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Studies Suggest Challenges Remain for Dried Blood Spot Protein Assays

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NEW YORK (GenomeWeb) – With their potential to enable easier patient sampling and significantly lower assay costs, dried blood spots are an area of growing interest among clinical proteomic researchers and companies.

Some recent studies indicate, however, that technical hurdles still stand in the way of clinical implementation of this sample collection technique.

For instance, in work presented last month at the Mass Spectrometry: Applications to the Clinical Lab (MSACL) and published last year in Proteomics – Clinical Applications, researchers from the University of Washington found significant differences in results determined via dried blood spot analysis compared to established clinical assays using matched blood collected using standard venipuncture.

Meanwhile, an October 2017 Clinical Chemistry study led by researchers from the University of Victoria found that certain amino acids in dried blood spot samples may be more susceptible to degradation due to heat and humidity than previously known.

Typically consisting of microliter volumes of blood spotted and dried on filter paper, dried blood spots offer potentially significant advantages compared to traditional blood draws. For instance, because dried blood spots use a finger prick as opposed to conventional phlebotomy, patients could conceivably draw samples themselves without needing to visit a doctor's office, which could open up a number of applications around, among other things, longitudinal monitoring of chronic conditions or even basic wellness monitoring.

Perhaps even more notable are the potential cost savings. Conventional blood draws require rapid cold shipping via services like FedEx, which is a significant contributor to assay costs. Dried blood spots, it is thought, could be shipped under less-controlled conditions including possibly through standard mail.

Dried blood spots have long been used in applications like drug development and newborn testing, but they have yet to make their way into clinical proteomic testing in a significant way. This is due in part to the fact that conventional immunoassays, which are widely used for protein measurements in the clinic, are poorly suited to analyzing dried blood spots given their limited sample volume.

The rise of mass spec as a clinical proteomic tool, however, has made them a plausible sample source for protein-based tests.

"Absolutely," said Paul Kearney, president and chief scientific officer at clinical proteomics firm Integrated Diagnostics, when asked if the company was exploring dried blood spots as a sample source. "I can't imagine anyone not looking into it. It's just simply a lot cheaper."

"A misconception I think people have is that mass spec tests are really expensive," Kearney said. "That's just not the case. It's the cost of getting the samples to your lab that is the big thing, the cost of goods. It's that costs of goods where you can really make a difference [in test cost], and filter paper is clearly the way to go."

The University of Victoria work suggests, though, that dried blood spot samples might be somewhat less stable than thought. In a study funded by the Bill & Melinda Gates Foundation, the researchers looked at the stability of 21 amino acids under various conditions, finding that a number of amino acids were degraded due to heat and humidity with histidine and tryptophan among the most affected.

Christopher Borchers, director of the University of Victoria-Genome BC Proteomics Centre and senior author on the study said that humidity, in particular, impacted amino acid stability, while temperature had a somewhat smaller effect, and sunlight exposure did not appear to be an issue.

High humidity "is really the most important thing you will want to avoid," when shipping and storing dried blood spot samples intended for protein analysis, he said.

While the findings indicate some care is necessary to optimize dried blood spot analysis, it doesn't present especially notable challenges to their use, Borchers said.

"I still think it is a great system. You just need to take care after you have spotted the blood on the piece of filter paper to put it into a plastic bag, add a [desiccant] gel, seal it, and ship it," he said. And while these samples would ideally be shipped under refrigerated conditions, that is still easier and less expensive than shipping samples frozen, as is typically done with standard blood draws.

Potentially more problematic are the findings from the University of Washington team, which suggest that dried blood spot samples may not always return the same results as venipuncture samples.

"We have a lot of collaborators who work on metabolic diseases, cardiovascular diseases, things like that. And some of them have a lot of dried blood spot samples, or would like to start using dried blood spots," said Clark Henderson, a research scientist in the UW department of laboratory medicine and first author on the study.

"There have been a lot of studies that have looked at dried blood spots and claimed that they're really good for quantitating a lot of different proteins," Henderson said. But, he added, there has been little published work looking at how protein measurements made in dried blood spot samples compare to measurements in conventional blood samples.

