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KWN ......................... -- MAY ------------------- & reij^L Reprinted front me Archives of Environmental Health Septcmocr 1956, Volume 17 Copyright 1568. American Medical Association 1, ;7 m '7 /( 327 PLAINTIFF'S EXHIBIT CAP-1387 Ferruginous Bodies in Human Lungs Prevalence at Random Autopsies Michael D. TJtidjian, MB; Paul Gross. MD; ar.d Robert T. P. deTreville, MD, Pittsburgh Ferruginous bodies with a transparent central In 1964 in Pittsburgh, Cauna, et al3 re core or no visible central tilament were present in ported finding 47% and 34%, respectively, 97 of 100 autopsies; they were found in 55 of 50 men (93%) and in 42 of 44 women (95.5%). The youngest subject with ferruginous bodies was a 24-year-old woman. The bodies ware generally more abundant in the lungs of the man. Thirty-two patients in this sories had some form of malignant disease, but there was no apparent association between tho malignancies and the relative abun dance of ferruginous bodies. There wore two cases in men and women autopsied. Tha same year, Anjilvel and Thurlback4 studied autopsies in Montreal, and found 57% in men and 34% in women. Meanwhile, Meurman3 was finding an overall prevalence of 70% in the urban ele ment of a regional survey for asbestos bodies in Finland. of primary lung cancer in man, but no case of In 1963, Ghezzi, et al reported finding mesothelioma, nor of asbestosis. 54% and 4-1%, respectively, in men and The significance of these findings from an epi demiologic point of view must await identification of the central core of the ferruginous bodies, but in any event, pulmonary fibrosis or neepiasm is rarely associated with thair presence in the lungs cf these autopsied city dwellers not occupationally exposed to asbestos dust. women autopsied in Milan, Italy. As it appears that all the series studied so far have been of comparable age range and occupational backgrounds, the most likely explanation for this trend is that pro gressively more intensive searches are being conducted. The techniques employed in the foregoing studies, with the exception of the In RECENT years studies have been con Finnish, have been basically the same: ex ducted in a number of cities of the prevalence of ferruginous bodies in the lungs of city dwellers at autopsy. In 1960 in Cape Town, South Africa, Thomson et all reported finding that 30% of amination of unstained air-dried smears of juice expressed from cut portions of fresh postmortem lung or scraped from a cut lung surface. Some took their samples from tha lung bases (Thomson et al1-2) and some the men and 20% of the women at autopsy from both upper and lower lobes (Cauna were found to have asbestos bodies present et al3). Anjilvel ar.d Thurlbeck* sampled only in their lungs. left lungs. Meurman5 examined 20 micron The following year, Thomson and Graves2 sections'stained for iron by Perle's stain in reported finding a similar prevalence in Mi ami, Fla. Men and women showed positive results in 31.6% and 20.4% respectively. Subnutted for publication April 2-", 1968; accepted May 9. From the Industrial Hygiene Foundation of Amer ica, Inc., 4400 Fifth Avo. Pittsburgh. Reprint requests to the Industrial Hvgiens Foun dation of America, Inc., 4400 Fifth Ave, Pittsburgh 15213 (Dr. Gross). his search for bodies. In all the above studies, their authors have defined the bodies morphologically in similar terms, and have described them as "asbestos bodies" on the grounds that they considered onty asbestos fibers to be respon sible for the formation of such bodies and to be at their core. However, as Meurman5 points out, bodies with a striking or even Arch Environ Health--VoI l~.,Sept 1963 l ASARCO ALV 0006620 328 FERRUGINOUS BODIES--UTIDJIAN ET AL complete resemblance to asbestos bodies have been found to have at their core, not asbestos, but elongated or fibrous particles of a variety of other minerals including biotite-fusain, hornblende-rutile, diatomaceous earth, graphite, and carborundum. More over, it has recently been shown by Gross et aIT that bodies morphologically indistin guishable from true asbestos bodies can be produced in hamsters by three different man-made, nonasbestos fibrous materials (namely, silicon carbide "whiskers", ceram ic aluminum silicate, and fibrous*glass). The term "ferruginous body" has, therefore, been adopted by the authors, to describe all such bodies unless and until the central fiber has been positively identified as asbestos. Tin's generic term encompasses both asbes tos and pseudo-asbestos bodies as previously employed. In this paper, the ferruginous body is defined as an elongated golden to reddish brown structure usually with clubbed ends; the shaft which often shows a segmented or beaded appearance is usually straight, but sometimes curvilinear with a tendency to wards symmetry; usually it is from 3 to 5/t in diameter and 20 to 100ft in length. The coating contains iron demonstrable by Perle's stain (Prussian blue reaction), and which is probably composed of ferritin or a ferritin-like protein material (Davis8 and Collet, written communication, June 19G6); it may cover the structure completely, mask ing the central fiber from direct view, or may be incomplete in the central portion of the shaft or in the interstices of the body, revealing an expanse of naked fiber. In this study, only ferruginous bodies with trans parent or no visible central fibers were counted. Materials and Methods The 100 lungs comprising the material for this study were supplied to us, formalin-fixed, through the central autopsy room of the Pres byterian University Hospital, Pittsburgh, and came from autopsies performed on patients who died in five metropolitan Pittsburgh hospi tals during the period from late 1966 through June 1967. The lungs were not, therefore, from consecutive autopsies and were unselected by us. There were 56 men, age range 37 to 87 years (mean age 65), and 44 women, age range 16 to SO (mean age 59). Thirteen of the men and six of the women were Negroes. Death had resulted from a wide variety of causes; 32 died from malignant con ditions, including two cases of primary lung cancer (both men). There was no evidence of mesothelioma in any of the patients at autopsy. The lungs were examined superficially for cal cified pleural plaques, and none were found; however, as some of the lungs were incomplete when received, there is a possibility that their presence may have been missed in some in stances. The lungs wore sliced longitudinally on a commercial electric "bacon sheer," into slices approximately 3 mm in thickness and a total of about 300 gm (wet weight) was sliced in this way in each case. The slices were (bated on the surface of about 2(2 inches of domestic laundry bleach, undiluted (active constituent 5.25% sodium hypochlorite), contained in individual plastic pails of 9(4 inches internal diameter at the base. Immediately as the lung tissue came into contact with the bleach, there was vigorous effervescence with the evolution of chlorine, which ceased after a few hours. Only the sur face in contact appeared to be attacked. The pails were left undisturbed in a fume hood for IS to 24 hours. At the end of this period the floating lung slices were seen to have shrunk considerably in area. The attacked undersur face was seen to be bleached yellowish white. At the bottom of the bucket, a dirty-looking grey to black sediment had formed. In some cases this was firmly adherent to the bottom and sides of the bucket as a greyish film. In others, the sediment appeared to be quite loose and readily resuspended in the spent bleach. In the former cases with an adherent film it was possible, after picking off and discarding the shriveled lung tissue, to decant all the spent bleach which was also discarded. The film was then dissolved off the inner surface of the pail by gentle agitation with about 200 ml of a 50% mixture of chloroform and 95% ethanol. The turbid, grey suspension so obtained was then decanted into a liter-separating funnel and shaken with an equal volume of clean water and allowed to stand. In the cases in which the sediment was nonadherent, about three fourths of the spent bleach only was carefully decant ed, leaving most of the sediment in the re maining one fourth bleach. This was then mixd in the bucket with a similar volume of the chloroform-alcohol mixture and the whole swirled around the pail a few times and then decanted into a separating funnel. On standing for ten minutes or so, the con tents of the separating funnel were seen to sepa rate into three layers. The lower, denser clilo- Arch Environ Health--Vol 17, Sept 196S FER oform layer was more or It ;olorcd and contained the c janic, insoluble, mineral r< gested portion of the lung, 1 bodies that might be presen layer was turbid and grey, um chloride, any undecora chlorite and other soluble j tion of lung tissue and bl variable amount of organ sion. At the interface be layers was invariably a d Fig 1.