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HIV Drug Resistance Training

HIV Drug Resistance Training. Module 8: Dried Blood Spots for HIVDR Genotyping. Topics. Overview of Dried Blood Spots (DBS) How to collect and dry DBS How to package and store DBS Valid and invalid DBS. Objectives. Describe the purpose of using DBS for HIVDR genotyping.

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HIV Drug Resistance Training

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  1. HIV Drug Resistance Training Module 8: Dried Blood Spots for HIVDR Genotyping

  2. Topics • Overview of Dried Blood Spots (DBS) • How to collect and dry DBS • How to package and store DBS • Valid and invalid DBS

  3. Objectives • Describe the purpose of using DBS for HIVDR genotyping. • Identify advantages & disadvantages of DBS vs. plasma. • Describe recommended procedures for collection, handling, transportation, and storage of dried blood samples. • Describe different methods for RNA extraction from DBS. • Identify modifications needed to sequencing procedure when using DBS.

  4. overview of dbs What are the advantages and disadvantages of using DBS for HIVDR genotyping? What do you need in order to use DBS?

  5. What Is a Dried Blood Spot (DBS)? • Whole blood • Collected on filter paper and dried • Used for routinely for metabolic, serology, and HIV diagnosis tests

  6. +/- of Using DBS for HIVDR Genotyping * if kept dry, for at least 2 weeks

  7. Special Considerations When Using DBS for HIVDR Genotyping • Lower sensitivity for amplification – input copy number lower, risk of amplification bias at low VL • Contribution from DNA in WBCs • RNA stability under various storage conditions • Risk for molecular contamination • Biohazard risk (if using scissors or other sharps to cut out DBS circles) • Viral load measured from DBS – is it equivalent to plasma?

  8. Required Supplies for DBS • Blood collection card (filter paper) • Sealable plastic bags • Desiccant packs* • Humidity indicator cards* *or combination dessicant+indicator

  9. Discussion • What are the advantages and disadvantages of using DBS for HIVDR genotyping? • What do you need in order to use DBS?

  10. how to collect and dry dbs How do you collect and dry dried blood spots?

  11. Venipuncture and collection into EDTA tube; manually spot blood on to paper using pipette This method is preferred since greater quantities can be obtained, but requires slightly more manipulation Direct spotting from finger prick or heel prick DBS Collection: 2 Primary Methods

  12. DBS Collection from EDTA Blood • Collect at least 2 ml blood in an EDTA (or ACD) Vacutainer or similar tube • If necessary, the EDTA anti-coagulated blood can be stored for up to 24 hours before DBS preparation, preferably at 4°C

  13. DBS Collection from EDTA Blood Clearly label card with identification number Pipette volume required to uniformly saturate entire circle (usually 50-75 μl) Four complete circles are required for genotyping

  14. DBS Collection from Finger Prick • Use Universal Safety Precautions • Clearly label card with identification number • Follow finger prick procedure • Uniformly saturate entire circle Four complete circles are required for genotyping

  15. DBS Collection: Tips • Do not touch the areas of the card that will be used to collect specimens. • Do not apply blood to both sides of the filter paper. • Do not apply blood more than once in the same collection circle. • Do not apply blood from more than one patient on the same collection card. • Do not press the filter paper against the puncture site. • Do not “milk” the finger.

  16. How to Dry DBS • Dry papers suspended, horizontal • Use a drying rack if possible • Room temperature • Fully air dry (≥4 hours, or overnight) • Do not touch or smear spots • Do not dry in direct sunlight • Do not heat, stack, or allow DBS to touch each other or other surfaces while drying

  17. Drying DBS

  18. Discussion • How do you collect and dry dried blood spots?

  19. how to package and store dbs What are the steps in packaging dried blood spots? What guidelines should be followed in storing and shipping dried blood spots?

  20. How to Package DBS for Storage

  21. 1. Insert Into Sealable Plastic Bag

  22. 2. Add Desiccant Packets

  23. 3. Add Humidity Cards and Seal Bag

  24. 5. Label Outside of Plastic Bag with Contents

  25. How to Store DBS • Keep packaged DBS (in sealable plastic bags) cool and dry until transported to reference laboratory with 2 weeks. • Do not leave in vehicle, as sun and heat will deteriorate DBS.

  26. How to Package DBS for Shipping Insert bundled DBS into rip-resistant envelope. Include appropriate documentation. Insert both into envelope and seal for shipment.

  27. What Are the Collection Site Responsibilities? • Collect valid specimens • Label and store appropriately • Transport appropriately for genotyping • Ensure records are properly maintained • Avoid transcription errors A test result is only as good as the specimen collected!

  28. Examples of "Invalid" DBS Cards Not dry Serum rings Scratched or abraded No blood

  29. Valid or Not?

  30. Discussion • What are the steps in packaging dried blood spots? • What guidelines should be followed in storing and shipping dried blood spots?

  31. Discussion • What are some reasons that dried blood spots may be invalid, and how can we spot them?

  32. More on DBS • If DBS need to stored for more than 2 weeks before genotyping, store at -20°C or lower • Summary of studies (handout bibliography) • WHO plans for validation and proficiency testing

  33. Genotype Assays: Generic Procedure Virus in plasma Virus in DBS RNA extraction (routine, often kit-based procedure) RNA+DNA extraction (special in-house procedure) viral RNA RT-PCR PR-RT DNA Sequencing Protein Sequence Selection Resistance Mutations Interpretation Prediction of Drug Susceptibility

  34. Methods for RNA extraction from DBS • In-house Boom extraction • Nuclisens (BioMerieux) • Qiagen (modified) • Abbott • Others? • All procedures involve capturing total nucleic acids on a matrix (e.g. silica), washing and eluting; some may be relatively more RNA-specific or automated

  35. Extraction of Nucleic Acid from Dried Blood Spots (Nuclisens: Manual-silica-based Method) CUT LYSE BIND WASH DRY ELUTE

  36. Discussion/Reflection • Are we going to use dried blood spots for HIVDR genotyping? • If so, what infrastructure do we need to put in place to ensure valid samples reach the lab? • Facilities • Equipment • SOPs • Personnel • Training

  37. Summary • Overview of DBS • How to collect and dry DBS • How to package and store DBS • Valid and invalid DBS

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