Review articleThe use of dried blood spots for characterizing children's exposure to organic environmental chemicals
Introduction
Exposure assessment is inherently the most difficult part of epidemiology, sometimes being called its Achilles’ heel (Kogevinas, 2011). While personal measurements and ecologic assessments are often used for assessing exposure, the measurement of chemicals and their metabolites or reaction products in biological matrices has several distinct advantages over these techniques. For example, biological measurements quantify chemicals that enter the body and integrate all routes of exposure into one measurement (Barr et al., 2005b; Calafat, 2012; Needham et al., 2005). With advances in analytic instrumentation and the recognized advantages of direct measurements in human matrices, biomonitoring of exposure has burgeoned over the past 20 years (Barr et al., 2005b; Needham et al., 2005). It remains one of the primary and most widely accepted tools for exposure assessment used today (Barr et al., 2005b; Calafat, 2012).
Biomonitoring has been used for decades to measure exposure to bioaccumulative organic chemicals, also called persistent organic pollutants (POPs) (Adgate et al., 2001; Barr et al., 2005b, 2007; Needham et al., 2005). These chemicals, such as polychlorinated biphenyls (PCBs), organochlorine (OC) pesticides and polybrominated flame retardants, are usually measured in serum or plasma (Barr et al., 2005b). These measurements provide excellent information on body burden and are considered the gold standard for exposure assessment for these chemicals. More recently, biomonitoring in serum and plasma is also being extended to a growing number of biologically non-persistent organic chemicals (NPOPs) such as bisphenol A, phthalates and current-use pesticides (Adgate et al., 2001; Aylward et al., 2010; Barr et al., 2004, 2005a, 2006, 2007; Barr and Angerer, 2006; Biggs et al., 2008; Bouchard et al., 2011; Hanari et al., 2006; Horii et al., 2010; Kannan et al., 2004, 2007; Tao et al., 2008; Wang et al., 2019). However, the use of serum and plasma matrices for biomonitoring has several distinct limitations, especially for children's studies: (1) they provide only current body burden information, thus the timing and magnitude of exposure is unknown; retrospective biomonitoring is only possible if serum or plasma samples have been previously and properly collected and archived; (2) the invasive collection of blood (usually ~10 mL) typically precludes the enrollment of infants and children in such studies; (3) for many NPOPs, serum is subject to contamination from pre-analytic processes (e.g., phthalate contamination from venipuncture) or the parent chemical can be quickly metabolized by blood enzymes; (4) sampling requires a trained phlebotomist; and (5) transfer of samples from a field location to the laboratory is cumbersome and costly (Barr et al., 2005b; Needham et al., 2005).
Dried blood spots (DBS) – drops of capillary whole blood obtained through heel prick in infants ≤ 6 months old and finger stick in those > 6 months old — collected on standardized filter paper represent a minimally invasive alternative to venipuncture. Since the 1960s, DBS have been collected routinely as a part of each US state's newborn screening (NBS) program to facilitate screening of newborns for congenital metabolic disorders (Guthrie and Susi, 1963; Mei et al., 2001) such as phenylketonuria (Carreiro-Lewandowski, 2002). The number of diagnostic tests performed and the number of blood spots (up to 15 spots) collected vary by state (Carreiro-Lewandowski, 2002), with some programs screening over 60 disorders [https://data.newsteps.org/newsteps-web/].
As a result of the successful NBS program, the pre-analytic and analytic procedures for the use of DBS in NBS have been fully vetted and validated (Adam et al., 2011; Chace et al., 1999; Denniff et al., 2013; Li et al., 2012; McHugh et al., 2011; Mei et al., 2001, 2010). For example, the filter papers or Guthrie cards are certified to meet performance standards for absorption and lot-to-lot consistency of the materials (Mei et al., 2010). Storage and shipping procedures have been evaluated to test for analyte stability under varied conditions (Adam et al., 2011; Bowen et al., 2011; Flores et al., 2017; Mei et al., 2011). Furthermore, testing procedures are guided and evaluated using a standardized quality assurance program developed and administered by the Centers for Disease Control and Prevention (CDC) for almost 40 years (De Jesus et al., 2010) that includes proficiency testing and quality control materials.