--End f naked ,fi ASARCO ALV 0006621 . AE .* men and six of the womei ath had resulted from a wid< 32 died from malignant con two cases of primary lung :>. There was no evidence of ny of the patients at autopsy, cammed superficially for callues, and none were found; of the lungs were incomplete ;re is a possibility that their :e been missed in some in- sliced longitudinally on a e "bacon slicer," into slices m in thickness and a total of t weight) was sliced in this 'he slices were floated on the j inches of domestic laundry (active constituent 5.25% .e), contained in individual inches internal diameter at ely as the lung tis.-ue came e bleach, there was vigorous the evolution of chlorine, a few hours. Only the sureared to be attacked. The cturbed in a fume hood for the end of this period the -e seen to have shrunk he attacked undersurbleached yellowish white, he bucket, a dirty-looking ent had formed. In some y adherent to the bottom .ket as a greyish film. In appeared to be quite loose Ic'd in the spent bleach. In h an adherent film it was :g off and discarding the . to decant all the spent i discarded. The film was inner surface of the pail th about 200 ml of a 50% a and 95% ethanol. The m so obtained was then r-separating funnel and 1 volume of clean water In the cases in which the rent, about three fourths . ily was carefully decanthe sediment in the re bleach. This was then ith a similar volume of mixture and the whole 1 a few times and then lting funnel. minutes or so, the conunnel were seen to sepaT't" lower, denser chlo FERRUGINOUS BODIES--UTIDJIAN ET AL 329 roform layer was more or less clear and amber colored and contained the denser, mainly inor ganic, insoluble, mineral residue from the di gested portion of the lung, plus any ferruginous bodies that might be present. Tiie upper watery layer was turbid and grey, and contained sodi um chloride, any undecoreposed sodium hypo chlorite and other soluble products of the diges tion of lung tissue and blood, formalin, and a variable amount of organic debris in suspen sion. At the interface between the two main layers was invariably a dark grey to jet black layer, from 1 mm to 2 cm in depth, according to the amount of carbonaceous material present in the original lung, consisting mainly of carbon Fig X.--End of naked fiber seen in concentrate smear from human lung in this series. Appearance implies fusion during formation of fiber (unstained, X 1100). together with a certain amount of lipidic ma terial (there was also evidence that some lipid material was held in solution in the chloro form). The lower layer only was run off into an elongated glass lube with ground-glass stopper, mixed with a similar volume of fresh clean wa ter and vigorously shaken. This time the emul sion formed was usually much more stable and was poured into 50-ml centrifuge tubes and spun for ton minutes at 2,000 rpm. Once more there was a throe-layer separation with rela tively little carbonaceous material at the chlo roform-water interface, plus a dense deposit at the very apex of the tube, from 1 to 3 mm in depth and ranging in color from dirty white through greyish pink to a distinct reddish brown. Occasionally this deposit was almost black, indicating the persistence of carbona ceous material adherent to the denser matter. In such cases, a further separation was per formed by resuspending the deposit in more chloroform-water emulsion and rcccntrifuging. Both chloroform and watery layers were finally decanted and discarded, and any carbonaceouslipid ring adherent to the walls of the tube re moved with cotton wool swabs before the tube was righted. The apical deposit was suspended in two or three drops of distilled water, plus two drops of dimethyl sulphoxide as a preserv ative and was then ready for microscopy as a smear. Smears were prepared from a drop of the suspension of as near constant size as possible, using uniform pipettes held vertically, and al lowing a free-falling drop to form and fall onto the slide. The smear was allowed to form by the natural spread of the drop only. It was found feasible to examine the smears initially in the wet state under low power (1G mm), un der which conditions the majority of the ferru ginous bodies were very readily seen and counted under transmitted light. Subsequently, as they became air dried, they were coverslipped with a mounting medium (Permount) and reexamined under high power for confirma tion of very small bodies or otherwise doubtful structures. Quantitative Estimation of Relative Abun dance.