Document zz064jZqyjYB2qNKeM1xK2GyR

/ Rtprinted from the Archives of Pathology May 1968, Vol 85 Copyright 1968, by American Medical Association Printteeda anad Published i%p* the United States of America <=\C^ tr to* Pulmonary Ferruginous Bodies Development in Response to Filamentous Dusts and a Method of Isolation and Concentration Paul Crow, JSD; and Robert T. P. deTnvillt, MD, DSc, Pittsburgh; Lewis J. Orallev, PhD, Cincinnati; and J. M. G. Davit, PhD, Cambridge, England Formation of ferruginous bodies should not be confused with pathogenicity. Failure to un derstand this differentiation may result in the erroneous generalisation that all fibrous dusts share the ability of abestos to produce lung damage. Such materials as fibrous aluminum silicate, silicon carbide whiskers, cosmetic talc, and glass fibers produce ferruginous bodies experimentally which are indistinguishable from those produced by asbestos fibers. A carried out in these cities. These bodies are similar to asbestos bodies, though the natures of the central fibers have not been identified. Asbestos bodies are golden-brown, ferro-coated formations found in the lungs of persons who have inhaled asbestos dust. They are generally described as sym metrical, segmented structures, usually method of isolation and concentration of fer with clubbed ends, 3p to 5p in diameter ruginous bodies from lungs of animals and and 20p to 50p long. The core is composed humans is described. Ferruginous bodies from asbestos fibers are much more pleomorphic than has generally been described, casting further doubt on morphological distinctions used in the pest in separating so-called as bestos bodies from pseudoasbestos bodies. wORLDWIDE attention was refo cused on ferruginous bodies by the publi cation of Thomson,' who found, this phenomenon in the lungs of more than 30% of unselected autopsied adult hospital patients in Capetown, South Africa. A of a transparent colorless asbestos fiber that is not always demonstrable. Apparently the only difference between an asbestos body and a pseudoasbestos body is that in the former, the central fi ber is composed of asbestos, and in the latter, of material other than asbestos. Since asbestos-like bodies can form in response to respirable, transparent, color less fibers deposited in the lungs and com posed of materials other than asbestos; and since basing classification of these similar percentage was noted in Miami,2 structures upon identification of the cen 43% in Pittsburgh,* and 48% in Mon tral fiber presents difficulties, a generic treal.* The higher percentages reported term, "ferruginous," has been proposed in Pittsburgh and Montreal are possibly for all bodies formed in response to the inherent in the more intensive searches presence (in body tissues) of a broad Accepted for publication on Oct 17. 1967. From the Industrial Hygiene Foundation, Pitts burgh (Dr. Gross and Dr. deTreville); the National Center for Urban and Industrial Health (Dr. Cralley); and the Department of Pathology, British Asbestosis Research Council, University of Cam bridge, Cambridge, England (Dr. Davis). Reprint requests to the Industrial Hygiene Foun dation, 4400 Fifth Ave. Pittsburgh 15213 (Dr. Gross). spectrum of fibers, including asbestos. Davis* and Collet (according to a letter in June 1966) demonstrated with the elec tron microscope that ferruginous bodies are formed within macrophages by gran ules of ferritin or a ferritin-like protein Arch Path--Vol 85. Maw 1988 HFM -013139 8005 1241 PRODUCED BY FORD SCF-FA-6095 MO FERRVOINOVS BODIES--GROSS ET Ah do not result in the forma tion of ferruginous bodies when inhaled. Respirable fibers, how ever, are apparently ubiq uitous.* They may be mineral, animal, or vege table in nature and of either natural or synthetic origin. They are dissemi nated by industrial proces sing, community activities, personal habits, and the action of natural forces. Fig 1.--Asbutos bodies from ttM lung of on asbestos worker to Illustrate some of the more simple forms thet may be found. In addition to some apparently naked Fibers, there are pale, nonsegmented, rodlike bodies with bipolar clubbing. The dust was probably chrysolite (concentration method; smear; unstained; X 1.100). Also, we have recently re ported experimental pro duction of ferruginous bodies with ceramic fi that are precipitated upon and around some foreign materials. An asbestos body, therefore, is only one kind of ferruginous body; one in which the central filament is an asbestos fiber. As will be seen later, the appearance and dimensions of these bodies are so varied as to defy the reasonably short description usually employed. The problem now confronting invest!