Document n9aLGg4v4ZyOQa0yyzQprkpXX

1>, 11.. Gilson. ' k. & McDon.: , chrysotileasbo-a - Archives of /:- 58-400 ' I' pleural ealeifica- ,ers. Annals ofthe . 351-367 Eifects of asbestos atJournal, iii, 574-- ,-g. L. (1943] Dust ,-luding roentgcno- nerican Journal of '.11-29 ' (1965) Proposed sis. Annals of 'k- 3"'3-378 on resulting fro" t Journal of Roem- genesis of pleural .. Pneumoconiosis, ference, JohannesUniversity Press, (1970) Circulating a asbestos workers. -c and its results in jconiosis. Proceed 'd', Johannesburg, tv Press, pp. 192- \t. (197! | Instuaison of a randf"* British Journal ' L. (1967) Pleura. . Environmental PLAINTIFF'S EXHIBIT Structure and composition of pleural plaques L. LE BOUFFANT,1 J. C. MARTIN,1 S. DURIF 1 & H. DANIEL 1 | The study described below is of human pleural at their lower edge by a thin network of elastic fibres .J' plaques taken from patients occupationally exposed which is stained by orcein and which itself lies on f to asbestos dusts; for purposes of comparison subjacent adipose tissue. The surface, which is ? plaques taken from patients with calcifications of bounded by endothelium, is frequently covered with f completely different etiology, such as tuberculosis or nodules in which collagen fibres are arranged con i emphvsema, were studied. J f centrically. like onion rings. There are only a few cellular elements, mainly ) MACROSCOPIC APPEARANCE fibroblasts, inside the plaque itself. Inflammatory % type cells are never encountered. On the other V% The specimens studied were taken preferably from hand, in the peripheral zone at the edge of the f the parietal pleura and had the well-known macro- plaque, small agglomerations of lymphopiasmocytes 1 scopic appearance of pearly-white plaques, firm to or histiocytes or of mast cells can be seen in the the touch, a few millimetres thick with an area vicinity of small vessels. ; ranging from a few square centimetres to about 50 The calcifications are usually situated in the col . cm2. In most cases the free side of the plaque had a lagenous zone; and in section, after staining with mammillated surface. '" yon Kossa stain or following micro-incineration, Seen in section both the plaque and the mam- they show up as Battened nodules or thin laminae of ? millae have a laminated texture which can clearly be varying area, or as more diffuse deposits. They are [ seen under the magnifying glass. Examination in preponderant in the central or deeper zone of the I. section also reveals possible calcifications. These plaque, and they are separated from the endothelium !! almost always appear in the median plane of the by a relatively thick layer of non-calcified fibro plaque and in the centre of the mammillae. If the hyaline tissue. calcification is already compact, it can be clearly differentiated from the neighbouring parts of the LLTRASTRUCTURE plaque and can be cut away from them. The calci fied zones may not be clearly visible in fresh tissue, but if they are kept in formalin for some time they The fibrous and calcified zones of the plaques will be considered separately. can be distinguished by a markedly yellow colour. Fibrous zone HISTOLOGY Pleural plaques are made up of fibrohyaline con nective tissue, practically free of cells, made up essen tially of thick bundles of collagen fibres separated by optically empty pseudolacunae. They are bounded : Centre d'Etudes et Reclierches des Charbonnages de France, 60550 Verneuil-en-Halatte. France. Whatever the size of this zone in relation to the pleural plaque as a whole, a certain number of com mon ultrastructural characteristics are found in all the cases examined, with only a few variations. Electron microscopy first of all confirms the struc tures that can be seen under the light microscope, i.e.. bundles of collagen fibres embedded in a hyaline matrix (Fig. 1). These fibres usually show the nor mal periodicity of collagen (64 nml: on the other hand, some of them have a markedly larger diameter - 249 - 250 BIOLOGICAL EFFECTS OF ASBESTOS than the general average (200 nm as compared to 30 70 nml (Fig. 2). Also, some cell debris is en countered embedded between the collagen layers. In addition to these organic components, numer ous electron-dense particles can be seen scattered in the fibrohyaline tissue (Figs. 3 & 4). Some of these particles are solid or laminated rounded bodies with a diameter between 0.1 and 0.5 (xm. Others, also com pact and of similar dimensions, have a marked fibrous external structure which consists of very fine needles with diameters ofthe order of 7.5 nm. Finally, other types of markedly differentiated dense particles are found, some of them in the form of spheroid or ovoid plaques of fragmented lamellated appearance with a diameter of the order of 1 um, and others as bundles of acicular crystals-of very varied dimen sions. These different types of