SMILE
Johns Hopkins University
Baltimore, MD USA
Preparation of Dried Blood Spots (DBS) - Guidelines
Name of Procedure
Guideline Number
Pro81-07-G
Effective Date
06 Feb 2009
Page
1 of 1
Supersedes
New
Preparation of Dried Blood Spots
Subject
Preparation of Dried Blood Spots - Guidelines
SMILE Comments: This document is provided as an example only. It must be revised to
accurately reflect your lab’s specific processes and/or specific protocol requirements. Users are
directed to countercheck facts when considering their use in other applications. If you have any
questions contact SMILE.
Background Information
Dried blood spot specimens are obtained by placing several drops of blood on to a filter
paper collection device. The blood is generally obtained from a heel stick (infant) or
finger stick (child or adult) but may be obtained from other sources such as an EDTA
venous specimen. Once the dried blood spot specimens are obtained they are dried
and transported to a central laboratory for testing.
The Dried blood spot (DBS) method of collecting blood samples is now routinely used
for collecting blood specimens from infants and adults for many different types of
laboratory testing including HIV PCR. Dried blood spots samples are often used as part
of NIH/DAIDS Clinical Trial protocols as the DBS method provides a convenient way of
collecting and transporting specimens from resource poor setting. However, it should
be noted that different protocols may have different procedures and very specific
requirements for the type of filter paper collections devices and storage bags to be used
for the protocol.
Resources
1. Mei, Joanne :CDC Power Point Presentation: External Quality Assurance for
HIV Rapid Tests Using Dried Blood Spots, downloaded from the internet ,10
December 2008 wwwn.cdc.gov/mlp/pdf/GAP/Mei.ppt
2.
IMPACT Protocol - P1060: A Site-Restricted, International Trial of the
International Maternal Pediatric Adolescent AIDS Clinical Trials Group version
2. 14 May 200 7, Appendix V.
3. Dried Blood Spot Testing : Downloaded 10 December 2008 from
wilipedia.org,,http://en.wikipedia.org/wiki/Dried_blood_spot _testing
4. Clinical Laboratory Standards Institute (CLSI): Blood Collection on Filter Paper
for Newborn Screening Programs - Fifth Edition. CLSI Document LA4-A 5
(ISBN 1-56238-644-1). Clinical and Laboratory Standards Institute, Wayne, PA
Preparation of Dried Blood Spots
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Preparation of Dried Blood Spots
Author(s), Name &
Title
Jo Shim MBA, MT(ASCP)
Document
Number
Effective Date
Pro81-07-SOP
06 Feb 2009
SMILE Comments: This document is provided as an example only. It must be revised to accurately
reflect your lab’s specific processes and/or specific protocol requirements. Users are directed to
countercheck facts when considering their use in other applications. If you have any questions contact
SMILE.
Name, Title
Approved
By
Signature
Date
Signature
Date
Penny Stevens
Name, Title
SOP
Annual
Review
Version # [0.0]
Revision Date
[dd/mm/yy]
Description (notes)
Revision
History
Name (or location)
# of copies
Name (or location)
# of copies
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Associated Forms:
Preparation of Dried Blood Spots
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I acknowledge that I have read, understand and agree to follow this SOP.
Name (print)
Preparation of Dried Blood Spots
Signature
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Date
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Purpose
This procedure provides general instructions for the preparations, collection and
preservation of dried blood spot (DBS) specimens obtained from heel sticks and venous
blood draws.
Scope
The collection of blood using dried blood spots is applicable for many types or testing
and particularly useful for screening tests for newborns and pediatric patients. DBS
specimens are required for many NIH/DAIDS HIV Clinical Drug Trials.
Responsibility
Lab Director
Laboratory Supervisor
All Laboratory technologists, technicians and phlebotomists trained to prepare dried
blood spots.
Definitions
DBS –Dried Blood Spots
DAIDS- Division of AIDS
NIH-National Institutes of Health
Safety precautions
It is essential that Standard Precautions be followed while working with DBS specimens.
Laboratory staff involved in obtaining DBS specimens should use appropriate personal
protective equipment in accordance with the safety procedure followed at [your
laboratory]. For safety reasons, glass capillary tubes are not recommended for
collecting blood for DBS testing.
Procedure
General Instructions
1. For DAIDS HIV network clinical trials, prepare dried blood spot (DBS) cards as
specified by specific protocol standards.
2. Ensure that the filter paper collection devices to be used have not expired.
3. Fill out the DBS card with the required patient information.
4. Prepare to fill the DBS cards by one of the following methods:
a. Blood drawn in an EDTA (purple top) tube
b. Performing a heel stick (infant)
c. Performing a finger stick (Child or adult)
5. Other specimens may be used such as cord blood but these are generally not
recommended for DAIDS/sponsored HIV clinical trials.
6. The DBS filter cards come with printed circles. Always apply blood to the inside
of the circles and fill the entire circle with blood.
Preparation of Dried Blood Spots
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7. Apply blood to only one side of the filter paper (the side with the printing).
An Example of a Filter Paper Collection Device
Place label here
Date of Sample Collection: _____________________
Type of Visit: ________________________________
Causes for Sample Rejection
1. Failure to wipe off alcohol residue as this may dilute the specimen and adversely
affect test results.
2. Milking or squeezing the puncture may cause hemolysis and will add a mixure of
tissue fluids with specimen
3. Use of a capillary tube to apply the specimen to the card. Application of blood
with a capillary tube results in scratching of the surface of the filter paper and
may adversely affect the results.
4. Layering or applying more than one layer of blood onto the filter paper collection
device may also cause failure. This practice causes uneven distribution of
analytes and may invalidate the specimen.
