JGK TRAINING PROGRAMME MODULE 2: METHOD VALIDATION AND UNCERTAINTY OF MEASUREMENT FOR VETERINARY LABORATORIES SAVC REF. ACCREDITATION NO.

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1 Registered as JKG Lab AfriQA (Pty) Ltd Reg. No. 2012/154570/07 P.O Box 1581 Potchefstroom Sylvia Street `` Potchefstroom 2531 Tel: Fax: Cell: : kangumbaj@gmail.com 2: joule@jgklab.co.za Website: JGK TRAINING PROGRAMME MODULE 2: METHOD VALIDATION AND UNCERTAINTY OF MEASUREMENT FOR VETERINARY LABORATORIES SAVC REF. ACCREDITATION NO.: AC/1551/16 DATE: AUGUST 2019 VENUE: ONDERSTEPOORT, PRETORIA MONDAY TUESDAY : THEORY ON PRINCIPLES OF METHOD VALIDATION & MEASUREMENT UNCERTAINTY 1. INTRODUCTION INTENDED PURPOSE OF THE ASSAY TO COVER MOST COMMON VETERINARY ASSAYS DESCRIPTION OF THE ASSAY AS ABOVE TEST METHOD PROTOCOL 2. DEVELOPMENT AND FEASIBILITY PRIOR TREATMENT OF SPECIMENS TO COVER MOST SAMPLES USED IN VETERINARY LABS REAGENT SELECTION ASSAY OPTIMIZATION FEASIBILITY TESTING REPEATABILITY ANALYTICAL SENSITIVITY ANALYTICAL SPECIFICTY 3. DIAGNOSTIC PERFORMANCE REFERENCE TEST (GOLD STANDARD) CONTROLS AND REFERENCE REAGENTS SELECTION OF REFERENCE POPULATIONS SAMPLING OF THE REFERENCE POPULATION DIAGNOSTIC SENSITIVITY DIAGNOSTIC SPECIFICITY PERFORMANCE OF TEST AGAINST REFERENCE TEST (GOLD STANDARD) ASSAY INTERPRETATION PRESENTATION OF RESULTS DISCUSSION OF RESULTS Africa s leader in Laboratory Quality Assurance Programs & Accreditation Page 1

2 4. TECHNOLOGY TRANSFER AND REPRODUCIBILITY REPRODUCIBILITY MONITORING VALIDITY OF ASSAY PERFORMANCE MAINTENANCE AND ENHANCEMENT OF VALIDITY CRITERIA 5. MEASUREMENT UNCERTAINTY WEDNESDAY : PRACTICAL ON METHOD VALIDATION PART 1: GUIDED PRACTICAL THURSDAY MORNING SESSION: PRACTICAL ON MEASUREMENT UNCERTAINTY PART 2: GUIDED PRACTICAL THURSDAY AFTERNOON SESSION PART 3: UNGUIDED PRACTICAL FRIDAY : RECAP CERTIFICATES GROUP PHOTO TEACHING METHODOLOGY Lectures PowerPoint Presentations (1) Presentation 1: Principles of Method validation (2) Presentation 3: Selection and use of reference samples and panels for method validation (3) Presentation 4: Measurement Uncertainty (4) Presentation 5: Validation of micro method for use in food industry Flip chart / White board (1) Used for writing and drawing Handouts (Flash discs) Content: (1) Presentations (2) Notes: Appendix 0: Principles and Method of Validation (OIE Terrestrial Manual) Appendix 1: International Reference Antibody Standards for Antibody Assays Appendix 2: Brucella Culture and Identification Appendix 3: Avian Influenza MultiS-Screen ELISA_ How the kit was validated by the manufacturer Appendix 4: Avian Influenza MultiS-Screen ELISA_ How the kit was validated under the SA conditions Appendix 5: IDEXX TB ELISA Validation_ how to write a validation experiment protocol Appendix 6: Validation Template for Extension of Existing Assays Appendix 7: Validation Template for Serological Assays Africa s leader in Laboratory Quality Assurance Programs & Accreditation Page 2

