Document 5bYmaGvjk3xbRvdE2Y7L2GoO4

FILE NAME: Avondale (AVD) DATE: 1973 Mar DOC#: AVD129 DOCUMENT DESCRIPTION: Journal Article - Identification of Asbestos in Human Tissues Identification o f Asbestos in Hum an Tissues A. M . U n$er, P kD ,; Richard Ashley, V ictor lad e n , Mi.D.? Carl Seritey, M.S.; L C. Hammond, Dr.5.; Anne O. M ackler, M .i; C. |. M a nie re , PH.D.; W. J. NichoUon, Ph.D.; A. N. K o lif PhO .; I B. Rubin) M .I.; A ntonio Sastre, M i ; and I f. S elilrnff, M_D R e cen t studies have established that occupational exposure to asbestrK dust ts associated w ith serious risks of lung scarring and neoplastic disease ,' 5 Under certain circumstances, albeit to a lesser degree, simitar hazards may xim 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 w ithin a half mile of an asbestos plant.6 7 Although exposure to 11 *tber varieties have been shown to be associated with disease, there (mam differences con ceming the relative hazards associated w ith each asbestos liber ty p e 1,J ll has been difficult to determine the quantities or dimensions of any one fiber type needed to produce disease* Studies of human exposures to crocidofite,' K fl chrysolite." 11 17 amosite," tremolitp.15 and anthophyllite,' 11J have been d if ficult to compare since they reflected d if 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 which pprm.t overlapping usage in thousands of products and applications 19 Fibers often tend to occur together m the same mineral deposit as mixturesi3 All ex posures therefore, whether occupational, indirect o r environmental, may. to some from rhe fn v ira ifw m a l Sciences ijtx x iu x v . Mount Sin* School of Mediano al (ho Citr Jmvot ufy o l New York. N Y Repnnt reqge&K to o n * ot Commumlv M e rit ine Ml S in * School of Medicine, C ily Uiw c r iIy of New York. 3th Ave <nd tOQh St New tu>k X520 (I> Langer) extent, be `mixed" in nature Essential inform ation about the fiber-disease relationship may be added by analyzing lung issues of individuals exposed to fibrous dusts However, recovery and (UeriliiiCdUoii uf asbestos particles from human tissues have been difficult and af fected by such factors as the nature of the recovery technique, fib er-type sought, amount of tissue available, level ol exposure, analytical technique used in identification, and size-sensitivity ol the method of examination In this report, we review and discuss a number of techniques developed and used in the past w ith limited 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 o f B uk 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 tw o im portant assumptions (1) each asbestos m ineral possesses a characteristic p ro pe rty 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 ifj wvo Initially, those in struments used by physical soenlists to characterize large bulk samples of "pute" asbestos mineral were used to analyze tissue extracts It follow ed lo gically that the tissue materials analyzed w ith these "b u lk instrumental techniques" were those obtained from individuals occupationally po sed to mineral fiber since m such cases, the cnnrpntratlon of mineral fiber in tissue was assumed to be high and relatively Journal of Occuwttonil Medione/Voi. 15, No 3/Msrch 1973 large quantities ot tissue materials would he available A. -- Extraction o f Inorganic M aterials 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 e le c tro n ic a lly activated oxygen hydrogen peroxide digestion and high temprature ashing Most o f these were developed to study silicosis, coal 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 slabilit) o f asbestos minerals differ from inorganic materials for w hich the techniques were onginally developed thus chrysotile fiber* may be reduced to amorphous silica 'ghosts" in extremely weak a ca s,16 *> the amphibole minerals have different degrees of stability in alkalis and acids ,a Ther^nre most tractio n 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 Nagelschmidt to remark that a good extraction technique 'w (he removal of asbestos fibers from human tissues had yet to be deve lo pe d 32 It was Nagelscbmidt's laboratory w hich used different techniques on equal aliquots o f the same lungs and obtained different minerai ex tracts w ith each technique. Most laboratories rely on oxidation of bulk tissues for recovery of inorganic mineral residues This may rovo/ve either 287 high temperature oxidation oi bulk, tissue m a muffle furnace o ' low temperature ondanon of- smafl quunlitit-. o f tissue m activated oxygen 8oth techmqoei. work w e ll w ith some preference tor use o i the l<jvs temperature technique since the inorganic phases arc less likely to be dehvdfoxylaled Oxidation mav be used m conjunction w ith a wet chemical digestion method if a sppritic liber is being sought for example potassium hydroxide digestion ot tissue may be used tor chrysottle tecosery as the pH of fte the system (at isodectnc 108 ,8 S*k coni enttaiion) is near pom ot the fiber ( ) Search for a single fiber type has the disadvantage mat the technique may not permit survival ot other mineral fibers which may be present and important biologically Evaluation ot at! currently used digestion techniques indicates .hat no one is suitable for extraction and recovery o a ll tiber types Some Iteration ot mineral phases w ilt occur, especially in ftder\ of small particle size However, even with thf^e diff-OJliiesi extractions and analyses o f bulk lung residues have been made B. X ra y D iffra c to m e try The x-ray powder patterns and crystal structures for in d ivid u a l asbestos minerals have been de'ismined in a number of studies !he*e data have been used purnanly w ith phase iden tification of sott'ort fibrous dusts41 *' or for quality co ntro l41 Some work has been done on tissue extracts w ith this technique w ith varying results 12 u ** Araphibofe asbestos fibers have been identified by this method but m no case has chrysolite been identified pven in cases k ro w n ro ftave been o c cupationally exposed to this variety of asbestos A number of analyses of lung tissues extracted w ith the low temperature ashing technique m conjunction w ith potassium hydroxide digestion have been made The x-ray diffraction pat terns obtained on extracted residues w w not dtdiaUeustic. oi the abestos fibers believed to have been present m the tissues on the basts oi occupational histones44 No successful identification was made on residues obtained from lungs of woriemen exposed ro chrvsutrte containing dusts Some representative x ray reflections were obtained from sam ples of dusts from men exposed to am- 288 1 Caffe tstocM i m l i w s te a m ( M M few a mfesaa s e ts * sa** Ifeer Sam St mM t M f l m i ^ M t U f e f t e i f t p i f e r k M f M t n M i i l M f l Han& featefa* aaafetr af filar* arc prete} afeKl an tais to 5 kract it itfc. Caaia af ierifc isfeestesb. phibole asbestos, e g , amosite The technique proved inadequate even when more sensitive powder camera techniques were used The reasons or lack o f 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 angles nf reflections extremely difficult, inhaled particles 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 poo r resolution the presence o f endogenous iron compounds (hemosiderin) tended to induce x-ray fluorescence obscuring Identificato a fcbutos in Honan Tissue/lani et al 4mall peak 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 mtaecil m ovie in addition to the am biguities which derive from complex mixtures diminished intensities and par ticle size pffects. marked alteration o f the particles m vtvo also lim it their iden- duration Biological degradation of asbestos has been lepurted previously 44- s* yhe extrem ely acid-sensitive chiysottle is most readily broken dow n m vivo, reduced in sire50 and depleted in magnesium O ur observations in dicate that even when the occupational history is known the x-ray diffraction technique is inadequate to characterize lung dust burden ft is o f limited value for am phibole asbestos exposures, especially if fiber types are mixed, and almost valueless if exposure is to chrysotile Although some reports in dicate some success in fiber iden- tificat'on, the x-ray technique should be recognized as having lim ited value i t s3 tCtta tfor ao fr m w M< fastretfst8oht-mtMfcpiaMcrtofcsariMkiuHsed< wtrepeet u*4MRriea ofit eenip*<$*t4efci feitmtyiutaAt** mSktetre EfeiypeIugfrcet maie^n Cu r * wt M k Ih ( c--tai C rt--rtTKM hw>| to rn ipn iw u iMumRA-- a r t npta< Iwto rawM* i* t Wt i i a raMat M q Fw Man a m etMnul m hs k u m by SgU ttm tef y. Cam <RaaMf tee| cMcar Uku- A friM rr m bpM t mftoa at tba caka a n aba tm 4 at H b w Journal of Occupiboftai Wadicine/VoJ 15, No. 3/Bareli 2973 C -- Infrared Spectroscopy th e infrared spectra o f asbestos minerals have been determined4** 55 54 As w ith x-ray diffraction, only lim ited success has been achieved for iden tification of mineral phases in tissue residues * u Ousts containing amphibote asbestos minerals yield inrrared spectra characteristic o f the amphibole mineral gtoup. 1 e , the spectrum ob tained u characiemt'C of the structural array of ail minerals in the group rather than one specific member of ihe group Specific amphibole minerals could not be identified The characteristic spectra of chrysotile obtained on pure mineral specimens were almost identical to the olher serpentine mineral phases lizardile and antigom e* making them virtually indistinguishable from each other In ad dition, the infrared spectra o f serpentine minerals ate teirraikably similar to other sheer silicates so that any admixtures of chrysotile w ith days or talc tor example make fiber identification impossible by infrared spectroscopy alone As m the case of x-ras diffraction alteration o l the asbestos fibers m vivo, o r during recovery frnm lissii? w o uld alter the diagnostic properties Although a high resolution spectrophotometer* was not used in our study, confounding effects existed w hich Jav outside the Instrument 289 design Complex mineral assemblages bodv safts organic debns and alteration o f particles in v n o restrict the use of this technique fix identification purposes D . -- D ifferentia) Thermal Analysts The thermal behavior* or various asbestos minerals have been deter mined4* 55 r *for a range of sample and instrumental variables The am phibol yield characteristic DTA patterns which are inadequate for