The American Journal of PATHOLOGY JUNE 1976 * VOLUME 83, NUMBER 3
Pulmonary Mineral Dust A Study of Ninety Patients by Electron Microscopy, Electron
M icroanalysis, and Electron M icrodiffraction J. P. Berry, PhD, P. Henoc, Ing, P. Galle, MD, R. Pariente, MD
The results of a study of 90 patients are presented. Intrapulmonarv mineral deposits were characterized by electron diffraction and electron probe microanalysis. Using this method, pneumoconioses mav be distinguished from other pneumopathies. In cases of pneumoconiosis, there exists a specific relationship between the etiolog,y of the dust exposure and the crvstallographic characteristics of the intrapulmonary deposits. The nature of the deposits mav be indicative of a specific type of pneumoconiosis. This method is particularly useful in differentiating between asbestos bodies and ferruginous bodies. The value of the method in general and its importance in the study of pneumoconiosis are discussed. (Am J Pathol 83:427-456, 1976)
NI YN STU DIES have been devoted to the direct analysis of intrapulmonary mineral deposits in histologic sections. The first studies carried out on ultrathin sections using electron microdiffraction were useful in determining the crystalline or noncrystalline character of the deposits;6 however, without supplementary information concerning the chemical element present, microdiffraction data alone are generally insufficient to identify the specific crystal present. Nloreover, electron diffraction is of no use in studying the numerous noncrvstalline mineral deposits present in human lungs: for example, black intrapulmonary pigment cannot be studied by this means. The first studies using the electron microprobe analy-zer of Castaing were made on thick tissue sections (3 ,)1.8 They showved that the black From the Labortoire de Biophysique. Faculte de \ledecine de Creteil. Creteil. C\ET. IssslesNIoulineaux. Hopital Antoine-WB-eclre. Clamart. France Supported by the INSERM Institute kA. T P 8. Pathologie broncho-pulmonaire et pollution). Taken from the thesis submitted by Jean-Pierre Berry to the Faculty of Medicine of Creteil in partial fulfillment of the requirements for the Doctorate degree in human biology. Xccepted for publication December 29. 1973. Xddress reprint requests to Dr J -P Bern-. Laboratoire de Biophysique. Faculte de \ledecine de Creteil. 6 rue du Ge&neral Sarrail. 94. Creteil. France
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intrapulmonarv pigment known as anthracosis pigment was composed, not of carbon as had been previouslyr suspected, but of a large number of mineral elements. The analysis of manv lung specimens subsequently showed that these same mineral elements could be found in all cases of intrapulmonary deposits whether occurring in normal lungs exposed to ordinary atmospheric pollution or lungs from patients having pneumoconiosis: electron probe microanalysis of thick sections, therefore, does not establish a differential diagnosis. These studies were extended to include the analysis of deposits in ultrathin sections (1500 A). Each deposit was analyzed by an electron probe microanalvzer coupled with an electron microscope and by electron microdiffraction. Preliminary results have been published Fl showing that only this combination of analytical methods establishes with certainty the chemical and crvstalline character of each mineral deposit present in the macrophages. This report presents the results of a study of 90 patients, some of whom were exposed only to normal atmospheric pollution and others who were affected with pneumoconiosis. Materials and Mefthods Tssue Spcmens All tissue specimens were obtained from suburban Paris hospitals (Pneumo-Phtisiology Unit of Professor Pariente, H6pital Antoine Becl&e, Clamart; and Pneumology Unit of Professor Chretien, Hopital Intercommunal, Creteil) w here the clinical diagnoses w-ere
made. The specimens were obtained either at biopsy, surgery (surgical removal), or autopsy. We examined tissue from patients with or without diagnoses of pneumoconiosis. Pulmonar- tissue was fixed in buffered glutaraldehyde (pH 7.4), postfixed in osmium tetroxide, and embedded in Epon. Blocks were sectioned on a Reichert ENMU 2 Ultramicrotome with a diamond knife, picked up on grids with reference positions, and stained with uranv-l acetate. Specimens From Patients Without Pneumoconiosis A total of 40 specimens was studied. In 20 cases, tissue was selected at random from the
following areas of the lung: anterosuperior, anteromiddle, anteroinferior, posterosuperior, posteromiddle, posteroinferior, subpleural, peribronchial. In 20 cases (lobectomv), tissue was selected from regions having the macroscopically dark appearance characteristic of dust accumulations. Specimens From Patients With Pneumoconiosis More than 50 specimens from patients with pneumoconiosis were examined. Specimens were obtained at biopsy, surgical lobectomy, or autopsy. Tissue w-as selected
from darkened regions of the specimens.
Techniques Sections were examined with a Philips EN :300 electron microscope. Electron probe microanalvsis was done using a CANMECA NMS 46 microanalyzer
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equipped with an electron microscope. Details of the analI-tical technique has-e been prev iousli published.9 Electron microdiffraction u-as done using a Siemens Elmiskop I electron microscope equipped with a goniometer stage and double condenser.
