How long should blood clot before centrifuge?

I. Clinical specimens for IgM and IgG antibody detection

Clinical samples for the diagnosis and surveillance of measles and rubella should be obtained at the first contact between the patient with the clinical case and the health care system, irrespective of the stage of disease at which the patient presents. Depending on the country, blood obtained by venipuncture, dried capillary bloodspots on filter paper and/or oral fluid may be used.

I.A. Whole blood for IgM and IgG antibody detection

Blood collection for serum by venipuncture and handling

Blood should be collected in a sterile tube (5 ml for older children and adults and 1 ml for infants and younger children) and labelled with the patient's name and/or identification number and the collection date.

Whole blood can be stored at 4–8°C for up to 24 hours before the serum is separated, but it must not be frozen.

Whole blood should be allowed to clot and then centrifuged at 1000 × gravitational units (g) for 10 minutes to separate the serum. If there is no centrifuge, the blood can be kept in a refrigerator (4–8°C) until there is complete retraction of the clot from the serum (no longer than 24 hours).

The serum should be carefully removed with a fine-bore pipette to avoid extracting red cells, and transferred aseptically to a sterile vial labelled with the patient's name or identifier, date of collection and specimen type.

A measles/rubella laboratory request form should be fully completed when the specimen is collected and must accompany all specimens sent to the laboratory (Fig. 1).

Fig. 1

Measles and rubella laboratory request and result form. Notes: a The Specimen No. should be exactly as written on the sample container.

Storage and shipment of serum samples

Serum should be stored at 4–8°C until shipment takes place, or for max. 7 days.

When kept for longer periods, serum samples should be frozen at −20°C or lower and transported to the testing laboratory on frozen ice packs. Repeated freezing and thawing of serum samples for IgM testing should be avoided, as it may have detrimental effects on the stability of IgM antibodies.

As a general rule, serum specimens should be shipped to the laboratory as soon as possible. The shipment should not be delayed for the collection of additional specimens.

Serum specimens, in their uniquely labelled, sealed vials, should be placed in sealable plastic bags or pouches containing absorbent materials such as cotton wool to soak up any leakage that may occur (Fig. 2).

Fig. 2

Example of packaging of a serum sample for shipment.

Styrofoam boxes or an insulating (vacuum) flask should be used to contain the sealed bags or pouches. The specimen form and investigation form for each specimen should be placed in a separate plastic bag and taped securely to the inner surface of the top of the styrofoam box or the outside of the vacuum flask (Fig. 3).

Fig. 3

Example of packing several sera into one container for shipment.

If ice packs (which should be frozen) are used, they should be placed at the bottom and along the sides of the styrofoam box. The samples should then be placed in the centre and more ice packs placed on top.

A shipping date should be arranged between the sample collectors and the laboratory. When arrangements have been finalized, the addressee should be informed of the time and manner of transportation. More details on the packaging and transportation of samples are provided in the Manual for the laboratory diagnosis of measles and rubella virus infection (1).

I.B. Dried blood spots for IgM and IgG antibody detection

Collection and handling of dried blood spots

Clean each participant's finger (or heel in the case of very young children) with alcohol and prick with a sterile, disposable microlancet.

Collect up to four drops of whole blood on standardized filter paper (such as Whatman Chromatography paper no 3®, Schleicher and Schuell #903® or another high-quality paper).

The filter paper should be marked up, either by hand or laser-printed, in a standard format that includes 14–15 mm circles within which to place the blood drops. Spaces should be marked to write the name, age and sex of the patient, with a space provided to write the laboratory or specimen number (Fig. 4).

Fig. 4

Example of dried blood spot filter paper collection form.

The filter paper should be allowed to dry thoroughly (for at least 60 minutes) at room temperature. Filter papers may be placed in a slide holder or similar receptacle during the drying process.

Storage and shipping of dried blood spots

Each dried filter paper should be wrapped individually in paper, foil or plastic to prevent possible cross-contamination.

Filter papers should be stored away from sunlight and inside a plastic bag to protect them from dust and moisture.

Dried blood spot samples are not considered biohazardous and can be shipped without special requirements or special documentation from the site of collection to the laboratory.

Although samples do not need to be kept refrigerated or frozen during transport, it is advisable to store them in a cool place and transport them to the laboratory as soon as possible.

I.C. Oral fluid for IgM and IgG antibody detection

Collection and handling of oral fluid

Crevicular fluid exuded from the interface between the gums and teeth contains low levels of IgM. A number of swab collection devices (such as the Orocol®) have been developed specifically to collect this fluid from the mouth.

The swabs are designed to be used like a toothbrush and should be rubbed along the gum until the swab is wet. This usually takes one minute.

The wet swab should be placed inside the clear plastic transport tube, which has an area on the outside to write the name and details of the patient and the date of collection.

Some devices have virus transport medium incorporated within the plastic transport tubes, while others require that a small volume of transport medium be added. Specific instructions provided by the manufacturer of the device should be followed.

