The Congo Red Stain Revisited*

M. TAREK ELGHETANY, M.D., ABDUS SALEEM, M.D., and KAYE BARR, H.T., A.S.C.P.

Departments o f Pathology, Baylor College o f Medicine, and The Methodist Hospital, Houston, TX 77030

ABSTRACT

The Congo red stain has undergone several modifications since it was first used by Bennhold1 in 1922 in order to increase the specificity for staining amyloid. Most of the laboratories in the United States use the method of Puchtler16 which uses alkaline Congo red solution. Some of the variables associated with the procedure were investigated by us. Our results showed the following: (1 ) amyloid showed green birefringence at all levels between 4 to 12 (x thick sections with better visualization of small deposits with increased thickness. Best results were obtained with 8 |x thick sections; (2 ) omission of the pretreatment with alkaline alcoholic solution of sodium chloride (NaCl) did not affect the sensitivity of the method; (3) the use of polar mounting media had no effect on amyloid and collagen birefringence; (4) 50 percent saturation of the Congo red staining solution with NaCl caused strong staining of collagen, elastic fibers and eosinophilic granules. In addition, collagen showed green birefringence and dichroism and its differentiation from amyloid became difficult; and (5) using the staining solution fully saturated with NaCl, no positive stain ing was seen with tissues other than amyloid. Collagen and elastic fibers showed red fluorescence which was of less intensity than amyloid. It is our conclusion that the method of Puchtler16 for detecting amyloid gives bet ter results if the staining solution is fully saturated with NaCl. The pre treatment step may be deleted without compromising the quality of stain ing. Improved staining of amyloid enhances the specificity of green birefringence, dichroism, and red fluorescence.

Introduction ized by the deposition of insoluble fibril lar protein in the extracellular spaces. The term amyloidosis describes a het Despite their chemical diversity, amy erogeneous group of diseases character- loid fibrils share the following diagnostic properties: (1) Positive Congo red stain ing with apple green birefringence on Address reprint request to: Abdus Saleem, M.D., Professor of Pathology, Baylor College of Medicine, polarization microscopy; (2) B-pleated One Baylor Plaza, Houston, TX 77030. sheet secondary structure; and (3) Fibril 190 0091-7370/89/0500-0190 $00.90 © Institute for Clinical Science, Inc. CONGO RED STAIN 191 lary quaternary structure with electron hydrate sections through graded alcohol microscopy. 3,7 The Congo red stain was to distilled water; (2) stain with Mayer’s first used by Bennhold1 in 1922 for the acid hemalum for 10 minutes; (3) wash in detection of amyloid in tissues. Despite three changes of distilled water; (4) stain several modifications of the stain, the in freshly alkalinized alcoholic solution of procedure has not been standardized. Congo red for 50 minutes; and (5) dehy Variables such as thickness of the sec drate quickly in three changes of abso tion, saturation of the staining solution lute alcohol, clear in xylene and mount. with sodium chloride (NaCl) and the In our laboratory, sections are routinely type of the mounting media could inter mounted in Harleco.f This mounting fere with the quality and specificity of medium contains the neutral resin ethyl the staining. 2,20,21,24 This work was done methacrylate. 14 to investigate some of the variables to Sections were examined under light get optimal staining of amyloid. and polarized microscopy with and with out red compensation for birefringence Materials and Methods and dichroism using American Optical microscope 1 1 0 with built-in analyzer Three cases of systemic amyloidosis, and attachable polarizer and red com including two cases of primary systemic pensator (AO 1153). Fluorescence was amyloidosis and one case of myeloma- examined using Microphot, FX, Nikon. related amyloidosis, were found in the Objectives, condensers, glass slides, autopsy files of The Methodist Hospital and cover glasses were tested to be between 1979 to 1986. A total of 20 sam strain free as follows: with the light ples of different organs, including heart, source turned on, glass slides and covers liver, spleen, kidney, lung, tongue, were examined with both polarizer and uterus, prostate, pancreas, peripheral analyzer in crossed position. The field nerves, bowel, and lymph nodes, were was completely dark without any light included in the study. All specimens spots. 19 were fixed in four percent buffered form Dichroism was examined using two aldehyde solution, routinely processed polarizing plates as follows;10 the polar in ethyl alcohol and xylene, and embed izer and analyzer are placed in crossed ded in paraffin. The following staining position. Either of them is rotated a solution was prepared: alkaline alcoholic small angle alternately clockwise and solution saturated with NaCl14 in which counterclockwise from the crossed posi two grams of NaCl were dissolved in 20 tion. The background and non-dichroic ml of distilled water followed by 80 ml of birefringent objects will brighten sym absolute alcohol. The solution was alka- metrically as one of the polarizing plates linized just before use by the addition of is rotated in either direction while a one ml of one percent sodium hydroxide dichroic object will brighten when polar (NaOH). Staining solution: 0.2 g of izer is rotated in one direction and Congo red* was added to the previous darken as it is rotated in the opposite solution. direction. Staining procedure (modified from Variables examined include: (1) thick Puchtler16 and Sheehan and Hrapchak23 ness of the section: serial sections of 4, 6 , was as follows: (1 ) deparaffinize and 8 , 1 0 , 1 2 (x in thickness were stained with the above procedure and mounted