Results Lungs From Patients Without Pneumoconiosis-"Normar Logs
Masses of dense intrapulmonary mineral deposits were found in the cv-toplasm of alveolar macrophages. licroanalysis of the entire group of deposits present in a macrophage showed the silicon, aluminum, iron, titanium, and carbon; these elements were found in all lungs (Figure 1). Ho wever, wvhen each inclusion X-vas analyzed separately in ultrathin sections, differences were found in chemical composition and ultrastructural morphology. Three main kinds of deposits could be distinguished according to these two criteria. The most frequently encountered deposits were made up of clumps of small, moderately electron-aense spheres surrounded by a halo of fine needles. This tvype of inclusion contained Si, Fe, and Al (Figure 2A). A second type of deposit was formed by clumps of very electron-dense spheres and contained only Ti (Figure 2B). The third tvpe wvas made up of large homogeneous electron-dense masses hav ing clear-cut straight edges. They wvere found to contain C and Fe (Figure :3). Another type of inclusion of irregular shape containing C and Fe was occasionally noted. Electron diffraction diagrams of all these inclusions showed the diffuse rings characteristic of amorphous structures. In certain cases, these observations wvere extended in the following way: the sections wvere again introduced into the electron probe microanalyzer and the same deposits wvhich on previous anal-sis had show n the presence of Si Xwere bombarded wvith a strong electron current. This treatment heats the sections to a high temperature. Afterwards, electron microdiffraction anal-sis showed the thin ring pattern characteristic of the crystalline structure of Si. Heating the deposit wvas thus responsible for the formation of silicon crystals. The same treatment carried out on deposits containing titanium resulted in the formation of titanium dioxide (recognized from its diffraction diagram). These observations lead to the conclusion that these intracellular mineral inclusions do not possess a crystalline structure. Lungs From Patent Wiffi Pneumoconiosis
In this case, crystalline patterns were obtained without prior treatment of the mineral deposits. However, the diffraction patterns were generally
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not diagnostic of a specific crvstal without the supplementary data on chemical composition provided by electron probe microanalysis. Table 1 shows the names of the different minerals which we have identified, and their chemical formulas and crystalline structure. In Table 1 it can be seen that only about twentv minerals recur in all the cases of pneumoconiosis examined. We found that certain minerals are found in the majority of cases of pneumoconiosis, while others are found only rarely and are often the product of special circumstances. The minerals which are found in most cases of pneumoconiosis can be divided into three categories: a) minerals associated with silicosis, b) asbestos bodies and ferruginous bodies, and c) other minerals frequently seen in cases of pneumoconiosis. Minerals Associated With Cases of Silicosis
The minerals most often encountered in silicosis are quartz, micas, and clavs. Quartz. In the cytoplasm of certain macrophages we observed dense, rounded masses with irregular but generally well-defined contours. Some of the masses were thick and protruded somewhat from the section, while others were thin and showed a slight opalescence when the objective lens current was varied. Electron probe microanalysis of these deposits showed that only silicon was present. The microdiffraction diagrams presented the particularity of bright Kikuchi lines. Measurement of the parameters led to the identification as of these deposits quartz (Figure 4). Micas. The morphologic aspect of these inclusions varied greatly. Thev formed either dense compact masses or elongated leaflets. Electron probe microanalysis showed that the composition of these crvstals also varied. Spectra showed the presence of Si, potassium, Al, and Fe as well as these same elements plus magnesium. With this information, the microdiffraction diagrams can be interpreted. Together they showed that the micas most often present in silicosis are muscovite (white mica) and biotite (black mica, ferromagnesium) which is associated with Mg (Figures 5 and 6). Clays. The morphology of these crvstals is extremely varied. They may occur as heterogenous masses of small, dense grains; thin, larger, elongated fibers; small, dense regions dotted with regularly spaced clear circles; dense blade-like configurations; or in masses formed of thick layers or chevrons. Electron probe microanalysis shows the complexity of the chemical composition of these crystals. Microdiffraction diagrams show, in general, a hexagonal pattern which
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reflects the projection of the basal plane of the crystal. Since this plane is the same for all clays, it is impossible to distinguish between them. When diffraction patterns are obtained from other crystal planes, it is possible to use the information obtained from microprobe analysis to interpret these diagrams. Three kinds of clays were frequently found: illites (which contain Ti) (Figure 7) made up of fine, undifferentiated particles; montmorillonite (which contains K) (Figure 8), very malleable and which swells in water; and pyrophyllite (composed of only Si and Al) which resembles talc. The leaflets of clays are fragile and the ultramicrotome knife often distorts them, producing a sort of "layering" of the structures. This artifact results in deformations of the diffraction diagrams (ring or oval figures). (Figure 9). Clays are the minerals most often found in all cases of pneumoconiosis. Asbestos Bodies, Ferruginous Bodies
We observed a large number of fibrous structures made up of an elongated monocrystal often surrounded by a ferruginous coating. Nature of the Coating. The coating is formed of dense masses of small granules arranged in successive layers of irregular contour around the monocrystal. Electron probe microanalysis shows the presence of Fe. The microdiffraction pattern of thin rings corresponds, in the Association Society for Testing Materials (ASTM) classification of inorganic compounds, to the formula for FeO(OH). The nature of the central monocrystal distinguishes asbestos bodies from ferruginous bodies. Asbestos Bodies. In these cases, the axial fiber was a monocrystal of asbestos. Depending on the plane of section, the fiber profile appeared as a long wide blade or a small central inclusion. Microprobe analysis shows the predominant presence of Fe, Si, and sodium which, combined with the electron diffraction patterns, identified these crystals as being crocidolite (blue asbestos) (Figure 10). The presence of other amphiboles was also shown. Ferruginous Bodies. Although their morphologic aspect was identical in these cases, the axial fiber was not asbestos (Figure 1 1). We have shown the presence of different varieties of monocrystals in the axial fiber. Depending on the case, it may be composed of carbon, sillimanite (aluminium silicate) (Figure 12), sericite (white mica), or clay. Other Minerals Which Occur Frequently in Pneumoconiosis
Talc. Morphologically, these crystals appeared as large, waffled areas which disintegrated easily in the electron beam. Si and Mg were found by
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electron probe microanalysis, thus simplifying the interpretation of the electron microdiffraction patterns (Figure 13). Cristobalite. These small inclusions have a very characteristic geometric form (Figure 14). As in the case of quartz, microprobe analysis showed that onlv Si was present; however, the parameters measured from the diffraction pattern clearly showed the crystalline structure corresponding to cristobalite. This crvstal is normally formed at high temperatures, in volcanic regions, for example. Chlorites. These inclusions were found as groups of thin flakes. Certain of them had a verv characteristic form which consisted of elongated, sinuous crystals, grouped in bundles. Microprobe analysis combined with electron diffraction shows the presence of Si, Al, Mg, and Fe in crystals of vermiculite (thermal insulation material used in building construction) (Figure 15). Micas. Biotite and muscovite were also found frequently in cases of penumoconiosis.
Spinelles. These inclusions do not have any particular characteristics. They are metallic oxides of various metals (Ca, Fe, manganese, etc.) Particular Deposits Seen in Coalworker's Pneumoconiosis
In these lungs we observed macrophages filled with carbon deposits. Most of the carbon deposits were, however, encircled by a well-defined ring of fine particles (granules). Electron probe microanalysis showed these particles to be composed of silicon (Figure 16). Less Frequently Encountered Minerals
These minerals were found either alone or in association with others. Most often, their presence was the result of particular circumstances. Some of these minerals, however, have a characteristic morphology, as in the case of leucite, which occurred as round masses of concentric lamellae (Figure 17). Discussion Two points will be discussed: the characteristics of the techniques emploved and their usefulness in the study of pneumoconiosis. Te
es
Two different kinds of methods can be used to study intrapulmonary mineral deposits: a) methods of whole-sample comprehensive analytical methods, and b) methods of in situ analysis.