unequivocal iden tification, even m the ' pure' mineral state 8oth exothermic and endothermic peaks tend to b * small <n magnitude and broadened over a long thermal range Chrysolite produces a well-defined pat tern but tends to be extremely sensitive to changes in sample preparation and penm ental conditions ss Variations m particle size and packing ol sample influence the endothermic dehydroxyUtton peak so that it may shill sc much as 100C The influence o f these factors on diffusion of vapor from the site of reaction** is w e ll 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 ex tracted lung dusts using this technique4* The major problem was interference of the residual organic phases w hich produced a senes o f slrong exothermic reactions m the thermal range m which characteristic asbestos reactions occur t *-- Summary--Sulk Techniques T lit use of a bulk uisU uuitiiial technique to characterize phases m re covered lung dusts presents many d if ficulties Even w ith the most useful technique, x ray diffraction, the results are marginally useful lo t amphtboles a -d of 10 value for chrysolite identification A numhor o f serious probtems are in herent m the analysis evidence demonstrates that chrysotile alters m vivo, thereby losing or changing its pfrysical and th tirm a l U w iau w istits needed for identification, extraction of particles from (issues may lead to ex tensive alteration of asbestos present ana lytica l instruments 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 cies o f asbestos body presences ranging making individual phase identification from 0-100% Evaluation o f these find d ifficu lt chrysotile asbestos closely ings is impossible because many fac resembles the platy serpentine minerals tors and study designs were not uniform as well as other sheet silicates, making The studies differed in such variables as its identification difficult >f not im age and sex, techniques o f material possible large quantities o f materials are jyeparation selection of kinds and needed fo r analysis so that only tissues am ounts o f b io lo g ic a l specim ens from occupationally exposed individuals examined, lung sites examined: number are of use and bulk tissue materials are of specimens examined per case areas needed for mineral fiber jecoveiy Tire scanned per case, magnifications used in results obtained on tissues from in scanning, type of microscope employed dividuals exposed to asbestos minerals fiber type involved duration o f ex are generally poo>, even w hen the ex posure, relative dustiness of trade and posure history >s well known work areas, type of histologic stain; pre paration used for digestion and cen trifugation o f bulk samples All studies Id e n tific a tio n o f S ingle Par ticles--Light M icroscopy assumed hom ogeneity o f body distribution in the tissue materials (a) Asbestos todies. -- in general, the Some erf the above differences may object most readily noted m lungs of not appear to be obvious drawbacks fo t workmen po sed to ashestos dust K the example asbestos bodies tend to form asbestos body It ts readily discernible by w ith different degrees o f efficiency m light microscopy and is commonly cited human lungs as a function o f fiber as an index o f asbestos exposure The type*" The same number o f asbestos non-specificity of the body has been bodies observed in tissues o f workmen recognized almost as long as the obiect exposed to different fibers need not in 'ts e ll45 t4J' V5 Asbestos bodies have been dicate similar exposure levels. Different used as asbestos maifcers in numerous fiber types tend to form aerosols with studies of groups ranging from oc rharactenstir size distributions* so that cupationally exposed workmen to the "th ic k " bodies, those easily seen by light genera) population. Bodies have been microscopy, are more likely to be counted m sputum, lung smears, lung nucleated on Iremolite anthophyllile or scrapes, digested bulk tissues and amosue. Chrysotile, the most commonly various preparations and modifications used fiber m the United States, tends to of histologic sections 11 W-92 form fibers o f small s i? readily These studies have reported frequen degrades in vivo, and resists formation of ItatifiefttiM el Asbestos m Human Tissue/Unger ct 1. asbestos bodies <I* (b ) AsbestCK tfb e n . -- lu ng tissues fro 3000 case, from ih g n m l population ot New York City were examined lo t asbestos bod> co nte ntB} The optically visible asbestos bodies represented only a small fraction of the total inorganic particles present m lung ti$ues In these m e tissues many 1no[game uncoated fibers were ob served, including many w ith diameters of less than 1 micron In 1,028 of the 2 000 cases these fibers were observed jo be piesent ^ The occurrence ot such thin fibers appears to correlate with asbestos body co u n t While the light microscope appears to be adequate for the search for asbestos in human lungs, there is much evidence to suggest that this is 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, relative 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 were 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 w ith 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 