1A - NordiQC

Mogens Vyberg 2012Immunohistochemistry in tumour diagnosis• IHC needed in 10-25% of malignant tumours• Anaplastic tumours (90% (re-)classified)• Malignant lymphomas (50% (re-)classified)• Important diagnostic information gained in 50%• Narrowing of possibilities• Specific diagnosisi• Unsuspected diagnosis• Diagnosis confirmed in 40%• Tumour phenotypes identified• New entities and classifications establishedTumours of unknown origin: Occurrence10 - 15% of all cancers+ ??% uncertain if primary or metastaticLiver, lung, bone, lymph nodes, brain, peritoneum . . .• Adenocarcinomas (80-90%)• Squamous cell carcinoma (5-10%)• ‘Undifferentiated’ neoplasms (5-10%)- carcinomas- malignant lymphomas-sarcomas- malignant melanomas- germ cell tumours . . . .Tumours of unknown origin: Implications- A correct histopathologic classification savestime, money and inconvenience for thepatients and clinicians- Specific treatment protocols with survivaladvantage exists for, e.g.- malignant lymphomas- carcinomas- breast, prostate, ovary, thyroid, liver, kidney, NE . . .- sarcomas- GIST, synovial sarcoma . . .- germ cell tumoursTumours of unknown origin: Diagnosis• Morphology- pattern recognition- current classificationsAncillary techniques- immunohistochemistry- electron microscopy- molecular genetics• Clinical information• Laboratory studies•Image studies• Expert opinionsdense core granulesPSAIHC – ParametersDecalcificationPreparationTissueType, Dimension,Laser resection,De-differentiationControlmentQuantificationReportingFixationTime, Type, VolumePreanalyticPostanalyticSectionThicknessStorageDryingWith 3 choices for 5variables in each phase = >4 million protocols….Pre-treatmentManualStainerPrimary antibodyClone, DilutionBuffer, Time, TempAnalyticVisualizationSensitivity, Specificity DevelopmentSensitivity,LocalizationInterpretationLocalizationPositive/Negative - cut-off level62 variables…..2

Mogens Vyberg 2012IHC – Preanalytical parametersIHC – Preanalytical parameters• Appropriate tissue fixation and processing– Problem 1: Delayed fixation– Problem 2: Too short fixation in NBF– Problem 3: Other fixatives than NBF– Too long fixation in NBF is not a problem !CourtesyBrian Hewlett, Ca• Appropriate tissue fixation and processing– Problem 1: Agressive decalcification– Problem 2: Deviation from SOP – e.g. section bakingFalse negative or false positive50%2hHelander, KG. Kinetic studies of formaldehyde binding in tissue.Biotechnique and Histochemistry. 1994; 69, 177 -179IHC – Preanalytical parametersIHC – Preanalytical parametersMinimum formalin fixation time for consistent estrogen receptor immunohistochemical staining of invasive breast carcinoma.Goldstein NS, Ferkowicz M, Odish E, Mani A, Hastah FAm J Clin Pathol. 2003 Jul;120(1):86-926 - 48h8 - 72h“ The minimum formalin fixation time forreliable immunohistochemical ER results is6 to 8 hours in our laboratory, regardlessof the type or size of specimen(core biopsy or resection)”. (mAb clone 1D5)IHC – Preanalytical parametersIHC – Preanalytical parameters13 hours versus 79 hours in 10% NBF (the week-end dilemma…..)101 breast carcinomas:99 % Concordance for ER (SP1)95 % Concordance for PR (1E2)98 % Concordance for HER2 (A0485)3

Mogens Vyberg 2012IHC – Preanalytical parameters4 h 24 h48 h 168 hBreast carcinoma 3+, HER-2 PATHWAY, rmAb 4B5IHC – Preanalytical parametersInternal4 h. NBF 24 h. NBF 48 h. NBF 168 h. NBFSISH validationTumour 1 - - - FNTumour 2 Amp + + + +Tumour 3 (?) - FN FNTumour 4 - - FN FNTumour 5 - - - -Tumour 6 Amp + + + +Tumour 7 - - - FNTumour 8 poly. - - - FNTumour 9 poly. - - - FNHER-2 ISH: 8/36 cores could not be assessed..!Breast carcinomas, Dual SISH CCrb ext, P3. 8 mIHC – Preanalytical parametersIHC – Preanalytical parameters4 h 24 h48 h 168 hBreast carcinoma, 1+ Dual SISH CCrb ext, P3. 8 mIHC – Preanalytical parametersIHC – Preanalytical parameters0 hr delay, Q-score 3 2 hour delay, Q-score 2 8 hour delay, Q-score 0ER, mAb clone 1D5CD4 – SP354

