fibrils and are there-fore polycrystalline in character. In some circum-stances, where the diffraction aperture defines alarge number of randomly orientated crystallites,Debye-Scherrer ring patterns are obtained. As themajority of selected area diffraction patterns fromchrysotile particles are polycrystalline, the character-istic features of the patterns are the curved reflectionswhich can be observed in Figures 3 and 4. If thecurved reflections are sufficiently pronounced andthe pattern is not too distorted, the distances betweendiametrically opposed reflections can be measuredand the interplanar spacings or 'd' values producingthe reflections can be calculated and compared withdata obtained from reference samples. Comparisonof the unknown diffraction pattern with the diffrac-tion patterns obtained from known particles ofchrysotile is also useful in order to compare theposition of reflections which appear as either arcs orsingle spots.The chrysotile particles in lung specimens were all

extremely variable as predicted from the examinationof dust particles of commercial chrysotile. Theyvaried from bundles or strands of chrysotile fibres ofvarious lengths, sometimes curved and twisted,sometimes straight, to single fibres of chrysotile,again of various lengths and in a variety of attitudes.Aggregated fibres were also found sometimes

containing only several small fibres, but also severalthousand fine fibres. Examples of these varioustypes of particles are illustrated (Figs 5 to 8).The great majority of chrysotile fibres detected in

lung tissue had developed no ferruginous coating.

- *4VVW, _

St

FIG. 5. Large aggregate of chrysotile fibres from a nonoccupationally exposed case.


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150 F. D. Pooley

fibres by some several thousand to one. An exampleof one classical asbestos body formed on a bundle ofchrysotile fibres is shown (Fig. 9).The examination of chrysotile particles in those

specimens prepared from ashed thin sections sug-

FIG. 6. Bundles of chrysotile fibre detected in an occu-pationally exposed case.

FIG. 8. Large, loose aggregates and long, single chryso-tile fibres of the type observed in occupationally ex-posed cases.

FIG. 7. Single chrysotile fibres characteristic of a nonoccupationally exposed case.

Classical asbestos bodies containing a chrysotilecore were very rare and were found only in thoselungs which had received a heavy exposure tochrysotile fibre. Free or uncoated chrysotile fibres inthe heavily exposed specimens outnumbered coated

9,

FIG. 9. Asbestos body formation (A) on a bundle ofchrysotile fibre, from an occupationally exposed case.

IA

I.7 Abl,.714:


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gested that some of the particles may have changed inappearance after deposition in the lung. Some of the A

less compact fibre aggregates could have been ,produced by opening of the fibre bundles under the _action of lung fluids or they could also have beenformed by the deposition of a large number ofparticles in one particular location. Fibres andbundles of fibres were often found conforming tothe shape of airways, while the majority of the .chrysotile particles were grouped together in large *clusters. These large clusters were often detected atthe bifurcation of small airways and illustrated thetendency that fibrous particles have to be pre-ferentially deposited at positions where their extremeshapes interfere with the ability to change theirdirection of motion, as proposed by Timbrell (1965).Besides these large clusters of particles, finerchrysotile fibres were also found scattered throughoutthe lung sections. Some examples of the variousdeposits from ashed, thin sections of lung tissue areillustrated (Figs 10 to 12).As expected from the examination of prepared FIG. 11. Ashed section of an occupationally exposed case

chrysotile dusts, the particles of chrysotile seen in showing both amphibole and chrysotile fibres.preparations of lung tissue of necropsy cases varieda great deal in character. Although it has not beenfound possible to apply any rigid dimensions to theparticles of chrysotile detected, it has been foundpossible to divide the lung specimens examined intotwo groups, in each of which chrysotile particles /were found mainly in a particular form.The findings in lungs of men with occupational NA

