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Global Polio Laboratory Network

Document version

(date)

Description of substantive revisions

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Guidance

Paper 3

Version 1, 28 Dec. 2016

Poliovirus antibody testing

for GPLN personnel using

Dried Blood Spot

3

TABLE OF CONTENTS

1. Background _________________________________________________________________________ 4

2. Objective ____________________________________________________________________________ 4

3. Scope ________________________________________________________________________________ 4

4. Responsibility ______________________________________________________________________ 4

5. Documentation _____________________________________________________________________ 4

6. Biosafety precautions ______________________________________________________________ 4

7. Materials needed __________________________________________________________________ 5

8. Collection of DBS from Fingerprick ______________________________________________ 6

9. Collection of DBS from whole blood in EDTA tube______________________________ 6

10. Drying of DBS specimens _________________________________________________________ 7

11. Storage of DBS specimens _________________________________________________________ 8

12. Shipping of DBS specimens ________________________________________________________ 9

13. Examples of invalid spots and invalid collection of DBS _____________________ 10

14. References ________________________________________________________________________ 12

15. Example of Documentation of DBS samples ___________________________________ 13

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1.Background

The rationale for implementing poliovirus antibody testing in Global Polio Laboratory

Network (GPLN) facilities is to document the immune status of polio laboratory staff against all

three types of poliovirus (PV), and to determine their potential need for immunization. It will

also allow meeting GAP III requirements for PV antibody testing and vaccine during the

Containment of PV2 in Phase II and Final Containment of PV in Phase III. Reference can be

made to the Biorisk Management Standards described in GAP III Annex 2, 3 and 6, Sub-

element 9.1.2 (Worker Health Programme) and Sub-element 9.2.1 (Vaccination of Personnel).

Polio seroimmunity can be assessed using either serum or dried blood spots (DBS).

While serum is relatively easy to collect and process, countries that face difficulties in

shipping liquid specimens may prefer to use DBS. DBS are prepared by applying a small amount

of whole blood onto filter paper cards and dried. Specimens can be shipped ambient as non-

dangerous goods. Care must be taken, however, to properly collect, dry, store and ship DBS to

ensure valid and accurate results.

2.Objective

To describe the procedure for collecting, packaging, storing and shipping samples collected

as dried blood spots (DBS) for poliovirus antibody testing of personnel in GPLN facilities.

3.Scope

This document applies to personnel of GPLN facilities (and other institutional personnel

considered to have significant risk of exposure to poliovirus materials) who cannot be tested

locally in a certified Polio-Essential Facility (PEF), or whose serum samples cannot be shipped

to a PEF of their choice. The determination of PV antibody titres shall be done annually.

DBS specimens will not be used for testing parameters other than polio antibody at

designated GPLN reference laboratory and the testing laboratory will not receive personal

information related to any specimens.

4.Responsibility

The Head of GPLN polio laboratory is responsible for the implementation of this guidance

document. He/she is responsible for :

• collecting valid specimens, timely shipment to designated reference laboratory,

and obtaining feedback of results;

• maintaining complete documentation of specimens and confidentiality of results

and records;

• implementing measures according to institutional policy for seronegative staff

or those with low-antibody titer (below protective level) by providing booster

vaccine, e.g. IPV vaccination if type 2 or bOPV vaccination if type 1 or 3, and

follow-up antibody response evaluation.

5.Documentation

A linelist of all specimens collected and referred should be maintained containing the

required identifiers such as Code of staff, Specimen ID, Date of Collection, Date of last OPV or

IPV dose, No. years of exposure to Poliovirus materials, Referral Lab, Date referred, Result of

antibody testing, Date Results Received, and Remarks (e.g. reimmunization, date, vaccine type,

condition of DBS). An example of the linelist is provided in Section 15.

6.Biosafety Precautions

It is essential that Universal Precautions be taken while working with or collecting DBS

specimens. DBS on filter paper are not considered to be a biohazard, but whole blood handled

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during specimen collection may be hazardous. Appropriate personal protective equipment

(PPE), including gloves and lab coat, should be worn to ensure safe handling of specimens.

