6What is “Immunohistochemistry (IHC)” technique Is the technique used to detect the antigens in the cells. The antigens may be at cell membrane, cytoplasm or nucleus. This technique uses the principle of antigen-antibody reaction. If there are antigens, the reaction will occur and can be seen by using light microscope.Detection systemPrimary AbAntigen-antibodycomplexTissue AgGlass slide6
8Quality Assurance (by CAP) The practice of assessing performance in all steps of the laboratory testing cycle including pre-analytic, analytic and post-analytic phases to promote excellent outcomes in medical care."…assessing performance…"Requires a standard and comparison against the standard"…to promote excellent outcomes…"Requires corrective action if standard is not met.CAR = Corrective Action RequestO = observationAdapted from Morken T & Ruegg P. NSH Symposium Seattle, WA. Validation and Quality Control for IHC
9e.g. false negative stain from over/under fixation Quality ControlAn integral component of quality assurance consisting of the aggregate of processes and techniques to detect, reduce, and correct deficiencies in an analytical process."…Detect…"Requires procedures that prevent possible non-conformances."…Reduce and Correct…"Leads to procedures that reduce or eliminate confounding results.e.g. false negative stain from over/under fixation
10Differences between QA and QC Achieve and/or maintain qualityInspect at each step and assess what changes may be neededInternal validation of protocolPerform on each batchGuarantee accuracy of every testQAImprove overall performances of testingCompare performance with other centers
11Quality ImprovementThe practice of continuously assessing and adjusting performance using statistically and scientifically accepted procedures."…continuously assessing and adjusting…"• Regular review of result data (frequency)• Investigate and correct of root cause (RCA)“…statistically and scientifically…"• Analyze data trends over a time period• Hypothesize root cause (fish bone analysis)• Eliminate confounding variables• Solve the problem
14Quality Management Better Medical Care QA, QC, QI Quality System Better QualityBetter Medical CareNon-conformity
15Quality Control and Validation Validation of all 3 laboratory phases Pre-AnalyticAnalyticPost-AnalyticProper Validation leads to good quality.
16Set Standard followed by everyone and regularly monitored Pre-Analytic Validation:Specimen: time to fixation, amount of tissueFixation: type and range of time in fixativeProcessing: several protocols?, decalcificationSectioning: thickness, drying, heatingStorage: unstained slide, blockInternal auditSet Standard followed by everyone and regularly monitored
17Delay to fixation Effects from fixation & fixative type Study design: Breast cancer size 4 cmstore at 4C for 4 dayscut as core needle biopsy, fixed in different fixatives for 1,2,3,4,5,6,7,8,9,10,11,12,24,48,72,168 hConclusion: fixative type used, fixation time 1 h-1wk, delay to fixation 4 days at 4C did not effect the accuracy of ER&PR.Apple et al. Am J Clin Pathol 2011Oyama et al. Breast Cancer 2007
18Delay to fixation Effects from fixation & fixative type Mimic large and un-opened specimenOyama et al. Breast Cancer 2007Oyama et al. Breast Cancer 2007
19Delay to fixation Effects from fixation & fixative type Oyama et al. Breast Cancer 2007Oyama et al. Breast Cancer 2007
21Fixative type: Formalin substitution กิ๊ก จะดีกว่า คนเก่า (แก่) จริงหรือ?Formalin: carcinogenic to humans IARC (gr. 1) & cross-link & not for proteomic testingMoelans et al. Am J Clin Pathol 2011Formalin-free fixatives have potential in routine and proteomic testingKothmaier et al. Arch Pathol Lab Med 2011
22Storage effects: section Jennifer et al. CEBP 2004
23Storage effects: block Timothy et al. JNCI 1996Section within 2 weeks, idealBlock at RT (20-25C) long periodWolff AC, et al. J Clin Oncol 2007;25:Bilous M et al. Mod Pathol 2003;16:
24Standard Protocol Analytic Validation: Validate Accuracy with typical cases• Sensitivity: expression range of positive cases, low to high• Specificity: positive versus expected negative cases/tissues• Best controls: normal or disease?, set your own settingDetermine Precision (Reproducibility)• Intra-run and Inter-runShould have similar staining pattern and intensity of all slidesStandard Protocol
