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

Nada R, Kumar A, Kumar VG, Gupta KL, Joshi K - Indian J Nephrol (2016 May-Jun)

Bottom Line: 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.This method showed good results on autopsy tissues archived for as long as 15 years.

View Article: PubMed Central - PubMed

Affiliation: Department of Histopathology, Post Graduate Institute of Medical Education and Research, Chandigarh, India.

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.

No MeSH data available.


Related in: MedlinePlus

Photomicrographs shows C3 staining, (a) C3 mesangial staining, (b) granular (arrow) staining along glomerular capillaries in membranous glomerulonephritis (GN), (c) coarse mesangial (2+) focal extending to capillaries in dense deposit disease, (d) peripheral glomerular capillaries as seen in C3 GN, (e) C1q staining glomerular capillaries which is segmentally negative, (f) C1q along tubular basement membrane (fluorescein isothiocyanate [a, c-f: ×20, b: ×40])
© Copyright Policy - open-access
Related In: Results  -  Collection

License
getmorefigures.php?uid=PMC4862263&req=5

Figure 2: Photomicrographs shows C3 staining, (a) C3 mesangial staining, (b) granular (arrow) staining along glomerular capillaries in membranous glomerulonephritis (GN), (c) coarse mesangial (2+) focal extending to capillaries in dense deposit disease, (d) peripheral glomerular capillaries as seen in C3 GN, (e) C1q staining glomerular capillaries which is segmentally negative, (f) C1q along tubular basement membrane (fluorescein isothiocyanate [a, c-f: ×20, b: ×40])

Mentions: Out of 43 autopsy cases, immune-complex mediated GN was confirmed in 18 cases (Lupus nephritis-11, IgAN-6, MGN-1). Complement-mediated diseases like DDD (n-1) and monoclonal disease was diagnosed in 4 cases (amyloidosis-3, cast nephropathy-1) [Table 2]. Immune-mediated injury excluded in 17 cases (focal segmental glomerulosclerosis [FSGS] with collapsing glomerulopathy-3, crescentic GN-6 (pauci immune-3, anti-GBM disease-3), Thrombotic microangiopathy-5, atherosclerosis-3. We had good staining with complements, that is, C3 and C1q in all possible patterns with different diagnosis in paraffin sections [Figure 2].


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

Nada R, Kumar A, Kumar VG, Gupta KL, Joshi K - Indian J Nephrol (2016 May-Jun)

Photomicrographs shows C3 staining, (a) C3 mesangial staining, (b) granular (arrow) staining along glomerular capillaries in membranous glomerulonephritis (GN), (c) coarse mesangial (2+) focal extending to capillaries in dense deposit disease, (d) peripheral glomerular capillaries as seen in C3 GN, (e) C1q staining glomerular capillaries which is segmentally negative, (f) C1q along tubular basement membrane (fluorescein isothiocyanate [a, c-f: ×20, b: ×40])
© Copyright Policy - open-access
Related In: Results  -  Collection

License
Show All Figures
getmorefigures.php?uid=PMC4862263&req=5

Figure 2: Photomicrographs shows C3 staining, (a) C3 mesangial staining, (b) granular (arrow) staining along glomerular capillaries in membranous glomerulonephritis (GN), (c) coarse mesangial (2+) focal extending to capillaries in dense deposit disease, (d) peripheral glomerular capillaries as seen in C3 GN, (e) C1q staining glomerular capillaries which is segmentally negative, (f) C1q along tubular basement membrane (fluorescein isothiocyanate [a, c-f: ×20, b: ×40])
Mentions: Out of 43 autopsy cases, immune-complex mediated GN was confirmed in 18 cases (Lupus nephritis-11, IgAN-6, MGN-1). Complement-mediated diseases like DDD (n-1) and monoclonal disease was diagnosed in 4 cases (amyloidosis-3, cast nephropathy-1) [Table 2]. Immune-mediated injury excluded in 17 cases (focal segmental glomerulosclerosis [FSGS] with collapsing glomerulopathy-3, crescentic GN-6 (pauci immune-3, anti-GBM disease-3), Thrombotic microangiopathy-5, atherosclerosis-3. We had good staining with complements, that is, C3 and C1q in all possible patterns with different diagnosis in paraffin sections [Figure 2].

Bottom Line: 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.This method showed good results on autopsy tissues archived for as long as 15 years.

View Article: PubMed Central - PubMed

Affiliation: Department of Histopathology, Post Graduate Institute of Medical Education and Research, Chandigarh, India.

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.

No MeSH data available.


Related in: MedlinePlus