Biomarkers can provide valuable information about biological processes ranging from normal to pathogenic that may contribute to clinically identifiable disease¹. In research settings, dried blood spots (DBS) are becoming increasingly preferred to venipuncture as a method for collecting biomarkers in whole blood. DBS collection is simple, relatively painless, less invasive then venipuncture, and requires minimal storage requirements (i.e. samples do not need to be immediately frozen and can be stored for a long period of time in a stable freezer environment before assay). DBS samples can be tested for a variety of different analytes, including cholesterol, C-reactive protein, glycosylated hemoglobin, Epstein-Barr virus antibodies, and several cytokines.

Venipuncture has long been the gold standard for the collection of biomarkers in blood. However, obtaining, storing, and shipping blood samples requires adherence to a number of protocols and health codes, which may not be possible in certain research protocols. Phlebotomists must be officially certified for venipuncture, while DBS sampling procedures are simpler and do not require official certification. Venous blood collection also requires more equipment than does DBS collection (i.e. needles, tubes, tourniquets), and more post-collection effort. Venipuncture samples must be processed (i.e. serum or plasma extracted from the blood sample via centrifugation or standing at room temperature for a given period of time) and frozen between -20 °C and -70 °C immediately in order to prevent the degradation of analytes, while DBS samples must simply be allowed to air dry and then may be stored at room temperature for a week or more depending on the analyte, before transfer to a freezer². DBS samples are less expensive to collect than venipuncture samples for reasons described above. DBS samples also take up less space, reducing shipping and storage costs prior to assay.

The use of saliva as biomarker has become key in biobehavioral studies. Saliva offers a simple, noninvasive method to collect biomarkers and has become the standard for field work aimed at measuring cortisol or cotinine^3-5. Urine is another specimen that can be collected noninvasively and can be used in population level studies for assays of melatonin or for toxicology. While both urine and saliva offer straight forward collection procedures that are able to be completed by participants themselves, not all analytes can be measured in these media.

DBS offer a simple, cost effective, alternative to venipuncture and are becoming an increasingly implemented research tool in field and population studies⁶.

The following protocol has been approved by the applicable institutional review board and was conducted in accordance with the Declaration of Helsinki.

Figure 1. Dried blood spot sample. A collection card is shown with four, valid collected spots in the process of drying. Click here to view larger image.

Figure 2. Good and bad dried blood spot samples. Results of successful (right) and unsuccessful (left) DBS collection are shown. Click here to view larger image.

Figure 3. Managers' attitudes & practices associated with employee cardiometabolic disease risk⁶.  This figure has been modified from Berkman, L. F., Buxton, O. M., Ertel, K. & Okechukwu, C. Manager's practices related to work-family balance predict employee cardiovascular risk and sleep duration in extended care settings. Journal of Occupational Health Psychology 115, 316-329 (2010). Employees exposed to managers with mid or low work-family scores of openness and creativity for helping with their employees' work and family need, have statistically significant increased odds of having two or more cardiovascular risks (current smoker, obesity, high blood pressure or hypertension, high cholesterol, and high glycosylated hemoglobin (Hb A1C) or diabetes diagnosis). Top and middle panels depict mean values ± standard deviations for total cholesterol assessed by dried blood spot and Hb A1C assessed by point of care device (which can also be reliably measured by dried blood spots). Bottom panel shows cardiovascular disease risk by manager score. Analysis restricted to nurses and other employees providing direct care indicate that exposure to managers with low creativity and openness related to work-family issues may be particularly important for these individuals (OR for low W-F score=6.4, p=0.01). All models control for age, gender, wage, education, race/ethnicity, and worksite. Click here to view larger image.

Biomarkers are extremely useful tools across multiple disciplines, including medicine, cell biology, genetics, psychology, sociology, demography, and social epidemiology. Within each discipline, early phase validity and reliability biomarker research was used to uncover both hidden disease risk and provide longitudinal prediction of health outcomes. In these instances, biomarkers that are accurate, durable (i.e. they can survive the lag between sample collection and implementation of the lab assay to extract the biomarker), and versatile (i.e. useful in a variety of models) are required.