* gators concerns the significance of the widespread finding of ferruginous bodies in the lungs of urban population groups. The solution of the problem is, of course, linked to the identity of the central fiber about which the ferruginous body forms and which is at present unknown. It is hoped that recent analytical advances, such as electron diffraction and micro probe will provide techniques for defini tive identification of the central fiber. bers of aluminum silicate. These bodies were indistinguishable by light microscope from many of those iso lated from an asbestotic lung of a known asbestos worker ' (Fig 1). Although nonsegmented, they were golden-yellow, sym metrical, clubbed bodies, staining deep blue with Peris' test and exhibiting a central transparent filament. The present paper discusses our further findings as follows: 1. --Ferruginous bodies are developed in the lungs of hamsters in response to the presence of "biologically inert" filament ous aluminum silicate, glass, and silicon carbide particles. 2. --A simple method is given for isolat ing ferruginous bodies end bare fibers (including asbestos bodies) from lungs. The method is given in detail and the re sults obtained are described briefly. Up to this point, the identification of ferruginous bodies in the lungs of un selected autopsied hospital patients as as bestos bodies1 has been based on the hypothesis that transparent fibers of re spirable size composed of materials other than asbestos either are not encountered in industrial and community environ ments, are not deposited in the lungs, or Hie Production of Ferruginous Bodies Groups of 12 hamsters each were in jected intratracheally with 3.5 mg of fibers contained in 0.5 ml of aqueous suspen sions. This was done under light ether anesthesia with the aid of an illuminated, self-retaining speculum that made the vo cal chords visible and allowed the inser- itrch Both--Vol 85. May 1968 8005 1242 PRODUCED BY FORD FERRUGINOUS BODIES--GROSS ET AL Ml tion of a long 18-gauge needle between the ruginous bodies and bare fibers are often vocal chords under direct observation. All associated with a sticky lipidic film ad of the following were injected: herent to the bottom of the container. The 1. Ceramic aluminum silicate fibers. stickiness allows one to pour off all the This is an uncoated ceramic fiber with a fluid and undigested lung tissue without median diameter of 2p. Fifty percent of loss of the bodies and fibers. Because of the fibers were under 75(i in length, and the presence of anthracotic pigment, the many filaments were shorter than 15p. No film is usually gray in color. The film is free silica was detected in the fibers. dissolved by vigorously washing with a 2. Silicon carbide whiskers. These were mixture of one volume of chloroform and 99.5-j-% SiC. Fiber diameter ranged from two volumes of approximately 50% ethyl 0.5(1 to S(i, and fiber length that ranged alcohol; the total volume should be the from 100)i to 750(1. minimal amount needed to remove all the 3. Glass fibers, uncoated. The fibers had film. The wash fluid is centrifuged at a mean diameter of 0.4(i and a mean 2,000 rpm for about five minutes. Because length of 4.4|i. pf their high specific gravity, the fer 4. Cosmetic talc. Fifty percent of the ruginous bodies and the insoluble mineral fibrous material in the talc was under 0.2(i particles settle to the bottom of the tube. in diameter and l(i in length. On the other hand, most of the carbonace 5. Attapulgite (fibrous clay mineral). ous materia] collects at the interphase be Fifty percent of the fibers were under tween the chloroform and the aqueous 0.1|i in diameter and l(i in length. alcohol. If too much alcohol is used, the 6. Chrysotile (95% of the asbestos used carbonaceous material will lose some of on this continent is chrysotile). Most of the water that lowers its specific gravity. the fibers were of ultramicroscopic di As a result, there will be no separation mensions. between the anthracotic material and the Isolation and Concentration of ferruginous bodies. In such cases, rehy dration followed by the addition of chloro Ferruginous Bodies form will usually effect a good separation. Samples of lung tissue cut into thin strips 3 to 4 mm thick, or fragments about 0.5 cc in volume, are placed in clean glass or plastic