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Methods of Whole-Sample Analysis
These methods involve the examination of residues of inorganic material after destruction of the organic constituents of the pulmonary samples by treatments such as incineration at 500 to 800 C, oxygenation, or acid or alkaline digestion.15 21 The mineral residues are subsequently analyzed by various physical techniques: fluorescence, x-ray diffraction, mass spectroscopy, differential thermal analysis, etc.2,6'7,16,18-. These methods have two major drawbacks: a) Minerals obtained in this way may have been greatly altered by the heat, acid, or alkali treatment, and their crystalline structure modified. Certain minerals may even be destroyed altogether.21'2"'3335 b) Because of the destructive nature of these methods, no morphologic correlations are possible. Methods of In Situ Anaysis
The second group of methods in which mineral inclusions were analyzed in tissue sections have the advantage of furnishing morphologic correlations. Three techniques were used: histochemical methods, electron microdiffraction, and electron probe microanalysis. There are only a few histochemical reactions for chemical elements, and the results are often nonspecific.5'" The utilization of electron microdiffraction alone on ultrathin sections yielded diffraction pattems which were very difficult to interpret since the small quantity of matter present in the section did not fumish sufficient information. Electron probe microanalysis used alone did not provide data for a differential diagnosis. Using thick sections (5 ;), the analysis of pulmonary deposits gave approximately the same results for all lungs analyzed. However, the combined use of electron probe microanalysis and electron diffraction makes a precise determination possible. The data from microprobe analysis provided the elementary chemical composition of each particle, and these data helped in the interpretation of the microdiffraction diagrams. Specifically, they narrow the choice to a single crystal from the many possible crystals which coincide with the parameters measured in the diffraction pattern.910'11 Consequently, the combination of data from electron probe microanalysis with that from electron microdiffraction seems, at the present time, to be the method of choice in determining the chemical and crystalline nature of mineral inclusions.
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the Sbtuy of Ps
In this area three points deserve emphasis: 1. Crystalline structures are not found in all cases. We have observed that deposits found in nonpneumoconiosis lungs were not in a crystalline state. This is so despite of the fact that almost all inhaled atmospheric dust particles are crystalline. It mav, therefore, be supposed that the macrophages of pulmonary tissue are capable of denaturing the dust particles and reducing them to an amorphous state. Certain similar processes have been noted in tissue culture."," Moreover, it is known that some mineral crystals undergo degradation in an acid milieu. Intracellular acids may be furnished by Iysosomes.-m In cases of pneumoconiosis, on the other hand, most mineral deposits are found to be in the crystalline state. It is, therefore, possible to establish a differential diagnosis by the simple presence of intrapulmonarv crvstals. 2. The determination of the nature of the axial fiber in asbestos or ferruginous bodies is difficult. The industrial use of asbestos and its possible role in carcinogenic processes have stimulated many studies 2 on the effects of asbestos fibers on the organism. In previous studies, inorganic residues were analyzed, as were fibers with or without their enveloping sheaths, without succeeding in localizing these inclusions in the tissue, thus leading to the following conclusions: a. The techniques used for isolating the fibers introduce the artifacts already mentioned above (alteration of crystal structure, etc.,). Moreover, contamination by atmospheric dust particles cannot be systematicallv eliminated. b. The identification of asbestos fibers is particularly difficult. The mineral asbestos is made up of two subgroups: the amphiboles and the serpentines. Almost all varieties of asbestos are found in the fibrous form of amphiboles which have very similar crystalline parameters. Their chemical formulas vary, however, making it possible to distinguish different groups (sodium-containing, non-aluminium-containing, and aluminium-containing). The sole representative of the serpentine group is chrysotile (magnesium silicate). This crystal structure undergoes almost complete denaturation when subjected to the treatments used to destroy organic material.2 The differentiation of asbestos from ferruginous bodies is also made verv difficult by the presence of the ferruginous coating which surrounds the axial fiber. It should be emphasized that ferruginous bodies are found not only in
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all cases of asbestos pathology but also in numerous other pneumopathies. Once again, analy sis of crystals in ultrathin sections in wvhich the chemical nature is determined by electron microprobe analysis and the crystal structure by electron microdiffraction is at the present time the only method by which the nature of these fibers can be accurately determined in pulmonary tissue. 3. There is a relationship between the characteristics of pulmonary mineral deposits and the nature of the minerals inhaled. It was established as a result of the studv of a large number of cases that pneumoconiosis of different origins present different and often characteristic mineral deposits. In certain cases of idiopathic fibrosis of unknowvn origin, the discovery of crystalline deposits max reveal a dust pathology which had been overlooked at the initial examination. The chemical, morphologic, and crystalline characteristics of the pulmonary mineral deposits reported here are of special interest in clinical studies where they may be of great use in establishing a diagnosis. References 1. Banfield WG, Tousimis AS. Hagerty JC, Padden IR: Electron probe microanaly-sis of human lung tissues, Chapt 9. X-Ray and Electron Probe Analysis in Biomedical Research. New York, Plenum Press, 1970. p 23 2. Berkley C, Langer AM, Baden V: Instrumental analysis of inspired fibrous pulmonary particulates. Trans NY Acad Sci 30::331-350, 1967 :3. Berry JP, Galle P. Stupfel N: Techniques d'etudes de la pollution automobile: Analyse des dep6ts intra-pulmonaires chez des rats exposes au gaz d'echappement de moteur automobile. Nouv Presse Med 2:1856-1858, 197.3 4. Cralley Ll: Identification and control of asbestos exposures. Am Indust Hy-g Assoc J 32:82-85. 