--It is appreciated that employing a technique of this kind, with possibilities of sam pling variation at almost every stage, only a very crude estimate as to the relative abun dance of ferruginous bodies in the original lung tbsue can be formed. However, the specimens did seem to form naturally into three catego ries. There were those in which examination of several smears (five was the maximum number practicable with the amount of material avail- Arch Environ Health--Vol 17, Sept 196S ASARCO ALV 0006622 330 FERRUGINOUS BODIES--UTIDJIAN ET AL able) was necessary to find a single ferruginous body. These were arbitrarily classified as "Abundance Category 1" and were taken to in clude those where' no more than one body was found in any one smear. Next, those specimens in which from two to five bodies were found in any one smear were placed in "Abundance Category 2" and lastly, those yielding over five bodies in any one smear were classified as "Abundance Category 3." The three negatives in this series yielded no bodies in five smears (approximately 2,000 lowpower fields). The specimen in category 3 with the most'bodies showed 32 in one smear. For comparison, a smear was prepared by the same technique, from the lung of a known clinical case of asbestosis. The resulting density of as bestos bodies was so high that they could not be counted with any accuracy, but would be of the order of many hundred in one smear. Routine histological sections stained with he matoxylin and eosin were prepared from 20 of the lungs showing the most ferruginous bodies by the digestion technique. There was definite histologic evidence of interstitial pulmonary fibrosis in only two of the lungs examined in category 3, but this was not of the type seen in asbestosis. The two cases of primary carcinoma of the lung were both in category 2. Naked Fibers.--The digest concentrates contain not only ferruginous bodies, but varying amounts of insoluble and relatively dense debris, denser, that is, than 1.5 gm/cc, the density of chloroform. Where carbon Fig 2.--One end of naked fiber show ing multiple longitudinal fission as seen in asbestos fibers (unstained, X 1100). Results Ferruginous Bodies.--Ferruginous bodies were found in all but one of the lungs taken from men (9S%), an 89-year-old white man. Ferruginous bodies were found in all but two of the lungs taken from women (95.5%). The two negative results were found in the lungs of white females: one 16 years of age, and the other 42. The Abundance Categories, mean ages in each category, and percentage in each cate gory with malignant disease for both sexes are given in the Table. The relative abundance of ferruginous bodies is significantly higher in men than in women (X.2 = 8.973, indicating that sex and abundance category are associated, at a 97.5% significance level). The age range for men was 37 to 87, and for women, 24 to 90. There seems to be a slight tendency for the relative abundance to increase with age, but the range of mean ages between the different abundance categories is small compared to the overall age ranges, and it was not possi ble to demonstrate statistical significance for this tendency. There does not appear to be any definite association between malignancy in general and relative abundance of ferruginous bod ies in the lungs in this series. Arch Environ Health--Vol 17, Sept I96S persists in the mented lungs, adhesion to de ticular interest, what appear, least, to be nr Nearly all are Their range of for the ferrugi; have fractured ally conchoidal ever, have fu crook" configur consider to be ther "mineral 'v which material terial which is of steam or air which shows t'. fission associat 2). Whether su come ferruginovening death of Fibrous dust part of the me least of