title: Molecular-assisted immunohistochemical optimization.
abstract: Immunohistochemistry (IHC) is an essential tool in diagnostic surgical pathology, allowing analysis of protein subcellular localization. The use of IHC by different laboratories has lead to inconsistencies in published literature for several antibodies, due to either interpretative (inter-observer variation) or technical reasons. These disparities have major implications in both clinical and research settings. In this study, we report our experience conducting an IHC optimization of antibodies against five proteins previously identified by proteomic analysis to be breast cancer biomarkers, namely 6PGL (PGLS), CAZ2 (CAPZA2), PA2G4 (EBP1) PSD2 and TKT. Large variations in the immunolocalizations and intensities were observed when manipulating the antigen retrieval method and primary antibody incubation concentration. However, the use of an independent molecular analysis method provided a clear indication in choosing the appropriate biologically and functionally relevant "staining pattern". Without this latter step, each of these contradictory results would have been a priori "technically acceptable" and would have led to different biological and functional interpretations of these proteins and potentially different applications in a routine pathology setting. Thus, we conclude that full validation of immunohistochemical protocols for scientific and clinical use will require the incorporation of biological knowledge of the biomarker and the disease in question.
authors.affiliation: Oncology Research Institute, National University of Singapore, Singapore.
authors.forename: Mohd Feroz
authors.initials: MF
authors.lastname: Mohd Omar
authors.forename: Ning
authors.initials: N
authors.lastname: Huang
authors.forename: Keli
authors.initials: K
authors.lastname: Ou
authors.forename: Kun
authors.initials: K
authors.lastname: Yu
authors.forename: Thomas C
authors.initials: TC
authors.lastname: Putti
authors.forename: Hiroyuki
authors.initials: H
authors.lastname: Jikuya
authors.forename: Tetsuo
authors.initials: T
authors.lastname: Ichikawa
authors.forename: Osamu
authors.initials: O
authors.lastname: Nishimura
authors.forename: Patrick
authors.initials: P
authors.lastname: Tan
authors.forename: Manuel
authors.initials: M
authors.lastname: Salto-Tellez
mesh_terms: D048868:Adaptor Proteins, Signal Transducing / Q000032:analysis; D000937:Antigen-Antibody Reactions; D014408:Biomarkers, Tumor / Q000032:analysis*; D001943:Breast Neoplasms / Q000737:chemistry; D051347:CapZ Actin Capping Protein / Q000032:analysis*; D002262:Carboxy-Lyases / Q000032:analysis / Q000378:metabolism; D002265:Carboxylic Ester Hydrolases / Q000032:analysis / Q000378:metabolism; D006801:Humans; D007150:Immunohistochemistry / Q000379:methods* / Q000592:standards*; D016612:Paraffin Embedding; D016601:RNA-Binding Proteins / Q000032:analysis; D015203:Reproducibility of Results; D016707:Tissue Fixation; D014174:Transketolase / Q000032:analysis / Q000378:metabolism; D014407:Tumor Cells, Cultured
chemical_list: D048868:Adaptor Proteins, Signal Transducing; D014408:Biomarkers, Tumor; C495845:CAPZA2 protein, human; D051347:CapZ Actin Capping Protein; C101633:PA2G4 protein, human; D016601:RNA-Binding Proteins; D014174:Transketolase; D002265:Carboxylic Ester Hydrolases; C022363:6-phosphogluconolactonase; D002262:Carboxy-Lyases; C022353:phosphatidylserine decarboxylase