Overall, newborn screening procedures for DBS blood collection in NBS are minimally-invasive, low cost, and can be carried out by people with minimal training (De Jesus et al., 2010). Unlike serum or plasma, DBS samples do not need to be centrifuged, separated, or immediately frozen following collection for use in NBS. A cold chain (i.e., a documented process ensuring appropriate refrigeration or freezing at each step in the pre- and post-analytic processes) from the point of sample collection to receipt in the laboratory is not required. Capillary blood is simply dripped, not pressed, onto filter paper, allowed to dry thoroughly, and then wrapped, stacked, and stored. Most analytes measured in NBS remain stable at room temperature for a week or more, providing considerable flexibility in procedures for sample collection and transport. In addition, DBS samples are stable in laboratory freezers for longer periods (>1 week up to years) of time and can be stacked in relatively small amounts of space. For example, a standard 27 cubic foot lab freezer can hold 8,000 to 10,000 DBS sampling cards. After testing has occurred on the DBS samples, the state health departments retain the DBS for a specified period of time, either a short duration (<3 years) or a long duration (indefinitely), for further diagnostic testing or for other novel uses (Therrell et al., 2011)[ https://data.newsteps.org/newsteps-web/]. In 2011, the Secretary of Health and Human Services convened an Advisory Committee on Heritable Disorders in Newborns and Children to develop recommendations on retention and future uses of DBS. These recommendations largely focused on genetic testing and confidentiality issues with no discussion of potential biomonitoring applications (Therrell et al., 2011; Vladutiu, 2010). However, the promise of increased repositories of residual DBS poses an opportunity for population-based research.
DBS offer unique opportunities for children's health studies. Because of their ease of collection, shipment and storage, great interest has been expressed in using DBS in broad-ranging applications including infectious (Iyer et al., 2018; Lange et al., 2017a, 2017b; van Loo et al., 2017; Won et al., 2018) and chronic disease screening (Bjornstad et al., 2018; Brindle et al., 2010; Henderson et al., 2017; Hu et al., 2015; Lacher et al., 2013; Maleska et al., 2017; McDade et al., 2004; McDonald et al., 2017; Miller and McDade, 2012; Nguyen et al., 2014; Samuelsson et al., 2015), genetic profiling (Bassaganyas et al., 2018; McDade et al., 2016; Segundo et al., 2018), pharmaco-management (Gallay et al., 2018; Page-Sharp et al., 2017; Schauer et al., 2018; Spooner et al., 2009), forensic testing (Hamelin et al., 2016; Perez et al., 2015; Sosnoff et al., 1996; Stove et al., 2012) and biomonitoring of chemical exposures (Archer et al., 2012; Basu et al., 2017; Batterman and Chernyak, 2014; Batterman et al., 2016; Chaudhuri et al., 2009; Funk et al., 2008, 2013; Kato et al., 2009; Kim and Kannan, 2018; Krishnan et al., 2013; Ladror et al., 2017; Ma et al., 2013, 2014b; Murphy et al., 2013; Pedersen et al., 2017; Spector et al., 2014; Spliethoff et al., 2008a). In fact, procedures for measuring over 100 analytes in DBS have been published including indicators of endocrine, immune, reproductive, and metabolic function, nutritional status and infectious disease status (McDade et al., 2007). On average, about 50–100 μL of whole capillary blood are collected onto each spot and measurement assays may use anywhere from a 6-mm punch to one or more entire spots. The use of a controlled 6-mm punch enables further use of the spot since about three to four 6-mm punches can be obtained from one DBS. When an entire spot is used, multiple analyses may be performed from the spot eluate if the general elution is compatible with multiple assays. Recently, more than 35,000 DBS samples have been collected by NIH-funded studies in the United States, including large surveys like the National Longitudinal Study of Adolescent Health (McDade et al., 2007; Nguyen et al., 2014) and the Health and Retirement Study (McDade, 2011; McDade et al., 2007; Sonnega et al., 2014). The National Health and Nutrition Examination Survey (NHANES), widely regarded as the gold-standard for assessing the health of children and adults in the United States, has initiated an effort to implement DBS sampling to complement the mobile examination centers currently used to collect biological specimens (Miller et al., 2015). These applications indicate that DBS sampling is feasible for large-scale research applications, and that it is generally acceptable to research participants. For example, in 2007–2008, the National Longitudinal Study of Adolescent Health implemented DBS sampling in a large, nationally representative sample of 24–32 year old adults across the United States and found that 94% of participants were willing to provide a finger stick DBS sample which was higher than the percentage that willingly reported their income (93%) (McDade et al., 2007). Similarly, in the recent Household Air Pollution Intervention Network Trial (2017–2021) (Barr et al., 2020; Clasen et al., 2020), a multi-national birth cohort study with 3,200 pregnant women, their children and other women in their households, longitudinal DBS were successfully collected in 91–99% participants (unpublished data), even in children from birth to 1 year of age.
