HIV Drug Resistance Training
Module 8:
Dried Blood Spots for
HIVDR Genotyping
1
Topics
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Overview of Dried Blood Spots (DBS)
How to collect and dry DBS
How to package and store DBS
Valid and invalid DBS
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Objectives
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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.
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overview of dbs
What are the advantages and disadvantages of using DBS for
HIVDR genotyping?
What do you need in order to use DBS?
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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
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+/- 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
Yes
No
Yes
Yes
Yes
1-5 ml
No
No
Yes*
No
No*
No
0.25-0.5 ml
* if kept dry, for at least 2 weeks
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Special Considerations When Using DBS for
HIVDR Genotyping
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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?
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Required Supplies for DBS
Blood collection card
(filter paper)
 Sealable plastic bags
 Desiccant packs*
 Humidity indicator
cards*

*or combination dessicant+indicator
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Discussion
What are the advantages and disadvantages of
using DBS for HIVDR genotyping?
 What do you need in order to use DBS?

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how to collect and dry dbs
How do you collect and dry dried blood spots?
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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
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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
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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
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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
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DBS Collection: Tips
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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.
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How to Dry DBS
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Dry papers suspended, horizontal
– Use a drying rack if possible
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Room temperature
Fully air dry (≥4 hours, or overnight)
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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
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Drying DBS
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Discussion
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How do you collect and dry dried blood spots?
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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?
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How to Package DBS for Storage
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2
3
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• Insert into sealable plastic bag
• Add desiccant packets
• Add humidity cards
• Label contents of bag and seal
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1. Insert Into Sealable Plastic Bag
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2. Add Desiccant Packets
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3. Add Humidity Cards and Seal Bag
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5. Label Outside of Plastic Bag with
Contents
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How to Store DBS
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Keep packaged DBS (in
sealable plastic bags)
cool and dry until
transported to
reference laboratory
with 2 weeks.
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Do not leave in vehicle,
as sun and heat will
deteriorate DBS.
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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.
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What Are the Collection Site Responsibilities?
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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!
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Examples of "Invalid" DBS Cards
Not dry
Serum rings
Scratched or abraded
No blood
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Valid or Not?
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Discussion
What are the steps in packaging dried blood
spots?
 What guidelines should be followed in storing and
shipping dried blood spots?
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Discussion
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What are some reasons that dried blood spots
may be invalid, and how can we spot them?
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More on DBS
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If DBS need to stored for more than 2 weeks
before genotyping, store at -20°C or lower
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Summary of studies (handout bibliography)
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WHO plans for validation and proficiency testing
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Genotype Assays: Generic Procedure
Virus in plasma
RNA extraction
(routine, often
kit-based
procedure)
Virus in DBS
viral RNA
RNA+DNA extraction
(special in-house procedure)
RT-PCR
PR-RT DNA
Sequencing
Protein Sequence
Selection
Resistance Mutations
Interpretation
Prediction of Drug
Susceptibility
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Methods for RNA extraction from DBS
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In-house Boom extraction
Nuclisens (BioMerieux)
Qiagen (modified)
Abbott
Others?
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All procedures involve capturing total nucleic
acids on a matrix (e.g. silica), washing and
eluting; some may be relatively more RNAspecific or automated
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Extraction of Nucleic Acid from Dried Blood
Spots (Nuclisens: Manual-silica-based Method)
CUT
LYSE
BIND
WASH
DRY
ELUTE
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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?
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–
–
–
–
–
Facilities
Equipment
SOPs
Personnel
Training
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Summary
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Overview of DBS
How to collect and dry DBS
How to package and store DBS
Valid and invalid DBS
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