Better samples, better science

Study explores integrity of research specimens

Published October 01 2019

Effective diagnosis and treatment of disease draw on painstaking research, which often relies on biological samples. The avalanche of studies used to better understand illnesses and design effective therapies cost billions of dollars and potentially affects millions of lives.

So it would seem reasonable to assume that the reliability of biological samples, on which accurate results depend, would be of paramount concern for the scientific community.

According to Chad Borges, a researcher in the Biodesign Institute’s Virginia G. Piper Center for Personalized Diagnostics at Arizona State University, that assumption is often wrong.

“One of the major reasons that there are so many discoveries of biomarkers in the literature but so few positive validations that confirm those findings is the fact that in many cases, during the discovery, samples were used that have a history or an integrity that’s simply unknown.”

Biological samples can be susceptible to changes over time, which often occur when they are removed from deep refrigeration. Degraded samples can produce spurious results in research. To address these concerns, Borges and his colleagues have designed a sensitive test that can be used to establish the integrity of blood plasma and serum, the most common biosamples used in medical research.

Ensuring such samples have been properly handled is the first step in careful research that meets the necessary high standards of reliability and reproducibility. The new test, which relies on accurate measurement of the relative proportions of two forms of the protein albumin present in blood, recently was described in the journal Molecular & Cellular Proteomics.

Houston, we have a problem

The immediacy of the issue of sample quality became apparent to Borges during his own research, which involved experiments on biological samples slated for distribution by the National Institutes of Health.

“We got a little suspicious that something wasn’t quite right about the sample set,” he said. Borges applied the newly designed test to the samples, with surprising results. “Lo and behold, there was a major difference between the cases and controls for this specimen integrity marker.”

Borges found that the freezer in which the control samples were stored had lost power for several days during a natural disaster. “That information is really important with regard to the quality of the samples and the stability of the markers that were in them.”

The implications of this discrepancy plainly went beyond his own research. “Who knows how many other markers are differentiated simply because of the way in which the cases and controls were handled,” Borges said.

Biological samples are ground zero in the quest for dependable science, yet researchers hoping to publish their work may have a disincentive to spend the time to probe the integrity of their specimens. Should they uncover a problem, it may throw their data into question and preclude publication—a serious setback, with little for the researcher to show for it. There is a danger of an ignorance-is-bliss mentality.

Addressing the problem requires two things, Borges noted. First, a regulatory body such as the NIH needs to issue strict guidelines that include detailed documentation of sample history and handling. Some scientific journals do require documentation of specimen storage conditions prior to publication, but such records are often inadequate for ensuring a high level of sample integrity. Secondly, researchers need reliable methods for testing their samples to ensure they meet exacting standards. The technique described in the current study is an important advance in this direction. Freezing samples

QC for blood

The new biomarker sets cutoff values for blood plasma and serum, allowing researchers easily to assess the quality of samples and their suitability for given experiments, even if a detailed record of sample handling and storage is unavailable. For the first time, plasma and serum —the most commonly used biospecimens for medical research — can be tracked with a reliable biomarker.

The biomarker, which relies on relative proportions of two isoforms of albumin, requires only a low volume of plasma or serum and minimal sample preparation. (Different isoforms of this protein are functionally similar, but an oxygen-induced modification that occurs to an abnormal extent outside of the body is used to identify mishandled samples.)

Albumin is the most abundant protein in blood plasma and serum, constituting roughly half of all protein content in these biofluids. Outside the body, the natural unmodified form of albumin becomes oxidized with time. This can be detected by observing a change in protein mass using mass spectrometry.

By describing a chemical rate law for this protein oxidation reaction that takes place in plasma and serum, the biomarker acts as a kind of molecular stopwatch that can gauge precisely the elapsed time a particular sample has remained in a thawed state.

The biomarker described is inexpensive, easy and rapid to use, and can be fully automated, making it a strong candidate to serve as the new gold standard for plasma and serum analysis. It is capable of detecting biospecimen exposure to room temperature conditions for as little as two hours, quickly and accurately identifying mishandled or poorly stored samples and preventing their inclusion in clinical research.

In addition to more conventional clinical research, the new biomarker is poised to make inroads in a variety of health-related investigations. It recently has been incorporated into a project sponsored by the Defense Advanced Research Projects Agency, which uses epigenetic markers in blood to identify exposure to weapons of mass destruction or their precursor chemicals. The new biomarker will be used to ensure the quality of blood samples, further establishing the power and versatility of this approach.

This article originally was published on the Arizona State University Biodesign Institute website. It has been edited for ASBMB Today.

Richard Harth Richard Harth is a senior science writer for the Biodesign Institute at Arizona State University.