* C.I. 22120, Sigma Chemical Company, St. Louis, MO. t EM Diagnostic System Inc., NJ. 192 ELGHETANY, SALEEM, AND BARR in Harleco medium; (2) pretreating the between 4 to 12 |x in thickness. Collagen tissue with alkaline alcoholic solution sat did not stain and showed a white-blue urated with NaCl before staining (pre birefringence and no dichroism. With treatment step): An 8 |jl thick section was sections, between four and eight jjl thick, pretreated for 2 0 minutes before staining more intense staining and green birefrin in the Congo red solution; (3) 50 percent gence of small deposits were seen with saturation of Congo red solution with increasing thickness. No appreciable NaCl: An 8 |x thick section was stained as effect on dense deposits was detected. previously stated except for using Congo Sections of more than 8 |x in thickness red staining solution which was 50 per were technically cumbersome and did cent saturated with NaCl, i.e., one g of not add any advantage in detecting amy NaCl instead of two g in the alkaline loid. The white-blue birefringence of alcoholic solution; (4) effect of mounting collagen was increased with thicker sec media: Sections of 8 jul thickness were tions. stained using Congo red solution with full and 50 percent saturation with NaCl. E f f e c t o f t h e P retreatment S t e p The sections were gradually rehydrated in 70 percent alcohol, 50 percent alco No appreciable difference was detect hol, and distilled water then mounted in ed in staining or birefringence with or Aqua Mount. $ This medium contains a without pretreatment step. Occasional high polar synthetic resin, polyvinyl sections showed slight increase in colla alcohol.8 Sections were compared with gen birefringence and slight decrease in those mounted in the neutral mounting Congo red staining of amyloid with no medium; and (5) unstained birefringence effect on green birefringence if the pre and fluorescence: An 8 |x thick sec treatment step was used. tion was deparaffinized and mounted unstained in Harleco medium and exam E f f e c t o f S a l t S a t u r a t io n ined. For controls, normal tissues were When the Congo red staining solution selected from the surgical and autopsy was used with 50 percent saturation with files representing fibrin (thrombi in arte NaCl, collagen, elastic fibers, eosino rial repair), nerves (sympathetic and phils, and amyloid were stained. Colla peripheral), smooth muscles (prostate, gen and amyloid showed green birefrin uterus, gall bladder), collagen (fibrocys gence and dichroism. Fibrin, smooth tic disease of breast, Dupytren’s contrac muscles, and nerves did not stain. When ture, fibrous thickening of pericardium), the Congo red staining solution was fully and elastic fibers (aorta and pulmonary saturated with NaCl, no positive staining artery from young autopsies). Several was seen with tissues other than amy sections of the control tissues were loid. Collagen showed a white-blue bire examined for each of the variables. fringence and no dichroism.

