parameters (i.e. results interpretation and reporting) (4, 5). In an EQA setting, by circulating serial sections to a number of laboratories to be stained for specific antigenic markers and assessing them in a standardized and objective way, the pre- and post-analytical parameters influencing the results of IHC can be taken into consideration, allowing a direct comparison of the laboratory performance and analytic parameters potentially influencing the staining quality. Establishment of NordiQC. A Brief History In 1999, Dako Nordic organized a meeting of pathologists from Denmark, Finland, Norway and Sweden to find a suitable means of optimizing methods and improving results of clinical IHC. Based on these meetings and a set of pilot runs, NordiQC was established on January 1, 2003, as an independent, scientific, non-profit organization for Nordic laboratories. The following year, the capacity was expanded to include a limited number of laboratories outside the Nordic countries, and currently, 130 laboratories from 20 countries are enrolled. The NordiQC EQA scheme currently comprises of a general module with three annual runs catering for a total of 15-18 markers selected for diagnostic purposes in pathology departments, particularly in the analysis of neoplastic lesions. Recently a breast module with two annual runs catering mainly for estrogen and progesterone receptors and HER2 has been initiated. Laboratories can only participate by submitting protocols detailing the technical variables in a web-based questionnaire. For tests, multi-tissue blocks made from several (usually 5-7) normal and tumor tissue samples selected to include cells with varying content of epitopes are used. Included are tissues containing critical stain quality indicators (CSQI); defined as cell or tissue epitopes that are weakly expressed, due to a low concentration and, therefore, easily lost in suboptimal protocols. For each marker to be demonstrated, a pair of unstained slides is circulated to the participating laboratories, which are requested to perform IHC stains using their standard protocols and return one of the slides. All slides are assessed anonymously by pathologists and technicians experienced in analyzing IHC slides. Each stain is by consensus marked as optimal, good, borderline or poor. The general results of each testing are presented on the Web site www.nordiqc.org in an aggregate fashion together with an analysis of the protocol data pointing out variables that are found to be of importance for the staining quality. Individual scores are sent to all participating laboratories confidentially using e-mail. In the case of borderline or poor marks, individual explanations and specifically tailored recommendations for protocol improvement are provided. Results of five years NordiQC work Between 2003-2007, NordiQC tested the immunohistochemical staining for 65 clinically relevant epitopes at least one to five times (refer to details at www.nordiqc.org). The over-all scores were almost evenly distributed between optimal, good and insufficient (i.e. borderline or poor). For some important markers like CD5, CD15, chromogranin A, cytokeratins, and immunoglobulin light chains, as many as 50-70% of the tests were insufficient in the first runs. The main causes of insufficient stains have been identified as: 1. less successful primary Abs, including ready-to-use (RTU) products, 2. insufficient, inappropriate or missing epitope retrieval, 3. improper calibration of primary Ab concentration, and 4. endogenous biotin reaction. Examples of optimal and insufficient staining results are illustrated in Figs. 1-3. Often a combination of several of the above-mentioned factors have been found. In about 90% of the insufficient results, the sensitivity of the applied protocols was too low resulting in too weak or false negative reactions. False-positive tests were found in about 10% of all insufficient results (giving a false-positive reaction or marked diffuse background). For tests carried out several times, the proportion of insufficient results declined in almost all cases. Among the laboratories following the NordiQC recommendations, improvement in a subsequent run was seen in about 70%, while among those who did not change their protocols, improvement was seen in less than 20%. In the