using VAMS® in the battle to fight COVID-19
by James Rudge, PhD, Technical Director, Neoteryx | 15 min read
When the COVID-19 pandemic hit in early 2020, people needed to shelter and isolate, whole countries were locked down and movement of individuals was reduced to an absolute minimum. For many, there was a temporary respite during the summer when infections dropped significantly, and life seemed to be returning to “nearly normal.” Yet, as the colder temperatures returned to the northern hemisphere, infections in many countries began to rise sharply. Throughout this pandemic, there has been huge demand for specimen sampling options to allow organizations to remotely monitor populations for COVID-19 infection and also to monitor patients with chronic illnesses who must stay safe at home. Additionally, remote sampling and telehealth communications have helped care providers and researchers reach vulnerable patients and volunteers for clinical trials.
The Mitra® microsampler based on VAMS® technology meets the needs of remote specimen sampling and is being used globally as the demand for remote patient-centric blood collection has skyrocketed. One area where demand for Mitra and VAMS (volumetric absorptive microsampling) has been particularly high is in serological and related testing to aid SARS-CoV-2 research. This mini-review of COVID-19 literature focuses on the use of VAMS in serological and related assays pertaining to the coronavirus pandemic.
The NIH Announces a Large Serological Survey Using Mitra and VAMS
In April 2020, the National Institutes of Health (NIH) announced they would conduct a serological study of at least 10,000 volunteers . The NIH researchers wanted to provide volunteers a remote option for collecting their own blood samples at home for mailing to the NIH lab. They decided to use Mitra® Blood Collection Kits from Neoteryx for this purpose. The researchers reported that Mitra devices had been a proven vehicle for easily collecting and transporting quantitatively precise Mitra samples for similar studies. In May 2020, the research team shared their initial findings, reporting that they had developed a robust ELISA for testing IgA, M and G antibodies for both spike and RBD proteins from Serum. Furthermore, they had also demonstrated an excellent correlation from Mitra blood sample extracts for IgG and IgM for both the Spike protein and the Receptor binding domain. In a recently published article in Nature Communications (January 2021, ) they stated:
“There was a strong linear correlation between the serum and [Mitra] microsampler results (spike = 0.991, RBD = 0.961), demonstrating that our method worked well with both sample types. This suggests that not only could detection of antibodies be performed reliably, but quantification of antibodies from mail-in sampling devices was possible.”
Summary of the NIH Mitra Extraction Method:
- Mitra tip (20 µL) added to PBS 1x (400 ul), BSA (1%) and Tween 20 (0.5%) in a 1 mL deep well plate;
- The plate was sealed with adhesive foil and incubated at 4 °C overnight;
- Eluate was stored at – 80 °C or analysed by ELISA using HRP detection (full details can be obtained from the original manuscript ).
Multiplex Assay Using Mitra to Simultaneously Measure >30 Strains of Influenza
The NIH researchers learned about the utility of Mitra devices and VAMS technology for serology studies from another well-respected research institution. A year prior to the COVID-19 study at the NIH, a research group at the University of Rochester Clinical and Translational Science Institute (UR-CTSI) led by Dr. Martin Zand conducted a small serological study on a cohort of 20 volunteers to examine seroprevalence of 30 influenza strains. This pilot study aimed to determine if it was possible to combine VAMS home sample collection with the FluPlex assay that Dr. Zand’s lab had developed. This bridging study showed good sample stability where Mitra dried blood samples were stored 21 days at ambient conditions in the lab. Samples remained stable even after being mailed to the lab at the height of summer when temperatures soared. Blood values corrected to serum levels (using an algorithm) with high concordance (R2 0.9721). Also, Mitra samples collected by a healthcare professional on day 1 correlated with equally high concordance (R2 0.976) . They stated in their report that:
“Finally, we also would like to note that this approach is not pathogen-specific, but could be adapted to estimating antibody mediated immunity after vaccination for any other viral pathogens, where the key antibody targets are known, and broad populations need to be surveyed. In addition, other biologic data could easily be obtained from a second VAMS device (another 10 μL of blood), including DNA samples for genomic sequencing of the host or blood-borne pathogens. Such methods are likely to be increasingly used in clinical trials in remote and rural areas, as well as newer ‘site-less’ clinical trials.”
It was these near prophetic words from Dr. Zand in his group’s study paper, and similar observations, which gave NIH researchers the confidence to run their large COVID-19 serological study in 2020 using remote Mitra devices with VAMS technology.