industr ment upon thei They give a loi vegetable, and and synthetic, forming fibers far as the naki are concerned, ruled out as b prolonged actiand also being form. However, coated to form well survive inf Ferruginous ferruginous bod ible central fila these are poton ly, asbestos b smears additio; seen with opa(Fig 3). A small nir were seen whic! of the central fi' ties. Figure 4 s ASARCO ALV 0006623 FERRUGINOUS BODIES--UTIDJIAN ET AL 331 persists in the concentrate from heavily pig mented lungs, it presumably does so by adhesion to denser mineral matter. Of par ticular interest, however, is the presence of what appear, in the light microscope at least, to be naked fibers of various types. Nearly all are translucent and rectilinear. Their range of dimensions is similar to that for the ferruginous bodies. Most appear to have fractured ends, and the fracture is usu ally conchoidal in appearance. Some, how ever, have fused ends or a "shepherd's crook" configuration (Fig 1). The latter we consider to be undoubtedly man-made, ei ther "mineral wool" or fibrous glass, both of which materials are made from fused ma terial which is spun and drawn out in a jet of steam or air. One naked fiber was found which shows the characteristic longitudinal fission associated with asbestos fibers (Fig 2). Whether such fibers were destined to be come ferruginous bodies, but for the super vening death of the host, we cannot say. Comment Fibrous dust particles would appear to be part of the modern urban environment, at least of industrial cities. Cralley et al com ment upon their ubiquity in a recent paper. They give a long list of materials of animal, vegetable, and mineral origin, both natural and synthetic, which have the capability of forming fibers of respirable dimensions. As far as the naked fibers in our concentrates are concerned, organic materials may be ruled out as being unlikely to survive the prolonged action of sodium hypochlorite, and also being of lower density than chloro form. However, once such a fiber has become coated to form a ferruginous body, it may well survive into the concentrate. Ferruginous Bodies.--In this series, only ferruginous bodies with transparent or invis ible central filaments have been counted, as these are potentially, though not necessari ly, asbestos bodies. However, in many smears additional ferruginous bodies were seen with opaque black central filaments (Kg 3). A small number of ferruginous bodies were seen which exhibit longitudinal fission of the central filament and multiple extremi ties. Figure 4 shows such a' body with one Fig 3.--Ferruginous body showing marked beading or segmentation in coating, and opaque central filament (unstained, X 1100). Arch Environ Health--Vol 17, Sept 1968 ASARCO ALV 0006624 332 FERRUG1SOUS BODIES--UTIDJIAN ET AL Distribution of Cases' Abundance No. of Category Cases Distrfbutton <%) Mean Age With Malig* nancy (%) Men 1 2 3 Total Women 1 2 3 Total 21 38 62 33 15 27 64 40 19 35 70 21 55 100 65 33 29 69 57 31 8 19 68 38 5 12 64 20 42 100 60 31 "Mean age, prevalence of malignancy by sex, and abundance of ferruginous bodies. Fig 4.--Ferruginous body showing bifurcation of shaft and one double end. Appearance strong* Iy sugoestive of asbestos body (unstained, X HOC). double end. Such bodies are almost certainly asbestos bodies, for we know of no other ma terial which undergoes longitudinal fission on this microscopic scale. However, full and positive identification of both central filaments and naked fibers will have to await the further development of physical or physicochemical techniques. Positive identification of asbestos fibers by their distinctive electron-diffraction pattern is already underway in this laboratory.' Significance of Findings.