Periodic Acid-Schiff-Light Green Stain to Detect Glomerular Protein Deposits by Routine Light Microscopy

Transcription

1 Periodic Acid-Schiff-Light Green Stain to Detect Glomerular Protein Deposits by Routine Light Microscopy CHARLES N. GAMBLE, M.D. Department of Pathology, Sutter Memorial Hospital, Sacramento, California ABSTRACT Gamble, Charles N.: Periodic acid-schiff-iight green stain to detect glomerular protein deposits by routine light microscopy. Am J Clin Pathol 63: , A simple, easily reproducible periodic acid-schifflight green stain (PAS-LG) for the detection of glomerular protein deposits by routine light microscopy is described. The deposits are selectively stained a deep blue and contrast sharply with the staining of adjacent glomerular structures. Correlation with immunofluorescent and electron microscopy has shown that it is possible with this stain to categorize accurately a large variety of glomerular lesions by light microscopy alone. (Key words: Periodic acid-schiff-light green stain; Renal biopsy; Glomerular protein deposits; Immune-complex deposits; Human glomerulonephritis.) DURING THE PAST DECADE, percutaneous needle biopsy has become an increasingly important and relatively common procedure for the diagnosis of renal disease. Its widespread use is largely due to an increased knowledge of the immunopathogenesis of human glomerulonephritis, which has resulted from correlation of detailed immunofluorescent and electron microscopic studies of human biopsy material with the experimental work of Dixon and others. 2-4,9 These studies have shown that the deposition of circulating antigen-antibody or immune complexes within glomeruli is the most common cause of glomerular injury. 6,8a2 The most complete understanding of immune complex-induced glomerular lesions is afforded by a combination of light, immuno- Received June 26, 1974; revised August 8, 1974; accepted for publication August 8, Address reprint requests to Dr. Gamble. 310 fluorescent, and electron microscopy. However, facilities for the latter two studies are not readily available in many institutions. Because of this, it was felt important to attempt to find a simple and easily reproducible histologic stain for the detection of protein (immune complex) deposits in routinely processed paraffinembedded sections of kidney tissue. Many special stains have been used in the past in attempts to better define various types of glomerular lesions in both biopsy and autopsy specimens. 1 ' 5,7,11,13-15 While many of these stains do assist in better defining the glomerular lesions in various types of glomerulonephritis, none has proven entirely satisfactory for the detection of protein deposits in immune complex-induced glomerulonephritis. This is primarily due to a lack of selective staining of the various components of the affected glomeruli or to a lack of easily reproducible selective staining.

