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Mogens Vyberg

Standardization of Tumour Type and Mutation Specific Protein Analysis:
NordiQC Results

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(see next handout below)

NordiQC has assessed the participants' laboratory performance in terms of detection of mismatch repair proteins MLH1, MSH2 and MSH6, and markers relevant for the differential diagnosis of lung adenocarcinoma vs. squamous cell carcinoma. Det general performance leaves room for improvement. Selection of more robust clones and optimization of protocols including enhanced demasking and use of a more sensitive visualization system has direct impact on the laboratory proficiency. The results from latest assessments (detailed on www.nordiqc.org) are as follows:

MLH1: 85 participants, 57% sufficient results, only 35% optimal. mAb clone ES05 was most successful, both as a concentrate and in an RTU system. HIER in alkaline buffer and the use of a 3-step polymer based detection system was important for an optimal result. A significant difference in the overall performance for MLH1 was also related to the IHC platform applied: Only 6 out of 25 (24 %) stains performed on the fully automated platform BenchMark XT or Ultra, Ventana, were sufficient, and none were optimal. In contrast, 10 out of 13 (77 %) stains performed on a similar fully automated platform Bond-max or Bond III were assessed as sufficient, out of which 8 (62 %) were optimal.

MSH2: 51 participants, 73 % sufficient results, only 20% optimal, mainly due to insensitive protocols (too low Ab concentration, insufficient HIER). There are several good Abs on the market (25D12, G219-1129, FE11), but all need a sensitive protocol.

MSH6: 90 participants, only 33% sufficient results. The new rmAb clones EPR3945 and EP49 appear to perform markedly better than the old mAb clones 44 and PU29. Efficient HIER in an alkaline buffer and a sensitive visualization system are mandatory.
 
TTF-1: 183 participants, 60% sufficient results. Excellent performance of clone SPT24 (88% sufficient) in contrast to clone 8G7G3/1 (3% sufficient). The marked difference in this challenge was the inclusion of a lung carcinoid, which could only stain positive with SPT24.
 
p63: 110 participants, 95 % sufficient results. Still many protocols could be optimized, particularly by increasing the Ab concentration.
 
CK-HMW: 163 participants, only 23% sufficient results, mainly due to the use of mAb clone 34BE12, which when used with HIER gives excessive cross reaction with a presumed CK-LMW.
 

External Quality Assessment for Standardization in Applied Immunohistochemistry – Experiences from NordiQC

Handout

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 reliability (1-5). While internal quality control (IQC) 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 (MTBs) 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.

Nordic Immunohistochemical Quality Control (NordiQC) was established 2003 as an independent, non-profit scheme for external quality assurance (EQA), currently attended by almost 400 laboratories from 40 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 ISH module. For the MTBs tissue samples are selected to include both high and low antigen expressor as well as non-antigen expressor cells and clinically relevant tumours. 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 www.nordiqc.org 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 10 years tested staining for about 80 of the clinically most relevant epitopes up to 15 times based on more than 25,000 multitissue sections (refer to details at www.nordiqc.org). 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 Abs, improper calibration of the Ab concentration, and insufficient epitope retrieval. Less successful primary Abs - identified as the major cause of suboptimal stains in 18% 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 39%. Insufficient, inappropriate or missing epitope retrieval was the major cause in 31%. 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. False-positive reactions were found in about 10% of all insufficient results.

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 systems and new challenges, but the performance of laboratories participating in the same tests several times are better than 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 (antigen retrieval, visualization system etc.), and staining 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 (1) is a central issue. Continuous EQA should be implemented in all IHC laboratories as well as diagnostics companies.

References

1. Torlakovic EE, Riddell R, Banerjee D, El-Zimaity H, Pilavdzic D, Dawe P, Magliocco A, Barnes P, Berendt R, Cook D, Gilks B, Williams G, Perez-Ordonez B, Wehrli B, Swanson PE, Otis CN, Nielsen S, Vyberg M, Butany J. Canadian Association of Pathologists-Association canadienne des pathologists National Standards Committee/Immunohistochemistry: Best practice recommendations for standardization of immunohistochemistry tests. Am J Clin Pathol. 2010 Mar;133(3):354-65.
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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CV

Dr. Mogens Vyberg graduated in 1973, completed residencies in Anatomic and Clinical Pathology at hospitals associated with University of Copenhagen, and was board-certified 1984. Since 1988 he has been senior consultant at the Institute of Pathology, Aalborg Hospital and was appointed as professor of clinical pathology at Aalborg University in 2012.
 
Dr. Vyberg’s main research fields and clinical expertise are immunohistochemistry and gastrointestinal, liver and brain pathology. He is author and co-author of more than 70 scientific papers and several book chapters in these fields as well as the text book Compendium of Applied Immunohistochemistry (Danish; English version scheduled for 2013). He is head of the Laboratory for immunohistochemistry/R&D, which carries out quality control and protocol optimization for the world’s leading diagnostics companies.

Dr. Vyberg is co-founder and scheme director of the international organization Nordic Immunohistochemical Quality Control (NordiQC), which was established in 2003 with Aalborg Hospital as its domicile and currently serves about 400 pathology laboratories from 40 countries.

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