Document 71mmNQRGjEwV55YrZexDJOejR

FILE NAME: Shipbuilders Council of America (SHIP) DATE: 1973 Mar DOC#: SHIP052 DOCUMENT DESCRIPTION: Journal Article - Identification of Asbestos Human Tissues Identification of Asbestos in Human Tissues A. M. Unger, PkD ,; Richard Ashley, Vidor Baden, M i.D.; Carl Berkley, M.S.; L C. Hammond, Dr.5.; Anne 0 . Mackler, M .i; C. |. Magpore, PK.D.; W. J. NichoUon, Ph.D.; A. N. Kolif PhO .; I B. Rubin) M X; Antonio Sastre, M i ; and I f. Selilrnff, M_D R e c e n t studies have established that occupational exposure to asbestr> dust is associated with serious risks of lung ^caning and neoplastic disease , ' 5 Under certain circumstances, albeit to a lesser degree, simitar hazard* may exisi with indirect occupational exposure,4 s with family contactb 7 and as the result of neighborhood and environmental ex posure The latter group might include residents within a half mile of an asbestos plant.6 7 Although exposure to all *tber varieties have been shown to be associated with disease, there remain differences con ceming the relative hazards associated with each asbestos liber typ e1,J ll has been difficult to determine the quantities or dimensions of any one fiber type needed to produce d ise a se * Studies of human exposures to crocidofite,' K fl chrysolite." 11 17 amosrte,M tremolitp.15 and anthophyllite' 11J have been dif ficult to compare since they reflected dif ferent exposures under different con ditions These uncertainties partly result from lack of .nformatron defining fiber ex posure The asbestos mineral group defined commercially is comprised of five fibrous hydrated siliratp minerals * - ,B Although the minerals range m chemical and physical propert'es, they possess some qualities w hich perm,! overlapping usage in thousands of products and applications 19 Fibers often tend to occur together m the same mineral deposit as m ixturesi3 All ex posures therefore, whether occupational, indirect or environmental, may. to some from the Environmental Sciences ijtxxiuxv. Mount Sma< School of Medicine of (tie Ciw Jmver ufy ol New York. N Y R*print reqge&K to o n * ot Commumtv M 4r ine Ml S in * School of Medicine, Cily UnrvcRity ol New York. 3th Ave and tOQh St Nwv tu>k 1>X)30 (I> Larger) extent, be " mixed' ' in nature Essential information about the fiber-disease relationship may be added by analyzing lung tissues of individuals exposed to fibrous dusis However, recovery and (OerilificdUon uf dstwilos particles from human tissues have been difficult and af fected by such factors as the nature of the recovery technique, fiber-type sought, amount of tissue available, level ol exposure, analytical technique used in identification, and size-sensitivity of the method of examination In this report, we review and discuss a number of techniques developed and used in the past with limned success New techniques ofier some promise for solution of a number o f unanswered questions These w ill be described and initial results presented Analysts of Buk Ousts A number of instruments have been used in attempts to identify asbestos fibeis m human tissues These make use of physical and chemical properties of asbestos minerals The accuracy of the procedures is predicated upon two im portant assumptions (1) each asbestos mineral possesses a characteristic property by w h ic h n may be distinguished from others as w ell as from other mineral species* (2) asbestos minerals are not altered chemically or physically in vivo Initially, those in struments used by physical so enlists to characterize large bulk samples of "pute" asbestos mineral were used to analyze tissue extracts It followed logically that the tissue materials analyzed with these " bulk instrumental techniques" were those obtained from individuals occupationally exposed to mineral fiber since m such cases, the cnnrpntratlon of mineral fiber in tissue w as assumed to be high and relatively Journal of Occuwttonil MedidfleAfoi. 15, No 3/Nardi 1973 large quantities ot tissue materials would he available A. -- Extraction of Inorganic Materials from Tissues \ number of techniques have been devised to extract inorganic particles from human tissues These tndude the formamide method,70" n hydrochloric acid digestion24 25 hot glacial acetic a c id 76 alkali digestion,77 a oxidation of tissue in electro n ically activated oxygen hydrogen peroxide digestion and high temperature ashing Most o f these were developed to study silicosis, coat miner's pneumoron.osis and other dust disease* of the lungs, Those used to investigate the asbestos problem have produced results o f questionable value TTie 3tdbilit> of asbestos minerals differ from inorganic materials for w hich the techniques were onginally developed thus chrysolite fiber* may be reduced to amorphous silica 'ghosts" in extremely w eak a c a s .,ff w 31 the amphfbole minerals have different degrees of stability in alkalis and acids ,a Therr^nre most attraction techniques commonly used m laboratories io study pneumoconioses are too vigorous for the asbestos mineral group Extraction of sub-micron fibers from human tissues by such methods may profoundly change the character of recovered minerals These observations prompted Nagetschmidt to remark that a good extraction technique 'w (he removal of asbestos fibers from human tissues had yet to be d evelop ed 32 It w as Nagelscbmidt's laboratory w h ich used different techniques on equal aliquots of the same lungs and obtained different mineral ex tracts with each technique. Most laboratories rely on oxidation of bulk tissues for recovery of inorganic mineral residues This may involve either 287 htgK temperature oxidation ot bulk tissue m a muffle furnace or low. temperature oxidation of-small quunlitn.