2 March 1975 PAS-LG STAIN FOR GLOMERULAR PROTEIN DEPOSITS 311 Early in our investigation of different histologic stains for the detection of glomerular protein deposits, we tried the periodic acid-schiff stain counterstained with light green SF yellowish (PAS-LG). 10 With this combination of stains, numerous distinct blue granules were visible in the cytoplasm of proximal convoluted tubular epithelial cells in sections of renal biopsies from patients with the nephrotic syndrome. The blue-staining granules corresponded in size, number and distribution to the brightly fluorescent protein resorption droplets in frozen sections of the biopsy specimens stained with fluoresceinconjugated (FITC) antihuman albumin. The PAS-LG stain was then used to stain thin paraffin-embedded sections of selected renal biopsy specimens showing a variety of glomerular lesions. Comparison of these sections with frozen sections of portions of the biopsy specimens stained with FITC antihuman immunoglobulins and with FITC antihuman &C (C3) revealed an identical pattern of granular glomerular staining that was deep blue in the PAS-LG-stained sections and brightly fluorescent in the FITC antisera-stained sections (Figs. 1, 2, and 3). The deep blue staining of the glomerular protein deposits in the PAS-LG-stained sections contrasted sharply with the magenta-staining glomerular capillary basement membranes and mesangial sponge or stalk fibers and the pale blue-green staining of glomerular cell cytoplasm and nuclei. It appeared from these observations that proteins in both resorption droplets within proximal convoluted tubular epithelial cells and immune-complex deposits within glomeruli were selectively stained a distinct deep blue color as a result of the combination of basic fuchsin and light green SF under the conditions of the staining procedure. To demonstrate this further, serial sections of several renal biopsy specimens were stained with the periodic acid- Schiff stain alone and the light green SF stain alone. In sections stained with PAS alone, the protein deposits stained a distinct fuchsia and were difficult to distinguish from adjacent normal glomerular structures, which stained a similar color. In the light green SF-stained sections, the deposits were medium blue-green and again were difficult to separate from similarly-staining normal structures. Sections stained with a combination of the stains revealed the characteristic deep blue staining of the deposits in a location identical to that found in companion frozen sections stained with appropriate FITC antisera. Following these initial studies, the PAS-LG stain was used as a routine stain in the examination of 360 subsequent renal biopsy specimens studied by thin-section light, immunofluorescent, and electron microscopy. The glomerular staining patterns observed in the PAS- LG-stained sections correlated extremely well with the glomerular findings obtained by both immunofluorescent and electron microscopy in cases of immune complexinduced glomerulonephritis. This correlation was especially striking between the PAS-LG-stained sections and FITC antisera-stained sections where the glomerular distributions of granular blue staining and fluorescent protein deposits were identical. Materials and Methods Solutions Sensitized Schiff Reagent: Basic fuchsin (C.I ) 1 Gm. Sodium metabisulfite 2Gm. 1 N hydrochloric acid 20 ml. Deionized water 80 ml. Shake the solution at intervals over a minute period until it is clear yellowbrown with no residue. Add 0.6 Gm. activated charcoal, shake for 2 minutes, and filter through coarse filter paper until the solution is colorless. The final ph of the solution should be The reagent may be used for two to three weeks if

3 312 A.J.C.P. Vol. 63 GAMBLE # 0 J* %» «* '*. -. * «* «. <"»* FIG. 1. IgA-IgG nephropathy with mesangial deposits. /J (top): hematoxylin-eosin-stained section shows only a slight increase in mesangial cells associated with a slight increase in mesangial volume (X285). B (middle): PAS-LG-stained section reveals numerous closely packed (deep blue-staining) protein deposits within mesangial and mesangiocapillary junction areas (X285). C (bottom): FITC antihuman IgA-stained section shows numerous IgA-containing deposits in an identical location (x285). *' *

4 March 1975 PAS-LG STAIN FOR GLOMERULAR PROTEIN DEPOSITS F I G. 2. Hypocomplementemic membranoproiiferative glomerulonephritis with basement membranedense deposits. A (top): hematoxylin-eosin-stained section shows early accentuation of lobules, variable mesangial cell proliferation, an increase in mesangial volume, and irregular thickening of capillary walls (x285). B (middle): PAS-LG-stained section reveals scattered coarse (deep bluestaining) protein deposits in expanded mesangial areas in association with prominent (deep bluestaining) pseudolinear subendothelial deposits involving the peripheral capillary walls (x285). C (bottom): FITC antihuman /3,C (C3)-stained section reveals numerous brightly fluorescent C3-containing deposits in identical locations (x285). 313

