HIV Drug Resistance Training Module 8 Dried Blood

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

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

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. § § § 3

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

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 § § 5

+/- of Using DBS for HIVDR Genotyping Feature Plasma DBS Requires venipuncture Requires centrifugation Stable at "room temperature" Biohazard for shipping purposes Dry ice required for shipping Routinely used for genotyping Volume range Yes No Yes Yes 1 -5 ml No No Yes* No No* No 0. 25 -0. 5 ml * if kept dry, for at least 2 weeks 6

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? 7

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

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

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

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

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 12

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 13

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 14

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. 15

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 16

Drying DBS 17

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

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? 19

How to Package DBS for Storage 1 • Insert into sealable plastic bag 2 • Add desiccant packets 3 • Add humidity cards 4 • Label contents of bag and seal 20

1. Insert Into Sealable Plastic Bag 21

2. Add Desiccant Packets 22

3. Add Humidity Cards and Seal Bag 23

5. Label Outside of Plastic Bag with Contents 24

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. 25

How to Package DBS for Shipping 1. Insert bundled DBS into rip-resistant envelope. 2. Include appropriate documentation. 3. Insert both into envelope and seal for shipment. 26

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! 27

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

Valid or Not? 29

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

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

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 32

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

Methods for RNA extraction from DBS § § § In-house Boom extraction Nuclisens (Bio. Merieux) 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 34

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

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 36

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