-- o f li'Mre m activated oxygen 8oih techniques work welt with some preference tor use 01 the l<jw temperature technique since the inorganic phases arc less likely to be dehvdfoxylaled Oxidation mav be used m conjunction with a wet chemical digestion method if a spprnic tiber is being sought for example potassium hydroxide digestion ot !is*ue may be used tor chrysotile recovery as the pH of the syyfpm (at S* com enuatiun) is near the isodectnc pomi ot the fiber (1 0 8 ) ,8 Search lor a single fiber type has the disadvantage mat the technique may not permit survival ot other mineral fibers w hich may be present and important biologically Evaluation ot alt currently used digestion techniques indicate*- -hat no one is suitable for extraction and recovery ot all tiber types Some alteration r mineral phases wilt occur, especially m fider\ of small particle size However, even with thf^e diff.culhes extractions and anafvses ot bulk lung residues have been made B. X ray D iffractom etry The x-ray powder patterns and crystal structures for in d iv id u a l asbestos minerals have been de'otmined in a number of studies ***> lhe%e data have been used purnanly with phase iden tification of settled fibrous dusts41 *' or for quality control41 Some work has been done on risvur extract*- with ibis technique with varying results 12 u ** Araphibole asbestos fibers have been identified by this method bui m no case has chrysolite been identified pven in ca ses k ro w n ro ftave been o c cupationally exposed to this variety ot asbestos A number of analyses of lung tissues extracted with the low temperature ashing technique m conjunction with potassium hydroxide digestion have been made The x-ray diffraction pat ferns obtained on extracted residues w w not clidiaUeiistic o' the abeslos fibers believed to have been present tn the tissues on the basis or occupational histones44 No successful identification was made on residues obtained from lungs of wot*;men exposed ro chrvsotite containing dusts Some representative x ray reflections were obtained from sam ples of dusts from men exposed to am- 288 l Caffe* tstocM Sm liw steam (M M few a rnfesaa a * * * iM tlfe liter Sam St wM tM flm i^ M tU fe fte iftp ife rk M fM tn M iilM fl Kara & feateafa* Matteraffilar* arc ffttttt afeick an tais t ie 5 skract it lt*fei. C**e af leefe isfecstesb. phibote asbestos, e g , amosite The technique proved inadequate even when more sensitive powder camera techniques were used The reasons lor lack of success with this technique are the p article population tended to be mixed so the xrav spectrum obtained from each sample was complex the x-ray reflections ten ded to be broad and ot low intensity making accurate determination of anqlns nf reflections extremely difficult, inhaled (articles tended lo be o' small size thus producing a 'line-broadened" x-ray sp e ctru m 4' w ith g e n e ra lly poor resoluiion the presence of endogenous iron compounds (hemosiderin) tended lo induce x-ray fluorescence obscuring Identification of fe b u to t in Hamm Tissue/laninr nt al 4mall peaks in a heightened background H>e iw n reported cases o f success using the x-ray technique*-' 15 were obtained on workmen exposed to the amphibole mineral am ovte fn addition to the am biguities which derive from complex mixtures diminished intensities and par ticle M7e pffects. marked alteration of the particles m vtvo also limit their iden- tmcation Biological degradation of asbestos has been jepurted previously 44- sa yhe extrem ely acid-sensitive chiysoftle is most readily broken down m vivo, reduced in sire50 and depleted in magnesium Out observations in dicate that even when the occupational history is known the x-ray diffraction technique is inadequate to characterize lung dust burden It is o f limited value for am phibole asbestos exposures, especially if fiber types are mixed, and almost valueless if exposure is to chrysoiile Although some reports in dicate some success in fiber iden- tificat'on, the x-ray technique should be recognized as having limited value 3 CkA** o f r w M <Mf 8h m pacros *Mm s 4 (n w * w fc m p*$*4 te chqruit* Sk*r E ip tu re its ttt fra m *f tttestot-tMfciMtf cadki (w pauR R fiten < t e fimt at* m t t * fey Igfct maie^n turn wt M k Ih ( c ita i C rtw rtn<M IW>|s m ij )wu ittew Rk m a a r t aptaR te ttlw to tr-- "tek1* ii a r*M tectorj Fm than am *tsnl m b* kum by SgU a ltro iiw . Cjhm (4**Rr ha| o u r <ak*m- A triaarr w kpH t Maptoaa *t a catoa an ate* ten* at ttri***y Journal of Occupiboftai ttedictne/Vol 15, No. 3/Morch 2973 C -- Infrared Spectroscopy the infrared spectra of asbestos minerals have been determined4** 55 54 As with x-ray diffraction, only limited success has been achieved for iden tification of mineral phases in tissue residues * u Ousts containing amphibote asbestos minerals yield infrared spectra characteristic of the amphibole mineral group, i ? , the spectrum ob tained u characteristic of the structural array of ail minerals in the group rather than one specific member ol ihe group Specific amphibole minerals could not be identified The characteristic spectra of chrysoiile obtained on pure mineral specimens were almost identical to the olher serpentine mineral phases lizardile and antigome* making them virtually indistinguishable from each other In ad dition, the infrared spectra o f serpentine minerals ate tenraikably similar to other sheer silicates so that any admixtures of chrysoiile with days or talc tor example make fiber identification impossible by inftared spectroscopy alone As m the case of x-ras diffraction alteration of the asbestos fibers m vivo, or during recovery frnm tissue would alter the diagnostic properties Although a high resolution spectrophotometer* was not used m our study, confounding efiects existed w hich Jav outside the Instrument 289 design Complex mineral assemblages bodv safts organic debns and alteration of particles in v n o restrict the use of this technique fix identification purposes D. -- Differentia) Thermal Analysts The thermal behavior* or venous asbestos minerals have been deter mined4* 55 r *for a range of sample and instrumental variables