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      Immunohistochemistry for Pathologists: Protocols, Pitfalls, and Tips

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          Abstract

          Immunohistochemistry (IHC) is an important auxiliary method for pathologists in routine diagnostic work as well as in basic and clinical research including exploration of biomarkers, as IHC allows confirmation of target molecule expressions in the context of microenvironment. Although there has been a considerable progress in automation and standardization of IHC, there are still many things to be considered in proper optimization and appropriate interpretation. In this review, we aim to provide possible pitfalls and useful tips for practicing pathologists and residents in pathology training. First, general procedure of IHC is summarized, followed by pitfalls and tips in each step and a summary of troubleshooting. Second, ways to an accurate interpretation of IHC are discussed, with introduction to general quantification and analysis methods. This review is not intended to provide complete information on IHC, but to be used as a basic reference for practice and publication.

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          Formaldehyde fixation.

          C Fox, F B Johnson, J. Whiting … (1985)
          Article 0 comments Cited 225 times – based on 0 reviews     Review now
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            Qualitative and quantitative analysis of nonneoplastic lesions in toxicology studies.

            Cynthia Shackelford, Gerald Long, Jeffrey Wolf … (2016)
            A pathology report is written to convey information concerning the pathologic findings in a study. This type of report must be complete, accurate and communicate the relative importance of various findings in a study. The overall quality of the report is determined by three Quality Indicators: thoroughness, accuracy, and consistency. Thoroughness is the identification of every lesion present in a particular organ or tissue, including spontaneous background lesions. Experienced pathologists familiar with background lesions may disregard certain types of lesions or establish a threshold or a severity above which background lesions are diagnosed. Accuracy is the ability to make, and precisely communicate, correct diagnoses. Nomenclature of lesions is a matter of definition and experienced pathologists generally agree as to what terms are to be used. Consistency is the uniform use of a specific term to record a defined lesion and implies that the same diagnostic criteria are being followed for each type of diagnosis. The relative severity of nonneoplastic lesions can be recorded either semiquantitatively or quantitatively. Semiquantitative analysis involves the application of defined severity grades or ranges for specific lesions. Quantitative analysis (counts and measurements) can be performed manually or electronically, utilizing image analysis and stereological techniques to provide numerical values. When both qualitative and quantitative parameters are applied in preparation of a pathology report, the recorded pathology findings can be interpreted and put into perspective. The use of this approach assures a reader that the pathology report meets the highest standards.
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              Selecting immunohistochemical cut-off scores for novel biomarkers of progression and survival in colorectal cancer.

              Russell Steele, Luigi Terracciano, Alessandro Lugli … (2007)
              Cut-off scores for determining positivity of biomarkers detected by immunohistochemistry are often set arbitrarily and vary between reports. To evaluate the performance of receiver operating characteristic (ROC) curve analysis in determining clinically important cut-off scores for a novel tumour marker, the receptor for hyaluronic acid mediated motility (RHAMM), and show the reproducibility of the selected cut-off scores in 1197 mismatch-repair (MMR) proficient colorectal cancers (CRC). Immunohistochemistry for RHAMM was performed using a tissue microarray of 1197 MMR-proficient CRC. Immunoreactivity was scored using a semi-quantitative scoring method by evaluating the percentage of positive tumour cells. ROC curve analysis was performed for T stage, N stage, tumour grade, vascular invasion and survival. The score with the shortest distance from the curve to the point with both maximum sensitivity and specificity, i.e. the point (0.0, 1.0), was selected as the cut-off score leading to the greatest number of tumours correctly classified as having or not having the clinical outcome. In order to determine the reliability of the selected cut-off scores, 100 bootstrapped replications were performed to resample the data. The cut-off score for T stage, N stage, tumour grade and vascular invasion was 100% and that for survival 90%. The most frequently selected cut-off score from the 100 resamples was also 100% for T stage, N stage, tumour grade, and vascular invasion and 90% for survival. ROC curve analysis can be used as an alternative method in the selection and validation of cut-off scores for determining the clinically relevant threshold for immunohistochemical tumour positivity.
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                Author and article information

                Journal
                Journal ID (nlm-ta): J Pathol Transl Med
                Journal ID (iso-abbrev): J Pathol Transl Med
                Journal ID (publisher-id): JPTM
                Title: Journal of Pathology and Translational Medicine
                Publisher: The Korean Society of Pathologists and the Korean Society for Cytopathology
                ISSN (Print): 2383-7837
                ISSN (Electronic): 2383-7845
                Publication date (Print): November 2016
                Publication date (Electronic): 13 October 2016
                Volume: 50
                Issue: 6
                Pages: 411-418
                Affiliations
                Department of Pathology, Asan Medical Center, University of Ulsan College of Medicine, 88 Olympic-ro, 43-gil, Songpa-gu, Seoul 05505, Korea
                Author notes
                Corresponding Author Chan-Sik Park, MD Department of Pathology, Asan Medical Center, University of Ulsan College of Medicine, 88 Olympic-ro, 43-gil, Songpa-gu, Seoul 05505, Korea Tel: +82-2-3010-5838 Fax: +82-2-472-7898 E-mail: csikpark@ 123456amc.seoul.kr
                Article
                Publisher ID: jptm-2016-08-08
                DOI: 10.4132/jptm.2016.08.08
                PMC ID: 5122731
                PubMed ID: 27809448
                SO-VID: 7219f487-7931-41ca-81f6-67b87c23c507
                Copyright © © 2016 The Korean Society of Pathologists/The Korean Society for Cytopathology
                License:

                This is an Open Access article distributed under the terms of the Creative Commons Attribution Non-Commercial License ( http://creativecommons.org/licenses/by-nc/3.0/) which permits unrestricted non-commercial use, distribution, and reproduction in any medium, provided the original work is properly cited.

                History
                Date received : 18 April 2016
                Date revision received : 5 August 2016
                Date accepted : 8 August 2016
                Categories
                Subject: Review

                Keywords: immmunohistochemistry,antigen-antibody reactions,immunostain,auxiliary test
                Data availability:
                Keywords: immmunohistochemistry, antigen-antibody reactions, immunostain, auxiliary test

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