Immunoperoxidase versus immunofluorescence in the assessment of human renal biopsies

Abstract

For half a century, immunofluorescence (IF) on frozen sections has been the gold standard for immunohistochemical evaluation of renal biopsy specimens. In routine diagnostic immunohistopathologic evaluation, traditional IF has been replaced to a large extent by immunoperoxidase (IP) methods applied to paraffin sections of formaldehyde-fixed tissue. This is caused in part by the practical disadvantages inherent in the IF method, eg, separate tissue specimen and handling, UV microscopy, fading and impermanence of the label-making archiving, and difficult later investigation. Our aim for the present study is to evaluate IP as an alternative to IF in the diagnostic assessment of renal biopsy specimens. Proteolytic antigen retrieval, antibodies effective on deparaffinized sections, a sensitive detection system (Dako EnVision HRP; Dako, Copenhagen, Denmark), and a standardized and rigorously controlled procedure were applied to a series of renal biopsy specimens (n = 81) previously classified by means of light microscopy (LM) and IF. Staining for immunoglobulin G (IgG), IgA, IgM, C1q, and C3c were recorded as positive or negative for IF and IP in paired proportions, presuming that IF was the test standard. Concordant observations were 71% for all (282 of 398 observations), 82% for IgG (65 of 79 observations), and 89% for IgA (72 of 81 observations). The majority of discordant observations (74 of 116 observations) were positive by means of IP, with mesangial deposits of IgM and C1q that were not found by IF. Statistically, there was no significant difference in outcomes between IF and IP for IgG, IgA, and C3c ( P > 0.2). In addition, IP staining allowed simultaneous evaluation of tissue by LM and therefore correlation between tissue structure and immune deposits not readily attained by IF. In the present study, it is documented that for the detection of IgG, IgA, and C3c, IP applied to protease-digested deparaffinized sections of formaldehyde-fixed renal tissue is, with few exceptions, equal to IF on frozen sections. The EnVision HRP method used here is several times more effective in terms of primary antibody dilution than earlier existing IP methods, and because the avidin-biotin system is not involved, very little nonspecific background staining will occur. Discordant observations (116 of 398 observations; 29%) were in the majority (91 of 116 observations) due to positive IP findings of IgM and C1q, which deserve additional investigation.