{"id":21131,"date":"2024-02-08T08:32:02","date_gmt":"2024-02-08T08:32:02","guid":{"rendered":"https:\/\/clinlabint.com\/?p=21131"},"modified":"2024-08-22T08:33:00","modified_gmt":"2024-08-22T08:33:00","slug":"getting-the-most-from-every-drop-new-advances-in-blood-separation","status":"publish","type":"post","link":"https:\/\/clinlabint.com\/getting-the-most-from-every-drop-new-advances-in-blood-separation\/","title":{"rendered":"Getting the most from every drop \u2013 new advances in blood separation"},"content":{"rendered":"
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Getting the most from every drop \u2013 new advances in blood separation<\/h1>\/ in E-News<\/a>, Featured Articles<\/a> <\/span><\/span><\/header>\n<\/div><\/section>
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Getting the most from every drop \u2013 new advances in blood separation<\/h3>\n

Alexandra Sommer, Senior Product Manager, Clinical Diagnostics at Tecan<\/strong><\/p>\n

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Liquid biopsies are poised to play a central role in the future of diagnostics, especially in oncology.1 Currently, a conventional tissue biopsy is the gold standard in the diagnosis of many malignancies, but this technique comes with many drawbacks, including its invasive nature, the risk to patients, the need to fix the samples, and the difficulty in applying it to all cancer types and patients. Liquid biopsy allows genomic and proteomic assessment of components in the blood, or other sampled fluids, as an alternative, less invasive and more convenient method of monitoring cancer patients during and after treatment. Following sampling, the buffy coat and plasma can be separated from the erythrocytes by centrifugation, and analysed to understand the health status of the patient, and also check sample quality. Individual biomarkers in both the plasma and buffy coat \u2013 including circulating tumour cells (CTCs), circulating tumour DNA and circulating tumour ribonucleic acid (ctDNA, ctRNA), and exosomes \u2013 can be analysed to provide further insights into a patient\u2019s cancer, offering a comprehensive view of tumour heterogeneity, and giving an overview of tumour characteristics such as progression, staging, heterogeneity, gene mutations, and clonal evolution. This can assist clinicians with devising personalised therapeutic regimens, as well as allowing continuous monitoring \u2013 by repeated sampling \u2013 to measure treatment efficacy and screen for therapeutic resistance.<\/p>\n

CTCs<\/strong>
\nCTCs are intact tumour cells that are released into the bloodstream from primary or secondary tumour sites, and their migration through the circulatory system results in the development of distant metastases. They can be collected in the buffy coat following blood separation by centrifugation, and analysis of CTCs provides diagnostic and prognostic information in a number of cancer types. Their detection and characterisation is an important tool for monitoring and preventing the development of overt metastatic disease, and CTCs have also shown potential as screening biomarkers for early detection of cancers.2<\/p>\n

ctDNA<\/strong>
\nCancer cells shed naked DNA molecules, known as ctDNA, into the bloodstream. This genetic material can be isolated from plasma, and has become one of the main markers to investigate the mutational status of cancers through the use of next generation sequencing. Comparison of ctDNA with cfDNA \u2013 a normal product of cell turnover that is detectable in healthy individuals \u2013 can be used to identify mutations in tumours are not an exact match to an individual\u2019s DNA, providing highly specific markers for various cancers.3 Recent advances in precision medicine have also allowed analysis of ctDNA to guide treatment decisions, predicting response and resistance to targeted therapies and immunotherapies. While most current clinical practice using ctDNA has been aimed at identifying druggable and resistance mutations for directing systemic therapy decisions, newer research is evaluating its potential as a marker of minimal residual disease, and as a useful screening tool to detect cancers in the general population.4<\/p>\n

Exosomes<\/strong>
\nExosomes are nano-vesicles released by various cell types, and carry a variety of \u2018molecular messengers\u2019 \u2013 including proteins, RNAs, DNAs and lipids. There is substantial evidence that exosomes are involved in intercellular communication by exchanging these messenger molecules between cells, and that they play important roles in cancer development. They can be isolated from plasma following blood separation, and recent studies have shown that exosomes are superior to CTCs and ctDNA for early diagnosis, disease monitoring, and prognostic prediction. However, they are more challenging to extract and study than other blood constituents, so improved strategies to isolate exosomes from bodily fluids \u2013 and to profile exosomal contents in a fast and sensitive way \u2013 are required to allow the practical application of exosome-based liquid biopsies for precision cancer medicine.5<\/p>\n