1971 .3. Policard A. Dovorow S: Etudes histochimiques de l'anthracose pulmonaire: DMductions pathogeeniques. Presse Med 55:903-909, 1929 6. Pregermain S, Henoc P, Normand-Reuet C: Etude par microdiffraction electronique des particules minerales d'un poumon de mineur de charbon. C R Acad Sci (Paris) 261:2019-2022, 1965 7. Harrison EG, Koses G, Andersen HA: X-rav diffraction and spectrographic analysis in pneumoconiosis. Arch Environ Health 14:412-423. 1967 8. Berr- JP Pariente R, Watchi JM: Analyse au microanalyseur d sonde electronique du "pigment noir pulmonaire.' Rev Fr Etud Clin Biol 14:915-918. 1969 9. Berrn JP. Henoc P, Galle P, Pariente R: Etude ultrastructurale des depos des poumons normaux et pathologiques: Resultats preliminaires. Analyse au microanalyseur d sonde electronique et en microdiffraction d'eectrons. J Microscopie 17:11-18, 1973 10. Berry JP. Galle P, Pariente R: Analyse des dep6s mineraux intrapulmonaires des macrophages alv&'olaires: Etude en microscopie electronique, microanalyse et microdiffraction d'electrons. Colloque sur les reactions broncho-pulmonaires aux polluants atmospheriques. Pont-d-Nlousson, France, Cahiers INSERM, 1974
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Bem- JP. Galle P. Pariente R: Analy-se des dep6ts min6raux intra-pulmonaires: R6le duI du macrophage alveolaire. Revue Francaise des Maladie Respiratoires. V-ol 2. Le
Niacrophage alveolaire. Sy-mposium Institut Pasteur de Lille, Paris. 1974 12. Galle P. Bern- JP: Cvtochimie elementaire ultrastructurale sur coupes ultrafines du poumon. Poumon Coeur 25:307-8_317, 1969 13. Pariente R. Berry JP Galle P. Cavrol E. Brouet G: Etude de l'empoussierage pulmonaire au microscope electronique et au micro-analI-seur d sonde electronique. Presse Med 78:162 7-1628. 1970 14. Pariente R. Bern- JP Galle P. Cayrol E. Brouet G: A study of pulmonary dust deposits using the electron microscope in conjunction with the electron sound analyzer. Thorax 27:80-82, 1972 13. Einbrodt HJ. Han KJ: Die V'erteilung von Kohle und Mineral in menschlichen Lungen. Int Arch Gewerbepathol 2-3:141-134. 1967 16. Gold C: Asbestos levels in human lungs. J Clin Pathol 22:307, 1969 (Abstr) 17. Gold C: A simple method for detecting asbestos in tissue. J Clin Pathol 20:674-673. 1967 (Abstr) 18. Gross P. de Treville RTP. Cralley Ll, Davis JMG: Pulmonary ferruginous bodies: Development in response to filamentous dusts and a method of isolation and concentration. Arch Pathol 83:539-346, 1968 19. Gross P. de Treville RTP. Cralley Ll, Granquist WT, Pundsack FL: The pulmonary response to fibrous dusts of diverse compositions. Am Indust Hyg Assoc J
:31:125-132, 1970
20. Langer AM, Ashley R. Baden V'. Berkley C. Hammond EC. Mackler AD. Maggiore CJ. Nicholson W'J. Rohl AN, Rubin IB. Sastre A. Selikoff IJ: Identification of asbestos in human tissues. J Occup Med 15:287-296. 1973 21. Rulttner JR. de Guervain F: Die Methodik der mikrolokalisat-reschen dastellung und kristalloplischen Identifizierung um Staubablagrerungen in silikolischen Geweben. Z Unfallmed Berufskr 40:73-77, 1947 22. Belt TH. Irwin D, King EJ: Silicon and dust deposits in tissues of persons without occupational exposure to silicon dusts. Can Med Assoc J 34:125-135. 1936 23. Catilina P. Champeix J: Corps asbestosiques pulmonaires: Apport de la microsonde electronique de Castaing a la connaissance de la gangue et d l'identification de la nature asbestosique de la fibre centrale. C R Soc Biol (Paris) 165:1899-1902. 1971 24. Goni J, Remond G, Jaurand MC, Bignon J. Bonnaud G, Brouet G: Possibilites actuelles d'identification des corps ferrugineux du poumon humain par la microsonde electronique et le microscope electronique a balavage. Rev Tuberc Pneumol (Paris) 36:1223-1236, 1972 23. Gross P. Cralley Ll, de Treville RTP: Asbestos bodies: Their nonspecificity. Am Indust Hyg Assoc J 28:341-542, 1967 26. Hargreaves A. Taylor WH: An x-ray examination of decomposition products of chrvsotile (asbestos) and serpentine. Mineral Mag 27:204-216. 1946 27. Le Bouffant L. Martin JC. Darif S. Normand C, Tichoux C: Isolement des particules fibreuses d'origine pulmonaire et leur identification par diffraction des rayons X et des electrons. Rev Tuberc Pneumol (Paris) 36:1237-1248. 1972 28. Miller A. Teirstein AS, Bader ME, Bader RA, Selikoff IJ: Talc pneumoconiosis: Significance of sublight microscopic mineral particles. Am J Med 50:393-402. 1971 29. Otto H. Bauer F: Infrarotspektroskopische Untersuchungen von Lungenstauben. Int Arch Gew-ebepathol 23:49-37, 1967 :30. Parsons DF: The examination of mineral deposits in pathological tissues by electron diffraction. Int Rev Exp Pathol 6:1-34. 1968 31. Rose Y: L'empoussi&age broncho-pulmonaire. Mted Audiovision 5:30-57. 1966 32. Ruttner JR. Spycher M1A. Sticher H: The detection of etiologic agents in interstitial pulmonary fibrosis. Hum Pathol 4:497-512, 1973
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:3:3. Ashcroft T, Heppleston AG: The optical and electron microscopic determination of pulmonary asbestos fibre concentration and its relation to the human pathological reaction. J Clin Pathol 26:224-234, 1973 34. Frost JK, Gupta PK, Erozan YS, Carter D, Hallander DH, Levin ML, Ball WC Jr: Pulmonary cytologic alterations in toxic environmental inhalation. Hum Pathol 4:321-536, 197.3 :35. Nagy B, Bates TF: Stability of chr-sotile asbestos. Am M\ineral 37:1055-1058. 1952 36. Martoja R, Cantacuzene A M, Ballan-Dufrancais C: Analyse critique des m6thodes de localisation des mineraux par substitution metallique. J Micros 14:66, 1972 .37. Allison AC, Harington JS, Birbeck NM: An examination of the cytotoxic effects of silica on macrophages. J Exp Med 124:141-154, 1966 :38. Mazliak P: Lysosomes, Glvoxvsomes, Peroxisomes. Paris, Edit Doin, 1975 39. Speil S, Leineweber JP: Asbestos minerals in modern technology. Environ Res 2:166-208, 1969
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Figure 9-Diffraction patterns of clays showing the rolling effect resulting from the interaction of the microtome knife with the layers of the clay structure. This interaction results in the simultaneous rotation of the layers on themselves and the tilting of the layers with respect to the central axis of the crystal. The result is that hexagons (A) turn little by little into circles (B and C), and the circles become ovals (D and E). The C axis of the crystal (the axis perpendicular to the layers) is shown in F. (A, x 10,000; B, x 10,000; C, x 10,000;D, x 10,000; E, x 10,000; F, x 10,000)
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Fgu 16-Coal miner's pneumoconiosis. A-Low power view of carbon deposits in macrophages; an elemental analysis spectrum is shown at lower left (x 10,000). B-At higher magnification, it can be seen that each carbon deposit (C) is surrounded by very fine granules of silicon (Si) (x 40,000).