in human tissue is lim ited com pared to smaller, sub-tight microscopic fibers. Observation o f the prevalence o f asbestos b odies w ith the optical microscopp may o r may not indicate the burden of asbestos fiber The search for and identification o f fibers must be made on the sub-light microscopic level Id e n tific a tio n o f S ingle Far* tkie s--Electron Seam Instram ents A greet num ber o f sub light mtcroscoptc asbestos fibers occur in lungs of workmen exposed to aerosols containing asbestos <m *><r <eth e smalt fibers appear to be present to amounts estimated from an equal amount to more than 1,000 urmts greater than those fibers visible by light microscopy This is o f im portance in studying exposure in the general population among whom there appear to be prim arily sub-light m ic ro*cppc fibers in lung},1" ** a find ing understandable in light of recent w ork dem onstrating sub-hght m ic ro s c o p ic ch ryso trle fib e rs as significant constituents in urban air sam ples ,m 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 or the sublight microscopic level, tissues should be prepared so they are compatible w ith the technique. Several methods currently available fo r this are f t ) direct microm am pulation o f objects from tissues on to appropnate substrates (2) digestion o f bulk tissue, (3) carbon ex traction o f partides from histologic sec tions (a ) Tissue Preparation. (1) The mirromanipuiator has been used to remove 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 n( Ihe tissue matnx by low -tem perature oxidation, placement o f the ashed slide on the miCTomantpulator stage and use o f a mechanical manipulation system to remove selected objects from the sec tio n The use o f the tight 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 rs digested by the alkali method or ashed in air or activated oxygen and the Joural ef O aiptiO M l MedicinefYol-15. No. 3/Metdi 1973 residues examined by Section beam in struments.* >3w w icn One procedure in volves several cyctex o f washing and ce ntrifugation (alkali digestion technique} and both require homogeneous dispersal of the residue by sonificaiton m an aqueous medium 4t*J pipetting the homogenate on to electron microscope grids The technique is ec- cellem ror tissues m which low con centrations o f asbestos dusts are present The m a jo r draw backs are the 'equiremem for bulk tissue samples and the partial destruction by atk!t o f some amphiboles. (3) A technique has been developed which permits using normal histologic sections m the search for asbestos particles * ns method is called the carbon extraction technique and consists of a procedure in which the tissue is ashed on a glass slide, impregnated w ith a water-soluble plastic removed from the slide, a layer of carbon deposited on the tissue sur face. and the plastic dissolved w ith hot wa'er leaving a floating carbon film w ith 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 wliere only slides are available all exposure (eve)} may be examined in this manner b> electron beam mstiuments (general population, or low intensity exposure cases, require m uch scanning tim e ), the carbon provides a stable medium and sup p orting film far electron beam examination o f all particles, the same section may be transferred to the vanous beam instruments for determining the range o f physical and chem ical parameters required for characterization. (b ) Transmission Electron M icroscopy -- 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 Qirysortle generally consists of broken open, often curved, "bundles" of unit fibrils 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 1 amorphous edge which increases in thickness w ith lim e o f exposure l the electron beam The relative dimensions of the units mas w ry Thesr* leatures have been described m the m in e ra to g ic a l11" - " 15 and m e d ical literature Occasionally chrysolite may be present in tissue as dum ps9* but more fivqiw^nth as individual ItbnU especially in individuals not oc- cupationaiiv exposed to the f i b e r 101 Chrysolite thar has been vigorously treated, such as in hammer- or ball- m illing, tends tr> detorm readilv under an electron beam suggesting that the crystalline structure at the edges ot the tibnU 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 charactefisnr o f the am- phibole asbestos fibers are remaricably similar, tending to be electron den* and either straight or bent Occasionally un der high mag'iification "cross-hatching" produced b\ diffraction contrast may be observed along the ubers w id th fhese 'b ands' may oscastonafly be observed to move up and do-vn the fiber as (he intensity of the electron beam t\ varied on the surface of the sample These ef fects re likely caused by thermal Stress and change ir con'/eigance of the elec tron beam It ( possible to differentiate the ampmbole asbestos minerals mor phologic a lly " Fibers are measured across ihotr w id th and a w id th distribution is determined Each am- phtbole tvfM? apparently possesses a relatively unique w idth distribution w hich mav be used as an identification to o l Current studies in the En vironmental Sciences Laboratory suggest that this may