External QA in Immuhohistochemistry

Transcription

1 Cadiz 13 th April 2018 External QA in Immuhohistochemistry Nordic Immunohistochemical Quality Control Jan Klos, MD Department of Pathology Stavanger University Hospital Norway

2 Conflict of interests NONE

3 IHC is a complicated procedure With 3 choices for 5 variables in each phase = > 4 million protocols. Decalcification Preparation Tissue Type, Size, Instruments used for resection, De-differentiation Control Quantification Reporting Fixation Time, Type, Volume Postanalytic Preanalytic Section Thickness Storage Drying Pre-treatment Manual Stainer Visualization Sensitivity / Specificity Interpretation Localization Positive/Negative/cut-off 3 level Primary antibody Clone, Dilution Buffer, Time, Temp Analytic Development Sensitivity, Localization

4 IHC has quality problem Staining quality varies between different laboratories depending on the individual selection of methods and the level of technical expertise The quality of antibodies, reagents and guidelines is varying Internal quality control will often not identify a poorly calibrated IHC system or varying quality of products giving insufficient or aberrant staining results External quality assurance is mandatory 4

5 About NordiQC Established 2003 by a group of Nordic pathologists with the base in Aalborg University Hospital as an independent organization. It became a part of Aalborg University Hospital in 2017 and is currently managed by an executive board. Currently we have almost 800 participating laboratories from the whole world. Aim The aim of Nordic immunohistochemical Quality Control (NordiQC) is to promote the quality of immunohistochemistry and expand its clinical use by arranging schemes for immunohistochemical proficiency testing and providing examples of recommended protocols, tissue controls and other relevant information including descriptions of epitopes and technical protocol parameters. Policy NordiQC is a professional and scientific organization independent of economical or political interests.

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8 Test material Multi-tissue FFPE blocks 10% NBF h (ASCO/CAP guidelines ) Normal and clinically relevant tumour tissues Different levels of antigen expression high, moderate, low, none 2 unstained slides for each marker send to the participants 1 stained slide returned for central assessment

9 Test material - example The slide to be stained for Bcl-6 comprised: 1. Tonsil, 24 h. 2. Tonsil, 48 h. 3. Follicular lymphoma, grade I 4. Follicular lymphoma, grade II 5. Diffuse large B-cell lymphoma HE NE Tissue selection High Low None Expressor LE 9

10 NordiQC - Assessment Central assessment with consensus between experienced pathologists and histotechnologists Correlates staining results with central protocol parameters in order to identify Successful and less successful Abs Appropriate and inappropriate protocol settings Staining platform issues Reliable control tissues Publish general results on an open website individual results to the participants Specific explanations for insufficient results Tailored recommendations for improvement 10

11 Assessments 89 runs so far ~ slides = ~225,000 tissue sections

12 Protocol analysis ~45,000 slides ~ ~ 22,5 mln. details of information

13 Assessment criteria Optimal staining Perfect or close to perfect in all of the included tissues. Good staining Fully acceptable in all of the included tissues. However, the protocol may be optimized to ensure the best staining intensity and signal-to-noise ratio. Borderline staining Insufficient, e.g., because of a generally too weak staining or a false negative staining of one of the included tissues, or a false positive staining reaction. Poor staining Very insufficient e.g., because of false negative staining of several of the included tissues, or a marked false positive staining reaction. Sufficient Insufficient

14 Feed-back PDF file ed to participants with assessment marks, explanations and recommendations 14

15 Epitope/assessment/protocol 15

16 Recommended protocol 16

17 Recommended protocol 17

18 NordiQC assessment results General module ~ 20,000 slides ( ~ core sections) Insufficient 32% 21% 11% 33% 35% Optimal Good Borderline Poor

19 NordiQC - general results Major causes of insufficient stains Less successful antibodies 18 % Inappropriate antibody dilution 39 % Inappropriate epitope retrieval 31 % Other inappropriate lab. performance 12 % Endogenous biotin reaction (EBR) Section drying-out after HIER Technical platform error.... Unexplained 19

20 NordiQC general results Less successful antibodies 18 % Poor antibodies Poor ready-to-use formats Less robust antibodies Platform dependent antibodies Other error-prone antibodies Lot-to-lot variation Mouse-anti-Golgi (MAG) reaction Poor cocktail composition. NordiQC regrets any offence caused to laboratories and companies 20

21 Poor antibodies 21

22 Poor antibodies (few examples) Antigen Clone High expressor Low expressor Non expressor CD5 CD5/54/F6 FN CD23 MHM6 FN CD31 1A10 ( ) FN CD31 SP38 * ( ) FN CD138 5F7 ( ) FN CDX2 SP54 * ( ) FN FP CDX2 CDX2-88 FN FP CEA TF-3H8-1 FP CGA DAK. A3 FN PR SP2 * FP SYP SY38 FN 22

