J. clin. Path., 1972, 25, 373-396 J Clin Pathol: first published as 10.1136/jcp.25.5.373 on 1 May 1972. Downloaded from Renal hyalin A study of amyloidosis and diabetic fibrinous vasculosis with new staining methods A. C. LENDRUM, W. SLIDDERS, AND D. S. FRASER From the University Department ofPathology, Royal Infirmary, Dundee SYNOPSIS This describes the sodium sulphate-Alcian Blue (SAB) method for staining amyloid in paraffin sections. Its value lies in the possibility of subsequent counterstaining and thus of revealing the structural relationships of amyloid. In the kidney the topical disposition of amyloid closely resembles the disposition of fibrin in the kidney of diabetics; this suggests that upset in vascular permeability plays a part in determining the site of the amyloid deposits. Furthermore, an aging process in amyloid can now be envisaged resembling the aging of extraluminal fibrin. Both materials proceed to a hyalin material that, staining like collagen, merits the name pseudo-collagen. This term we apply to a hyalin, staining like collagen, for which,we can postulate a specific precursor. copyright. The I ight microscopist has generally accepted Another study meanwhile captured our attention. amyloid as a hyalin substance, an acellular firm gel, This, the formulation of new trichromic methods for situated interstitially, and distinguished from other staining fibrin (Lendrum, Fraser, Slidders, and hyalins by particular staining reactions, notably the Henderson, 1962; Lendrum, Fraser, and Slidders, metachromatic reaction with methyl violet and an 1964; Slidders, 1968), led to the discovery that these http://jcp.bmj.com/ affinity for Congo red. Neither of these methods is methods could graphically demonstrate and elucidate ideal for the study of the structural relationships of changes in the diabetic kidney and in many other amyloid. vascular lesions (Lendrum, 1963). On turning our Our attempts to demonstrate and study amyloid, attention again to amyloid, one of us (W.S.) found as seen in postmortem material, were progressing a way of using the new, and still current, Alcian unprofitably when suddenly one divagation revealed Blue. This we could now complement with new and A evolved apt counterstains derived from our studies on possibilities. rationally although perhaps on September 25, 2021 by guest. Protected inaccurate idea led one of us (W.S.) to produce a fibrin staining; and the changes seen in the diabetic modified Alcian Blue solution. This enabled us to kidney proved an illuminating counterpart to what stain amyloid, in sections from paraffin, with a dye we were now finding in the amyloid kidney. that could be stabilized in situ and thus allow a variety Our present purpose is to describe the new of counterstainings. Although, as with most other methods, and the facts made manifest in the amyloid dye-staining methods, the results were not chemically kidney. This study with the light microscope specific for amyloid, with suitable counterstaining revealed an unexpected similarity with the diabetic they proved sufficiently selective to allow study of the kidney not only in the topical disposition of the two precise situation of the amyloid deposits. Unfor- deposits but also in the indication that amyloid, like tunately the dye makers, in 1955, changed the interstitially deposited fibrin, undergoes changes on constitution of the dye, and our results altered aging and loses its characteristic staining reactions, miserably. becoming just anonymous hyalin. When obviously Please address requests for reprints to: Professor Lendrum, Depart- derived from fibrin we called this material pseudo- ment of Pathology, The University, Dundee. collagen (Lendrum, 1961; Lendrum et al, 1962, Received for publication 13 January 1972. 1964) because with our trichromic methods it stains 373 J Clin Pathol: first published as 10.1136/jcp.25.5.373 on 1 May 1972. Downloaded from .374 A. C. Lenidrli,m, W. Slidders, Ci,id D. S. Fraser as collagen, and similarly, we now believe that molecule the properties of a cationic (basic) dye. On amyloid also can become a pseudo-collagen. textiles Alcian Blue is fixed and rendered fast by At a time when world-wide intent is converging on after-treatment with alkali; this, by causing the the experimental production of amyloid, and on its solubilizing groups to decompose, deposits the strikingly profitable study by the electron micro- copper phthalocyanine pigment within the fibre. scope (Mandema, Ruinen, Scholten, and Cohen, The earlier and somewhat different dye, Alcian 1968), it is worthy of note that at this same sym- Blue 8GS, was the form of Alcian Blue introduced by posium Cohen (1968), summing up a day's pro- Steedman (1950) as a stain for mucin. The fact that ceedings said, 'One must return to more classical both mucin and amyloid are revealed by metachro- light-microscopic studies to give the proper per- matic dyes first prompted us to test Alcian