not be *s straightforward as previously indicated Also in many nmoccupationally exposed cases ihe number of such fibers observed in tissues is small so that lire usefulness of differential w idth 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 secondary electron, o f a bo ut 0,2 on a specific, fiber type basis o nly m micron* X-ray spectrometers"' ,lJ are cases where exposure is "p u re ' Such equipped w ith crystals w hich serve as exposures are generally rare so that only dispersive x-ray analyzers The system the amphibofe group may be identified may detect dements down to and m usually not the fiber species cludtng atomic number 5 (boron) and (<) Selected Area electron D if fraction. -- Most transmission electron achieve a concentration sensitivity or 50100 ppm depending on instrumental and mirroscope^ today am equipped w ith sample conditions controls w hich make possible change <n We have analyzed asbestos bodies instrument geometry so that diffracted and fibers bv means of the electron electrons may be brought mto focus on the image screen Crystalline materials mtCTOProbe55 5,1-51 The possibility of suc cessful analysis of fibrous cures of p a lte r electrons in patterns represen asbestos bodies and uncoated fibers was tative of their crystal structure and m- predicated upon chemical differentiation terpldnar spacing* Single crystal Laue o f the asbestos fiber types The chemical geometry (fixed film and crystal) applies composition of single fibers was deter so that both dimension and angle mined on the probe Asbestos fiber characteristics may be measured direc- types, especially amphibotes, could be tfy t * fro m each asbestos fiber type a rlw rn ra lly differentiated and idpnhtieri charactenstic single crystal pattern may be obtained97 This technique permits on a single fiber basts * * 55 s* A number of interesting observations have been more exact identification o f fibers when morphological characteristics are not made, chrysolite fibers are not stable m the biological environment and lend 10 definitive. lose magnesium single amphtbole fibers 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 may be identified on a chemical basis, amphibole fibers which occupy the cores o f asbestos bodies are not degraded chemically whereas uncooted materials have demonstrated the im fibers appear to have lost some magnesi 97 portance of the technique as a too! for identification of chrysolite ~ O n e report recently proposed that it 15 possible to differentiate among amphibole asbestos type* w ilh the dif 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 fraction technique ,[W However, these asbestos bodies only when the fiber o b se rva tion s have re c e n tly been protrudes from the coating material In criticized because several patterns ap' dividual species of the amphibole fibers pear which are not accounted for b y the mineral's space group symmetry 1,0 Recent work has shown that reflec tions obtained by the electron diffraction may be d iffe re n tiate d w ith this technique (e) 5ummafy--Electron Seam In struments. * Preparation techniques method indexed according to known are new available which make possible space groups fo r Ihe am p hib o le examination o f any human tissues m minerals, show heretofore unsuspected electron beam instruments W ith the car structural modifications " uTherefore, the b on e x tra c tio n tech n iq u e , gross amphibole mineral group possesses histologic structures arc recognizable structural characteristics w hich make the and inorganic particles preserved in their mineral group but n ot the individual original anatomical sites No physical or members o f the group, idenlifiable chemtcal artifacts are created during (d) Electron Mkroprobe Analysis. -- tissue preparation This preparation The electron microprobe analyzer makes forms a stable matrix for the materials possible x ray spectrochemical analysis and allows long time examination of of small particles under an electron particles under an electron beam The beam The imaging system Is a lim ited mounting o f the preparation on an efec d epth o f fie ld scanning electron tron microscope gnd permits its trans microscope w ith spatial resolution, using ference mto any beam instrument and identfficatitn of Asbestos In Human Tissue/Unger et al. j allows multiple analyses to top per formed by one or several instruments Asbestos fibers may be Identified on the basis o f morphological, structural and chemical criteria Chryyoble 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 ur laboratory has now examined nssues from some 150 individuals w ith exposures to asbestos (occupational to 'general population") w ith electron beam instruments The tissue preparation and idenimcation techniques have m dudod a il those described in this presen lation In all cas 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 oc cupationally exposed to amphibole fibers as w ell as chrysotile In the latter case however, the small fibers (fibnls in many case) were present in numbers about 100-1 OOOx that of the larger libers this size distribution existed m the tissue since the cartoon extraction technique was used m tissue preparation. Ex tra pu lm o n ary tissues have been examined, eg. spleen, color, 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 601) 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 a r'tc le p op u la tio n Decause of utilization of a bulk tissue digestion technique, if was assumed that this might be an artifact size distribution produced dunng extraction However this size part-|p was observed fo predominate in relic tissues prepared toy the carbon extraction technique in another study.