following, some details from selected examples of test results are described. Estrogen receptor (Fig. 1) The rate of sufficient test results increased considerably during five runs, from 32 of 71 laboratories (45%) in the first run to 61 of 73 (84%) in the latest. Optimal staining results could be obtained with all the three mAb clones SP1, 6F11 and 1D5. However, with the latter a generally higher proportion of suboptimal results occurred, indicating that this clone may be less robust, if other protocol parameters are not optimal (frequently insufficient Heat-Induced Epitope Retrieval (HIER)). This is in accordance with the results obtained by United Kingdom National External Quality Assessment Scheme for Immunocytochemistry (UK NEQAS-ICC). (http://www.ukneqasicc.ucl.ac.uk/neqasicc.shtml) and the recently published results by Phillips et al. (7). These evaluations are based on staining intensity and are not clinically correlated. Laboratories that changed their estrogen receptor protocol more or less according to the NordiQC recommendations, improved their staining result much more frequently than those not changing their protocol (Table 1). An example of optimal vs. insufficient estrogen receptor staining is shown in Fig 1. Laboratory Number of labs advised Number of labs improved Recommendation followed 39 32 (82%) Recommendation not followed 24 7 (29%) Table 1. Effect of NordiQC recommendations to laboratories with an insufficient estrogen receptor staining Connection 2008 | 46 April 2008, VOL 11 Connection IHC STANDARDIZATION AROUND THE WORLD In this issue Q&A with Patho Contents APRIL 2008, VOL 11 2 3 4 Editorial George L. Kumar, PhD Featured Laboratory The Osamura Editorial Immunohistochemistry (IHC) Standardization Around the World George L. Kumar, PhD Managing Featured Laboratory The Osamura Laboratory Laboratory headed by Prof. Robert Yoshiyuki Osamura, MD, Q&A Ask the Experts: On Immunohistochemistry (IHC) Standardization Professor Leong is Medical Di time interval between removal of tissue and immersion in fixative, the temperature of tissue storage Connection: How would you like to standardize IHC in Australia? As discussed above, standardization Connection: Antigen retrieval is essential in immunohistochemistry (IHC) in order to restore epitope Connection: Is a universal image analysis system feasible? No, not until we standardize the all impo Q&A Fernando Soares, MD, PhD University of São Paulo, São Paulo, Brazil Dr. Fernando Soares is F Connection: In your opinion, what is the biggest hurdle for standardization? Probably education in l There are no standards in Latin America. We always follow the manufacturers protocol during our firs Q&A Bryan R. Hewlett, ART, MLT Consultant Technologist to the Anatomic Pathology EQA program of There is no standardization of AR steps circumstances, it would be impossible toand, undera the pre Connection: How would you rate European (UK NEQAS, NordiQC) and US IHC standards to Canadian IHC sta Q&A Prof. Chen Jie Prof. Cui Quancai Peking Union Mediacl College Hospital, Beijing, China Prof. Connection: According to Goldstein et al. Appl Immunohistochem Mol Morphol 2007;15:124­133 Immunohis Connection: Is it true that a particular histology feature may be better demonstrated by other fixat Connection: What constitutes standardization of image analysis as applied to immunohistochemistry (I Q&A Dr. Tanuja Manjanath Shet Dr. Vani Parmar Tata Memorial Hospital, Mumbai, India Tanuja Manj ... the biggest hurdle in India is suboptimal fixation and processing of tissues. Though I agree th Connection: Is it true that a particular histology feature may be better demonstrated by other fixat Connection: Can you comment on the internal and external quality control (EQC) procedures followed i Q&A Prof. Robert Yoshiyuki Osamura Department of Pathology, Tokai University School of Medicine Connection: In your opinion, what is the biggest hurdle for standardization? The biggest hurdle for the standardization of image ... appropriate for pre-screeninganalysis is of the staining quality. Connection: Why is standardization of image analysis in diagnostic pathology important? Because the Q&A James F. Happel, DLM (ASCP), HTL Technical Director of Surgical Pathology, Research and Deve Connection: United Kingdom National External Quality Assessment