Summary of the FluPlex Assay Extraction Method:
- Tips (10 µL) soaked in 200 µL of extraction buffer (PBS+1% BSA +0.25 Tween) in 1mL 96 well plates, shaken overnight;
- Sample further diluted 1:250 to give a final dilution of 1:5000;
- The diluent (200 µL) was then added to a clear bottomed 96 well plate. For the immunoassay extraction (MAGPIX Multiplex Reader);
- A bead mix (50 µL, containing 30 separate recombinant HAs was added to each well of the plate);
- Plates were then incubated with gentle shaking for 2h at room temperature then 3 x washed with (PBS + 1% Bris + 0.1% BSA) using a magnet to immobilise the beads;
- IgG-PE secondary Ab was added, and the plates were incubated for another 2h;
- Three more washes were carried out and then the beads were resuspended in drive fluid (Luminex Co., TX).
Massachusetts General Publishes Roche Serology Assay from Remote Mitra Samples
Both the NIH and University of Rochester papers demonstrated that it was possible to successfully develop serology assays from Mitra devices, and both of these groups used “home-grown” assays. The NIH team designed their assay so that anyone with standard lab equipment could adopt their assay for conducting ELISA. The FluPLex assay requires the use of high-sensitivity equipment that not all serology labs possess but it has the advantage of being a multiplex assay. In November 2020, researchers at Massachusetts General Hospital and Partners Healthcare Group published work on adapting an FDA-approved Emergency Use Authorization (EUA) assay on the Roche Cobas® 8000 e801 Immunoassay Analyzer. They were the first to demonstrate such an assay on a large serology analyzer from Mitra extracts. The assay was validated to measure IgG, IgM and/or IgA antibodies raised from the nucleocapsid protein of the SARS-CoV-2 virus . The group validated the assay including a comparison of results two different facilities with high concordance (R2 0.97). When comparing a cohort of matched serum (from venepuncture) and dried extracts (capillary collection in the field), both showed seroprevalence of 24% (98/402 serum) and 97/402 for dried samples. This translated to a concordance of 99% (397/402). The team concluded the following:
“Extracts of dried blood from Neoteryx Mitra® devices acquired in a community seroprevalence study showed near identical sensitivity and specificity in detection of SARS-CoV-2 antibodies as compared to neat sera using predefined thresholds for each specimen type. Overall, this study affirms the use of the Neoteryx Mitra® dried blood collection device with the Roche Elecsys® SARS-CoV-2 total antibody assay for remote or at-home testing as well as large-scale community seroprevalence studies.”
Summary of the Extraction Methodology for the Adapted Roche Method:
- 20 µL Mitra, sampled and dried for at least 24h;
- Extracted in Autoracks with 200 μL/well of extraction buffer (1% BSA, 50 mM Tris (pH 8.0), 140 mM NaCl, and 0.05% Tween-20);
- It was reported that BSA was used to stabilize antibodies and the addition of Tween 20 was used to facilitate extraction and inactivate the virus;
- The plate was Shaken for 2h at 600 RPM;
- The assay was run on a Roche Cobas® 8000 e801 Immunoassay Analyzer, which also measured hemolysis and lipemia indices.
A small drawback of the Roche assay was that it had been developed to detect antibodies raised against nucleocapsid protein. This was a highly sensitive assay showing a great specificity for seropositivity to those who had developed COVID-19. However, the authors commented that this assay wouldn’t be able to detect immunity to antibodies raised from the spike and receptor binding domain (RBD) which are both targets for vaccine development and deployment. They commented that if such an assay were to be developed on spike and/or RBD, there would be the option to differentiate between convalescence/natural immunity and immunity raised by vaccination.
Comprehensive Analysis of SARS-CoV-2 Antibody Dynamics in New Zealand
In December 2020, a research team at the University of Auckland reported an investigation into the longitudinal immunity to SARS-CoV-2 by measuring not only raised antibody profiles, but also which of those were neutralizing. Using the same analytical platform as Rochester (Luminex MagiPlex), the Auckland team were able to develop multiplex assays to examine Spike, RBD and Nucleocapsid (NC) antibodies of the classes, IgM, IgA, IgG, as well as subclasses of IgG 1, 2, 3 and 4 over a 250-day period . They were able to demonstrate that, unsurprisingly, neutralizing antibodies were raised against Spike and RBD but not NC. Furthermore, IgG showed the highest protective longevity, which led the team to conclude such antibodies are present for 4-8 months after infection. In terms of classes of the IgG antibody, IgG1 and IgG3 were more prevalent and lasted longer than IgG2 and IgG4, which aligns with what is known about viral infection compared to immunological challenges .