* In a number of cases, fibers have been found w hich could o nly 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 is chrysotile Small amphtoole 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, canitot be discounted as a major pathogenic agent m individuals w ith disna<p HguiPt 1-5 The relationships o f fiber size and type in association w ith human disease are complex Arbitrary simple assumptions u< c o n c lu s io n s based on lig h t microscopy that one specittc fiber type o ' size is responsible for disease are un warranted The ml'ii**d spectiophorometet used w*` a fwkin fin Model 21 cqupped witf* VaO off'* 'Cwi>|xvti -Juiir m Ihe Enviionmendl S<lence"laboraion lo be puWiRvec Compel) vJiKh fcSt - to pUbhshed References i 5**liki>if II al Asbestose and neoplasia, Amet I Med 42 487 I9h7 Z Eniicknap | and Smilher VVI Peritonea! tumors rr. asbe'iosis Bn1 I irx/usi Med 21 20 1964 3 belikoh i| Hammond fC and Churg I Asbesto exposure, smoking and neoplasia /AMA 204 20, 1%B 4 Harruy PC ArbeMOs h/jtri-. in isj'.ii sfapyarris Ann Ocu/> Hy 11 135 l`W) 5 MrFwen } el al Mesothelioma in Scotland fini tord four 4 575, W7D h Newhojse M and Thompson H Misnihelioma ol pleura ami pettioneum totlowmg exposure (n asbestos m the London area Bn( i Indus; JMed 22 261, 1%> Ix-ben J and fiistawka H Mesothifionia and asbestos qxAutt; Aicb in vm n Health 14 5*3 1%7 8 Wagner K Sleggs CA arid Maictuixi P Diffuse pleural mesciheliom? and asbestos exposure m North VVesiem Cape Province Bn< I M u n toed 17 2MJ I960 9 Mviiunto R Pleural cakifiratiix as a roen'gprKjlogic sign nt non-occupational en dem anihophvltite ast>esosrs Aua Radioing 19140{tA) sy1m96p0osium held m Carcfctf, Wales, 7-9 tAecjxisil rs19a/0sbsei-xtou-sstiIsfeieUieTwvaihs (m!*uch dtscyss<ro(n ibi'oloihgiscalmheaeztianrgds caosnsocecirantiendg wtihihe trheelatdeisl*lrearnegrt ' afrsibimesto'asll maiitn- eartail-u-fibtMersoiogOuaplutvu'noins only some fibers are active Projjert*. of single liber ivpes were also discussed in cluding fiber length Irate metals assocrated will die litjyi sixbeti hvO'ixatbons etc No agreement as reached as m the importance <m each of these properties btolngicafh n Orewsen wC, DallasalIP |M Edwards Tl Milter NV aixj Sayers R* A Study of Asbrsiosis <n ihr* Asbestos Texiile industry U5PHS Public IteaKh Bull no 341, Augusj 12 1938 Doll R Mortality from king cancer in asUrvus workers Crn I loduir M id 12 139 1955 14 Setiknit l|, Hammond EC and Seidman M Axbesios disease associated ssith insulation work in shipsaids in the United Slates fnlf Symp on Saictv and Health in Shipyards and Ship Repairing Helsinki August, 1971, tn Press 15 Kl<->n(tdd M ol Mortality among tak mux r- and millers ix New y/xk srare. Arch inwron Hlth U h b i t%7 Ti. Hf+vdrv S\V The geology occum-tvces and major usesnf asbc-siiK Arm NY Atadic 1TJ t7 t%5 17 Gaze ft fhe phvsical ant4 molecular structure o' asbestos Ann VY Arad Scr 132 23 1%S 18 Spcif b and Lemeweber j Asbestos mirteroU m nicxiem tcchnntogy toviron kes 21 W> 1%9 19 RiAdtii DV AsU'sil l(s Indusirut Applicaiions Rhembild Pub 0 214 1959 20 Thomas K and Stfjt>mann H for mamitit methods of umuo digeslion ol 5iIicixh lungs Senraj;*" Stfik Fon<h 28 I, 1954 21 UMerU H Lmbrixll HJ and Klosierboller W Gram si/e and irnn*rtal uiriti-ni <ji Jung dusi ot coal miners compared with m iw dus! Inhaled Particle- and Vapours 2nd toll 5\m r Dam's i Pergamon Press 301, 1967 22 Ulrich l and lurk A fate ol submKTnrvir mhiled du I m Ihe lungs inhaled Particles jnd Vapours 2nd Ini! 5>mp Davies Ed Pergamnn Pte>< 419 f%7 23 Steifx i W Einbrcxfi Ht and Kfosti-dcoller W Quaniilaiive siudips in dust relenimo in animal and human lungs after chrome journal of Occupattonat Medicma/Vot 15, No 3iM*rch 1973 233 inHalaiion tnha'ed p^rtir!-. arvd Vapours 2nd Inii Syrup D<v-> l . i , rv-ij4<TKin Prr.-> 409 196' 24 Caflwriqhi ) Ai h>xv ituvt in i oaf mint"- It' paoii le-e selection charac Ipfisiics of dust <4mphnR insiMitwnts Inhaled Particle*- and Vapour*. 'nd Inti Svmp Daw s i d . Peinamon IV " - 393 1967 25 Mums TC t i ai Comparison oi ticnl Henfein in '(x-citic palKi^en tree and `-an dard tats inhaled I'annh. ant- \apnuts 2nd Inti Svmp Davos f d fVigamon Press JOS I% " 26. Borgmann l Ibe detpmiinaiwn nt toal m formalin fixed pncurooroniriric limps a rw*w method 1 itssue diqpst'on u*mg gfat ial acetic acid Anaivr Qhem 38+*i 19% X~ Ruvims O H at Dus) <nnltml iadtolm\ and patholog) in simple poeumocomoso oi coal workers Part I General observation** PaP 2 Dialled analysis nl the da!a 8/ / tflduilr M /17 07 JS60 28 Rossiier Q et al Dust conteni radioing) and pathology m simple pneumoconiosis or coal wixlu-rs inhaled Pa' lirfes and Vapour. 