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by James Rudge, PhD, Technical Director, Trajan on May 5, 2022 9:30:00 AM
Finger-stick collection of capillary blood for dried sample analysis is a highly convenient and minimally invasive alternative to conventional phlebotomy.
The increased availability of remote specimen collection tools like Mitra® devices based on VAMS® technology, which offer the ability to capture a quantitative blood sample from virtually anywhere, has broadened the scope for research.
Numerous research institutions have already adopted these devices for “remote microsampling studies,” in which study volunteers can participate by self-collecting their own samples at home.
Applications of Dried Blood Spot (DBS) Sampling and Associated Analytical Challenges
There are now more than 200 manuscripts in the research literature that describe highly successful validations and clinical studies using dried blood samples collected remotely with Mitra-VAMS devices. The studies include therapeutic drug monitoring on both small and large drugs.
Microsampling devices have also shown great utility in pharmacokinetics for preclinical and clinical pharmaceutical studies. Increasing success in these areas has allowed for more studies to onboard dried blood sampling for new drug trials.
Also, the Covid-19 pandemic illustrated the utility of remotely collected samples for serological research assays and, in so doing, helped validate the approach for related assays.
Additionally, several research papers have focused on remote sampling for therapeutic drug monitoring (TDM). Further, research into clinical toxicology and sports doping has shown that these devices can be used not only for measuring traditional drugs but also for measuring biological doping agents such as transgene vectors.
Research into biomarkers has shown that dried blood samples collected with devices based on volumetric absorptive microsampling can be used to measure specific analytes.
For example, studies measuring hormones and other current biomarkers, such as creatinine, HbA1c (available only on specific analyzers), and thiamine, have shown promise using dried Mitra-VAMS samples.
Moreover, there have been great strides made in novel biomarker research. For example, papers using Mitra-samples have been published in the fields of molecular diagnostics, proteomics, and metabolomics.
Finally, there has been interest in using dried blood samples for research into environmental and biological toxins, such as metals and exposure to ‘forever’ chemicals like Per- and Polyfluoroalkyl Substances (PFAS) that may be found in drinking water and household items.
Analytical Limitations and Diagnostic Issues in Dried Blood Spot (DBS) Measurement
Given the surprising variety of research assays that have shown utility on Mitra devices with VAMS and on the HemaPEN, it may be surprising to know that there are certain limitations that prevent dried blood microsamples from being used to measure all analytes. There are two primary reasons for this.
- Physiological incompatibility: Where the drying and processing of blood damages the analytes in the matrix (or damages the matrix itself) in such a way that they are not measurable in any meaningful way — or even at all!
- Analytical incompatibility: Where current / popular instrumentation is incompatible with microsamples and or dried blood extracts. An example of this is common blood tests, such as liver function enzymes and lipids, which are measured using clinical chemistry analyzers.
Key Assays Challenged by Dried Blood Spot (DBS) Workflows: Analytical and Diagnostic Limitations
Below is a list of common assays currently incompatible with dried blood samples.
Serum lipids: Such as cholesterol, LDL, and HDL. Cell walls contain cholesterol, and when dried blood is hydrated, the cells lyse. In doing so, the cells release lipids into the serum, leading to a false-positive result. The measurement of apolipoproteins may serve as an alternative to cholesterol or LDL/HDL measurements from dried blood samples for assessing cardiac risk. However, these do not fully correlate with standard lipid biomarkers and so are not yet standard practice.
Whole blood count / fraction: Typically, blood counts are measured using flow cytometry and require intact cells. Drying and rehydrating whole blood lyses cells, destroying the sample for analysis.
Liver function: The most popular assay for measuring liver function is to measure enzymes, released by the liver into the blood when damaged. This assay requires centrifugation of clotted blood to form serum (a clear liquid). Following this, a substrate is added to the serum, which reacts specifically with the liver enzyme to produce a pigmented product that can then be measured with spectrophotometry.
Dried blood, when hydrated, is fully lysed. Even after centrifugation, the extract is highly pigmented (primarily with hemoglobin). This leads to false positives in the assay.