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Pulmonary Mineral Dust A Study of Ninety Patients by Electron Microscopy, Electron
M icroanalysis, and Electron M icrodiffraction J. P. Berry, PhD, P. Henoc, Ing, P. Galle, MD, R. Pariente, MD
The results of a study of 90 patients are presented. Intrapulmonarv mineral deposits were characterized by electron diffraction and electron probe microanalysis. Using this method, pneumoconioses mav be distinguished from other pneumopathies. In cases of pneumoconiosis, there exists a specific relationship between the etiolog,y of the dust exposure and the crvstallographic characteristics of the intrapulmonary deposits. The nature of the deposits mav be indicative of a specific type of pneumoconiosis. This method is particularly useful in differentiating between asbestos bodies and ferruginous bodies. The value of the method in general and its importance in the study of pneumoconiosis are discussed. (Am J Pathol 83:427-456, 1976)
NI YN STU DIES have been devoted to the direct analysis of intrapulmonary mineral deposits in histologic sections. The first studies carried out on ultrathin sections using electron microdiffraction were useful in determining the crystalline or noncrystalline character of the deposits;6 however, without supplementary information concerning the chemical element present, microdiffraction data alone are generally insufficient to identify the specific crystal present. Nloreover, electron diffraction is of no use in studying the numerous noncrvstalline mineral deposits present in human lungs: for example, black intrapulmonary pigment cannot be studied by this means. The first studies using the electron microprobe analy-zer of Castaing were made on thick tissue sections (3 ,)1.8 They showved that the black From the Labortoire de Biophysique. Faculte de \ledecine de Creteil. Creteil. C\ET. IssslesNIoulineaux. Hopital Antoine-WB-eclre. Clamart. France Supported by the INSERM Institute kA. T P 8. Pathologie broncho-pulmonaire et pollution). Taken from the thesis submitted by Jean-Pierre Berry to the Faculty of Medicine of Creteil in partial fulfillment of the requirements for the Doctorate degree in human biology. Xccepted for publication December 29. 1973. Xddress reprint requests to Dr J -P Bern-. Laboratoire de Biophysique. Faculte de \ledecine de Creteil. 6 rue du Ge&neral Sarrail. 94. Creteil. France
427
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American Journal of Pathology
intrapulmonarv pigment known as anthracosis pigment was composed, not of carbon as had been previouslyr suspected, but of a large number of mineral elements. The analysis of manv lung specimens subsequently showed that these same mineral elements could be found in all cases of intrapulmonary deposits whether occurring in normal lungs exposed to ordinary atmospheric pollution or lungs from patients having pneumoconiosis: electron probe microanalysis of thick sections, therefore, does not establish a differential diagnosis. These studies were extended to include the analysis of deposits in ultrathin sections (1500 A). Each deposit was analyzed by an electron probe microanalvzer coupled with an electron microscope and by electron microdiffraction. Preliminary results have been published Fl showing that only this combination of analytical methods establishes with certainty the chemical and crvstalline character of each mineral deposit present in the macrophages. This report presents the results of a study of 90 patients, some of whom were exposed only to normal atmospheric pollution and others who were affected with pneumoconiosis. Materials and Mefthods Tssue Spcmens All tissue specimens were obtained from suburban Paris hospitals (Pneumo-Phtisiology Unit of Professor Pariente, H6pital Antoine Becl&e, Clamart; and Pneumology Unit of Professor Chretien, Hopital Intercommunal, Creteil) w here the clinical diagnoses w-ere
made. The specimens were obtained either at biopsy, surgery (surgical removal), or autopsy. We examined tissue from patients with or without diagnoses of pneumoconiosis. Pulmonar- tissue was fixed in buffered glutaraldehyde (pH 7.4), postfixed in osmium tetroxide, and embedded in Epon. Blocks were sectioned on a Reichert ENMU 2 Ultramicrotome with a diamond knife, picked up on grids with reference positions, and stained with uranv-l acetate. Specimens From Patients Without Pneumoconiosis A total of 40 specimens was studied. In 20 cases, tissue was selected at random from the
following areas of the lung: anterosuperior, anteromiddle, anteroinferior, posterosuperior, posteromiddle, posteroinferior, subpleural, peribronchial. In 20 cases (lobectomv), tissue was selected from regions having the macroscopically dark appearance characteristic of dust accumulations. Specimens From Patients With Pneumoconiosis More than 50 specimens from patients with pneumoconiosis were examined. Specimens were obtained at biopsy, surgical lobectomy, or autopsy. Tissue w-as selected
from darkened regions of the specimens.
Techniques Sections were examined with a Philips EN :300 electron microscope. Electron probe microanalvsis was done using a CANMECA NMS 46 microanalyzer
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equipped with an electron microscope. Details of the analI-tical technique has-e been prev iousli published.9 Electron microdiffraction u-as done using a Siemens Elmiskop I electron microscope equipped with a goniometer stage and double condenser.
Results Lungs From Patients Without Pneumoconiosis-"Normar Logs
Masses of dense intrapulmonary mineral deposits were found in the cv-toplasm of alveolar macrophages. licroanalysis of the entire group of deposits present in a macrophage showed the silicon, aluminum, iron, titanium, and carbon; these elements were found in all lungs (Figure 1). Ho wever, wvhen each inclusion X-vas analyzed separately in ultrathin sections, differences were found in chemical composition and ultrastructural morphology. Three main kinds of deposits could be distinguished according to these two criteria. The most frequently encountered deposits were made up of clumps of small, moderately electron-aense spheres surrounded by a halo of fine needles. This tvype of inclusion contained Si, Fe, and Al (Figure 2A). A second type of deposit was formed by clumps of very electron-dense spheres and contained only Ti (Figure 2B). The third tvpe wvas made up of large homogeneous electron-dense masses hav ing clear-cut straight edges. They wvere found to contain C and Fe (Figure :3). Another type of inclusion of irregular shape containing C and Fe was occasionally noted. Electron diffraction diagrams of all these inclusions showed the diffuse rings characteristic of amorphous structures. In certain cases, these observations wvere extended in the following way: the sections wvere again introduced into the electron probe microanalyzer and the same deposits wvhich on previous anal-sis had show n the presence of Si Xwere bombarded wvith a strong electron current. This treatment heats the sections to a high temperature. Afterwards, electron microdiffraction anal-sis showed the thin ring pattern characteristic of the crystalline structure of Si. Heating the deposit wvas thus responsible for the formation of silicon crystals. The same treatment carried out on deposits containing titanium resulted in the formation of titanium dioxide (recognized from its diffraction diagram). These observations lead to the conclusion that these intracellular mineral inclusions do not possess a crystalline structure. Lungs From Patent Wiffi Pneumoconiosis
In this case, crystalline patterns were obtained without prior treatment of the mineral deposits. However, the diffraction patterns were generally
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not diagnostic of a specific crvstal without the supplementary data on chemical composition provided by electron probe microanalysis. Table 1 shows the names of the different minerals which we have identified, and their chemical formulas and crystalline structure. In Table 1 it can be seen that only about twentv minerals recur in all the cases of pneumoconiosis examined. We found that certain minerals are found in the majority of cases of pneumoconiosis, while others are found only rarely and are often the product of special circumstances. The minerals which are found in most cases of pneumoconiosis can be divided into three categories: a) minerals associated with silicosis, b) asbestos bodies and ferruginous bodies, and c) other minerals frequently seen in cases of pneumoconiosis. Minerals Associated With Cases of Silicosis