exposure differed from the control groups in two

~~~AA~~ ~ ~

FIG. 12. Fibre aggregate (A) detected in an ashed sectionfrom a non occupationally exposed case.

ways. First, there was a great deal more asbestos inAi.fr / the occupational group and, secondly, the kind of

l 'rfibres and aggregates of fibres was different. In theFIG. 1O. An area ofan ashed section from an occupation- occupational group the fibre was practically alwaysally exposed case showing the tissue ash and associated found in the form of strands, i.e., bundles of singlechrysotile fibre. chrysotile fibres. These strands of fibre varied in


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152 F. D. Pooley

thickness, i.e., in the number of singlcontained, and were often twisted antheir ends. Single fibres of chrysotile wein these cases, but the single fibres werevery long in relation to their diametrarely less than 1 micron in length. Aithese occupationally exposed cases

(Fig. 13). It was only in these occupatioilungs that asbestos bodies containircores were found.

In the control groups of specimerfibres were always very sparse. A nui

parations from these groups, which incfrom industrial, rural, and urban e

were found to be free of chrysotile. Mthe chrysotile occurred in two main folarge aggregates of several hundredsecondly, as dispersed, very short indor small bundles of chrysotile. These tillustrated (Figs 5 and 7). The obsLanger and colleagues (1971) from a goccupationally exposed cases confirmpicture presented above. Their resulpredominance of single chrysotile fibresthe majority of the particles detectlength: diameter ratio of less than 10:1Both aggregates and short, single fi

bundles are apparently unsuitable vel

formation of ferruginous or asbestcthe classical type. In general, a prethe formation of an asbestos body is a

WW

tributed throughout the tissue ash of an

exposed case.

le fibres theyid bushed atre also found- generally allter and weren example ofis illustratednally exposedng chrysotile

exceeding several microns in length; rarely did thechrysotile fibres in the non occupationally exposedgroup have this particular characteristic and in factno asbestos bodies containing chrysotile cores werefound in this group, although bodies on amphibolefibres were detected.

DiscussionVorwald et al. (1951) observed that, by reducing the

as, chrysotile length of the chrysotile dust injected into guinea-pigmber of pre- lungs from 20 to less than 3 microns, they reduced-luded people the formation of asbestos bodies to practically nilnvironments, and those which were observed were found to haveVhen present, formed on the few long fibres which were contained)rms: first, as in the -3 micron dust. Similar observations with

fibres and, amphibole fibres confirm the dependence of theLividual fibres formation of the classical asbestos bcdy on fibretwo types are length.servations of The differences in character between chrysotilegroup of non dust particles contained in the lungs of occupationallyi the general and non occupationally exposed persons are not tooIts show the difficult to comprehend when the production of dustover bundles, particles is considered. Occupationally exposeded having a individuals generally handle raw fibre which has

been treated in such a way that the long fibrousibres or fibre nature of the material is retained as much asiicles for the possible. Chrysotile asbestos fibre is only commercial)s bodies of because it can be produced in long fibre or strandrequisite for form. It is therefore safe to say that the majority ofstraight fibre dust particles produced by the handling of raw

commercial fibre will be fibrous (i.e., strand-like)as the amount of mechanical handling it receives iskept to a minimum to preserve this property.The chrysotile dust particles found in the lungs of

non occupationally exposed persons will have beenderived from different sources than in occupa-tionally exposed persons. The sources will be themany asbestos products containing chrysotile andfrom which dust particles are produced by far moremechanical action than the raw fibre ever receives.It is likely, therefore, that the character of particlesproduced by the secondary handling of chrysotileasbestos products will be different.Langer et al. (1971) found that chrysotile asbestos

is biologically affected by its residence in the lung,resulting in a loss of its magnesium content, anobservation also reported by Morgan and Holmes(1970). Of the chrysotile particles detected in the 300cases examined and subjected to diffraction analysis,all were found to give the characteristic. chrysotile

Aim diffraction pattern. Studies by Morgan and Pooley(1970) of magnesium-leached chrysotile have shownthat the characteristic chrysotile diffraction patterncan be obtained from fibres with up to 50% of the

s found dis- magnesium removed. The fibres found and tested inoccupationally the cases reported in this paper could therefore have

lost up to about 50% of their magnesium content,


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but leaching, it appears, had not proceeded beyondthis point.The detection of chrysotile dust particles in the

