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Histochemistry of the Acid Mucopolysaccharides in Cutaneous Calcification* Waine C

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Histochemistry of the Acid Mucopolysaccharides in Cutaneous Calcification* Waine C View metadata, citation and similar papers at core.ac.uk brought to you by CORE provided by Elsevier - Publisher Connector HISTOCHEMISTRY OF THE ACID MUCOPOLYSACCHARIDES IN CUTANEOUS CALCIFICATION* WAINE C. JOHNSON, M.D.,** JAMES H. GRAHAM, M.D.** AND ELSON B. HELWIG, M.D.*** Several theories have been suggested in recentwas used for the standard. It is recognized that a years regarding the mechanisms involved inpH meter is not entirely accurate at values below physiological and pathological tissue calcification,pH 1, but in our experience, the readings on these but none are widely accepted at this time. Certainsolutions have been consistent and reproducible. evidence indicates that the development of calci-In addition to hematoxylin and eosin-stained fication in cartilage differs from that in bone, andsections the following histochemical studies were that both of these processes may be unlikeperformed: pathological calcinosis. However, the concept of 1. Colloidal Iron Technic, With and Without a matrix substance acting to bind calcium hasBovine Testicular Hyaluronidase Digestion. Modi- received attention from workers studying thefications of the Hale iron method as described by various types of tissue calcification. The purposeMowry (1) were performed, using a van Gieson of this paper is to report our observations on thecounterstain. The pH of the working solution was histochemistry of the acid mucopolysaccharidesin the range of 1.5 to 1.7 and the pH of the fer- rocyanide hydrochloric acid (HC1) solution was which may act as matrix materials in the patho-1.1. The hyaluronidase (Nutritional Biochemicals) genesis of cutaneous calcinosis. was prepared immediately before use with 150 U.S.P. units to 1 ml of phosphate buffer solution MATERJALS AND METHODS at pH 6.8. A circle was drawn about the tissue sec- tion using a diamond pencil, and the area was Formalin-fixed paraffin-embedded tissue sec-covered with hyaluronidase solution and placed tions from examples of generalized cutaneousin a covered dish lined with moistened filter paper calcinosis (so-called metastatic calcinosis), lo-for digestion for 1 hour at 37° C. Controls for calized (dystrophic) calcinosis, basal cell car-hyaluronidase activity were processed as above using only the buffer solution. Since iron com- cinoma and adnexal carcinoma with focal areaspounds in the tissue may produce a false positive of calcification, calcified epidermoid cysts, andcolloidal iron reaction, a ferrocyanide-HC1 re- calcinosis occurring in pseudoxanthoma elasticumaction was used to detect the presence of any iron. were studied. The number of specimens studied 2. Colloidal Iron Stain With Ribonuclease Di- from each type of calcinosis varied from 2 to 6. gestion. Digestion was carried out for 2 hours at 37° C using a 0.1% solution of ribonuclease buf- The procedures used were performed as out-fered at pH 6.8. The digested sections and controls lined in the "Manual of Histologic and Specialwere then processed using the colloidal iron Staining Technics" (1) except where otherwisemethod. stated. The pH values referred to in this paper 3. Hydrochloric Acid and Potassium Ferrocya- nide Method for Iron With a Nuclear Fast Red were determined by a Zeromatic Beckman pHCounterstain. meter. Buffer solutions of pH 1, 2, 3, 4, 6, and 7 4. Alcian BlueS GS Stain at pH 2.5 and 0.4 With obtained from Fisher Scientific Company, werea Nuclear Fast Red Counterstain. Phosphate-HCI used to standardize the instrument. The buffersolutions were prepared at pH values of approxi- mately 2.5 and 0.4, and these were used for treat- solution nearest the pH value of the test solutionment of the tissue sections prior to staining, for *FromThe Skin and Cancer Hospital of Phil-staining with 1% alcian blue, and for the first adelphia,** Department of Dermatology, Templerinse. The usual method using 1% alcian blue in University School of Medicine,** Philadelphia,3% acetic acid (pH 2.5 to 3.0) was also performed. Pa., and The Armed Forces Institute of Pathol-A 0.5 normal HCI and 0.1 molar sodium monobasic ogy, ***Washington,D.C. 20305 phosphate solution gave a pH reading of 0.4. It is Thisinvestigation was sopported in part byrecognized that such a pH reading may not rep- research training grant No. 2A-5289 (C1), from the National Institute of Arthritis and Metabolicresent an absolute value, but for the purposes of Diseases, Public Health Service, Bethesda 14,this report the stain prepared from it will be re- Maryland. ferred to as alcian blue pH 0.4. This solution can Presented by title at the Twenty-fourth Annualbe prepared by using 42 ml of concentrated HCI Meeting of The Society for Investigative Derma-and 13.8 grams of sodium monobasic phosphate tology, Inc., Atlantic City, N. J., Jone 17—20, 1963. (F.W. 138), made up to 1 liter with distilled water. 