containers. To the tissue is added about 20 times the tissue volume of commercial 5% sodium hypochlorite solution. This is allowed to stand undis turbed at room temperature for several hours until all chemical action has ceased. More hypochlorite solution is then added at frequent intervals until the tissue has been digested. For small lungs, such as those of rats and guinea pigs, the frequency and amount of addition of fresh hypochlorite solution should be such that all lung tissue is digested in approximately 24 hours. For human lungs, unless quantitative recovery of ferruginous bodies is desired, complete digestion is not necessary. All of the fluid and the carbonaceous, more viscid material above the sediment at the bottom of the tube are discarded, and the walls of the tube are cleaned of the adherent anthracotic material. The sediment is washed several times with water to remove all hypochlorite and other water-soluble materials. It is then stored in an aqueous or alcoholic medium. When smears are made of the suspen sions, it may be advisable to dehydrate the smear and use a mounting medium to render much of the mineral dust associ ated with the ferruginous bodies less con spicuous. The naked fibers remain visible. In the case of small animal lungs, chloroform and the supernatant fluid were poured into centrifuge tubes in a propor tion of T.2. After centrifuging, the super natant fluid was carefully removed and In the case of human lungs, the fer discarded except for about 1 ml left un- Arch Path--Vol 65, Mav 1966 8005 1243 PRODUCED BY FORD M2 FERRUGINOUS BDDJES--GROSS 7 AL disturbed on the bottom. The film on the from an asbestotic lung of a worker bottom of the original container in which known to have been exposed to chrysotile the lung tissue was digested was then re- asbestos dust for SO years (Fig 1). moved, as in the case of human lungs. Ibis The ferruginous bodies that formed in fluid was added to a pool of whatever response to chrysotile asbestos were sediment was obtained from the super- smaller than those that formed in response natant fluid. The subsequent procedure to aluminum silicate and glass, and also was the same as with human lungs. different from the latter two in being seg- We have worked with formalin-fixed mented (Fig 4 bottom left). The ferrugin- tissue only, but there appears to be no ous bodies were more readily found in the reason why this method should not work lung sections of hamsters injected with equally well with fresh lung tissue. aluminum silicate filaments than in lung sections of animals Injected with filament- Results ous glass or chrysotile dust. No ferrogin- Paraffin Sections.--Initially the fer- ous bodies were found in the lung sections ruginous bodies were sought only in hamsters injected with silicon carbide paraffin sections that had been stained filwnents at this time (one month after with hematoxylin and eosin or Peris' test bijection) for iron. Because of the paucity and small Examination of lung sections of hamsize of these bodies in the sections, the sters killed six months after the intra hematoxylin made the search more difficult. Subsequently, replicate sections that were given the Peris' test only, or also lightly counterstained with eosin and cleared, proved to be satisfactory. tracheal injection of the filamentous dusts . revealed no more ferruginous bodies than were encountered five months earlier. No segmented forms were found except in association with chrysotile dust. No fer- The injected fibers generally were con- ruginous bodies were seen in lung sec- fined to the air spaces, where they were 110115 * hamsters injected with silicon associated with free macrophages. Some carbide. Dust-containing alveoli found in of the filaments passed through the bodies sections of lung from a hamster injected of one to three, and even four, macro- telc were easily identified because phages so that these cells appeared to be of 11)6 blue coloration caused by the pres- impaled as though upon a spit (Fig 3 bot- enc* of iron'. however, ferruginous bodies tom left). When the sections had been coula not ** identified. Similarly, no stained for iron, the dust-containing areas, ferruginous bodies were found in lung under low magnification, were usually sections from hamsters injected intramarked by a granular deep blue colora- tracheally with attapulgite. tion. Lung Digests.