The minerals most often encountered in silicosis are quartz, micas, and clavs. Quartz. In the cytoplasm of certain macrophages we observed dense, rounded masses with irregular but generally well-defined contours. Some of the masses were thick and protruded somewhat from the section, while others were thin and showed a slight opalescence when the objective lens current was varied. Electron probe microanalysis of these deposits showed that only silicon was present. The microdiffraction diagrams presented the particularity of bright Kikuchi lines. Measurement of the parameters led to the identification as of these deposits quartz (Figure 4). Micas. The morphologic aspect of these inclusions varied greatly. Thev formed either dense compact masses or elongated leaflets. Electron probe microanalysis showed that the composition of these crvstals also varied. Spectra showed the presence of Si, potassium, Al, and Fe as well as these same elements plus magnesium. With this information, the microdiffraction diagrams can be interpreted. Together they showed that the micas most often present in silicosis are muscovite (white mica) and biotite (black mica, ferromagnesium) which is associated with Mg (Figures 5 and 6). Clays. The morphology of these crvstals is extremely varied. They may occur as heterogenous masses of small, dense grains; thin, larger, elongated fibers; small, dense regions dotted with regularly spaced clear circles; dense blade-like configurations; or in masses formed of thick layers or chevrons. Electron probe microanalysis shows the complexity of the chemical composition of these crystals. Microdiffraction diagrams show, in general, a hexagonal pattern which
PULMONARY MINERAL DUST
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reflects the projection of the basal plane of the crystal. Since this plane is the same for all clays, it is impossible to distinguish between them. When diffraction patterns are obtained from other crystal planes, it is possible to use the information obtained from microprobe analysis to interpret these diagrams. Three kinds of clays were frequently found: illites (which contain Ti) (Figure 7) made up of fine, undifferentiated particles; montmorillonite (which contains K) (Figure 8), very malleable and which swells in water; and pyrophyllite (composed of only Si and Al) which resembles talc. The leaflets of clays are fragile and the ultramicrotome knife often distorts them, producing a sort of "layering" of the structures. This artifact results in deformations of the diffraction diagrams (ring or oval figures). (Figure 9). Clays are the minerals most often found in all cases of pneumoconiosis. Asbestos Bodies, Ferruginous Bodies
We observed a large number of fibrous structures made up of an elongated monocrystal often surrounded by a ferruginous coating. Nature of the Coating. The coating is formed of dense masses of small granules arranged in successive layers of irregular contour around the monocrystal. Electron probe microanalysis shows the presence of Fe. The microdiffraction pattern of thin rings corresponds, in the Association Society for Testing Materials (ASTM) classification of inorganic compounds, to the formula for FeO(OH). The nature of the central monocrystal distinguishes asbestos bodies from ferruginous bodies. Asbestos Bodies. In these cases, the axial fiber was a monocrystal of asbestos. Depending on the plane of section, the fiber profile appeared as a long wide blade or a small central inclusion. Microprobe analysis shows the predominant presence of Fe, Si, and sodium which, combined with the electron diffraction patterns, identified these crystals as being crocidolite (blue asbestos) (Figure 10). The presence of other amphiboles was also shown. Ferruginous Bodies. Although their morphologic aspect was identical in these cases, the axial fiber was not asbestos (Figure 1 1). We have shown the presence of different varieties of monocrystals in the axial fiber. Depending on the case, it may be composed of carbon, sillimanite (aluminium silicate) (Figure 12), sericite (white mica), or clay. Other Minerals Which Occur Frequently in Pneumoconiosis
Talc. Morphologically, these crystals appeared as large, waffled areas which disintegrated easily in the electron beam. Si and Mg were found by
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electron probe microanalysis, thus simplifying the interpretation of the electron microdiffraction patterns (Figure 13). Cristobalite. These small inclusions have a very characteristic geometric form (Figure 14). As in the case of quartz, microprobe analysis showed that onlv Si was present; however, the parameters measured from the diffraction pattern clearly showed the crystalline structure corresponding to cristobalite. This crvstal is normally formed at high temperatures, in volcanic regions, for example. Chlorites. These inclusions were found as groups of thin flakes. Certain of them had a verv characteristic form which consisted of elongated, sinuous crystals, grouped in bundles. Microprobe analysis combined with electron diffraction shows the presence of Si, Al, Mg, and Fe in crystals of vermiculite (thermal insulation material used in building construction) (Figure 15). Micas. Biotite and muscovite were also found frequently in cases of penumoconiosis.
Spinelles. These inclusions do not have any particular characteristics. They are metallic oxides of various metals (Ca, Fe, manganese, etc.) Particular Deposits Seen in Coalworker's Pneumoconiosis
In these lungs we observed macrophages filled with carbon deposits. Most of the carbon deposits were, however, encircled by a well-defined ring of fine particles (granules). Electron probe microanalysis showed these particles to be composed of silicon (Figure 16). Less Frequently Encountered Minerals
These minerals were found either alone or in association with others. Most often, their presence was the result of particular circumstances. Some of these minerals, however, have a characteristic morphology, as in the case of leucite, which occurred as round masses of concentric lamellae (Figure 17). Discussion Two points will be discussed: the characteristics of the techniques emploved and their usefulness in the study of pneumoconiosis. Te
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Two different kinds of methods can be used to study intrapulmonary mineral deposits: a) methods of whole-sample comprehensive analytical methods, and b) methods of in situ analysis.
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Methods of Whole-Sample Analysis
These methods involve the examination of residues of inorganic material after destruction of the organic constituents of the pulmonary samples by treatments such as incineration at 500 to 800 C, oxygenation, or acid or alkaline digestion.15 21 The mineral residues are subsequently analyzed by various physical techniques: fluorescence, x-ray diffraction, mass spectroscopy, differential thermal analysis, etc.2,6'7,16,18-. These methods have two major drawbacks: a) Minerals obtained in this way may have been greatly altered by the heat, acid, or alkali treatment, and their crystalline structure modified. Certain minerals may even be destroyed altogether.21'2"'3335 b) Because of the destructive nature of these methods, no morphologic correlations are possible. Methods of In Situ Anaysis
The second group of methods in which mineral inclusions were analyzed in tissue sections have the advantage of furnishing morphologic correlations. Three techniques were used: histochemical methods, electron microdiffraction, and electron probe microanalysis. There are only a few histochemical reactions for chemical elements, and the results are often nonspecific.5'" The utilization of electron microdiffraction alone on ultrathin sections yielded diffraction pattems which were very difficult to interpret since the small quantity of matter present in the section did not fumish sufficient information. Electron probe microanalysis used alone did not provide data for a differential diagnosis. Using thick sections (5 ;), the analysis of pulmonary deposits gave approximately the same results for all lungs analyzed. However, the combined use of electron probe microanalysis and electron diffraction makes a precise determination possible. The data from microprobe analysis provided the elementary chemical composition of each particle, and these data helped in the interpretation of the microdiffraction diagrams. Specifically, they narrow the choice to a single crystal from the many possible crystals which coincide with the parameters measured in the diffraction pattern.910'11 Consequently, the combination of data from electron probe microanalysis with that from electron microdiffraction seems, at the present time, to be the method of choice in determining the chemical and crystalline nature of mineral inclusions.
