The analysis of serum specimens for the presence of measles- or rubella-specific IgM antibodies is traditionally regarded as the gold standard for laboratory confirmation. The enzyme immunoassay (EIA) is the method recommended for the WHO measles and rubella laboratory network for the detection of virus-specific IgM antibodies in serum. The EIA kits that are commercially available vary in sensitivity and specificity, but many kits have been evaluated by the laboratories in the GMRLN. Additional guidelines for selection of EIAs are provided in Chapter 4. Antibody detection methods for laboratory confirmation of measles, rubella, and CRS. The guidelines are based on analyses of the results obtained from network laboratories that have participated in the proficiency testing programme over many years. Several commercial measles and rubella IgM EIA kits (and in-house assays) have been utilized by network laboratories [2].
Blood collection requires trained personnel, dedicated equipment for collection and processing, and a reliable reverse cold chain. Any staff involved with the collection of patients’ blood specimens should be fully trained in the process, the appropriate use and disposal of needles and syringes. Refer to WHO guidelines on drawing blood: best practices in phlebotomy [3]. The availability of a serum specimen from suspected cases provides a means to conduct testing using similar serologic assays for other causes of febrile rash illness. The specific testing performed for other rash illnesses varies among laboratories but may include diseases that are commonly encountered in a particular age group (e.g., young children and parvovirus B19) or diseases that are endemic in the area (e.g., dengue, zika virus).
Two topics are discussed below. The first, “3.3.1, Timing of blood collection for measles and rubella IgM detection”, covers the recommendations for optimal results for antibody detection and the circumstances under which the collection of a second serum specimen may be appropriate. The final subsection, “3.3.2, Collection, processing, storage and transport of serum specimens”, covers the technical and logistical issues for provision of quality serum specimens.
3.3.1 Timing of blood collection for measles and rubella IgM detection
Blood is collected at first contact with a suspected case of measles or rubella. In most instances, a single serum specimen will be sufficient to classify a suspected measles or rubella case based on the presence or absence of virus-specific IgM. For surveillance purposes, an adequate serum sample for measles or rubella is one that is obtained within 28 days after the onset of rash. However, when serum is collected within the first few days following rash onset, a proportion of infected individuals may test negative for IgM due to low levels of virus-specific IgM that are undetectable by EIA (see discussion below). Refer to Chapter 8. Laboratory testing in support of measles and rubella surveillance in elimination settings for guidance regarding laboratory confirmation including serologic testing that is specifically recommended for elimination settings.
Measles virus-specific IgM may not be detectable by EIA in serum that is collected in the first 72 hours (≤3 days) after rash onset in approximately 30% of measles cases [4]. In the study on which this guidance is based, day 1 was defined as ≤24 hours after onset of rash. Of 28 serum specimens collected on day 1, there were 20 (71%) that were IgM positive using an IgM capture EIA. By day 3 (48-72 hours after rash onset), the IgM positivity rate increased to 77% (20/26). For those patients whose second serum specimen was IgM positive, 100% of the serum specimens collected from day 4 through day 11 were IgM positive by the measles capture EIA.
Rubella-specific IgM may be undetectable by EIA in up to 50% of rubella cases from serum specimens that are collected on the day of rash onset. In >90% of rubella cases, serum that is collected at 5 days after rash onset will be IgM positive [5]. A discussion of the timing of blood collection for confirmation of CRS and CRI cases is provided in section 3.6.
During outbreaks that involve large numbers of cases, determination of epidemiologic links to laboratory-confirmed cases should be utilized as an adjunct for case confirmation in order to prioritize laboratory testing for suspected cases that may arise in new locations or in previously unaffected groups. Ongoing coordination between the laboratory and the field staff can reduce unnecessary blood collection and testing. However, periodic collection of clinical specimens for molecular testing during the course of ongoing outbreaks (extending beyond one month) is recommended to monitor the genotype over time (see Chapter 7. Molecular epidemiology of measles and rubella).
In the absence of an epidemiologic link to a laboratory-confirmed case, collection of an additional serum specimen may be required to classify the suspected case. However, in outbreak settings where measles is endemic, resources should not be used to collect follow-up serum specimens. The guidelines below are primarily intended for countries in near or post-elimination settings. More information for testing in these settings is available in Chapter 8. Laboratory testing in support of measles and rubella surveillance in elimination settings. However, regardless of the phase of disease elimination, collection of a second serum specimen may be required under some circumstances to provide laboratory confirmation for suspected cases that may occur in locations outside of the initial outbreak area or when a suspected case has been identified in a previously unaffected group.
