Paraffin immunofluorescence for detection of immune complexes in renal biopsies: an efficient salvage technique for diagnosis of glomerulonephritis in dogs

Abstract

BackgroundRenal biopsy is an essential tool for the diagnosis of proteinuric kidney diseases in dogs, and evaluation of immune complexes (IC) by immunofluorescence (IF) of frozen sections (IF-F) is required for the diagnosis of IC-mediated glomerulonephritis (ICGN). However, the use of frozen sections from renal biopsies can have limitations. The aim of this study was to develop a reliable IF method using formalin-fixed and paraffin-embedded (FFPE) sections to detect ICs in dog ICGN.MethodsRenal biopsy specimens were obtained from dogs with protein-losing nephropathies. FFPE sections were prepared, and eight antigen retrieval pretreatment protocols were performed: digestion with trypsin, microwave (MW) heating in citrate buffer (MW-CB; pH 6.0), MW heating in Tris-EDTA buffer (MW-TEB; pH 9.0), as well as combinations of the above, and a non-treated control.ResultsA combination of trypsin for 30 min (Try-30) and MW-TEB; pH 9.0 was the most effective antigen retrieval pretreatment, with clear positive signals for IgG, IgA, IgM, and C3 detected by IF-FFPE. Granular signals, an important diagnostic indicator of ICGN, were clearly observed by both IF-F and IF-FFPE after combined pretreatment with Try-30 and MW-TEB, and IgG, IgA, IgM, and C3 signals were almost completely matched in all samples by IF-F and IF-FFPE.ConclusionIF-FFPE with Try-30 and MW-TEB pretreatment is a valuable technique for the diagnosis of renal diseases in dogs. This method could be an efficient tool when standard IF-F cannot be used, or does not provide useful results due to lack of glomeruli in the specimens for IF-F.

Paraffin Immunofluorescence: A Valuable Ancillary Technique in Renal Pathology

“…Because formalin fixation induces protein cross-linking, which generally blocks antigenicity, an antigen-retrieval step that allows for increased penetration of antibodies to the antigens “masked” by formalin fixation is required in IF-P. 5 , 6 This step involves incubating the paraffin sections with a proteolytic enzyme or heating the sections before incubation with fluorescein isothiocyanate–conjugated antibodies against Igs and complement components. Multiple proteolytic enzymes have been used in IF-P, including trypsin, 7 , 8 , 9 , 10 , 11 , 12 pronase E (protease XIV), 13 , 14 , 15 , 16 , 17 proteinase XXIV, 18 and proteinase K. 13 , 19 , 20 , 21 , 22 Successful results were also obtained by heat treatment with Tris or citrate buffers 15 and with dual microwave heating in EDTA antigen-retrieval solution. 23 In our laboratory, we use the pronase technique, which was originally described by Fogazzi et al.…”

“…Studies performed as early as the 1970s have shown that IF-P is a valuable salvage technique in renal pathology when frozen tissue is inadequate (e.g., lacks glomeruli) or not available. 8 , 10 , 14 , 18 , 20 , 21 Overall, diagnostic results by IF-P can be obtained in >80% of cases, 14 , 18 , 20 , 21 but the diagnostic yield varies depending on 3 factors: (i) The antigen-retrieval method used: proteinase K, pronase, proteinase XXIV, and dual microwave heating appear to be more sensitive than the other methodologies, although a systematic study comparing the diagnostic yield of these methods side by side on the same cohort of cases has yet to be performed. (ii) The disease type: the best results are obtained in dysproteinemia-associated kidney diseases ( Table 2 ).…”

Diagnostic Accuracy of Direct Immunofluorescence Test on Paraffin-Embedded Blocks in Comparison with Frozen Section Blocks in Renal Biopsies

“…For the purpose of antigen retrieval, the enzymes trypsin, pepsin, protease VII, pronase, protease XXIV, and proteinase K are employed for different periods, temperatures, and concentrations. Achieving ideal digestion to unmask the antigen locations is the most important step in using an enzyme technique to perform DIF on formalin-fixed renal biopsies [ 22 , 24 ].…”

Ten Approaches That Improve Immunostaining: A Review of the Latest Advances for the Optimization of Immunofluorescence

“…Likewise, several innovations have also been made for immunofluorescence, examples of these strategies include increasing antibody permeabilization [2,15,24,25]; remotion of aldehyde excess [15]; background reduction, avoiding the artifact's increase of the signal-to-noise ratio, as well as antibody specificity [2,15]; epitope retrieval by proteolytic and/or heat treatments [24,26,27]; fixation improvements and organic degradation [28,29]; avoiding the dephosphorylation of proteins [30]; deep antibody penetration into "hidden" structures [31]; and enhancing antibody signals in an entire and well-preserved retina structure [32].…”

“…Drawbacks to Face Disadvantages to face that are presented in (Figure 1): 7-9, 14 and 15. Yabuki et al [26] applied a combination of heat and proteolytic antigen retrieval methods. One of the most common consequences of the formaldehyde fixation is the crosslinking of methylene bridges between formaldehyde and proteins [56], making it impossible for antibodies to reach the epitopes of proteins, leading to weak specific signals of immunofluorescence staining and favoring the high background given by false-positive staining if an antigen retrieval step is not applied before the immunofluorescence method.…”