How stable are analytes in dried blood?
by James Rudge, PhD, Microsampling Technical Director 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 — specifically 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 there are delays in logistics, risking a thawing event that can lead to potential sample degradation.
Do dried blood samples remain stable during shipping?
It is generally well known that analyte stability is often significantly improved when the matrix is dry compared to wet. There are several reasons for this. For example, drying samples can often put a halt to 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 there is progressive reduction in analyte peak areas 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 there are certainly a lot of changes that occur in the first 24 hours of the blood sample drying process. Most changes occur during this timeframe, but they can sometimes continue over the first 7 days or more.
This phenomenon was elegantly demonstrated by the pharmaceutical organization Merck & Co., Inc. in a 2018 paper where they showed, for a small drug molecule, an incomplete extraction recovery from a Mitra® microsampling device (40-52%) for a direct extraction method. As a result, the group observed a significant lost 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?
Issues with sample loss can also be a concern with 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 experienced an 11% drop after 24 hours of sample 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 amount of time.
Conversely, an opposite scenario was seen by researchers at Ghent University 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 measuring across 0.5 to 56 hours, DPS samples decreased from 59-32% (8 µg/µL) and 60-41% (80 µg/µL). The corresponding Mitra samples decreased from 101-87% at the lower concentration and only 102-97% at 80 80 µg/µL.
From these three examples different stabilities can be seen on the DBS and Mitra chemistries. This is one of the reasons why Trajan Scientific and Medical offers microsampling solutions that include both paper substrates for precise-volume DBS microsampling (using the hemaPEN® device) and polymer material for volumetric absorptive microsampling (using the Mitra® device).
Sample Stability and the Journey of the Sample
It is important to establish that sample loss is a result of degradation and 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 for details on how to optimize your extractions). It is important to garner an understanding pertaining to under what conditions the sample is stable and for how long. One way is to get test temperatures and conditions that the sample may be exposed to when being 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 that had been collected at home by study subjects and mailed back to the laboratory and samples collected onsite by study personnel.
The blood samples were collected for a multiplex serology application for influenza. It is remarkable that the remote Mitra samples showed good sample 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 for measuring tacrolimus remotely. In this example the remotely collected samples that were mailed to the lab showed good stability.
Conclusion: Blood Sample Stability
To date, there are over 370 peer reviewed publications featured in the Microsampling Resource Library on Trajan’s Neoteryx.com website, which show successful validations and applications of microsampling. A good proportion of these papers show excellent stability data for a wide variety of analytes and panels sufficient for the intended use of the assay.
When embarking on your validation work, it is highly recommended that you visit this online library to review the microsampling literature in your field of interest. Such a review will give you an idea of what to expect with the class of analyte(s) that you are investigating. You might also peruse our microsampling blog page where we regularly summarize microsampling research articles, including some that discuss analyte stability.
Explore microsampling for infectious disease studies here.
If you have questions, please contact our microsampling specialists to assist you.
Image Credits: iStock Photos, Trajan, Neoteryx
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