Appendix 4. Laboratory tests

Laboratory confirmation is based on testing for specific antibodies to the measles virus (ELISA detection of IgM antibodies). Tests based on virus detection (RT-PCR, sequencing and culture) are used not for diagnosing cases but for studying the genome (global measles surveillance) or for isolating the virus. 

The specimen collection techniques are the same regardless of test type.

4.1 Sample collection

4.1.1 Collecting capillary blood on filter paper

Medical supplies

MODULE DRIED BLOOD SPOT (DBS) & TRANSPORT 2017 [KMEDMSAMDBS3]:

• CHLORHEXIDINE 2%, 70% isopropyl alcohol SWAB/WIPE [DEXTCHLHA2W], 1st choice
• HUMIDITY INDICATOR CARD, 10 - 60 % [ELABHUMI2C-]
• SILICA GEL, granulated, with saturation indicator, 5 g, bag [SLASSILI1C5]
• SAFETY LANCET, high flow, blade 1.2 x 1.5 mm, pink, s.u. [STSSLANCSH3]
• SAMPLE COLLECTION CARD, 5 circles perforated (Munksjö) [STSSSACC2]
• RACK for drying [STSSSACC101]
• BAG, plastic, impervious to gas, zip lock [STSSSACC102]

Additional materials

• ALCOHOL-BASED HAND RUB, solution/gel, 500 mL, bot. [DEXTALCO5S-]
• SUCROSE, 24% oral solution, 2 mL, vial [SDDCSUCR2V2], for children < 6 months
• COMPRESSE, NON WOVEN, 4 plies, 7.5 cm, non sterile [SDRECOMN7N-]
• COTTON WOOL, hydrophilic, roll, 500 g [SDRECOTW5R-]
• SHARPS CONTAINER [SINSCONT+++]
• GLOVE, EXAMINATION, latex, s.u. non sterile [SMSUGLOE1--]

Sample collection procedure

• Perform hand hygiene and, if taking sample from a finger, ask the patient to also perform hand hygiene with soap and water.
• Put on non-sterile gloves.
• The sample collection card must be labelled with the patient’s unique identification number and the collection date. Be careful not to touch the circles.
• Choose the puncture site (finger or heel if child < 6 months). Angle the patient’s hand downward, palm up and choose the finger (middle or ring finger).
• Apply intermittent pressure to the chosen finger or the foot.
• For infants (< 6 months), consider giving an oral sucrose solution 2-3 minutes before the stick; if the procedure lasts more than 5 minutes, a second dose can be given. A sheet or towel can be used to hold the child’s arms, if necessary.
• Thoroughly disinfect the puncture site with a chlorhexidine wipe, using a back and forth motion, for 30 seconds (except in newborns - use warm water and gauze/cotton wool only). Let dry.
• Massage around the area to be pricked before and during collection (not on the collection area itself). Do not squeeze the finger/foot.
• Remove the protective cover on the lancet. Hold the finger firmly and place the lancet on the side of the distal phalanx. For heel sticks, flex the patient’s foot and hold it in position with your non-dominant hand, placing one finger on the arch of the foot and your thumb below the puncture site at the ankle.
• Press the top of the lancet firmly to prick the puncture site and discard the lancet.
• Wipe away the first drop of blood with a piece of gauze or dry cotton wool and then let the blood flow (ideally it should flow ‘on its own’) onto the circles printed on the sample card.
• The blood should saturate the paper and completely fill the number of circles required by the reference laboratory.
• Alternatively, transfer 50 microliters of whole blood using a pipette onto the circles after collecting venous blood (into an EDTA/purple tube).
• Note: If molecular testing is planned, automated pipette tips with filters must be used.
• Apply a compress to the puncture site and press until bleeding stops.
• Take off non-sterile gloves and throw them with the other waste into the appropriate trash bins.

After collecting the sample

The blood-impregnated sample card should dry naturally, in a horizontal position, for 3 to 4 hours in a place where it is protected from direct sunlight, dust, insects, and draughts. Do not allow filter papers to touch each other, especially before the sample is completely dry. There are drying racks like the rack that comes in the kit.

Once dry, each DBS card should be stored in an airtight, transparent transport bag with silica gel packets to absorb moisture and a humidity indicator card. The DBS cards should ideally be stored in the cold chain (+ 2 °C to +8 °C) or at less than 25 °C with no light or humidity, as soon as possible after drying.It is important to ensure that DBS cards are completely dry before packaging. Otherwise, test quality may be poor.

The humidity level of the DBS cards should be checked daily. If the level reaches 30%, the humidity indicator and silica gel should be changed.

