Handout - NordiQC
Handout - NordiQC
Handout - NordiQC
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CIQC/CAP-ACP Seminar in<br />
Diagnostic Immunohistochemistry<br />
July 08-09, 2010<br />
Immunohistochemical laboratory<br />
proficiency and innovation<br />
– the role of external quality assessment<br />
Mogens Vyberg, MD<br />
Assoc professor<br />
Scheme director, <strong>NordiQC</strong><br />
Inst. of Pathology, Aalborg Hospital<br />
Aarhus University Hospital<br />
Denmark<br />
2
Immunohistochemistry: A highly complex analysis<br />
Decalcification<br />
Preparation<br />
Tissue<br />
Type, Dimension,<br />
De-differentiation<br />
Necrosis<br />
Heat cauterisation<br />
Control<br />
Quantification<br />
Reporting<br />
Fixation<br />
Time, Type, Volume<br />
Preanalytic<br />
Postanalytic<br />
Section<br />
Thickness<br />
Storage<br />
Drying<br />
3 choices for 5 variables<br />
in each phase:<br />
4 million different protocols !<br />
Pre-treatment<br />
Manual<br />
Stainer<br />
Visualization<br />
Sensitivity, Specificity<br />
Primary antibody<br />
Clone, Dilution<br />
Buffer, Time, Temp<br />
Analytic<br />
Interpretation<br />
Localization<br />
Positive/Negative - cut-off level<br />
Development<br />
Sensitivity,<br />
Localization
External Quality Assurance: Estrogen Receptor<br />
Correct<br />
False negative<br />
½<br />
Control<br />
Lab. A<br />
Lab. B<br />
4
<strong>NordiQC</strong> EQA: Estrogen Receptor 2003<br />
Sufficient<br />
Insufficient<br />
½<br />
32 / 71 labs = 45% 39 / 71 labs = 55%<br />
5
External Quality Assurance<br />
• Staining quality varies greatly between different<br />
laboratories depending on the individual selection of<br />
methods and the technical expertise<br />
• Internal quality control will often not identify a<br />
poorly calibrated IHC system giving insufficient<br />
staining results<br />
• Standardization of staining methods is not possible<br />
but standardization of staining results is mandatory<br />
6
Establishment of a Nordic EQA program for IHC<br />
Nordic Immunohistochemical Quality Control<br />
<strong>NordiQC</strong> founded 2003 by Nordic pathologists<br />
• Independent, scientific, not-for-profit organisation<br />
• Institute of pathology<br />
Aalborg Hospital<br />
• General module:<br />
3 annual runs<br />
~16 different markers/tests<br />
• Breast cancer module:<br />
2 annual runs<br />
HER-2 IHC & BRISH, ER/PR<br />
7
Website: www.nordiqc.org<br />
8
<strong>NordiQC</strong> Participants<br />
9
Over-all assessment results<br />
<strong>NordiQC</strong> consensus marks (average 2003-10)<br />
40 runs = 25,000 slides<br />
• Optimal: 36 %<br />
• Good: 33 %<br />
• Borderline:<br />
}<br />
too weak / false neg.: ~ 90 %<br />
31 % {<br />
• Poor:<br />
over-stained / false pos.: ~ 10 %<br />
10
<strong>NordiQC</strong> over-all assessment results<br />
Major causes of insufficient stains:<br />
• “Less successful” antibodies 18 %<br />
• Inappropriate antibody dilution 39 %<br />
• Inappropriate epitope retrieval 31 %<br />
• Other inappropriate lab. performance 12 %<br />
• Inappropriate calibration / home brews<br />
• Endogenous biotin reaction (EBR)<br />
• Section drying-out after HIER<br />
• Technical stainer errors<br />
• . . . .<br />
• Unexplained<br />
11
Results of <strong>NordiQC</strong> tailored recommendations<br />
Lab response to 419 advices (11 markers)<br />
No. Improved %<br />
Positive 268 195 73<br />
Negative 151 21 14<br />
12
<strong>NordiQC</strong> EQA: Estrogen Receptor 2003-11<br />
200<br />
180<br />
160<br />
140<br />
No. participants<br />
120<br />
100<br />
80<br />
67<br />
84<br />
67<br />
72<br />
79 81<br />
74<br />
67<br />
90<br />
60<br />
45<br />
40<br />
20<br />
0<br />
8 10 13 B1 B3 B5 B7 B8 B10 B11<br />
2003 2006 RUN 2009 2011<br />
PASS RATE (%)<br />
No. OF PARTICIPANTS
External IHC Quality Assurance<br />
•Almost 1/3 of all IHC stains produced by <strong>NordiQC</strong><br />
participants are still insufficient !<br />
• New labs<br />
• New antibodies, techniques, platforms<br />
• Increasing demands<br />
•How many IHC stains produced by labs not<br />
participating in an EQA scheme are insufficient ?<br />
•How many scientific publications are based on<br />
insufficient IHC stains ?<br />
•What are the consequences for the patients ?