The am phiboles yfefd characteristic DTA patterns which are Inadequate for uru'q uivrval iefetiftcation, even m the ' pure' mineral state 8oth exothermic and endothermic peaks tend to b0 small in magnitude and broadened over a long thermal range Chrysotile produces a well-defined pat tern but tends to be extremely sensitive to changes in sample preparation and expenmental conditions ss Variations m particle size and packing of sample influence the endothermic dehydroxyUtton peak so that it may shift sc m uch as 100C The influence of these factors on diffusion of vapor from the site of reaction** is w ell documented In addition, instrumental variables such as ctiange m heating rate and instrumental sensitivity may alter peak position and configuration of thermal curves W e had no success characterizing e x tracted lung dusts using this technique4* The major problem was interference of the residual organic phases w hich produced a senes of strong exothermic reactions m the thermal range m which characteristic asbestos reactions occur t *-- Summary--Sulk Techniques T lit use of a bulk uisU uintiilal technique to characterize phases m re covered lung dusts presents many dif ficulties Even with the most useful technique, x ray diffraction, the results are marginally usetul for amphtboles a-d of 10 value for chrysotile identification A number rvf m io m prohliw*. are inherent n the analysis evidence demonstrates that chrysotile alters m vivo, thereby losing or changing its pfiyyicaf and chemical U w ia u w isfits needed for identification, extraction of particles from (issues may lead to ex tensive alteration of asbestos present ana lytica l instrum ents w h ich have routinely achieved acceptable results for pure mineral samples may not produce comparable results on mixed samples or unknowns: the amphibole asbestos 290 minerals tend to overlap in properties making individual phase identification difficult chrysotile asbestos closely resembles the platy serpentine minerals as well as other sheet silicates, making its identification difficult if not im possible large quantities of materials are needed for analysis so that only tissues from occupationally exposed individuals are of use and bulk tissue materials are needed for mineral fiber jecovety Tire results obtained on tissues from in dividuals exposed to asbestos minerals are generally poo>, even when the ex posure history >s well known Iden tification of Single Par ticles--light Microscopy (a) Asbestos todies. -- in general, the object most readily noted m lungs of workmen exposed to ashestos dust k the asbestos body It ts readily discernible by light microscopy and is commonly cited as an index of asbestos exposure The non-specificity of the body has been recognized almost as long as the obiect 'tse il4 5 Asbestos bodies la v e been used as asbestos matkers in numerous studies of groups ranging from oc cupationally exposed workmen to the general population. Bodies have been counted m sputum, lung smears, lung scrapes, digested bulk tissues and various preparations and modifications of hisiologic sections 11 These studies have reported frequen cies o f asbestos body presences ranging from 0-100% Evaluation of these find ings is impossible because many fac tors and study designs were not uniform The studies differed m such variables as age and sex, techniques of material jxeparauon selection of kinds and amounts of biological specimens examined, lung sites examined: number of specimens examined per case areas scanned per case, magnifications used in scanning, type of microscope employed Tiber type involved duration of ex posure, relative dustiness of trade and work areas, type of histologic stain; pre paration used for digestion and cen trifugation of bulk samples All studies assum ed hom ogeneity of body distribution in the tissue materials Some erf the above differences may not appear to be obvious drawbacks for example asbestos bodies tend to form w ith different degrees of efficiency m human lungs as a function of fiber t y p e T h e same number o f asbestos bodies observed in tissues o f workmen exposed to different fibers need not in dicate similar exposure levels. Different fiber types tend to form aerosols with rharactenstir size distributions* so that "th ick" bodies, those easily seen by light microscopy, are more likely to be nucleated on tremolite anthophyliile or amostie. Chrysotile, the most commonly used fiber m (he United States, tends to form fibers of sm all sizes readily degrades in vivo, and resists formation of ItatifiefttiM el Asbestos m Human Tissue/Unger ct l. asbestos b odies <I* (b ) AsbestCK tfb e n . -- lung tissues (rom 3000 c a s e , from ih g n m l population ot New York City were examined lot asbestos bod> contentB } The optically visible asbestos bodies represented only a small fraction of the total inorganic particles present m lung ti$ues In these same tissues many 1no[game uncoated fibers were ob served, including many w ith diameters of less than 1 micron lr 1,028 of the 2 000 cases these fibers were observed jo be piesent ** The occurrence ot such thtn fibers appears to correlate with asbestos body co unt While the light microscope appears to be adequate for the search for asbestos in human lungs, there is much evidence to suggest that this s not so Fven after light microscopy indicates the presence ol ' fibers" and 'bodies", important questions remain unanswered What are the uncoated fibers, specifically', whal lies at the cote uf "asbestos bodies"7 Light microscopy furnishes information about the morphology ol a particle, its color, telative crystallinity if polarized light optics are employed, and the in dices of refraction if immersion oils are used This may provide information tor fiber identification w ere one certain that the particle had not altered in vivo More important, the major drawback in using the light microscope to search for and identify asbestos m human lungs