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

Abstract

Aim: To describe the technique of immunofluorescence on paraffin embedded tissue sections and discuss the potential pitfalls with an in depth review of literature.

Methods: Immunofluorescence 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.

Results: Over 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. Non-diagnostic 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). Comparing cases with both routine IF and IF-P, 35 of 37 showed either equal intensity or a minor difference in intensity of staining (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.

Conclusion: As a "salvage" technique, immunofluorescence on paraffin embedded renal biopsies is of great diagnostic utility, however not without pitfalls.

Keywords: Enzymatic digestion; Immunofluorescence on paraffin section; Proteinase K; Renal biopsy; Salvage technique.

Figure 1

Examples of technically adequate and inadequate digestion. A: Immunofluorescence staining on a paraffin embedded tissue section in a case of diffuse proliferative lupus nephritis after enzymatic digestion with proteinase K. Note the adequate digestion evidenced by disappearance of tubular epithelial cells (arrow) (FITC IgG, × 200); B: Immunofluorescence staining in a case with inadequate digestion with visible tubular epithelial cells (arrow). Note the antibody sticking to the surface of the capillary wall (FITC IgG, × 200). FITC: Fluorescein isothiocyanate.

Figure 2

Comparable staining pattern on immunofluorescence on frozen and paraffin embedded tissue. A case of dense deposit disease showing bright C3c deposition 3+ (0-3+ scale) on IF-F (A, FITC C3c, × 200). Note the comparative coarse granular capillary wall staining of C3c (3+) on paraffin embedded tissue section after enzymatic retrieval (B, FITC C3c, × 200). FITC: Fluorescein isothiocyanate; IF-F: Immunofluorescence on fresh frozen tissue.

Figure 3

Glomerulonephritis diagnosed on immunofluorescence on paraffin. A: IgA nephropathy: There is predominantly mesangial deposition of IgA (FITC IgA × 100); B: Class IV or diffuse lupus nephritis: Immunofluorescence reveals coarsely granular deposition of immunoglobulins in both mesangium and in the peripheral capillary wall (FITC IgG × 200); C: Membranous nephropathy: Immunofluorescence reveals fine granular capillary wall deposition of IgG (C, FITC IgG × 200); D: Post infectious glomerulonephritis: Garland pattern with elongated peripheral loop deposits is depicted, along with occasional mesangial deposits (D, FITC C3c, × 200); E: C1q nephropathy with mesangial deposition of C1q (FITC C1q × 100); F: Diabetic nephropathy with linear accentuation of glomerular capillary wall and tubular basement membrane (FITC IgG × 200). FITC: Fluorescein isothiocyanate.

Figure 4

Immunofluorescence on paraffin to demonstrate monoclonal deposits. A case of light chain deposition disease with kappa light chain restriction. There is nodular mesangial, capillary wall and tubular basement membrane deposition of kappa light chain (A, FITC kappa, × 100) while no deposition of lambda is noted (B, FITC lambda, × 200); C: A case of primary amyloidosis with lambda light chain restriction. The lambda deposition is noted in the mesangium (FITC lambda × 200); D: There is no deposition of kappa (D, FITC kappa × 200); E: A case of cast nephropathy with kappa light chain restriction. Note the brightly positive casts for kappa (FITC kappa × 200) with no traces of lambda (F, FITC lambda × 200). FITC: Fluorescein isothiocyanate.