THIOFLAVIN-T FOR AMYLOID DETECTION

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1 THE AMERICAN JOURNAL OP CLINICAL PATHOLOGY Copyright 67 by The Williams & Wilkins Co. Vol. 7, No. Printed in U.S.A. THIOFLAVIN-T FOR AMYLOID DETECTION S. M. SAEED, M.D., AND GERALD FINE, M.D. Department of Laboratories, Henry Ford Hospital, Detroit, Michigan A number of special staining procedures for the detection of amyloid deposits in tissue are in use, but because of the complexity and heterogeneity of amyloid, results are inconsistent and frequently not diagnostic. This has resulted in repeated search for more specific methods to detect amyloid. The nuorochrome dye, thioflavin-t, introduced by Vassar and Culling in and investigated by others,, appears to be the most promising of the stains to date. This study was carried out to determine the specificity of thiofiavin-t for amyloid and to compare it with other staining procedures as an amyloid screening method. MATERIALS AND METHODS For a double blind study, cases, nonamyloid and 6 of amyloid of various types diagnosed by the distribution and staining characteristics in hematoxylin-eosin preparations, were selected; tissue blocks, representing with and 7 without amyloid, were examined. The type and organ distribution of the amyloid cases are shown in Table and the nonamyloid tissues are listed in Table. Phosphate buffered formalin, % (ph 7.), fixation was used in all cases, and special fixatives alcohol (SO % and %), formol calcium acetate, Carnoy's fluid, Bouin's fluid, Helly's fluid, and Zenker's solution with and without acetic acid were used in two cases with amyloid. Consecutive 6-M sections utilizing blocks, 7 representing different organs or tissues with amyloid and representing different conditions without amyloid, were stained with crystal violet, Congo red, van Gieson, and thioflavin-t. The remaining blocks, from the amyloid group and 6 Received, June, 66. This paper was presented in part at the joint annual meeting of the American Society of Clinical Pathologists and College of American Pathologists, Chicago, Illinois, October to, 6. from the nonamyloid group, were stained only with thioflavin-t. Thioflavin-T staining and examination. Deparaffinized sections were stained for min. with alum-hematoxylin, differentiated for sec. in acid alcohol to abolish nuclear autofluorescence, stained for min. with % thioflavin-t freshly filtered to eliminate dye precipitate, differentiated for min. in % aqueous hydrochloric acid, and mounted in Paragon aqueous mounting medium. In some instances the staining time was varied from to min. and the differentiation time from to min., and gelatin, glycerine-saline, and Permount were tried as alternative mounting media. Sections were examined with a Zeiss ABO fluorescent microscope equipped with a -watt Osram mercury vapor light source, a BG exciter filter, and Zeiss No. 7, 7/6, and barrier filters. The effect of staining amyloid-bearing tissue Avith hematoxylin and eosin, methenamine silver, silver nitrate, or crystal violet before thioflavin-t was also studied in instances. RESULTS Superior thioflavin-t specificity for amyloid is indicated in Table, whereas greater Congo red and crystal violet affinity for nonamyloid hyalin tissue gives rise to a greater number of false-positive results than does thioflavin-t. The only false-positive result with thioflavin-t was the aortic wall polysaccharide of Marfan's disease. Fluorescence, approaching the brilliance seen with amyloid, was observed with other structures (Table ); some of these were also stained by other methods used in the study. The location, morphology, and less intense staining of these structures serve to distinguish them from amyloid (Fig. ). The brilliant fluorescence of amyloid after thioflavin-t staining makes it more easily Downloaded from on March