Precaution should be taken to avoid needle injury. If a needle puncture should occur, the

institutional policy for handling work-related injuries should be followed. Wash hands after

handling DBS samples. Sharps and other contaminated materials should be disposed according

to the laboratory’s biosafety procedures.

7.Materials needed

7.1. The following materials will be supplied through the WHO Regional Office (Fig 1):

• Whatman Protein Saver Card #903* (Whatman 10531018) - 5 spots

• Cardboard drying rack, 10 cards slots (Whatman 10537173)

• Desiccant packs (Whatman 10548234)

• Humidity Indicator Cards 5%, 10%, 60% RH (DESCO 13859)

• Sealable (Zip-lock) plastic bags

Important:

Whatman Protein Saver Card #903, Desiccant packs and Humidity Indicator Cards

should be kept securely sealed in a dry area of the laboratory that is protected from

heat (e.g. direct sunlight, motor, etc..) and other adverse conditions during storage.

7.2. The following materials will be provided/prepared by the laboratory:

• Supplies for blood collection, e.g. single-use blood lancets for Fingerprick or

syringe with needle and EDTA Vacutainer tube (purple top) for venipuncture

collection.

• Alcohol pad

• Bandage

• Gloves for specimen collection and handling of filter paper cards

• Discard bins

• Waterproof marker for labelling

Fig. 1. Materials to be supplied by WHO for DBS specimen collection

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8.Collection of DBS from Fingerprick

8.1. Clearly label each Whatman Protein Saver Card #903 with Specimen ID and Date of

collection. Do not include personal identifiers other than Specimen ID.

8.2. Follow the Fingerprick procedure and the additional steps below (Fig. 2):

• Before skin puncture, massage finger to enrich the blood flow towards the

puncture site.

• Clean the puncture area with an alcohol pad (or a suitable disinfectant) and allow

to dry. Do not touch the puncture site or allow to come into contact with any non-

sterile item or surface.

• Puncture the skin with a single-use blood lancet.

• Apply gentle pressure to the finger and allow a large drop of free-flowing blood to

collect in the puncture site. Do not press the filter card against 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 uniformly soak through until the circle is full or saturated. A

completely filled or saturated spot will contain 100 µl of blood.

• Repeat the procedure 4 times to fill all 5 circles of the card.

• Do not touch the DBS circle once blood is applied.

• Proceed to Section 10: Drying of DBS specimens.

Important:

• Do not squeeze or “milk” the finger/puncture site excessively as this might cause

hemolysis of the specimen or result in collection of tissue fluids with the

specimen.

• Perform a second puncture on another finger if more blood is needed to fill all the

required circles.

• Do not layer successive drops of blood in the same collection circle.

• See example of a valid DBS specimen in Fig. 4 and bad spots in Section 13.

Fig. 2. Collection of DBS from Fingerprick

9.Collection of DBS from whole blood collected in EDTA Vacutainer tube

9.1. Clearly label each Whatman Protein Saver Card #903 with Specimen ID and Date of

collection. Do not include personal identifiers other than Specimen ID.

9.2. Follow the procedure for venipuncture and the additional steps below:

• Collect blood in EDTA tube to the recommended volume so that the anticoagulant

is at the proper dilution.

• Gently invert the tube several times to mix the blood and anticoagulant

thoroughly.

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• Using a pipette, gently apply 100 µl of blood to the center of one circle of the filter

card until the circle is fully saturated (Fig. 3). Caution: Do not touch the surface of

the card with the pipette tip.

• Repeat the procedure 4 times to fill all 5 circles of the card.

• Proceed to Section 10: Drying of DBS specimens.

Important:

• Do not apply blood more than once in the same circle.

• Do not touch the DBS circle once blood is applied.

• See example of a valid DBS specimen in Fig. 4 and bad spots in Section 13.

.