Service (UK NEQAS) helps to ensure t Connection: The American Society of Clinical Oncology (ASCO) and the College of American Pathologist would recommend that standardization Ibegin with identifying a reliable and trustworthy source ... Interview Immunohistochemistry for Oestrogen and Progesterone Receptors Dr. Andrew Lee Consultant H Connection: What is the difference between the H score and the Allred score? Which is better? What d Connection: Can you comment on the burden in the laboratory, if one changes from a current ER/PR ass Opinion & Interview IHC Standardization: A Dako Perspective Dr. Ole Rasmussen R&D Director, Connection: Dako has developed Readyto-Use Antibodies for in vitro diagnostic applications. How is t Articles UK NEQAS-ICC & ISH: Historical perspective, current role, future directions Andy Dodso UK NEQAS-ICC in the 1990s In his first year as Scheme Organiser, Keith oversaw the transition to sub The application could be argued to represent a field change in terms of the rigour with which the an Assessment teams consist of four assessors, who view slides around a multi-headed microscope and sco The archive which UK NEQAS holds, both in terms of stained slides and methodological data, must sure For Immunocytochemistry and FISH RESULT: RUN 80L SLIDE: NEQAS Laboratory No: XXX Mr. A. Scientist De Figure 6. Feedback on results has always been given high priority, and for many years this has been a b c d Figure 7. The antigen chosen by Gerry Reynolds for his very first assessment run was kap Bibliography Selected UK NEQAS-ICC & ISH papers. Ibrahim M, Dodson AR, Barnett S, Fish D, Jasani Articles Nordic Immunohistochemical Quality Control (NordiQC) An Organization for External Quality A parameters (i.e. results interpretation and reporting) (4, 5). In an EQA setting, by circulating ser CD79a (Fig. 2) Among 112 laboratories submitting stains in the latest run, most used mAb clone JCB11 References 1. Rhodes A, Jasani B, Barnes DM, Bobrow LG, Miller KD. Reliability of immuno-histochemic Fig. 2. CD79a A. Optimal CD79a staining of the tonsil using the monoclonal antibody (mAb) clone JCB Standardization of Ki-67 Immunohistochemical Staining for Diagnosing Grade of Gastrointestinal Strom was conducted in 49 GIST cases. The concordance rate for the evaluation results at three laboratorie CB pH6 a b c d TE pH9 e f g h Autoclave 121° C/10 min Water bath 95° C/40 min Microwave 50 Table 1. Correlation between NCC and STD methods R2=0.9483 Categories of proportion NCC Method 3 Opinions Importance of Standardization for Predictive Prognosis David J. Dabbs, MD Chief of Pathol is documented and serves as a surrogate marker for the initial exposure to formalin. Since the first Opinions The New Era for ER and PRIts time to Standardize! Dr. Ian Ellis, B.Med.Sci. BM, MS, FRCpa et al 2001). The main reason for false-negative results is due to inefficient heat-induced epitope ( Standardization of HER2 TestingInconsistency Raises Questions Opinions Sunil S. Badve, MD, FRCPath rence seen in these trials is in the order of 50%. This is the major reason for all the excitement a which now requires expression of HER2 by at least 30% of tumor cells (instead of 10%). It has also r IHC CONSENSUS MEETING, JANUARY 27 2008, SANTA BARBARA, CA, USA , IHC CONSENSUS MEETING, JANUARY 27, Richard Cartun, PhD, Sunil Badve, MD Jon Askaa, PhD Søren Nielsen, HT, CT, Mogens Vyberg, MD Elizab Dako Abstracts Abstracts presented at the 30th Annual San Antonio Breast Cancer Symposium December Dako Abstracts Amplification of ESR1 may predict resistance to adjuvant tamoxifen in postmenopausal Dako Abstracts Abstracts presented at the United States and Canadian Academy of Pathology (USCAP) A Dako Abstracts Metastatic Pancreatic Endocrine Tumors in the Liver Express KOC Briones AJ, Bourne P Dako Abstracts Merkel Cell Carcinomas Express K Homology Domain Containing Protein Overexpressed in Dako Abstracts Immunohistochemical Analysis of KOC/IMP3 in Malignant Pleural Mesothelioma Xu H, Sim Dako Abstracts KOC, TTF-1 and CDX2 Discriminate Small-Cell Carcinoma from Carcinoid and Pancreatic Dako Publications Publications Co-authored by Dako: In Press Li L, Xu H, Spaulding B, Cheng L, Si Dako Meetings 2007 - National Society for Histotechnology Meeting. 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