2 May 67 THIOFLAVIN-T FOR AMYLOID DETECTION S TABLE Type of Amyloid Primary Secondary! Multiple myeloma Hvalinized islets of Langerhans (diabetic) Medullary carcinoma of thyroid o. of Cases *. 7 :in and Soft Tissue in 7 TYPE AND ORGAN DISTRIBUTION IN CASES OF AMYLOID celetal Muscle c/ ongue H eart *. ungs, Trachea and Bronchi -J iver a S }leen w idney i S drenal Gland < 7 ancreas (X hyroid Gland H _ astrointestina Tract vj ymph Nodes ~~ iscellaneous* f% s otal No. of Tissues H' 7 Total * Central nervous system, pituitary gland, urethra, urinary bladder, ovary, testis. f Chronic infection, cases, carcinomatosis, cases. discernible than the staining produced by other methods, permitting the detection of amyloid deposits that are inconspicuous or unnoticed with the other stains (Fig. ). Fluorescence color varied with the barrier filter; it was yellow-green with the No. 7/6 filter and orange-green with the No. filter. Increasing the staining and differentiation time to to min., respectively, and the use of various water-soluble mounting media or Permount with prior dehydration and clearing did not noticeably affect thioflavin-t amyloid fluorescence. On the contrary, Permount reduced nonspecific fluorescence of peritubular fibrous tissue in a case of diabetic nephropathy. The variety of fixatives used in two cases of amyloid (one secondary and one primary) did not influence amyloid staining by any of the methods, except for that of Helly's and Carney's fluid; the former reduced and the latter increased the intensity of all staining reactions. DISCUSSION Amyloid staining by thioflavin-t is thought to be due to its affinity for proteinbound polysaccharide; thus it might be expected that false-positive stains would be encountered. With minimal experience, one can easily recognize the positively stained nonamyloid structures by their location and morphology and less intense fluorescence. The intensity of amyloid fluorescence is in sharp contrast to that of hyalinized collagen, vessel wall hyalinization, and the glomerular changes of diabetes and glomerulonephritis, and the distinction is much more readily made after thioflavin-t staining than after Congo red, crystal violet, periodic acid- Schiff, and van Gieson stains (Pig. ). The intense, red staining of collagen and yellow staining of amyloid with the van Gieson method makes the method useful in distinguishing these two, but it is of little or no value in differentiating vessel wall or glomerular amyloid deposits, since smooth muscle and the glomerular hyalin of diabetes and glomerulonephritis are stained yellow by the picric acid in the van Gieson stain. Limited experience with recently introduced cotton dyes ' 6 (Sirius red FBA, Sirius supra scarlet GG-CF, and Sirius supra blue FRA), and the Congo-red-polarization technic in in- Downloaded from on March

3 SAEED AND FINE Vol. /,7 TABLE NONAMYLOID CONDITIONS Conditions No. of Cases Congo red False-Positive Crystal violet Staining with van Gieson Thioflavin- T Vocal cord polyps Oral mucosal hyperplasia and fibrosis Rectal mucosa Chronic dermatitis, fibrosis, and elastosis Epidermal cyst Pleural () and pulmonary () fibrosis Arteriosclerotic heart disease Organized thrombi (), ventricular () and aortic () aneurysms Splenic vascular sclerosis Vascular nephrosclerosis Diabetic (Kimmelstiel-Wilson syndrome) nephrosclerosis Membranous glomerulonephritis Acute glomerulonephritis Necrotizing arteritis (kidneys, mesentery ) Cerebral atrophy and gliosis Postmenopausal ovary Thrombotic thrombocytopenic purpura (liver, lung, spleen, pituitary) Marian's disease (aorta) Rheumatoid nodule Cirrhosis of the liver Multiple myeloma (kidneys, spleen, bones, heart, lungs, lymph node, adrenal, liver, pancreas ) Macroglobinemia (Waldenstrom) (liver, lymph node, lung, spleen, kidney ) Hodgkin's disease (spleen, lymph nodes, liver) Carcinomata (lung, breast, ovary, thyroid ) o' Total 7 TABLE SIMULTANEOUS AMYLOID STAINS No. of Tissues Positive with Congo Red Positive with Crystal Violet Positive with both Congo Red and Crystal Violet Positive (Lack of Staining) with van Gieson Positive with Thiorlavin-T Amyloidosis Nonamyloid tissues 7 7 (.%) (.7%) (7.6%) 7 (.%) 7 (.%) None (.%) (.%) (7.%) 7 (.%) (.%) stances where the Congo red stain results were negative or equivocal, yielded results not significantly better than those obtained with Congo red staining, and inferior to those obtained with thioflavin-t. Polarization was helpful in distinguishing amyloid deposited in glomeruli, adrenal gland, liver, and other parenchymal organs, but it was of Downloaded from on March