215 TABLE I Results of histochernical procedures for acid mucopolysaccharides in cutaneous calcification Colloidal Iron Iron Stain Alcian Blue Aldehyde-Fuchsin PAS Toluidine Blue Method Without With hyalu- With ribo- Without With hyalu- 1" 2,5 pH 0.4 pH 1.7 pH 1.0 3.0 pH 1.5 ronidase ronidase nuclease diastase diastase Generalized (metastatic) +++ + +++ ++ — +++ +++ + — Calcinosis Cutis to to z ++ Localized (dystrophic) Cal- +++ + +++ ++ — +++ +++ + — C cinosis Cutis to to to — ++ + ++ Epidermoid Cyst +++ ++ +++ - ++ - - - ++ ++ + - to — +++ C Pseudoxanthoma Elasticum ++ + ++ - + - +++ +++ ++ ++ + — to to to to (elastic +++ ++ +++ ++ tissue) BasalCellCarcinomaand +++ +++ +++ + ++ + +++ ++ ++ ++ ++ + Adnexal Carcinoma (2 sped- mens) C C Key: +++ Strong positive ++ Moderate positive + Weak positive Trace — Negative HISTOCHEMISTRY OF CUTANEOUS CALCIFICATION 217 A 0.03 normal HC1 and 0.1 molar phosphate buffer pH 1.0 was prepared by adding concentrated HCI, solution gave a pH reading of 2.5. This solutionand the 80% alcoholic rinse used with the stain can be prepared by using 30 ml of 1 normal HC1was also adjusted to approximately pH 1.0. and 13.8 grams of sodium monobasic phosphate 7. 'I'oluidine Blue for Metachromasia at pH 3.0 made up to 1 liter with distilled water. The ac-and pH 1.5. Buffer solutions of the same pH as curacy of measurement of solutions and reagentsthe stains were used for rinsing. Since meta- as well as the pH of the distilled water used maychromasia may be lost in dehydration, wet sec- give rise to a minor variation in the pH of the finaltions were examined immediately, then air-dried solutions. Results of the routine alcian blueand mounted in Permount. A 0.01 normal HC1 technic (3% acetic acid at pH 2.5 to 3.0) were es-and 0.1 molar sodium monobasic phosphate solu- sentially the same as that obtained with the phos-tion gave a pH reading of 1.5. This solution can phate-HC1 stain at pH 2.5 and are reported to-be prepared by using 100 ml of 1 normal HC1 and gether as alcian blue pH 2.5 in Table I. An attempt13.8 grams of sodium monobasic phosphate made was made to evaluate the possibility of acidup to 1 liter with distilled water. Ten ml of 1 groups other than the acid mucopolysaceharidesnormal HCI and 13.8 grams of sodium monobasic such as phosphate groups or the carboxyl groupsphosphate made up to 1 liter with distilled water of citric acid playing a role in producing a falsegave a pH reading of 3.0. positive alcian blue reaction. Spot tests with 8. Danielli's Diazo Reaction for Amino Acids. calcium monobasic and dibasic phosphate, andThe method as described by Lillie (2) using H citric acid were performed and the alcian blueacid was followed. stain did not show affinity for them. 9. Alizarin Red Stain for Calcium. The tech- 5. Periodic Ae.id-Schiff (PAS) Reaction Withnic of McGee-Russell was used (3). and Without Diastase Digestion With a Picric Acid 10. Von Kossa Stain for Salts of Calcium. Gounterstain. Diastase digestion was used for removal of glycogen. The pH of the 0.5% periodic RESULTS AND INTERPRETATION acid and that of the Schiff's solution were both approximately 2.0. The results of the histochemical procedures for 6. Aldehyde-Fuchsin Stain at pH 1.7 and 1.0acid mucopolysaccharides are summarized in With a Metanil Yellow Counterstain. The methodTable I. The basis for our interpretation of of Gomori (1) was followed and the pH of the prepared working solution was approximately 1.7.these methods have been published (4, 5) and In addition, a working solution of approximatelyonly the interpretation will be given. ! a!;•L I "4 RC !a , ! t. # •4S . IAfS 4! FIG. 1. Basal cell carcinoma. Colloidal iron reaction with hyaluronidase digestion. The colloidal iron reaction is strongly positive at calcified foci and the reactive material is not removed by hyaluronidase digestion. X 425. 218 THEJOURNAL OF INVESTIGATIVE DERMATOLOGY Colloidal Iron Technic, With and WithoutAt pH 2.5, most acid mucopolysaceharides give Hyaluronidase Digestion. Acid mucopolysac-a blue color and nucleic acids may produce a charides produce a blue to green color and nucleicless intense color reaction. At pH 0.4 only strongly acids sometimes produce a similar but lessacid substances such as the sulfated acid muco- intense reactioa. In paraffin-embedded tissuepolysaccharides will give a positive reaction. In sections other acid substances do not remain ingeneral, this method is not as sensitive as the quantities sufficient to produce a color reaction.colloidal iron technic. We interpret a positive Bovine testicular hyaluronidase digestion for 1reaction at pH 2.5 and 0.4 as seen in calcified hour at 37°C will usually remove most of thesites of basal cell carcinoma and adnexal carci- hyaluronic acid, but does not digest ehondroitinnoma to indicate the presence of a sulfated acid sulfate B. In our experience (4), digestion asmueopolysaecharide. We have reported (6) the carried out in this study does not remove sig-presence of sulfated acid mucopolysaceharides, nificant amounts of chondroitin sulfates A andpresumably a chondroitin sulfate, in certain C from tissue sections.
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