NEW YORK (GenomeWeb) – Researchers in Germany have found that Norgen Biotek's blood storage tubes performed the best among four companies' tubes for minimal cell-free microRNA hemolysis and cell-free (cf-DNA) extraction.
According to the group, led by researchers from the University Hospital of Heidelberg, which published their results last week in the International Journal of Molecular Sciences, BSTs from Streck performed the worst because of its high rate of hemolysis induction.
The team set out to assess the ability of different blood storage tubes (BST) developed by four manufacturers to preserve blood samples under time-based conditions. More specifically, they wanted to find the tool that best stabilized hemolysis and breast cancer-associated cf-miRNA biomarkers, in addition to two cancer-associated genes for potential downstream clinical diagnostic applications. Initially, the scientists tested the limits of conventional EDTA tubes used for conventional testing over a 12-hour period.
In addition to Norgen and Streck, the researchers looked at BSTs from Roche Diagnostics and PreAnalytix. According to Sarah Schott, senior author of the study and head of translational women's health at UHH, the team decided to use BSTs from the companies because they were the only four groups on the market that promised preservation tubes that lasted at least seven days.
"For us, it was important that the quality of samples would remain stable over long periods so that our experiments and tests could be reproducible in other labs," Schott explained. "It would [also] be important to collect blood that could be stored up to 24 hours and can be shipped to our hospital or an external analytic center for further processing."
To determine the stability of cf-miRNAs after storage in conventional blood storage tubes, the researchers drew blood from six healthy individuals and stored them at either room temperature or 4 degrees Celsius for up to 18 hours.
The group analyzed several biomarkers, including miR-16 and MiR-451 which act as hemolysis markers, and miR-148b, -652, -376c, and -200c, which act as breast cancer biomarkers. For samples stored at room temperature, the team saw a significant increase in miR-148b and -652 in half of the samples after 18 hours, which did not occur in samples stored at 4 degrees Celsius. However, the team found that circulating miRNAs were relatively stable when stored in tubes for up to 12 hours before processing them for downstream applications.
In the second part of the study, Schott and her team tested the long-term stability of cf-miRNAs by comparing BSTs from the four companies: Streck's Cell-Free DNA Tube, Roche's Cell-Free DNA Collection Tube, PreAnalytiX's (PAX) PAXgene Blood ccfDNA Tube, and Norgen's Cf-DNA/Cf-RNA Preservative Tube. The researchers noted that all companies advertised their tubes' ability to stabilize cf-DNA for at least seven days by using a liquid that preserves nucleated blood cells.
Collecting 108 milliliters from eight patients for three different blood tubes, Schott and her team stored a total of 128 samples in the tubes at room temperature for up to seven days.
Extracting cf-miRNA from 300 microliters of plasma, the team performed qRT-PCR on 2 microliters of the plasma and synthesized cDNA using the universal Taqman Advanced mi-RNA cDNA synthesis probe on the samples at four time points: four hours, 24 hours, five days, and seven days.
Schott explained that the team quantified the total cf-miRNA concentrations using Thermo Fischer Scientific's Qubit Fluorometer 3.0. After that, the team determined the expression of the hemolysis markers miR-451 and -15, as well as the four breast cancer-associated miRNAs.
While the total cf-miRNA content remained stable in blood stored for up to seven days in Streck, PAX, and Norgen's tubes, the team noticed that Roche tubes had a slight increase in hemolysis after five days, and then a significant jump in miR-451 after seven days.
Schott and her team also noticed a twentyfold increase in blood cell contamination of miR-451 in Streck tubes after five days, as the miR-451 amount skyrocketed compared to the initial four-hour sample the team initially tested.
The researchers then observed the different tubes' ability to maintain cf-DNA stability in plasma from five patients over an extend period.
After waiting seven days, the team extracted 2 milliliters of plasma and extracted cf-DNA using a QIAmp circulating nucleic acid kit, quantifying each sample using the Qubit dsDNA assay. Comparing blood samples on the four-hour and seven-day time points, the team saw that DNA content remained stable in all conditions, with the most cf-DNA extracted from samples stored in Norgen and PAX tubes.
The team then examined the size of cf-DNA in the different tubes, as Schott noted that the size profiles "of extracted cf-DNA might give additional information about the capacity of blood stabilization."
