HISTOCHEMISTRY OF THE ACID MUCOPOLYSACCHARIDES OF SKIN IN NORMAL AND IN CERTAIN PATHOLOGIC CONDITIONS

Transcription

1 THE AMERICAN JOURNAL OF CLINICAL PATHOLOGY Vol. 40, No. 2, pp. 233 August, 963 Copyright 963 by The Williams & Wilkins Co. Printed in U.S.A. HISTOCHEMISTRY OF THE ACID MUCOPOLYSACCHARIDES OF SKIN IN NORMAL AND IN CERTAIN PATHOLOGIC CONDITIONS WANE C. JOHNSON, M.D., AND ELSON B. HELWIG, M.D. Armed Forces Institute of Pathology, Washington 25, D.G. A histochemical study of the acid mucopolysaccharides of the skin was made to gain information regarding the nature of the mucin in normal skin and in certain pathologic conditions that exhibit an increased amount of mucin. Results of the histochemical procedures are discussed and correlated, when possible, with reported chemical analyses. Because of the confusion and lack of uniformity in terminology used to describe various types of mucin, certain terms used in this paper are defined in the next 5 paragraphs.. Mucopolysaccharide. A hexosaminecontaining polysaccharide, which may or may not be combined with protein, including mucins of connective tissue and of epithelial origins. 2. Acid mucopolysaccharide. A mucopolysaccharide that is acid in character. This includes Group III substances (Table ), as well as Groups I and II. 3. Arninopoly saccharide. A polysaccharide that contains amino sugar without a protein moiety. 4. Sialomucin. An acid mucopolysaccharide containing sialic acid; it is found in many mucins of epithelial origin. 5. Blood group substances. Neutral mucopolysaccharides composed of glucosamine, galactosamine, galactose, fucose, and amino acids. CLASSIFICATION An entirely adequate classification of Received, December 22, 9G2; accepted for publication Ma}', 003. Dr. Johnson is Assistant, Professor of Dermatology and Assistant Professor of Dermal Pathology, Temple University School of Medicine; Research Pathologist and Assistant Director of Laboratory, The Skin and Cancer Hospital of Philadelphia. Dr. Helwig is Chief, Department of Pathology, and Chief, Skin and Gastrointestinal Branch, Armed Forces Institute of Pathology. mucopolysaccharides is not possible with the present state of knowledge, but a working classification is essential in discussing data in this field. The classification presented (Table ) includes only the acid mucopolysaccharides and is an adaptation of work presented by Meyer and associates, 6 BettelheimJevons, 3 Zachariae and Dyrlye, 25 and Werner. 24 The table is not intended to be complete but to serve as a guide for the material to be presented in this report. MATERIAL, METHODS, AND INTEHPUETATIONS Formalinfixed, paraffinembedded tissue from normal skin and various lesions was used for the histochemical procedures. The number of specimens of each pathologic condition studied varied from 3 to.0. There is a difference of opinion as to the meaning of a positive reaction in some of the procedures used in this study; therefore, outinterpretation of a positive reaction is stated after each method. Fixation of the tissue, processing, and details of the procedure are important factors that may give rise to variable results among different workers. One of the most important factors in some of the methods is the pll. at which the reaction is performed. The procedures used in these studies were performed as outlined in the "Manual of Histologic and Special Staining Technics," 5 except where otherwise stated. Colloidal Iron Stain, With and, Without Hyaluronidase Digestion Modifications of the Hale iron stain as described by Rinehart and AbulHaj' 2 and Mowry 8 were used, with a van Gieson counterstain. The ph of the working solutions of both methods was in the range of.5 to.7. The ph of the ferrocyanidehcl 23 solution was.. Bovine testicular hyaluronidase was performed at 37 C. for hr. Controls using heatdeactivated Downloaded from on 25 February 208