5 314 GAMBLE A.J.C.P. Vol. 63 stored at 4 C. in a brown bottle. Approximately halfway through this study, we found that Harleco Schiff reagent, ph 1.6 (Hartman-Leddon Company, Philadelphia, PA), worked extremely well, and since that time we have used this Schiff reagent exclusively to insure standardization of the reagent and to save preparation time. Stock Light Green Solution: Light green SF, yellowish (C.I. 670) 0.2 Gm. Deionized water 100 ml. Glacial acetic acid 0.2 ml. Working Light Green Solution: For use, a 0.05% solution is made by adding 10 ml. of the stock solution to 40 ml. of deionized water. The final ph of the solution should be approximately 3.6. Method Fix specimen in Helly's solution for 30 minutes to one hour. Wash specimen for one hour in running tap water and place in 10% neutral buffered formalin for a minimum of two hours. Process in the usual manner and embed in paraffin. Cut sections 2 to 3 micra in thickness, deparaffinize, and pass through graded alcohols to water. Treat with Lugol's iodine solution for 7 minutes, wash in water for 6 minutes, and then place in 5% sodium thiosulfate for 3 minutes and again wash in water for 6 minutes. (1) Place sections in 0.5% periodic acid solution for 5 minutes and wash in five changes of deionized water. (2) Place in Schiff reagent for 15 minutes and wash in running tap water for 10 minutes to develop a fuchsia color. (3) Dip slides, one at a time, in 0.05% light green SF solution until sections turn green (40 to 50 dips). (4) Differentiate in 95% alcohol until sections become a pale blue-green (4 to 5 dips). (5) Dehydrate through 95% alcohol and two changes of absolute alcohol and check stain under the microscope. The background should be pale blue-green. If necessary, the sections can be returned through 95% alcohol to deionized water. If the sections are too dark, water will remove excess light green stain or, if they are too pale, the light green SF stain can be repeated until a proper background stain is obtained. (6) When differentiation is complete, dehydrate slides through a third change of absolute alcohol for 2 minutes, clear in three changes of xylene, and mount in Permount. Results (1) Glomerular immune complex deposits deep blue. (2) Basement membrane magenta. (3) Mesangial sponge fibers (matrix) magenta. (4) Cell cytoplasm, nuclei and nucleoli pale blue-green. (5) Erythrocytes medium bluegreen. (6) Tubular protein resorption droplets deep blue. (7) Tubular casts blue-green to reddish-blue depending on CHO content. (8) Fibrin deep blue. (9) "Hyaline" deep blue to reddishblue. (10) Amyloid light magenta to bluishred. Discussion Although we have used 10% formalinfixed specimens with some success, the use of Helly's fixed tissue results in much clearer definition of glomerular protein deposits. Helly's fixation also results in much sharper histologic and cytologic detail with the hematoxylin and eosin, AB-PAS, and PASM stains that are routinely used to stain renal biopsy specimens in this laboratory. The sections used for the PASM stain, however, require additional mordanting in 10% formalin prior to staining. The Helly's fixative that

6 March 1975 PAS-LG STAIN FOR GLOMERULAR PROTEIN DEPOSITS *»»> ^ «* FIG. 3. Focal sclerosing glomerulopathy. A (top): hematoxylin-eosinstained section shows prominent segmental sclerosis of approximately half of the glomerulus (x285). B (middle): PAS-LG-stained section reveals prominent (deep blue-staining) homogeneous and granular subendothelial protein deposits involving the capillary walls in the area of segmental sclerosis (x285). C (bottom): FITC antihuman /3,C (C3)stained section reveals prominent staining for C3 in an identical location (x285). * * 315

