HIV Drug Resistance Training

1. HIV Drug Resistance Training Module 11: Quality Control and Quality Assurance

2. Topics • Quality Assurance vs. Quality Control • Quality Assurance Before, During, and After Testing • Quality Control • EQA

3. Objectives • Identify requirements for QC/QA. • Identify types of quality controls that must be in place and the rationale for each. • Given individual lab needs, develop a plan for setting up an internal QC program. • Identify types of quality assurance programs and their purpose. • Given individual lab needs, develop a plan for setting up an external QA program.

4. quality assurance vs. quality control What is the difference between QA and QC?

5. Terms • Quality Control • Quality Assurance • Quality Assessment

6. Definitions

7. Quality Assurance vs. Quality Control

8. Quality Control is Part of Quality Assurance "The aim of quality control is simply to ensure that the results generated by the test are correct. However, quality assurance is concerned with much more: that the right test is carried out on the right specimen, and that the right result and right interpretation is delivered to the right person at the right time.” Quality Assurance

9. QA or QC? • HIV-negative plasma control • SOP writing • Personnel records • Phylogenetic analysis • DBS condition inspection • Instrument calibration • Proficiency testing • New operator training • Assay validation • Lot release testing

10. The Quality Assurance Cycle • Data and Lab Management • Safety • Customer Service

11. Discussion What is the difference between QA and QC?

12. quality assurance before, during, and after testing What are some examples of errors that occur before, during, and after testing, and what can we do about them?

13. The Quality Assurance Cycle • Data and Lab Management • Safety • Customer Service

14. Pretesting • Data and Lab Management • Safety • Customer Service

15. Preventing and Detecting Errors Before Testing Lab personnel training and certification Sample criteria set and used Specimen labeling, collection and transport Specimen storage and processing Check storage and room temperature Lab set up Reagent inventory and expiration dates SOPs for all related procedures Preventive maintenance and equipment checks External Quality Assessment (proficiency testing)

16. During Testing • Data and Lab Management • Safety • Customer Service

17. Preventing and Detecting ErrorsDuring Testing Conducting test according to SOPs Quality Control (QC) (in-process controls) Safety precautions Prepare reagents Correctly interpreting test results Time management and planning

18. Post-testing • Data and Lab Management • Safety • Customer Service

19. Preventing and Detecting Errors After Testing Re-check patient/client identifier Write legibly Clean up and dispose of contaminated waste Complete quality control review Positive and negative control results Contamination check (phylogeny) Other QA checking (e.g. SQUAT) Transcribing, reporting and interpreting results Critical review of results and interpretation Record Maintenance

20. SQUAT: Sequence Quality Assessment Tool • QA tool developed for WHO by Dr. Rami Kantor and colleagues, Brown University • Open-source and freely available • To be used by genotyping labs before sending sequences to national HIVDR working groups for surveillance and monitoring activities • Labs should perform initial investigations and take corrective actions if required • Sequences can be assessed by WHO for external quality assurance as part of ongoing accreditation process

21. Sequence alignment HIV PR and/or RT sequence identification Mutation identification and generation of export file for uploading to database Flagging of problematic sequences ambiguous bases (mixtures) stop codons insertions/deletions frameshifts genetic distance anomalies atypical and ambiguous amino acids SQUAT Functions

22. Quality Assurance is Everywhere! • Data and Lab Management • Safety • Customer Service

23. Find It, Then Fix It • Use a problem-solving team. • Investigate root causes. • Develop and implement appropriate corrective actions. • Examine effectiveness. • Record all actions and findings.

24. Discussion Why do errors occur? What are some common errors that might occur with HIVDR Genotyping? Where is QA applied in a HIVDR testing site? What are some steps to take before, during, and after testing to assure the quality of results? Describe the impact that errors will have on public health.

25. Quality Control What do we need to do to set up a quality control program for our genotyping?

26. What is quality control (QC)? • Monitoring the test quality itself • Ensuring that the assay is working appropriately and accurate test results are reported with confidence. • Prevention • Detection • Correction

27. QC: Two Levels/Two Types • Levels • Testing of samples with known results to verify the procedure is working properly • Interpreting the presence or absence of controls. • Types • Internal quality control • External quality control

28. Internal QC • Internal QC samples with known reactivity are included with the following HIVDR test kits. • TruGene • ViroSeq • For in-house assays, prepare and qualify your own internal QC samples • Test the IQC samples as you would test patient specimens. • Include positive and negative controls in every run. • Know what to do in case of unexpected results

29. Genotyping Assay Procedures Positive and negative controls Positive control Patient virus RNA Extraction cDNA synthesis (RT) cDNA amplification (PCR) Sequencing reactions Electrophoresis Data Analysis (base calling) Report (sequence, mutations, interpretation)

30. Guidelines for HIVDR Genotyping Internal QC Policies • Negative controls • HIV-negative plasma, at extraction step • Water, at RT step and at PCR step • If any negative control shows a detectable PCR product, all samples must be discarded • Investigate to locate source of contamination • Positive controls • HIV-positive plasma, at extraction step • HIV DNA at PCR step (low copy number) • If positive control does not produce a PCR product, investigate to determine cause; handling of products from test samples may depend on outcome • HIV DNA at sequencing step • SOPs required

31. External QC • External QC samples do not come with the test kit and are provided by an external source • Regional reference laboratory • Commercial supplier • Another laboratory that has prepared validated quality control in-house samples • Test the EQC samples as you would test patient specimens.