2 TABLE CLASSIFICATION OF ACID MUCOPOLYSACCHARIDES Substance Uronic Acid Amino Sugar Sulfate Groups Acetyl Groups Effect of Hyaluronidase Bovine testicular Bacterial Natural Sources (Human Unless Otherwise Noted) Group I. Simple Aminopolysaccharides* (Group I and Group II constitute the acid mucopolysaccharides as defined by Meyer) Hyaluronic acid Chondroitin Glucosamine Galactosamine 0 0 Ground substance of connective tissue in skin, blood vessels, heart, umbilical cord, synovial fluid, and vitreous humor Bovine cornea Group II. Sulfated Amine/polysaccharide Complexes^ Chondroitin sulfate A Chondroitin sulfate C Chondroitin sulfate B Keratosulfate Heparin Iduronic (Galactose) Galactosamine Galactosamme Gallic tosamme Glucosamine Glucosamine Up to 3 0 (test tube) or ± (tissue) (test tube) or ± (tissue) Cartilage, bovine cornea, bovine aorta, bone Type A major component Type C minor component "Whole" human and pig skin, calf skin, rat skin, human aorta, and J pig tendon Bovine cornea, calf bone Mast cells in various tissues Group III. Complex Compoundst (Mucoprotein, glycoprotein of Meyer; niucoprotein, mucopolysaccharide of BettelheimJevons; mucosubstance) Constituents Natural Sources (Human Unless Otherwise Noted) Amino acids Amino sugars Glucosamine Galactosamine Other acid groups Sialic acids Muranic acid (bacterial cell walls only) Unknown groups Neutral sugars Galactose Man nose Fucose Epithelial mucous secretions Apocrine and eccrine sweat glands Histochemical reactions suggest a mucosubstance of the sialic acid type Salivaiybovine. Sialic acid isolated Mammary gland. Sialic acid isolated Gastrointestinal. Sialic acid isolated Gastrointestinal mucin has been reported to have a small amount of sulfated mucopolysaccharide Respiratory, cervical, ovarian Sialic acid isolated Urine. Sialic acid isolated Blood plasma. Sialic acid isolated Egg white (ovomucin). Sialic acid isolated Sialic acids, acylated derivatives of neuraminic acid. These acids are widely distributed in animal mucosubstances Sialidase {neuraminidase), enzyme derived from Vibrio cholerae and influenza viruses which will hydrolyze those mucosubstances containing sialic acid, especially if located as end group * Unbound to protein or loosely bound to relatively small amounts of protein. (An aminopolysaccharide is a polysaccharide containing amino sugar without any protein.) t Usually associated with relatively large amounts of protein through "weak" ionic linkage. t "Strongly" bound to protein through covalent linkage. Probably does not contain uronic acids or ester sulfate. Present nomenclature based on tissue of origin. 24 Downloaded from on 25 February 208

3 Aug. 968 ACID MUCOPOLYSACCHARIDES OF SKIN 25 hyaluronidase and iron stains were performed. Interpretation. The reaction is probably based on binding of colloidal iron by acid groups. Acid mucopolysaccharides produce a blue to green color, and nucleic acids a less intense blue to green; few other acid substances remain in sufficient quantity after processing to produce a reaction. Hyaluronidase by the above technic will remove most of the hyaluronic acid but does not remove significant amounts of chondroitin sulfates in tissue sections. This is in contrast to of chondroitin sulfates A and C by testicular hyaluronidase in testtube experiments. 7 Colloidal Iron Stain with Ribonuclease Digestion Digestion was performed for 2 hr. with a 0. per cent solution of ribonuclease (Worthington) buffered at ph 6.8. The digested sections and controls were then stained with the colloidal iron methods stated above. Interpretation. Most ribonucleic acid is removed, but acid mucopolysaccharides remain and yield a positive reaction. Alcian Blue Stain A per cent solution of alcian blue 8GS in 3 per cent acetic acid (ph 2.7 to 3.0) was prepared and used with a nuclear fast red counterstain. In addition, phosphatehcl buffers were prepared at ph 0.4,.0, 2.0, and 3.0, and used for staining ( per cent alcian blue 8GS) and for the first rinse. Interpretation. A positive reaction is based on the presence of acid groups. 