using VAMS microsampling for mercury biomonitoring
by James Rudge, PhD, Technical Director, Neoteryx | 6 min read
An article by Stefan Rakete et al at the Institute and Clinic for Occupational, Social and Environmental Medicine, University Hospital in Munich, Germany and three related institutions was published in the March 2022 issue of the Journal of Environmental Monitoring and Assessment. It reported on the development of a direct atomic absorption assay to measure mercury levels from dried blood samples collected on Mitra® devices with VAMS® technology. The paper is entitled “Mercury biomonitoring in German adults using volumetric absorptive microsampling.” It describes the development and validation of an assay comparing mercury (Hg) levels in dried VAMS blood samples and paired with venous blood.
The co-authors saw a strong correlation between both sample types and reported that VAMS sampling used in combination with direct mercury analysis was an accurate and viable alternative for human biomonitoring of Hg. A critical part of the study was to measure sample stability and they found that the choice of storage medium was key to keeping the samples stable.
Mercury Exposure and Toxicity to Humans
Mercury (Hg) is a pollutant and neurotoxin that is harmful to humans and poses a significant health concern, as highlighted by the World Health Organization (WHO) and United Nations Environment Programme (UNEP). Exposure to mercury can occur a number of ways, including through ingestion of seafood containing methylmercury and through mining (especially mining mercury). Exposure to the element may also occur through certain dental amalgams, some skin whitening creams, or as a result of burning of fossil fuels.
Exposure to Hg can cause many physiological effects, leading to disorders in the cardiovascular and nervous systems. Indeed, prenatal exposure to Hg is of great concern as it may negatively impact healthy brain development in fetuses. Moreover, Hg exposure can cause negative effects in the kidneys and reproductive systems.
A recent paper published in Nature Communications estimated that the global human health cost of exposure to methylmercury alone was $117 billion and accounted for 29,000 deaths. Methylmercury is an organometallic form of mercury, formed by specific microorganisms. Once synthesized, the methylmercury enters and accumulates in the food chain. Two other forms of mercury are elemental Hg, and inorganic mercury compounds. Elemental Hg is found in several sources, such as in fluorescent light bulbs and emissions from burning fossil fuels. Inorganic mercury compounds or salts can occur naturally or for use in industry, as well as in some skin whitening creams.
Monitoring Mercury Exposure and Toxicity Levels in Humans
Although mercury can be detected and analyzed in urine and hair, the gold standard for monitoring mercury is venous blood. However, Stefan Rakete et al highlighted in their published paper that venous blood sampling has been associated with relatively high logistical costs for human biomonitoring, especially in remote African, Asian, and Latin American countries. The study authors underlined the fact that access to a trained phlebotomist for venous blood draws in remote areas and logistics for cold chain shipping of samples were the main challenges for biomonitoring.
Due to these challenges, Rakete et al proposed that microsampling might be of great interest as dried microsamples can often be collected remotely, finger-stick blood collection is minimally invasive, and dried samples do not often require cold-chain shipping. Although dried blood spot (DBS) with filter cards has been the most popular microsampling method to date and has shown some success in Hg analysis, the authors also noted that there have been reports of background contamination and hematocrit dependency. They stated that volumetric blood collection with VAMS has a number of benefits over DBS, including the fact that “VAMS enables relatively easy and ergonomic blood collection.”
Because Mitra devices with VAMS had not been previously used for Hg monitoring under field conditions, the research group embarked on comparing paired venous and capillary blood from non-occupationally exposed adults using a direct mercury analysis technique.
Mercury Biomonitoring Study Design and ResultsStability Experiment
- Dried VAMS blood samples (20µL measured at 23 µL) were stored 1, 2 and 4 weeks in either plastic zip-lock bags ((22 × 16 cm) from Buerckle) or 1.5 µL plastic screw cap vials that had been acid washed to reduce trace metal contamination. To test environmental conditions, the samples were stored at –20° C, room temperature, or 40° C.