lungs of persons handling the mineral is to beexpected in every case under the optical microscope.The detection of chrysotile dust particles in the lungsof non occupationally exposed persons would not, itwould seem, be very likely. This is simply because thefibres are smaller in diameter, and near the power ofresolution of the optical microscope, and also becausethere is little body formation on such fibre.

It is likely that the chrysotile dust inhaled byasbestos workers was freshly made and containedparticles of a relatively high sedimentation rate,whereas the control cases would be less likely to beexposed to fresh dust and therefore slow sedimen-tation or resuspendable particles would pre-dominate in their lungs. This is confirmed by theobservations performed on the control cases whereaggregates and short individual fibres form thepredominant chrysotile particle types detected.Both of these particulate forms possess very muchlower sedimentation rates when compared with thefibre bundles found in the occupationally exposedcases.

ConclusionsChrysotile fibres in lung sections and residues pre-sent a wide variety of patterns due to their smalldiameter, great variation in length, curved shape, andthe tendency to aggregate into bundles of fibrils. Nosimple description can adequately describe thevariety of appearance, but the high specificity of thediffraction pattern enables positive identification ofindividual fibres which can be selected for examina-tion with the aid of the electron microscope.The hollow appearance characteristic of single

chrysotile fibrils is also useful in substantiating anidentification but cannot in itself be considered to bea feature on which positive identification can bemade. As in all mineralogical studies, identificationmust be based on the observation of a number ofphysical and chemical properties of the material.The character of chrysotile dust particles found in

the lung differs between occupationally and nonoccupationally exposed individuals. In the formergroup, the particles are generally found as strands ofsingle fibres, mainly grouped together in discretelocations in the lung. In the latter group, the majorityof the chrysotile particles are found as aggregatestogether with short single fibres and fibre bundlesdistributed about the lung.

Ferruginous bodies or asbestos bodies have beenfound to form very rarely on chrysotile particles and

only on particles suitable for their formation, i.e.,straight fibre bundles over several microns inlength. Chrysotile dust particles are therefore notalways suitable to act as a nucleus for a classicalasbestos body shape. The majority of asbestos bodiesseen under the optical microscope may be said to beformed on amphibole asbestos fibres.

This work has been supported by the Medical ResearchCouncil and the International Agency for Cancer Re-search. I should like to thank Dr. J. C. Gilson and Dr.J. C. Wagner for their help in the preparation of thismanuscript.

References

Gold, C. (1967). A simple method for detecting asbestos intissue. J. clin. Path., 20, 674.

Gross, P., deTreville, R. T. P., and Haller, M. N. (1969).Pulmonary ferruginous bodies in city dwellers. Arch.environm. Hlth, 19, 186-188.

Langer, A. M. (1970). In Applied Seminar on the LaboratoryDiagnosis of Disease Caused by Toxic Agents, edited byW. F. Sunderman and F. W. Sunderman, Jr., pp. 126-136.Warren H. Green, St. Louis.

, Selikoff, I. J., and Sastre, A. (1971). Chrysotile asbestosin the lungs of persons in New York City. Arch. environm.Hlth, 22, 348-361.

Lynch, J. R., and Ayer, H. E., (1968) Measurement ofasbestos exposure. J. occup. Med., 10, 21-24.

Maser, M., Rice, V. R., and Klug, H. P. (1960). Chrysotilemorphology. Amer. Mineralogist, 45, 680-688.

Morgan, A., and Holmes, A. (1970). Neutron activationtechniques in investigations of the composition andbiological effects of asbestos. In Pneumoconiosis: Proc.Int. Conference, Johannesburg, 1969, edited by H. A.Shapiro, pp. 52-56. Oxford University Press, Cape Town.

- and Pooley, F. D. (1970). Private communication withHealth Physics Section Atomic Energy Research Estab-lishment, Harwell.

Pooley, F., Oldham, P., Um, Chang-Hyun, and Wagner,J. C. (1970). The detection of asbestos in tissues. InPneumoconiosis, Proc. Int. Conference, Johannesburg, 1969,edited by H. A. Shapiro, pp. 108-116. Oxford UniversityPress, Cape Town.

Speil, S., and Leineweber, J. P. (1969). Asbestos minerals inmodern technology. Environ. Res., 2, 166-208.

Timbrell, V. (1965) The inhalation of fibrous dusts. Ann.N.Y. Acad. Sci., 132, 255-273. .

, Pooley, F., and Wagner, J. C. (1970). Characteristicsof respirable asbestos fibres. In Pneumoconiosis, Proc.Int. Conference, Johannesburg, 1969, edited by H. A.Shapiro, pp. 120-125. Oxford University Press, CapeTown.

Vorwald, A. J., Durkan, T. M., and Pratt, P. C. (1951).Experimental studies of asbestosis. Arch. industr. Hyg.,3, 1-43.

Yada, K. (1967). Study of chrysotile asbestos by a highresolution electron microscope. Acta crystallogr., 23.704-707.

Received for publication July 15, 1971.

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electron microscope characteristics of inhaled chrysotile ... · department of mineral...

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