Reliability of dried blood spot (DBS) cards in antibody measurement: A systematic review

Background Increasingly, vaccine efficacy studies are being recommended in low-and-middle-income countries (LMIC), yet often facilities are unavailable to take and store infant blood samples correctly. Dried blood spots (DBS), are useful for collecting blood from infants for diagnostic purposes, especially in low-income settings, as the amount of blood required is miniscule and no refrigeration is required. Little is known about their utility for antibody studies in children. This systematic review aims to investigate the correlation of antibody concentrations against infectious diseases in DBS in comparison to serum or plasma samples that might inform their use in vaccine clinical trials. Methods and findings We searched MEDLINE, Embase and the Cochrane library for relevant studies between January 1990 to October 2020 with no language restriction, using PRISMA guidelines, investigating the correlation between antibody concentrations in DBS and serum or plasma samples, and the effect of storage temperature on DBS diagnostic performance. We included 40 studies in this systematic review. The antibody concentration in DBS and serum/plasma samples reported a good pooled correlation, (r2 = 0.86 (ranged 0.43 to 1.00)). Ten studies described a decline of antibody after 28 days at room temperature compared to optimal storage at -20°C, where antibodies were stable for up to 200 days. There were only five studies of anti-bacterial antibodies. Conclusions There is a good correlation between antibody concentrations in DBS and serum/plasma samples, supporting the wider use of DBS in vaccine and sero-epidemiological studies, but there is limited data on anti-bacterial antibodies. The correct storage of DBS is critical and may be a consideration for longer term storage.

Introduction spot' OR 'Guthrie card' AND 'antibody'. The database was last searched on the 15 th October 2020.
The PRISMA checklist and the full search string are available in the S2 Method.

Eligibility criteria
The studies that were considered eligible for inclusion were original research articles, concerning infectious diseases in humans, comparing antibody titres/concentrations in serum/ plasma to DBS or describing stability of antibodies in DBS from longitudinal studies of storage at different temperatures. We included studies from all countries. Opinion pieces, reviews, comments, letters and conference abstracts were excluded. Studies that used animals to compare antibody levels in serum/plasma and DBS were also excluded. Studies that had insufficient data (absence of two or more of the following: number of participants, age, sensitivity, specificity, correlation of antibody levels in matched DBS-serum/plasma samples) were also excluded. Additionally, we included all studies that investigated the stability of antibodies in DBS samples.

Study selection
Two independent reviewers (FA and EA) screened the titles and abstracts of the identified studies. After the initial screening, the reviewers obtained full texts of reports and they independently reviewed each article to determine whether it would be included in the final review. Disagreement on studies were resolved in discussion with a third reviewer (KLD).

Data extraction
The reviewers (FA and EA) independently extracted the data from the included studies using PICO (patient, intervention, comparison and outcome) [11]. The following information was extracted from the selected studies: author, publication year, country, journal, infectious disease, aim of research, study design, duration of study, number of participants, mean age, sensitivity, specificity, method of sample collection, method of sample storage, laboratory tests used for confirmation, elution method and outcome. The country income of the included studies was classified by using their respective gross domestic product (GDP) using the World Bank [12]. All studies were either classified as a low-middle income country (LMIC) (which consisted of low-income, lower middle-income and upper middle-income countries) or as a highincome country (HIC).

Data synthesis and analysis
Due to the high heterogeneity of study design, participants and outcomes, we were only able to conduct a narrative synthesis of included studies, summarising the findings with respect to each infectious disease. We calculated the pooled estimates of specificity, sensitivity and correlation coefficient using the accuracy data (true positive, true negative, false positive and false negative).

Risk of bias
The risk of bias was assessed by FA using the Cochrane Risk of Bias for non-randomised studies (ROBINS-I) tool [13]. This included information on bias due to confounding, bias in selection of participants into the study, bias in classification of interventions, bias due to deviations from intended interventions, bias due to missing data, bias in measurement of outcomes and bias in selection of the reported results. Due to the nature of the interventions considered in this review, the study's participants could not be blinded.

Results
We identified 1,508 studies from the electronic databases published between 1 st January 1990 to 15 th October 2020. Using our search term, we sourced 789 papers from Medline, 667 from Embase and 52 from Cochrane. After the removal of duplicates, 837 studies were identified for abstract screening. A total of eighty-eight full text studies were assessed for eligibility, forty studies met the criteria for inclusion (Fig 1). An additional five papers investigating only antibody stability were also included.