2nd Inti Svmp Davies Ed Pergamcm Press m 196" 29 Berkley C Cf'un' ! and 5Itknii It Defr- non and locaii/anw ol mineral libers m lisyue, Ann NY Acad Set 132 48 1%S 30 \ag> B and Bales i f Stabiliiv r rhrv'soi!(* asbestos Amor Mineral 37 ffl>S 31 Hargreaves 4 and fa>lnr VVH An s-on exammaitno ol fW om poilion products oi chr>sonic (asbestos) .tmf serpentine. Mineral Mig 27 04, 1956 32 NagpKchrnirlt (i Some ibsfrvanini' ol if*' dus' sooieni and ^ompnsmon in lungs with asbesiosis made dunog work on coal miners pciounv oru*] Ann NY At ad S' i 132 64 1965 33 8nvsicsO Asi)f"k>s UWMButt 4039' 19V 34 GiUon (C Wyeo memorial trflurc Health hazards oi asbi stos Keren! sijdK-- on its hmlogtral t*nt>cfs Tram Srx Or r up Med 16o2 1%6 35 Ernst WG Amphifooles crW al cbenmtrv phase lelatmns and occurrence New Turk 5pimgn Vmty* 125 19145 36 fimixe'l V Owodemncs oi the OiCC standard re'e't-nce sample o' Jibcsios Pneumoconiosis Pror of the Inti Coni, |h, 1060 H A Ahapun id Oxford (JnivOfstiV Press Capetown >5 1970 37 Mgodtinski H and Ktince G Die roitcheiwruciur 'k-s cbrysotils 1 Neues lahrbuch iUr Mineral Nyv 4 5 95 1951 US iagndzmskt H anrl Kunze G Die roikhenvirurlu' de. rhrv-odiS II Neoc*- lahr buch fur Mineral No 6 112 1954 39 belfndge GC An x-ra) and optical in vesligahnn nt the serpentine minctais Amp Mineral 21 461 1936 40 WfortaVer ?JW and iussman J The cturail(vi3l>or> o' vprprnlirc minerals by x- ray diffraction Mrne/al Mag 31 107, ;95641 Gable IV and Knott MJ Application oi 294 x rav difiraction lo the determirutton of chr^ncilc in bulk or settled dusts, Ame/ ln(j >s AiiO / 27 383 m u 42 OaWe (V. and kuvoh M} QuanWatisex rav cj)tiiai.tuxv analysis oi corodniite and amosito ,n bytk or srole<1 dust samples. Ajnw Ind Hw Ann / 27 449, i<*fc 41 Badrjilet m 5 and MuGourt> P tricn lilicaiion ot minerals assrv talcd wilh asbestos b x rav dilriaction paderns Tram Can Mm and Meioit Suli 6 1 109. 1950 44 N'agelschrmdi C Personal com munication Work d<"cubed in laboratory oi Saletv Mines Research EstaWnfmenf Shut rn-frt I onHon 196" 45 5undius N and flv^den A Der Staubinhait erruM Ashestosn Lur^e und dir Beschaih-nheit rk*r sogennannter AsTtPStosisKofpcicncn, Arch Grwerbi'path Cewprbohy^ 8 i)h 1938 46 Berkley C Linger AM and Baden V Instrumental Analysis of inspired fibrous pulmonao pamcutaies NY Acad So Trans 33 1 1967 47 Cufliiy BO Clements ol x-ia> diffraction Addison-Wesle) Pub Co Reading, Mass 514, 1967 48 MoiRin A and lolnves A Neutron at tivation techniques in investigations ot the composition and biological elfects o< asbestos Pneumoconiosis Pnx of the liul% G yil |nh, HA Shapiro, Ed Oxtrvd Untv Pr<*.s Gapetow n 52 1970 49 langer AM Rubm I and Sehkoll tl elec tron Microjitobo analysts ol asbestos bodies Pnx of the Pneumoconiosis C m i, Rth HA 5hd|Mro. Cd Oxford Umv Press Capeiown 57 1970 50 Suzukr V. and Churg i Sttuciute and devplopmem of tlx* asbestos bod> Amw / Path 55 79 1%9 51 tanker AM cltclior microprubo anafyvv (studs of aslxtstos fibers and bodies from lung tissue! Uh. Diag nl Disease*, caused b>. lone Agems, FW Sundcmvarv and bunderman f w , Eils WH Green and Co 5i I rxjic 126 1970 52 Ungrv AM. Ru'xo I Seliknil H Chemical *haracten7atu>n o( asbestos body cores b> flecirnn microprobe analss HiiUxbem and Cytochrm 20 723 1972 53 Unger AM. et al Chemical chatac tenzauon oi uncoiled asbestos libers from the lungs of asbestos workers by electron microDrobe analysis / Hurochem anti Cytochew 735, 1972 54 Beadtc and Knox F/ Studies of mmciai onient and partide size disinbulion in the tur^s I avfacv-os lexiilp workers- Davies, CN Id inhaled PanlOes ami vajiuuis, Ppfgamon Press Inc, NY 419, 1%1 55 SAartinea Chrysotile xsbeslos retafion sht|i <i the surface and thermal properties to the crystal structure Iramacunrti, W 414 1966 56 Brindley GW and Zussman | Intmred abvorpjjpn da|a for serpen!j/ir* minerals, A m y Mineral 44 185. 1959 57 arttvjley GW, and iussman $ a stoic- lunf sludy of the ihetmal Irans/ormalion m soipontiru mtneraiv to forvti'iiin Amer Mineral, 42 461, 1957 58 Faust CT and fahey B it** serpenune group minerals 5 Cecil Sun Prof Paper 394-A92 1962 59 Langer AM and K<*rr H tvaluanon oi kaotmite and quait; dilhvential ihermal curves with a new high trmjKnature cdl, Amtx Mineral 52 509, 1967 60 Cooke Wfc A` l>0'-io- dull and cuntui bodies found in pulmixurv asltesios^ Bm Med I 2 S7fl 1929 61 Williams E `Curious Itodios tourvl in the lungs o'coal-workers lancer 2541 1933 62 5dxlle< F Adsnrplionsersrheirujrtgen an glauben m vivo pseudo asbeMrm* unci 5chjumankorperthen A*ch Gnwerliepaih Ce6w3erhvpohnygRu1t4tn6cr64Ifl19G56ber Vbestosound Pseudoasbestosekiiipcichen Schweiz Z M r Path, 15 628 !9 U 64 Utidjian MD, Cross P and D-Tf<*vi)le Ri Ferruginous btxlies tn human lungs Arc6 Lnv Heakh 17 32?, i960 65 Gross P Crailev LI, and lef evilie RTP Asbestos' Ixxfies 7hci non-speciiiotv / A w r fndust Hyg Assoc, 28 >41 i%7 66 Siewdii M|, ansi Fladow A I'ecufeat bodies in ouimonarv asbestosa / Path Sau 32 172 1929 67 Simscm FW and Srrarhan AS Asbesnw hnries m sputum / Path Bad 34 1 1931 66 M inn WB <t ol Asbeslots Part 111 Special Bulletin So 42 Penn Depi of Latux anWd fndPmagtrev RHCarrisSbuprugiumPennina p1u9lJm5onary asbestos!