A second or follow-up serum may be appropriate under the following circumstances:
- Detection of virus-specific RNA by RT-PCR is either unavailable or the results were inconclusive
- The first serum specimen was collected ≤3 days after rash onset and is negative for measles IgM, or is negative in serum collected ≤5 days for rubella IgM by EIA
- Repeat testing of the initial serum specimen fails to resolve an equivocal result for IgM
- The IgM is positive but there is suspicion that the result is false positive, and the rash is due to another etiologic agent (see Chapter 4. Antibody detection methods for laboratory confirmation of measles, rubella, and CRS, section 4.2.2)
Depending on the situation, it may be acceptable to delay blood collection so that the appropriate interval exists between the initial and second specimen to evaluate a diagnostically significant rise in IgG. A repeat IgM test is then performed with the follow-up serum. If necessary and feasible, IgG titres can be evaluated by a quantitative IgG EIA using paired serum specimens collected 10-21 days apart. The interval between the two serum specimens can be reduced if virus-specific IgG was not detected in the first serum specimen. When a seroconversion can confirm the case, the interval between serum collections can be shorter than 10 days but the second serum should not be collected until at least 9-10 days after rash onset when virus-specific IgG should be detectable. Refer to Chapter 4. Antibody detection methods for laboratory confirmation of measles, rubella, and CRS for additional information regarding testing strategies for case classification by IgM and IgG antibody testing.
3.3.2 Collection, processing, storage and transport of serum specimens
Collection of blood is performed by venepuncture using a sterile, plain collection tube (red-top tube) or serum separator tube without additives. Although a volume of 4-7 ml is typically collected from adults (1 ml will yield typically about 400 μl of serum), collection of 0.5 ml- 1ml is acceptable from infants. Whole blood can be stored at (+)4−8°C (never freeze whole blood) for up to 24 hours or for 6 hours at (+)20-25°C before the serum is separated from the clotted blood through centrifugation. After this time, whole blood must be transported to a facility equipped to separate the serum in order to avoid haemolysis.
Blood should be transported with adequate insulation on cold packs. After arrival at the laboratory, the blood tubes should be allowed to clot in an upright position, usually 30-60 minutes at room temperature. After the blood has clotted, the stoppered tube is centrifuged at 1000 × g for 10 minutes to separate the serum. The serum should be removed carefully without disturbing the red cell layer and transferred to a sterile, labelled vial. The vial should have a durable label attached with the patient’s name or identifier, date of collection and specimen type.
Procedures for processing blood without a centrifuge:
If a serum separator tube has been used, serum can be pipetted out aseptically after the red blood cells have separated (about 6 hours at +4°C). If the blood was collected in a plain tube, the blood may be refrigerated until there is complete retraction of the clot from the serum (no longer than 24 hours). At (+)20-25°C, the blood will clot in about 20 minutes. The serum should be carefully removed with a fine-bore pipette, to avoid extracting red cells, and transferred aseptically to a sterile, labelled vial.
Serum should be held at refrigeration temperature (4 to 8°C) until shipment, but ideally should not be stored at 4-8°C for longer than 7 days*.
*Note: The guidelines for storage of serum at refrigeration temperatures vary in regard to protecting the integrity of antibodies in serum. Some manufacturers of EIA kits specify that 2-3 days is the maximum time and others indicate serum maybe be stored at 4-8°C for up to 28 days. However, when a delay is anticipated in shipping or in testing, the serum specimens should be frozen at ‑20°C, or colder. A frost-free freezer, which has a periodic warming cycle to prevent ice build-up, should not be used.
The frozen serum specimens should be transported to a testing laboratory packed with frozen cold/ice packs, in a sufficiently insulated container (see details below). Since repeated freezing and thawing can have detrimental effects on the integrity of IgM antibodies, these cycles should be avoided, and aliquots of important serum specimens should be prepared prior to freezing. Generally, serum specimens should be shipped to the laboratory as soon as possible and shipment should not be delayed for the collection of additional specimens.
The requirements for shipping serum may vary according to local conditions and regulations, and depend on whether shipment of serum is within national borders or must be shipped internationally (see section 3.1, above). However, all serum specimens should be transported in properly labelled vials. The vials should be packaged with absorbent material and protected against crushing of the contents. The laboratory request form and any other paperwork for each specimen should be placed in a separate plastic bag. An insulated container with cold packs may be required. For transit times of several days, the insulated contained should be lined with frozen cold packs with the samples placed in the centre of the container with more cold packs placed on top. The methods and details of the shipping (courier, date of shipping) should be arranged or communicated prior to shipment between the sender and the receiving laboratory.
As an alternative to shipping a refrigerated package, whole blood can be spotted on filter paper in place of serum if a cold chain or an economical method for shipment is not available (see next section on alternative samples for IgM testing). Efforts by the laboratory network to substitute dried serum spots using existing serum samples for confirmatory testing has not proven to have consistently acceptable performance for measurement of IgM. The procedure for preparation, shipment, and extraction of DBS samples is provided in Annex 3.2.