microsampling research: analytes that are challenging to measure from dried blood

Finger-stick collection of capillary blood for dried sample analysis is a highly convenient and minimally invasive alternative to conventional phlebotomy. With the increased availability of remote specimen collection tools like Mitra^® devices based on VAMS^® technology, hemaPEN^® based on precision DBS, and other user-friendly options, 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” where study volunteers can participate by self-collecting their own samples at home.

Areas of Research Where Dried Blood Samples Are Useful

There are now more than 200 manuscripts in the research literature that describe highly successful validations and clinical studies using dried blood samples that were remotely collected with Mitra-VAMS devices or the hemaPEN. The studies include therapeutic drug monitoring on both small and large drugs.

Microsampling devices have also shown great utility in pharmacokinetics for pharmaceutical pre-clinical studies and clinical trials. 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 on serological research assays and in so doing, has helped to validate the approach for other related assays. 

Additionally, there have been several research papers focused on using remote sampling for therapeutic drug monitoring (TDM). Further, research into clinical toxicology and sports doping have shown that these devices can be used not only for measuring traditional drugs but also can be used for measuring biological doping agents such as transgene vectors.

Research investigations into biomarkers have, in recent years, shown that dried blood samples collected with devices based on volumetric absorptive microsampling can be used for measuring specific analytes. For example, studies measuring hormones and other current biomarkers such as creatinine, HbA1c (specific analyzers only), and thiamine have shown promise using dried Mitra-VAMS samples.

Moreover, there have been great strides gained in novel biomarker research. For example, papers using Mitra-VAMS and hemaPEN 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.

The list of programs in which dried blood sampling can be applied continues to grow as research scientists become increasingly skilled in handling microsamples and as analytical instruments and techniques becoming increasingly more specific and sensitive.

Limitations of Dried Blood Microsamples for Whole Blood Research Assays

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 there are certain limitations that prevent dried blood microsamples being used to measure all analytes. There are two primary reasons for this.

1. 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!
   
   
2. 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.

Popular Assays Currently Incompatible with Dried Blood Microsamples

Below is a list of some of the common assays that are currently incompatible with dried blood samples.

1. 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 spill lipids into the serum, causing a false-positive result. The measurement of apolipoproteins may become an alternative approach for the measurement of cholesterol or LDL/HDL 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.

2. 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.

3. 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 from the extraction step will act to spill these enzymes into the sample matrix leading to further false positives when compared to serum where the cellular contents are removed by centrifugation prior to sampling.

There are however attempts to find new omics markers that could be compatible with dried blood, but these are still at the experimental stage.

4. 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 release cellular K into the serum, causing a significant false-positive when conducting an electrolyte test.

5. Vitamins: Vitamin D and B1 have been successfully evaluated on Mitra devices. However, vitamins are typically very difficult to measure. This is partly a result of their antioxidant properties. In short, they risk oxidative degradation in the dried matrix.

6. Metals: There have been several papers published on Mitra devices with VAMS used for metal analysis in blood samples taken from people who had received metal prostheses. Some papers have shown successful validations, including a recent paper measuring mercury.

However, surfaces (including the surfaces of VAMS tips on Mitra devices) are easily contaminated with metals, so high background levels of other metals such as lead, have been reported by experts analyzing Mitra samples. There are ways to mitigate this, but it is important to consider contamination before embarking on research of metals on dried blood samples.

Concluding Thoughts on Measuring Analytes From Dried Blood

As a rule of thumb, if you are looking to develop a method using dried matrix sampling, then either 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 for analysis from much more complicated 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 these, please look at these resources (24, 25, 26, 27) in the list of source links below.

Image Credits: Neoteryx, Trajan Scientific and Medical