HIV Drug Resistance Training

1. HIV Drug Resistance Training Module 13: Equipment and Supplies

2. Topics • Overview of Responsibilities • Equipment and Supplies Needed for Genotyping • Equipment Use and Care

3. Objectives • Identify specialized equipment and supply needs for a genotyping lab. • Describe how to ensure quality through appropriate care and use of equipment.

4. 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

5. Functioning Equipment is Vital to Quality Service Produces reliable test results Quality Results Lowers repair costs Functioning Equipment Prevents delays in testing Maintains productivity

6. overview of responsibilities What are the responsibilities of lab management? What are the responsibilities of lab staff/technicians?

7. Management Responsibilities

8. Staff/Technician Responsibilities • Attend training and learn all procedures • Properly use the instrument by following SOPs • Perform routine maintenance • Routine function check • Troubleshooting • Maintenance log • Take corrective actions: notifying lab manager when problems arise • Keep records on maintenance and problems

9. Discussion • How will you assign responsibilities for equipment among different members of management and staff?

10. equipment and supplies needed for genotyping What equipment and supplies do all labs need for HIVDR testing? What equipment and supply decisions depend on the testing method?

11. Core Laboratory Equipment • Microcentrifuge • Vortex • Biological safety cabinet • Agarose gel electrophoresis • Computer, printer, Internet connection • UV Imaging system • Reliable power supply (Uninterruptible power supply, UPS, for short outages)

12. Core Laboratory Supplies • Micropipettes • Agarose gel electrophoresis, power supply • Refrigerators and freezers • Distilled, deionized water supply • Sink • Ice machine • Timers

13. Equipment for Genotyping May be influenced by platform (ViroSeq, TruGene, or In-house) • DNA sequencer • PCR machine • Dead-air cabinet (PCR workstation) • Computer attached to sequencer • Specialized software dependent on Platform used • Sequencing kit

14. Laboratory Supplies • Sequencer supplies: capillary array, polymer, buffers • PCR supplies: tubes, plates, trays • RNA/DNA purification: columns, buffers • Electrophoresis: Agarose, ethidium bromide or other stains, buffers, MW markers/standards • Sterile, RNAse-free water • Pipette tips (aerosol-resistant for pre-PCR) • Polaroid film or special printing paper • Disposable gloves • Lab coats (disposable or not)

15. Types of Pipettes • Single, multichannel, automated, repeater • Use disposable, single-use, pipette tips • Positive displacement and aerosol barrier tips recommended for pre-PCR work

16. Equipment: Pre-Amplification Area, In-house Assay • Essential: • Bench with sink / tap water • Biohazard flow, class IIb • Dead air cabinet (master mix preparation) • -20°C freezer and 4°C refrigerator • Microcentrifuge 12,500-15,000 g • Vortex • Timers • Dedicated set of micropipettes for master mix and RNA extraction • Optional: • Ultracentrifuge 21,000-25,000 g • Dead air cabinet (RNA extraction) • -80°C freezer

17. PCR Workstations (Dead-air Cabinets)

18. Equipment: Post-Amplification Area,In-house Assay • Essential: • Bench with sink / tap water • Dead air cabinet (nested reaction) • Thermal cyclers (e.g. ABI, Bio-Rad, Hybaid, etc) • Agarose gel apparatus • Photo documentation of agarose gel • DNA sequencer (e.g. ABI, Beckman) • Computer + programs (editing) • Microcentrifuge 450-550 g • Vortex • Dedicated set of micropipettes • Timers • -20°C freezer • 4°C refrigerator • Optional: • -80°C freezer

19. ABI Sequencing Instruments

20. Alternative Sequencing Instruments Beckman CEQ8000/8800 TruGene

21. Remember Workflow! • Consider lab layout (module 4) when planning equipment needs and placement

22. Reflection • What is our “shopping list?”

23. equipment use and care What can we do to make sure our genotyping equipment is properly cared for?

24. Maintenance • Properly maintained equipment is needed for quality results • Maintenance: Routine and biannual or yearly scheduled maintenance • Reduces breakdowns • Reduces costs in the long run • Minimizes "down time"

25. Introducing New Equipment • Calibrate or verify specified performance upon installation • Pipettes • Centrifuge • Vortex • Etc. • Perform assay equivalence testing • Key components of the genotyping process (sequencer, PCR machine) • 10-20 samples previously reported and verify results within expected range of variability

26. Maintenance: Function Checks • Verify that equipment is working properly • Performed routinely • Daily, weekly, monthly • After adjustment or repair • Example: • Monitoring refrigerator temperatures • Verifying pipette accuracy • Checking centrifuge speed

27. Refrigerator and Freezer: Use and Care • Keep organized • Periodically clean inside and outside • Ensure door is completed sealed when closing • CAUTION! – DO NOT store food items or beverages in laboratory refrigerator or freezer

28. Monitor daily Refrigerator: 4°C (2°C to 8°C) Freezer: -20°C (+/- 5°C) or -80°C (+/- 10°C) If possible install electronic monitoring and alarm system Avoid "frost-free" freezers for storage of critical reagents (enzymes, specimens, etc.) Refrigerator & Freezer:Temperature Checks

29. Refrigerator & Freezer:Temperature Log

30. Keep a Log for All Maintenance Activities

31. Exercise: Create a Maintenance Activity List • Purpose: • Use what you have learned in this module and create a maintenance checklist specific to your test site • Process: • Work in groups of 3-4 (or by test site) • Create a list of maintenance activities on a daily, weekly, monthly, and yearly basis • You can use the handout as a starting point. • Activity time = 10 minutes

32. Discussion • What equipment and supplies do all labs need for HIVDR testing? • What equipment and supply decisions depend on the testing method? • What challenges do you face in your setting regarding equipment and inventory supply?

33. Summary • Overview of Responsibilities • Equipment and Supplies Needed for Genotyping • Equipment Use and Care