6 At ph 2.7 to 3.0 most acid mucopolysaccharides produce a blue color, and nucleic acids produce a less intense blue. At low ph values (0.4) only strongly acid substances such as the sulfated acid mucopolysaccharides will yield a positive reaction. Periodic AcidSchiff Reaction xvith Diastase Digestion Diastase was used for removal of glycogen. The PAS procedure was followed with a hematoxylin counterstain. The ph of the 0.5 per cent periodic acid used was 2.0, as was that of the Schiff's solution, also. Interpretation. The Schiff's reagent reacts with aldehydes formed by the periodic acid, producing red to magenta compounds. Most mucopolysaccharides, except the acid aminopolysaccharides (hyaluronic acid, chondroitin sulfates), will yield a positive PAS reaction. 5» S In our experience hyaluronic acid does not react selectively; cartilage and mast cell granules are PAS positive, however, presumably because of the chondroitin sulfate complex and heparin complex, respectively. Substances other than mucopolysaccharides may contain adjacent hydroxyl groups and yield a positive reaction. AldehydeFuchsin Stain The technics as described by Gomori 8 and AbulHaj and Rinehart were used. The ph of the working solution by both of these methods was approximately.7. A working solution at ph.0 was prepared by adding concentrated HC. The 80 per cent alcoholic rinse used with this stain was also adjusted to ph.0. Metanil yellow or orange G counterstain was used. Interpretation. Evidence has been presented 2 22 to indicate that aldehydes, as well as certain sulfated compounds, will yield a positive reaction. AbulHaj and Rinehart believed the reaction to be specific for sulfated mucopolysaccharides and related organic sulfurcontaining substances. In our experience, at ph.7 sulfated aminopolysaccharides, as well as most mucins of epithelial origin, yield a positive reaction, whereas substances rich in hyaluronic acid do not react, or yield only a faint reaction. At ph most mucin of epithelial origin loses its affinity for aldehydefuchsin, but mast cells, cartilage, and blood vessel walls remain positive. Inasmuch as mucin of breast, colon, respiratory tract, and so on, has not produced significant quantities of sulfates on chemical analysis 24 but does yield a positive aldehydefuchsin reaction at ph.7, we doubt that the reaction is based solely on the presence of sulfatecontaining substances. At lower ph values, only strongly acid compounds such as the sulfated acid mucopolysaccharides yield a positive reaction. Toluidine Blue for Metachromasia PhosphateHCl buffer solutions of 0. per cent toluidine blue were prepared at ph Downloaded from on 25 February 208

4 26 JOHNSON AND HELWIG Vol. 40 values varying from.5 to 6.5. The rinse was performed at the same ph as the staining. Sections were examined immediately, then airdried and mounted in Permount. Interpretation. The production of metachromasia is dependent upon the presence of acid groups. Acid mucopolysaccharides, nucleic acids, and other acids, if present in sufficient quantity, will cause a positive reaction. At pli values of 2.0 and below, only strongly acid compounds, which in tissue sections seem to be limited to the sulfated acid mucopolysaccharides, yield a positive reaction. This method is not as sensitive in detecting small quantities of acid substances. Danielli's Diazo Reaction for Amino Acids The technic as described by Lillie, 4 using H acid, was followed. Interpretation. Iiistidine, tryptophan, tyrosine, and the purines and pyrimidines of nucleic acids yield a positive reaction. RESULTS The results of the various histochemical procedures are listed in Tables 2, 3, 4, and 5. COMMENTS Normal Skin Inter fibrillar ground substance. The histochemical reactions are interpreted as indicating the presence of a nonsulfated hyaluronidaselabile acid mucopolysaccharide; i.e., hyaluronic acid. This differs from the current concept that the ground substance is composed of equal parts of hyaluronic acid and chondroitin sulfate B. The current concept is based on chemical analysis of whole skin, 20 which includes the fibers, blood vessels, mast cells, and adnexal structures, which may be the actual source of the sulfated material. Mast cells. The reactions indicate the presence of a sulfated aminopolysaccharide complex. The procedures used will not produce satisfactory distinction between the different compounds in Group II (Table ). It is generally agreed that the sulfated mucin present is heparin. Previous