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Mogens VybergProfessor of Clinical PathologyDirector of NordiQCAalborg University Hospital, Aalborg, Denmark
NordiQC External Quality Assurance in
Immunohistochemistry
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AALBORG (~ 200.000 inhabitants)
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Aalborg University Hospital
Institute of Pathology
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Serial sections stained for Estrogen receptor
Lab. A Lab. B
Optimally processed ductal breast carcinoma tissue
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Serial sections stained for Estrogen receptor
Lab. A Lab. B
False neg.
High expressor
Low expressor
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Serial sections stained for Estrogen receptorUterine cervix
Lab. A Lab. B
False neg.
Tonsil
Controls
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Serial sections stained for Estrogen receptor
Clone 6F11 in 15/37 labsClone SP1/EP1/1D5 in 225 labs
False pos.
ExternalQualityAssurance !
Uterine cervixTonsil
Controls
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Control
correct false negative
External Quality Assurance – ER
Craig Allred
“Through the inquiry, the public learned that between 1997 and 2005 nearly 400 of about 1,000 breast cancer patients receivedincorrect test results of the ER status of their breast tumors.”
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Suboptimal IHC assays may be due to:• Preanalytical issues
• Fixation too short, too late, decalcification too soon…• Analytical issues:
• Less successful / too dilute antibody clones/RTUs• Insufficient epitope retrieval• Insensitive visualization systems• Platform problems
• Post-analytical issues • Interpretation criteria, interobserver variation …
The challenge of IHC
Should beidentified
with proper controls
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• International organization for proficiency testing of IHC• Founded 2003 by Nordic pathologists • Independent, scientific, not-for-profit organisation • Institute of Pathology, Aalborg University Hospital, DK
• General module: 3 runs/year• 15-18 different marker challenges
• Breast cancer IHC module: 2 runs/y• HER-2, ER/PR, Ki67/E-Cad …
• HER-2 ISH module: 2 runs/year• BRISH, FISH
• Companion module: 2 runs/year• PD-L1 …
Nordic immunohistochemical Quality Control
www.nordiqc.org
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Free PMC Article
Nordic immunohistochemical Quality Control
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Nordic immunohistochemical Quality Control
NordiQC Participants
Nordic labs
2015-18
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WWW.NORDIQC.ORGFREE ACCESS
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C.ORG
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Participant site
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Multi-tissue FFPE blocks10% NBF 24-48 h (ASCO/CAP guidelines …)• Normal and clinically relevant tumour tissues • Different levels of antigen expression
• high, moderate, low, none
Test material
2 unstained slides for each marker send to the participants1 stained slide returned for central assessment
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The slide to be stained for Bcl-6 comprised:1-2. Tonsils, 24 h/48 h
3. Follicular lymphoma, grade I
4. Follicular lymphoma, grade II
5. Diffuse large B-cell lymphoma
Test material
HE
LE
NE
Tissue selection:• High Expressor
• to confirm antibody• Low Expressor
• to ensure sensitivity• No-Expressor
• to ensure specificity
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Open website
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PDF file e-mailed to participants with assessment marks and – when needed – explanations and recommendations
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35%
33%
21%11%
OptimalGoodBorderlinePoor
NordiQC assessment results 2006 – 2015
General module ~ 20,000 slides ( ~100.000 core sections)
Insufficient 32%
too weak / false neg.: ~ 90%over-stained / false pos.: ~ 10%
{Insuff.
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58%21%
9% 12%
OptimalGoodBorderlinePoor
NordiQC assessment results 2006 – 2015
Breast cancer module ~ 9,000 slides (~35,000 core sections)
Insufficient 21%
too weak / false neg.: ~ 90%over-stained / false pos.: ~ 10%
{Insuff.
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Major causes of insufficient stains in ~ 9,000 slides
Less successful antibodies/RTUs 17 %
Inappropriate antibody dilution 20 %
Inappropriate epitope retrieval 27 %
Inappropriate detection kit 19 %
Other inappropriate lab. performance 17 %
Endogenous biotin reactionSection drying-out after HIERTechnical platform error. . . . Unexplained
NordiQC general results 2006 – 2015
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Go for Low antigen expressors ~
Critical Assay Performance Controls (CAPCs)
essential to evaluate sensitivity
essential to assure consistency
Normal tissues - easier to ensure the quality
90 % of insufficient staining results in EQA are caused by weak/false negative results
often related to the use of inappropriate positive tissue controls......
IHC – Biomarker controls
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Publications
AIMM 2015, 23:1
AIMM 2014, 22:241
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AIMM 2016-17
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NordiQC EQA: Estrogen Receptor in 13 runs
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8 10 13 B1 B3 B5 B7 B8 B10 B11 B13 B15 B17PASS RATE (%)
70
281122
141197
Participants
45%
87%
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2003 2005 2007 2009 2011 2013 2015
Estrogen receptorAntibody clone selection
SP1
EP16F111D5
NordiQC EQA ER
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2-step polymer 3-step polymer
IHC – Optimal performance
ER 1D5 1:100HIER Ci pH 6
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Pass rate (optimal + good) by participant status
Estrogen receptorNew participants ’Old’ participants
Run 10, 2004 57% 71%
Run B15, 2010 70% 86%
Run B19, 2015 51% 73%
Average 59% 77%
Results of NordiQC recommendations
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HER-2 staining results in 17 runs
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NordiQC runs for HER2 IHCCK7
Ampl. 3+ Ampl. 2+ Unampl. 2+ Unampl. 0
Optim
al
Ampl. 3+ Ampl. 1+ Unampl. 1+ Unampl. 0
Poor
* *
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NordiQC runs for HER2 IHCCK7
Ampl. 3+ Ampl. 2+ Unampl. 2+ Unampl. 0
Optim
al
Ampl. 3+ Ampl. 2+ Unampl. 3+ Unampl. 1
Poor
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NordiQC runs for HER2 IHC
Every $1 saved by laboratories by using cheaper reagents could potentially result in approximately $6 additional costs to the healthcare system.
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Lung ALK
ALCL
Lung adenocarcinoma
ALK15A4
Optimal
Poor
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Lung ALK
The immunoassay must fit for the purpose:
• Identify the antibody useful for the specific task
The right external controls must be used:
• Tissue with high epitope expression to identify
the right antibody
• Appendix
• Tissue with low epitope expression to assure the sensitivity:
• ALK-positive lung adenocarcinoma
• Tissue with no epitope expression to assure the specificity
• e.g., liver
Appendix - ALK
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Lung ALK – run 45, 176 labs
Appendix
Lung adenocarcinoma
ALK15A4
Optimal Poor
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419 advices for 11 markers
No. Improved %
Positive 268 195 73
Negative 151 21 14
Results of NordiQC recommendations
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Almost 1/3 of all IHC stains produced by NordiQC participants are still insufficient ! New labs New antibodies, techniques, platforms Increasing demands
How many IHC stains produced by labs not participating in an EQA scheme are insufficient ?How many scientific publications are based on insufficient IHC stains ?What are the consequences for the patients ?
Perspective
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When you believe in automation and stop thinking
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Mogens VybergProfessor of Clinical PathologyDirector of NordiQCAalborg University Hospital, Aalborg, Denmark
NordiQC External Quality Assurance in
Immunohistochemistry