New biomarker-based test to assess the integrity of stored blood samples Evolving Science For The Future | Articles

Written by Roshini Beenukumar, PhD

The long-standing issue of research reproducibility

Several recent studies have highlighted the issue of reproducibility of experiments in multiple scientific disciplines. In medical research, one of the major causes of this problem is the poor integrity of stored samples. In most cases, sample storage, handling and maintenance conditions determine the usability of samples for downstream analysis. For example, paraffin-embedded tissues stored at room temperatures show poor nucleic acid quality rendering them unsuitable for research and diagnostic uses. Storing such samples at lower temperatures or using a formaldehyde-free fixative improves nucleic acid quality.

For blood plasma and serum samples, multiple parameters could affect integrity and downstream analysis. These include collection tube type, degree of tube filling, number of tube inversions, degree of hemolysis, duration before centrifugation and duration in a thawed state.

The need for robust quality assurance

As the number of biospecimens collected around the world increases, the need for robust quality control checks also rises. How can biorepositories ensure the quality of biospecimens and eliminate institute-dependent biases? One approach would be to have an independent auditing body to ensure the implementation of certain set quality standards, such as those set by the International Standards Organization (ISO). However, compliance with these standards is currently left to the discretion of individual biobanks.

For blood plasma and serum samples, quality control biomarkers, such as the ones described in this publication could be used to ensure quality and integrity. The advantage of a biomarker assay is that it can be applied to thousands of samples with speed and ease. An ideal QC biomarker assay for blood plasma and serum samples should fulfill most or all of the following criteria:

• Native blood analyte
• Low sample volume requirement
• Minimal sample preparation
• Fully automatable from start to finish
• Cheap and fast

An endogenous biomarker-based quality control assay for serum plasma samples

A group of scientists from Arizona State University lead by Dr. Chad Borges has designed a highly sensitive biomarker-based quality control assay to check the integrity of serum plasma samples.

Albumin, a highly abundant plasma/serum protein, is susceptible to ex vivo oxidation at temperatures greater than −30°C, but stable when the temperature is maintained at −80°C. Oxidation of albumin results in S-cysteinylation of its single free cysteine residue giving rise to S-cysteinylated albumin (S-Cys-Alb). The developed biomarker assay is based on “measuring S-Cys-Alb before and after an intentional incubation period that causes S-Cys-Alb to hit its maximum value, the difference between these values, ΔS-Cys-Alb, is readily interpreted as inversely proportional to the degree of ex vivo oxidation that occurred prior to the first measurement of S-Cys-Alb.” In simpler terms, the test measures the abnormal (oxidized) form of albumin (with an altered mass) using mass spectrometry. The more the exposure to thaw the more the oxidation.

Case study: New biomarker test identified poor sample integrity due to freezer malfunction

Dr. Borges and colleagues applied the new biomarker test on a set of serum samples from stage I lung cancer patients and controls. These samples were collected and handled by experienced investigators using well-defined standard operating procedures. Surprisingly, the biomarker values were significantly different between the cases and controls. This suggested an anomaly in integrity between the two sets of samples. Upon further investigation, it came to light that the −80°C freezers with the control samples malfunctioned for about 3–4 days during a natural disaster.

"Who knows how many other markers are differentiated simply because of the way in which the cases and controls were handled," asks Dr. Borges

Conclusion

The study describes a novel endogenous quality control biomarker – ΔS-Cys-Alb – that measures the cumulative sample thawed time of serum plasma samples. It fulfills most of the criteria for an ideal QC biomarker for plasma and serum such as low sample requirement, automatable, speed and low cost. Hence, this biomarker test has the potential to become the gold standard for plasma serum analysis.