Storage and shipping of oral fluid

Once a sample has been collected, the device should be sealed according to the manufacturer's instructions.

If the daily ambient temperature is below 22°C, samples should be shipped to the laboratory within 24 hours.

At higher temperatures samples should be kept in a refrigerator (4–8oC) until they can be shipped to the laboratory on ice.

The samples are usually not considered biohazardous and can be shipped without special requirements or special documentation from the site of collection to the laboratory.

II. Clinical specimens for virus isolation

Clinical samples for virus isolation should be collected as soon after onset of the rash as possible, and at least within seven days of onset.

II. A. Urine for isolation of measles and rubella virus

Collection of urine samples

It is preferable to obtain the first urine passed in the morning. Urine (10–50 ml) should be collected in a sterile container and held at 4–8°C before centrifugation.

Urine must not be frozen before the concentration procedure is carried out. A refrigerated centrifuge is recommended, but otherwise start with urine that has been chilled at 4°C.

Urine should be centrifuged at 500 × g (approximately 1500 rpm) at 4°C for 5–10 minutes, preferably within 24 hours after specimen collection. The supernatant should be discarded and the sediment resuspended in 2–3 ml sterile transport medium, tissue culture medium or phosphate-buffered saline.

If centrifugation facilities are not available, whole urine can be shipped directly to the laboratory in well-sealed containers at 4°C immediately after collection. Do not freeze.

Storage and shipping of urine samples

The resuspended pellet may be stored at 4°C and shipped within 48 hours to a measles reference laboratory.

Alternatively, it may be frozen at −70°C or lower in viral transport medium and shipped on dry ice in a well-sealed screw-capped vial to protect against CO2 contamination.

II. B. Nasopharyngeal specimens for isolation of measles and rubella virus

Collection of nasopharyngeal samples

Nasopharyngeal specimens may be taken as follows (in order of increasing yield of virus):

• nasal aspirates are collected by introducing a few millilitres of sterile saline into the nose with a syringe fitted with fine rubber tubing and collecting the fluid in a screw-capped centrifuge tube containing viral transport medium;

• throat washes are obtained by asking the patient to gargle with a small volume of sterile saline and collecting the fluid in viral transport medium;

• nasopharyngeal swabs are obtained by firmly rubbing the nasopharyngeal passage and throat with sterile cotton swabs to dislodge epithelial cells; the swabs are placed in a sterile viral transport medium in labelled screw-capped tubes.

Storage and shipping of nasopharyngeal samples

Nasopharyngeal specimens should be refrigerated and shipped at 4–8°C to arrive at the testing laboratory within 48 hours.

If arrangements cannot be made for rapid shipment, swabs should be shaken in the medium for elution of the cells and then removed.

The medium or nasal aspirate should be centrifuged at 500 × g (approximately 1500 rpm) at 4°C for five minutes and the resulting pellet resuspended in cell culture medium.

The suspended pellet and the supernatant should be stored separately at −70°C or lower and shipped to the testing laboratory on dry ice in well-sealed screw-capped vials to protect against CO2 contamination.

II.C. Whole blood for isolation of measles and rubella virus

Collection of whole blood for virus isolation

Measles virus is often detectable in peripheral blood mononuclear cells (PBMC) from a few days before to at least seven days after onset of rash. Samples collected for virus isolation should normally be collected as soon as possible and within two days of onset of rash.

For isolation of PBMC for subsequent virus isolation, blood should be collected by venipuncture in a sterile tube supplemented with ethylenediaminetetraacetic acid (EDTA). A minimum blood volume of 5 ml is recommended.

The plasma fraction can be used to determine the measles-specific IgM antibodies. The tube should be labelled with the patient's identification number and the date of collection.

Storage and shipment of whole blood

Whole blood samples may be shipped in well-sealed tubes at 4°C.

EDTA-supplemented whole blood should be processed for virus isolation within 48 hours after collection and must not be frozen at any time prior to processing.

III. Samples for reverse transcription processing of specimens

Although not recommended as a primary screening test, several laboratories have the capacity to use reverse transcription polymerase chain reaction (RT-PCR) for measles and/or rubella as a supplementary or confirmatory test.

Any sample collected for virus isolation and transported to the laboratory can be used for RT-PCR analysis.

Measles and rubella virus can often be detected by RT-PCR in whole blood (PBMC) for three to four days after onset of rash, and in urine and nasopharyngeal samples for a few days longer.

Oral fluid and dried blood spots can be used for RT-PCR analysis if they have been collected within seven days of onset of rash (oral fluid even longer) and transported to the laboratory under nondenaturing conditions.

IV. Processing of specimens on arrival at the laboratory

As each specimen is logged in, a laboratory identification number and information about the patient and the specimen should be recorded in the spreadsheet. The specimen information may be helpful in identifying problems that may contribute to difficulty with antibody detection and/or to loss of virus and inability to make isolations. Problems in shipment or with the samples should be reported to the sender.

The following important data should be recorded:

Reference