Combined Plasmatic and Tissue Approach to Membranous Nephropathy—Proposal of a Diagnostic Algorithm Including Immunogold Labelling: Changing the Paradigm of a Serum-based Approach Vincenzo L’Imperio, MD,* Federico G. Pieruzzi, MD,† Renato A. Sinico, MD,† Manuela Nebuloni, MD,‡§ Antonella Tosoni, BSc,‡§ Antonio Granata, MD,∥ Domenico Santoro, MD,¶ Irene Capelli, MD,# Maurizio Garozzo, MD,** Stefano Casano, BSc,* Andrew Smith, PhD,†† Antonella Radice, MD,‡‡ and Fabio Pagni, MD*§ Abstract: Membranous nephropathy represents the most frequent cause of nephrotic syndrome in the adult, leading to end-stage renal disease in one third of all the patients. In the last years, the discovery of circulating autoantibodies against phospholipase A2 receptor 1 (PLA2R) and thrombospondin type-1 containing 7A domain (THSD7A), shed light on the pathogenesis of idiopathic forms, be- ing responsible for 70% and 3% of all the cases, respectively. These identifications allowed the development of serological and histologic tests to detect autoantibodies and relative targets for diagnostic and prognostic purposes. Rising evidences suggest that serum titer cor- relates with disease activity and response to therapy. For these reasons, for patients with nephrotic syndrome, a serum-based ap- proach has been proposed, reserving renal biopsy only in cases with doubtful/negative serology. However, the recent introduction of useful criteria for the interpretation of PLA2R/THSD7A im- munohistochemistry could lead to high values of sensitivity and specificity for the in situ detection of target antigens. The present multicentric study on a series of membranous nephropathy cases with available serum/histologic correlation will show the importance of the crosstalk among the different techniques, recovering the possible role of electron microscopy in challenging situations. Key Words: renal biopsy, PLA2R, THSD7A, membranous nephropathy, nephropathology (Appl Immunohistochem Mol Morphol 2019;00:000–000) M embranous nephropathy (MN) represents the most frequent cause of nephrotic syndrome in the adult, leading to end-stage renal disease (ESRD) in one third of all the patients. 1–3 The majority of cases are idiopathic, the re- maining being secondary to infections, autoimmune diseases, drugs, and malignancies. 4,5 In the last years, the discovery of circulating autoantibodies against 2 transmembrane podocyte antigens, M-type phospholipase A2 receptor 1 (PLA2R) and thrombospondin type-1 containing 7A domain (THSD7A), clarified the pathogenesis of idiopathic forms, being respon- sible for 70% and 3% of all the cases, respectively. 6,7 These identi fications allowed the development of serological and histologic tests to detect autoantibodies and relative targets for diagnostic and prognostic purposes, 8–11 with rising evi- dence that serum titer correlates with disease activity 12–14 and that the phenomenon of “epitope spreading” can explain the reduced likelihood of remission in some cases. 15 The wide routine utilization of these tools, along with the high speci- ficity of serum autoantibody testing, 9,16 led to the proposal of a serum-based approach to nephrotic syndrome–presenting patients, suggesting to perform a renal biopsy only in cases with doubtful/negative serology. 17 However, it is well known that the so-called “kidney as a sink” effect is responsible for a higher rate of serum false negatives in the early phase of the disease when only the immunohistochemical (IHC) staining on renal biopsy can show the deposits. 18,19 Moreover, sero- conversion may occur later in the course of the disease, ex- plaining a significant rate of seronegative MN. 8 Finally, the recent introduction of useful criteria for the interpretation of PLA2R/THSD7A IHC could lead to high values of sensi- tivity and speci ficity for the in situ detection of target Received for publication November 17, 2018; accepted February 4, 2019. From the *Department of Medicine and Surgery, Pathology, San Gerardo Hospital; †Department of Medicine and Surgery, Nephrol- ogy Unit; ††Department of Medicine and Surgery, Proteomics and Metabolomics Unit, University of Milano-Bicocca, Monza; ‡Department of Biomedical and Clinical Sciences L. Sacco; §Research Center for Renal Immunopathology, University of Milan, Milan; ‡‡Microbiology and Virology Department, San Carlo Borromeo Hospital, Milan, Italy; ∥Department of Nephrology, San Giovanni Di Dio Hospital, Agrigento; ¶Department of Clinical and Experimental Medicine, University of Messina, Messina; #University of Bologna, Nephrology Unit, Sant’Orsola Malpighi Hospital, Bologna; and **Renal Unit, Santa Marta e Santa Venera Hospital, Acireale, Catania, Italy. The study was conducted following the ethical standards stated in the Declaration of Helsinki. The authors declare no conflict of interest. Reprints: Fabio Pagni, MD, Department of Medicine and Surgery, University of Milano-Bicocca, Via Cadore 48, Monza 20900, Italy (e-mail: fabio.pagni@unimib.it). Copyright © 2019 Wolters Kluwer Health, Inc. All rights reserved. RESEARCH ARTICLE Appl Immunohistochem Mol Morphol  Volume 00, Number 00, ’’ 2019 www.appliedimmunohist.com | 1 Copyright r 2019 Wolters Kluwer Health, Inc. Unauthorized reproduction of this article is prohibited.