For shipping, the DBS cards should be left in their transport bag with a humidity indicator, but with new silica gel packets. DBS cards are exempt from IATA regulations.

4.1.2 Collecting venous blood and preparing serum and plasma (no longer standard)

Collecting venous blood

Medical supplies

• CHLORHEXIDINE 2%, 70% isopropyl alcohol, SWAB/WIPE [DEXTCHLHA2W], 1st choice
• MARKER, permanent, black, fine point LABMARK1B-](tube Ø 13/15 mm, 5 mL) RACK [ELABTUBE12R]
• TOURNIQUET, elastic, 100 x 1.8 cm [EMEQTOUR1--]
• TRAY, DRESSING, 30 x 20 x 3 cm, stainless steel [EMEQTRAD3--]
• SUCROSE, 24% oral solution, 2 mL, vial [SDDCSUCR2V2], for children < 6 months
• COMPRESSE, NON WOVEN, 4 plies, 7.5 cm, non sterile [SDRECOMN7N-]
• COTTON WOOL, hydrophilic, roll, 500 g [SDRECOTW5R-]
• ADHESIVE TAPE, fabric, 2 cm [SDRETAPA025]
• SHARPS CONTAINER [SINSCONT+++]
• GLOVE, EXAMINATION, latex, s.u. non sterile [SMSUGLOE1--]
• For serum: (blds. syst.) TUBE, VACUUM, plastic, K2EDTA, 2 mL, purple [STSSBSVT2E-]
• Pour plasma:
  • TUBE, VACUUM, plastic, SERUM, 2 mL, red [STSSBSVT2S-]
    or TUBE, VACUUM, plastic, SERUM, 4 mL, red [STSSBSVT4S-]
• HOLDER for VACCUM TUBE with needle ejector [STSSBSVVH1- ]
• NEEDLE, sterile, 21G (Vacutainer®) [STSSBSVVN21]
• (SAMPLING SET, with wings, 23G (Vacutainer®) [STSSBSVVN23W]

Sample collection procedure

• Position the patient comfortably with their arm angled downward and supported by an armrest.
• Perform hand hygiene.
• Label the tubes with the patient’s unique identification number and the collection date and time.
• Locate the vein: while this step is not essential, it may turn out to be necessary if the patient’s veins are difficult to see.
• Apply the tourniquet to the chosen limb (four finger widths above the venepuncture site for adults and two finger widths above for newborn and paediatric patients).
• Choose the venepuncture site by palpation. Ask the patient to clench/unclench their fist. Once the vein has been identified, remove the tourniquet.
• Connect the collection tube (Vacutainer®) holder to the blood collection needle.
• For infants (under 6 months):
  • Consider giving an oral sucrose solution 2-3 minutes before venepuncture; if the procedure lasts more than 5 minutes, a second dose can be given.
  • Consider immobilising the child with a towel or asking an assistant for help.
• Perform hand hygiene and put on a pair of non sterile gloves.
• Disinfect the patient’s skin with an antiseptic solution-soaked compress, using a back and forth motion, for 30 seconds. Let dry.
• Apply the tourniquet.
• Uncap the needle and turn it so the bevel faces up. Secure the vein by applying traction with your thumb, taking care not to touch the insertion site.
• Using your dominant hand, in one smooth motion, insert the needle into the vein at an angle of about 15 to 30°.
• Reduce the insertion angle of the needle as soon as you feel it pierce the wall of the vein (or blood flows into the tubing, if using a winged needle), then slightly advance the needle into the vein, if possible.
• Begin collecting the sample by pushing the first vacuum tube into the tube holder until its cap is pierced.
• Fill, according to the vacuum/to the mark indicated, the number of tubes needed; avoid moving the needle in the vein when switching the tubes in the holder. Remove the tubes once the required amount of blood has been drawn.
• When the last collection tube has been filled, loosen the tourniquet before detaching that final tube (if it is a small vein and the tourniquet has been kept in place).
• Place dry cotton wool on the puncture site and remove the needle. Apply enough pressure on the cotton wool to stop the bleeding. If bleeding lasts more than a minute, you can place medical tape over the cotton wool. The nurse can also ask the patient to hold the dressing until the bleedings stops. Never bend the elbow (this increases the risk of haematoma).
• Immediately place the needle in a sharps collector and properly dispose of the other waste using the usual procedures.
• Gently invert the tubes 5 to 10 times.
• Remove the non-sterile gloves and throw them with the other waste into the appropriate trash bins.
• Perform hand hygiene.

After collecting the sample

• Place the tubes into the specimen transport bag or box.
• Store the tubes away from direct sunlight. Follow the usual procedure for transporting samples to the laboratory.

