Over the last two years we have published around sixty blogs summarizing some of the amazing research papers that have been published about studies using dried blood samples collected on Mitra® based on VAMS® technology and the hemaPEN® microsampling devices. These blogs have covered research focused on clinical trials, therapeutic drug monitoring, environmental exposure monitoring, omics, serology studies, and related topics.
Additional validated methods using Trajan’s Neoteryx dried blood devices have also involved small drug molecules such as antiepileptics and large drug molecules, such as monoclonal antibodies (MAbs). It must be noted, however, that not all analytes can be effectively measured from dried blood extractions.
This is due to both physiological and/or instrumentational incompatibilities, as discussed in a previous blog. As a result, extraction methodology and analysis need consideration with respect to the analyte, matrix (usually dried capillary blood), and choice of analytical instrumentation.
It is recommended that all assays follow regulatory guidance. The vast majority of papers which we have reviewed align with one or more guidance documents issued by the US (United States) Food and Drug Administration (FDA), European Medicines Agency (EMA), and the International Association of Therapeutic Drug Monitoring and Clinical Toxicology (IATDMCT). Much of the guidance is around assessing typical bioanalytical parameters, such as stability, accuracy, imprecision, and linearity.
However, the major difference when comparing plasma to dried blood is the presence of the packed cell volume or hematocrit (HCT) in the blood. Indeed, it is recommended to measure the impact that different % HCT has on a number of your analytical parameters. HCT typically ranges from 25-65% and the usual value for most healthy people is around 45%.
However, the %HCT can impact the way blood spots spread on dried blood spot (DBS) cards, which can lead to analytical biases. Furthermore, %HCT can impact how easily analytes are extracted from any dried blood medium, whether collected on DBS cards or other dried blood microsampling devices.
This issue is outlined in the IATDMCT guidance document and mentioned in a previous blog reviewing a paper from SK Hall on variances in DBS observed for neonatal screening. Finally, the age of the sample also has an impact on sample extractability, which is comprehensively covered in the IATDMCT guidance document.
Plasma is the straw-colored liquid that is seen when blood has been carefully centrifuged to avoid hemolysis, and serum is the clear fluid left from blood that has been left to clot and then centrifuged.
The reason it is important to consider these two compartments (extracellular and intracellular) is because, depending on the analyte’s physicochemical properties and in some cases biochemical properties, they will either partition equally between both compartments or they will preferentially partition into one compartment over the other.
If analytes partition equally between the intracellular and extracellular compartments, this can have little to no impact or contribution to any observed analytical bias, when compared to a standard plasma or serum assay. However, if analytes accumulate in or on the cellular portion, then whole blood extraction, rather than processing plasma or serum, is conducted irrespective of whether the blood is wet or dry.
This is the case with analytes such as phosphatidyl ethanol (PEth) or the calcineurin inhibitor Tacrolimus. Indeed, for these analytes, concordance between wet and dried blood is often highly correlative with minimal bias reported.
There are two scenarios to consider when analytes are portioning into the plasma compartment, and these include complete or partial partitioning.
Complete partitioningComplete partitioning often occurs with certain biomarkers, such as steroid hormones and natural antibodies, are found only in the extracellular compartment and do not partition into the HCT. However, certain drugs such as monoclonal antibodies can also be totally excluded from the cellular fraction too. When this occurs, negative biases are seen in the whole blood when compared to plasma — sometimes upwards of -50%. As a result, there are three main ways to mitigate this natural bias:
Incomplete partitioning is where an uneven proportion of the analyte will partition into one compartment or the other. This is sometimes seen with small drug molecules for example and is discussed in a review on the analysis of antiepileptic drugs from volumetric absorptive microsampling) extracts.
This phenomenon can also be concentration- and time-dependent. Indeed, researchers at Astra Zeneca published an approach that intended to mitigate some of this dynamic observed for some molecules.
When developing an assay, it is important to spike the blood (allow enough time for any partitioning to occur), then sample with a dried blood microsampling device and separate out the plasma for comparative analysis. If partitioning has occurred, then a negative bias will be observed from the dried blood extract. However, it is important to ensure high extraction efficiency has been achieved as % recovery will cause negative biases too. Please refer to the Mitra Microsampling User Guide as well as the IATDMCT guidance document for advice on optimizing extractions from dried blood samples.
It must be noted that if plasma and blood is spiked into each matrix at the same concentration and then extracted separately, then blood to plasma partitioning bias will not be observed. Blood to plasma partitioning can only be observed when the analyte is spiked into the blood and plasma is harvested from the same blood samples, after capillary microsampling.
For an excellent deep dive into the impact of blood to plasma partitioning, the reader is encouraged to read a paper by Gary Emmon and Malcom Rowland entitled “Pharmacokinetic considerations as to when to use dried blood spot sampling.”
This blog includes content curated from published study papers that were summarized for our readers by James Rudge, PhD, Microsampling Technical Director. To learn more about the important research discussed here, please visit our Microsampling Resource Library, where we provide access to the original, third-party papers.
Image Credits: Trajan, Neoteryx, iStock, Dreamtime