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Driven by Potential Cost Savings, Convenience, Proteomics Explores Dried Blood Spot Analysis

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Used for decades as a sample collection method for newborn screening, dried blood spots have in recent years drawn interest as a potential approach for drug development and clinical work, where the format could offer improved convenience and cost savings compared to conventional blood samples.

Much of this recent interest has focused on analysis of small molecules for purposes like pharmacokinetic assays. However, proteomics researchers have begun looking into dried blood spots, as well, with some suggesting that they could prove a key technology for establishing widespread, regular proteomic testing.

Dried blood spots typically consist of microliter volumes of blood spotted and dried on filter paper. They can be stored and shipped without refrigeration and analyzed by a variety of methods including immunoassays and mass spectrometry.

With regard to proteomic analysis of such samples, there have been questions about how amenable they are to the detection of proteins, and particularly low-abundance proteins. However, recent research efforts suggest they could indeed prove suitable for such work.

For instance, Leigh Anderson, CEO of clinical proteomics firm SISCAPA Assay Technologies, has recently begun running his company's library of mass spec-based protein quantitation assays against a collection of more than 100 dried blood spot samples he has collected over the last four years.

The data from this work is "looking very interesting," Anderson told ProteoMonitor. "For instance, if you have a cold, you can actually see huge inductions of the immune response in [the sample]. There is real biology revealed in these things exactly as we hoped there would be."

Anderson and his SAT colleagues began looking into dried blood spots as an alternative to conventional plasma and serum samples "because they are just so easy to collect and very robust," he said.

Thus far, he noted, they have managed to develop methods for reliable trypsin digestion of proteins in such samples and have implemented essentially the same automated LC-MS/MS workflow they use for quantitating proteins in traditional blood or plasma samples, which offers throughput of roughly 400 samples per day.

In terms of what proteins the researchers have been able to detect, Anderson said they could measure in dried blood spots more or less any proteins that would be detectable in a conventional plasma sample of equivalent volume, 10 microliters.

He added that they plan to publish initial results from the work in the near future and have also established several collaborations with vendors of equipment for dried blood spot analysis.

Anderson and SAT are not alone in looking at dried blood spots for proteomic work. In 2011, nanotechnology firm NanoInk began offering dried blood spot analysis for protein biomarker research as a service on its NanoArray Assay System (PM 9/23/2011).

And, also in 2011, scientists from King’s College London and Guy’s and St Thomas’ NHS Foundation Trust launched SpotOn Clinical Diagnostics, a firm using multiple-reaction monitoring mass spec analysis of dried blood spots to screen newborns for hemoglobin disorders (PM 12/9/2011).

In addition to hemoglobinopathy screening, SpotOn has also developed assays for proteins linked to nephrotoxicity, which it hopes to offer for drug-development work; and assays to track renal and cardiovascular health.

Another firm using dried blood spots for protein assays is Chandler, Ariz.-based clinical proteomics outfit Healthtell.

The company, which was spun out of the lab of Arizona State University Biodesign Institute researcher Stephen Johnston in 2010, uses immunosignaturing – a technique to broadly profile antibody levels using random-sequence peptide arrays – to identify antibody expression patterns indicative of disease states in samples including dried blood spots.

In a paper published last year in the journal Clinical and Vaccine Immunology, Johnston and his colleagues demonstrated the stability of dried blood spots, finding that samples sent through the mail yielded usable immunosignatures, and that dried blood spots stored at high temperatures remained usable for immunosignaturing (PM 4/27/2012).

In an interview following the release of the paper, Johnston told ProteoMonitor that ultimately he hoped to develop the technology into a direct-to-consumer product that would allow people to monitor their health status by regularly mailing in dried blood spot samples.

"Our goal is that everybody will monitor their health on a regular basis," he said. "So if you're going to do that you want to have some really robust way for people to send you their samples. So we wanted to look into that carefully to make sure the idea that someone would send in a drop of blood or spit on a regular basis was practical."