studies 2 on mast cells in mastocytosis suggest that the heparin is associated with protein in a complex, and hyaluronic acid could not be TABLE 2 HISTOCHEMICAL REACTIONS FOB ACID MUCOPOLYSACCHARIDES OP THE NORMAL SKIN* Colloidal Iron Alcian Blue AldehydeFuchsin Toluidine Blue Material Without hyaluronidase With hyaluronidase hr. at 37 C. ph 2.7 to 3 ph0.4 tol PAS, Diatase Resistant ph.7 ph ph 3 pi 2 Ground substance Mast cells Blood vessel walls Apex of cell and lumen, apocrine Secretory granules, apocrine Cyfcophism of secretory cells, eccrine Interstices of epidermal cells Dermal papilla of antigen hair Nerves t to ± t to ± to ± _ to to ± to to ± _ * = strongl}' positive; = moderately strongly positive; = moderately positive; = weakly positive; ± = trace reaction; = negative, f Except those containing iron. (tort Downloaded from on 25 February 208

5 Aug. 963 ACID MUCOPOLYSACCHARIDES OF SKIN 27 TABLE 3 HlSTOCHEMICAL REACTIONS FOR ACID MUCOPOLYSACCHARIDES OK THE MYXOID GROUP* Condition Without hyaluronidase Colloidal Iron With hyaluronidase hr. at 37 C. Alcian Blue ph 2 to 3 ph 0.4 to PAS, Diastase Resistant AldehydeFuchsin ph.7 ph Toluidine Blue ph 3 ph 2 Reaction Localized myxedema Papular mucinosis Myxoid cyst Focal mucinosis Alopecia mucinosa 4f to ± to ± to ± to ± to ± 4f to ± to ± to ± to ± to ± * 4f = strongly positive; 3f = moderately positive; f = weakly positive; ± = trace reaction; negative. TABLE 4 HLSTOCHEMICAL REACTIONS FOR ACID MUCOPOLYSACCHARIDES OF THE MISCELLANEOUS GROUP* Material Colloidal Iron Without hyaluronidase With hyakironidase hr. at 37 C. Alcian Blue ph 2.7 to 3 ph 0.4 to PAS, Diastase Resistant AldehydepH.7 ph Fuchsin Toluidine Blue ph 3 pii2 Senile elastosis Pseudoxanthoma elasticum Calcinosis cutis Cartilage rest Acute lupus erythematosus Scleroderma and morphea (between fibers) Keloid (between fibers) f to 4f ± to 2f 3f 3f to 3f to 3f to 4f ± to to ± to ± f to ± to f to 3f f to 3f to ± 2f to 4f 4f (elastica) 4f (elastica) to ± to ± f to ± to ± f to ± (elastica) to ± (elastica) 2f 4f f to 2f f to 2f to * 4f = strongly positive; 3f = moderately strongly positive; 2f = moderately positive; f weakly positive; ± = trace reaction; = negative. demonstrated in mast cells by histochemical methods. Blood vessel walls. The larger vessels in the lower corium and subcutaneous tissue contain mucin which is interpreted as being a combination of hyaluronic acid and a sulfated aminopolysaccharide (chondroitin sulfate). On chemical analysis the aorta has been reported to contain hyaluronic acid and chondroitin sulfate B. 6 Apocrine gland, apex of cell and, lumen. The material is a nonsulfated hyaluronidaseresistant acid mucopolysaccharide (Group III type mucosubstance). These reactions are consistent with a sialomucin. Apocrine gland, secretory granules. The granules do not react with stains for acid substances and, therefore, probably represent a neutral mucopolysaccharide or glycolipid. The neutral nature of this material Downloaded from on 25 February 208

6 28 JOHNSON AND HELWIG Vol. 40 TABLE 5 HISTOCHEMICAL REACTIONS FOR AdD MUCOPOLYSACCHARIDES OP THE TUMOR GROUP* Tumor Without Colloidal Iron With hyaluronidase hr. at 37 C. With ribonuclease Alcian Blue ph 2.7 to 3 ph 0.4 PAS, Diastase Resistant Aldehyde Fuchsin ph.7 ph Toluidine Blue ph 3 ph2 Danielli Reaction Adenoid basal cell carcinoma Adenocystic carcinoma Mucinous carcinoma of breast metastatic skin Bronchial carcinoma metastatic skin Carcinoma colon metastatic skin Extramammary Paget's disease Malignant melanoma, cytoplasm ± to ± to ± to to ± ± to to ± * = strongly positive; = moderately strongly positive; weakly positive; ± = trace reaction; = negative. moderately positive; has been previously reported by Goltz and colleagues. 7 Eccrine gland, cytoplasm of secretory cells. Within the cytoplasm or attached to the cell membranes of the socalled dark cells, or both, there is abundant material that reacts as a Group III type of mucosubstance. These histochemical findings suggestive of a sialomucin are supported by the chemical analysis of eccrine sweat by Pallavicini and associates. 