26Intra run vs Inter run Validation Testing CD 3Testing CD 310 runs 1 slide each1 run…10 slidessimilar staining pattern and intensity of all slides
27• Interpretation guidelines • Acceptance/Rejection criteria Post-Analytic Validation• Define: scoring/interpretation, rejection criteria,reporting parameters• Document: pathologist interpretation• Determine: variations from standard over time of- reagent performance- interpretation• Interpretation guidelines• Acceptance/Rejection criteria
28Sensitivity Analytic Sensitivity: • Lowest amount of substance detected by the testDiagnostic Sensitivity:• Ability of the test to determine true diagnostic positiveversus false negative (higher % FN = less sensitive)• Requires comparison to a previously validated antibodyIHC Sensitivity:• Lowest antibody used that still detects the target.NOTE: depend on antibody and detection system!(adapted from: Theoretical and Practical Aspects of Test Performance, in Immunomicroscopy, Taylor & Cote, 2005)
29Specificity Analytic Specificity Diagnostic Specificity • Accuracy on tests of known positive and negative controlsDiagnostic Specificity• Ability of a test to determine true diagnostic negative versusfalse positives (Higher % FP = less specific)• Requires comparison to a previously validated antibodyIHC Specificity• Ability of an antibody to bind to its particular antigen notother molecules(adapted from: Theoretical and Practical Aspects of Test Performance, in Immunomicroscopy, Taylor & Cote, 2005)
31Controls Positive Tissue Control - Ideally, same specimen type as sample• Not always possible, some antigens decrease in tumor.• Normal tissue should have consistent level of antigen.- Fixed/processed with same procedures as sample- Best practice: put on same slide as sample(one control per run, per antibody, acceptable)- Use internal positive controls- Low expressers are ideal to avoid false negatives.- Multi-tissue blocks with positive and negative, idealNegative Tissue Control- Tissue known to lack the antigen, Multi-tissue blocks
32Controls Positive Reagent Control: Primary Antibody - Must be positive on the positive control tissue, indicatingreactivity, specificity of primary antibody- Controls with range of expression will indicate sensitivity.- Internal control on sample help determine tissue reactivity.- Must be negative on negative control tissue or sample
33Controls Negative Reagent Control: • Replace primary antibody with: - Pre-immune serum from same animal (very rarely available)- Isotype-specific negative control antibody- Irrelevant primary antibody from same species (expensive)- Non-immune whole serum from same species (most common)- Antibody diluent or Wash buffer only• Must be negative on both positive & negative tissue, indicate- specificity (no cross reaction)- problems with detection system (non-specific binding)- problems with blocking reagents (not working?)- patient tissue problems (fixation, processing, etc)
34Because there are variable procedures and results among laboratories StandardizationInter-Laboratory and Intra-Laboratory? StandardizationBecause there are variable procedures and results among laboratoriesWhose responsibility?
35+ CQI Standardization Whose responsibility? 0rganization Laboratory Inter-Laboratory and Intra-LaboratoryWhose responsibility?+0rganizationLaboratoryCAPUK-NEQASNordiQCCQIJoinLong effort to promote standardizationLargely failed in US due to lack of consequencesUK-NEQAS took 20 years to achieve.Driven by oncologists, due to variable Her2 resultsRecommendations:Appl Immunohistochem Mol Morph (2); ,Arch Pathol Lab Med V.131 Jan 2007, pp.18-43
36Intra-Lab Standardization • Re-search current protocol for BEST result • Once validated & optimized the protocol must be followed: Every time!!! By everyone!!! • Record and report deviations from protocol • Review regularly and do CQIInter-Lab Standardization (EQA)• Participate regularly with an acceptable EQA organization• Track trend by analyzing the EQA results• RCA: protocol/practice if EQA results are out of range.
37How to make it becomes true? ConclusionQA, QC, QIQuality SystemBetter QualityBetter Medical CareHow to make it becomes true?