Document dawL77pYR9wVjK124NX0JMaoG

REFERENCES BaJ^^ M....................................... 1957. J. Clin. Invest., 30, 871. Bothwell, T. H., Pirzio-Biroli, 1958. J. Lab. Clin. Med., 51, 24. G., and Finch, C. A. Bratley, F. G., Burroughs, 1931. Amer. J. Med. Set., 182, 507. H. II., Hamilton, D. M., and Kern, C. Cruz, W. O., Hahn, F. F., and 1941-42. Amer, J, Physiol., 135, 595. Bade, W. F. Giblett, E. II., Motors ky, A. G., 1950. Blood, 11, 1118. Casserd, F., Houghton, B., and Finch, C. A. Jandl, J. H., and Kaplan, M. E. 1900. J. Clin. Invest., 39, 1145. Kaldor, I......................................... 1954a. Austral. J. Exp. Biol. Med. Sci., 32, 795. 1954b. Ibid., 32, 801. II M Kaldor, ........................ * * 1958. I, and Powell, 1957. Ibid., 30, 173. Ibid., 35, 123. Marjorie Laurell, C.-B................................. 1952. Pharmacol. Rev., 4, 371. Morcan, E. H.f and Carter, G. . 1900. Austral. Ann, Med., 9, 209. Mum, R., and Dunn, J. S. 1914--15. This Journal, 19, 417. I* M H it 1916-10. This Journal, 20, 41. Palmer, J. G., Eiciiwald, E. J., 1953. Blood, 8, 72. Cartwright, G. E., and Wint- robe, M. M. Ramsay, IF. N. M.......................... 1958. In Advances in clinical chemistry, cd. by H. Sobotka and C. P. Stewart, New York and London, vol. 1, p. 2. SCHADE, A. L., AND StENGLE, J. M. 1959. J. Lab. Clin. Med., 54, 593. Shoden, A., Gabrio, B. W., and 1953. J. Biol. Cltem., 204, 823. Finch, C. A. Shoden, A., and Sturgeon, P. . Walsh, R. J., Arnold, Barbara J., Lancaster, H. O., Coote, 1958. 1953. Amer. J. Path., 34, 1130. Austral. Med. Res. Counc. Spec. Rop. Ser. no. 5, Sydney. Maroaret A., and Cotter, Helen / '|TM. HISTOCHEMICAL DEMONSTRATION OF HYAL URONIC ACID IN PLEURAL MESOTHELIOMAS it J. C. Wagner*, D. E. Munday * and J. S. Harinoton Pathology Division, Pneumoconiosis Research Unit of the Councilfor Scientific and Industrial Research, Johannesburg (Plats XXIII) yi Difficulties in histological diagnosis experienced during an in vestigation of diffuse pleural mesotheliomas and their possible associa tion with exposure to asbestos (Wagner, Sleggs and Marchand, 1960) led to the present study. In many cases the diagnosis had to be made on biopsy material, and often the tumours mimicked adenocarcinomas. ' bix undoubted peripheral pulmonary adenocarcinomas have been' ; detected in our cases of asbestosis. Meyer and Chaffee (1839, 1940), Blix (1951) and Winslow and Taylor (1960) reported the association of hyaluronic acid with mesotliclial tumours. Our earlier attempts to demonstrate hyaluronio acid'1 , liistochemically in these tumours were only partially successful. This 1 ) was probably due to the fact that the majority of the specimens had' been fixed in 10 per cent, formol-saline in which hyaluronio acid is soluble, as it is in most aqueous media. In this investigation an * * attempt has been made to develop a standard histochemical procedure in the diagnosis of mesotheliomas. In five of our cases it has been possible to confirm the histochemical findings by the ohemical isolation of byaluronio acid from the pleural fluid. Material and methods Experiments with various fixatives showed that formol-sublimate, alcohol, formol-alcohol-acetic, and Zenker's, Helly's, Bourn's or Camoy's fluids were all suitable. It was decided to use formol-alcohol-acetic (Tellyesniczky, 1698) a* the standard fixative for this investigation, because it was found that, after several days of immersion, both the histology and the secretion were well preserved. This point was important because some specimens took up to three days to reach the laboratory. For this study tissue from 14 mesotheliomas fixed in forraol-eloohol-aoetlo was used. Two further specimens which had been fixed in 10 per cent, formollatino but still retained a stainable secretion were Included. Control material rumo from 4 peripheral pulmonary adenocarcinomas, 4 colloid carcinomas from Iho gastro-intostinal tract, healthy umbilical cord and 2 metostatio adeno carcinomas from tho omentum. The site of origin of the last two tumours is unknown, but one was initially thought to be a peritoneal mesothelioma. Blocks of tissue from all these specimens were processed and sections were ilnincd by the following methods ! 1. Mayer's alum-hnmatoxylin and eoein. * Preiont addreasi Pneumoconiosis Research Unit, Llandough, South Woles. 1 m * jvmiiui ,mu u lu'irui'.i s (rj;..r)) mi--,i.i HUiimng ronction. 'IJIFoi Molulidii of ii/ui'u "A" MucNeut (Uovoctor) in 'JO por cent. wu.s ^^Klnlc's colloidal iron mothod (Hole, 1940). At tho suggestion of Dr D. J. Winslow, Monjy',i modified! ion (1958) of this teehniquo was adopted. 4. McManus' (I!) Hi) periodic aciil-Scliiff method (P.A.S.). In ft fow case* tlio results obtained by this method were confirmed by tho uso of tho Hotchkiss (1948) technique. 