Fig. 3. Collection of DBS from a Vacutainer tube

Fig. 4. Example of a valid DBS specimen

10.Drying of DBS Specimens

10.1. Allow the blood spot to fully air dry without the card flap covering the spots, in a clean,

dry place, e.g. Drying rack (Fig 5) or biosafety cabinet, that is protected from insects,

rodents, and direct sunlight for at least 4 hours or overnight (in areas with higher

humidity). DBS change from bright red to dark red or brownish colour as they dry

(Fig. 4).

10.2. When drying is complete (i.e., no red areas are visible), tuck in the flap of the Whatman

Protein Saver Card #903 as indicated on the card.

10.3. Seal the card in a zip-lock bag containing at least two desiccant packs and one

Humidity Indicator Card (HIC). Store only one specimen card in a single bag (Fig. 5).

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Important:

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

• Do not use electric dryer or oven to dry the cards.

• Keep away from direct sunlight. 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 process.

• Care must be taken to avoid exposing DBS to environmental conditions that may

compromise the integrity of the specimens.

Additional tip:

• Ensure that the bag is properly sealed to prevent moisture from entering.

• HIC and desiccant packs can be re-charged before use. If the HIC is pink at 10%

level, recharge card and desiccant packs by heating at 50-60°C for 3-4 hours in a

drying oven. Heat only the HIC and desiccant packs, and not the DBS. Cool for 10

minutes and IMMEDIATELY RETURN card and desiccant packs to sealable plastic

bag.

Fig. 5 Example of drying DBS specimens in Whatman Cardboard drying rack and

storage of DBS

11.Storage of DBS Specimens

11.1. DBS cards in sealed bag (with desiccant and HIC) may be stored in a refrigerator (2-

8°C) or at ambient temperature (15 - <30°C) for up to 14 days. However, for optimal

preservation, long term storage (>90 days) at ≤ 20 °C is recommended.

11.2. Store DBS cards (preferably in a Styrofoam box) in a cool, dry place that has no

exposure to direct sunlight or dripping water (e.g., malfunctioning refrigerator,

ceiling), free of insects and rodents, and where ambient temperature will not exceed

30°C until shipped to a reference laboratory.

11.3. Check HIC in bags weekly while DBS are stored at ambient temperature or in a

refrigerator. Change (or recharge) desiccant packs and HIC, if HIC colour changes

from blue to pink and document date.

11.4. Make sure the zip-lock bags are sealed tightly.

12.Shipping of DBS Specimens

12.1. Ship DBS specimens to Centers for Disease Control and Prevention (CDC) or other

polio serology reference laboratory of choice, as soon as possible.

12.2. DBS cards that are stored at room temperature may be shipped at ambient

temperature.

12.3. DBS cards stored in a refrigerator or freezer must be equilibrated to room

temperature (minimum of 30 minutes) prior to shipping under ambient conditions.

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12.4. Inspect DBS specimens for contamination or humidity change, and replace or add

desiccants prior to shipping. Record any observation (good or bad condition).

12.5. DBS are exempt biological specimens according to ICAO and IATA . They can be

shipped via courier (or EMS Postal Service) as non-dangerous goods, avoiding arrival

on weekend or holidays.

• Use the basic triple-packaging system where the primary container is the filter

paper that contains the DBS, the sealable (zip-lock) bag enclosing the individual

DBS card (with biohazard marking) as the secondary container . The individual

DBS card in zip-lock bag may be placed in a bigger sealable plastic bag with

additional desiccant packs (5-10 pcs. depending on the number of cards) and HIC

to provide additional protection against humidity before placing in tertiary

container..

• Place in a tertiary container that is sturdy and of high quality paper, e.g. shipping

envelope supplied by courier.

• Enclose a linelist of DBS specimens containing the following information:

Laboratory name, Specimen ID and Date of collection. Ensure that the list matches

the specimens in the package.

• No biohazard sign or content marking is required.

• Clearly indicate delivery and return address.