23 Poor antibodies: CD5 CD5 N Sufficient* Optimal* 4C7 conc % 49% SP19 conc 11 91% 46% CD5/54/F6 conc 28 4% 0% * With optimal protocol settings 23

24 Poor antibodies: CD5 SP19 TP Tonsil B-CLL CD5/54/F6 FN TP FN 24

25 Poor antibodies: CD31 JC70A 1A10 Optimal (16%)

26 Poor antibodies: CD31 JC70A 1A10 Optimal (16%)

27 Poor antibodies: CD31 JC70A 1A10 Optimal (16%) Haemangiosarcoma

28 Poor RTU formats 28

29 Poor RTU formats: CD5 CD5 Run 24 N Sufficient* Optimal* SP19 conc 11 91% 46% SP19 RTU Dako 3 100% 100% SP19 RTU VMS 14 79% 14% CD5 Run 34 N Sufficient* Optimal* SP19 RTU VMS 33 97% 97% * With optimal protocol settings FN

30 Poor RTU formats: CGA Medullary carcinoma LK2H10 REF pab RTU Company 1 mab LK2H10 RTU Company 2 mab LK2H10 RTU Company 3

31 Poor RTU formats: CGA Small cell carcinoma LK2H10 REF pab RTU Company 1 mab LK2H10 RTU Company 2 mab LK2H10 RTU Company 3

32 Platform dependant antibodies 32

33 Platform dependent antibodies Antigen Clone XT / Ultra automated Bond-max automated Autostainer semiautomated CD4 1F6 FN Weak SP35 CD56 123C3 FN Weak MRQ-42? CD79a JCB117 Weak SP18 BSAP/Pax5 24 FN Weak SP34 BCL6 PG-B6p FN Weak GI191E/A8 SYP 27G12 Weak MRQ-40 33

34 Platform dependent antibodies: PAX5 Hodgkin lymphoma NS clone SP34 RTU VMS/CM x200 clone 24 RTU VMS/CM x200

35 Inappropriate epitope retrieval & Misleading data sheets 35

36 Inappropriate retrieval (31%) AE1/AE3 + HIER TP Liver RCC FN AE1/AE3 + proteolysis TP 36 FN

37 IHC - NordiQC 2014 AE1/AE3 : Optimal results only obtained by HIER in NordiQC runs Dako: RTU HIER Leica: RTU Proteolysis Thermo: VMS: RTU - Proteolysis Misleading data sheets + Wrong control material used Conc: Proteolysis or HIER Conc: HIER Conc: HIER Quanto Proteolysis UltraVision 37

38 Misleading datasheets Antigen Clone Company Datasheet Result CGA Lk2H10 VMS No retrieval FN CK8 5D3 Leica RTU: HIER Conc: proteolysis Confound FN CK19 RCK108 BioGenex Proteolysis FN CK19 B170 Leica Proteolysis FN CKPan AE1/AE3 VMS/Dako Proteolysis FN CD34 QBEnd 10 Leica RTU: HIER Conc: proteolysis CD34 QBEnd 10 VMS Changed from no retrieval to HIER Confound FN CD68 KP1 Thermo Proteolysis FN DES DE-R-11 Cell Marque Proteolysis FN PLAP PL8-F6 BioGenex No retrieval FN VIM 3B4 VMS Proteolysis FN FN / OK WT1 6F-H2 Dako RTU: HIER Conc: proteolysis Confound FN 38

39 Tailored recommendations 39

40 Tailored recommendations Replace less successful antibodies (conc./rtu) Calibrate the antibody concentration Use HIER (instead of proteolysis or no retrieval) Increase HIER time / temperature / buffer ph For 95% of epitopes ph 8-9 is preferable to ph 6 Use a non-biotin based vizualisation system Use FDA approved kits instead of home-brews..... Improve the internal QC: Identify the right controls Select well defined normal low expressor cells/tissues 40

41 Results of NordiQC recommendations 352 labs were advised in 6 challenges with repeated tests (CGA, Calr, CD5, CD15, CD23, CK-LMW) Protocols No. Improved % Changed Not changed

42 Perspective 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?

43 Conclusions External Quality Assurance (EQA) Provides objective evidence of lab performance Identifies methodological errors Provides directions for improvements & controls The results of the NordiQC work indicate that Improvement of IHC is strongly needed. EQA schemes, industry and KOL must align describing the requirements for optimal IHC performance. 43