Liquid biopsy applications also go beyond cancer. For example, non-invasive prenatal testing (NIPT) is becoming a preferred choice for many mothers undergoing testing for chromosomal abnormalities, as a safer alternative to amniocentesis. This is an increasingly important consideration as the average age of mothers continues to increase,6 prompting significant research interest in this area. Liquid biopsy approaches are also showing promise for monitoring patients following organ transplant. For example, a number of biomarkers \u2013 including donor-derived cell-free DNA, microRNAs and exosomes \u2013 are showing high potential for determining the long-term prognosis for kidney transplantation patients.7<\/p>\n

The challenge of separating blood samples<\/strong>
\nBlood collected for liquid biopsy must be separated into its constituents \u2013 plasma, buffy coat and erythrocytes \u2013 in order to study the markers described above. Traditional manual separation techniques are slow, and are susceptible to human error, even when performed by an experienced technician. Following centrifugation, the delicate buffy coat forms a thin layer between the plasma and erythrocytes, and manual isolation \u2013 usually carried out with a Pasteur pipette \u2013 carries the risk of contamination between the phases. Even when automation technology is available, it can be challenging to identify liquid-liquid interfaces, because barcodes and labels may interfere with optical sensors. With the increasing number of applications for liquid biopsies \u2013 from cancer monitoring to NIPT \u2013 laboratories need ways to streamline the workflow and overcome the bottleneck of blood separation.<\/p>\n<\/div><\/section><\/p><\/div>

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\u00a0(A) centrifuged blood<\/em><\/p>\n<\/div><\/section><\/p><\/div>

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(B) after plasma separation<\/em><\/p>\n<\/div><\/section><\/p><\/div>

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(C) after buffy separation<\/em><\/p>\n<\/div><\/section><\/p><\/div>

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Presenting Phase Separator\u2122<\/strong><\/p>\n

Phase Separator from Tecan is an advancement in liquid-liquid separation technology that offers precision and speed, helping to streamline workflows and improve efficiency in both research and clinical laboratories. It is a unique, pressure-based detection technology for the Air Flexible Channel Arm\u2122 (Air FCA) on Tecan\u2019s flagship liquid handling platform, the Fluent\u00ae<\/sup> Automation Workstation. This novel method addresses the critical challenge of detecting liquid-liquid interfaces and effectively separating neighbouring phases, while avoiding the risk of contamination, making it ideal for removing plasma or buffy coat layers from centrifuged blood samples.<\/p>\n

Phase Separator can achieve remarkable speeds, working in either tubes or plates, by combining phase detection with the pipetting action. Processing speed is further enhanced when all eight channels on the Air FCA are used in parallel \u2013 enabling the aspiration of plasma from 24 tubes of centrifuged blood in under 10 minutes* \u2013 and twice as fast again on dual-arm systems. These benefits come with no loss of bench space, and no additional equipment to maintain. Crucially, the technology detects liquid levels with pinpoint precision inside the tube or plate, so there is no interference from barcodes or other labware markings.<\/p>\n

Proven against traditional methods<\/strong><\/p>\n

The value of Phase Separator for sample preparation in pre-natal testing has been investigated, in efforts to optimize the quality and quantity of cfDNA. Donated blood from women in different stages of their pregnancy was collected in different tube types \u2013 including Cell-Free DNA BCT\u00ae<\/sup> (Streck) and PAXgene\u00ae<\/sup> Blood ccfDNA Tube (PreAnalytiX\u00ae<\/sup>) \u2013 and the plasma was separated using either the standard, laboratory-validated method (labelled PM) or the Phase Separator. Between five and seven millilitres of plasma was separated per sample, based on input volume and tube type.<\/p>\n

The Phase Separator was also tested with samples that had undergone a second centrifugation step after plasma separation, to determine the impact on cfDNA extraction, presented as percentage of foetal fraction isolated from total cfDNA in a sample (Figure 2). Different separation conditions using the Phase Separator were tested, and compared with an established and validated manual separation and cfDNA extraction protocol:<\/p>\n