- When stored in plastic bags at room temperature, Hg recoveries increased up to 708% after 4 weeks. It was hypothesized that this may be due to contamination from the bags or possibly due to the gas permeability of the plastic. Interestingly, this phenomenon was not observed at -20° C where at 1 week recovery = 113%, 2 weeks = 92% and 4 weeks = 99%. At 40° C, all three time periods showed recovery of between 142% and 158%. Similar results had also been seen in a previously reported DBS paper. In contrast to that previous study, this phenomenon was practically eliminated in the study reviewed here when samples were stored in glass vials. However, blank samples did not show any difference in background Hg Levels in any of the tested conditions.
- Paired venous (7mL) and capillary (VAMS x 4) samples (n=69) were collected from 65 adults, and samples were collected from 2 volunteers at more than one timepoint.
- For VAMS collection, contamination was minimized by taking great care with handwashing, disinfection, discarding the first drop of blood, and not touching the device tip.
- After drying for two hours, the tips were removed from the Mitra sampler bodies and placed in either vials or zip-lock bags.
- Direct Hg analysis was conducted by atomic absorption spectroscopy (253.5 nm) by adding either venous blood (100 µL) or 1-2 Mitra device tips to quartz sample boats.
- The group commented that the advantage of this technique compared to inductively coupled plasma mass spectrometry (ICP-MS) is that no lengthy sample preparation was needed.
- After excluding 1 sample (below LLOQ), 68 samples were compared.
- 91% of samples showed a <20% RSD compared to all venous samples, where 100% were <20%.
- Mean RSD values were 8.7% and 3.4% for VAMS and venous samples, respectively. It was concluded that the higher RSD was related to the smaller blood volume compared to venous blood. Indeed, they saw that RSD improved with increasing Hg levels, which had also been previously observed with DBS.
- Both samples showed a strong linear relationship (Spearman-Rho, R2 = 0.958, p < 0.001). However, below 0.1%, the results deviated from the identity line.
- Recoveries were high from the Mitra-VAMS devices and apparent high recoveries of below 0.4 µg/L may have been due to potential contamination in the samples or possible wicking volume differences.
- Bland-Altman data showed a positive bias for the VAMS samples at 0.11 μg/L. However, 65 out of the 68 samples were within 95% confidence interval (-0.1- 0.31 μg/l) showing good agreement between both methods.
- When samples (n=24) were < 1 μg/L, two samples (VAMS) were analyzed simultaneously in the sample boat to help boost sensitivity. Although this didn’t help median recovery, it did improve precision, leading the group to conclude that this approach would be helpful in future studies.
- This was the first study to measure Hg using paired VAMS blood samples and venous blood samples using direct analysis, negating the need for sample preparation.
- The study thoroughly investigated storage conditions.
- Sensitivity was a challenge for 24% of the VAMS samples (@ <0.5 µg/L) when measured as individual samples, but this was improved by measuring 2 samples simultaneously. The authors concluded that either employing ICP-MS or 30 µL tips would help with sensitivity.
- When collected, capillary blood is a mix of venous and arterial blood as well as a chance of some interstitial fluid, so differences could be seen between the two matrices, but recent studies suggest this is not an issue.
- The authors were able to demonstrate that the sampling method they used “produces reliable and accurate results and can therefore be used for Hg exposure assessment in studies with human subjects with limitations for low exposed individuals (Hg levels < 0.4 μg/l).”
Neoteryx Thoughts and Observations
Measurement of environmental contaminants for public health and epidemiological studies are key to helping to improve the health of millions globally. An efficient way to collect samples from ‘hard to reach’ regions and from vulnerable populations (such as children) is critical to the success of future studies.
The study conducted by S. Rakete et al demonstrated that such an approach using VAMS offers efficiency and has real merit. However, it is important to minimize unwanted environment sample contamination prior to and during studies. For example, it was observed in this study that using a zip-lock bag, compared to an acid-washed vial, introduced unwanted contamination during a stability study. Use of gas impermeable bags (including desiccant), as supplied in Mitra Collection Kits, may help to mitigate this issue. Furthermore, use of desiccant is crucial especially in highly humid environments to prevent microbial damage of the sample.
This study paper was summarized for our readers by James Rudge, PhD, Neoteryx Technical Director. This is curated content. To learn more about the important research outlined in this review, visit the original article published in the Journal of Environmental Monitoring and Assessment.
Image Credits: mercury level testing-iStock, Neoteryx
You can access this microsampling study and others in our Technical Resource Library.