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by James Rudge, PhD, Technical Director, Trajan on Dec 11, 2023 9:00:00 AM
One of the great benefits of dried blood microsampling is the fact that these samples can be remotely collected almost anywhere, at almost any time and by almost anyone. However, one of the concerns facing remote microsampling, is ensuring that the molecular integrity of the sample will remain intact during the shipping process.
The Challenge with Transporting Blood Samples
When considering wet blood samples, compound and matrix stability can be compromised by shipping conditions, particularly at elevated temperatures (this is analyte-dependent).
As a result, many wet blood samples are transported under special temperature-controlled conditions using dry ice or refrigeration. However, cold shipping is costly and can be problematic when logistics are delayed, risking a thawing event that could lead to sample degradation.
Do dried blood samples remain stable during shipping?
It is well known that analyte stability is often significantly improved when the matrix is dry rather than wet. There are several reasons for this. For example, drying samples can usually halt specific enzymatic degradative events. In addition to enzymatic degradation, analytes in wet matrices can also be exposed to non-specific degradation, such as hydrolysis.
In many cases, drying the blood samples prevents or slows down analyte degradation. Dried sampling then allows for a wider temperature stability, enabling the journey from the sampling event to the laboratory with the analytes intact.
But how do we know if an analyte of interest will survive the journey from the sampling location to the lab? This blog explores methods to detect analyte degradation and good laboratory practice to assess the sample integrity during 'journey of the sample' experiments. It also provides examples in the literature showing results of such experiments.
Degradation or extraction efficiency?
Sample degradation can be suspected when analyte peak areas decrease progressively over time. However, we must also consider another common reason for this phenomenon, which can resemble instability — temporal extraction bias.
It is well known that, for many analytes, the older the blood becomes, the lower the extraction recovery. The mechanism behind this is not fully understood, but many changes occur during the first 24 hours of the blood sample drying process. Most changes occur during this timeframe, but they can sometimes persist for the first 7 days or longer.
This phenomenon was elegantly demonstrated by the pharmaceutical organization Merck & Co., Inc., in a 2018 paper, where they showed that, for a small drug molecule, incomplete extraction recovery (40-52%) was observed from a Mitra® microsampling device using a direct extraction method. As a result, the group observed a significant loss of method accuracy over 7 days.
However, by switching to a liquid-liquid extraction (LLE) method for the same analyte, they saw a vast improvement in extraction efficiency (~90%). The method remained accurate for at least 35 days at room temperature (and –20 °C) for 35 days. Moreover, the assay also remained accurate at 40 °C for 14 days.
The researchers demonstrated a relationship between extraction recovery and accuracy, which we called 'temporal extraction bias.'
Several years ago, the Neoteryx microsampling research and development team saw a similar phenomenon when optimizing a corticosteroid assay on Mitra® devices with VAMS® technology. In that case, the team saw analyte recovery drop from ~90% to less than 70% after leaving the VAMS tips to dry for 20 hours, when compared to 3 hours. However, this issue was solved by increasing the vortexing time (see figure 1).
Figure 1. Recovery of corticosteroids under two different extraction conditions (Method B has a longer vortexing time).
Different microsampling device chemistries?
Sample loss can also be a concern across different matrices. For example, when measuring the diabetes marker c-peptide, researchers at Swansea University in Wales (UK) reported good stability for c-peptide on dried blood spots (DBS). When assessing C-peptide in liquid blood samples in their lab, the group observed an 11% decrease after 24 hours of storage at room temperature. In contrast, the group experienced only a 0.1% drop after storing samples collected on Mitra devices with VAMS for the same period.
Conversely, researchers at Ghent University observed the opposite when measuring HbA1c on dried blood samples. They found that HbA1c failed suitability requirements on both DBS cards and Mitra devices with VAMS over a few days. However, in this case, the stability of HbA1c on the DBS matrix was better than on the VAMS matrix.
Another example of sample stability is found in research published in Australia in 2016 on the antibiotic vancomycin. In this study, the research group compared dried plasma spots (DPS) with plasma dried on Mitra devices. When measured over 0.5 to 56 hours, DPS samples decreased from 59% to 32% (8 µg/µL) and from 60% to 41% (80 µg/µL). The corresponding Mitra samples decreased from 101-87% at the lower concentration and only 102-97% at 80 80 µg/µL.
Sample Stability and the Journey of the Sample
It is essential to establish that sample loss results from degradation, not a temporal extractability-mediated bias. If it is the latter, this can often be solved by optimizing extraction methodology (see the Mitra Microsampling User Guide below for details on how to optimize your extractions).
It is important to understand under what conditions the sample is stable and for how long. One way is to obtain test temperatures and conditions to which the sample may be exposed when sent via standard mail.
When validating methods, some groups will compare remote samples mailed to the lab from home to samples that have be collected onsite. For example, in 2019, researchers at the Clinical and Translational Science Institute, University of Rochester Medical Center, in New York, demonstrated a high correlation (R2 = 0.9496) between Mitra samples collected at home by study subjects and mailed back to the laboratory and those collected onsite by study personnel.
The blood samples were collected for a multiplex serology application for influenza. Remarkably, the remote Mitra samples showed good stability, even though they were sent during the height of summer in August, when they would have been exposed to elevated temperatures.
In another example, researchers at Oslo University Hospital in Norway conducted a similar experiment to measure tacrolimus levels remotely. In this example, the remotely collected samples that were mailed to the lab showed good stability.
Understanding Analyte Stability in Dried Blood Microsamples
With hundreds of peer-reviewed publications in the Microsampling Resource Library, researchers across disciplines have validated dried blood microsampling as a robust method for diverse analytes.
Learn More About Microsampling Advantages
If you are exploring stability, usability, or the broader scientific value of microsampling, we recommend visiting our in-depth page, Microsampling Capillary Blood Advantages. This page provides a clear overview of the technology, advantages, challenges, and real-world applications across clinical trials, pediatrics, TDM, population studies, and more.
Image Credits: iStock Photos, Trajan, Neoteryx
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