* Amec j Med Set 189(1)44 1935 70. Herat ? of ai AsSsiOv hodfes m sputum samples, f Sara Med Assoc 12 387 1961 71 Plameydi H I A^hnsiiw Finches m sSpyord wmkefs Meet T Crn^tik 100 49 1964 72 Surs-Cremet GG Asbeslty. in South Afm.0 Ann NV Acad 5ci 132 215 t%3 73 Stumphus 1 and Meyer PB AVsestos bodies and mesothelioma Ann Qcr HvRiene I I m 1966 74 Thomson IG Kaschi-la K and MacDonald R Avljetios as a mrvtern iicfvtn haurd South M rtan Mf / 27 77, t% 3 ?5 G uru 0 Tonen RS and Cross P Asbestos bodice at autopsy AMA 192371 1%$ 76 Anjtlvel Itfy and Thurlbeck WM In cidence oi asbestos bodies at random necropsies m Montteal Can Med asuk l 9S7171.7i9hnn1i%so6n }G, and Corves WM Asbcsio as an urban air contaminant, Arch Pith 01 456, 1966. 78 Ghecci I, Molrem G, and Puccetn U Asbestos bodies m lur^js of inhabitants of Milan. Med avorn 58 No. 3 223 1%7 79 Roberts GH Asbestos bodies in lun?s at necropsy, / Chn Path, 20 570, 1967 80 Ashcroft F Asbes'i bodies in routine autopsies on fynevde Bm Med} i 614 1%& ol Asbestos in Human Tissue/Unftr et I. j 51 Gibson -AAM tH 1 E|*dt"nfolog> o> rrvesothetrrxna tn Scotland Hex X 2nd imi Coni ot) Biological Efforts ni Ashesrns Dresden Meeting, 21-25 Apnl, Paper (/} 82 Avril | and Champeu f Results ' jsb^'.lo v evp osu /e ir> Franc P n riim n tn n H m * 1ml Coni Johannesburg H A Shapiro EH , Perganioti i'rtfss Capetown 10-1 1470 83 Baden V' and Athwart/ I Oemomlration ni asbestos bodies Comparison of available ir-fh'iutut'*. ('for ot >{ {nil Coni on Biological Effect of Asbestos, Dresden Meting 21 2S April l%8 Paper h 84 Prfiiack A and Sacks M Asbesios bodies m ha -at Jong smiurs, k w l j MJ St i 4.*23 l%8 85 Peacock PR Long smear for asbestos foxl"*"., furofi ) Caned 5 135 1%9 8b Xipell fManti Bhaihal PS A lb u m Path 1 327 1%9 87 Firm*. PC MrQughev W7 ami vVadi O t D iffuse itx^othcHtoma of ihe pleura ami asbesio 8m M a i 1 1 J50 l%3 0 0 H ounhanc D O B B io p 'y Kne*- in mesothelioma A ite m p is to ick-nitty sixmos A n n a / N Y A ca d Set 1J2 M - f9 M 89 Meurman LO Asfxsio, bodies ami pleura plague* m a Ftomsh e*tffs Acta Path A-ficiob Scanti Sop/i/ 181 1102 19bh 90 CoUiMem 8 ml Rendall KtB fetrutjmous bodies Pneumoconiosis Inil Con* Job., H A Shapiro Ed PwK.imim Press Cap 'own 40 1970 91 Bcger PI Obff tlie AstioMosiskbr- pe-hen Virchow. AMh Path Aruf 290280 193J 92 Cnii C Data presented m Symposium on llx* issue rt*ponsi to asliestos 7 9 April 970. Cd'Cut Walt's 1970 93 I'ooley FD Asbestos bodies thcic for mation composition and charade* four R<* S 3 f ii. 1<*T2 94 IimbreJI D Prle-y fD ancf Wagner C ChawumsiKs oi Pneumoconiosis respirable px of the aInsrbfesot>osm hV(xorhs , H A Shipuo Cd, Orfoaf IJnn Pi h Capetown 120 1970 95 larger AM. (4 al Inorganic fibers, m dudicg chiysmile. in lungs al autopsy preliminary report Inhaled Particles HI Proc Inti Bin. Ore Hyg Sex' S>mp londiyn 1970 W H Wahon Ed IJowm Bros lid , London 2 ) 1975 % Poolev F al the cMecuon of asbestos in tissues PncumocomosK Proc o* the Inti Coni Joh J9h9. HA Shapro Ed, Oxford Unn 97 LParergsser CAaMpcdPrxosolnev1F0D8 I1d9e7n0tification oi sihinegllnef{dtCbtosnioi sounbci-hits*inBihnunEfufnorttsisosurtAPsbroesctoisx Lyon fiance i ft October 197> Pr No 19 972 96 Pooley FD EJpriron Microscope charar teiiMics ot inhaled cJvysotife abes|K fiber Bat i 99 Imi UAndg/er29A1M4ft S1e9li7k2oft l) and Sastre A ChrysotiJi asbestos m the lungs (>f persons in Now York City, Arch to y Heart/123 , 448 1971 100 5eliLoii l| N'jcbokon WJ and Unger AM Asbestos au pollution, Arch fnv Health 5 1 1972 101 Unger AM and SelikoP H Chrs-sili* Ost'yi* MPiraochi2(nlxd JInulrJiesU"oIarnt-AKirteCiiol'nignreSstsswVars^hli DUrCery DErelcs A19r7a0d PMreM's NEenwgltmVodrandflWfT 1 1 5 197} 102 Ht-ndeison Wi vt M \a!t and car cinoma nf Ihe tivary am! ctt\w / O/nlrt and Cyncof (Bin Commonweallhl 78 iftft 1971 103 Clifloo RA Huggins CW and Shell HR fiOllOW CfWHXiie filXTs Alton Mut 51 jOO, l% ft 1E>4 Bates 7F CmK'r )| Eurfhet nbsenraiions on ihe morpholnjjy of ciitysofde and halloysrte Proc Sixth Nad Coni Uayv and Clav Mrn> NA5-NRC >37 1959 105 Ydtfe K StixJi >x chry'Wde asfxMlos a fagh resolunon efernon microscope, Acu Crysi 23 704 19^7 tOft McConnell 1DC Dwtnx mun/ssojo and electron dnfraction Physical Method m Oeiernimatike Miniffalogy, 1 2ussirtan, id Acad Pss M 5U I9h7 107, 7v> ijim BB Heriron Dnfraction Anahsis of CUs Mineral Struclures Tram S lyse Plenum Pn*ss n v 3b4 I9ft7 >08 2ussman L Brindley CW and Comer f mE!<inilemranls, AdirilrraMciiinon42 stu33d,ies>95o7t swpentine 109 9ktkr Ml lalbta Hi and Rwvtall REC Electron duiraft'On patterns ot U l C C AsbtMtn Sample in v f&i 4 141. 1971 110 Poolev FD Personal comrruntcaiton, April 1972. wrwk to prngnws, DoiMnmenl ol Mineral trf^rxtalion Umvsv of Souih Wales and Monmnuthsbrp Cardiff 111 Buk* IS tli Iron profx rnKJoanalysis Monograph ot Anal Cbem and us ^ppii<jit<xK /nir^scieoi e PuM NY. 17 253 19M 112 Anderseti C \ An iniccxturtKm t<* the Hectmo p-obe rmcrnanaly/ anti iK ap(lhcaJinn to bmchemisifs Methods ni Buxfrem Anal O Click Ed Initnctence Publ NY 15 147 1%/ 113 M eutm jn I f ) Ktviloolo R and Kalcama M Mudalih and morbidth ot nployees of atMho\>h>1lile asbeMOs m min* of CmUnrt lnil Crxit Btolog nis nt Asbixios Pr 11 Lynn. OCI 2-ft 1972 kurM l of Occupation! MadiciM/Vol. 15, No 3/Mscch 1973 295