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Nada R, Kumar A, Kumar VG, et al. Unmasking of complements using proteinase-K in formalin fixed paraffin embedded renal biopsies. Indian J Nephrol. 2016;26(3):182-7doi: 10.4103/0971-4065.159558.
Nada, R., Kumar, A., Kumar, V. G., Gupta, K. L., & Joshi, K. (2016). Unmasking of complements using proteinase-K in formalin fixed paraffin embedded renal biopsies. Indian journal of nephrology, 26(3), 182-7. https://doi.org/10.4103/0971-4065.159558
Nada, R, et al. "Unmasking of complements using proteinase-K in formalin fixed paraffin embedded renal biopsies." Indian journal of nephrology vol. 26,3 (2016): 182-7. doi: https://doi.org/10.4103/0971-4065.159558
Nada R, Kumar A, Kumar VG, Gupta KL, Joshi K. Unmasking of complements using proteinase-K in formalin fixed paraffin embedded renal biopsies. Indian J Nephrol. 2016 May-Jun;26(3):182-7. doi: 10.4103/0971-4065.159558. PMID: 27194832; PMCID: PMC4862263.
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Indian J Nephrol. 2016 May-Jun;26(3):182-7. doi: 10.4103/0971-4065.159558.

Unmasking of complements using proteinase-K in formalin fixed paraffin embedded renal biopsies.

Indian journal of nephrology

R Nada, A Kumar, V G Kumar, K L Gupta, K Joshi

Affiliations

  1. Department of Histopathology, Post Graduate Institute of Medical Education and Research, Chandigarh, India.
  2. Department of Nephrology, Post Graduate Institute of Medical Education and Research, Chandigarh, India.

PMID: 27194832 PMCID: PMC4862263 DOI: 10.4103/0971-4065.159558

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.

Keywords: Antigen retrieval; complement; direct immunofluorescence; formalin-fixed paraffin-embedded; membranoproliferative; proteinase-K

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