Immunofluorescence on paraffin embedded renal biopsies: Experience of a tertiary care center with review of literature

Abstract

AIMTo describe the technique of immunofluorescence on paraffin embedded tissue sections and discuss the potential pitfalls with an in depth review of literature. METHODSImmunofluorescence is integral to diagnostic renal pathology. Immunofluorescence on paraffin embedded renal biopsies (IF-P) after enzyme treatment has been described in literature, however has not found widespread use in renal pathology laboratories. In our laboratory proteinase K digestion of paraffin embedded renal biopsy material was standardized and applied prospectively in cases where immunofluorescence on fresh frozen tissue was non contributory or not possible. Diagnostic utility was assessed and in a cohort of cases comparison of intensity of staining with routine immunofluorescence was performed. RESULTSOver the 5-year study period, of the 3141 renal biopsies received IF-P was performed on 246 cases (7.7%) and was interpretable with optimal digestion in 214 cases (6.8%). It was of diagnostic utility in the majority of cases, which predominantly included glomerular disease. Nondiagnostic IF-P was found in membranous nephropathy (2 of 11 cases), membranoproliferative glomerulonephritis (2 of 32 cases), lupus nephritis (1 of 25 cases), post infectious glomerulonephritis (1 of 11 cases) and chronic glomerulonephritis (3 of 8 cases Observational Study(1+) for the diagnostic immunoglobulin/complement. Technically assessment of immunofluorescence on the paraffin embedded tissue was found to be easier with clearly observed morphology, however a false positive staining pattern was observed in under-digested tissue. CONCLUSIONAs a "salvage" technique, immunofluorescence on paraffin embedded renal biopsies is of great diagnostic utility, however not without pitfalls.

Paraffin Immunofluorescence: A Valuable Ancillary Technique in Renal Pathology

“… 20 , 22 We have also observed good results in other paraprotein-associated glomerulopathies, including immunotactoid glomerulonephritis, type 1 cryoglobulinemic glomerulonephritis ( Supplementary Figure S1 ), and proliferative glomerulonephritis with monoclonal Ig deposits ( Figure 1 e). Good results can also be obtained in most immune-complex–mediated proliferative glomerulonephritides, including lupus nephritis (96%–100%) 14 , 17 , 18 , 21 ( Figure 1 b), IgA nephropathy (88%) 14 ( Figure 1 a), cryoglobulinemic glomerulonephritis types II and III (100%) 14 ( Figure 1 d), C1q nephropathy, 21 and fibrillary glomerulonephritis (100%) 14 ( Figure 1 c; Table 2 ). IF-P has a lower sensitivity than IF-F in primary membranous nephropathy (50%–78%), 14 , 21 C3 glomerulonephritis, 20 , 21 and anti–glomerular basement membrane (GBM) nephritis.…”

“…Good results can also be obtained in most immune-complex–mediated proliferative glomerulonephritides, including lupus nephritis (96%–100%) 14 , 17 , 18 , 21 ( Figure 1 b), IgA nephropathy (88%) 14 ( Figure 1 a), cryoglobulinemic glomerulonephritis types II and III (100%) 14 ( Figure 1 d), C1q nephropathy, 21 and fibrillary glomerulonephritis (100%) 14 ( Figure 1 c; Table 2 ). IF-P has a lower sensitivity than IF-F in primary membranous nephropathy (50%–78%), 14 , 21 C3 glomerulonephritis, 20 , 21 and anti–glomerular basement membrane (GBM) nephritis. 14 , 20 In our laboratory, we use a case of diffuse proliferative lupus nephritis as our positive control for IF-P. (iii) The antigen tested: Good results can be obtained for IgG, kappa, and lambda (aside from cases of primary membranous nephropathy and anti-GBM nephritis), IgA, IgM, and C1q.…”

“…Because formalin fixation induces protein cross-linking, which generally blocks antigenicity, an antigen-retrieval step that allows for increased penetration of antibodies to the antigens “masked” by formalin fixation is required in IF-P. 5 , 6 This step involves incubating the paraffin sections with a proteolytic enzyme or heating the sections before incubation with fluorescein isothiocyanate–conjugated antibodies against Igs and complement components. Multiple proteolytic enzymes have been used in IF-P, including trypsin, 7 , 8 , 9 , 10 , 11 , 12 pronase E (protease XIV), 13 , 14 , 15 , 16 , 17 proteinase XXIV, 18 and proteinase K. 13 , 19 , 20 , 21 , 22 Successful results were also obtained by heat treatment with Tris or citrate buffers 15 and with dual microwave heating in EDTA antigen-retrieval solution. 23 In our laboratory, we use the pronase technique, which was originally described by Fogazzi et al.…”

“…Studies performed as early as the 1970s have shown that IF-P is a valuable salvage technique in renal pathology when frozen tissue is inadequate (e.g., lacks glomeruli) or not available. 8 , 10 , 14 , 18 , 20 , 21 Overall, diagnostic results by IF-P can be obtained in >80% of cases, 14 , 18 , 20 , 21 but the diagnostic yield varies depending on 3 factors: (i) The antigen-retrieval method used: proteinase K, pronase, proteinase XXIV, and dual microwave heating appear to be more sensitive than the other methodologies, although a systematic study comparing the diagnostic yield of these methods side by side on the same cohort of cases has yet to be performed. (ii) The disease type: the best results are obtained in dysproteinemia-associated kidney diseases ( Table 2 ).…”

“… 37 (vi) The various IF-P techniques described are based on formalin fixation. 4 , 9 , 14 , 15 , 20 , 21 The performance characteristics of IF-P on non–formalin-based fixatives (Bouin, Duboscq-Brasil, and other fixatives) used in approximately 25% of renal pathology laboratories 38 , 39 are not known.…”