External Quality Assurance – ER<br />
correct<br />
false negative<br />
“Through the inquiry, the public<br />
learned that between 1997 and<br />
2005 nearly 400 of about 1,000<br />
breast cancer patients received<br />
incorrect test results of the ER<br />
status of their breast tumors.”<br />
Control<br />
15<br />
Craig Allred
External Quality Assurance<br />
“Less successful” antibodies 18 %<br />
• Poor antibodies<br />
• Poor ready-to-use formats<br />
• Less robust antibodies<br />
• Other error-prone antibodies<br />
• Mouse-anti-Golgi (MAG) reaction<br />
• Lot-to-lot variation<br />
• Platform dependent<br />
• Poor cocktail composition<br />
• ….<br />
16
Poor antibodies – CD31
Poor antibodies – CD31<br />
JC70A<br />
1A10<br />
Optimal (16%)
Poor antibodies – CD31<br />
JC70A<br />
1A10<br />
Optimal (16%)<br />
Haemangiosarcoma
Poor antibodies – MLH1<br />
MLH1 clone ES05<br />
MLH1 clone EPR3894
Poor RTU formats – CD79a<br />
JCB117 optimal JCB117 RTU 760-2630
Less robust Abs – Estrogen receptor alpha
Less robust Abs – Estrogen receptor alpha<br />
Ductal carcinoma I<br />
Optimal<br />
? Ab/dil./HIER/Viz.
Less robust Abs – Estrogen receptor alpha<br />
Ductal carcinoma II<br />
Optimal<br />
? Ab/dil./HIER/Viz.
Less robust Abs – Estrogen receptor alpha<br />
EP1<br />
SP1<br />
rmAb EP1, 1:15 Epitomics<br />
TRS High pH/20M/EnV-FLEX<br />
rmAb SP1, RTU Ventana<br />
CC1M/32M/36˚C/UV
Platform dependent antibodies<br />
Antigen Clone XT / Ultra<br />
BSAP Pax5 24<br />
FN<br />
BCL6 PG-B6p<br />
FN<br />
CD4 1F6, 4B12 FN<br />
CD5 4C7 FP<br />
CD56 123.C3<br />
FN<br />
CD79a JCB117 FN<br />
OCT-2 OCT-207 FN<br />
ASMA 1A4<br />
FP<br />
SF-1 NR5A1<br />
FN<br />
SMAD4 BC/B8 FN<br />
Alternative<br />
SP34<br />
GI191E/A8<br />
SP35<br />
SP19<br />
MRQ-42<br />
SP18<br />
MRQ-2<br />
None<br />
None<br />
None<br />
26
Platform dependent antibodies – PAX5
Platform dependent antibodies – PAX5<br />
Hodgkin lymphoma NS<br />
clone SP34<br />
RTU VMS/CM<br />
x200<br />
clone 24<br />
RTU VMS/CM<br />
x200
Poor cocktail composition – AE1/AE3 Leica<br />
Appendix<br />
IHC F – Enzyme 1, 5 min, RTU<br />
IHC F – BERS 2, 20 min. RTU<br />
IHC F – BERS 2, 20 min. AE1AE3 1:75, DAKO<br />
Ref.: AE1AE3 1:75, DAKO – BenchMark Ultra
Poor cocktail composition – AE1/AE3 Leica<br />
Liver<br />
IHC F – Enzyme 1, 5 min, RTU<br />