5. Failure to collect the required # if blood spots can also invalidated a specimen.
6. Touching the area inside the filter paper circles before the blood is applied.
7. Insufficient drying before placing the specimen in the designated storage bag.
8. See Appendix A (page 9) for examples of correctly and incorrectly collected
DBS specimens.
Preparation of Dried Blood Spots
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Preparation of DBS Using a Heel stick Specimen
Equipment Needed:
1.
2.
3.
4.
5.
6.
Heel stick blood draw apparatus
Alcohol swab
dry sterile gauze pad
Filter paper collection device
Plastic storage bag
Desiccant pack
Procedure:
1. Prepare infant’s heel for a heel stick blood draw according to the established
procedure [provide reference to your lab’s phlebotomy, specimen management
or other appropriate SOP].
2. Following the performance of the heel stick, use a sterile gauze pad to wipe away
the first drop of blood. The blood is then allowed to flow and large drop should
form prior to blood collection.
3. Collect the blood on the designated filer paper collection device by gently
touching the filter paper to the blood drop. Allow a sufficient quantity of blood
soak through and fill each circle. Do not press the filter paper against the
puncture site on the heel.
4. If you need to obtain more blood gently squeeze the infant’s heel but do not “milk
the heel as this has the effect of mixing interstitial fluid with the blood
5. Once the required number of blood spots are obtained raise the infant’s leg and,
bandage the heel according the established procedure [for your lab].
6. Do not touch the DBS circle once blood is applied.
7. The DBS filter paper collection device should be placed in a horizontal position
and allowed to air dry in a clean dry place that is protected from rodents and
insects for at least 4 hours (over night may be necessary in areas with higher
humidity). The DBS collection device should then be placed in a storage bag.
The type of plastic bag will be designated by the protocol.
8. General recommendations for DBS storage indicate that DBS specimens should
not be stored airtight leak-proof bags such as plastic or foil bags. The lack of air
exchange in airtight bags may lead to heat build-up and moisture collection which
may be detrimental to the DBS specimens.
9. Dry thoroughly before enclosing in storage bag. Desiccant packs should be
added to the selected storage bags to prevent the accumulation of moisture.
Protocol participants should follow the instructions for DBS storage provided in
specific protocols.
Preparation of Dried Blood Spots
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Baltimore, MD USA
10. For NIH/DAIDS studies store no more than one card per storage bag. Once in
the bag, DBS specimens can be stored at room temperature until ready for
processing or shipping.
Preparation of DBS Using Venous Blood Collected in EDTA Tube
Equipment Needed:
1. Vacationer Evacuated Blood Collection Tubes. These tubes are designed to be
filled with a predetermined volume of blood by vacuum. The rubber stoppers are
color-coded according to the additive that the tube contains. Total volume required
per card will be 250 micro liters to fill five spots each at 50 microliters per spot.
Under no circumstances should these purple top tubes be placed in the refrigerator.
2. Alcohol swab
3. Tourniquet
4. Bandage/Plaster
5. Vacutainer Needle
6. Vacutainer Needle Holder
7. Filter paper collection device
8. Storage bag
9. Desiccant pack
Procedure:
1. Draw an EDTA tube following the established procedure for venipuncture. Fill
the EDTA blood collection tube to the recommended volume so that the
anticoagulant is at the proper dilution.
2. Gently invert the tube (5-10 times) to mix the blood thoroughly.
3. After the blood is completely mixed, remove the cap, and using a 50uL micro
pipette and a new tip transfer 50 microliters of whole blood and apply to a single
spot on the designated filter paper collection device.
4. This transfer of blood should be performed with a micropipette and a disposable
tip. Care should be taken not touch the DBS circle once blood is applied.
5. Place the blood spot collection device in a horizontal position. It should be
allowed to air dry in a clean dry place that is protected from rodents or insects.
The DBS card should be dried for at least 4 hours (over night may be necessary
in areas with higher humidity).
6. General recommendations for DBS storage indicate that DBS specimens should
not be stored airtight leak-proof bags such as plastic or foil bags. The lack of air
exchange in airtight bags may lead to heat build-up and moisture collection which
may be detrimental to the DBS specimens.
7. Dry thoroughly before enclosing in storage bag. Desiccant packs should be
added to the selected storage bags to prevent the accumulation of moisture.
Protocol participants should follow the instructions for DBS storage provided in
specific protocols.
Preparation of Dried Blood Spots
Version#: 1.0
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SMILE
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Baltimore, MD USA
8. For NIH/DAIDS studies store no more than one card per storage bag. Once in
the bag, DBS specimens can be stored at room temperature until ready for
processing or shipping.
References
1. Mei, Joanne :CDC Power Point Presentation: External Quality Assurance for HIV
Rapid Tests Using Dried Blood Spots, downloaded from the internet ,10
December 2008 wwwn.cdc.gov/mlp/pdf/GAP/Mei.ppt
2. IMPACT Protocol - P1060: A Site-Restricted, International Trial of the
International Maternal Pediatric Adolescent AIDS Clinical Trials Group version 2.
14 May 200 7, Appendix V.
3. Dried Blood Spot Testing : Downloaded 10 December 2008 from
wilipedia.org,,http://en.wikipedia.org/wiki/Dried_blood_spot _testing
4. Clinical Laboratory Standards Institute (CLSI): Blood Collection on Filter Paper
for Newborn Screening Programs - Fifth Edition. CLSI Document LA4-A 5 (ISBN
1-56238-644-1). Clinical and Laboratory Standards Institute, Wayne, PA
Appendices:
1. Appendix A : Dried Blood Spot Collection Examples
Preparation of Dried Blood Spots
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Appendix A – DBS Collection Examples
Preparation of Dried Blood Spots
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