--There is an ur gent need for more data in order to form any estimate of the significance of ferru ginous bodies and naked fibers in the human lung in terms of pathogenesis, actual or po tential. The definite facts are few. It is now widely accepted that asbestos dust, if in haled in sufficient quantity such as only oc curs in those occupationally exposed in the absence of adequate dust control, may be carcinogenic to the human lung. In rats, the inhalation of chrysotile asbestos dust in high concentrations has been associated with a 35% prevalence of lung cancer.10 There is also strong evidence that crocidolite, a par ticular variety of asbestos largely mined in South Africa and little used in the United States, may cause pleural mesothelioma--a rare tumor in man, after a time lag of many years. Lastly, there is the established fact that primary carcinoma of the lung has increased in incidence in most industrialized countries over the past 50 years. However, little or nothing is known at present about the quan titative relationships involved. In the case of asbestos, what is the minimum pathogenic or carcinogenic dose? Tire only data on this question are afforded by the observation that the excess experience of lung cancer shown by certain asbestos industry workers in the 1930's and 19-10's has been greatly rc- Arch Environ Health--Vol 17. Sept 196S ASARCO ALV 0006625 FERRUGIS'OUS BODIES--UTIDJIAX ET AL 333 dticed or altogether eliminated by the imple mentation of dust-control measures in the plant.11 However, it is unlikely that.such dust-control measures have reduced the lev el of exposure of the workers to respirable asbestos dust as low as that of the general population. Is the asbestos fiber rendered completely and permanently innocuous to the human body once it has become coated to form an asbestos body so that only the naked asbestos fibers are pathogenic? Which if any of the nonasbestos fibers, coated or uncoated, are pathogenic to the human lung and in what quantity? Can ability of non asbestos fibers to provoke ferruginous body formation be related in any degree to patho genicity? There is evidence from animal ex periments that it cannot. Gross et al7-12 have shown that fibrous glass, ceramic aluminum silicate fibers, and silicon carbide whiskers are relatively inert in animal lungs, beyond the fact of ferruginous body formation. In conclusion it can be said that at this time there is no evidence that the presence of asbestos dust particles or dust particles of other fibrous materials, coated or uncoaled with ferruginous material, in the lungs of individuals not occupationally exposed to these dusts in industrial urban communities, is making any contribution to pulmonary disease, inflammatory or neoplastic. This study was supported in the Department of Health. Education and Welfare Public Health Serv ice contract PH 85-CG-156 from the Bureau of Disease Prevention and Environmental Control. Chairman Emmanuel Forber, MD, Department of Pathology, University of Pittsburgh School of Medi cine, provided the lungs investigated in this study. References 1. Thomson, J.G.; Kaschula, R.O.C.; and Mac Donald, RR.: Asbestos as a Modem Urban Hazard, S Air Med J 37:77-81 (Jan 19) 1963. 2. Thomson. J.G., and Graves, W.M.: Asbestos as an Urban Air Contaminant. Arch Path 81:458-464 (May) 1966. 3. Cauna, D.; Totten, R.S.; and Gross, P.: Asbes tos Bodies in Human Dungs at Autopsy, JAMA 192:371-373 (May 3) 1965. 4. Anjilvei, L., and Thurlbeck, W.M.: The Inci dence of Asbestos Bodies in the Lungs at Random Necropsies in Montreal, Canad Med Assoc J 95:1179-1182 (Dec 3> 1966. 5. Meurman, L.: Asbestos Bodies and Pleural Plaques in a Finnish Series of Autopsy Cases, A.cta Path Microbiol Scand, supp 181, 1966. 6. Ghezzi, I.; Molteni, G.; and Puccett, U.: Asbes tos Bodies in the Lungs of Inhabitants of Milan, Med Lauoro 58:223-227 (March) 1967. 7. Gross, P., et al: Pulmonary Ferruginous Bod ies, Development in Response to Filamentous Dusts and a Method of Isolation and Concentration, Arch Path 85:539-546 (May) 1968. 8. Davis. J.M.G.: Electron-Microscopic Studies of Asbestosis in Man and Animals, Ann NY Acad Sci 132:98-111 (Dec 31) 1965. 9. Cratiey, L.J., et al: Source and Identification oi Respirable Fibers, A.IHA 29:123-13$ (March-Aprii) 1968. 10. Gross, P., et al: Experimental Asbestosis: The Development of Lung Cancer in Rats with Pulmo nary Deposits of Chrysolite Asbestos Dust, Arch Environ Health 15:343-355 (Sept) 1967. 11. Knox. J.F.; Doll, R.S.: and Hill, I.D.: Cohort Analysis of Changes in Incidence of Bronchial Car cinoma in a Textile Asbestos Factory, Ann NY Acad Sci 132:526-535 (Dec 31) 1965. 12. Gross. P,, et ai: The Effect of a Synthetic Ce ramic Fiber Dust Upon the Lungs oE Rats, Arch Industr Health 13:161-166 (Feb) 1956. Arch Environ Health--Vol 17, Sept 1958 Printed and Published in the United States of America '[ I ASARCO ALV 0006626