electron-dense bodies represent incipient calcifications. They are soluble in acids, and most of them produce an electron dif fraction pattern corresponding to that of whitlockite in the case of the compact or lamellar ovoid forms and of apatite in the case of acicular crystals. Examination under high magnification after lead staining shows dark granular patches with incipient crystallisations or impregnating crystalline masses in the course of growth. ' It should be noted finally that the nascent calcifi cations, particularly those of the whitlockite type, are situated mostly in zones containing cell debris. However, early calcification of the apatite type can also be seen along the length of the collagen fibres. An important point to note is the similarity be tween these structures and those encountered in the same subjects in the thickened visceral pleura, where fibrohyaline tissue enclosing cell debris and rounded electron-dense bodies made up essentially of whit lockite can also be observed. Calcified zone This zone is characterised by an invasion of the fibrohyaline tissue by acicular apatite crystals, among which whitlockite nodules persist (Fig. 5). The crystals apparently grow along the length of the collagen fibres whose underlying periodicity is still visible in many places. Phosphotungstic acid or hydrochloric acid dis solve the apatite and allow the collagen fibres to be seen (Fig. 6). Lacunae may persist among the agglomerations of apatite crystals: in these a fairly loose-woven fibro hyaline tissue can be seen which is found with !e.:a staining to consist of a fine reticulation. These lacunae also contain isolated apatite crystals. Comparison ofpleural plaques and pulmonary calcifi cations of different etiologies As was stated above, there is a marked similarity between the calcifications found in the fibrous zone of a parietal pleural plaque and those seen in the thickened visceral pleura of the same individual. To determine whether this similarity applied to other types of calcified lung tissue, we examined three specimens from pathological calcifications of totally different origins, i.e.. (i) a fibrohyaline plaque from a subject whose occupation apparently involved no exposure to asbestos dust (a search for asbestos fibres, made on the basis of the method described later, proved negative); (ii) a gas cyst; and (iii) a tuberculous caseification. In all three cases electron-microscopy revealed the presence of electron-dense rounded bodies against a background of cell debris and of collagen or elastic fibres, according to the type of lesion. The spheroid calcifications sometimes had a laminated appearance. When oriented crystals were present, these needles grew among the structured elements of the fibrous weft, the collagen or the elastic fibres. Altogether these calcifications showed a marked similarity to those of pleural plaques. COMPOSITION or THE PLEURAL PLAQUES Fragments of pleural plaques were analysed for theircontents ofcollagen, phosphate, calcium, sulphur and hexosamines. Measurements were made separ ately in the fibrous zone and in the calcified zone. Collagen Specimens were first decalcified, and the collagen level was determined after acid hydrolysis by mea surement of hydroxy proline. Table 1 shows the col lagen values in the decalcified tissue and, forpurposes of comparison, the concentrations in a fibrohyaline plaque from a subject not exposed to asbestos dust. There appears to be no significant difference be tween the collagen levels in the two zones of the calcified pleural plaques. Moreover, the proportion J with lea < on. These tals. ary calcifi- J similarity brous zone seen in the ividual. applied to nined three is of totally ject whose \posure to s, made on ter, proved evealed the es against a n or elastic he spheroid tppearance. :ese needles the fibrous J a marked QL'ES .nalysed for urn, sulphur made separtied zone. the collagen sis by mell ows the col'or purposes librohi aline bestos dust, itference beones of the e proportion "*r Fig. 2. Parietal pleural plaque--fibrous zone--collagen fibres with large diameter. !! t Fig. 3. Parietal pleural plaque--fibrous zone--incipient calcifications--A -- whitlockite; B -- apatite; C collagen Fig of cc F;g. 4 Parietal pleural plaque--fibrous zone--lamellated structures. l ! i ?& ,,;ri- 7JK.4-#?' '%*p ::* .1 Fig 8. Parietal pleural plaque---calcified 2cne--chrysotile fibres visible after treatment with phosphotungstic acid and trypsin. 