Results E f f e c t o f M o u n t in g M e d ia

T h ic k n e s s o f t h e S e c t io n The use of a water soluble polar mounting medium required the sections The green birefringence and dichro- to be rehydrated and washed in distilled ism were seen at all examined levels water, resulting in apparent decrease in the intensity of staining of amyloid. Nev t Lemer, CT. ertheless, green birefringence and CONGO RED STAIN 193

dichroism of amyloid and the white-blue with NaCl. Amyloid stains red and shows birefringence of collagen were still green birefringence, dichroism, and red detected. When compared with neutral fluorescence. Neural tissue , 2 colla mounting media, polar media showed no gen, 12 22 exogenous polysaccharide mate difference in birefringence of collagen rial such as vegetable material and cotton and amyloid. fibers4,6 are stained by Congo red and show green birefringence. Eosinophilic granules, 6 elastic fibers, 4,18 and fibrin9 U n s t a in e d S e c t io n s are congophilic with no green birefrin gence. Our results confirm that satura Both unstained amyloid and collagen tion of the staining solution with NaCl is have a white positive birefringence. an important step in the staining tech With the red compensator, both have nique to increase the specificity of the purple-blue color when parallel to the method. The current authors agree with slow axis of the red compensator and Carson and Kingsley2 that with full satu orange-red when perpendicular to it. ration of the staining solution with NaCl, Unstained elastic fibers, smooth mus no false positive staining, green birefrin cles, fibrin, sympathetic and peripheral gence and dichroism was seen and colla nerves did not show birefringence. gen showed white-blue birefringence. Using 50 percent saturation with NaCl introduced false positive staining of elas F luorescence tic fibers, collagen and eosinophilic gran The Congo red powder showed red ules. In addition, collagen showed green fluorescence. Unstained collagen and birefringence and dichroism, and its dis elastic fibers had white-blue fluores tinction from amyloid became impossi cence. Unstained amyloid gave blue flu ble. The addition of salt had been known orescence. Congo red-stained amyloid to have an effect on the staining proper had a bright red fluorescence. Collagen ties of some dyes . 11 The binding of and elastic fibers stained with Congo red Congo red to amyloid occurs by non solution fully saturated with NaCl had ionic bond . 13,17 The ionizing groups of white-blue fluorescence. With the use of amyloid exert a repelling force against 50 percent saturation of NaCl in the the anionic Congo red. This repulsive staining solution, elastic fibers and sepa effect is neutralized by the addition of rate collagen bundles showed red fluo NaCl allowing the linear amyloid mole rescence which was of lesser intensity cule to align itself along the filaments of than Congo red stained amyloid. amyloid. 13,17 It had been suggested that the hydro gen bonding is the major way of linking Discussion amyloid to Congo red . 15 However, dye bath manipulation indicates that a com The use of the Congo red stain is still bination of van der Walls forces, hydro the mainstay for the identification of philic and hydrogen bonding are respon amyloid in tissues. The method most sible for the dye-amyloid binding.5 The commonly used is the alkaline Congo red red fluorescence had been described to described by Puchtler. 16 This method be specific for Congo red stained amy required pretreating tissues with alka loid. 16,17 Unstained collagen and elastic line alcoholic solution saturated with fibers have white-blue fluorescence. NaCl followed by staining in alkaline False red fluorescence of collagen and alcoholic solution of Congo red saturated elastic fibers occurs with false positive 194 ELGHETANY, SALEEM, AND BARR

