6 6 What 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. Glass slide Detection system Primary Ab Tissue Ag Antigen-antibody complex
Pre-analytic Tissue handling Time to fixation Fixation time Fixative type Tissue processing Sectioning Post-analytic Interpretation Reporting elements Scoring system Image/manual Analytic Validation Equipment Protocol Staff competency Antigen retrieval Test reagents Control Why QA, QC? Quality?
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. Adapted from Morken T & Ruegg P. NSH Symposium Seattle, WA. Validation and Quality Control for IHC. 2010 Quality Assurance (by CAP) CAR = Corrective Action Request O = observation
An 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. Quality Control e.g. false negative stain from over/under fixation
Differences between QA and QC QA Improve overall performances of testing Compare performance with other centers QC Achieve and/or maintain quality Inspect at each step and assess what changes may be needed Internal validation of protocol Perform on each batch Guarantee accuracy of every test
"…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 Quality Improvement The practice of continuously assessing and adjusting performance using statistically and scientifically accepted procedures.
Quality Management QA, QC, QI Quality System Better Quality Better Medical Care Non- conformity
Pre- Analytic Analytic Post- Analytic Quality Control and Validation Validation of all 3 laboratory phases Proper Validation leads to good quality.
Set Standard followed by everyone and regularly monitored Pre-Analytic Validation: Specimen: time to fixation, amount of tissue Fixation: type and range of time in fixative Processing: several protocols?, decalcification Sectioning: thickness, drying, heating Storage: unstained slide, block Internal audit
Effects from fixation & fixative type Oyama et al. Breast Cancer 2007 Delay to fixation Apple et al. Am J Clin Pathol 2011 Study design : Breast cancer size 4 cm store at 4C for 4 days cut 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 h Conclusion: fixative type used, fixation time 1 h-1wk, delay to fixation 4 days at 4C did not effect the accuracy of ER&PR.
Effects from fixation & fixative type Oyama et al. Breast Cancer 2007 Delay to fixation Mimic large and un-opened specimen Oyama et al. Breast Cancer 2007
Effects from fixation & fixative type Oyama et al. Breast Cancer 2007 Delay to fixation Oyama et al. Breast Cancer 2007
Moelans et al. Am J Clin Pathol 2011 Fixative type: Formalin substitution กิ๊ก จะดีกว่า คนเก่า (แก่) จริงหรือ? Formalin: carcinogenic to humans IARC (gr. 1) & cross-link & not for proteomic testing Kothmaier et al. Arch Pathol Lab Med 2011 Formalin-free fixatives have potential in routine and proteomic testing
Storage effects: section Jennifer et al. CEBP 2004
Storage effects: block Wolff AC, et al. J Clin Oncol 2007;25:118-145. Bilous M et al. Mod Pathol 2003;16:173-182 Timothy et al. JNCI 1996 Section within 2 weeks, ideal Block at RT (20-25C) long period
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 setting Determine Precision (Reproducibility) Intra-run and Inter-run Should have similar staining pattern and intensity of all slides Standard Protocol
Intra run vs Inter run Validation …10 slides Testing CD 3 1 run 10 runs 1 slide each similar staining pattern and intensity of all slides
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
Analytic Sensitivity: Lowest amount of substance detected by the test Diagnostic Sensitivity: Ability of the test to determine true diagnostic positive versus false negative (higher % FN = less sensitive) Requires comparison to a previously validated antibody IHC 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) Sensitivity
Analytic Specificity Accuracy on tests of known positive and negative controls Diagnostic Specificity Ability of a test to determine true diagnostic negative versus false positives (Higher % FP = less specific) Requires comparison to a previously validated antibody IHC Specificity Ability of an antibody to bind to its particular antigen not other molecules (adapted from: Theoretical and Practical Aspects of Test Performance, in Immunomicroscopy, Taylor & Cote, 2005) Specificity
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, ideal Controls Negative Tissue Control - Tissue known to lack the antigen, Multi-tissue blocks
Positive Reagent Control: Primary Antibody - Must be positive on the positive control tissue, indicating reactivity, 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 Controls
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) Controls
Standardization Inter-Laboratory and Intra-Laboratory ? Standardization Because there are variable procedures and results among laboratories Whose responsibility?
Standardization Inter-Laboratory and Intra-Laboratory Whose responsibility? 0rganizationLaboratory CAP UK- NEQAS NordiQC Long effort to promote standardization Largely failed in US due to lack of consequences UK-NEQAS took 20 years to achieve. Driven by oncologists, due to variable Her2 results Recommendations: Appl Immunohistochem Mol Morph 2007 15(2);124-133, Arch Pathol Lab Med V.131 Jan 2007, pp.18-43 + Join CQI
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 CQI Intra-Lab Standardization Inter-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.
Conclusion QA, QC, QI Quality System Better Quality Better Medical Care How to make it becomes true?