Metabolon’s Validation of Dried Blood Spot (DBS) Cards for Metabolomics Analysis
DBS is a valuable and convenient sampling option to drive metabolomics research forward.
The COVID-19 pandemic has created a challenge for researchers who need to access patients’ blood plasma and serum, slowing or preventing research across the world. Metabolon realized it had a valuable matrix option at hand to drive metabolomics research forward. The ability to sample and ship small volumes of blood on a dried blood spot (DBS) card represents an alternative to in-clinic visits, enabling uninterrupted monitoring of vulnerable patients and sampling in remote populations, without escalating sampling costs. DBS allows sampling of small volumes with less invasive devices, making it an ideal solution for studies requiring frequent collections, such as human longitudinal studies and animal pharmacokinetics and pharmacodynamic studies where sampling may be daily- or even hourly.
DBS consists of a few drops of capillary blood from a finger or heel prick, which is applied to a card directly from the puncture site. Metabolon’s validation of DBS (collected on a Whatman™ 903 Protein Saver cards) on the Precision Metabolomics™ platform provides a valid solution for exploratory research on blood metabolites when access to, or appropriate storage and handling of, blood plasma and/or serum is limited or not possible.
Researchers are naturally inclined to wonder whether DBS is a suitable alternative to plasma, asking questions like, “can I use DBS samples for metabolomics instead of plasma?” and “how stable are the metabolites in DBS samples?” To address these frequently asked questions, we recently published two whitepapers: “Comparison of metabolomic profiles in plasma versus dried blood spot cards”1 and “Stability of metabolite profiles using dried blood spot cards”2, both of which are based on our extensive validation of DBS using Metabolon’s global metabolomics technology.
In the Q&A section below, we asked Kelli Goodman, Staff Scientist at Metabolon, to address some of the common questions regarding the use of DBS samples for metabolomics analysis.
Q: What makes DBS and plasma different matrices, and how do their metabolomic profiles compare?
A: Unlike plasma, DBS samples contain whole blood, which is composed of approximately 55% plasma and 45% blood cells. The presence of cellular matter in DBS samples introduces cellular-specific metabolites that are present at low levels, or not at all, in plasma. DBS samples are also dried on filter paper prior to storage, rather than stored in liquid form like plasma; the effect of which results in oxidative and/or hydrolytic degradation of some susceptible compounds. The drying effect also halts many of the enzymatic reactions that continue to occur in liquid samples, thereby preserving the stability of compounds at room temperature in DBS samples.
In order to compare the metabolomic profiles of DBS and plasma, we collected donor-matched venous EDTA plasma and DBS card samples from 49 volunteers, of which half fasted and the remainder ate at libitum prior to sample collection. The data were clear and, together with results from other studies conducted at Metabolon, support our conclusion that the metabolic profiles are indeed similar between matrices:
- Even with a reduction in metabolite coverage in DBS compared to plasma (~700 – 900 vs ~1200 metabolites, respectively), all major pathways and more than 95% of the metabolic sub-pathways that we routinely detect in plasma are detected in DBS.1
- Metabolic signatures of disease that have been well-characterized in plasma are maintained in DBS.1
Q: What areas of study are DBS cards appropriate, and/or better suited, for than plasma?
A: Researchers should consider DBS over plasma when plasma is impractical to collect and/or store. With that said, there are some compound classes that perform better in DBS due to the addition of cellular material and cellular-bound metabolites. The following classes contain metabolites that can be more easily detected in DBS than in plasma: carbohydrates (nucleotide sugars; glycolysis/gluconeogenesis/pyruvate metabolism), amino acids (glutathione and histidine metabolism), nucleotides (purine metabolism, particularly adenine- and guanine-containing), cofactors and vitamins (NAD+ and ADP-ribose), and some known inflammatory markers (such as eicosanoids and docosanoids).
Conversely, DBS might not be well-suited for studies with a strong focus on metabolites that are susceptible to oxidation and/or hydrolysis, such as unsaturated fatty acids. Note that storing DBS samples in a sealed, gas-impermeable bag with desiccant at -80°C will help preserve said biochemicals.1,2
Q: Are DBS suitable for monitoring biological changes in metabolite levels?