7 316 GAMBLE A.J.C.P. Vol. 63 we use is made by adding 1 ml. of 100% formalin to 20 ml. of Zenker base (mercuric chloride, 50 Cm., potassium chloride, 25 Gm., sodium sulfate, 10 Gm., deionized water, 1,000 ml.). Although Helly's fixative penetrates tissue slowly, this does not present a problem with percutaneous needle biopsy specimens, which average only 1 to 1.5 mm. in diameter. Open renal biopsy specimens and autopsy specimens should be cut no thicker than 1.5 mm. to allow adequate penetration of fixative in the suggested time. It is important to cut the sections at 2 to 3 micra to obtain the best results. Thicker sections (4 to 6 micra) result in superimposition of glomerular structures and too intense staining, both of which obscure fine detail. A nuclear stain should not be used, since nuclear staining does not contribute additional information and, when attempted by us, has tended to obscure finer tinctorial detail. The deep blue staining of the glomerular protein deposits with this stain is a result of the combined staining of protein with both basic fuchsin and light green SF. Proteins other than the immunoglobulin and complement components of immune complexes also stain a similar color. In most instances, this does not present a problem in interpretation because much of the similarly-staining material is located in specific extraglomerular sites such as protein resorption droplets within the cytoplasm of proximal convoluted tubular epithelial cells, casts within the tubular lumina, and "hyaline" within the walls of small arteries and arterioles. With regard to similar staining within the glomeruli, "hyaline" droplets in the glomerular epithelial cells are quite easily distinguished from immune-complex deposits, because of their characteristic location within the cytoplasm of hypertrophied epithelial cells and their relatively large size, considerably larger than even aggregated deposits. Fibrin, which may be present in association with immune complexes in areas of severe glomerular injury, is impossible to distinguish from the protein components of the deposits. This is not felt to be important in the proper interpretation of immune complex-induced glomerular lesions. Fibrin and other serum proteins in exudative glomerular lesions that are not related to the deposition of immune complexes present a greater problem. However, the staining of glomerular exudative cap lesions, such as occur in diabetes mellitus and accelerated hypertension, usually differs sufficiently so that a distinction can be made. In many of the exudative glomerular lesions, exuded fibrin and other serum proteins are located within the concavities of the endothelial side of the glomerular capillary loops and are very smooth in appearance. In many instances, discrete vacuoles are visible within the blue-staining homogeneous material. This contrasts with the granular character of the immune-complex deposits, which are within the glomerular capillary walls, overlying their epithelial convexities, or are present within mesangial areas. Glomerular amyloid, even in early cases of primary nephrotic amyloidosis, can be distinguished from immune-complex deposits by the light magenta to bluish-red color of the amyloid and its characteristically striated appearance. Diabetic intercapillary glomerulosclerosis, especially the early nodular and the diffuse forms, can also be distinguished on the basis of the distinct magenta staining of the increased intercapillary material. Acknowledgment. Mrs. Rebecca LeFevre, Mrs. Shirley Harnar, and Mr. Harry Low provided technical assistance. References 1. Churg J, Prado A: A rapid Mallory trichrome stain (chromatrope-aniline blue). Arch Pathol 62: , 1956

8 March 1975 PAS-LG STAIN FOR GLOMERULAR PROTEIN DEPOSITS Dixon FJ: Glomerulonephritis and immunopathology. Hosp Practice 2:35-43, Dixon FJ: The pathogenesis of immunologically induced nephritis. Third Int Congr Nephrol, Washington, D.C. Volume 2. Basel, Karger, 1967, pp Dixon FJ: The pathogenicity of antigen-antibody complexes, Pathology Annual. Edited by SC Sommers. New York, Appleton-Century- Crofts, 1969, pp Ehrenreich T, Espinosa T: Chromatrope silver methenamine stain of glomerular lesions. Am J Clin Pathol 56: , Germuth FG, Rodriguez E: Immunopathology of the Renal Glomerulus: Immune Complex Deposit and Antibasement Membrane Disease. Boston, Little, Brown, 1973, pp Jones DB: Glomerulonephritis. Am J Pathol 29:35-51, Kincaid-Smith P, Mathew TH, Becker RL (editors): Glomerulonephritis: Morphology, Natural History and Treatment. Parts I and II. New York, John Wiley and Sons, 1973, pp Lerner RA, Glassock RJ, Dixon FJ: The role of antiglomerular basement membrane antibody in the pathogenesis of human glomerulonephritis. J Exp Med 126: , Luna LG (editor): Manual of Histologic Staining Methods of the Armed Forces Institute of Pathology. Third edition. New York, McGraw Hill, 1968, pp Masson PJ: Trichrome stainings and their preliminary technique. J Techn Methods 12: 75-90, McCluskey RT, Klassen J: Immunologically mediated glomerular, tubular and interstitial renal disease. N Engl J Med 288: , McManusJFA: The periodic acid routine applied to the kidney. Am J Pathol 24: , McManus JFA, Mowry RW: Staining Methods, Histologic and Histochemical. New York, Hoeber, 1960, pp Movat HZ, More RH: The nature and origin of fibrinoid. Am J Pathol 28: , 1957