While DBS collection is a relatively straightforward procedure, several techniques can help maximize volume of collected spots. Before collection begins, ensuring a participant's hand is warm, and therefore that blood vessels are dilated, will help blood flow once the finger is punctured. Using hand warmers or having a participant run their hands under warm water can help with this process. Milking the finger before collection will pull blood to the finger tip and warm the hand to help blood flow. Milking is a process that improves with practice. Collectors should use whatever technique they find most comfortable and effective, but the most common approach involves milking the participant's hand with the collector's opposite hand. This method will allow the collector's thumb to work deep into the participant's palm while the opposite hand can stabilize the participant's wrist. As an example, if one were collecting blood from a finger on the participant's left hand, the collector would stabilize the participant's wrist with their left hand and milk with their right hand. Common mistakes made while milking include not using a firm grip, pulling the skin above the hand rather working into the muscles to pull blood towards the finger tip. It is also important to avoid pinching the finger with the milking hand or tightly gripping the participant's wrist with the nonmilking hand, both of which impede blood flow.

When puncturing the finger, make the puncture as deep as possible by firmly pressing the lancet into the finger before discharge. A shallow puncture results in less blood flow and quicker clotting. After puncturing the finger, working quickly is key. Thin blood leads to easier collection and larger spots. After puncture, the blood becomes more and more viscous until clotting. The less time required to collect the blood spots on the card the greater their volume.

Ideally the participant's blood will be thin enough that drops will fall of their own weight, but this will not be the case for all participants, as some people's blood is thicker or clots faster than others. If the blood is too thick to fall from the finger, the filter card must be brought to the blood for collection. When bringing the card to the blood it is important to hold the card in contact with the drop while it is hanging from the finger for as long possible, rather than just dabbing the blood to the card. This will help pull blood from the finger to maximize the size of the spot. This technique requires steady hands to maintain contact between the blood and the card. Using both hands, one to steady the participants hand and one to hold the card, and keeping both hands in contact with one another helps to stabilize the card and the blood together.

Advanced research areas, such as cardiometabolic risk assessment, have long relied upon biomarkers such as C-reactive protein (CRP), insulin, and glycosylated hemoglobin (HbA1C) for early risk detection and prevention efforts, as well as biomarkers of "hard endpoints" for cardiovascular disease (CVD) risk and CVD-associated mortality¹². Clinical epidemiology frequently assesses biomarkers to prospectively predict cardiometabolic risk outcomes such as heart disease and Type II diabetes. Perhaps the most widely used of all biomarkers is the Framingham risk score, a composite that includes blood pressure, HDL cholesterol levels and total cholesterols levels that has demonstrated utility in predicting CVD¹³. However, the Framingham score has experienced some technical controversies, and has limitations in studies focused on linking social and environmental factors to health, suggesting the need for more accurate and specific biomarkers related to cardiovascular research.

There are a number of commercially available kits using ELISA or similar techniques available for the assay of DBS samples. Necessary for the assay of DBS samples are the use of specific and optimized elution steps to bring the dried samples into solution². This is an expanding area of research as assays for more analytes are currently being developed¹. Among the assays already validated and being widely used are cholesterol²,CRP^10,14,15, IgE16, EBV^9,15, leptin¹⁷, and cyotkines¹⁸. There are a number of laboratories specializing in the assay of DBS, including the University of Washington Department of Laboratory Medicine (UW Lab Med) Biomarker Laboratory (Director: Mark Wener).

Bearing in mind that a panel of biomarkers, rather than a lone biomarker, is most useful in the fields of cardiovascular and diabetes research, biomarkers need to be considered along with a variety of contextual factors in order to maximize their utility. Biomarker research can be expensive however, both financially and in time for data collection. Thus, while it is vital to choose biomarkers that are accurate, durable, and versatile, work and family biomarker research must be rooted in practicality by selecting biomarkers that are inexpensive, appropriate, and can be collected quickly with ease. Furthermore, practical considerations often result in an inability to simultaneously maximize price, quality, and speed within one procedure. Herein lie the next steps in health biomarker research within field and population studies. The field of biomarker research is currently in a period of dramatic growth; however, most of the work is yet to come.

Orfeu Buxton has received an investigator-initiated grant from Sepracor Inc (now Sunovion; ESRC-0977, ClinicalTrials.gov Identifier NCT00900159); serves as a consultant and expert witness for Dinsmore LLC, on the Scientific Advisory Board of Matsutani America, and received consulting fees from the Wake Forest University Medical Center.