QA, QC in IHC & Key to Success อบรมฟื้นฟูวิชาการ พยาธิวิทยากายวิภาค ครั้งที่ 11 วันที่ 25-27 กุมภาพันธ์ 2558 จังหวัดตรัง QA, QC in IHC & Key to Success รศ. ปลื้มจิต บุณยพิพัฒน์ ภาควิชาพยาธิวิทยา คณะแพทยศาสตร์ มหาวิทยาลัยสงขลานครินทร์ หาดใหญ่ สงขลา

ก่อนสู่บทเรียน

Antibody & reagent selection Polyclonal Ab Monoclonal Ab

ชิ้นเนื้อมีความหนา 4 ซม จะต้องใช้เวลานานเท่าไร ตำแหน่งตรงกลางของชิ้นเนื้อจะถูก fix ด้วยฟอร์มาลิน

Accuracy vs Precision ปาลูกดอก 10 ครั้ง

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. Detection system Primary Ab Antigen-antibody complex Tissue Ag Glass slide 6

Quality? Why QA, QC? Post-analytic Pre-analytic Analytic Interpretation Reporting elements Scoring system Image/manual Why QA, QC? Pre-analytic Tissue handling Time to fixation Fixation time Fixative type Tissue processing Sectioning Analytic Validation Equipment Protocol Staff competency Antigen retrieval Test reagents Control Quality?

Quality 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 Request O = observation Adapted from Morken T & Ruegg P. NSH Symposium Seattle, WA. Validation and Quality Control for IHC . 2010

e.g. false negative stain from over/under fixation Quality Control 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. e.g. false negative stain from over/under fixation

Differences between QA and 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 QA Improve overall performances of testing Compare performance with other centers

Quality Improvement The 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

ผังก้างปลา (Cause & Effect Diagram) การวิเคราะห์หาสาเหตุ ผังก้างปลา (Cause & Effect Diagram) เป็นแผนผังที่แสดงสมมติฐานของความสัมพันธ์อย่างเป็นระบบ ระหว่างหลายๆ สาเหตุ ที่ส่งผลต่อปัญหาหนึ่งปัญหา สาเหตุหลัก สาเหตุหลัก สาเหตุรอง สาเหตุรอง สาเหตุย่อย สาเหตุย่อย ปัญหา สาเหตุรอง สาเหตุรอง สาเหตุย่อย สาเหตุย่อย สาเหตุหลัก สาเหตุหลัก

การวิเคราะห์หาสาเหตุ ผู้ฟัง ผู้บรรยาย พูดน่าเบื่อ หลับไหล แชตดึก หลังอาหารเที่ยง ไม่มีเทคนิค สื่อไม่น่าสนใจ ผู้ฟังไม่สนใจการบรรยายหัวข้อนี้ ไม่น่าสนใจรู้แหล้ว…. เสียงรบกวน เมาท์กันแหลก เสียงโทรศัพท์ ไลน์ ซ้ำกับหัวข้อก่อนหน้านี้ เนื้อหา สิ่งแวดล้อม

Quality Management Better Medical Care QA, QC, QI Quality System Better Quality Better Medical Care Non-conformity

Quality Control and Validation Validation of all 3 laboratory phases Pre-Analytic Analytic Post-Analytic 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 Set Standard followed by everyone and regularly monitored

Delay to fixation Effects from fixation & fixative type 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. Apple et al. Am J Clin Pathol 2011 Oyama et al. Breast Cancer 2007

Delay to fixation Effects from fixation & fixative type Mimic large and un-opened specimen Oyama et al. Breast Cancer 2007 Oyama et al. Breast Cancer 2007

Delay to fixation Effects from fixation & fixative type Oyama et al. Breast Cancer 2007 Oyama et al. Breast Cancer 2007

ปล่อยให้ชะตาฟ้าลิขิต สรุป Fix ทันทีดีที่สุด ถ้ามีเหตุจำเป็นไม่ควรเกิน 1 ชม, แช่ใน 10% NBF ปริมาตร 10 เท่า, ให้หน้าตัดสัมผัสน้ำยามากที่สุด, นาน 6-48 ชม Garbage in Garbage out อย่า!!! ปล่อยให้ชะตาฟ้าลิขิต

Fixative type: Formalin substitution กิ๊ก จะดีกว่า คนเก่า (แก่) จริงหรือ? Formalin: carcinogenic to humans IARC (gr. 1) & cross-link & not for proteomic testing Moelans et al. Am J Clin Pathol 2011 Formalin-free fixatives have potential in routine and proteomic testing Kothmaier et al. Arch Pathol Lab Med 2011

Storage effects: section Jennifer et al. CEBP 2004

Storage effects: block Timothy et al. JNCI 1996 Section within 2 weeks, ideal Block at RT (20-25C) long period Wolff AC, et al. J Clin Oncol 2007;25:118-145. Bilous M et al. Mod Pathol 2003;16:173-182

Standard 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 setting Determine Precision (Reproducibility) • Intra-run and Inter-run Should have similar staining pattern and intensity of all slides Standard Protocol

Accuracy vs Precision ปาลูกดอก 10 ครั้ง

Intra run vs Inter run Validation Testing CD 3 Testing CD 3 10 runs 1 slide each 1 run …10 slides similar staining pattern and intensity of all slides

• 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

Sensitivity 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)

Specificity Analytic Specificity Diagnostic 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)

CD3 testing Present Absent Test pos True pos (a) False pos (b) All pos (a+b) Test neg False neg (c) True neg (d) All neg (c+d) All pos (a+c) All neg (b+d) Calculation Sensitivity = a/(a+c) Specificity = d/(b+d)

Controls 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 Negative Tissue Control - Tissue known to lack the antigen, Multi-tissue blocks

Controls 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)

Because there are variable procedures and results among laboratories Standardization Inter-Laboratory and Intra-Laboratory ? Standardization Because there are variable procedures and results among laboratories Whose responsibility?

+ CQI Standardization Whose responsibility? 0rganization Laboratory Inter-Laboratory and Intra-Laboratory Whose responsibility? + 0rganization Laboratory CAP UK-NEQAS NordiQC CQI Join 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

Intra-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 CQI 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.

How to make it becomes true? Conclusion QA, QC, QI Quality System Better Quality Better Medical Care How to make it becomes true?

Heart, Hand, Humanity, Honesty Keys to Success 5M+4H+1N Ne t wo r k 4 H 1 N - Man (3C: Competency, Concentration, Coporation) - Method (KPI: Knowledge, Performance, Improvement (using PDCA)) - Materials (3E: Enough, Efficiency, Economy) Management (QA, QC, QI, International, Good governance) - Money (Sufficiency, Flexible) + + Heart, Hand, Humanity, Honesty

ขอบคุณค่ะ