Preparing plasma or serum 

Medical supplies

• FORCEPS, BRUCELLE, 14 cm, straight, inox [ELABFOBR1--], to remove tubes from the centrifuge
• MARKER, permanent, black, fine point [ELABMARK1B-]
• PIPETTE, TRANSFER, graduated, plastic, sterile, s.u. [ELABPIPT1S-]
• CRYOTUBES, 2.0 mL, conical, ext. thread, sterile. DNA/RNAse free [ELABTUMC20EP]
• STORAGE BOX, PP, 9x9 microtubes 1-2 mL, autoclavable [ELABTUMB81PP]
• RACK, PK, 6x4 microtubes, autoclavable [ELABTUMR24PK]
• CENTRIFUGE, hand-operated + 4 tubes 15 mL [ELAECENE1M-], in case electrical centrifuge is not available
• CENTRIFUGE, electrical (Hetich EBA 200), 8 tubes, 230V [ELAECENE9--], spare parts and electrical protection

For serum:

• TUBE, VACUUM, plastic, SERUM, 2 mL, red [STSSBSVT2S-]
• TUBE, VACUUM, plastic, SERUM, 4 mL, red [STSSBSVT4S-]

For plasma: 

• TUBE, VACUUM, plastic, K2EDTA, 2 mL, purple [STSSBSVT2E-]
• TUBE, VACUUM, plastic, K2EDTA, 4 mL, purple [STSSBSVT5E-]
• TUBE, VACUUM, plastic, Li-HEPARINE, 2 mL, green [STSSBSVT2HL]
• TUBE, VACUUM, plastic, Li-HEPARINE, 4 mL, green [STSSBSVT5HL]

Preparing serum

• After collecting blood in a dry/red tube, leave the tubes on the bench for at least 20 minutes to allow the blood to clot completely before centrifugation.
• The tubes should then be spun at 1000 g for 10 minutes. With the Hettich EBA 200, this corresponds to about 3200 RPM (revolutions per minute). As with all centrifuges, be sure to balance by placing tubes of equal weight directly opposite each other. Manual centrifuges have four slots for tubes. The centrifuge must be well-balanced to prevent damage to the rotor. Manual centrifuges can reach a speed of 3000 RPM.
• If there is no laboratory or no available centrifuge, leave the tube at room temperature for 1 hour and then place it in the refrigerator (+2°C to +8 °C) in a vertical position until the clot has completely retracted (leaving the translucent yellow serum). The sample can be left in the refrigerator for a maximum of 24 hours before separating the serum (for ELISA tests).
• Label a cryotube with the patient’s unique identification number and the collection date.
• Transfer the serum into the cryotube with a pipette.

Preparing plasma

• After collecting blood in a tube with anticoagulant (such as: EDTA/purple, heparin/green) thoroughly mix the blood with the anticoagulant by gently and completely inverting the tube 5 to 10 times.
• After that, the tubes can be spun at 1000 g for 10 minutes (or 3200 RPM with the Hettich EBA 200 centrifuge).
• Just like when preparing serum, a manual centrifuge can be used.
• Label a cryotube with the patient’s unique identification number and the collection date.
• Transfer the plasma into the cryotube using a sterile pipette.

Notes :
If molecular testing is planned, sterile pipettes or automated pipette tips with filters must be used.

It is important that the sample not be haemolysed, because haemolysis makes analysis impossible. To prevent that, do not transport the sample collection tube before centrifuging it and separating the plasma. If that is impossible, reduce the risk of haemolysis by placing the tubes in sponges during transport (to reduce jolting).

4.2 Storing samples 

4.3 Transport, packaging, and shipping

Blood on filter paper

These samples are not considered ‘dangerous substances’ according to IATA regulations. However, DBS shipped via transporter require:

• Triple packaging: individual bag + Zip-lock bag+ outer packaging (envelope or cardboard box) measuring at least 10 cm x 10 cm. The mention « Exempt human specimen » must appear on the package (outer packaging) and on the Air Waybill

Whole blood, serum or plasma

Protect each tube in specific triple packaging that meets the regulations for transporting Category B infectious substances, UN 3733.

Before shipping

Verify that:

• The containers are tightly sealed
• The information is entered in the laboratory register
• The patient information form is inside the package

When shipping

• Write the exact address (including the service and the name of the addressee).
• For whole blood or serum: fill out the information on the outer packaging (3373).
• Attach all necessary shipping documents.
• Record the shipment to allow follow-up (receipt at lab and transmission of results).
• Alert the services concerned that the sample(s) have been shipped.

4.4 Reference laboratories

Send specimens to national laboratories, if possible; if not, use the closest WHO LabNet laboratory or contact laboratory advisors.