Earlier this year, Healthtell CEO Bill Colston told ProteoMonitor that the ability of the platform to use dried blood spots was "one of the big things" that convinced him to join the company in 2011.

"Our vision is that you can take a blood sample less than the size of a glucose measurement on a piece of tissue and mail it in to [Healthtell] and it will be stable with temperature and shipping and all that," he said.

"If you look at these [clinical] labs, a huge portion of their expense is FedEx bills," Colston said. "First [patients] have to go to some place to draw the blood, and then once they draw the blood they have to FedEx it, and that is an expensive process. So if you can just mail these things through regular mail, it just makes it much more attractive for the diagnostics market."

Anderson likewise noted the cost savings and opportunities for long-term monitoring that dried blood spots could offer.

"If it's possible to do at home conveniently and people can get used to it, then it's going to be practical to do longitudinal sampling ... and that would do a much better job with showing changes associated with, for example, chronic diseases," he said, adding that he was very intrigued by DTC genomics models established by companies like 23andMe.

Anderson suggested that the ability to send samples via standard mail could prove particularly attractive to large clinical reference labs like Laboratory Corporation of America and Quest Diagnostics, given the high volume of samples these companies process.

Richard Reitz, medical director and chair of the Endocrine Division at Quest's Nichols Institute, agreed, telling ProteoMonitor that "it's pretty obvious that if you could just stick some filter paper in an [envelope] and use the US mail, the cost is going to be considerably lower."

Quest has not begun looking into dried blood spots as a sample source for protein assays, but, Reitz said, he and several of his colleagues "are interested in beginning to get our toes in the water on this."

Stanford researcher Michael Snyder noted that in addition to the potential cost and convenience benefits, dried blood spot proteomics could open up a large number of new samples to researchers.

Snyder has not explored dried blood spots for protein measurements but has, with fellow Stanford researchers Gary Shaw and Hugh O'Brodovich, used them successfully for DNA analysis.

"States like California have been [collecting dried blood spots] for years," he told ProteoMonitor. "There are millions of these blood spots for different people, and other states have [similar collections]. It's an amazing resource that could be tapped."

In a similar vein, Anderson said he was in discussion with several Scandinavian dried blood spot repositories about obtaining samples from those collections. "They keep all their neonatal dried blood spot cards permanently, so they have [around] 30 years of them so far on everyone born [in that period]," he said, adding that proteins appeared to hold up "extremely well" under such storage conditions.

A number of mass spec vendors market workflows for dried blood spot analysis, but mainly for small molecule work, not proteins.

"We certainly have thought about applications in the large-molecule arena, but there is no project at the moment," Dennis Nagtalon, LC/MS clinical research manager at Agilent, told ProteoMonitor.

He added, however, that he thought such assays were technically possible and could offer significant benefits. "I would love to be able to offer this because for many practical purposes it really is a conduit to changing the current paradigm of how you do sample and also how you end up archiving certain biological samples," he said.

One technical issue, Nagtalon noted, is that because Agilent's dried blood spot methods are currently designed for small-molecule work, they actually remove all proteins and peptides from these samples.

"The design of the component[s] has actually been optimized to strip out everything that proteomics is interested in," he said. "So there would have to be some changes in terms of the chemistry to make it more applicable to a proteomics workflow."

Nagtalon added that vendor interest in developing workflows for dried blood spot analysis of proteins would likely track with the growth in protein biomarker and proteomic testing in general. "At this stage of the market, we're still not seeing a lot of proteins and peptides being used as clinical assays," he said.

In addition, a variety of technical questions remain to be answered, Anderson said.

"We're going to have to show [for instance] that the quality of the protein analytical results we get from dried blood spots have the same sort of clinical parameters as existing tests in plasma," he said. "What are the limitations going to be? Are there things we can't analyze effectively?"

"The big issue is: Can you get off a blood spot any protein or peptide you might be interested in?" Quest's Reitz said. "We would have to validate it on several different assays and prove to the company that this is a robust way of doing things."

"Right now I think that's still to be decided, but I think the concept and potential savings are certainly worth the investigation," he said.

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