Procedure for finger prick and DBS samples collection [1, 2]

1. Position hand palm-side up. Choose 2. Apply intermittent pressure to the finger
whichever finger is least calloused to help the blood flow

3. Clean the fingertip with alcohol. Start in the 4. Hold the finger and firmly place a new
middle and work outward to prevent sterile lancet off-center on the fingertip
contaminating the area. Allow the area to dry

5. Firmly press the lancet to puncture the 6. Wipe away the first drop of blood with a
fingertip sterile gauze pad or cotton ball

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 7. Allow a large drop of free flowing blood to 8. Apply a gauze pad or cotton ball to the
collect. Quickly and gently touch the card with puncture site until the bleeding stops
blood and let blood soak through. Repeat until
all circles are filled

10. Label the card with the appropriate and 11. Let the cards dry 24h at room temperature,
useful information (the patient ID number, the then put the DBS cards into sealable plastic
date of collection, and the study and visit bags (several cards in one bag plus desiccant)
identifier)

Tips from our lab to improve spot size and quality

- Warm up finger by massage, kneading it / rotating arm

- Press lancet down firmly, use at least 2mmx1.5mm lancet

- Hold finger below height of heart while waiting for drop to form

- If blood flow stopped, wipe the finger, maybe it was already clotted

- Softly (!) press the finger to increase blood flow

- Find which finger works best (index finger is not recommended)

- Maybe ask for a second prick on another finger

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 Procedure for venous blood and DBS samples collection [3, 4]
(for serum samples, heparinized tubes are required)

1. Select the site, preferably at the antecubital

area (i.e. the bend of the elbow). Warming the 2. Apply a tourniquet, about 4–5 finger widths
arm with a hot pack, or hanging the hand down above the selected venipuncture site
may make it easier to see the veins. Palpate the
area to locate the anatomic landmarks. DO NOT
touch the site once alcohol or other antiseptic
has been applied

3. Ask the patient to form a fist so that the 4. Disinfect the site using 70% isopropyl
veins are more prominent alcohol for 30 seconds and allow drying
completely

5. Anchor the vein by holding the patient’s 6. Enter the vein swiftly at a 30 degree angle
arm and placing a thumb BELOW the
venipuncture site

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 7. Once sufficient blood has been collected, 8. Withdraw the needle gently and then give
release the tourniquet BEFORE withdrawing the patient a clean gauze or dry cotton-wool
the needle ball to apply to the site with gentle pressure

9. The tubes should be labeled with the 10. If the tube does not have a rubber
patient ID number, the date of collection, and stopper, press the plunger in slowly to
the study and visit identifier reduce hemolysis

11. Place the stopper in the tube

12. Invert gently 5-10 times the sample in

order to mix the additives with the blood

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13. Pipette 50 μl to each circle to fully saturate it.
It is possible to use a Pasteur pipette.

15. Label filter paper card with appropriate identification information

of the patient

16. Let the cards dry 24h at room temperature

17. Put the DBS cards into sealable plastic bags

(several cards in one bag plus desiccant)

Appropriate DBS handling, drying and storage [1, 2]

- Clearly label card with the subject name, date, ID number and visit

- Do not press the filter paper against the puncture site

- Apply blood to only one side of the filter paper

- Do not apply blood more than once in the same collection circle

- Uniformly saturate entire circle

- Avoid touching or smearing the blood spots

- Allow the specimen to fully air dry overnight at room temperature

- Keep away from direct sunlight

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

- Desiccant packets serve as a drying agent. In places with difficult drying conditions, (humid or foggy)
desiccant packs should be placed in each bag.

- Dried DBS cards should be packaged in sealable plastic bags (several cards per bag, plus desiccant)
and stored at -20°C (or refrigerated if not possible) until analysis

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 Valid DBS Specimen

Blood has been collected in each circle completely saturating or filling the circle.

Blood is soaked through to the other side of the card.

Invalid DBS Specimen [5]

This specimen is invalid because the quantity of blood is insufficient
for testing. Possible cause:
- Removing filter paper before blood has completely filled the
circle of before the blood has soaked through the filter paper
- Applying blood to the filter paper with a capillary tube
- Filter paper coming in contact with gloved hand or
substances (lotion, powder) before or after blood specimen
collection
This specimen is invalid because it appears scratched or abraded.
This may have been caused by applying blood with a capillary tube.

This specimen is invalid because the specimen was not dry before
packaging (let dry 24h at room temperature).

This specimen is invalid because the blood appears clotted or layered.

Possible cause:
- Touching several drops of blood to the same circle
- Filling the circle on both sides of the filter paper
This specimen is invalid because it appears hemolysed, discolored or
contaminated. Possible cause:
- Squeezing or “milking” the punctured fingertip excessively
- Filter paper coming in contact with gloved hand or
substances (lotion, powder) before or after blood specimen
collection
- Exposure of blood spots to direct heat
This specimen is invalid because it inhibits serum rings (serum
separated from cells). Possible cause:
- Not allowing alcohol on finger to dry completely before skin
puncture
- Filter paper coming in contact with gloved hand or
substances (lotion, powder) before or after blood specimen
collection
- Squeezing or “milking” the punctured fingertip excessively
- Not allowing the specimen to dry completely (24h at room
temperature)
- Applying blood to filter paper with a capillary tube

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 Example from our lab: valid/invalid DBS Specimen
1: Valid DBS card

2: Invalid DBS card

3: Invalid DBS card

4: Valid DBS card except spot nr. 5

5: Valid/invalid DBS card

- Valid spots: nr. 3, 4
- Invalid spots: nr. 1,2,5,6

6: Invalid DBS card

REFERENCES

1. WHO. Module 8. Blood Collection-Finger Prick. 2005. Last accessed: 14.11.2012; Available from:
http://wwwn.cdc.gov/dls/ila/hivtraining/section4.aspx.
2. WHO. Module 14. Blood Collection and Handling –Dried Blood Spot (DBS). 2005. Last accessed: 14.11.2012;
Available from: http://wwwn.cdc.gov/dls/ila/hivtraining/section4.aspx.
3. WHO. WHO Manual for HIV Drug Resistance Testing Using Dried Blood Spot Specimens. 2010. Update July
2012. Last accessed: 28.11.2012; Available from: http://apps.who.int/iris/handle/10665/75829.
4. ACTG and IMPAACT. Processing of Dried Blood Spots. Standard Operating Procedure. 2012. Last accessed:
28.11.2012; Available from: https://www.hanc.info/labs/labresources/procedures/ACTGIMPAACT Lab
Manual/55-DriedBloodSpots-LTC-SOP-v2.0-19Mar2012.pdf.
5. The New York State Department of Health. Unsatisfactory Specimens. Simple Spot Check. 2007. Last
accessed: 13.09.2012; Available from:
http://www.whatman.com/References/NeonatalScreeningSimpleSpotCheck%281%29.pdf.

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