A: Early on in our R&D endeavors we sought to answer this question by conducting a 30-day repeated-measures study, in which 22 Metabolon volunteers self-collected their DBS samples once daily, at their own home, and recorded daily metadata, including diet and drug prescription/supplementation usage and exercise habits. This study illustrated the inherent variability (or for many biochemicals, the homeostatic nature), of the human metabolome within an individual over time and across individuals.1
In this proof-of-concept study in DBS card samples, we were able to observe many of the biological signatures that we usually observe in plasma, including exercise biomarkers, dietary biomarkers and even the cyclical trend of the female hormonal biomarkers. These results indicate that DBS is a suitable matrix for conducting metabolomics experiments.
Even in this small population of “healthy” individuals, these DBS data provided evidence of the homeostatic regulation of metabolite levels occurring within our bodies and the biological diversity that exists across individuals (often referred to as a metabolic fingerprint that is unique to each individual). This biological precision per individual and diversity across individuals was demonstrated across many pathways.
Q: How should DBS samples be handled?
A: Like any other matrix, handle all DBS samples (control and treatment groups) as consistently as possible. The preferred method for storage and shipping is with dry ice (-80°C) and desiccant packs, sealed in a gas-impermeable zip-top bag. Based on our data, temperature rather than storage duration has the biggest impact on sample stability: warmer temperatures increase the frequency (number of affected biochemicals) and the delta response (from collection); however, most of these stability effects occur rapidly and cease by the third week of storage (i.e., affected biochemicals stabilize at the new level within this period). For this reason, samples will not be analyzed until they have been stored for at least three weeks post-collection; and together with ongoing stability data of at least 3.5 years, storage duration is not an issue for archived DBS samples.
If similar storage conditions cannot be guaranteed across samples, then increased group sizes would be recommended to help account for any increased sample variability. If areas of metabolic focus are of interest or critical need, reaching out to study directors is advisable to ensure client expectations can be met.2
Q: What’s the best way to collect samples for DBS cards, and what’s important to know?
A: DBS samples, like other biological samples, must be collected and stored consistently. Regarding collection, the most important tip is to ensure your hand and fingers are warm prior to performing the puncture. Read the “Tips to Improve Blood Flow” and the collection guidelines reported in “Best Practices for Dried Blood Spot Card Collection.” Drink water 30 minutes prior to collection and stand with your hand below your waist before and during collection. If the puncture site is not properly prepared, then a cascade of issues might arise, including spots being too small and/or hemolysis of blood cells caused by “milking” the finger. The second most important collection tip is to allow a LARGE blood droplet to form at the puncture site prior to applying the drop to the DBS card- the droplet will be large enough once it comes close to dripping off the finger. This will ensure a large enough spot is collected (>7 mm diameter; a pencil eraser). Do not apply more than one droplet per spot; if it seems too small, wait for a larger droplet to form then spot the next outlined circle. Two spots are required per analysis. Finally, regarding handling and shipping, for the most accurate metabolomic data we recommend storing and shipping samples as cold as possible, keeping in mind to do so in a consistent manner across all the samples.
To discuss your needs and how our dedicated team can support your research, contact us or email us at hello@metabolon.com.
Kelli Goodman
Kelli has been a part of Metabolon’s technology R&D team for five years where one of her key contributions has been leading the company initiative to better understand self-collection blood strategies for metabolomics applications, an area that has gained considerable interest during the COVID-19 global pandemic. She managed the ISO-9001-compliant launch of Metabolon’s metabolomics capabilities in dried blood spot (DBS) card samples, which included a full analytical and biological validation for > 700 metabolites. Using her extensive experience in targeted bioanalytical LC-MS/MS method development and GLP/CLIA-CAP validations, she also leads the development of quantitative assays on the untargeted global platform. Kelli graduated from North Carolina State University with a B.S. in Zoology. Her previous experience involved developing ELISAs at an Immunotoxicology CRO before transitioning into LC/MS.
References:
- McCulloch, Scott D., et al. “Comparison of metabolomic profiles in plasma versus dried blood spot cards” Metabolon Inc, Morrisville, North Carolina, March 18, 2021
- McCulloch, Scott D., et al. “Stability of metabolite profiles using dried blood spot cards” Metabolon Inc, Morrisville, North Carolina, March 18, 2021