Mogens Vyberg 2012IHC – Preanalytical parametersIHC – Preanalytical parametersDelayed fixation…………..Kappa; Burkitt´s LymphomaCD10 – 56C6Lambda; Burkitt´s LymphomaBy courtesyStavanger IHCIHC – The Influence Of Formalin FixationColon: MLH1, ES05IHC – The Influence Of Formalin FixationTonsil: S100, polyclonalPathos – 3h NBF, 6h prog.Pathos – 24h NBF, 6h prog.Pathos – 3h NBF, 2h prog.Pathos – 24h NBF, 2h prog.Pathos – 48h NBF, 6h prog.Pathos – 168h NBF, 6h prog.Pathos – 48h NBF, 2h prog.Pathos – 168h NBF, 2h prog.IHC – The Influence Of Formalin FixationTonsil: CD5, SP19 - HIER in CC1, 99˚C, 24 min. vs 48 min.IHC – Preanalytical parametersHIER 48 min.HIER 48 min.HIER 24 min.Strong acid: Mild acid: Calcium chelate:10% HCl 10% Formic acid 5 - 10% EDTAPathos – 24h NBF, 6h prog.Pathos – 3h NBF, 6h prog.Fast Intermediate SlowIHC+ IHC++* IHC+++DNA(+) DNA+/++ DNA++* e.g. CD79a, clone JCB117 reduced, Elastase, clone NP57 extracted5

Mogens Vyberg 2012IHC – Preanalytical parametersIHC – Preanalytical parametersProstate – Ki67, rmAb clone 30.910 % NBF 24h → 24h 10 % form. acid 10 % NBF + 10 % form. acid 24hHER-21 μm 3 μm 8 μmIHC – Preanalytical parametersIHC – Preanalytical parameters2 weeks at room-temp.Otherwise -20 / -80°C60°C 1h. HER-2: 3+ 80°C 16h. HER-2: 1+Baking just before use.Coating with paraffin is notconfirmed to be beneficialIHC – Preanalytical parametersParaffin Section Storage and Immunohistochemistry: Effects of Time, Temperature,Fixation, and Retrieval Protocol with Emphasis on p53 Protein and MIB1 AntigenWester, Kenneth Ph.D.; Wahlund, Eva B.L.T.; Sundström, Christer M.D., Ph.D.;Ranefall, Petter Ph.D.; Bengtsson, Ewert Ph.D.; Russell, Pamela J. Ph.D.; Ow, KimT. M. Sc.; Malmström, Per-Uno M.D., Ph.D.; Busch, Christer M.D., Ph.D.AIMM :Volume 8(1), March 2000, pp 61-702 weeks at room-temp.Otherwise -20 / -80°CBaking just before use.Coating with paraffin is notconfirmed to be beneficialIHC – Analytical parametersThe basal fundament for a technicaloptimal IHC performance:• Appropriate tissue fixation and processing• Appropriate and efficient epitope retrieval• Appropriate choice of antibody/clone• Robust, specific & sensitive detection system• Appropriate choice of control material6

Mogens Vyberg 2012IHC – Analytical parameters• Appropriate and efficient epitope retrieval– Problem 1: Too short effcient HIER period– Problem 2: Use of a non-alkaline HIER buffer– Problem 3: Wrong epitope retrieval typeFalse negative or false positiveIHC – Analytical parametersHeat Induced Epitope RetrievalOptimized temperature-time-pH-buffer system‘Heating condition’ = temperature × time:121ºC/1 min 100ºC/20 min 95ºC/40 min 60ºC/24 h.Device:Water bathMWOPressure cookerPressure cooker & MWOAutoclaveSteamConsiderations:EfficiencyStandardizationTissue damagePerformanceIHC – Analytical parametersIHC – Analytical parametersRef. A.J. Balaton et al. Appl. Immunohistochem. 4(4):259-263,1996MWO20Pressure cooker10HIER in EDTA pH 8 bywaterbath at 95°CDomesticPolar PatentMilestonePrestigePascalMinimum formalin fixation time for consistentestrogen receptor immunohistochemical stainingof invasive breast carcinoma.Goldstein NS, Ferkowicz M, Odish E, Mani A,Hastah FAm J Clin Pathol. 2003 Jul;120(1):86-92IHC – Analytical parametersTon 24h 10 % NBF – CD23 rmAb SP23IHC – Analytical parametersTon 168h 10 % NBF – CD23 rmAb SP23HIER CC1 – short 8 min.HIER CC1 – mild 30 min.HIER CC1 – short 8 min.HIER CC1 – mild 30 min.HIER CC1 – standard 60 min.HIER CC1 – extended 90 min.HIER CC1 – standard 60 min.HIER CC1 – extended 90 min.7

