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Unmasking of complements using proteinase-K in formalin fixed paraffin embedded renal biopsies
1Department of Histopathology, Post Graduate Institute of Medical Education and Research, Chandigarh, India.
Insights
Standardizing direct immunofluorescence (DIF) on formalin-fixed, paraffin-embedded (FFPE) renal biopsies using proteinase-K improves complement staining. This technique aids in diagnosing various glomerulonephritis types, even in long-archived autopsy samples.
Area of Science:
- Nephrology
- Pathology
- Immunofluorescence
Background:
- Renal biopsy interpretation traditionally relies on histopathology, direct immunofluorescence (DIF), and electron microscopy.
- Standard DIF requires fresh or frozen tissue, which is not always available.
- Complement staining in formalin-fixed, paraffin-embedded (FFPE) tissues using DIF has been historically unsatisfactory.
Purpose of the Study:
- To standardize DIF staining for complement components (C3 and C1q) in FFPE renal biopsies using proteinase-K for antigen retrieval.
- To evaluate the efficacy of this standardized technique in diagnosing various glomerulonephritis (GN) subtypes, including immune-complex and complement-mediated diseases.
- To assess the utility of this method on archived autopsy tissues.
Main Methods:
- Standardization of DIF protocol using proteinase-K for antigen retrieval on FFPE renal biopsy tissues.
- Pilot study on known cases of membranous glomerulonephritis (MGN), membranoproliferative glomerulonephritis type-1 (MPGN-1), immunoglobulin A nephropathy (IgAN), and anti-glomerular basement disease (anti-GBM).
- Application of the standardized technique to 75 renal biopsies and 43 autopsy cases, utilizing an immunofluorescence panel including IgG, IgA, IgM, C3, C1q, kappa, lambda, and fibrinogen.
Main Results:
- The standardized proteinase-K-based DIF method successfully unmasked complement deposition (C3 and C1q) in FFPE tissues.
- In autopsy cases (n=43), immune-complex GN was confirmed in 18, complement-mediated dense deposit disease (DDD) in 1, and monoclonal diseases in 4.
- In renal biopsies (n=75) lacking frozen samples, the technique classified 52 cases with mesangiocapillary patterns as MPGN type-1 (46), DDD (2), and C3GN (4).
- The method demonstrated good results on autopsy tissues archived for up to 15 years.
Conclusions:
- Proteinase-K enzymatic digestion is effective for antigen retrieval in FFPE renal biopsies, enabling satisfactory complement staining via DIF.
- This standardized DIF technique is valuable for diagnosing immune-complex and complement-mediated glomerulonephritis, particularly when frozen tissue is unavailable.
- The method shows promise for retrospective studies using archived renal tissues, extending diagnostic capabilities in nephropathology.
Abstract:
Renal biopsy interpretation requires histopathology, direct immunofluorescence (DIF) and electron microscopy. Formalin-fixed, paraffin-embedded tissue (FFPE) sent for light microscopy can be used for DIF after antigen retrieval. However, complement staining has not been satisfactory. We standardized DIF using proteinase-K for antigen retrieval in FFPE renal biopsies. A pilot study was conducted on known cases of membranous glomerulonephritis (MGN), membranoproliferative type-1 (MPGN-1), immunoglobulin A nephropathy (IgAN), and anti-glomerular basement disease (anti-GBM). Immunofluorescence panel included fluorescein isothiocyanate (FITC) conjugated IgG, IgA, IgM, complements (C3 and C1q), light chains (kappa, lambda) and fibrinogen antibodies. After standardization of the technique, 75 renal biopsies and 43 autopsies cases were stained. Out of 43 autopsy cases, immune-complex mediated glomerulonephritis (GN) was confirmed in 18 cases (Lupus nephritis-11, IgAN-6, MGN-1), complement-mediated dense deposit disease (DDD-1) and monoclonal diseases in 4 cases (amyloidosis-3, cast nephropathy-1). Immune-mediated injury was excluded in 17 cases (focal segmental glomerulosclerosis -3, crescentic GN-6 [pauci-immune-3, anti-GBM-3], thrombotic microangiopathy-5, atherosclerosis-3). Renal biopsies (n-75) where inadequate or no frozen sample was available; this technique classified 52 mesangiocapillary pattern as MPGN type-1-46, DDD-2 and (C3GN-4). Others were diagnosed as IgAN-3, lupus nephritis-2, MGN-4, diffuse proliferative glomerulonephritis (DPGN)-1, Non-IC crescentic GN-1, monoclonal diseases-3. In nine cases, DIF on FFPE tissue could not help in making diagnosis. Proteinase-K enzymatic digestion of FFPE renal biopsies can unmask complements (both C3 and C1q) in immune-complexes mediated and complement-mediated diseases. This method showed good results on autopsy tissues archived for as long as 15 years.
