While serum-based IgM detection is recommended by WHO for routine surveillance for measles and rubella, the use of dried blood spots (DBS) and oral fluid (OF) have proven to be acceptable alternative samples for antibody detection when logistical barriers exist for collection, proper processing and transport of serum specimens. However, external quality control programmes have not yet been established for DBS and OF. Guidelines for the use of DBS and OF are discussed in the specific sections (3.4.1, 3.4.2) that address each type of sample.
An evaluation and recommendations for the use of DBS and OF in network laboratories as diagnostic samples for measles and rubella was published in 2008 [6]. The figures based upon the evaluation are included below (Figures 3.1, 3.2).
Some of the advantages these alternative specimens provide compared to serum collection are listed below:
- An equivalent combined cost for collection, extraction and testing
- The potential to greatly reduce transportation cost (a reverse cold chain generally is not required for DBS)
- DBS and OF can be used to detect both specific IgM and RNA* in the same sample
- OF may extend the window of opportunity for RNA detection post rash onset
- DBS samples are equivalent to serum for detecting IgG and offer versatility for use in sero-epidemiology studies
* RNA detection by real-time RT-PCR is more sensitive than conventional end-point RT-PCR. Although nested RT-PCR is often found to be more sensitive, it also imposes a greatly increased risk of cross-contamination and is not recommended to be used in a routine diagnostic setting. Refer to Chapter 6. Detection of viral RNA by RT-PCR for the confirmation of measles and rubella infection.
3.4.1 Dried blood spots on filter paper
As an alternative to serum, whole blood specimens that have been dried on filter paper (i.e., dried blood spots, DBS) have been used for a range of epidemiological studies. The use of DBS may be considered where a reliable cold chain is not available or logistical barriers for efficient transport of serum exist [6]. While routine blood collection (venipuncture) can be used to prepare DBS, in general, DBS samples are prepared using capillary blood, a less invasive blood collection method that may be perceived as more acceptable, particularly for young children and infants. Antibodies (IgM and IgG) are stable once the blood has dried on filter paper, making this method a good alternative option for collection of blood specimens in remote areas without a reliable cold chain. The added advantage is that DBS can also be utilized to detect both measles and rubella RNA when collected soon after disease onset (section 3.5).
To meet the surveillance requirement for an adequate sample of DBS, the blood volume collected must be sufficient to completely fill three of the four marked circles on a standardized filter paper card designed for blood collection (e.g., Whatman 903). However, efforts should be made to fill all four marked circles (14-15 mm). This provides for a separate spot/extraction for measles testing, rubella testing, retesting, and an additional spot for referral if necessary. A drop of whole blood (50 μl) is necessary to fill each circle. As is true for serum specimens for antibody testing, the blood sample for DBS must be collected from suspected measles or rubella cases within 28 days of rash onset to meet the laboratory surveillance requirements.
Blood collection can be performed by finger- or heel-prick using a sterile, preferably using a single use, disposable lancet. The lancets currently available include a safety feature for activation and produce a consistent puncture depth (e.g., BD Microtainer 366594). Blood drops are then applied to marked circles on the filter paper card. For an example, see Figure 3.3, Example of an appropriate format for filter paper used for dried blood spots (DBS).
Blood specimens that have been spotted on filter paper must be allowed to air dry completely. The time for complete drying will vary according to humidity levels, but is usually 2-4 hours. Once dried, individual cards can be wrapped in wax paper, and placed in a sealable plastic bag with a desiccant pack. Ideally, the desiccant should have a colour indicator so the ability of the desiccant to absorb moisture can be monitored.
For elution of the serum, a standard hole punch is used to remove three 6mm discs from one of the filled filter paper circles. Each 6mm disc punched out from the DBS contains approximately 5μl serum. The remaining DBS are stored at -20°C if re-testing is necessary or for confirmatory testing.
Although properly prepared DBS samples are stable for a limited time at room temperature, ideally the DBS should be stored at 4°C until they can be shipped to the laboratory. Transport of DBS at ambient temperatures up to 42 °C is acceptable if the sample is delivered to the laboratory within 3 days [7]. Upon arrival to the laboratory the DBS should be held at 4°C until the serum can be extracted from the filter paper. The recommended protocols for the preparation, shipment and extraction of serum from DBS are provided in Annex 3.2
3.4.2 Oral fluid samples
The use of oral fluid (OF) samples has been successfully implemented in the UK since the early 1990s and is used almost exclusively for measles, rubella and mumps laboratory-based surveillance. An OF specimen includes saliva and secretions from the parotid, submaxillary and sublingual glands. OF is a minimally invasive sample, is relatively easy to collect, and is more acceptable to both clinicians and patients compared to blood collection. Because OF is stable at moderate ambient temperatures, these samples may be easily and economically shipped if moderate ambient conditions exist with reliable local postal or delivery services. In the UK, the use of OF for laboratory testing for suspected cases of measles and rubella has resulted in a more comprehensive sampling of suspected cases.
Collection devices for OF are not commonly available in many regions and would need to be provided to health-care facilities by the surveillance programme. The Oracol™ device has been widely used in the GMRLN for OF collection (See Figure 3.4).
Oral fluid sampling devices which use a preservative for stabilising IgM (such as OraSure™) should not be used for collection of OF samples that would be tested by RT-PCR. The collection of an OF sample does not require professional medical personnel but does require instructions on the proper collection to obtain a good quality OF sample.
The technique for OF collection is intended to allow collection of gingival crevicular fluid at the gum and dentine interface which contains antibodies from the capillary beds along the gingival crevice. An adequate OF sample is one that is collected by gently rubbing along the base of the teeth and gums for at least 1 minute, which should allow the sponge to absorb about 0.5ml of crevicular fluid [7]. After collection, the sample is placed inside a microtube which is included. An OF sample for antibody testing is considered an adequate sample for surveillance if collected within 28 days of rash onset.
Note: There is evidence for the effectiveness of a new OF collection and extraction device (Oralight), although as of this writing the data is pending publication. The use of OF samples for virus-specific RNA detection is discussed in Chapter 6. Detection of viral RNA by RT-PCR for the confirmation of measles and rubella infection.
A summary is provided in Table 3. 1, Expected success rates for IgM or virus detection using serum, DBS and OF.
If the daily ambient temperature is below +22°C, OF samples may be shipped without refrigeration but ideally should be shipped to the laboratory within 24 hours. At higher temperatures, the OF samples should be kept at 4-8°C until the samples can be shipped to the laboratory on cold packs. The OF samples are not considered a biohazard and can be shipped without special documentation from the site of collection to the laboratory.
Oral fluids must be extracted from the swab as soon as possible after receipt in the laboratory. The processing required for the OF includes an extraction step (Annex 3.3, Processing oral fluid samples). External quality assessment programmes, such as those currently required for testing serum, have not yet been established for OF and assays must be validated for testing OF. However, one method of assessing the quality of the OF for use in IgM assays is to verify that a minimum level of total IgG is present in the sample (protocol included Annex 3.3).