Mogens Vyberg 2012IHC – Analytical parametersIHC – Analytical parametersA: CD20, cl. L26B: Ki67, cl. MIB1C: HMB45(D: MOC31)Tonsil24 h NBFHIERPascal PCTE pH 9CD3 PS1 CD19 LE-CD19 PMS2 A16-46 8 - 9Modified from: Shi et al. J Histochem Cytochem 1995 43:193-201Ci pH 6IHC – Analytical parametersIHC – Analytical parametersCD10Proteinase K 5 minHIER TRS S1699Ber-EP4OptimalExcessive45Colon adenocarcinomaIHC – Analytical parametersPLAP antibodyTonsilClone PL8-F6 - Internal validation and protocol set-upIHC – Analytical parametersclone PL8-F61:50 Recommendation 1:50 Own optim. + HIERPlacentaSeminomaMB Ab test 18

Mogens Vyberg 2012IHC – Analytical parametersParametres to be considered related to the primary antibody:MB Ab test 1IHC – Analytical parametersParametres to be considered related to the primary antibody:MB Ab test 1• Antibody incubation time• 15 - 30 min. (up to 24 hours at 4°C)• Antibody incubation temperature•Typically y room temp. or 37°C15 min. RT 30 min. RT 30 min. 37°C 24 H 4°C1:25 1:50 1:75 1:200Typically…..IHC – Analytical parametersIHC – Analytical parametersMB Ab test 1Parametres to be considered related to the primary antibody:• Sensitivity to endogenous peroxidase blocking MB Ab test 1DOG1: rmAb SP31, RTU CMCC1M/16M/37°C/UVDOG1: rmAb SP31, RTU CMCC1M/32M/37°C/UVStaining for CD4 in a tonsil using the clone1F6Left: endogenous peroxidase blockingin 3 % H2O2 after HIER and beforeincubation in primary Ab..Right: endogenous peroxidase blockingin 3 % H2O2 before HIER and beforeincubation in primary Ab.Staining for CD4 in a tonsil using the clone4B12Left: endogenous peroxidase blockingin 3 % H2O2 after HIER and beforeincubation in primary Ab..Right: endogenous peroxidase blockingin 3 % H2O2 before HIER and beforeincubation in primary Ab.IHC – Analytical parametersIHC – Aanalytical parametersThe basal fundament for a technicaloptimal IHC performance:• Appropriate tissue fixation and processing• Appropriate and efficient epitope retrieval• Appropriate choice of antibody/clone• Robust, specific & sensitive detection systemBiotin baseddetection systemsshould not be usedfor IHC….!• Appropriate choice of control material9

Mogens Vyberg 2012IHC – Analytical parametersIHC – Analytical parameters• EnVision Flex / Flex +OptiView+amp (M+R)• UltraView• OptiView• UltraVision LP• UltraVision One• Refine• ImPress• Super SensitiveSensitivityCo omplexityQuanto (M+R)Hi Def. (M+R)EnV.Fl.+ (M+R)Refine (M)Super Sens. (M)OptiView (M+R)MACH 4 (M)• Super Picture• Hi Def• Quanto• MACH 4TATPriceThe choice of a polymer / multimer based systemUlt.Vis. ONEEnV. Fl.UltraViewSensitivityUltraView+amp (M+R)• .......1:25 1:50 1:150 1:500IHC – Analytical parametersPAX5 SP34 RTU – 32 min in primary, HIER CC1 standard:IHC – Analytical parametersThe basal fundament for a technicaloptimal IHC performance:3-step Multimer system (UltraView + Amplification)Tonsil & Hodgkin• Appropriate tissue fixation and processing• Appropriate and efficient epitope retrieval• Appropriate choice of antibody/clone• Robust, specific & sensitive detection system• Appropriate choice of control material2-step Multimer system (UltraView)IHC – Analytical parametersIHC – Analytical parameters2011• Appropriate choice of control material -requirements:1. Control material to evaluate the performance of thebiomarker in the range of laboratory tissue processingprocedures2. Control material to evaluate the diagnostic use of thebiomarker (sensitivity / specificity)3. Control material to evaluate the consistency of thebiomarker assay - day to day4. Control material evaluate the performance of thebiomarker - laboratory to laboratory10