4 May J 67 THIOFLAVIN-T FOR AMYLOID DETECTION TABLE SOURCES OF FALSE-POSITIVE THIOFLAVIN-T STAIN FOR AMYLOID Autofluorescence Elastic tissue Cotton fibers Staining RBC Hemosiderin and lipochrome pigment Salivary and pancreatic zymogen granules Renal juxtaglomerular cell granules Nissl granules (occasionally) Renal casts, prostatic and pulmonary corpora amylacea and thyroid colloid Colloid droplets of renal tubular cells Mucin; gastric, colonic no value in distinguishing amyloid from naturally polarizing collagen, stained with Congo red or unstained. Differentiation is a critical step in the crystal violet, Congo red, and van Gieson methods, and slight overdifferentiation may eliminate or greatly reduce specific staining. This has not been a problem with thioflavin-t; intentional overdifferentiation had no effect upon the intensity of amyloid fluorescence in the few cases attempted by us. SUMMARY A double blind study to determine the relative efficacy and specificity of thioflavin-t staining of amyloid as compared with the Congo red, crystal violet, and van Gieson technics is reported. The greater accuracy and ease of interpretation of the thioflavin-t stain make it the method of choice for detection of amyloid. REFERENCES. Heller, H., Missmahl, H.-P., Sohar, E. and Gafni, J.: Amyloidosis: its differentiation FIG.. Well demarcated, brightly fluorescent amyloid is unmistakable. A, (upper, left.) secondary amyloidosis of renal glomerulus; B (upper, right), secondary amyloidosis of liver; C (lower, left) primary amyloidosis of a coronary arteriole; D (lower, right), amyloid tumor of bronchus. Thioflavin-T stain. X. Downloaded from on March

5 F I G.. Serial sections of a hyalinized diabetic islet of Langerhans show vast superiority of thioflavin T over other stains. A, hematoxylin and eosin; B, crystal violet; C, Congo red; D, thioflavin-t. X. F I G.. Nonamyloid staining in A (left), renal casts; B (right), Leydig cells (lipofuscin). Thioflavin-T stain. X. Downloaded from on March

6 May 67 THFLAVIN-T FOR AMYLOID DETECTION into peri-reticulin and peri-collagen types. J. Path. & Bact., : -, 6.. Hobbs, J. R., and Morgan, A. D.: Fluorescence microscopy with thioflavine-t in the diagnosis of amyloid. J. Path. & Bact., 6: 7-, 6.. Kurban, A. K.: Fluorescent stain for amyloid. An easy histological differentiation between cutaneous amyloidosis, colloid milium, and senile elastosis. Bull. Johns Hopkins Hosp., 7: -, 6.. Puchtler, H., Sweat, F., and Levine, M.: On the binding of Congo red by amyloid. J. Histochem. Cytochem., : -6, 6.. Sweat, F., and Puchtler, H.: Demonstration of amyloid with direct cotton dyes. Arch. Path., : 6-6, Vassar, P. S., and Culling, C. F. A.: Fluorescent stains, with special reference to amyloid and connective tissues. A. M. A. Arch. Path., 6: 7-,. 7. Vassar, P. S., and Culling, C. F. A.: Fluorescent amyloid staining of casts in myeloma nephrosis. Arch. Path., 7: -6,. Downloaded from on March