Discussion
To our knowledge, this is the first comprehensive systematic review to summarise the utility of DBS considering the key aspects of storage, assay methods and card handling which are all important considerations for vaccine trials and serological studies. Overall, the diagnostic accuracy and precision was high when comparing serum/plasma to DBS, indicating that DBS are a useful alternative to serum.
In a review of anti-HCV antibodies eluted from DBS, Vazques-Moron et al reported a sensitivity of >96% and a specificity of >99% for anti-HCV antibodies in DBS samples [57]. Their reported figures are similar to the pooled diagnostic performances we have shown. However, our pooled results indicate that there may be differences in both sensitivity and specificity depending on the pathogen type. A study of SARS-COV-2 antibodies demonstrated that sensitivity of matched plasma and DBS was 98.9% [52]. This is potentially useful knowledge during  the pandemic as DBS could be used as an alternative to blood samples for national surveillance. DBS sampling would be more convenient for sampling as it does not require attendance at clinic to collect samples and samples can be easily sent back by post.
There are no guidelines on how DBS samples should be stored for short-and long-periods of time and this is evident from the variable storage described in the studies we reviewed. We have demonstrated that storage at room temperature (22-28˚C) is acceptable for up to 28 days; making the transportation of DBS samples straightforward, especially in environments lacking cold chain. However, we also report that longer term storage should be at refrigerated or frozen temperatures after 28 days at room temperature, as antibodies degraded significantly thereafter. Overall, our results indicate that -20˚C is the optimum temperature to store DBS samples for prolonged periods and it may be necessary for this to be factored into trials where samples may be stored for several years prior to use. This is consistent with the national laboratory guidelines in Denmark [58], Scotland [59], US [60] and Germany [61], which all recommend long term freezer storage. Williams et al [62] re-quantified anti-HIV antibodies in four high positive controls that were initially spotted 23 years prior to the analysis. The antibody concentrations obtained were the same as those measured 23 years prior when stored at -20˚C. Yel et al's [44] study found that IgA antibodies were stable for up to 14 days at room temperature and at 4-8˚C, however, IgA antibodies were stable for up to 10 days when stored at -20˚C. Going forward, it may be useful to determine the stability of the different antibody isotypes, as certain antibodies may be more stable than others on DBS.
Whilst this review provides support for the use of DBS for the investigation of immunity to several pathogens, we found only four studies which investigated the antibody concentration against bacterial infections. It is vital that more research is undertaken to understand the stability of antibodies against bacterial infections in DBS samples and how antibody concentrations compare to serum or plasma samples. This is of particular importance if DBS are to be used in vaccine trials against bacterial pathogens, which are required to reduce the impact of the continued spread of antibiotic resistance.
There is of considerable urgency as many bacterial infections are becoming increasingly antibiotic resistant and DBS could be used as part of studies to measure vaccine or natural immunity to bacterial infections [63]. There are several limitations to this systematic review. Firstly, the quality of the studies included in this review were generally low (S1 Table), which precluded a meta-analysis of the data. The filter papers, blood volume collected, size of dried blood spots, elution process and the assays used for antibody quantification also differed amongst the studies, compounding the limited translation of results. Furthermore, the variability of the study designs may have also contributed to the heterogeneity which has restricted direct comparisons and prevented any metaanalysis of data, even of the same pathogen. Secondly, differences in specificity, sensitivity and correlation were noted for different pathogens, suggesting that antibodies against some pathogens in DBS may be less stable than others. Thirdly, the studies did not investigate the effect of humidity on DBS, which is often an issue in sub-Saharan Africa and Asia. Finally, we acknowledge that heterogeneity exists when different cut-off levels are applied between the studies.
Further data are needed to demonstrate the stability of DBS for different pathogens, especially bacteria, under different field transport and storage conditions likely to be encountered in low resource settings, including the effect of high ambient temperature or humidity levels.
Consideration of the use of DBS sampling in clinical vaccine or sero-epidemiological studies will depend on both healthcare setting and available infrastructure. The current lack of guidelines for the adaptation of assays from serum to DBS and on the optimal pre-analytical treatment of specimens makes quality control challenging. With optimal storage, DBS can be a useful adjunct to serological analysis due to their relative simplicity to take and requirements for a less rigorous cold chain, saving time and reducing costs.  Funding acquisition: Kirsty Le Doare.