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UsefuesS of This Method
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the Sbtuy of Ps
In this area three points deserve emphasis: 1. Crystalline structures are not found in all cases. We have observed that deposits found in nonpneumoconiosis lungs were not in a crystalline state. This is so despite of the fact that almost all inhaled atmospheric dust particles are crystalline. It mav, therefore, be supposed that the macrophages of pulmonary tissue are capable of denaturing the dust particles and reducing them to an amorphous state. Certain similar processes have been noted in tissue culture."," Moreover, it is known that some mineral crystals undergo degradation in an acid milieu. Intracellular acids may be furnished by Iysosomes.-m In cases of pneumoconiosis, on the other hand, most mineral deposits are found to be in the crystalline state. It is, therefore, possible to establish a differential diagnosis by the simple presence of intrapulmonarv crvstals. 2. The determination of the nature of the axial fiber in asbestos or ferruginous bodies is difficult. The industrial use of asbestos and its possible role in carcinogenic processes have stimulated many studies 2 on the effects of asbestos fibers on the organism. In previous studies, inorganic residues were analyzed, as were fibers with or without their enveloping sheaths, without succeeding in localizing these inclusions in the tissue, thus leading to the following conclusions: a. The techniques used for isolating the fibers introduce the artifacts already mentioned above (alteration of crystal structure, etc.,). Moreover, contamination by atmospheric dust particles cannot be systematicallv eliminated. b. The identification of asbestos fibers is particularly difficult. The mineral asbestos is made up of two subgroups: the amphiboles and the serpentines. Almost all varieties of asbestos are found in the fibrous form of amphiboles which have very similar crystalline parameters. Their chemical formulas vary, however, making it possible to distinguish different groups (sodium-containing, non-aluminium-containing, and aluminium-containing). The sole representative of the serpentine group is chrysotile (magnesium silicate). This crystal structure undergoes almost complete denaturation when subjected to the treatments used to destroy organic material.2 The differentiation of asbestos from ferruginous bodies is also made verv difficult by the presence of the ferruginous coating which surrounds the axial fiber. It should be emphasized that ferruginous bodies are found not only in
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American Journal of Pathology
all cases of asbestos pathology but also in numerous other pneumopathies. Once again, analy sis of crystals in ultrathin sections in wvhich the chemical nature is determined by electron microprobe analysis and the crystal structure by electron microdiffraction is at the present time the only method by which the nature of these fibers can be accurately determined in pulmonary tissue. 3. There is a relationship between the characteristics of pulmonary mineral deposits and the nature of the minerals inhaled. It was established as a result of the studv of a large number of cases that pneumoconiosis of different origins present different and often characteristic mineral deposits. In certain cases of idiopathic fibrosis of unknowvn origin, the discovery of crystalline deposits max reveal a dust pathology which had been overlooked at the initial examination. The chemical, morphologic, and crystalline characteristics of the pulmonary mineral deposits reported here are of special interest in clinical studies where they may be of great use in establishing a diagnosis. References 1. Banfield WG, Tousimis AS. Hagerty JC, Padden IR: Electron probe microanaly-sis of human lung tissues, Chapt 9. X-Ray and Electron Probe Analysis in Biomedical Research. New York, Plenum Press, 1970. p 23 2. Berkley C, Langer AM, Baden V: Instrumental analysis of inspired fibrous pulmonary particulates. Trans NY Acad Sci 30::331-350, 1967 :3. Berry JP, Galle P. Stupfel N: Techniques d'etudes de la pollution automobile: Analyse des dep6ts intra-pulmonaires chez des rats exposes au gaz d'echappement de moteur automobile. Nouv Presse Med 2:1856-1858, 197.3 4. Cralley Ll: Identification and control of asbestos exposures. Am Indust Hy-g Assoc J 32:82-85. 1971 .3. Policard A. Dovorow S: Etudes histochimiques de l'anthracose pulmonaire: DMductions pathogeeniques. Presse Med 55:903-909, 1929 6. Pregermain S, Henoc P, Normand-Reuet C: Etude par microdiffraction electronique des particules minerales d'un poumon de mineur de charbon. C R Acad Sci (Paris) 261:2019-2022, 1965 7. Harrison EG, Koses G, Andersen HA: X-rav diffraction and spectrographic analysis in pneumoconiosis. Arch Environ Health 14:412-423. 1967 8. Berr- JP Pariente R, Watchi JM: Analyse au microanalyseur d sonde electronique du "pigment noir pulmonaire.' Rev Fr Etud Clin Biol 14:915-918. 1969 9. Berrn JP. Henoc P, Galle P, Pariente R: Etude ultrastructurale des depos des poumons normaux et pathologiques: Resultats preliminaires. Analyse au microanalyseur d sonde electronique et en microdiffraction d'eectrons. J Microscopie 17:11-18, 1973 10. Berry JP. Galle P, Pariente R: Analyse des dep6s mineraux intrapulmonaires des macrophages alv&'olaires: Etude en microscopie electronique, microanalyse et microdiffraction d'electrons. Colloque sur les reactions broncho-pulmonaires aux polluants atmospheriques. Pont-d-Nlousson, France, Cahiers INSERM, 1974
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Bem- JP. Galle P. Pariente R: Analy-se des dep6ts min6raux intra-pulmonaires: R6le duI du macrophage alveolaire. Revue Francaise des Maladie Respiratoires. V-ol 2. Le
