Standardization and Validation of Antigen Retrieval Methods for Immunofluorescence on Formalin-Fixed, Paraffin-Embedded Biopsies

Introduction Direct immunofluorescence (DIF) performed on fresh-frozen tissue is regarded as the reference standard for detecting immune deposits in renal and skin biopsies. However, limitations, such as inadequate frozen tissue, technical constraints, and the inability to perform retrospective analysis, necessitate alternative approaches. Immunofluorescence on formalin-fixed, paraffin-embedded (FFPE) tissue, combined with effective antigen retrieval methods, has emerged as a potential substitute. This study aimed to standardize and validate different antigen retrieval techniques for immunofluorescence on FFPE tissue and to assess their diagnostic performance in comparison with frozen section immunofluorescence. Materials and methods A total of 100 biopsies, comprising 50 native renal biopsies for glomerulonephritis and 50 skin biopsies for immunobullous disorders, were included. The corresponding frozen section immunofluorescence served as the reference standard. FFPE sections were subjected to antigen retrieval using proteinase-K, pronase, trypsin, and dual microwave methods. Fluorescein isothiocyanate (FITC) conjugated antibodies against IgA, IgG, IgM, C3, kappa, and lambda were applied. The staining intensity was assessed using a semi-quantitative scale ranging from 0 to 3+. Accuracy, positive predictive value, negative predictive value, sensitivity, and specificity of each FFPE-based method were calculated in comparison with frozen section immunofluorescence. Results Among the antigen retrieval techniques evaluated, proteinase-K demonstrated the highest sensitivity across immunoglobulins and complement components in both renal and skin biopsies. Repeated-measures one-way analysis of variance (ANOVA) showed a statistically significant difference in sensitivity among methods (p = 0.0280), while specificity did not differ significantly, with all methods showing near-perfect specificity. Conclusion Immunofluorescence on FFPE tissue using proteinase-K antigen retrieval provides high sensitivity and excellent specificity, closely approximating frozen section results. This method represents a reliable and practical alternative, particularly when frozen tissue is unavailable, and serves as an effective salvage technique in routine diagnostic pathology.