9 They found sialic acid, galactose, and galactosamine to be the predominant carbohydrates with small amounts of mannose, glucose, fucose, and glucosamine. In addition, discrete diastaseresistant, PASpositive, and aldehydefuchsin (ph.7) positive granules are sometimes seen. Interstices of epidermal cells. There is a variable amount of partially hyaluronidaselabile nonsulfated acid mucopolysaccharide present. We interpret this to indicate the presence of hyaluronic acid, although other types of mucin may also be present. Dermal papilla of anagen hair. In this location and along a narrow zone about the outer root sheath of the lower part of the follicle the mucin reacts as a sulfated aminopolysaccharide. It is probably one of the chondroitin sulfates, but the type can not be distinguished with the procedures used. Nerves. Within the cutaneous nerve sheaths there is a relatively large amount of mucin that reacts as hyaluronic acid. Myxoid Group The conditions studied in this group are localized myxedema, papular mucinosis, myxoid cyst (synovial cyst), focal mucinosis, and alopecia mucinosa. The term "focal mucinosis" is being used tentatively to describe a condition that ordinarily appears as a single nodule or cyst in sites on the trunk and extremities, usually not in proximity to the joints. Microscopically the lesion appears similar to an individual lesion of papular mucinosis. The mucin in all of these conditions exhibits histochemical reactions that we interpret to indicate hyaluronic Downloaded from on 25 February 208

7 Aug. 963 ACID MUCOPOLYSACCHARIDES OF SKIN 29 acid. No evidence of sulfated mucin is noted. A small amount of protein is present in association with the mucin, as determined by the Danielli reaction. Chemical analysis of "whole skin" was reported by Watson and Pearce 23 from a patient with localized myxedema. They found the amount of hyaluronic acid greatly increased and the amount of chondroitin sulfate increased to a lesser extent. They noted that the skin taken for examination appeared almost like a keloid. It is possible that chondroitin sulfate found by chemical analysis in localized myxedema, as well as in normal skin, may be an integral part of the fiber material, inasmuch as it has been demonstrated in bovine skin that more chondroitin sulfate is associated with the large, coarse collagen fiber in the lower dermis than in the upper dermis, where there is a greater amount of ground substance. Miscellaneous Group Senile elastosis. Near and between the abnormal elastica there is an increased amount of mucin that manifests reactions we interpret as indicating hyaluronic acid. The mucin is not completely hyaluronidase labile and, therefore, may contain other mucopolysaccharides in addition to hyaluronic acid. The aldehydefuchsin reaction can not be used for evaluation of the mucin, as it normally reacts positively with elastic tissue. In some examples there is only a slight increase in hyaluronic acid, but in others it is quite conspicuous. Pseudoxanthoma elasticum. The abnormal elastic fibers in the middle and lower corium yield a positive reaction to stains for acid mucopolysaccharide, presumably from material with which their outer surfaces are coated. The histochemical reactions indicate that this material is predominantly hyaluronic acid. Calcinosis cutis. The reactions are interpreted to indicate the presence of hyaluronic acid. In the various conditions with calcium deposition in skin that we have studied, such as that in pseudoxanthoma elasticum, tumors, and calcinosis cutis, we have found acid mucopolysaccharide present. This finding suggests that elaboration of mucopolysaccharide as a tissue response to injury is an intermediate stage in the pathologic changes resulting in calcification, owing to the affinity of the acid mucins for cations. The colloidal iron reaction for acid mucopolysaccharides is based on this affinity for the ferric ion. Cartilage rest. The reactions are interpreted to indicate the presence of a sulfated aminopolysaccharide. On chemical analysis cartilage has been demonstrated to contain chondroitin sulfate A and C. Acute lupus erythematosus, scleroderma, morphea, and keloid. In all