3 Appendix 8: Validation Template for Nucleic acid Assays Appendix 9: Sample, Sampling, Random and Systematic Sampling Appendix 10: Glossary of Terms Appendix 11: Validation Template (Simplified) Appendix 12: Proficiency Testing Appendix 13: Antimicrobial Residues in Eggs Appendix 14: Method for Measurement of B. abortus Antibody Appendix 15: Validation of Micro Method for use in Food Industry Appendix 16: Evaluation of CFT Performance Limitations Appendix 17: Measurement Uncertainty - OIE Notepads (1) Supplied to participants for taking notes Demonstration Worked examples Several examples will be used to teach the module, including but not limited to: (1) Method for the monitoring of antibiotic residues in eggs Appendix 13 (2) Methods for the measurement of Brucella abortus antibodies Appendix 14 (3) Validation of microbiological techniques for use in food industry_salmonella Appendix 15 (4) Comparative evaluation of Enzyme-linked immunosorbent assay and Complement fixation test in the laboratory diagnosis of brucellosis Appendix 16 (5) Isolation and identification of Brucella abortus Appendix 2 (6) Avian influenza MultiS-Screen Enzyme-linked immunosorbent assay (Influenza A) Antibody kit: review of how the test was validated in Europe Appendix 3 (7) Avian influenza MultiS-Screen Enzyme-linked immunosorbent assay (Influenza A) Antibody kit: review of how the test was validated under South African conditions Appendix 4 (8) Bovine tuberculosis ELISA Antibody kit: review of how the validation study was designed and carried out in South Africa Appendix 5 (9) Fluorescence polarization assay: review of how the preliminary validation study was designed and carried out in South Africa (10) Interlaboratory test comparison by proficiency testing: Rose Bengal test and Compliment fixation test for bovine brucellosis Appendix 12 (11) Interlaboratory test comparison by proficiency testing: Trichomonas foetus and Campylobacter fetus in sheath washes Appendix 12 Guided practical: Setting of repeatability and reproducibility conditions (1) Intra-plate and inter-plate variations (2) Between analysts variations (3) Between days variations (4) Mean (5) Standard variations (6) Coefficients of variations Quantitative data (1) (1) to (5) as above (2) Conversion of data into logarithmic values (3) Z-score calculation (4) 95% Confidence interval calculations Performance characteristics of a test (1) Diagnostic sensitivity calculations 2 x 2 contingency table (2) Diagnostic specificity calculations 2 x 2 contingency table Sample size calculations (1) Table for size calculation (2) Random and systematic sampling frames Diagnostic Se and Sp estimates (1) Allowable errors in the DSe and DSp estimates (2) Confidence levels Interpretation of test results (1) Positive predictive value calculations 2 x 2 contingency table Africa s leader in Laboratory Quality Assurance Programs & Accreditation Page 3

4 (2) Negative predictive value calculations 2 x 2 contingency table Data and templates with instructions will be provided Appendixes 1, 3 and 11 (1) Participants will follow the instructions and will be guided to: Develop validation protocols Analyze data Compile simple validation reports Data for the practical will include, but not limited to: - Avian influenza Haemagglutination Inhibition test and Enzyme-linked immunosorbent assay Appendix 3 - Diagnostic bacteriology Appendix 2 Own data secondary validation of existing assays 1) Participants will pick own assay(s) among those currently used at the laboratory. These can be already validated assays, or candidate tests for validation 2) Using the supplied templates with instructions, participants will review the validation of the assays (if already validated) or design a validation study for the assay (if not validated yet). Appendixes 1, 6, 7, 8 and 11 Evaluation of a validation study (i.e. to determine if a validation study was designed and carried out efficiently) 1) The following templates will be used to evaluate the validation status of assay(s) Template for extension of existing assays (adapted from SCAHLS) Appendix 6 Template for serological assays (adapted from SCAHLS) Appendix 7 Template for nucleic acid assays (adapted from SCAHLS) Appendix 8 Measurement of uncertainty Appendix 17 1) Mean 2) Standard deviation 3) Relative standard deviation and coefficient of variation 4) Uncertainty and expanded uncertainty 5) 95% Confidence interval and range Group discussions o Team 1: Diagnostic bacteriology / Molecular o Team 2: Microbiology o Team 3: Diagnostic serology PARTICIPANTS EVALUATION Tests Assignments Group discussions: breakaway session and report back COURSE EVALUATION Questionnaire TOOLS FOR PARTICIPANTS Supplied by JGK (1) Notepads (2) Pens (3) Pencils (4) Highlighters Africa s leader in Laboratory Quality Assurance Programs & Accreditation Page 4

5 (5) Erasers (6) Flash discs (7) JGK Calculators for: Mean Standard deviation Relative standard deviation and coefficient of variation Uncertainty and expanded uncertainty 95% Confidence interval and range Log values Z-scores Not supplied, but essential for the course (1) Calculator (2) Computer/laptop: Excel, Word, PowerPoint required Africa s leader in Laboratory Quality Assurance Programs & Accreditation Page 5