Module 14

Blood Collection
and Handling –
Dried Blood Spot (DBS)

Purpose To provide you with the skills to collect and handle dried blood
spots (DBS) for EQA purposes.

Pre-requisite Module 6: Safety at HIV Rapid Testing Site

Modules
Module 8: Blood Collection – Fingerprick
Module 13: EQA

Learning At the end of this module, you will be able to:

Objectives

Collect dried blood spots (DBS)

Package and store DBS in a way to maintain specimen
integrity

Maintain DBS records

Distinguish between valid and invalid DBS

Content Outline Required supplies

How to collect and dry DBS
How to package and store DBS
Valid and invalid DBS
Hands-on practice

Handout Example Specimen Transfer Log for Re-Testing

Notes on
Customization

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 What Is a Dried Dried Blood Spots (DBS) are whole blood collected on filter paper
Blood Spot and dried. They are made directly from the client’s whole blood.
(DBS)?
DBS are used for re-testing at a reference laboratory, which may
be part of your country’s External Quality Assessment plan.
Testing site results are compared to reference laboratory results.
DBS samples are useful for re-testing as they are easy to collect,
store, and transport.

EQA Re-testing Remember…Two types of specimens may be collected at the

site for referral to a reference lab for re-testing: serum/plasma or
dried blood spots (DBS).

What Are Your Your EQA responsibilities include:

Responsibilities?
• Collecting valid specimens
• Labeling and store appropriately until transported for re-
testing
• Ensuring records are properly maintained
• Avoiding transcription errors
A test result is only as good as the specimen collected.

EQA Specimen An example EQA specimen transfer log is provided for your
Transfer Log reference at the end of this module.

Data recorded on transfer logs must be accurate. Care should be

taken in transferring information from test records to EQA transfer
log. Errors that are typically made include:

Specimen ID on transfer log sheet doesn’t match
specimen ID filter paper

Final result at testing site incorrectly transcribed from test
records

Required When collecting DBS, you

Supplies for DBS need the following items:
• Blood collection card (filter
paper)
• Glycine weighing paper
• Sealable plastic bags
• Humidity cards
• Desiccant packs

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 Other supplies include safety supplies such as discard bins and
supplies for performing a fingerprick. While the collection card
may include 5 circles, only one patient/client’s blood may be
collected on one card.

How to Collect Always use Universal Safety

DBS Precautions. This includes:

Treat all blood samples as
though they are infectious

Wash hands

Wear gloves and apron/lab
coat

Take precaution to avoid
needle injury
It is critical that entire circle
• Dispose of contaminated be uniformly saturated.
sharps and waste
appropriately

Clearly label each card with appropriate identification number – It

is unacceptable to submit a blood card for testing that has not
been properly labeled.

Follow the fingerprick procedure and the additional steps below:

Apply gentle pressure to the finger and allow a large drop
of free flowing blood to collect at the puncture site.

Working quickly, hold the filter paper by the edges and
touch the filter paper gently against the large drop of
blood and in one step allow a sufficient quantity of blood
to soak through and completely fill or saturate a circle. A
completed saturated spot will contain 100 µl of blood.

Repeat, until you have collected enough blood to fill at
least 3 circles on the blood collection card.

Completing filling the circle is important because the
laboratory will need to use a hole puncher to punch a
section of the circle of blood for testing

If collecting spots using a pipette, collect 100 µl of blood
and gently apply to filter paper.

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 Additional tips:
• DO NOT press the filter paper against the puncture site.
• Apply blood to only one side of the filter paper.
• Do not layer successive drops of blood or apply blood
more than once in the same collection circle.

Do not “milk” the finger as excessive milking or squeezing
the puncture site might cause hemolysis of the specimen
or result in collection of tissue fluids with the specimen,
which might adversely affect the test result. (NOTE:
“hemo-” means red cells, “-lysis” means destruction,
“hemolysis” means destruction of red cells.)
NOTE: Two complete circles are better than five incomplete
ones! While the collection card may include 5 circles, only one
patient/client’s blood may be collected on one card.

How to Dry DBS Important tips for drying DBS:

Avoid touching or smearing the blood spots

Allow the specimen to fully air dry horizontally (at least 3
hours) at room temperature

Keep away from direct sunlight - Care should be taken to
avoid exposing DBS to environmental conditions that may
compromise the integrity of the specimen. DBS should
not be dried near an open window as sunlight, dust and in
some cases flying insects may come in contact with the
DBS during the drying procedure.

Do not heat, stack or allow DBS to touch other surfaces
during the drying process

Dry Completely There are several ways in which you can effectively dry DBS
Before
Packaging

The image on the left is one that was taken in India. DBS are
dried horizontally between bamboo.
The image on the right can be obtained from Schleicher &
Schuell Bioscience, Inc. (Manufacturer of DBS collection cards)
DBS change from bright red to dark red as they dry.

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 How to Package Follow these steps:
DBS for storage:
1. Appropriately stack DBS
An overview
2. Insert into sealable plastic bag
3. Add desiccant packets
4. Add humidity cards
5. Label contents of bag and seal.

Step 1 - Stack Place weighing or glycine paper between DBS cards before
DBS transport to prevent cross-contamination.

Place filter paper between Fold weigh ends of weighing

sheets of weighing paper paper

Step 2 - Insert You can typically place up

Into Sealable to 15 DBS cards in each
Plastic Bag plastic bag. The bag should
be just the right size to hold
the cards. Avoid using bags
that are too big as the cards
will shuffle inside the bag.

Sandwich bags will not work as many of these are of the type
where you fold the top. The bag should be a sealable heavy duty
plastic bag, one that will prevent moisture from entering.