is the fact that it is size and resolution limited Identification of particles ts based upon correlation w ith standard asbestos fibers, the optical properties of which were determined on much latger fibers, fibers recovered from tissues tend to be much smaller and the optical properties associated with larger fibers are infrequently observed in smaller ones Optical microscopy should be considered preliminary because size and resolution limitations allow only narrow perspective o f the total lung burden of particles The number of light-visible fibers m human tissue is limited com pared to smaller, sub-fight microscopic fibers. Observation of the prevalence of asbestos bodies w ith the optical microscopp may or may not indicate the burden of asbestos fiber The search for and identification of fibers must be made on the sub-light microscopic level Iden tification of Single Far* tkles--Electron Seam Instruments A greet num ber of sub light microscopic asbestos fibers occur in lungs of woriemen exposed to aerosols containing asbestos 5* **<w <r <ethe smalt fibers appear to be present to amounts estimated from an equal amount to more than 1,000 times greater than those fibers visible by light microscopy This is of im portance in studying exposure in the general population among whom there appear to be primarily sub-light m ic roscpptc fibers in lungs,'" ** a find ing understandable in light of recent w ork dem onstrating sub-hght m icro scop ic ch ryso tile fibers as significant constituents in urban air sam ples The search for asbestos fiber should be made by a method w hich per mits companion on all exposure levels and fur all fiber types, electron beam in struments may be employed tor such purposes To examine specimens on the sublight microscopic level, tissues should be prepared so they are compatible w ith the technique. Several methods currently available for this are ft ) direct micromanipufatton o f objects from tissues on to appropnate substrates (2) digestion of bulk tissue, (3) carbon ex traction of partides from histologic sec tions (a ) Tissue Preparation. ( 1} The mirromampuiator has been used to rem ove and transfer objects (torn histologic sections to appropriate sub strates for examination bv electron beam instruments 51 w This technique requires localization of objects w ith a light microscope- removal o( the tissue matnx by low-tem perature oxidation, placement of the ashed slide cm the miCTomantpulator stage and use of a mechanical manipulation system to remove selected objects from the sec tion The use of the fight microscope tn the search for these objects precludes, by definition, sub-light microscopic par ticles from analysis Asbestos bodies so removed tend to be nucleated on am- phibole fihpr*.52 7i Uncoated fibers are only rarefy removed from tissues by this method, (2) Occasionally bulk tissue is digested by the alkali method or ashed in a ir or activated oxygen and the Joural el OaipJttonil MedicinefYol-15. No. 3/Metdi 1973 residues examined by electron beam in struments.* >3v w icn O ne procedure in volves several cyrt#*s of washing and centrifugation (a lka li digestion technique) and both require homogeneous dispersal of the residue by sonlficaiton m an aqueous medium and pipetting the homogenate on to electron microscope grids The technique is eccellem ror tissues m which low con centrations of asbestos dusts are present The m ajor draw backs are the 'equiremem for bulk tissue samples and the partial destruction by aik!t o f some amphiboles. (3) A technique has been developed w hich permits using normal histologic sections m the search for asbestos particles" * - ' * n * s method is called the carbon extraction technique and consists of a procedure in which the tissue is ashed on a glass slide, impregnated with a water-soluble plastic removed from the slide, a layer of carbon deposited on the tissue sur face. and the plastic dissolved with hot w a'er leaving a floating carbon film with relic tissue and particles 55 * The importance of the carbon ex traction technique cannot be overstated it may be used for all tissue materials, even in cases wlrere only slides are available all exposure (eve)} may be examined in this manner b> electron beam instalments (general population, or low intensity exposure cases, require m uch scanning tim e), the carbon provides a stable medium and sup porting film far electron beam examination of ail particles, the same section may be transferred to the vanous beam instruments for determining the range of physical and chem ical parameters required for characterization. (b) Transmission fteclron Microscopy -- The examination of tissue preparations by transmission elec tion microscopy makes visible all fiber in the tissues and the morphological characteristics by w hich they may be identified Chrysolite generally consists of broken open, often curved, "bundles" of unit fibnis or o f isolated fibrils These fibrils are 20O-4OOA m diameter and are made up of an electron translucent cen ter (a capillary), electron dense "w aits" (crystalline structure containing the capillary) and an electron translucent 291 amorphous edge w hich increases in thickness with lim e o f exposure to ihe electron beam The relative dimensions of the units mas w ry Thesn leatures have been described m the m in e ra lo g ic a i11" - " 15 and m ed ica l literature Occasionally chrysolite may be present in tissue as d um ps9* but more frvcjiienth as individual ItbnU esp ecia lly in in d iv id u als not oc- cupationaiiv exposed to the f i b e r 101 Chrysolite thar has been vigorously treated, such as in hammer- or ball- millrng, tends tr>deiorm readilv under an electron beam suggesting that the crystalline structure at the edges ot the fibrils have been disrupted ^ The mor phological characteristics ot ftbrilv ob served m human tissue vary to some degree vet thev are distinctive enough for unique identification *7 99 At first glance the overall mor phological charactefisnrs of the am- phibole asbestos fibers are remaricably similar, tending to be electron dense and either straight or bent Occasionally un der high mag'iification "cross-hatching" produced b\ diffraction contrast may be observed along the ubers width fhese 'b and s' may occasionally be observed to move up and do-vn the fiber as (he intensity of the electron beam is varied on the surface of the sample These ef fects are likely caused by thermal Stress and change ir con'/eigance of the elec tron beam It i possible to differentiate the ampmbole asbestos minerals mor- phologually Fibers are measured across ihotr w id th and a w id th distribution is determined Each am- phtbole tvfM? apparent!