Given the ease of implementation into population-based studies and the biorepository of archived blood spots available in each state, the implications of the use of DBS in children's health studies are profound. Blood sample collection in infants or small children is difficult, at best, but more often infeasible, essentially precluding them from participation in many important studies. DBS represent a potential opportunity to include this vulnerable segment in more population-based studies as parents are more likely to allow a less painful, minimally-invasive heel or finger prick sample be taken than the more invasive venous blood draw (Bell et al., 2018; Ghassabian et al., 2018; Yeung et al., 2016), albeit the DBS measurements would be subject to the same limitations as venous blood in interpretation. Furthermore, the existence of perinatally collected spots may enable retrospective analysis of early indicators or markers of exposure or disease that may occur later in life. In fact, over the last two decades, archived DBS have been tested for feasibility for the analysis of environmental chemical exposures (Burse et al., 1997; Kato et al., 2009; Ma et al., 2013; Spliethoff et al., 2008a). While these studies have shown that the physical measurement of a chemical or metabolite in the DBS is feasible, despite the low volume of blood provided by a single spot, few studies have taken the process past the laboratory measurement stage to also test and validate the impact of field sampling, shipping and storage techniques on the viability of DBS as a matrix for biomonitoring. A recent review by Parsons et al. provided a historical view of DBS analysis for inorganic environmental chemicals in biomonitoring studies and summarized the current challenges and limitations in measuring inorganic chemicals in archived DBS with recommendations and technical justifications for improvement (Parsons et al., 2020). In this paper, we provide an overview of the published work on the use of DBS in exposure biomonitoring applications for organic pollutants and suggest some critical gaps that need to be addressed before DBS should be routinely used in biomonitoring studies, especially those that include children. Although other reviews have reported on feasibility of analysis of environmental and clinical biomarkers (Freeman et al., 2018; McClendon-Weary et al., 2020; McDade, 2011; McDade et al., 2007; Sharma et al., 2014), we specifically provide a laboratory-based viewpoint that addresses issues involved in proper measurement and interpretation of DBS-based biomonitoring data.
Section snippets
Biomonitoring of organic environmental chemicals in DBS
To interrogate the literature on the use of DBS to measure organic environmental chemicals, we used the Preferred Reporting Items for Systematic Reviews and Meta-Analyses approach (PRISM) (Moher et al., 2009). We searched PubMed, Web of Science, and Google Scholar databases; the search terms we used were “dried blood spots,” “environ*,” combined with one of the following terms: “chemical,” “PCB,” “BPA,” “PFAS,” “PFC,” “PBB,” “PBDE,” “pesticide,” “phthalate,” or “persistent organic pollutant.”
Detection of chemicals versus metabolites and adducts
As indicted above, the use of plasma, serum and DBS analysis methods are best suited to chemicals that persist in the body, with long half-lives. Short-lived chemicals will indicate recent exposures. Adducts that are formed by reactive metabolites on blood proteins such as hemoglobin (Funk et al., 2008) and albumin (Xue et al., 2016) will persist following exposure, and accumulate following repeated exposure. These biomarkers can provide an integrated dosimeter, based on the lifespan of the
Overall recommendations
Before routine use of DBS in epidemiologic studies, full validation procedures should be conducted. These validations should include:
- 1)
Laboratory measurement feasibility and validation studies:
- a.
Measurement feasibility on instrumentation should be evaluated. This can first be accomplished by injecting amounts of the target analytes anticipated in 50–100 μL blood on instrumentation. Given the ultra-sensitivity and selectivity required at such low concentrations, confirmatory analytical
- a.
Conclusions
Although DBS have been demonstrated to be a feasible matrix for environmental exposure assessment, many areas related to their use remain non-standardized or unvalidated. In general, DBS analyses have been shown to be most promising for measurement of POPs. Given that POPs are typically measured in blood or blood serum/plasma and are inherently stable, this may be an acceptable way of capturing exposure measures in children or evaluating retrospective exposures. The use of DBS for other
Contributions of authors
DBB, YC, KK, LM and EMF conceived and designed the paper. DBB, YC, KK and EMF performed literature searches and extracted the data for the paper. DBB and KK analyzed and interpreted the studies. DBB, KK, and EMF drafted the manuscript and DBB, KK, YC, LM, LMF, JDM, TDF, and EMF revised and critically edited the manuscript. All authors approved of the final version.
Declaration of competing interest
The authors declare that they have no known competing financial interests or personal relationships that could have appeared to influence the work reported in this paper.
Acknowledgements
We thank the quality assurance working group of the Children's Health Exposure Analysis Resource (CHEAR) for their assistance in developing the concept of this manuscript, in particular, Dr. Patrick Parsons. We also thank the CHEAR Steering Committee for their thoughtful review. Research reported in this publication was supported, in part, by the National Institute of Environmental Health Sciences Awards U2CES026560 (DBB), P30ES019776 (DBB), R21ES023927 (DBB), U2CES026542 (KK), U2CES026561 (LMP)
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