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NordiQC Background

Nordic Immunohistochemical Quality Control (NordiQC)

Immunohistochemical (IHC) assays are highly complex analyses increasingly used in diagnostic pathology to aid in the accurate identification and characterization of tissue and tumour types. Standardization of IHC staining reactions is vital for reliable and comparable results. This is emphasized by the increasing use of stand-alone IHC assays in the identification of prognostic and therapeutic markers in, e.g., breast cancer. Yet, staining quality varies greatly between laboratories compromising diagnostic reliability1-6. While internal quality control procedures are essential for IHC reproducibility in the individual laboratory, they will often not identify a poorly calibrated IHC system giving insufficient staining results.

The multiple parameters influencing the results of IHC can largely be ascribed to:
1. Pre-analytical parameters (tissue material, tissue handling before submission to the laboratory, tissue processing in the laboratory);
2. Analytical parameters (antigen retrieval procedure, primary Ab, visualization system, control selection, validation); and
3. Post-analytical parameters (interpretation, cut-off levels, reporting)4,5.

By circulating serial sections of multitissue blocks (TMAs) with well characterized routinely processed tissues to a number of laboratories to be stained for specific markers and assessing the stains in a standardized and objective way, variations in the pre- and post-analytical parameters influencing the results of IHC can be excluded, allowing a direct comparison of the laboratory performance as regards the analytical parameters potentially influencing the staining quality.

NordiQC was established 1st of January 2003 as an independent, non-profit scheme for external quality assurance (EQA), currently attended by more than 700 laboratories from more than 80 countries. Annually about 16 tests are performed in a general module, 6 tests in a breast cancer IHC module and 2 tests in a HER2 in situ hybridization (ISH) module. For all tests slides from TMAs are circulated. The TMA tissue samples are selected to include both high and low antigen expressor as well as non-antigen expressor cells in normal and clinically relevant tumour tissues. All slides returned are assessed by a board of experienced pathologists and technicians in a blinded fashion. Each stain is by consensus marked as optimal, good, borderline or poor. The general results of each run are presented in real time on the website  in an aggregate fashion together with an analysis of the primary antibodies (Abs) and central protocol parameters, pointing out variables that are found to be of importance for the staining quality. Individual scores (including explanations and recommendations, when needed) are sent to the participating laboratories confidentially.

NordiQC has during 13 years tested for about 85 of the clinically most relevant epitopes up to 15 times based on more than 25,000 TMA sections (refer to details at The over-all scores have been almost evenly distributed between optimal (36%), good (33%) and insufficient (i.e., borderline or poor, 31%). The causes of insufficient stains could be identified in the large majority of cases, mostly ascribed to less successful primary antibodies (Abs), improper calibration of the Ab concentration, insufficient epitope retrieval, and less successful detection kit.

Less successful primary Abs - identified as the major cause of suboptimal stains in 17% of the cases – comprise poor Abs, poor ready-to-use (RTU) formats, less robust Abs, and other error-prone Abs (mouse-anti-Golgi reaction, lot-to-lot variation, platform dependence, inappropriate cocktail composition). Interesting, while RTU formats previously gave relatively poor results, they now tend to perform better than stains based on concentrates used with in-house validated protocols. Improper calibration of primary Ab concentration (mostly a too dilute Ab) was the major cause in 20%. Insufficient, inappropriate or missing epitope retrieval was the major cause in 27%, and in 19% of the cases a too insensitive detection kit seemed a major issue. In 12% of the cases other causes could be identified, such as endogenous biotin reaction, excessive retrieval or drying out phenomena. In about 90% of the insufficient results, the sensitivity of the Abs/protocols was too low resulting in too weak or false negative reactions while the rest showed false-positive or combined false negative/false positive reactions.

Among laboratories with insufficient results in a specific test, improvement in the following test for the same marker was significant for those changing Ab and/or adjusting their protocol parameters according to the tailored NordiQC recommendations and the data presented on the webpage: Over-all, complying laboratories improved in the following test in more than 70%, while non-responding laboratories improved in about 15%. Over-all improvement is seen for some tests, e.g., estrogen receptor, where the proportion of sufficient results has increased from 45% to 85%. Generally, the results over time cannot be compared directly due to new participants, new Abs/protocols/platforms and new challenges, but the performance of laboratories participating in the same tests several times are markedly better than that of laboratories participating for the first time.

For the majority of IHC tests, optimal staining results can be obtained with different primary Abs, protocols and stainer platforms. Individual choices are prerequisites for development of new reagents, systems and instruments. However, this complicates the approach to standardization. Hence, for proper calibration of the systems, the identification of appropriate control tissues containing critical stain quality indicators is a central issue1. Continuous EQA should be implemented in all IHC laboratories as well as diagnostics companies.

1. Torlakovic EE, Nielsen S, Francis G, Garratt J, Gilks B, Goldsmith JD, Hornick JL, Hyjek E, Ibrahim M, Miller K, Petcu E, Swanson PE, Zhou X, Taylor CR, Vyberg M. Standardization of positive controls in diagnostic immunohistochemistry: recommendations from the International Ad Hoc Expert Committee. Appl Immunohistochem Mol Morphol. 2015 Jan;23(1):1-18.
2. Rhodes A, Jasani B, Barnes DM, Bobrow LG, Miller KD. Reliability of immuno¬histochemical demonstration of oestrogen receptors in routine practice: interlaboratory variance in the sensitivity of detection and evaluation of scoring systems. Clin Pathol 2000;53(2):125-30
3. Taylor CR. The total test approach to standardization of immunohistochemistry. Arch Pathol Lab Med. 2000;124(7):945-51.
4. O'Leary TJ. Standardization in immunohistochemistry. Appl Immunohistochem Mol Morphol. 2001;9(1):3-8.
5. Taylor CR. Standardization in immunohistochemistry: the role of antigen retrieval in molecular morphology. Biotech Histochem. 2006 Jan-Feb;81(1):3-12.
6. Hsu FD, Nielsen TO, Alkushi A, Dupuis B, Huntsman D, Liu CL, van de Rijn M, Gilks CB. Tissue microarrays are an effective quality assurance tool for diagnostic immunohistochemistry. Mod Pathol. 2002 Dec;15(12):1374-80.

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