A further complication that may occur when measuring some enzymes (such as the liver enzyme Aspartate transaminase, for example) is that these can also be found in red blood cells. Cell lysis during the extraction step can spill these enzymes into the sample matrix, leading to further false positives when compared to serum, where cellular contents are removed by centrifugation prior to sampling.
There are, however, attempts to identify new omics markers compatible with dried blood, but these are still at the experimental stage.
Serum electrolytes: Two key electrolytes in blood are sodium (Na) and potassium (K). The body pumps K into cells and Na into the plasma while maintaining a constant osmotic balance. Lysing cells from dried blood extracts releases cellular K into the serum, leading to a significant false-positive result when conducting an electrolyte test.
Vitamins: Vitamin D and B1 have been successfully evaluated on Mitra devices. However, vitamins are typically very difficult to measure. This is partly due to their antioxidant properties. In short, they risk oxidative degradation in the dried matrix.
Metals: Several papers have been published on Mitra devices with VAMS for metal analysis of blood samples from people who had received metal prostheses. Some papers have shown successful validations, including a recent paper measuring mercury.
However, surfaces (including the VAMS tips on Mitra devices) are easily contaminated with metals, leading to high background levels of other metals, such as lead, reported by experts analyzing Mitra samples. There are ways to mitigate this, but it is important to consider contamination before embarking on research on metals on dried blood samples.
Critical Assessment: Measurement Challenges and Diagnostic Accuracy in Dried Blood Spot (DBS) Analysis
As a rule of thumb, if you are looking to develop a method using dried matrix sampling, LC-MS/MS, or ligand-binding assays are often the best place to start. Due to good sample clean-up before analysis, these techniques often allow analysis of much more complex matrices, such as whole dried blood extracts.
However, if there are physiological reasons why dried blood samples are incompatible (such as K), then even these techniques will not help. As a result of these challenges, wet blood assays are the only realistic alternative for many analytes.
To develop a greater understanding of what is possible with dried blood assays and how to develop methods for them, please consult the resources listed below.
Resource Links:
- https://www.neoteryx.com/analytes-detected-vams-microsampling
- https://pubmed.ncbi.nlm.nih.gov/33675301/
- https://www.mdpi.com/1424-8247/14/7/627
- https://www.sciencedirect.com/science/article/abs/pii/S0021967320307214
- https://academic.oup.com/jat/article-abstract/45/6/581/5901577?redirectedFrom=fulltext
- https://www.neoteryx.com/microsampling-blog/using-mitra-with-vams-to-monitor-steroid-hormone-levels
- https://www.neoteryx.com/microsampling-blog/using-vams-to-measure-drug-level-and-renal-function
- https://pubs.acs.org/doi/10.1021/acs.analchem.0c05018
- https://www.neoteryx.com/microsampling-blog/mitra-and-dbs-used-in-epo-transgene-doping-detection
- https://pubmed.ncbi.nlm.nih.gov/30198258/
- https://pmc.ncbi.nlm.nih.gov/articles/PMC7261863/
- https://www.sciencedirect.com/science/article/pii/S2376999817300181
- https://pubmed.ncbi.nlm.nih.gov/35166117/
- https://www.semanticscholar.org/paper/Biofluid-Collection-in-Metabolomics-by-the-of-the-a-Meesters/d9b73806b097cfb1513dbd262f469e51579f88de?p2df
- https://orbi.uliege.be/handle/2268/268010
- https://www.sciencedirect.com/science/article/abs/pii/S0003269718300241?via%3Dihub
- https://www.sciencedirect.com/science/article/abs/pii/S0952327819300523
- https://pubmed.ncbi.nlm.nih.gov/27692372/
- https://link.springer.com/article/10.1007/s10661-022-09962-1
- https://www.sciencedirect.com/science/article/abs/pii/S0003267018310626?via%3Dihub
- https://pubmed.ncbi.nlm.nih.gov/31268966/
- https://pubmed.ncbi.nlm.nih.gov/31218897/
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