Although most of the work conducted by the Auckland team was from serum samples, they also cross-validated the assay on Mitra devices for measurement of total IgG. In this part of the study, Mitra extracts taken from whole blood (n=19) were compared with paired serum extracts. Like the NIH assay, the researchers assumed 50% adjustment to account for the HCT. Again, very high correlations were observed for NC (R2 = 0.9957), RBD (R2 = 0.9929), and Spike (R2 = 0.9918). This led the researchers to conclude:
“The strong correlation between anti-RBD IgG and NAbs, combined with the demonstration in this study and by others that anti-RBD can reliably be measured from dried blood finger-prick samples, provides feasibility for future SARS-CoV-2 immunokinetic studies that incorporate RBD-IgG-based assays. The importance of conducting such studies at scale during vaccine roll-out is particularly relevant in settings like New Zealand, where there is potential to gain novel insight on vaccine responses given the lack of circulating SARS-CoV-2 in the community.”
Summary of the Mitra Extraction Method:
- 20 µL tip was extracted in 400 ul of PBS + 1% of BSA + 0.05% Tween 20 shook overnight at 300 rpm 4° C;
- Serum equivalent dilution of eluents calculated at 1:40;
- Extracts were then analyzed by the MagiPlex system.
Using Mitra for Lateral Flow Immunoassay and Chip-based Arrays
The majority of serology testing is conducted using immunoassay. Either ELISA is employed or an immunoassay instrument, such as the Luminex or Roche Cobas, as discussed in the examples above. However, another option is to bring the lab to the home and conduct a lateral flow test which works rather like a pregnancy test. Although it can be argued that such tests lack the quantitation of other types of immunoassay, they have the benefit of portability. Indeed, such a test was reported where Mitra device tips and DBS spots were compared to ELISA. Surprisingly, the IgG results were very comparable, though the IgM were not as congruent .
Summary of the Extraction Methodology for the Lateral Flow Test:
- Mitra tip was removed from sampler bodies for extraction;
- Dried samples were sonicated for 10 min 1 in 100 µL EDTA;
- Following a 1 min spin-down at 625 × g, 25 L of the extract was subjected to RayBiotech lateral flow analysis.
Promising research has also been published on development of a nanoarray immunoassay which can run 1,024 samples per device with a specificity of 100% and a sensitivity of 98%, based on the analysis of 289 human serum samples . Extraction conditions were PBS (1% BSA) or PBS (1% BSA, 0.5% Tween-20) overnight incubation at 4°C and the authors found that the addition of 0.5% Tween-20 greatly improved the assay, which has also been used in other extraction methodologies of SARS-CoV-2 assays on Mitra. Mitra and other devices tested were stored for 1 day at 55 °C. For Mitra, this resulted in very little sample degradation after 6 days of storage at room temperature. A slight sample degradation was observed when stored at 55 °C for an additional day.
Using Mitra for Direct Antigen Testing
Presently, there are two popular methods for SARS-CoV-2 testing. The first is direct viral testing using PCR (looking at the viral RNA levels), and the other is measuring immunity after a COVID-19 infection. Currently, PCR requires a nasal and/or throat swab, which is extremely uncomfortable. The disadvantage of PCR testing is that it does not indicate past infections or whether an individual has been recently infected by the virus. The disadvantage of antibody testing is that it takes up to two weeks after a viral infection for antibodies to be generated. Moreover, antibody testing does not indicate if an individual is still infected. However, presence of IgM and IgA and rising IgG antibodies in the blood may give a physician insight into whether or not the patient is still infected. Quanterix, a life sciences company based in Massachusetts in the United States, has developed a very high sensitivity method for directly measuring viral antigen (nucleocapsid) from blood . The remarkable achievement of Quanterix is that this can be achieved from extracts of Mitra, where plasma and Mitra extracts where highly correlative (R2 = 0.9924). The preliminary results seem to suggest the assay is at least as sensitive as PCR testing, demonstrating that both antibody and antigen testing can be conducted from one Mitra cartridge using the same platform. Their paper reports the following:
“We have demonstrated detection in capillary blood using the Neoteryx Mitra® dried blood spot (DBS) collection device, which enables at-home and point-of-care sample collection. Using [Mitra] DBS samples, we successfully monitored disease status of staff and residents in the presence of active COVID-19 infections with clinical sensitivity comparable to molecular testing in our preliminary experiments. Higher concentrations of N-protein associated with increased disease severity and mortality, and vice-versa clearance of the antigen associated with greater recoverability.”