* possesses a relatively unique w id t h distribution w hich mav be used as an identification tool Current studies in the En vironmental Sciences Laboratory suggest that this may not be s straightforward as previously indicated Also in many nonoccupationally exposed cases the number of such fibers observed in tissues is small so that lire usefulness of differential width distributions is re- stneted Also, mixed fiber exposures, apparently more the rule than the ex ception make the results equivocal It appears that, on a morphological basis, chrysolite may be differentiated from all other inorganic silicate fibers m The amphibole fibers may be identified on a specific, fiber type basis only m cases where exposure is "p u re ' Such exposures are generally rare so that only the amphibofe group may be identified usually not the fiber species (<) Selected Area electron D if fraction. -- Most transmission electron m irfoscopf* today am equipped with controls w hich make possible change <n instrument geometry so that diffracted electrons may be brought Kilo focus on the image screen Crystalline materials scatter electrons in patterns represen tative of their crystal structure and mterpldnar spacing* Single crystal Laue geometry (fixed film and crystal) applies so that both dimension and angle characteristics may be measured directfy t * from each asbestos fiber type a charactenstic single crystal pattern may be obtained97 This technique permits more exact identification of fibers when morphological characteristics are not definitive. Chrysolite yields a unique diffraction pattern arid may be identified On this basis as well as by its morphology507 ,0* Diffraction studies of fibers m tissue materials have demonstrated the im portance of the technique as a too! for identification of chrysotile t>7~ O n e report recently proposed that it is possible to differentiate among amphibole asbestos types wilh ihe dif fraction technique ,[W However, these observations have recently been criticized because several patterns ap' pear which are not accounted for b y the mineral's space group symmetry 1,0 Recent work has shown that reflec tio n s obtained by the electron diffraction method indexed according to known space groups for Ihe amphibote minerals, show heretofore unsuspected structural modifications , "J Therefore, the amphtbole mineral group possesses structural characteristics w hich make the mineral group but not the individual members o f the group, identifiable (d) Electron Mkroprobe Analysis. -- The electron microprobe analyzer makes possible x ray spectrochemical analysis of small particles under an electron beam The imaging system Is a limited depth of field scanning electron microscope w ith spatial resolution, using secondary electrons, of about 0,2 micron* X-ray spectrometers"' ,lJ are equipped with crystals w hich serve as dispersive x-ray analyzers The system may detect dements down to and m eluding atomic number 5 (boron) and achieve a concentration sensitivity or 50100 ppm depending on instrumental and sample conditions W e have analyzed asbestos bodies and fibers bv means of the electron mtCTOprobe55 5,1-51 The possibility oi suc cessful analysis of fibrous cures of asbestos bodies and uncoated fibers was predicated upon chemical differentiation of the asbestos fiber types The chemical composition of single fibers was deter mined on the probe Asbestos fiber types, especially amphibotes, could be rlw rn ra lly differentiated and idenhtieri on a single fiber basts * * 55 s* A number of interesting observations have been made, chrysotile fibers are not stable m the biological environment and lend to lose magnesium single amphtbole fibers may be identified on a chemical basis, amphibole fibers which occupy the cores of asbestos bodies are not degraded chemically whereas uncooted fibers appear to have lost some magnesi um from their structures w If is possible to chemically charac tenze and identify uncoated asbestos fibers in human tissues Success has also been obtained on fibrous cores of asbestos bodies only when the fiber protrudes from the coating material In dividual species of the amphibole fibers m ay b e differentiated w ith this technique (e) Summary--Electron Seam In struments. * Preparation techniques are new available which make possible examination of any human tissues m electron beam instruments With the car bon extraetton technique, gross histologic structures arc recognizable and inorganic particles preserved in their original anatomical sites No physical or chemical artifacts are created during tissue preparation This preparation forms a stable matrix for the materials and allows long time examination of particles under an electron beam The mounting of the preparation on an efec tron microscope gnd permits its trans ference mto any beam instrument and identfficatitn of Asbestos In Human Tissue/Linger et al. j allows miiluplp analyst* to top per formed by one or several instruments Asbestos fibers may be Identified on the basis of morphological, structural and chemical criteria Chryyottle may be identified on the basis of morphology and selected area diffraction pattern The amohibote minerals, when occunng as single fibers, need