staining. The intensity of the false red 2. C a r s o n , F. L. and K in g s l e y , W. B.: Non-amy loid green birefringence following Congo red fluorescence was less than with amyloid. staining. Arch. Pathol. Lab. Med. 104:333-335, The red fluorescence may be a function 1980. of the Congo red stain itself since Congo 3. C o h e n , A. S . , S h i r a h a m a , T., S i p e , J . , and SK IN N ER , M.: Amyloid proteins, precursors, red powder had red fluorescence. Wol- mediators and enhancers. Lab. Invest. 48:1-4, man and Bubis24 reported that the green 1983. birefringence of amyloid was only 4. C o o p e r , J. H.: An evaluation of current methods for the diagnostic histochemistry of detectable in tissue sections between amyloid. J. Clin. Pathol. 22:410-413, 1969. five and 1 0 |m. thick. 5. D a v i e s , J. D . and M e r a , S. L.: Congo blue In this study, green birefringence was staining: A histochemical problem. Bristol Med. Chir. J. 98:90, 1983. seen at all levels between four and 1 2 |x 6. D e L e l l i s , R . L . and B o w l i n g , M. C.: The use thick. Increase in the thickness between of Sirius red and Congo red staining in routine four and eight fx was accompanied by histopathology. Hum. Pathol. 2:655, 1970. 7. E anes, E. D. and G lenner, G. G.: X-ray dif increase staining and green birefrin fraction studies on amyloid filaments. J. Histo- gence of small amyloid deposits but chem. Cytochem. 26:673-677, 1968. had no effect on heavy deposits. Sensi 8. F e d e r , N.: Polyvinyl alcohol as an embedding medium for lipid and enzyme histochemistry. J. tivity was not increased with section of Histochem. Cytochem. 70:341-347, 1962. more than eight |x in thickness. The 9. G l e n n e r , G . G . , E a n e s , E . D ., T e r m i n e , eight |x thickness is the one used in our J. D., et al.: The structural characteristics of some proteins having the properties of Congo- lab and gives excellent results. Adding red stained amyloid fibrils. J. Histochem. Cyto the pretreatment step did not have an chem. 22:406, 1973. appreciable effect on the staining of nor 10. G o l d s t e i n , D. J.: Detection of dichroism with mal and amyloid-containing tissues. the microscope. J. Microsc. 89:19-36, 1969. 11. H o r o b i n , R . W. and G o l d s t e i n , D . J .: The Occasional sections showed slight influence of salt on the staining of tissue sections decrease in the intensity of staining of with basis dyes: An investigation into the general applicability of the critical electrolyte theory. amyloid with no effect on green birefrin Histochem. J. 6:599-609, 1974. gence. The use of polar mounting media 12. K l a t s k i n , G.: Non-specific green birefringence had no effect on the birefringence of in Congo red-stained tissues. Am. J. Pathol. 56:1-12, 1969. amyloid and collagen. The disadvantage 13. L e n d r u m , A. C., S l i d d e r , W., and F r a s e r , of water soluble polar medium was that D. S. : Renal hyaline: A study of amyloidosis and the sections were rehydrated with diabetic fibrinous vasculosis with new staining some degree of stain “wash out”. There methods. J. Clin. Pathol. 25:373-396, 1972. 14. L i l l i e , R . D.: Histopathologic Technic and is disagreement between us and Rom- Practical Histochemistry, 3rd ed. New York, hanyi20,21 who reported that collagen McGraw-Hill Company, 1965, Chapter 96, pp. 517-518. birefringence was avoided by the use of 15. Lillie, R. D.: H. J. Conn’s Biological Stains, polar mounting media. 9th ed. Baltimore, The Williams & Wilkins It is our conclusion that selecting the Company, 1977, pp. 147-148. 16. P uC H T L E R , H. and S w e a t , F.: Congo red as a staining solution is a critical step in the stain for fluorescence microscopy of amyloid. J. specific identification of amyloid in tis Histochem. Cytochem. 13:693-694, 1965. sues. Better staining greatly increases 17. P u c h t l e r , H., W a l d r o p , F., and M e l o a n , S.: A review of light, polarization and fluorescence the specificity of green birefringence, microscopic methods for amyloid. Appl. Pathol. dichroism, and red fluorescence. 4:5-17, 1985. 18. P u c h t l e r , H ., Sw e a t , F., and L e v in e , M .: On the binding of Congo red by amyloid. J. Histo References chem. Cytochem. 10:355-364, 1962. 19. R o c h o w , T . G. and R o c h o w , E . G.: An intro 1. B e n n h o l d , H.: Ein spezifische Amyloid-far- duction to microscopy by means of light, elec bung mit Kongort. Mnch. Med. Wschr. trons, x-rays or ultrasound. New York, Plenum 69:1537-1538, 1922. Press, 1978, p. 97. CONGO RED STAIN 195

20. R o m h a n y i , G., D e a k , G., and B u k o v in s z k y , A.: amyloid detection. Am. J. Clin. Pathol. 47:588- Collagen-specific topo-optical staining reaction 593, 1967. with Congo red and its ultrastructural interpre 23. S h e e h a n , D . C. and H r a p c h a k , B . B .: Theory tation. Acta Morphol. Hung. 25:261-282, 1970. and Practice of Histotechnology, 2nd ed. S t. 21. R o m h a n y i, G.: Selective differentiation between Louis, The C. V. Mosby Company, 1980, pp. amyloid and connective tissue structures based 175-178. on collagen specific topo-optical staining reac tion w ith C ongo red . V irchow s A rch. [A] 24. W o l m a n , M. and B u b i s , J. J.: The cause of 354:209-222, 1971. green polarization color of amyloid stained with 22. S A E E D , S . M. and F i n e , G.: Thioflavin-T for Congo red. Histochemistry 4:351-356, 1965.