--The smears of the sedi- In sections of lungs of hamsters killed ment derived from the digestion of the one month after an intratracheal injection lungs from hamsters injected six months of aluminum silicate and glass fibers, oc- previously with aluminum silicate, glass, casional ferruginous bodies were found, and chrysotile, respectively, consisted These were non-segmented, light-yellow largely of naked filaments, but many ferstructures with bipolar clubbing and a ruginous bodies were also seen. Although transparent central filament (Fig 2 top left, and 3 top left). These, subsequent to the Peris' test, took on a deep blue color that often obscured the central filament, Very similar non-segmented bodies were most of the ferruginous bodies that had formed in response to aluminum silicate and glass fibers were non-segmented, a number of segmented forms were also found (Fig 2 top right, bottom left, bottom seen in suspensions of isolated and con- right; and 3 top right, bottom left, bottom centrated human asbestos bodies derived right). A more prolonged search of the Arch Path--Vot 8$. Mow JS6 8005 1244 PRODUCED BY FORD FERRVCWOVS BODIES--GROSS ET AL S43 sediment irom lungs of hamsters injected tion of fibers in the ambient laboratory with silicon carbide was necessary before air. several ferruginous bodies could be found. The sediment from the human ashes- This search was facilitated by applying totic lung was a rusty, red-brown color. Peris' test for iron to the smear. The bod In the smear, a wide variety of asbestos ies were mostly nonsegmented, and except bodies were seen in addition to innumer for the clubbed ends, the coating on the able naked fibers. As previously indicated, filaments was thin. One silicon carbide a large number of the asbestos bodies fiber was found with a club-shaped coat were pale, thin, and nonsegmented. Also ing at one end, a fusiform coating near of interest was the fact that many of the the other end, and a very slight thicken largest bodies did not have smooth sur ing near the middle (Fig 4 top left). Two faces, but were spiculeted. The spicules segmented ferruginous bodies that had formed around silicon carbide fibers were observed. Unfortunately, the diffusion of the blue pigment of the body rendered its outline indistinct (Fig 4 top right). Fig 2.--Ferruginous bodies formed In response to aluminum silicate filaments In a hamster killed five months after an Intratracheal Injection of 3.5 mg of the dust, bottom right* naked filament fconcentrstion method; unstained smear; X MOO). The sediment from a hamster lung in . .. v ... - . jected with talc was composed largely of crystalline plates; but upon careful search, a number of non-segmented ferruginous bodies were also found. These too had a transparent central filament (Fig 2 top left, and 3 top left). The Prussian blue re action for iron had also been used in this * M fsmear, and the outlines of these bodies were indistinct because of the diffusion of the pigment (Fig 4 bottom left). ^02 Unexpectedly, ferruginous bodies were more difficult to find in lung sections after chrysotile injections than after aluminum silicate injections. The same finding was s / observed with the concentration method. Although few in number, all asbestos bod ies were segmented (Fig 4 bottom ripht). One of the most interesting observations was that, when stained for iron, filaments that passed through the bodies of one or more macrophages showed a coating of ferritin or ferritin-like material only on the intracellular portions of the filament (Fig 3 bottom left). 10/* ft /; ^ ^j The sediments from the lungs of ham sters injected with attapulgite revealed no ferruginous bodies. The failure to find # : if* ' ferruginous bodies in these sediments may . be interpreted as a negative control to > 0 indicate that our positive findings were - v*..-: ... a.jv-s*: ~* - I\Jtr not attributable to the accidental inhala Arc)) Pflth--Vol 45, May J9B8 8005 1245 PRODUCED BY FORD M4 FERRUGINOUS BODIES--GROSS ET AL were often coarse with squared ends. A few of the bodies lacked symmetry. Some pear-shaped bodies without a central fila ment were also seen. Although most of the bodies were more or less rectilinear, many curvilinear forms were also present. Some of the latter appeared to measure between 180 to 270". In the larger specu lated forms, the central fiber was often obscured by the thick, dense, red-brown coating. Some of the very long thin fibers (50p to lOOp) often had segments of ferro- protein at either end with relatively long stretches of naked fiber in between. In an attempt to free asbestos bodies from adherent fine