chemical charac terization as wed for unique iden tification Some Observations O u r laboratory has now examined nssues from some 150 individuals with exposures to asbestos (occupational to 'general population") with electron beam instruments The tissue preparation and ideninnation techniques have in dudod ail those described in this presen lation In all c a s w lieie (lie occupational history indicated asbestos exposure, fibers were observed m the lung tissues Some of these were large enough to be seen by light microscopy However, most fibers present were w ell below the resolution of the light micioscope This w k nhcervpri in w orkm en o c cupationally exposed to amphibole fibers as w ell as chrysottle In the latter case however, the small fibers (fibnls in many casta) were present hi numbers about 100-1 OOOx that of the larger nbers this size distribution existed m the tissue since the cartoon extraction technique was used m tissue preparation. Ex trapulm onary tissues have been examined, eg . spleen, colon, pancreas, liver Primarily sub-light microscopic nbers were present in the New York O ty cases from the 'general population" chrysotile fibers were found m the lung tissues of 76 of 126 (about 601b ) casts f The amount oi tissue examined in this study was ex tremely small larger amounts would surely have increased the positive oc currences Here too, small fibers and fibrils of chrysotile predominate in the p ar'tcle population Because of utilization of a bulk tissue digestion technique, if was assumed that this might be an artifact size distribution produced dunng exlraction However this size part-|p w as observed fo predominate in relic tissues prepared toy the carbon extraction technique in another stu d y.* In a number of cases, fibers have been found w hich could only be detected by electron microscopy However even >n these cases, there exists some uncertainty as to whether the size distribution reflects biological degradation or the origin*) inhaled aerosol The prominent fiber in these cases s chrysotile small amphtoote fibers less than 01 microns in width have been observed m the lungs of workmen occupationally ex posed to amosite dusts The proportion of >ub-light microscopic fibers to light visible fibers ranges from about 2 to 100 hmes and is apparently a function of the anatomical site in tlx1 lung.* There are few published data suggesting that the majority o f small fiber* wete produced by biological degradation The biological effects observed in tissues in w hich these fibers occur have often been attributed to the associated larger particles The concomitant sub-light microscopic partides, once demonstrated, canr-ot be discounted as a major pathogenic agent m individuals w ith disna<p Fjguips 1-5 The relationships of fiber size and type tn association with human disease are complex Arbitrary simple assumptions u< co n c lu s io n s based on light microscopy that one speottc fiber type o ' size is responsible for disease are un warranted *Th ml'ii**d spectiopfroionx*iet used w * ` a fw kin firm * Model 21 cqupfied wii* S aG W " '' 'CwiiptviJ studr m Ihe Ennionmenial &irnce*. Ijbortion lo be puWiihec ` Competed viiith fcSi - to tie published References i 5**liki>if II at Asbestos!** and neoplasia, Amei I Med 42 487 t%7 Z Eniicknap |B and Smilher VVI Peritonea! tumors rr. asbe'iosiv flrn I fm/toi Med 21 20 1964 3 belrkoff i| Hammond fC and Churg I Asbesio, exposure, smoking and neoplasia IAMA 204 20, 1%8 4 Mamie PC ArbeMOs ki/jtri'. in sfapyartls Ann Ocui> tfyg 11 135 I96fl 5 MrFwen } el al Mesothelioma in Scotland But Med four 4 575, W7D h Newhoose M and Thompson H Misnihelnma ol pleura ami fx'itioneum (ollOMtng exposure in asbestos m the London area Br>( i Indus; JMed 22 261, 1%> Ix-hen J and Pistawka H MesothMioma and asbestos wqxAute Aicb Environ He*fth 14 ^**9 1%7 8 Wegner K Sieges CA arid M aictoixi P Diffuse pleural mesrthetiixna and asbestos exposure m S o nh VVesiem Cape Province Bn< I M u n toed 17 260 I 960 9 Xisduoto R Pleural cakif'fation a ' a roen'gpnologtr sign nt non-ocrufwtirxijl en dein anihophvttiie ast>es)osrs Aua Radioing 1 94 { i ) 1960 10 A symposium held in Cardiff, Wales, 7-9 A jx il 19/0 ti/iu-CtisoJ w tlii (I*- LntXuKHal 1ftecis rs asbe-tos Ifieie was much discussion i ' ifiis meeting concerning the relative biological hazards associated w iih the dillerert asbestos mineral fibers Opinions ranger4 from 'all ait- ati-u- iunloguaiiv' in only some 'ibers are acm e Projjetues of single liber types were also discussed in cluding fiber length to te metals asvocraied i(h it** fiWi ' orbed hvO'ixarbons etc No agreement a s rearfieri as m the importance <m each ol tbesp properties btolngicalh n Dreessen wC, Dallas alIP |M Edwards Tl Milter NV ai>d Sayers RR A Study of Asbr^iose <n ihr* Asbestos Textile industry U 5PHS Public I Ittalih Bull no 341, Augysj 1938 12 D o ll R Mortality from k in g cancer in asbestos iM xk ers grit I Indus f M id 12 139 1955 14 Selilum l|. Hammond EC and Seidman M AstoAios disease associated ss ith insulation work in ships aids in the United Stales fnlf Symp on Saietv and Health in Shipyards and Ship Repairing Helsinki August. 1971, m Press 15 Klt-mltHd M et al Mortality among talc mux r- and millers ot New n x k stare, Arch Environ Hfrh 14 661 t% 7 Tfe Hefidrv N\V The geology occurreivce' and major usesnf asbc-siiK ^nn N't A t a d 5c 1T J 17 1%5 17 Gaze ft fhe phssical ant4 mo/erulai structure ' asbestos Ann V Y A rad Sc 132 23 1% S 18 Sped b and Lem eweber j Asbestos m in erols tn nicxiem twfm rrfrjBy tn v irn n kes 2166 l%9 19 RizhJIu DV AsU 'siin Us Indosirul Appfications Rhembiid Pub Co 214 1959 20 Thom as K and Stfjt>mann H 6>rmamnit methods of tissue digeslion ol 5iIic<xh lungs fieuraije Sthk Punch 28 t, 1954 21 LrMertU H Einbnxll H) and KlosiMboller W Gram si/f and inmi-tal tonleni Jung dusi ot coal miners compared with mtne dusl In h ilrd Particle, and Vapours 2nd Inll S m r Dam's Pergamon Press 301, 1967 22 Ulrich J and lurk A fate ol submicrorur mhalod du I in ihe lungs inhaled Particles jn d Vapours 2nd Ini! 5ymp Davies Ed Pergamon Press 419 f% 7 23 5li|f)(i W &nbxfi H} and KfosU'dtoller W Quaniilaiive siiidips in riie-t relenlmn in animal and human lungs after chromr journal of Occupational Medicine/Vot 15, No 3fMarch 1973 233 inHatalion Inha'ed P^flirlt-s and Vapours 2nd Inii Syrup D<v-> l . i , rv-ij4<TKin Prr*-, 409 196' 24 Canwriqht ) Ai h>x* ituvt in i oaf mint". the parin ie-n'e yetecnor charac len'tics of duN sampling insiiurnentx Inhaled Particles and Vapour*. 