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

Table 4 Studies using the technique of immunofluorescence on enzyme digested paraffin embedded tissue in literature

Ref.	Year	Enzyme used	Cases (n)	IF panel applied	Significant results
[2]	1976	Trypsin for 120 min	NA	Immunoglobulins and complement	Feasible to demonstrate immunoglobulins but not complement Reduced background immunofluorescence
[5]	1979	Trypsin	52 renal biopsies	IgG, IgA, IgM, C3, Fibrinogen	Accurate detection of immunoglobulins (90%) and complement (75%) in comparison with IF on frozen
[6]	1980	Trypsin	21 (LN, MN, IgAN)	IgG, IgM, IgA	IF on trypsin-digested tissue was as sensitive as IF-F for immunoglobulins but less sensitive for complement
[7]	1980	Pepsin (0.4%) and trypsin	Experimental mice model of anti GBM disease	IgG	Pepsin +/- trypsin digestion better than trypsin alone Enzyme digested tissue showed trivial decrease in sensitivity but good preservation in comparison with IF on frozen
[8]	1989	Pronase (0.75 g/L for 60 min at 37 °C)	IgAN (10), MN (8), Proliferative LN (10)	IgG, IgA, IgM, C3, C1q	Correct diagnosis possible in all cases Better structural details and less fading of IF Lower intensity staining for C3 Retrospectively performed digestion on 1 and 2 yr old blocks, satisfactory in 86% cases
[9]	2005	Microwave treatment (10 min) followed by Protease VII (0.05% for 30/60 min) Trypsin (0.25% for 120 min)	IgAN (7), LN (7), MN (7), MPGN (3)	IgG, IgA, IgM, C3	Microwave treatment followed by protease digestion better than trypsin digestion Diagnostic immunoglobulin found in more than 80% cases
[10]	2006	Pronase (0.75 g/L for 60 min at 37 °C)	MN (8), MPGN (5), LN (5), PIGN (5), IgAN (8), Cryo GN (5), Fibrillary GN (5), Anti GBM (5), Cast nephropathy (5), Amyloid (5), LCDD (5), LCFS (10)	IgG, IgA, IgM, C3, C1q, kappa and lambda	Diagnostic utility in 83% cases Useful in dysproteinemia related renal disease particularly LCFS Less sensitive for staining with C3 in MPGN type I, Cryo GN, PIGN Less sensitive for IgG in MGN and anti-GBM disease
[11]	2007	Proteinase XXIV	LN (5), antiGBM (5), MN (9)	NA	IF-P on proteinase XXIV is more sensitive than IF-P with pronase In LN, better intensity staining for C1q and IgG In anti GBM, 80% sensitivity for detection of IgG In MGN, 55% sensitivity for detection of IgG
[12]	2009	Microwave treatment and/or Proteinase K – (30 or 60 min)	IgAN (24), MN (22), LN (24)	IgG, IgA, IgM, C3	Rate of agreement between immunofluorescence on paraffin sections and immunofluorescence on frozen sections with respect to the presence of IgA was 56.5%, IgM - 44.4%, IgG - 73.9%, and C3 - 51.5% IF-P may be used as a salvage technique when frozen tissue is not available
[13]	2011*	Trypsin (30 min), Pepsin	IgAN (20), MN (25)	IgA, IgG, HBsAg, HbcAg	Trypsin digestion better than pepsin digestion IF-P slightly weaker signal than IF-F
[14]	2012	Heat - Tris/Citrate buffer Pronase RTU ( 60 min at 37 °C)	LN (15), MN (11), IgMN (10), MPGN (2), IgAN (2)	IgG, IgA, IgM, C3, C1q	Heat based retrieval using Tris buffer showed superior results Pronase digestion shows less sensitivity for detection of immunoglobulins and complement
[15]	2015	Proteinase K for 20 min	304 cases (207 cases as salvage and 97 cases for antigen unmasking )	IgG, IgA, IgM, C3, C4, C1q, fibrinogen, kappa and lambda	Not only a good salvage technique but prevents misdiagnosis due to masked immune complex or light chain deposition

LN: Lupus nephritis; MN: Membranous nephropathy; IgAN: IgA nephropathy; IgMN: IgM nephropathy; MPGN: Membranoproliferative glomerulonephritis; anti GBM: Anti glomerular basement membrane nephritis; PIGN: Post infectious glomerulonephritis; Cryo GN: Cryoglobulinemic glomerulonephritis; LCDD: Light chain deposition disease; LCFS: Light chain fanconi syndrome; RTU: Ready to use; HBsAg: Hepatitis B surface antigen; HbcAg: Hepatitis B core antigen; IF: Immunofluorescence.