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 Step 3 - Add
Desiccant
Packets

Desiccant packets serve as a drying agent. At least 5 desiccant

packs should be placed in each bag. Some sites may need more
or fewer packets in each bag depending on environmental and
storage conditions.
Desiccant packets vary in size. The ones seen here are the
smaller ones.

Step 4 - Add Often humidity cards must be

Humidity Cards recharged before use. If the
and Seal Bag humidity card is pink at the
30% level, you must:

Recharge card and
desiccant pack by heating
at 50-60○C for 3-4 hours in
a drying oven.

Cool 10 minutes.

IMMEDIATELY RETURN Note: If a drying oven is not
card and desiccant pack to available, place cards and
sealable plastic bag. excessive number of desiccant
packets in a sealable bag or
envelope

Step 5 - Label A permanent marker

Outside of should be used to clearly
Plastic Bag with label the contents on the
Contents bag.

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 How to Store Keep packaged DBS (in sealable plastic bags) cool and dry until
DBS transported to reference laboratory. It is also acceptable to store
DBS in a Styrofoam box.
Avoid leaving it in a vehicle, as sun and heat will deteriorate DBS
Avoid placing spots in an malfunctioning refrigerator where water
may drip on or soak the spots

How to Package
DBS for Shipping

Follow these steps:

1. Insert bundled DBS into rip-resistant envelope. The
sealable plastic bag has a label on the outside indicating
bio-hazardous contents.
2. Include appropriate documentation (such as EQA
Specimen Transfer Log) with the shipment
3. Insert both into brown envelop and seal for shipment.

Valid DBS This specimen is valid because:

Specimen
• 100 µl of blood has been
collected in each circle
completely saturating or filling
the circle
• The filter paper card has been
labeled with appropriate
identification.
• Blood is soaked through to
the other side of the card.

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 Valid DBS On the card with MB/KP/120,
Specimen the blood is spreading from one
circle to another due to the
anemia (anemic blood is more
fluid). This is still considered a
valid specimen. Blood has
completely filled the circle.
Notice that the third and fifth
circles have been punched
(hence the white area in the
middle).

Invalid DBS
Specimen

This specimen is invalid because quantity of blood is insufficient

for testing. This may have been caused by::
• Removing filter paper before blood has completely filled
circle or before blood has soaked through to the other
side
• Applying blood to filter paper with a capillary tube
• Filter paper coming in contact with gloved or ungloved
hands or substances such as hand lotion or powder,
either before or after blood specimen collection

Invalid DBS
Specimen

This specimen is invalid because it appears scratched or

abraded. This may have been caused by applying blood with a
capillary tube or other device.

Invalid DBS
Specimen

This specimen is invalid because the specimen was not dry

before mailing. DBS must dry a minimum of 4 hours before
packaging and shipping.

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 Invalid DBS
Specimen

This specimen is invalid because the specimen appears clotted

or layered. This may have been caused by:
• Touching the same circle on the filter paper to blood drop
several times
• Filling circle on both sides of filter paper
The volume of specimen will not be uniform between spots
resulting in errors during the testing process.

Invalid DBS
Specimen

This specimen is invalid because the specimen appears

hemolyzed, discolored, or contaminated. This may have been
caused by:
• Squeezing or “milking” of area surrounding the puncture
site
• Allowing filter paper to come in contact with glove or
ungloved hands or substances either before or after blood
collection
• Exposing blood spots to direct heat

Invalid DBS
Specimen

This specimen is invalid because the specimen exhibits serum

rings – in other words, serum becomes separate from cells. This
may have been caused by:
• Not allowing alcohol to dry at puncture site before making
skin puncture
• Allowing filter paper to come in contact with alcohol, hand
lotion, etc.
• Squeezing area surrounding puncture site excessively
• Drying specimen improperly
• Applying blood to filter paper with a capillary tube

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 Invalid DBS
Specimen

This specimen is invalid because no blood was applied to the

filter paper.

DBS are used for re-testing at a reference laboratory,

which may be part of your country’s External Quality
Assessment plan

Care must be taken to collect, dry, store, and ship dried
blood spots used for re-testing.
Key message

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 Module Review

Find out how much you have learned by answering these questions.

Describe the procedures for collecting DBS.

__________________________________________________________________

__________________________________________________________________

__________________________________________________________________

Describe the procedures for drying DBS.

__________________________________________________________________

__________________________________________________________________

__________________________________________________________________

Describe the procedures for packaging DBS for storage.

_____________________________________________________________________

_____________________________________________________________________

____________________________________________________________

Describe the procedures for storing DBS.

__________________________________________________________________

__________________________________________________________________

__________________________________________________________________

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 Module Review

Find out how much you have learned by answering these questions.

Describe the procedures for packaging DBS for shipping.

__________________________________________________________________

__________________________________________________________________

__________________________________________________________________

Describe the criteria for valid DBS samples.

_____________________________________________________________________

_____________________________________________________________________

_____________________________________________________________________

What may have caused a DBS sample to be invalid?

__________________________________________________________________

__________________________________________________________________

__________________________________________________________________

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 Example Specimen Transfer Log for Re-testing
[Insert Name of Referring Testing Site,
Contact Name
Address and Phone Number]

Date: _______________________ Referring Testing Site _________________________________

Specimen Test Subject Final Result Date Specimen Collected by Referral Lab Date to Date Conf Result of
†
Tracking ID* (Testing Specimen Type Req referral lab Result Re-test
Number Site) Collected (DBS or Completed Received
Serum) )
(

*ID = Identification
†
Lab Req = Laboratory Requisition

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