IHC F – BERS 2, 20 min. RTU<br />
IHC F – BERS 2, 20 min. AE1AE3 1:75, DAKO<br />
Ref.: AE1AE3 1:75, DAKO – BenchMark Ultra
Inappropriate antibody dilution<br />
31
Inappropriate antibody dilution – CD79a<br />
JCB117 appropriate<br />
JCB117 too dilute<br />
Plasmacytoma<br />
32
Inappropriate antibody dilution – Ig light chains<br />
IgK: Dako pAb A0191<br />
~1:300 ~1:3.000 ~1:30.000
Inappropriate antibody dilution – Ig light chains<br />
239 IgK tests, 12 Abs:<br />
• 12% optimal<br />
Dako pAb A0191:<br />
• 17% optimal<br />
+TRS/Ci 3.000-16.000:<br />
• 29 % optimal<br />
All other Abs:<br />
• 0% optimal<br />
Alternative: FLOW cytometry
Inappropriate epitope retrieval<br />
35
Inappropriate retrieval – CK PAN<br />
HIER<br />
Proteolysis<br />
AE1/AE3<br />
AE1 detects CK8 after HIER only<br />
AE1 does not detect CK18<br />
AE3 does not detect CK8/CK18<br />
36
Inappropriate retrieval – CK PAN<br />
Colon AC<br />
Renal CC<br />
HIER<br />
Proteolysis
Inappropriate vendor information<br />
Misleading datasheet:<br />
AE1/AE3/PCK26<br />
2011<br />
38
Inappropriate vendor information<br />
Misleading datasheet: AE1/AE3<br />
2011<br />
39
Confusing vendor information<br />
Correct datasheet:<br />
AE1/AE3<br />
40
Package inserts<br />
Datasheet changed according<br />
to <strong>NordiQC</strong> recommendation:<br />
mal.melanoma<br />
mal.melanoma<br />
RP2/18/22 X16/99
Inappropriate package inserts – CK-LMW<br />
Specificity<br />
Protocols<br />
CK types<br />
Vendor recomm.<br />
Aalborg opt.<br />
5D3<br />
8 + 18<br />
Thermo: Trypsin<br />
HIER TE 9<br />
TS1<br />
8<br />
Thermo: Ci pH 6<br />
HIER TE 9<br />
DC10<br />
18<br />
NC: High temp.<br />
HIER TE 9<br />
C51<br />
8<br />
18<br />
ZM: Ci pH6 / Pepsin<br />
NC: High tp. / Trypsin<br />
HIER TE 9<br />
35BH11<br />
8<br />
Dako: Proteolys./heat<br />
Thermo: Pepsin<br />
HIER TE 9<br />
CAM 5.2<br />
8 (+7)<br />
BD: Trypsin II<br />
Proteinase K
Inappropriate epitope retrieval – CK8: 35BH11<br />
Pepsin<br />
↑ Proteinase K<br />
HIER TE9
Inappropriate epitope retrieval – CK8: 35BH11<br />
1:40<br />
Proteinase K<br />
1:50<br />
HIER TE9<br />
1:20<br />
1:25
S100 Package – Breast inserts x 200 – S-100<br />
Breast<br />
pAb PA0900, Leica RTU, Bond III<br />
Mal. melanoma<br />
mAb clone 4C4.9, Ventana RTU, Ultra<br />
Bond: Enzyme 1 Ventana: No retrieval (!?)