carbon particles in an otherwise "dean" suspension, it was sub jected to ultrasonic vibrations for a few seconds. Unexpectedly, only naked fibers remained, and no asbestos bodies could be found in the suspending fluid. The distinction heretofore made be tween asbestos bodies and pseudo-asbestos bodies may have been based in part on Fig Ferruginous bodies formed In response to filementous glut oust Bodies (top, left eng right, end bottom right) ere unstained. Four mecropheges ere faintly outlined. Only the Jntrecetluler portions of the fiiement ere cooted with Iron (Peris' stein, x 1.100). what the observer believed to be an ap pearance consistent or inconsistent with that of asbestos bodies. More often, how ever, this distinction was based on the observer's knowledge that the host had or had not been exposed to respirable asbestos fibers. Regardless of the nature of the central fiber, the bodies that result in response to the presence of filamentous dust in the lung have as a common feature a coating of iron-containing protein (ferritin or fer ritin-like) . Furthermore, it appears, from a study of the pleomorphism of human asbestos bodies, which was so well illus trated by Gloyne and Merewether,1 that differences in size, segmentation, or other morphologic features of the ferruginous coating probably would not serve to dis tinguish between ferruginous bodies of asbestotic origin and those of nonasbestotic origin. Thomson has proposed to differentiate between asbestotic and nonasbestotic fer ruginous bodies on the basis of the trans parency or opacity of the central fiber.5 This proposal seems inappropriate, inas much as we have demonstrated that a number of transparent fibers of respirable size other than asbestos are capable of producing ferruginous bodies that are in distinguishable from those produced by asbestos. A At any rate, it is obvious from the pro duction of ferruginous bodies in hamsters in response to respirable, colorless, trans parent filaments of aluminum silicate, glass, and silicon carbide, that such bodies Arch Path--Vo! 8i, May 1968 8005 1246 PRODUCED BY FORD FERRUGINOUS BOD1ES-CROSS ET Ah MS represent a general reaction to filamentous particles and are not a specific reaction to asbestos fibers. To what extent our find ings may have application to the fer ruginous bodies that are being found with increasing prevalence in human lungs1-4 remains to be determined. The applicabil ity depends, of course, upon the identity of the central fiber in the ferruginous bodies. The method of isolation and concentration of uncoated fibers and ferruginous bodies from human lungs which is described in this paper may help in the study of the nature, identity, and prevalence of inhaled filamentous particles and associated pul monary ferruginous bodies. It is highly probable that the composi tion of the ferruginous body is altered by the method used for isolating it, since it is not likely that with the destruction of proteins leading to the liquefaction of all tissue elements in the lung, the protein in the ferruginous bodies should be spared. In all likelihood, the protein constituent of the bodies is destroyed, and only the iron and other inorganic components re main to retain and maintain the form of the bodies. The fragility of this chemically altered ferruginous coating is indicated by the ease with which it is removed when subjected to ultrasonic vibrations, thereby rendering the central fiber naked.. This may further facilitate the identification of these fibers. There appears an increasing tendency to consider all filamentous dusts in the same category as asbestos with regard to their pathogenic potential. This reasoning is probably based on a mechanistic con cept of the pathogenicity of fibrous dust in general, and asbestos dust in particular. Such a mechanistic concept holds that the pulmonary fibrosis in asbestosis is the tis sue response to mechanical trauma pro duced when pulmonary cells are perfor ated or impaled by the fine points of the asbestos fibers. It need only be recalled that a similar mechanistic concept of the pathogenicity of crystalline silica was abandoned many years ago because of overwhelming evi dence against its validity. Just as the proven biologic `'inertness" of diamond dust was the coup de grace for the mecha nistic pathogenetic concept of silicosis* so should the proven biologic "inertness" of filamentous aluminum silicate ,0 have dis credited the mechanistic pathogenetic con cept of asbestosis. Extremely thin flakes of glass may also be considered to have sharp cutting edges; yet, glass has also been found biologically Fig 4.