Mogens Vyberg 2012IHC – Analytical parametersAnti-CD45 test:Protocol set-up:IHC – Analytical parameters• Low antigen expressors• Critical Staining Quality Indicators (CSQI)– essential to evaluate consistencyLiver 6h– essential to evaluate sensitivity• qualitative IHC biomarkers (CD markers etc)• quantitative IHC biomarkers (ER, HER-2..)1. Not NBF dependent, 2. Liver as control,3. IHC protocolMB Ab test Liver 1 72hIHC – Analytical parametersIHC – Analytical parametersHigh ExpressorsChromogranin AHighExpressorPancreas endocrine carcinomaOptimalInsufficientOptimalInsufficienti Non-expressorNon-expressorLow ExpressorLow ExpressorCritical stain quality indicator: Peripheral nervesCritical stain quality indicator: Peripheral nervesIHC – Preanalytical parameters• Low antigen expressors• Critical Staining Quality Indicators (CSQI)• CGA = peripheral nerves• Run 31, NordiQC CGA – 42/170 laboratories failed (25%)IHC – Preanalytical parameters• Low antigen expressors• Critical Staining Quality Indicators (CSQI)• CGA = peripheral nerves• Run 31, NordiQC CGA – 128/170 laboratories passed (75%)• 12/36 used pancreas as control (High expressor)• 12/36 used carcinoid as control (High expressor)• 5/36 used tumour as control (?? expressor)• 5/36 used appendix as control (High and low expressor)• 2/36 used Multi block as control (High and low expressor)24/36 = 66% of the laboratories failing the CGA assessment usedcontrol with only high antigen expression• 7/108 used pancreas as control (High expressor)• 14/108 used carcinoid as control (High expressor)• 3/108 used tumour as control (?? expressor)• 27/108 used appendix as control (High and low expressor)• 58/108 used Multi block as control (High and low expressor)85/108 = 79% of the laboratories passing the CGA assessment usedcontrol with low antigen expression11

Mogens Vyberg 2012IHC – Preanalytical parametersIHC – Preanalytical parametersBiocareBioGenexCell MarqueEpitomicsLeicaNeoMarkersRocheAalborg method:4 multi-tissue control-blocks for app. 180/220 markersMB1: Appendix, liver, tonsil, pancreasMB2: Brain, striated muscle, skin, melanomaMB3: Lung, prostate, placenta, thyroideaMB4: Thymus, kidney, bone marrow, tonsilSpec. ALK1, Hel., CA125, Hormones, etchttp://www.nordiqc.org/Techniques/Recommended_control_tissue.htmIHC – Preanalytical parameters“Ideal” daily control for themajority of routine markers:AppendixLiverTonsilPancreasCGA123/03CD3Appendix Hepar Tonsil PancreasLE - Nerves NE - Livercells NE - Lymphocyt. HE - Langerh. IsletsCD4 HE - T-cells. LE - Kupffer. LE - GC macroph. NE - Epithelial cellsIHC – Preanalytical parameters• Increased capacity (app. 30 - 40%) – previous1 pos control for each antibody1 neg control for each patient block• Established level of required sensitivity of theindividual antibodies (irrespective of the method used!)• Each slide has a positive control• Quality control is performed by the histotechnologistsfacilitated by use of normal tissue.IHC – Preanalytical parameters• Increased workload to produce multi-blocksGrossing, embedding etcQualifying, pre-screening• Increased reagent volume on all slides (300 ul)• No negative control - antibodies in panel serve asnegative control (+ multible tissues in control)• The identification of CSQI is an ongoing process…12