ANNALS OF CLINICAL AND LABORATORY SCIENCE, Vol. 8, No. 1 Copyright 1978, Institute for Clinical Science Light Microscopical Examination of Glomerular Basement Membrane in Systemic Lupus Erythematosus J. V. KLAVINS, M.D., Ph.D.,*f Z. WESSELY, M.D.f and P. PICKETT* Departments o f Pathology, Queens Hospital Center, Long Island Jewish-Hillside Medical Center Affiliation, Jamaica, NY 11432 and Duke University Medical Center, Durham, NC 27710 ABSTRACT To obtain as much information as possible from light-microscopy about changes in the glomerular basem ent-mem brane, a thin section microtome attachm ent is necessary in order to produce 1 to 3 micron sections. The most informative staining m ethod of paraffin em bedded tissue is the Müller-Mowry-Masson trichrome (M-M-M) combination stain. The deposits, e.g., in systemic lupus erythematosus (SLE), stain red, the basem ent m em brane light orange and the endothelial cell cytoplasm light pink. W ith this stain, as with the Müller-Mowry stain, normal glomeruli show an uninterrupted line of blue acid m ucopolysaccharides along the basem ent m em brane, w here it is joined by foot processes of the epithelial cells. In contrast, in glomeruli from patients with SLE, focal loss of acid mucopolysaccharides is noted. Introduction Light microscopy, w hen utilizing special staining techniques and a thin section microtome attachm ent, can provide significant information about the glomerular basem ent m em brane, e.g., in system ic lupus erythem atosus (SLE). D uring th ese stu d ie s, a new technique the M üller-m owry-m asson trichrom e com bination was developed. * Address for reprint requests: J. V. Klavins, M.D., Ph.D., Department of Pathology, The Catholic Medical Center of Brooklyn-Queens, 88-25 153rd St., Apt. 2-J, Jamaica, NY 11432. Materials and Methods Fifty cases clinically classified as SLE were studied. The paraffin blocks were at first sectioned w ith the conventional m i crotome and approximately five to six micron thick sections w ere stained with hematoxylin and eosin (H&E). These sections includedthe entire tissue sample that was em bedded in paraffin. Areas for more detailed studies were then selected from the preliminary H&E slides and were cut out from the paraffin blocks to provide a field approximately 5 x 5 mm. Such small blocks w ere necessary to obtain uniformly 74
STA IN IN G O F BA SEM EN T M EM BRANE 75 F ig u r e 1. Mü'llerMowry-Masson trichrome stain x 1380. Portion of glomerulus from patient w ith system ic lupus erythematosus. Red de posits can be differenti ated from a light orange basem ent m em brane, blue podocyte area and light pink cytoplasm of endothelial cells. th in sections on su b se q u e n t cu ttin g w ith a th in section attach m ent to th e m icrotom e, according to th e m eth o d by C h u rg an d G rishm an.2 T h ese sections m easu red a p proxim ately 1 to 3 x in thickness. M u l l e r -M o w r y -M a s s o n T r i c h r o m e C o m b i n a t i o n S t a i n (M -M -M ) This was done by first u tilizin g steps 1 th ro u g h 6 o f th e M uller-m ow ry colloidal I I t ( I #1^1 f i f / ^ «r / * ' : S ^ * V % j m r \ -ts.fi»'*«' TV: f* -T }, i n S <a>jv i d * *' *' * \ r 5 v J&! - f> t ' *%» / m. y * i *< ^ F igu re 2. Muller-Mowry colloidal iron stain x 800. Portion of normal glomerulus. Acid mucopoly saccharides are present uninterrupted along the entire basement membrane, where on electron-microscopic examination the foot processes of the epithelial cells join the basement membrane.
76 KLAVINS, W E S SE L Y AND P IC K E T T iron stain7 followed by a modification of the original M asson s trichrom e stain,6 namely: (1) Staining with Ponceau acid fuchsin solution for 5 m inutes; (2) Rinsing in 5 percent water; (3) Placing in 10 percent phosphom olybdic acid for 5 m inutes; (4) Rinsing in acidified water; (5) Staining in light green solution for5 m inutes; (6) Rins ing in acidified water for 3 to 4 m inutes; (7) D ehydrating in 95 percent alcohol, abso lute alcohol and xylol and (8) Mounting. Reag ents 1. Ponceau acid fuchsin solution A. O n e g o fp o n c e a u d e x y le d in e a n d 1 g of acid fuchsin in 100 ml distil led water. B. Exactly 0.5 g of azophloxine G.A. in 100 ml of distilled water. C. Precisely 0.5 ml of acetic acid in 100 ml of distilled water. 2. W orking solution o f Ponceau acid fuchsin A. A d d l0 m lo fs o lu tio n A a n d 2 m lo f solution B to 88 ml of solution C. 3. Light green solution A. One goflight green to 100m lof0.5 percent acetic acid solution. 4. Acidified water A. Exactly 0.5 g of glacial acetic acid to 100 ml of distilled water. In addition, the following staining pro cedures w ere applied: Masson trichrom e stain;6 W ilder s re ticulum stain;5 M uller-m owry colloidal iron m e th o d ;7 P e rio d ic a c id S ch iff technique (PAS);4 F eulgen tech n iq u e;4 and Periodic acid-m ethenam ine-silvermasson trichrom e sequence (PA-MS-M).1 F i g u r e 3. Mtiller-Mowry colloidal iron stain x 1600. Portion of glomerulus from patient with systemic lupus erythematosus. Focal absence of acid mucopolysaccharides over the glomerular basement membrane.
