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SISCAPA Assay Tech Exploring Dried Blood Spot Sampling for Mail-in, Longitudinal Proteomic Testing

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Clinical proteomics firm SISCAPA Assay Technologies is exploring dried blood spots as a sample source for targeted proteomics assays.

As a potentially less expensive, more convenient alternative to conventional blood samples, dried blood spots could enable "a completely different interface with the patient," said SAT CEO Leigh Anderson, opening up possibilities such as in-home and long-term longitudinal proteomic testing.

Anderson noted the company's foray into dried blood spots last year in an interview with ProteoMonitor. Last week, at the Mass Spectrometry Applications to the Clinical Laboratory annual meeting in San Diego, he presented results from some of this work, describing an assay the company used to perform longitudinal measurements of 22 proteins.

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.

To date, much of the interest in dried blood spots as a sample source has focused on analysis of small molecules for purposes like pharmacokinetic assays. Recent work by Anderson and others, however, suggests that they could prove suitable for proteomics assays, as well.

In his MSACL talk, Anderson presented data he and his colleagues collected using analysis of dried blood spots to measure levels of lipopolysaccharide-binding protein – which is involved in immune response – in an individual over time. The data identified rises in an individual's LPS-binding levels caused by common colds and a bout of pneumonia, providing a demonstration of the method's potential usefulness for longitudinal analyses.

Such analyses are, of course, theoretically possible using conventional blood draws, but, as Anderson noted, the ease of dried blood spot sample collection offers significant advantages.

"The idea that you can [take the sample] at home whenever you need to, that storage is not critical, and that it can be sent in [to a lab] by mail, just makes it a completely different equation in the mind of the user," he told ProteoMonitor.

He brought up the example of diabetes, where it is recommended that patients have regular blood tests to check their levels of hemoglobin A1c.

"That's the standard thing to do, but it is limited in its use because of the cost and complexity of getting everyone to come in and give their samples," he said.

With dried blood spots, on the other hand, patients could sample themselves at home with a finger prick, and then mail it in.

"If you can do [sampling] in a simpler way, then this whole longitudinal paradigm can actually work," Anderson said.

As he noted during his MSACL presentation, longitudinal sampling is particularly desirable due to the high levels of biological variability within populations, which can make it difficult to determine appropriate biomarker cut-points for individuals.

Such sampling would allow clinicians to "build personalized baselines," Anderson said, noting that this was probably the approach's "primary medical value."

It could also, though, offer improvements in terms of non-longitudinal sampling, he said, citing as an example the case of patients who live far away from medical centers where they get testing done.

For instance, a patient may live 100 miles from the hospital where they are scheduled to have an elective surgery, and, if, the day before the surgery the doctors need to do blood work, the patient might have to drive in to give blood, then drive back, only to go back again the next day for the surgery.

"It degrades medical care not being able to get samples when and exactly where you need them," Anderson said. "So, in such a case, [dried blood spots] are a great way to do it. Anything where you don't need an answer in the next two hours, it could make a huge contribution."

Anderson noted that dried blood spot analysis might also play a role in facilitating collection of clinical samples, a process that has emerged as one of the key bottlenecks in biomarker research and molecular diagnostic development.

"If we can demonstrate that all the measurements you would like to make – small molecules and proteins – will work in dried blood spots, then the economics of doing large clinical studies going forward is changed," he said. "Because the cost of collecting the samples [and] storing the samples is completely different."

With this in mind, Anderson and his SAT colleagues are looking into taking existing sample collections and spotting them onto dried blood spot cards, "so that you could preserve at least some of the existing collections in a more cost effective way going forward."

For its dried blood spot work, SAT employed the company's SISCAPA workflow, which uses stable isotope standards along with antibodies to enrich for target peptides after trypsin digestion, a process that improves the sensitivity and throughput of traditional multiple-reaction monitoring mass spec assays.

Using an Agilent 6490 triple quadrupole, the researchers measured in a single dried blood spot punch the levels of 22 proteins with abundances spanning eight orders of magnitude. The lowest abundance protein measured was mesothelin, which was present at levels as low as 1 ng/mL.

A key question regarding clinical proteomic assays from dried blood spots is how to normalize for sample-to-sample variations in volume and hematocrit content, an issue the SAT team tackled by making measurements of certain plasma-specific and red blood cell-specific proteins. While the company is still "in the process of optimizing" this approach, it "does a substantial amount of normalizing of those [sample-to-sample] differences," Anderson said.

SAT's current commercial focus is the development of standard SISCAPA assays for clinically important proteins and for customer targets, said Gus Salem, a member of the company's board. But, he added, "it is clear to us that the value of the SISCAPA technology combined with a mass spectrometer and a dried blood spot is a unique proposition."

"We're very much focused on the fact that this creates a very different experience for the patient to be sampled locally," he said. "We're still working through the logistics of that, but that is a specific direction we want to take the company."

Anderson cited chronic disease and monitoring of cancer recurrence as two specific areas where SAT believes mass spec-based proteomic analysis of dried blood spot samples could prove particularly useful.

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.

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.

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, which 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.

Researchers at the Cambridge Centre for Neuropsychiatric Research are also exploring the approach as part of their collaboration with Myriad Genetics on proteomic tests for psychiatric disorders.

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