'nd Inti SvTnp DavMA Ed . Pfigamon I V " 393 1967 25 Mum* TC ti ai Companion oi dud retention in '(x-cuic fwtKi^fn tier and ' n dard tat4- InhaltM Pannu--am. \apnuts ind Inti Svmp Davits fd IVigamon Prc-sy J0> I%" 26. Bofgmann l Ibe determination of foal in formalin iod pncurooronintir l<tnf*s a iv>w method l tissue digpst'on u*mg gfai 13I acetic acid Anafvt Qhem 38 441 19% X~ Rim><*- D w ai Dum tnniMU ladtoiogv and pathology in sunpfe poeumocomoso ot coal workers Part 5 G w ra l observation*. Par* 2 Derailed analysis fit the data Hut / tflduiir M /17 07 JSftO 28 Rossiter C l ei ai Dust content radiology and pathology m simple pneumoconiosis ot coat workers inhaled Par (irf*-. and Vapour- 2nd Inti Symp Davits Ed Pergamcm Pros*. 410 196" 29 Berkley C Chun* ! and 5 Itknfi t| Defe non and Incah/dtuxi o! mineral libers m lisyue, Ann NY Acad Set 132 48 1%S 30 Nagy B and Bales if Swbilitv <>t rhrvwMili* asbestos Amor Mineral 37 ffl>S l 2 31 Hargreaves 4 and fa>lnt VVH An *-on exanttnatino u< tieownpoilton product 01 chfy'onlc (asbestos) .iwf serpentine*. Mineral Mag V 204, 1956 32 NagpKchmirk (1 Some obsfrvafton' 01 if*' du'* yonitnt and ^omixniion in lung*, with asbostous made* during work n ro jl miners pneumor on.**]* Ann NY At ad S' 1 132 64 19h5 33 8ow,le**0 -V-bro-tos D$fMfluff 403 9 ' 19V 34 GiUon 1C Wyee. memorial Ircturn Hpaltli hazards o' asb< stos Keroni Mjdies on its biological en is Tram $rx Off u/> Med 16o2 1%6 35 trnst W G Am phi bob*** crystal cbenmtrv phas*. iHatmns and occurrence New tork 5jnnign Vmlag 125 19C41 36 rimbtc'l V Ovw-k i i-umic* oi iht' OtCC standard rr'e'twc* sample* o' Jibe-sins Pneumoconiosis Prof of (he Inti Coni, |h , mho H A AKifiur) fd Oxford (Jnivofsnv Pins Capetown >5 1970 37 lagodeimki H and Ktin/e C f)ie roilchnwruciur 'k-s rhrv>otik I Nt*ues lahrbueh i8r Mineral S>yy 4 5 45 1451 US Jagodzifi'-ki H anti Hunzt* G Ole roUcheminjrlti' ,1e, rhrv-od,^ I) Neue*- llhr bueh RJi Mineral No b I* 3 1954 39 belhidge GC An x-iay and optical in vesligahnn nl fhn v*ip.'nlim* minerals Amp* Mineral 21461 1936 40 WhuiaVer ifN and iussman J The cturailfvialion o' vpfprnlirc minerals by x- ray dilfiaclion Mux-ial Mag 31 107, ;95641 Gable IV and Knott MJ Application 01 294 x rav diiiraction to (he dHermtrutKKt of chry'ncilc in bulk or settled riusU, Ame/ Intf >S AiiO / 27 383 m u 42 CtaWe and hisotf M} QuanWahsex ra> dftfiai.txi analyse, ni corodniite and anvy-ito m bulk or srolerl dust samples. Ajnw Ind Hyq Ann / 27 440, |%A 41 Badoiiet M5 and MtGourty fP trien niicaiion ot minerals asst iju x f w iih asbesios b* x ras dilrcaction patterns Tram Can Mm and Meurt Su(J 6 1 169, 1950 44 Nagelschnidi C Personal com- munication Work ck*s<frfx*d in laboratory 01 Safety Mines Research Estabiofmeiit Shut iiMrt I nndon 196" 45 5imdius N and fly^den A Der SaubinhiK e*tv*r Ashestosp Lunge und di< Beschaih-nheit rk*r sogennannter Asbe$tosn> Kotpcitncn, Arch Cpvvoibnpath Cewprbohyg 8 i)h 1938 46 Berkley C lAiger AM and Baden V Instrumental Analysts of inspired fibrous pulmonao pamculaiei NY Acad So Trans 39 331 1967 47 Cufliiy OO Clements of v ia ) diffraction Addiyon-WediA Pub Co Reading, Mass 514, 1967 48 Morgan A and 1iolmes A Neylrwi at Kvalion technique's in irtvestigalions or the composition and biological elfecls of asbestos Pneumoconiosis Pnx of the Inil. Conf |nh, H A Shapiro, Ed Oxtvd Univ Pr<*.s Capetow n 52 1970 49 langerAM Rubni I and Sehkoli tl Clot* iron Microiitobo analysis 0! asbesios bodies Ptoc of the Pn?mnconiosis C m f, Rih HA Shdjiiro. CcJ Oxford Uniy Press Opeimvn 57 1970 50 Suzuki V. and Churg 1 Struciutf and deyplf^jmeni of rfx* asbestos bod> A n w / Path 55 79 1%9 51 ton^rr AM Ejection mirroprubo dnatyvy (studs n( astjesios fibers and bodies from lung tissue! U h . Diag nl Disease*, caused bv Toxic Agents, F W Sundcttoary and bunderman f w , Eds W H Green and Co * I imiis 126 1970 92 langw AM. Ru'nd I Seliknff H Chemical harauen7atu)n of asbestos body cores b> I'leciron mictoprobe analySK HiiUxbem and Cytochrm 20 723 1972 S5 Unger AM. al Chemical chatac tenzaiton 01 uncoiled asbestos fibers from the lungs of asbestos workers by electron microDtobe analysis / Hutochem anti Cytocherr J t 735, 1972 54 Beatitc and Knox F/ Studies of imnciai onient and partitk* size disinbution in the lure' s of ashes-os lexiik* workers- Davies, C M fd inhaled PanlOes and vajiuuix, f^tgamon Press Inc, N Y 419, 1%1 55 Martinez Chfysotiie asbeslos relation ship <i the surface and thermal properties to the crystal structure Iramacunrti, 69 414 1966 56 Bfindiey C W and Zussman | Infrared abiorppwi daja (nr serpen!inn minerals, A m y Mineral 44 185. 