• Refer to Polio Lab Manual 4th Ed. for other relevant shipping arrangements.

12.6. Email an electronic copy of the linelist at least 2 weeks in advance of shipping date.

• For shipments to CDC, send email to Dr William Weldon < [email protected]> and

Dr Steve Oberste <[email protected],>.

Address the package to:

Dr William Weldon

Centers for Disease Control and Prevention

Division of Viral Diseases

Polio and Picornavirus Laboratory Branch

1600 Clifton Rd., N.E., Mailstop G17

Atlanta, Georgia 30329

Email address: [email protected]

Tel. No: +1-404-639-5485

• For all shipments, copy the following in email communications:

WHO Regional Laboratory Coordinator and WHO HQ (Dr Ousmane Diop

<[email protected]> and Dr Fem Paladin <[email protected]>)

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13.Examples of Invalid Spots and Invalid Collection of DBS

• Sample with variable volume, the last two circles are not usable.

• Spot with incomplete absorption resulting to insufficient blood volume.

Possible causes:

- Removing the filter paper before blood has completely filled the circle or before

blood has soaked through the other side.

- Applying blood to the DBS card with a capillary tube.

- DBS paper coming in contact with gloved hands or substances such as hand lotion or

powder, either before or after blood specimen collection.

• DBS specimen appears scratched or abraded

Possible causes:

- Blood sample was potentially rubbed against the finger

- Applying blood with a capillary tube or other device.

• DBS specimen appears supersaturated

Possible causes:

- Applying excess blood to filter paper

- Applying blood on both sides of filter paper

• DBS specimen not dry before shipment.

Possible cause:

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- DBS not allowed to air dry completely before placing in zip-lock bag or before

mailing. DBS must dry for a minimum of 4 hours or overnight, especially in areas

with high humidity before packaging.

• DBS specimen appears clotted or layered. The volume of blood will not be uniform

between spots resulting in errors during the testing process.

Possible causes:

- Touching the same circle on the filter paper to blood drop several times

- Filling circle on both sides of filter paper

• DBS with diluted, hemolyzed or contaminated blood

Possible causes:

- Squeezing or “milking” the area surrounding the puncture site

- Allowing filter paper to come in contact with gloved or ungloved hands or

substances such as alcohol, water, hand lotion, or powder, etc. before or after blood

specimen collection

- Exposing blood spots to direct heat,

• DBS specimen exhibits serum rings

Possible causes:

- Not allowing alcohol to dry at puncture site before making the 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

• DBS card where no blood was added

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14.References

• WHO Global Action Plan (GAP III) to minimize poliovirus facility-associated risk after

type-specific eradication of wild polioviruses and sequential cessation of oral polio

vaccine use. WHO/POLIO/15.05 .

http://polioeradication.org/Portals/0/Document/Resources/PostEradication/GAPIII_2

014_CH.pdf

• ACTG/IMPAACT Lab Tech Committee, Processing of Dried Blood Spots Standard

Operating Procedure (LTC-SOP-55 v2.0), Users of the ACTG/IMPAACT Laboratory

Manual, 19Mar2012.

• Module 14: Blood Collection and Handling.

cdrwww.who.int/diagnostics_laboratory/documents/guidance/pm_module14.pdf

• Nico Grüner1, Oumaima Stambouli1, R. Stefan Ross. Dried Blood Spots - Preparing and

Processing for Use in Immunoassays and in Molecular Techniques. J. Vis. Exp. (97),

e52619, doi:10.3791/52619 (2015).

• GE Healthcare Life Sciences. Simple Spot check. Oct 15,

2016.https://www.gelifesciences.com/gehcls_images/GELS/Related%20Content/Files/

1392818611307/litdoc28984392_20161015134119.pdf

• Centers for Disease Control and Prevention. Shipping Guidelines for Dried-Blood Spot

Specimens.

https://www.cdc.gov/labstandards/pdf/nsqap/Bloodspot_Transportation_Guidelines.p

df

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15.Example of Linelist for Documentation of DBS samples