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

Table 1 Protocol for immunofluorescence on paraffin embedded renal biopsies

Cut formalin fixed paraffin embedded tissue at 3-4 μ thickness on poly-L-Lysine coated slides

Deparrafinize and rehydrate tissue sections

Immerse in Tris EDTA pH 9 for 30 min at room temperature

Perform enzymatic digestion with proteinase K 1.25 mg/mL (Sigma Aldrich, United States) at room temperature for 15 min1

Stop digestion by immersing in Tris EDTA at 4 °C

Leave in Tris EDTA for 40 min at 4 °C

Rinse in PBS for 10 min

Apply FITC conjugated polyclonal rabbit antibodies directed against IgG (dilution 1:50), IgM (1:60), IgA (1:60), C3 (1:30), C1q (1:30), kappa (1:25), and lambda (1:40) (BIOSB, Santa Barbara, CA, United States). Incubate for 2 h in a moist chamber in the dark

Rinse with PBS

Mount in glycerine

Examine slides under a dark field immunofluorescence microscope

¹Varied based on room temperature. PBS: Phosphate buffered saline; FITC: Fluorescein isothiocyanate.

Table 2 Renal pathologies diagnosed by immunofluorescence on paraffin embedded biopsies

Diagnosis	Total number of cases	Number of cases with non diagnostic IF-P (%)	Remarks
MPGN	32	21 (6.2%)	Classification into immune complex mediated MPGN ( 18 cases) and complement mediated MPGN ( 12 cases ) was possible 1In two cases C3 was not demonstrated and electron microscopy showed features of dense deposit disease
Membranous nephropathy	11	21 (22.2%)	In one case staining intensity of IgG was only 1+, however staining pattern was classical 1In 2 cases significant fine granular immunofluorescence was not noted
Lupus nephritis	25	11 (4%)	Classification into Class II ( 2 cases), Class III/IV ( 15 cases) and Class V (5 cases ) was possible Two cases of lupus podocytopathy were diagnosed 1In one case of lupus nephritis only IgM was demonstrated significantly, though electron dense deposits were noted on electron microscopy
Diffuse proliferative glomerulonephritis - post infectious glomerulonephritis	12	11 (8.3%)	1In one case only 1+ IgG and trace C3 deposition noted No tissue for electron microscopy was available
Pauciimmunecrescentic glomerulonephritis	7	-	-
IgAN	39	-	In 64 cases (minimal change morphology, mesangial proliferation or FSGS), IgAN was excluded by IF-P In one case of diabetic nephropathy IF-P was used to exclude secondary IgAN
C1q nephropathy	2	-	-
Light chain deposition disease	1	-	Tubular basement membrane and vascular deposits were also noted in addition to the glomerular deposits
Amyloidosis	4	-	2 cases of AL amyloid (demonstrating light chain restriction) and 2 cases of AA amyloid
CGN	8	3 (37.5%)	The immune complexes could not be demonstrated in 3 cases of chronic glomerulonephritis, one of these was a case of biopsy proven MPGN and the other was a case of IgAN. In one case of CGN no immune complexes were seen, however no previous renal biopsy record was available
Cast nephropathy	2	-	One case also demonstrated light chain restriction
Tubulointerstitial nephritis	9	-	Associated immune complex mediated glomerular disease was excluded

¹Non diagnostic cases and details of their immunofluorescence pattern. MPGN: Membranoproliferative glomerulonephritis; CGN: Chronic glomerulonephritis; IgAN: IgA nephropathy; FSGS: Focal segmental glomerulosclerosis.

Table 3 Comparison of immunofluorescence intensity on fresh frozen and paraffin embedded renal biopsies

Disease	Number of cases with no difference in intensity of diagnostic immunoglobulin/complement (%) IF-F = IF-P	Number of cases with difference in intensity of diagnostic immunoglobulin/complement (%) IF-F > IF-P		Total number of cases
		Difference of 1+	Difference of 2+
IgA nephropathy	7 (78%)	2 (22%)	-	9
C-MPGN	1 (25%)	2 (50%)	1 (25%)	4
IC-MPGN	4 (100%)	-	-	4
Lupus nephritis	3 (50%)	3 (50%)	-	6
C1q nephropathy	2 (100%)	-	-	2
Membranous nephropathy	3 (43%)	3 (43%)	1 (14%)	7
Post infectious glomerulonephritis	1 (100%)	-	-	1

IF-F: Immunofluorescence on fresh frozen tissue; IF-P: Immunofluorescence on paraffin embedded tissue; C-MPGN: Complement mediated membranoproliferative glomerulonephritis; IC-MPGN: Immune complex mediated membranoproliferative glomerulonephritis.

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.