Blue 8GS spective'. We are prompted to offer our observations as a stain for amyloid. on the grounds that the pathologist, particularly, is Our investigations soon revealed that Alcian likely to maintain his interest in both the wood Blue 8GS in aqueous-acetic solution had little and the trees. tendency to colour amyloid in sections from paraffin and efforts were directed to methods of activation. PART I Eventually, one of us (W.S.) by adding sodium sulphate to the usual aqueous-acetic solution of the The Staining of Amyloid dye found a distinctly adjuvant effect. The pro- duction of Alcian Blue 8GS was discontinued around I I THEORETICAL 1955 and thereafter Alcian Blue 8GX became the The demonstration of amvloid has for the light standard product, although several other Alcian microscopist always been visually unsatisfactory. Blues, two Alcian Greens, and an Alcian Yellow Of the generally used methods the metachromatic were also made available during this time. stain may show good colour contrast but gives only When Alcian Blue 8GS was replaced by Alcian poor definition. Staining with Congo red is often Blue 8GX a marked deterioration in our results feeble, and, even at their best, neither of these diverted our interests away from amyloid. On our classical methods nor the new fluorochrome method return, W.S. found that sodium sulphate-in'ducedcopyright. permits subsequent counterstaining of a type to staining of amyloid could be obtained with the new demonstrate the intimate relationship of amyloid dye if the solvent contained alcohol; unfortunately with the stromal tissue. this brought about a loss of selectivity regained, Given well fixed tissue a trichrome of the Mallory- however, by increasing the acetic acid content of the Masson family can show in the kidney the charac- stain. The present version of the method was men- teristic strong blue of collagen and of basement tioned and illustrated but not detailed in 1963http://jcp.bmj.com/ membrane, contrasting moderately well at high (Lendrum). power with the pale grey-blue of the amyloid The original, apparently irrational manoeuvre of substance (Figs. 1, 2, and 6, and colour Fig. 14). adding sodium sulphate to Alcian Blue was prompted Some use van Gieson in a similar fashion, the pale by an appreciation, albeit imperfect, of the factors buff colour of amyloid contrasting with the fuchsino- involved in the dyeing of textile fibres. phil collagen. These rather negative methods of The staining methods commonly used by histolo- offered the nearest to a carto- gists are regarded as comparable with the dyeing of staining approach on September 25, 2021 by guest. Protected graphic method to show amyloid's relation to the wool and thus as depending primarily on the forma- kidney's preexistent structure. Clearly, however, tion of salt-like linkages between cationic (basic) or something better is needed, some way of obtaining anionic (acid) dyes and, respectively, ionized acidic a positive staining of amyloid, and of fixing the stain (eg, carboxyl) or basic (eg, amino) groups in the so that counterstaining of the preexistent structures tissues. can be added without ousting or degrading the colour On the other hand, in the dyeing of cotton with the fixed in the amyloid. Such a method is the sodium so-called 'direct' dyes (large-molecule anionic dyes sulphate-Alcian Blue (SAB) method described by used without mordants) interaction of the dye and Lendrum, Slidders, and Fraser (1969) and repro- fibre occurs despite the fact that the only available duced below. ionizing groups in cellulose (hydroxyl) are themselves Alcian Blue (Alcian Blue 8GX, Colour Index anionic, although weak. Attachment of the dye in designation Ingrain Blue 11) is prepared by Messrs this instance is considered by some to depend on ICI from their copper phthalocyanine pigment hydrogen-bond formation. Bonding of this sort could Monastral Fast Blue B by the introduction of certain occur between, for example, the hydrogen atom of novel solubilizing groups; these confer on the the hydroxyl groups in the fibre and the nitrogen 'See part I v, Identification Of dVeSttlfs (the coloUr index). atom of an azo group in the dye. J Clin Pathol: first published as 10.1136/jcp.25.5.373 on 1 May 1972. Downloaded from Renal hyalin 375 To initiate this non-ionic bonding the dyer instance ionizing groups in the amyloid material itself generally has to add sodium sulphate or chloride to exert a repelling force on the dye ions, in the first the dyebath, the salt being thought to act by annul- instance against cationic Alcian Blue, and in the ling the antagonistic ionic forces, thereby permitting second against anionic Congo red; and it may be that the close contact, of dye and fibre, essential for these mutual ionic repulsions are annulled by the hydrogen bonding. salt in the solution, thereby encouraging non-ionic Even in the dyeing of wool, when using anionic dye binding to take place.