256 BIOLOGICAL EFFECTS OF ASBESTOS studied the fibres were those of chrysotile. In the sections treated with phosphotungstic acid and tryp sin the fibres are surrounded by a thin sheath of an unidentified substance. The results given in Table 4 show that pleural plaques found in persons who have been exposed to asbestos dusts may contain considerable quantities of very fine fibres whose presence can be demonstrated only after calcium deposits and collagen fibres have been eliminated. Table 4. Results of asbestos fibre counts No of fibres per cm3 of tissue | Case No 1 Case No 2 of these minerals, so that a more thorough stud; would appear to be necessary to clarify this point. Organic matter This consists essentially of fibres and of amorphous matter. The collagen fibres are of normal period icity with a small proportion of wide diameter fibres. Ultrastructuraliy, the amorphous matrix shows a microfilamentous organisation similar to that de scribed for the ground substance of collagen tissue. Furthermore, biochemical analysis demonstrates the presence of hexosamines in greater quantities in the calcified zone than in the fibrous zone; and this is confirmed more specifically for the acidic muco polysaccharides by histochemical methods. Lung tissue Pleural plaque fibrous zone calcified zone i io x i j 1 6*10 j 30x10 Diameter of fibres : 25-30 nm Length of fibres : 0.1-2 jxm 3.6x10 40x10 DISCUSSION The organisation of pleural plaques as seen in the light microscope has already been described and calls for no particular comment. On the other hand, infrastructural and biochemical study and electron diffraction techniques have thrown light on a certain number of points: Calcifications Microscopic nodular or acicuiar calcareous struc tures can be seen scattered throughout the fibrous part of calcified plaques as well as in fibrohyaline plaques. These structures are similar to those which form large areas of mineralisation in the calcified zones, thus indicating that these different tissues are related and essentially differ only in their degree of calcification. Nascent calcifications are encountered in the fibrous zone mainly in the sectors which contain cell debris, or within the hyaline substance. In the cal cified zone, the growth of the crystals is orientated in part by the collagen fibres. The calcifications are essentially of the whitlockite and apatite type. Significant differences are found, however, in the phosphorus and calcium composition Asbestos fibres The presence of numerous asbestos fibres in the pleural plaques of persons exposed to asbestos dust constitutes an important finding. Tn the cases examined the fibres were all of very small size and hence completely invisible under the light micro scope; moreover, their detection by electron micro scopy requires a suitable technique. It should be noted that they are present in a much higher concen tration in the calcified zone than in the fibrous zone. Histogenesis of the pleural plaques Data on this are still very incomplete and thus we are unable to sketch (he probable process for the formation of pleural plaques. It may, however, be noted in regard to the condi tions under which fibrohyaline plaques appear that they are not the specific result of exposure to asbestos and that they can be observed in other circumstances. On the other hand, it is interesting to note that the only pleural plaques showing massive calcifications contained large amounts of asbestos fibre, and that the calcified zone contained more abundant fibres than did the surrounding fibrous zones. At a more basic level, the initiation of these calcifi cations and their shape seem to be dependent on the substrate on which their nucleus is formed. To sum up. although the appearance of fibro hyaline tissues and of calcifications are both rela tively common phenomena in general pathology, it may be assumed that the occurrence of these two processes together in this particular site suggests a definite relationship with the risk of asbestosis. 1 stuc v> im. 'rphous periodt fibres, shows a hat de1 tissue, ales the .'S in the .1 this is : muco- s in the tos dust e cases size and ; microi microould be concenus zone. STRUCTURE AND COMPOSITION OF PLEURAL PLAQUES 257 SUMMARY \ -.uidy ha-. been made of the ultrastructure of fibroInalme tissue and calcification in pleural plaques. The microscopic calcifications present in the fibrous zone of these plaques are comparable to those in the calcified zone which hate the structure of whitlockile and apatite; however, their P:Ca ratio is different from that of those phosphates, particular^ in the highly calcified zone. The level of hexosammes. and particularly of acidic muco polysaccharides, is higher in the calcified zone than in the fibrous zone. Calcified pleural plaques from persons exposed to asbestos dust contain numerous fibres of that mineral. ACKNOWLEDGEMENTS The authors are indebted to C. N'ormand and G. Tichoux for their technical assistance. REFERENCE Boas. Y. F. (1Q53) Method for the determination of hexosamine in tissues. Journal of Biological Chemistry, 204, 553 thus we for the e condi'ear that asbestos istances. :that the tications and that at fibres -.e cttlcifint on the of fibrooth rclaology. it hese two jggests a isis. t f