Summary of the Extraction Methodology for Antibody & Direct Antigen Testing:
- Tips were used to absorb 20 μl of whole blood and were then allowed to dry for at least 16 hours in a pouch with desiccant;
- Tips were extracted into 250 μl of assay diluent with shaking at 400 rpm overnight at 2 - 8℃, resulting in a 12.5-fold sample dilution.
Quanterix Has Also Validated an Antibody Test on the Same Simoa® Platform
In addition to the ground-breaking direct antigen test, Quanterix has also developed an antibody test to measure IgG levels raised for the spike protein. In an application note published in 2020, they were able to demonstrate an impressive correlation between dried blood on Mitra devices with VAMS compared to wet plasma (R2 = 0.9996) . Moreover, when they corrected for HCT by measuring hemoglobin as a HCT predicate biomarker, an approach used by the Rochester group , the correlation even with HCT correction was equally impressive (R2 = 0.9987). The group also conducted an extensive stability study and demonstrated that even when storing the VAMS samples at 37 °C they observed a less than 20% decrease of SARS-CoV-2 protein IgG activity within one week. This led the group to conclude:
“This suggests that the Neoteryx dried blood VAMS tip samples are stable under extreme environmental conditions (37°C) during transportation for up to 1 week.”
The work conducted by Quanterix demonstrates that both SAR-CoV-2 antigen and antibody testing is possible from the same analytical system (albeit two separate assays). They have also shown that both assays are compatible with Mitra home sampling. One intriguing thought is that both antigen and antibody testing could be conducted from one drop of blood collected onto two Mitra samplers in one cartridge shipped to the same facility for testing. With the incredible sensitivity of the Simoa platform and its multiplex capability, antigen detection and a full antibody profile (including perhaps measurement of neutralizing antibodies) could, in theory, be developed.
Use of Mitra in High Throughput Environments for Mass COVID-19 Testing
One of the benefits of using Mitra devices (samplers) as vehicles for the ease of home blood collection and transport to laboratories is that the samplers are designed to work in both low and high throughput environments. Indeed, the NIH is using the samplers for their large cohort (n=> 10,000) serology survey and have developed a semiautomated ELISA platform to support the study . Another example is from LGC in the UK. The research group at LGC’s Cambridge-based Immunogenicity Centre of Excellence have developed a SARS-CoV-2 serology testing service with a capability of processing >40,000 Mitra samples per day in their high throughput laboratory. The Assay holds the CE Mark and they have applied for ISO17025 accreditation .
Using Mitra in Clinical Trials to Fight COVID-19
One of the huge benefits of remote testing using Mitra devices for microsampling is that drug levels can be monitored without requiring patients to visit clinics. This advantage has been demonstrated in the field of organ transplantation where Mitra devices have been deployed to pediatric transplant patients at home to remotely monitor their drug and biomarker levels [12,13]. This remote patient monitoring approach reduces the risk of transplant patients catching COVID-19 in hospitals, which is critically important since it has been reported that mortality of kidney transplant patients who contract this disease is high . Mitra also has been used extensively in clinical trials by the likes of Eli Lilly, Astra Zeneca and UCB Pharma, all of whom have discussed the use of Mitra and remote testing in clinical trials [15,16,17,18]. Indeed, Mitra was recently used in a clinical trial to test the efficacy of the antimalarial drug Hydroxychloroquine (HCQ) to determine if it was effective as a prophylactic against COVID-19. As part of the trial, blood measurements were taken from healthcare workers using Mitra devices for remote blood collection . Although the trial showed that HCQ showed no protective effect, it did demonstrate the ease and usability of the device in such trials.
Conclusion: A Year of Hard Lessons and Useful Insights
Two thousand twenty was a year that transformed societies and brought into sharp focus the fragility of human beings to viral outbreaks. As the SARS-CoV-2 virus continues to propagate globally, the medical and clinical science communities have responded to this health emergency with an unprecedented speed in assay development, medical treatment, vaccine development and deployment. The Mitra Microsampler powered by VAMS has proved to be a useful tool to have in the toolbox to fight this disease. In 2020, we learned that remote testing is not just a ‘nice to have’ technology but a necessary one for delivering clinical samples in times of crisis. We also learned that Mitra devices with VAMS technology provide a platform that can be used in a post-COVID-19 global society, enabling us to practice research and medicine across borders and without barriers.