1959 57 aruvjley GW, arel iussman \ a struc tural study of (he (heimal Irans/ormabon ot sorpontme minerals to forxtemn Am**r Mineral, 42 461, 1957 58 Faust CT and fahey B Ttx* serpentine group minerals U 5 Geol Sun Prof Paper 3*4*A92 1962 59 Linger AM and Kerr H tvatuanon ol kaoimite and quart; di/f.veotul thermal ctm.es with a nc high tpmjieTalure edi. Amts Mineral 52 509, 1967 60 Cooke WE A*bfr<-in* dull and cuntut bodies found in pulmonary asljcstoso Bm Med I 2 S7fl 1929 61 Williams E 'Curious irodtos loutvi n ibe lungs o'coal-workers laneer 2541 1933 62 Schiller F Adsorplionserstht-inungen an 5taubert m vivo Pseudo asbestose und 5chj umankorpert hen A*ch Gew erlrepam Cewerbpbyg 14 664 1956 63 von Ruttncr Ifl Gber VbertQsc und Pseudoasbestosekflipcictsen Schweti Z M r Path, 15 628 t9 U 64 Uhdjian MD, Cross P and DeTreville Kf Fetruginous btxlies tn human lungs Aith Lnv Health 17 327, I960 65 Gims P Crattey LI, and tier esille RTP Asbesky.' Ixxftes 7hci non-speciticits / Amer fndust Hyg Assoc, 28 >41 i% 7 66 Stewart M|, and Haddow A i'ecufeat bodies <n nuimnnarv' asbesfosA / Parti gau 32 172 1929 67 &mscm FW and Srrachan S$ Asbesio- hnries in sputum / Path Bad 34 1 1931 68 Fullnn WB <1 ol Asbeslots Part 111 Special Bulletin S o 42 Penn Depi of Lalnx and fndusjrv Harrisburg Penna 19J5 69 Page RC Spuium in pulmnnari Uesb Artier i Med 50 i8*Ht)44 m s 70. Herat ? ot af AsSstOy hodn m spuium samples, i Sara Med Assoc 12 387 1%1 71 Plameydi Hf A*.he\in< horlifn, m shipyard woikcrs Med T Crn*tfk 100 49 1964 72 Sluis-Cremet GC* Asbesttyvis in South Moca Ann NV Acad 5ci 132 215 1%3 73 Stumpbus 1 and Meyer PB AVsesios bodies and mesothelioma Ann Qcr Wvfiwn* l i m 1966 74 Thomson |G KaschUa K and MacDonald R Adjetins as a modern n<fwn haurd South M nan Mwi / 27 77, i% 3 ?5 Cauna 0 Toijen RS and Cross P Asbestos bod** at autopsy /AMA 192371 1%5 76 Anjilvel Itly and Thurlbeck WM In cidence 01 asbestos bodies at random necropsies hi Montreal Can Med Assoc /, 9S1179 1%6 77. Tbonison !G, and Craves WM Asbcsio as an urban air contaminant, Arch Path 01 456, 1966. 78 Gherci I, Molrem G, and Puccetn U Asbestos bodies m lur^s ot mhabnams of Milan, Med Lavoro 58 No. 3 22) l%7 79 Roberts GH Asbestos bodies m lungs at necropsy, ! Chn Path, 20 570, 1967 80 Ashcroft T Asbes'os bodies in routine autopsies on fynevde But Med} 1 614 l% a ol Asbestos in Human Tissue/Unftr et *1. A 51 Gibson -AAM tH al Epidemiology o> mesothelioma in Scotland ex ot 2nd imi Coni ot) Biological Efforts ni Ashestns Dresden Meeting, 21-25 Apnl, Paper (/} 82 Avnl | and Champett f Resulfo o> jib^ '.lo v e vpo sure ir> f n n c p P n riim n tn n H m * 1ml Coni Johannesburg H A Shapiro EH , Poigamon i'rtfss Capetown 10-1 1470 83 Baden V' and Crhwart/ I Oomomlration i)< asbestos bodies Comparison td available ir-chnutut'*. ('for ot >{ {nil Coni on Biological Effects of Asbestos, Dresden Meeting 21 2S April l%8 Paper h 84 Priliack A and Sacks M Ashesios bodies m ha -at lunq k w l j MJ U i 4 223 >%8 85 Pearork PR Long smear. tor asbestos foxlres. fuiofi i Canta 5 135 1%9 8h Xipell fMand Bhaihal PS A lb u m Pdlh 1 327 1969 87 Eirot*. PC MrQughev W7 ami Wadi Ot Diffuse ine*nthrtioma of ihe pleura ami asbestos 8m M ai f 1 J50 l%3 00 Hounhanc DOB Biofey s-ot. in mesothelioma Anempis to ick-nitty asfx>si>s Anna/ NY Acid Set 0 2 M- 89 Meurmin LO Asfxsio, bodies ami pleura plague*- m a Finnish w^ies Acra Path Mirrob Scsnel Supjil 181 1102 19bh 90 CoUiMem 8 *ml Rrndalf KtB ferruginous bodies Pneumoconiosis Inti Con* Job., H A Shapiro Ed Penjamun Press Capi n m n 40 1970 91 Beget PI ObCt die VdxMosuWn- peffhen Virchow. Ar(b Path Aruf 290280 191J 92 Cnii C Data presorted m Symposium on llx* lissuo response to asljeslns 7 9 April 970. Cd'Cnt Walt's 1970 93 Pooley FD Asbestos bodies their for mation composition and character fo u r Re* S 3fii. 9T2 94 (imbrell D Prle-y FD ancf Wagner C ChaiatintsiKs ai respirable asbeMns hl*rs Pneumoconiosis px of the Inti Com (oh, H A Uiipuo Cd, Orf/xrt Unn Pih v O petirwn 120 1970 95 langer AM. (4 al Inorganic fibers, m cluduig chiysmile. in lungs at autopsy preliminary report Inhaled Particles til Proc toll Bin. O r i* Hyg 3oc S>mp londrxv 1970 W H W aiiiKi Ed Unwin Bros Itd .lo n d o n 2 683 1975 % Poolev F al the detection o( asbestos in tissues Pneumoconiosis Proc o* the Inti Conf Joh J9h9, H A Shapro E d , Oxford Unis Prt-ss CapcHoss n 108 1970 97 Langcr AM Poolev FD Idenirfication oi singlt*d<be<trA iibc-i'tn hunuft nssut Proc t* the Inif Corn on iht* Bin Efforts ot Asbesiov lv*n France 2 ( October 197> Pr No 19 I972 96 Pooley FD EJprirnn mtaosrope charar tefi` tic- m inhaled chrysotifo asbeslcx fibur flm / Imi Med W 14ft 1972 99 Unger AM Seltkoft 1J and Sastre A Chrysotik asbestos m the lungs (>f persons m Now York City, Arch toy Heart123 448, 1971 100 5oltkoii <1 N'Kholsim W| and Unger AM Asbestos au pollution, Arch Inv Health 25 1 1972 101 Unger AM anti Selikoi* U ChrssiXile e U M ir- m (lx lung* <,l u-'k W-hI ' m St s Vr'rli O'v Proc 2nd Inll Clean Air Congress Wash. D C Dec 1970 MM Engltmd and W T Urcry Erls Arad Pro's New lo rn 1f1 1b5 197} 102 lUnd*tsojt Wl ft M lalt and caronnma n< Ihe <^arv am! cti\u / O/nirt and Qyncoi (But Commonweallhl 78 26b 1971 103 Clifloo RA Huggins CW and Shell HR Hollow dWHAikf fillers Aiun .Vfut 51 jOO, 1%6 1E>4 Bates 7F CmK'r )| further obtenraiions on lhe morpholrigy of ciitysolile and halloysrte Proc Sixth Nad Conf Uayv and Clav Mins NA5-NRC >37 1959 105 Ydtki K StixJs m chryswile asbestos hy a hgb resolunon eternon fmeroset^e, ACU Oyst 23 704 19^7 ton MrConneli 1DC Deunxi miui/w.M}jy and electron dnlraciion Phvsical Methods m Oeiernimatike Miniffjlogy, 1 2ussman, fd Acad Pss M 5U W>7 107, 7v> yjfin BB Heriron Dnlraciion Analysis of C1a% Mineral Structure* Tram 3 Lyw Plenum Pn*ss NV 3b4 19h7 >08 2usvnan L Brindley CW and Comer If Ei<dmn dntraciton studies ot serpentine mm4K, Arr Min 42 I3J, 1957 109 9ktkr Ml lalbta iH and Ridall REC Eloclron duiracnon patterns ot U l C C AsbtMtn Samples inv f&i 4 141. 1971 110 Poolev FD Personal fomiruntcaiton, April 1972. wiwk in prngnws, DoEMnmenl of Mineral tf*lation Umvefsv of South Wales and Monmnuthshre Cardiff 111 tl'ik* IS tli rtn>n probt micjoaiMlysis Monograph ot Anal Chem and s ^p- pticaitorK /nii^scieni e Publ NY. 17 293 19M 112 Andersen C \ An irucodurtKtn to the Hectron p'obe mtcrnanalycfr and iK ap(lficaJinn lo biochemisirs Atj-lhnds of Buxfrem Anal O Click Ed /rrtmcience Publ NY 15 147 1%/ 113 Meutmjn I f ) Kiviluoto R and Kalcama M Mortality and mnrbidih ot employees of aitfhopMilt* isbeMCA n mines 1 Finland lml Coot Btolog Etteos rt AsbeOov Pr 11 Lynn. Oci 2-6 1972 kurMl of Occupabonil MadiciM/Vol. 15, No 3/Msrch 1973 295