5. Steednmn's (1950) alciun blue method. Later Mowry's modification (19GG) was used. G. The method of Dempsey et al. (1947) for demonstrating motachromasia in varying pH levels, witti solutions of 0-1 per cent, az.uro "A" buffered to u reaction of pH 2 0, 3 0, 3 5 and 4 0 with Walpole's HCl/acotato buffer. Hyaluronidase treatment. Two enzyme preparations were used : (i) Testicular enzyme (Bcnger's Hyalaso), at a strongth of 75 turbidity, reducing units (TRU) per ml. in M/5 acetate buffer at pH C'6. Sections were incubated for 18 hr at 37 C. (ii) Streptococcal enzyme (generously donated by Lederlo Ltd, New York). This was used at a strength of 50 THU per ml. in M/5 acetate buffer at pH 5-G. Sections were incubated at 37 C. for 4$ hr. After treatment with tho nbovo solutions, control and digostod sections were stained by both Hale's colloidal iron and tho motnchromntic techniques. x Chemical dctcrminrition of hyaluronic acid. This depends upon tho production of a mucin clot on the addition of acetic acid to diluted pleural fluid, a method i very kindly communicated to us by Dr Kurl Meyer. Samples of fluid wero diluted j with 2-5 volumes of water and GO per cent, acetic acid added to a concentration of 0-2 per cent. Tho presence of hyaluronic acid is shown by tho formation of a \ typical syncretic clot ; this is inhibited by previous incubation of tho fluid with ) hyaluronidnse for 30 min. at 38 C. Wo have also used an unpublished method, f also provided by Dr Meyer, for tho quantitative isolation of hyaluronic acid. This ( involves the precipitation of tho hynluronic acid (with its protein), digestion with \ pepsin and trypsin, and subsequent isolation of tho polysaccharide. BB 0005527 t t J I I { * t j_ | Results From the tabic it may be seen that the secretion in all IC of tfio mesotheliomas was metachromatic, and stained by Hale's method and alcian blue. The metachromasia and Hale-staining were abolished after incubation with tcsticnlar hyaluronidase. Only 2 of the 1G were P.A.S.-positive. In contrast the sections of the adeno carcinomas gave strongly positive results with P.A.S., Hale's method, and alcian blue. Metachromatic secretion was observed in most of the adenocarcinomas studied. In none of tho adenocarcinomas with mctaohronmtic or Hale-positive secretion was the staining reversed by treatment with either hyaluronidase. Streptoooccal hyaluronidnse applied to the sections removed the metachromasia in 13 out of the lii mesotheliomas and markedly reduced it in the remaining 3. In tho mesotheliomas the secretion stained a magenta colour (a " gamma " metachromasia), whereas in the adenocarcinomas the metachromalio substance stained purple (" beta ''metachromasia). The metachromasia exhibited by the mesotheliomas is presumably due to the presence of * Hvai.ijronk: mid in Mu.urriiia.iouAt Pm. 1.--Mrnollieliorna stained by J bile`a method. ;< 120. NOTE: THIS DOCUIWENTDI& NfF COME FROM PPG FILES Klu. 2.--AnolIti-r section from the flame block, Hluiuod by Hide's method aftel' treatment with tefllkulnr hyidurunidase. X 120. carboxyl groups in the hyaluronic acid molecule, whv^Bbsults in a weaker metachromatio effect than that observed, fo^vxample, in cartilage and other tissues containing acid polysaccharide. The amount of metachromatio substance in the mesotheliomas was extremely variable and followed no definito pattern. For example, in some tumours large cystio spaces were filled with stained secretion, in others this Becretion was seen in the smaller spaces but not in the larger. In the more cellular tumours only a few clefts containing this dibstance were seen. Table Histochemical staining of secretions Type of tumoar mesOoitlhTeiulicoma Pcuaarldmceinononoma- ray inGatedaessnttironoa l carcinoma Meatdaesntoatlo carcinoma .Vo. of tumours examined . P.A.S. positive, not removed by diastase Metachromatio with azure 10 3 10 433 433 4 S 8- Motachromasia removed by 10 000 testicular hyaluronidase Metachromasia romoved by 13 slroptocuccal hyaluronidase (markodly 0 0 0/ atminishod In 3 others) Halo positive . . , 10 432 l{alo-positivo secretion re 16 000 moved by testicular hyalu {markedly ronidase diminished In 1 other) Alcian blue positive . . . 10 433 NOTE: THIS DOCUMENT DIO NOT COME FROM PPGFILES BB 0005523 The cffeot of pH on metaohromasia was tested in 7 mesotheliomas *nd 3 of the peripheral pulmonary adenocarcinomas. The differentia tion was not as clear as was expected, for although 6 of the meso theliomas showed no metaohromasia below pH4, one was metachromatio at pH 3-5. Two ofthe 3 adenocarcinomas were metachromatio at pH 1, tut the third became so only at pH 4. At the time of our earlier work on this subjeot we were impressed by tho stromal metaohromasia exhibited by some of tho mesotheliomas. Wo now oonslder this as a non-specific feature of the rapidly growing connective tissue and its ground-substance (Taylor and Saunders, 1057). Chemical studies (Watson and Pearce, 1950) indicated that this itromal substance consisted of a hyaluronic-acid chondroitin-sulphate complex. We were able to confirm this in part, by showing that the ilromal substance in the mesotheliomas and in the proliferating fibrous tissue provoked by certain earcinomas had its metaohromasia and Hale's colloidal iron staining completely removed by testicular byaluronidoso but only diminished by the streptococcal enzyme. MENT DID BB 0005529 - C'^^ad estimations. Hyaluronic acid was isolated from i|lr pleu.