- NIH Begins Study to Quantify Undetected Cases of Coronavirus Infection
https://www.niaid.nih.gov/news-events/nih-begins-study-quantify-undetected-cases-coronavirus-infection (Last accessed 07 Jan 2021)
- Standardization of ELISA protocols for serosurveys of the SARS-CoV-2 pandemic using clinical and at-home blood sampling
https://www.nature.com/articles/s41467-020-20383-x (Last accessed 07 Jan 2021)
- Application of volumetric absorptive microsampling (VAMS) to measure multidimensional anti-influenza IgG antibodies by the mPlex-Flu assay
https://www.cambridge.org/core/services/aop-cambridge-core/content/view/31604C8B8015E4B1DFEDC7AD75BDFBE0/S2059866119004102a.pdf/application_of_volumetric_absorptive_microsampling_vams_to_measure_multidimensional_antiinfluenza_igg_antibodies_by_the_mplexflu_assay.pdf (Last accessed 07 Jan 2021)
- Remote fingerstick blood collection for SARS-CoV-2 antibody testing
https://meridian.allenpress.com/aplm/article/doi/10.5858/arpa.2020-0713-SA/449019/Remote-fingerstick-blood-collection-for-SARS-CoV-2 (Last accessed 07 Jan 2021)
- Comprehensive analysis of SARS-CoV-2 antibody dynamics in New Zealand
https://www.medrxiv.org/content/10.1101/2020.12.10.20246751v1 (Last accessed 07 Jan 2021)
- IgG subclasses and allotypes: from structure to effector functions
https://www.frontiersin.org/articles/10.3389/fimmu.2014.00520/full (Last accessed 07 Jan 2021)
- Can dried blood spots (DBS) contribute to conducting comprehensive SARS-CoV-2 antibody tests?
https://onlinelibrary.wiley.com/doi/epdf/10.1002/dta.2816 (Last accessed 07 Jan 2021)
- A high-throughput microfluidic nano-immunoassay for detecting anti-SARS-CoV-2 antibodies in serum or ultra-low volume dried blood samples
https://www.medrxiv.org/content/10.1101/2020.10.07.20208280v3.full.pdf (Last accessed 07 Jan 2021)
- SARS-Coronavirus-2 nucleocapsid protein measured in blood using a Simoa ultra-sensitive immunoassay differentiates COVID-19 infection with high clinical sensitivity.
https://www.medrxiv.org/content/10.1101/2020.08.14.20175356v2 (Last accessed 07 Jan 2021)
- Application of Volumetric Absorptive Microsampling Devices to the Simoa® SARS-CoV-2 IgG Antibody Test
https://www.quanterix.com/whitepapers-appnotes/application-of-volumetric-absorptive-microsampling-devices-to-the-simoa-sars-cov-2-igg-antibody-test/ (Last accessed 07 Jan 2021)
- LGC Group Serology Assay https://www.lgcgroup.com/media/2331/lgc_dds_covid19_serology_assay_02_10_2020_update.pdf (Last accessed 07 Jan 2021)
- CountOnMe®, Information for patients and families
https://www.emeesykidney.nhs.uk/resources-for-families/countonme (Last accessed 07 Jan 2021)
- Assessment of tacrolimus and creatinine concentration collected using Mitra microsampling devices
https://journals.sagepub.com/doi/10.1177/0004563220948886 (Last accessed 07 Jan 2021)
- COVID-19 Infection in Kidney Transplant Recipients: Disease Incidence and Clinical Outcomes
https://jasn.asnjournals.org/content/31/10/2413 (Last accessed 07 Jan 2021)
- DIY Blood Sampling for Pediatric Clinical Trials—The Patients Perspective
https://www.appliedclinicaltrialsonline.com/view/diy-blood-sampling-pediatric-clinical-trials-patients-perspective (Last accessed 07 Jan 2021)
- A pharmacokinetic study of radiprodil oral suspension in healthy adults comparing conventional venous blood sampling with two microsampling techniques
https://bpspubs.onlinelibrary.wiley.com/doi/full/10.1002/prp2.459 (Last accessed 07 Jan 2021)
- Giving patients choices: AstraZeneca’s evolving approach to patient-centric sampling
https://www.future-science.com/doi/abs/10.4155/bio-2020-0105 (Last accessed 07 Jan 2021)
- Novel LC–MS/MS method for the determination of selumetinib (AZD6244) in whole blood collected with volumetric absorptive microsampling
https://www.future-science.com/doi/abs/10.4155/bio-2020-0062 (Last accessed 07 Jan 2021)
- Hydroxychloroquine as Pre-exposure Prophylaxis for Coronavirus Disease 2019 (COVID-19) in Healthcare Workers: A Randomized Trial
https://academic.oup.com/cid/advance-article/doi/10.1093/cid/ciaa1571/5929230 (Last accessed 07 Jan 2021)
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