Roche Innovation
PMS2<br />
Tonsil<br />
UltraView<br />
OptiView
Melan A, clone A-103<br />
Mal. melanoma<br />
UltraView<br />
OptiView
Melan A, clone A-103<br />
Adrenal gland<br />
UltraView<br />
OptiView
Melan A, clone A-103<br />
Granulosa cell tumour<br />
UltraView OptiView
EpCAM<br />
Normal kidney<br />
UltraView, EP4, CC1 OptiView, MOC31, TRS 6
EpCAM<br />
Clear cell renal cell carcinoma<br />
UltraView, EP4, CC1 OptiView, MOC31, TRS 6
Tailored recommendations<br />
53
Tailored recommendations<br />
• Replace less successful antibodies (conc./RTU)<br />
• Calibrate the antibody concentration<br />
• Use HIER (instead of proteolysis or no retrieval)<br />
• Increase HIER time / temperature / buffer pH<br />
• For 95% of epitopes pH 8-9 is preferable to pH 6<br />
• Use a non-biotin based viz. system<br />
• Use FDA approved kits in stead of home-brews<br />
• . . . . .<br />
• Improve the internal QC: Identify the right controls<br />
• Select well defined low expressor cells/tissues<br />
54
IHC – The Technical Test Approach<br />
• Low antigen expressors<br />
• Critical<br />
Staining<br />
Quality<br />
Indicators (CSQI)<br />
– essential to evaluate consistency<br />
– essential to evaluate sensitivity<br />
• qualitative IHC biomarkers (CD markers etc)<br />
• quantitative IHC biomarkers (ER, HER-2..)
IHC – The Technical Test Approach<br />
High Expressors<br />
Chromogranin A<br />
Optimal<br />
Insufficient<br />
Non-expressor<br />
Low Expressor<br />
Critical stain quality indicator: Peripheral nerves
IHC – The Technical Test Approach<br />
High<br />
Expressor<br />
Pancreas endocrine carcinoma<br />
Optimal<br />
Insufficient<br />
Nonexpressor<br />
Low Expressor<br />
Critical stain quality indicator: Peripheral nerves
IHC – The Technical Test Approach<br />
Run 31, <strong>NordiQC</strong> CGA test:<br />
42/170 laboratories failed (25%)<br />
36 controls submitted:<br />
• 12 used pancreas as control (High expressor)<br />
• 12 used carcinoid as control (High expressor)<br />
• 5 used tumour as control (?? expressor)<br />
• 5 used appendix as control (High and low expressor)<br />
• 2 used Multi block as control (High and low expressor)<br />
• 24-27 = 66-75% of the laboratories failing used<br />
control with only high antigen expression
IHC – The Technical Test Approach<br />
Run 31, <strong>NordiQC</strong> CGA:<br />
128/170 laboratories passed (75%)<br />
108 controls submitted:<br />
• 7 used pancreas as control (High expressor)<br />
• 14 used carcinoid as control (High expressor)<br />
• 3 used tumour as control (?? expressor)<br />
• 27 used appendix as control (High and low expressor)<br />
• 58 used Multi block as control (High and low expressor)<br />
• 85-88 = 79-81% of the laboratories passing used<br />
control with low antigen expression
IHC – The Technical Test Approach<br />
Biocare<br />
BioGenex<br />
Cell Marque<br />
Epitomics<br />
Leica<br />
NeoMarkers<br />
Roche
Nordic immunohistochemical Quality Control<br />
Conclusion I<br />
• External Quality Assurance (EQA)<br />
• Provides objective evidence of lab proficiency<br />
• Identifies methodological errors<br />
• Provides directions for improvements<br />
• The results of the <strong>NordiQC</strong> work indicate that<br />
• Improvement of IHC is strongly needed<br />
• EQA may have a major impact on lab proficiency<br />
• External quality assurance of staining results through<br />
laboratory proficiency testing is mandatory
Nordic immunohistochemical Quality Control<br />
Conclusion II<br />
• If IHC testing is not done properly, innovation may<br />
not be based on a solid foundation.<br />
• EQA groups must work closely with manufactures in<br />
order to accelerate the companies’ innovative efforts<br />
by early and constructive feedback on new or<br />
candidate new products …<br />
…including improved data sheets<br />
62
Nordic immunohistochemical Quality Control<br />
Thank you<br />
for your<br />
attention !<br />
Aalborg Hospital<br />
63
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