^Huids of 5 cases of con linnet! mesothelioma of tho pleura in concentrations varying from 0-02 to 1-25 per cent. In 3 of these caws the yield of the acid increased as the disease progressed ; in a fourth, a steady rise was observed until about two months before death, when none could be recovered. The disappearance of hyaluronic acid in such eases might be due to the formation of a gel of polysaccharide which could no longer be aspirated along with tho pleural Quid, Xo false positives for hyaluronic acid have yet been obtained from a large number of pleural iluids including specimens from idiopathic effusion!., tuberculous infections and from malignancies other than mesotheliomas. Dr Ivarl Meyer (personal communication) has found the polysaccharide in pleural fluids from ten cases of mesothelioma. It j J I j j I I-- | Discussion In reviewing the sections of pleural tumours that had been sub mitted to various pathological laboratories throughout South Africa prior to 19513, a number that had originally been diagnosed as adeno carcinomas were found to have histological features consistent with those of mesotheliomas. It is possible that a similar situation has existed elsewhere, as in a number of cases of carcinoma complicating asbestosis the tumours were described ns being pleural adenocarcinomas in which no primary site of origin had been found in the lung (Owen, 1951 ; Bonser, Faulds and Stewart, 1955). Similarly, no primary site of origin was found in several of the peritoneal tumours described by Krai in I960. It is hoped that the methods described will help to give a dearer indication of the number of mesotheliomas that have occurred in association with exposure to asbestos dust. The main difficulty in applying the histocheinical tests described here is the circumstance that the majority of specimens are received in formol-saline unless the presence of a mesothelioma has been pre vious^ suspected on clinical and radiological grounds. In formalinfixed tissue we have found the P.A.S. method most useful in dif ferentiating between peripheral pulmonary adenocarcinomas and mesotheliomas. In our experience pulmonary adenocarcinomas have always contained P.A.S.-positive secretion, whereas the mesothelioma* are usually negative. In addition to thoso mesotheliomas mentioned in the table, we have used tho P.A.S. method on a further 20 of these tumours that had a compound tubular structure whioh could be confused with that of adenocarcinoma, and in which tho tissue bud been fixed in formol-saline. Of these only 5 contained P.A.S.-posilive secretion and in every case this was removed by incubation with diastase. It is only in the 2 cases previously described that the P.A.S.positive send ion was not removed by this method. One of tlu'.w was similar to the mesotheliomas of " mesenchymal type " described by AleCanghey (195S). The other caso although classified os a mc.sotheliumn has several other features that would suggest that it was ft **.1 4 4 4UAJA U'iUj 'll peripheral adenocarcinoma : tho Halc-positivo secret ^Hras not entirely removed by incubation with either enzyme, and meuKiromasia appeared belowpH 4. Tho specimen was from a man who had worked on an asbestos mine for many years, and at thoracotomy the appearance of the tumour was typical of a mesothelioma. A lung biopsy taken at tho time showed the histological features of asbestosis. It is not yet possible to say whether the methods described in this paper will differentiate between peritoneal mesotheliomas and metastatic carcinoma. The isolation of hyaluronio acid from pleural fluid has been a useful diagnostic aid in some cases, but has been negative in a number of cuscs in which tiro diagnosis has subsequently been proved histologically in id histochemically. It i3 clear that only positive results are of any u.se. No false positives have been obtained in human cases in our series, nil hough several were obtained in pleural fluids from mice that had died of a streptococcal pneumonia. Hyaluronic acid was demonstrated histochemically in the acute pleural exudate of these animals. Summary Diffuse pleural mesotheliomas secrete material that stains as an acid mucopolysaccharide and can be removed by prior digestion with either testicular or streptococcal hyaluronidasc. Chemically, this material can bo quantitatively precipitated by acetic acid, and itsj presence confirmed by prior treatment of pleural fluid with hyaluropfcf duse. These methods have been used to differentiate between diffuso pleural mesothelioma and peripheral pulmonary adenocarcinoma, especially in asbestosis. Wo wish to thank Dr Ian Wobator, tho Sub-director of this unit, for alloy us facilities for carrying out this investigation, and Miss Tony Le Marc tivitnical assistance. t REFERENCES Rux, G...................................... 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