STAINING O F BASEM ENT MEMBRANE 77 Results Cross sections of a normal glomerular basem ent membrane were clearly dem onstrated with all stains as a dense uninterrupted homogeneous line. They were best delineated by staining with m ethenam ine silver. W hen kidney containing wireloops were cut in five or six micron sections, a single thick m em brane form ing the glomerular loops was apparent with H&E and PAS stains. However, w hen the sections w ere cut w ith the th in section attachm ent to the microtome, a clear distinction could be made betw een the basem ent m em brane and the deposits which, when using electron microscopy, appeared as m ore electron-dense osm iophilic m aterial. The deposits appeared less intense than the basem ent membrane although they had the same color. With Masson trichrome stain, the deposits appeared red while the basem ent mem brane appeared light orange. When the PA-MS-M sequence was used, the deposits stained red and the basem ent membrane dark brown or black. In a few instances, the deposits were also F e u lg e n positive. U tilizin g the M-M-M sequence, the basem ent m em brane stained light orange, the area of podocytes blue, the deposits red and the endoth elial cell cytoplasm light pink (figure 1). W hen norm al control kidneys w ere stained with the Muller-Mowry colloidal iron m ethod, an uninterrupted line of acid m ucopolysaccharides was seen b e tw een the basem ent membrane and the epithelium (figure 2). This corresponded to the electronm icroscopic fin d in g s3 which described a layer of colloidal iron positive material that covered the podocyte surface exposed to the urinary space. W hen the Muller-Mowry colloidal iron m ethod was applied to the SLE kidneys, frequent absence of this material was encountered (figure 3). These findings were also present with the M-M-M staining sequence. A pplying the M-M-M sta in in g se quence, the deposits were identified in all three predom inant sites of localization in relationship to the glom erular basem ent membrane: subendothelially, subep ith elially and w ithin the basem ent membrane where the deposits appeared globular or cylindrical in shape. In addition, there was segmental interruption of the deposit by basem ent membrane-like m aterial. O ther changes co n sisted of thickening, fragm entation, splitting or focal loss of basem ent membrane. Discussion T he anatom ical m an ifestatio n s of SLE, p a rticu la rly those of the ren al glomerulus, have been the subject of innum erable studies by light microscopy, electron microscopy and im m unofluorescence. It has b een established by electron m icroscopy and im m unofluorescence that antigen-antibody complexes are deposited along the basem ent m em brane of the glom erular capillary in a characteristic location, either betw een the basem ent m em brane and the endothelium or betw een the basem ent m em brane and the epithelium, w ithin the basem ent membrane, within the mesangium or in any combination of these. These deposits and their location were demonstrated using a thin section microtome attachm ent and the M-M-M staining sequence, which were developed in our laboratories. This technique provided the most useful information. In our cases, as in those described earlier, the preferential location of the deposits occurred in the subendothelial and m esangial areas in active progressive disease. Clinically, inactive lesions w ere more prone to have subepithelial or m e sangial deposits and basem ent membrane thickening.
78 KLAVINS, W ESSELY AND PICKETT The state of acid mucopolysaccharides (AMP) in kidney disease has not been investigated as widely as the other param eters. AMP was present betw een the basem ent membrane and the epithelium in glomeruli of normal control kidneys, and was stained with a M-M-M combination stain. There was spotty absence of AMP from the epithelial side of the basem ent m em brane in some cases of SLE. Our light microscopic findings in the normal control kidneys reflected the electronmicroscopic studies of Jones3 w herein a layer of colloidal iron positive material was described, covering the podocyte surface exposed to the urinary space. To the best of our knowledge, no comparable studies have been done in kidneys of patients afflicted with SLE. From our studies, it is not apparent w hether the absence of histochem ically dem onstrable AMP in d i cates loss of foot processes or some other alteration in ultrastructurally visible sites of the epithelial cells. References 1. BURKHOLDER, P. M.: Atlas of Human Glomerular Pathology. New York, Harperand Row, 1974. 2. Ch u r g, J. and G rishm an, E.: Application of thin sections to the problems of renal pathology. J. Mt. Sinai Hosp. 24:736-744, 1957. 3. J o n e s, D. B.: Mucous substance of the glomerulus. Lab. Invest. 21:119-125, 1969. 4. LlLLIE, R. D.: Histopathologic Technique and Practical Histochemistry, 3rd ed. New York, McGraw-Hill Book Co., 1965. 5. M a n u a l o f H is t o l o g ic St a in in g METHODS OF THE AFIP, 3rd ed. American Registry of Pathology. New York, McGraw- Hill Book Co., 1968. 6. MASSON, P.: Some histological methods; trichrome staining and their preliminary technique. J. Techn. Methods 12:75-90, 1929. 7. M o w r y, R. W.: Improved procedure for the staining of acidic polysaccharides by Muller s colloidal (hydrous) ferric oxide and its combination with the Feulgen and the periodic acid Schiff reactions. Lab. Invest. 7:566-576, 1958.