of these conditions there is a variable increase in the amount of interfibrillar ground substance. This substance manifests reactions we interpret as indicating the presence of hyaluronic acid. We do not believe that these changes are specific but that connective tissue injury from many causes will result in an increased production of hyaluronic acid. Increased ground substance in disseminated lupus erythematosus and dermatomyositis was reported by Cawley and coworkers, 4 but they did not find any significant increase in morphea and generalized scleroderma. Tumor Group Adenoid basal cell epithelioma. The mucin present manifests reactions of a sulfated aminopolysaccharide. The epithelial cells are intimately associated with the mucin, but in the 5 to 6M sections available for study, the site of elaboration could not be definitely established. The possibility exists that connective tissue cells may play a role in the production of the mucin. The fact that the mucin is similar to that seen in the dermal papilla of hair follicles could be used to support the concept that this variety of basal cell epithelioma is differentiating from or toward pilar structures. Adenocystic carcinoma. The reactions are interpreted to mean that the mucin present is a Group III mucosubstance, but that it differs from the others in this group in that the aldehydefuchsin (ph.7) is essentially negative. Mucinous carcinoma of breast, bronchus, and colon. In these conditions the mucin in the primary tumor and that in the cutane Downloaded from on 25 February 208

8 30 JOHNSON AND HELWIG Vol. 40 ous metastasis reveals the same histochemical reactions. The histochemical reactions were those of a Group III type of mucosubstance, either sialic acidcontaining complexes or other acids not yet recognized. Histochemical procedures are of value in distinguishing between mucuscontaining cutaneous metastases of these neoplasms and mucuscontaining adnexal and basal cell epitheliomas, which have a sulfated type of mucin. Chemical analysis by Werner 24 of mucins produced by the trachea, bronchi, small intestine, large intestine, and ovarian duct revealed the presence of sialomucin and bloodgroup substances. He did not find evidence of a sulfated complex in any of these sites. The bloodgroup substances are neutral in character and should not cause a positive reaction with the histochemical procedures for acid substances. The presence of the bloodgroup substances admixed with sialomucins could not be detected or ruled out by the methods we used. Extramammary Paget's disease. The mucin exhibits reactions of a Group III type of mucosubstancc, presumably a sialomucin. It is not affected by ribonuclease nor mild acid. Malignant melanoma. In the cytoplasm of melanoma cells, especially in those containing fine melanin granules, the colloidal iron stain, the alcian blue stain at ph 2 to 3, and the PAS reaction frequently yield positive results. Most of the reactive material is removed by ribonuclease. We doubt that these reactions indicate the presence of acid mucopolysaccharide and regard the material as more likely being ribonucleic acid or intermediate compounds, or both, in the tyrosinetomelanin reaction. There is some hyaluronidaselabile material between the melanoma cells, but whether this material is elaborated by connective tissue fibroblasts or melanoma cells can not be determined. The negative aldehydefuchsin reaction in melanoma cells and the positive reaction in extramammary Paget cells offers an excellent histochemical aid in distinguishing between these 2 conditions, as previously reported by Helwig and Graham. 