--Ferruginous bodies formed in response to other filamentous dusts end stained for iron (Peris* test). The central fiber (top, left and tight) is silicon carbide; and (bottom left) tremoiite (talc) (X 1.110). Arch Pcth--Vol 65, May 2968 B005 1247 PRODUCED BY FORD 546 FERJtVamOVS BODIES--GROSS ET AL "inert" when inhaled or injected into the lungs of animals. Silicon carbide is noted for its hardness, sharp edges, and points, which make it an Ideal abrasive. When inhaled as a fine dust or injected intra tracheally, it has caused a pulmonary re sponse likewise classified as biologically "inert."11,18 Gaining increased attention is a newer concept that the potential of extraneous trace metals and other materials associ ated with fibrous minerals can cause the injury previously attributed to fibers. The respirable fibers may provide a transport mechanism for dosing tissues with injuri ous materials associated with the fibers. Cralley et al11 have shown that asbestos textile workers in the past have been ex posed to appreciable concentrations of nickel, chromium, and manganese associ ated with the fibrous mineral, and abraded from the alloy metal in asbestos process ing equipment. They state further that there is evidence that this phenomenon exists in relation to a number of other fi brous minerals. There is also some indi cation that the biological response in the formation of ferruginous bodies may be related to the nature and extent of the layer of metal solute surrounding the fi ber. Additional research needed in these areas is currently underway. Unless the above facts are kept in mind, the finding that ferruginous bodies are formed in response to aluminum silicate, silicon carbide, and glass filaments in the lungs, may be used as still another reason for erroneously classifying these dusts with asbestos in their ability to produce lung damage. This investigation was sponsored by JohnsManvilie Corporation, New York, and supported in part by Public Health Service research grant U1 86-66-156 from the National Center tor Urban and Industrial Health. All experimental materials used, except chrysotile (supplied by Johns-Manville Corporation. New York) were obtained from and analyzed by the US Department of Health, Education and Welfare, Public Health Service Center for Urban and In dustrial Adairs, Occupational Health Program. References 1. Thomson, J.G.; Kaschula, R.O.C.; and Mac Donald, RR.: Asbestos as a Modem Urban Hazard, S Air Met J 31:77-81 (Jan) 1K3. 2. Thomson, J.G.; Path, F.C.; and Graves, TM.: Asbestos as an Urban Air Contaminant, Arch Path 81:C8-4W (May) IKS. 3. Cauna, D.; Totten, US.- and Gross, P.: As bestos Bodies In Human Lungs at Autopsy, JAMA 111:371-373 (May) 1K5. 4. Anjilvel, L,, and Thurlbeck, W.M.: The Inci dence of Asbestos Bodies in the Lungs at Random Necropsies in Montreal, Caned Med Amoc J 35: 1179-1182, IKS. 5. Davis, JM.G.: Electron-Microscope Studies of Ashestosis in Man and Animals, Ann NY Acad Sri 132:98-111, IKS. C. Cralley, LJ., et al: Source and Identification of Respirable Fibers, read before the annual meet ing of American Industrial Hygiene Conference, Chicago, May 1967, JA1HA, to be published. 7. Gross, P.; Cralley, LJ.; and deTrevUle, R.TP.: "Asbestos*' Bodies: Their Nonspeciflclty, JAIHA 28:541-542 (Nov-Dec) 1967. 8. Gloyne, SR., and Merewether, ERA.: "As bestos," International Labour Office Supplement, p 7 (Jan) 1838. 9. King. EJ.-, Yoganathan, M.; and Nagelschmidt. G.: The Effect of Diamond Dust Alone and Mixed With Quartz on the Lungs of Rats, Brit J Industr Med 15:82-95. 1958. ID. Gross, P,, et al: The Effect of a Synthetic Ceramic Fiber Dust upon the Lungs of Rats, Arch Indust Health 13:161-166 (Feb) 1956. 11. Gardner, L.U.: Studies on the Relation of Mineral Dusts to Tuberculosis, Part : The Rela tively Early Lesions in Experimental Pneumoconio sis Produced by Carborundum Inhalation and Their Influence on Tuberculosis, Am Rev Tuberc 7:344, 1923. 12. Gross. P.; Westrick, ML.; and McNemey, J. M.: Experimental Tubeiculopneumoconiosis, Arch Indus! Health 19:320-334 (March) 1959. 13. Cralley, LJ.; Keenan. R.G.; and Lynch, JR.: Exposure to Metals in the Manufacture of Asbestos Textile Products, read before the annual meeting of American Industrial Hygiene Conference. Chicago, May 1967. JAIHA 28:452-461 (Sept-Oct) 1K7. I I HFM -013146 Arch Path--Vol 85, Map 1968 8005 1248 PRODUCED BY FORD