32. Internal or External Control Failure • If QCs fail, repeat the run. • If problems or errors occur, then corrective actions should be taken before results distribution.

33. Planning Your Internal QC Program • Incoming samples • Incoming reagents and kits • Methodology or instrument comparison

34. Incoming Samples • Upon arrival, all incoming sample shipments should be evaluated for proper collection, transport and storage

35. Incoming Reagents and Kits • Evaluate all incoming reagents and kits upon arrival. • Perform lot-to-lot comparisons. • Assay previously tested patient samples or controls on both the previous lot and the new lot and compare the results. • When a new lot has been evaluated and ‘passed,’ label container “Ready to Use.”

36. Lot-to-lot Comparison for Critical Reagents • Lot-to-lot comparison for critical reagents: • For commercial kits: • Perform parallel run with new vs. existing kits • Define acceptance criteria for reagent lot to lot comparison • For home-brew assays: • Perform parallel run with new vs. existing lots • New lot of primers, RT-PCR enzymes, sequencing reagents • Source of specimens • Positive controls • Previously tested samples • Old proficiency panels

37. Critical Reagent Quality Control Extraction buffers RNA Purification Enzymes (RT, DNA polymerase) RT-PCR Primers Sequencing reactions Reaction buffers Capillary matrix polymer Electrophoresis Results

38. Methodology or Instrument Comparison • For tests that have multiple instruments available in the laboratory for use, compare instruments. • Results should be comparable. • Compare and document results routinely (depending on lab throughput). = ?

39. Small Group Discussion • What do you need to do to set up a QC program for genotyping? What components should be included? • If a RT-PCR negative control becomes positive, what should you do? • How can you perform lot-to-lot comparisons in your lab ? • What is critical to include in a QC SOP? • Once you are done with a lot-to-lot comparison and obtained satisfactory results, what should you do for the next step?

40. EQA What is EQA and why is it important? Which EQA Methods should we employ? Proficiency Testing On-Site Evaluation Re-testing

41. A Systems Approach to Laboratory Quality Organization Personnel Equipment Stock Management Quality Control Data Management SOP, Documents & Records Occurrence Management Assessment Process Improvement Specimen Management Safety & Waste Management

42. External Quality Assessment (EQA):Definition and Purpose • Objective assessment of a test site’s operations and performance by an external agency or personnel • Allows comparison of performance and results among different test labs • Provides early warning for systematic problems associated with kits or operations • Provides objective evidence of testing quality • Indicates areas that need improvement • Identifies training needs

43. Management Responsibilities • Determine policies for EQA (WHO, WHAT, WHEN, HOW) • Assign responsibility • Establish & maintain a system and schedule • Receive EQA results and support corrective action measures • Manage corrective action efforts • Monitor and maintain records • Investigate deficiencies • Communicate outcomes

44. Testing Personnel’s Responsibilities: Overview Participate in the EQA program Take corrective actions Maintain EQA records Communicate outcomes to supervisors

45. EQA Methods

46. What is Proficiency Testing? • Panels of specimens are sent to multiple test labs by reference laboratory/EQA provider • Test labs perform tests and report results • Results indicate quality of personnel performance and test lab operations • Results are often compared across several testing labs

47. Proficiency Panels for HIVDR EQA • VQA (NIH-supported, Rush University, Chicago1) • TAQAS (TREAT Asia2) • ENVA/QCMD3 • ANRS France • DigitalPT/Accutest • Acrometrix • Others? 1. Huang et al. J Clin Microbiol.43(8):3963-70 Aug 2005 2. Land et al. Journal of Virological Methods 159(2): 185-193; Aug 2009 3. Pandit et al. J Clin Virol. 43(4): 401-6; Dec 2008

48. What is On-site Evaluation? • Periodic site visits to perform systematic assessment of lab practices • Focuses on how the lab monitors its operations and ensures testing quality • Provides information for internal process improvement

49. What is On-site Evaluation? – Cont’d • Also referred to as audits, assessments, or supervisory visits • Learn “where we are” • Part of every lab quality system • Measures gaps or deficiency • Collect information for: • Planning & implementation • Monitoring • Continuous improvement

50. What is Re-testing? • The process by which a random selection of specimens are collected from the routine workload at the test lab and sent to the reference laboratory for validation • Used to detect errors