Niacrophage alveolaire. Sy-mposium Institut Pasteur de Lille, Paris. 1974 12. Galle P. Bern- JP: Cvtochimie elementaire ultrastructurale sur coupes ultrafines du poumon. Poumon Coeur 25:307-8_317, 1969 13. Pariente R. Berry JP Galle P. Cavrol E. Brouet G: Etude de l'empoussierage pulmonaire au microscope electronique et au micro-analI-seur d sonde electronique. Presse Med 78:162 7-1628. 1970 14. Pariente R. Bern- JP Galle P. Cayrol E. Brouet G: A study of pulmonary dust deposits using the electron microscope in conjunction with the electron sound analyzer. Thorax 27:80-82, 1972 13. Einbrodt HJ. Han KJ: Die V'erteilung von Kohle und Mineral in menschlichen Lungen. Int Arch Gewerbepathol 2-3:141-134. 1967 16. Gold C: Asbestos levels in human lungs. J Clin Pathol 22:307, 1969 (Abstr) 17. Gold C: A simple method for detecting asbestos in tissue. J Clin Pathol 20:674-673. 1967 (Abstr) 18. Gross P. de Treville RTP. Cralley Ll, Davis JMG: Pulmonary ferruginous bodies: Development in response to filamentous dusts and a method of isolation and concentration. Arch Pathol 83:539-346, 1968 19. Gross P. de Treville RTP. Cralley Ll, Granquist WT, Pundsack FL: The pulmonary response to fibrous dusts of diverse compositions. Am Indust Hyg Assoc J
:31:125-132, 1970
20. Langer AM, Ashley R. Baden V'. Berkley C. Hammond EC. Mackler AD. Maggiore CJ. Nicholson W'J. Rohl AN, Rubin IB. Sastre A. Selikoff IJ: Identification of asbestos in human tissues. J Occup Med 15:287-296. 1973 21. Rulttner JR. de Guervain F: Die Methodik der mikrolokalisat-reschen dastellung und kristalloplischen Identifizierung um Staubablagrerungen in silikolischen Geweben. Z Unfallmed Berufskr 40:73-77, 1947 22. Belt TH. Irwin D, King EJ: Silicon and dust deposits in tissues of persons without occupational exposure to silicon dusts. Can Med Assoc J 34:125-135. 1936 23. Catilina P. Champeix J: Corps asbestosiques pulmonaires: Apport de la microsonde electronique de Castaing a la connaissance de la gangue et d l'identification de la nature asbestosique de la fibre centrale. C R Soc Biol (Paris) 165:1899-1902. 1971 24. Goni J, Remond G, Jaurand MC, Bignon J. Bonnaud G, Brouet G: Possibilites actuelles d'identification des corps ferrugineux du poumon humain par la microsonde electronique et le microscope electronique a balavage. Rev Tuberc Pneumol (Paris) 36:1223-1236, 1972 23. Gross P. Cralley Ll, de Treville RTP: Asbestos bodies: Their nonspecificity. Am Indust Hyg Assoc J 28:341-542, 1967 26. Hargreaves A. Taylor WH: An x-ray examination of decomposition products of chrvsotile (asbestos) and serpentine. Mineral Mag 27:204-216. 1946 27. Le Bouffant L. Martin JC. Darif S. Normand C, Tichoux C: Isolement des particules fibreuses d'origine pulmonaire et leur identification par diffraction des rayons X et des electrons. Rev Tuberc Pneumol (Paris) 36:1237-1248. 1972 28. Miller A. Teirstein AS, Bader ME, Bader RA, Selikoff IJ: Talc pneumoconiosis: Significance of sublight microscopic mineral particles. Am J Med 50:393-402. 1971 29. Otto H. Bauer F: Infrarotspektroskopische Untersuchungen von Lungenstauben. Int Arch Gew-ebepathol 23:49-37, 1967 :30. Parsons DF: The examination of mineral deposits in pathological tissues by electron diffraction. Int Rev Exp Pathol 6:1-34. 1968 31. Rose Y: L'empoussi&age broncho-pulmonaire. Mted Audiovision 5:30-57. 1966 32. Ruttner JR. Spycher M1A. Sticher H: The detection of etiologic agents in interstitial pulmonary fibrosis. Hum Pathol 4:497-512, 1973
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BERRY ET AL
American Journal of Pathology
:3:3. Ashcroft T, Heppleston AG: The optical and electron microscopic determination of pulmonary asbestos fibre concentration and its relation to the human pathological reaction. J Clin Pathol 26:224-234, 1973 34. Frost JK, Gupta PK, Erozan YS, Carter D, Hallander DH, Levin ML, Ball WC Jr: Pulmonary cytologic alterations in toxic environmental inhalation. Hum Pathol 4:321-536, 197.3 :35. Nagy B, Bates TF: Stability of chr-sotile asbestos. Am M\ineral 37:1055-1058. 1952 36. Martoja R, Cantacuzene A M, Ballan-Dufrancais C: Analyse critique des m6thodes de localisation des mineraux par substitution metallique. J Micros 14:66, 1972 .37. Allison AC, Harington JS, Birbeck NM: An examination of the cytotoxic effects of silica on macrophages. J Exp Med 124:141-154, 1966 :38. Mazliak P: Lysosomes, Glvoxvsomes, Peroxisomes. Paris, Edit Doin, 1975 39. Speil S, Leineweber JP: Asbestos minerals in modern technology. Environ Res 2:166-208, 1969
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Figure 2A-The most frequently encountered aspect of mineral deposits in the lungs of patients without pneumoconiosis. Note the accumulation of small spheres surrounded by fine needles. The results of electron probe microanalysis of these deposits are shown in the lower left. N = nucleus, C = cytoplasm, M = mitochondria. (x 23,000) B-Very dense rounded deposits contain titanium (arrow). The spectrum of titanium obtained by electron probe microanalysis is shown in the lower right (x 20,000) Inset-A higher magnification photograph of the titanium deposits is shown. (x 60,000)
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Figure 7-The cytoplasm of macrophages is dotted with small, very dense particles of illite; a larger particle is also seen (arrows). The ovoid diffraction diagram (upper left) is similar for most clays. Electron microprobe analysis (upper right) shows the presence of numerous elements, among them, titanium (Ti) a characteristic element in the illites. (x 25,000)
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Figure 8-Different morphologic aspects of montmorillonite. A deposit formed by the stacking of thick layers is seen in the lower left of the photomicrograph (arrows). The projection of the basal crystal plane (hexagonal array) is shown in the uppe left; the ovoid diffraction pattern which is interpreted to be that of the clay montmorillonite is shown in the upper right The electron microprobe analysis spectrum is shown in the upper center. (x 20,000)
Figure 9-Diffraction patterns of clays showing the rolling effect resulting from the interaction of the microtome knife with the layers of the clay structure. This interaction results in the simultaneous rotation of the layers on themselves and the tilting of the layers with respect to the central axis of the crystal. The result is that hexagons (A) turn little by little into circles (B and C), and the circles become ovals (D and E). The C axis of the crystal (the axis perpendicular to the layers) is shown in F. (A, x 10,000; B, x 10,000; C, x 10,000;D, x 10,000; E, x 10,000; F, x 10,000)
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Fgu 16-Coal miner's pneumoconiosis. A-Low power view of carbon deposits in macrophages; an elemental analysis spectrum is shown at lower left (x 10,000). B-At higher magnification, it can be seen that each carbon deposit (C) is surrounded by very fine granules of silicon (Si) (x 40,000).
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