While most of the tubes produced cf-DNA with peak sizes ranging from 168 to 180 base pairs, Norgen's tubes produced DNA with an average length of 146 to 150 base pairs at both investigated time points.
Because of the cf-DNA's small base pair length contained in each Norgen tube, coauthor and UHH molecular biologist Tania Witte argued that Norgen's BSTs performed better at stabilizing DNA that wraps around a histone as part of a nucleosome complex compared to the three companies' tubes.
In the last part of the study, the researchers applied digital droplet PCR to measure how much TP53 and PIK3CA — two cancer relevant genes — were in the patients' blood samples. They saw that while Norgen tubes had the least amount of TP53, all four tubes contained similar amounts of PIK3CA.
Importantly, the group noted that plasma prepared from whole blood can be used to simultaneously quantify both cf-miRNAs and cfDNA. According to the study authors, PAX and Norgen tubes "performed the best at stabilizing cf-miRNAs and are, therefore, the most suitable candidate BCTs for clinical use when performing liquid biopsies."
The researchers emphasized that future studies will need to integrate cf-miRNA and cf-DNA markers for disease diagnosis. They also highlighted that future investigations should examine issues regarding the varied amount of copies of DNA among the blood tubes.
In terms of cf-DNA stability, the team found that Norgen outperformed the other manufacturers in the amount of isolated DNA. While Norgen's tubes can extract both cf-DNA and cf-miRNA, the study authors noted BSTs from other companies require researchers to use separate tubes for mi-RNA extraction, making other companies' tubes more expensive "not only financially but also in terms of the sample amount available."
Schott acknowledged that her team did encounter some challenges during the study regarding hemolysis, sample size, quality control, and real-world applications. Importantly, she pointed out errors that could have occurred when preparing the samples for collection.
"We observed [hemolysis] occurring at early time points for some tubes … but we believe it could also come from inter-individual differences," Witte added. "This can come from the time point when you draw the blood, that you immediately need to do these rotations so that the stabilization reagent preservative will mix homogenously with the blood. If this is not done properly … the samples will present inter-individual variations, and you will have variability in the same patient when looking at different time points."
However, Schott noted that cf-miRNA and cf-DNA amounts did not widely vary among samples from each company's tube, and the team therefore did not worry about slight variety in the amount of the circulating nucleic acids.
Schott also said that her team worked with a very small cohort of patients and only looked at a few miRNAs, rather than covering the whole miRNome in the study. The group is planning a future multicenter study that will evaluate the tubes on a much larger patient cohort, which Schott hopes will examine clinical implementation and see if the tubes can be used in "everyday clinical practice."
In addition, the study authors pointed out that they could not include other variables relevant to the real-work practice in the study, such as the effects of sample shipment and extreme temperatures. The team could not predict how physical forces like movement or temperature changes could "affect the yield and stability of circulating nucleic acids."
Norgen CEO and President Yousef Haj-Ahmad highlighted that the firm's use of technology that integrates cf-DNA and cf-miRNA in the sample tube helps minimize any chance of biological error during sequential extraction. If any variation or error occurs, he believes researchers can be confident that it stemmed from sample collection instead of the tubes themselves.
In addition, Haj-Ahmad noted that Norgen's tubes contained strands of DNA that were 146 to 150 bps in length in the study, which he emphasized that researchers and users seek for downstream applications like identifying cancer and other diseases.
A Streck representative said in a statement that the firm would like to thank the researchers for including the company in its study, and to "be on the lookout for new, innovative products" in 2019.
Witte believes that Norgen's BSTs might be useful for downstream applications, as researchers are currently determining the cell of origin and mechanism of cf-DNA release into blood in a patient's bloodstream. For example, she noted that during follow-up appointments of patients after tumor resections, the clinicians may need to correctly detect the driving mutations in cf-DNA samples to ensure whether the patient is tumor-free or still has evidence of cancer.
"When analyzing cf-DNA, it is crucial to assess the correct fragment size. Otherwise the experiments could be biased by signals coming from other cf-DNA fragments," Witte said. "Moreover, the fragment size can [be defined] if the released DNA is coming from the tumor or from non-cancer DNA fragments."