To investigate this question, Henderson and his colleagues developed mass spec-based dried blood spot assays to glycated hemoglobin-β, apolipoprotein A-I, and apolipoprotein B and compared them to existing clinical assays (immunoassays for ApoA-1 and ApoB and HLPC-UV for HbA1c) using conventional plasma and whole blood samples.

Looking at samples from 36 patients, the researchers found "a large discrepancy between the values we were getting from the dried blood spots and the [conventional] plasma [samples]," Henderson said. They also found a discrepancy between whole blood drawn via venipuncture and then spotted to make dried blood spots and paired capillary dried blood spot samples.

In some cases, Henderson said, the discrepancy was large enough that it would have changed clinical decision making, for instance, reclassifying a patient with normal HbA1c according to the standard assay as pre-diabetic based on the dried blood spot score.

"The clinical assays are solid," Henderson said. "We know that for sure. They are all based off of traceable standards and the CV of the hemoglobin A1c is 2 percent and less than 5 percent for both the immunoassays. We're very confident in those numbers."

The UW researchers also ran their mass spec assays on conventional blood samples and found that these measurements showed much better agreement with the clinical immunoassays, suggesting that the issue lay in the dried blood spots rather than the mass spec workflow.

Hematocrit bias is a potential issue with dried blood spots, where variations in sample hematocrit levels can lead to variations in the amount of blood actually being taken from a dried blood spot for analysis.

Henderson noted that he and his colleagues corrected for potential bias, but "couldn't see that it was causing a significant problem."

"We never did nail down what we thought was going on," he said, adding that given their findings, the lab isn't currently pursuing dried blood spot protein work.

"It may be something we revisit in the future if we see that there are promising technologies, but at this point we have no reason to continue to pursue it," he said. "It's a fantastic idea, and if it works it would be wonderful. Less invasive, better sample stability, all that sort of stuff. It would be great. But, right now, I think it's just not quite there."

However, SISCAPA Assay Technologies (SAT) CEO Leigh Anderson, a proponent of dried blood spot sampling for protein assays, said he and his colleagues have had success using mass spec to measure proteins including ApoA-1 and ApoB in such samples.

"We are getting total workflow CVs with replicate dried blood spot samples on the order of 2 percent, and they calibrate very tightly with the clinical assays done on serum at the same time," he said.

Anderson said the UW study raises what is "a very important question to resolve — what is the parallelism between normal clinical samples, venipuncture serum or plasma, versus capillary blood dried on a card?"

"There are reasons to believe there could be issues there, but when we test it, the results come out very highly parallel over six or seven orders of magnitude from hemoglobin all the way down to proteins that are really low abundance," he said.

Noting the University of Victoria findings, Anderson said that his lab stores its dried blood spots under refrigeration and with desiccants to reduce their exposure to humidity.

"They are shipped at room temperature but stored under refrigerated conditions and that model is perfectly practical for a clinical kind of assay system," he said.

SAT has developed a fully automated mass spec workflow for running dried blood spot samples, Anderson said, adding that the objective in the near term is to implement multiplex panels of existing clinical analytes as laboratory-developed tests offered out of a CLIA facility.

"The systems we are running now are ready to do it clinically," he said. "We have gotten to the stage where the instruments are fixed, and it is walkaway automation with no manual steps involved."

To the extent that improvements in dried blood spot workflows are still needed, Anderson said he believes they are around the sampling cards themselves.

"There is a lot of effort going on in that area," he said, noting some 20 different devices he is aware of that are aiming to improve over standard dried blood spot cards. However, he said, in many cases, these devices are not addressing what he sees as the main limitations of the existing technology.

In particular, he said, "We want more volume than [is provided by] one dried blood spot. We would like to have 100 to 150 microliters of whole blood. And we really want the volume precision to be 1 or 2 percent instead of 4 or 5 or 10 percent, which is what it typically is now. Our assays are good to 2 percent, and so we don't want to lose our precision just based on the volume of sample."

"These are doable things," he added, "and people are beginning to think in that direction."