0 SUMMARY This paper deals with the results of a histochemical study of the acid mucopolysaccharides of normal skin, and a survey of certain inflammatory diseases, metabolic diseases, and tumors. A working classification of the acid mucopolysaccharides was designed and is included. Histochemical procedures for the acid mucopolysaccharides are described and their interpretation discussed. Results are correlated with chemical analyses of similar material when possible. SUMMAUO IN INTEKLINGUA Iste communication concerne le resultatos de un studio histochimic del mucopolysaccharides acide de pelle normal e de lor comportamento in certe morbos inflammatori, metabolic, e noeplastic. Esseva establite un classification del mucopolysaccharides acide a usos practic. Isto es includite in le communication. Methodos histochimic pro le determination del mucopolysaccharides acide es describite. Le interpretation del resultatos es discutite. In tanto que possibile, le resultatos es ponite in correlation con analyses chimic de simile materiales. REFERENCES. ABULHAJ, S. K., AND RINEMART, J. F.: Fuchsinaldehyde staining of sulfated mucopolysaccharides and related substances. (Proc. Histochem. Soc.) J. Nat. Cancer Inst., 3: , BANGLE, R., JR.: Gomori's paraldehydefuchsin stain. I. Physicochemical and staining properties of the dye. J. Histochem., 2: 29299, BETTELHEIMJEVONS, F. R.: Protein carbohydrate complexes. Advances Protein C'hem., 3: 3505, CAWLEY, E. P., MCMANUS, J. F. A., LUI'TON, C. H., AND WHEELER, C. E.: An examination of skin from patients with collagen disease utilizing the combined alcian blueperiodic acid Schiff stain. J. Invest. Dermat., 27: , DA VIES, D. V.: Specificity of staining methods for mucopolysaccharides of the hyaluronic acid type. Stain Techn., 27: 0570, FUSARO, R. M., AND GOLTZ, R. W.: A comparative study of the periodic acidschiff and alcian blue stains. J. Invest. Dermat., 37: , GOLTZ, R. W., FUSARO, R. M., AND JARVIS, J.: The demonstration of acid substances in normal skin by alcian blue. J. Invest. Dermat., 3: 8390, GOMORI, G. L.: Aldehydefuchsin, a new stain Downloaded from on 25 February 208

9 Aug. 963 ACID MUCOPOLYSACCHARIDES OF SKIN 3 for clastic tissue. Am. J. Clin. Path., 20: , HELWIO, E. B., AND GRAHAM, J. H.: Anogenital (extramammary) Paget's disease; a clinicopathologic study. Cancer, 6: , HOFFMAN, P., LINKER, A., AND MEYER, K.: The acid mucopolysaccharides of connective tissue. II. Further experiments on chondroitin sulfate B. Arch. Biochem., 69: , HOOGHWINKEL, G. J. M., AND SMITS, G.: The specificity of the periodic acidschiff technique studied by a quantitative testtube method. J. Histochem., 5: 2026, JOHNSON, W. C, AND HELWIG, E. B.: Solitary mastocytosis (urticaria pigmentosa). Arch. Dermat. (Chicago), 84: 80685, LEBLOND, C. P., GLEGG,. E., AND EIDINGER, D.: Presence of carbohydrates with free,2glycol groups in sites stained by the periodic acidschilt technique. J. Histochem., 5: , LILLIE,. D.: Histopathologic Technic and Practical Histochemistry. New York: The Blakiston Co., Inc., 954, pp Manual of Histologic and Special Staining Technics, Ed. 2. New York: McGrawHill Book Co., Inc., The Blakiston Division, MEYER, K., DAVIDSON, E., AND HOFFMAN, P.: The acid mucopolysaccharides of connective tissue. Biochim. et biophys. acta, 2: 50058, MEYER, K., AND RAPPORT, M. M.: The hydrolysis of chondroitin sulfate by testicular hyaluronidase. Arch. Biochem., 27: , MOWRY,. W.: Improved procedure for the staining of acidic polysaccharides by Midler's colloidal (hydrous) ferric oxide and its combination with the Feulgen and the periodic acidschiff reactions. Lab. Invest., 7: , PALLAVICINI, J. C, GABRIEL, O., DI SANT' AGNESE, P. A., AND BUSKIRK, E..: Isolation and characterization of carbohydrateprotein complexes from human sweat. Ann. New York Acad. So., 06 (Art. 2): , PEARCE,. H., AND WATSON, E. M.: The mucopolysaccharides of human skin. Canad. J. Res. (Sec. E.), 27: 4357, RiNEHART, J. F., AND ABULHAJ, S. K.: An improved method for histologic demonstration of acid mucopolysaccharides in tissues. A. M. A. Arch. Path., 52: 8994, SCOTT, H.., AND CLAYTON, B. P.: A comparison of the staining affinities of aldehydefuchsin and the SchilT reagent. J. Histochem., : , WATSON, E. M., AND PEARCE, II. H.: The mucopolysaccharide content of the skin in localized (pretibial) myxedema. II. Am. J. Clin. Path., 9: , WERNER, I.: Studies on glycoproteins from mucous epithelium and epithelial secretions. Acta Soc. med. upsal., 58: 55, ZACHARIAE, F. G., AND DYRLYE, M. D.: Methods for isolation and identification of acid mucopolysaccharides in connective tissue. Acta Rheum. Scandinav., 5: , 959. Downloaded from on 25 February 208