{"id":41226,"date":"2023-02-20T14:45:53","date_gmt":"2023-02-20T15:45:53","guid":{"rendered":"https:\/\/peymantaeidi.net\/stem-cell\/?p=41226"},"modified":"2023-02-20T16:41:33","modified_gmt":"2023-02-20T16:41:33","slug":"liquid-biopsy-test-protocol-and-steps","status":"publish","type":"post","link":"https:\/\/peymantaeidi.net\/stem-cell\/2023\/02\/20\/liquid-biopsy-test-protocol-and-steps\/","title":{"rendered":"Liquid Biopsy Test: Protocol and Steps"},"content":{"rendered":"<div>\n<div id=\"placeholder-audio-player\" class=\"sc-kIeSZW qPHQt\">\n<div class=\"sc-hOqruk GSyvG\">\n<div class=\"sc-dtwnrN ikvCsJ\">Listen with <\/p>\n<p>Speechify\n                <\/p>\n<\/p><\/div>\n<\/p><\/div>\n<p>        <a target=\"_blank\" href=\"https:\/\/speechify.com\/?utm_campaign=inline-player&amp;utm_source=Technology+Networks&amp;utm_medium=speechify-api&amp;landing_url=https%3A%2F%2Fspeechify.com%2Ftext-to-speech-online%2F\" rel=\"noopener\"><\/p>\n<p>        <\/a>\n            <\/div>\n<div class=\"audio-article-overlay audio-article\">\n<div class=\"audio-article-overlay-inner\">\n<p>\n                    Register for FREE to listen to this article\n                <\/p>\n<div>\n<p>        <button type=\"\" class=\"gtm-event-click\" aria-label=\"\" data-eventcategory=\"none\"><br \/>\n                Listen Now<br \/>\n        <\/button>\n<\/p>\n<\/p><\/div>\n<\/p><\/div>\n<\/p><\/div>\n<p>Thank you. Listen to this article using the player above. <span>\u2716<\/span><\/p>\n<\/div>\n<div>\n<div class=\"top\">\n        <\/p>\n<div class=\"base_section_title\">\n<p>\n            Want to listen to this article for FREE?\n        <\/p>\n<\/div>\n<p>\n            Complete the form below to unlock access to ALL audio articles.\n        <\/p>\n<\/p><\/div>\n<\/div>\n<div id=\"inner_content_body_text\">\n<p class=\"MsoNormal\"><span lang=\"EN-US\">In this article, explore some of the advanced molecular techniques that are most used for detecting the main biomarkers involved in liquid biopsy.<\/span><\/p>\n<h2><b><span>How does a liquid biopsy work?<\/span><\/b><\/h2>\n<p class=\"MsoNormal\"><span lang=\"EN-US\">A <a href=\"https:\/\/www.technologynetworks.com\/diagnostics\/articles\/liquid-biopsy-guide-applications-and-techniques-328957\" target=\"_blank\" rel=\"noopener\">liquid biopsy<\/a> consists of the isolation and characterization of tumor-derived materials \u2013 such as intact cancer cells or nucleic acids \u2013 in a sample of body fluid collected from a cancer patient to gain information about their disease.<sup><a href=\"https:\/\/doi.org\/10.1186\/s12943-022-01543-7\" target=\"_blank\" rel=\"noopener\">1<\/a><\/sup><\/span><\/p>\n<div data-id=\"RelatedContentLinkFeature\" class=\"related-content-section\">\n<div class=\"linkFeature\"><a target=\"_blank\" href=\"https:\/\/www.technologynetworks.com\/diagnostics\/articles\/what-is-a-liquid-biopsy-369925\" rel=\"noopener\"><\/p>\n<div>\n<div>\n<div>\n<div>\n<div>\n<div>\n<div>\n<div>\n<div>\n<div>\n<div>\n<div>\n<div>\n<div>\n<div>\n<div>\n<div>\n<div>\n<div>\n<div>\n<div>\n<div>\n<div>\n<div>\n<div>\n<div>\n<div>\n<div>\n<div>\n<div>\n<div>\n<div>\n<div>\n<div>\n<div>\n<div>\n<div>\n<div>\n<div>\n<div>\n<div>\n<div>\n<div>\n<div>\n<div>\n<div>\n<div>\n<div>\n<div>\n<div>\n<div>\n<div>\n<div>\n<div>\n<div>\n<div>\n<div>\n<div>\n<div>\n<div>\n<div>\n<div>\n<div>\n<div>\n<div>\n<div>\n<div>\n<div>\n<div>\n<div>\n<div>\n<div>\n<div>\n<div>\n<div>\n<div>\n<div>\n<div>\n<div>\n<div>\n<div>\n<div>\n<div>\n<div>\n<div>\n<div>\n<div>\n<div>\n<div>\n<div>\n<div>\n<div>\n<div>\n<div><img decoding=\"async\" src=\"https:\/\/peymantaeidi.net\/stem-cell\/wp-content\/uploads\/2023\/02\/what-is-a-liquid-biopsy-369925-480x270-1.jpg\" alt=\"What Is a Liquid Biopsy?\" \/><\/div>\n<\/div>\n<\/div>\n<\/div>\n<\/div>\n<\/div>\n<\/div>\n<\/div>\n<\/div>\n<\/div>\n<\/div>\n<\/div>\n<\/div>\n<\/div>\n<\/div>\n<\/div>\n<\/div>\n<\/div>\n<\/div>\n<\/div>\n<\/div>\n<\/div>\n<\/div>\n<\/div>\n<\/div>\n<\/div>\n<\/div>\n<\/div>\n<\/div>\n<\/div>\n<\/div>\n<\/div>\n<\/div>\n<\/div>\n<\/div>\n<\/div>\n<\/div>\n<\/div>\n<\/div>\n<\/div>\n<\/div>\n<\/div>\n<\/div>\n<\/div>\n<\/div>\n<\/div>\n<\/div>\n<\/div>\n<\/div>\n<\/div>\n<\/div>\n<\/div>\n<\/div>\n<\/div>\n<\/div>\n<\/div>\n<\/div>\n<\/div>\n<\/div>\n<\/div>\n<\/div>\n<\/div>\n<\/div>\n<\/div>\n<\/div>\n<\/div>\n<\/div>\n<\/div>\n<\/div>\n<\/div>\n<\/div>\n<\/div>\n<\/div>\n<\/div>\n<\/div>\n<\/div>\n<\/div>\n<\/div>\n<\/div>\n<\/div>\n<\/div>\n<\/div>\n<\/div>\n<\/div>\n<\/div>\n<\/div>\n<\/div>\n<\/div>\n<\/div>\n<\/div>\n<\/div>\n<\/div>\n<\/div>\n<\/div>\n<p><\/a><\/div>\n<\/div>\n<p class=\"MsoNormal\"><span lang=\"EN-US\">Since most tumors are in contact with the bloodstream, liquid biopsies often involve blood sample analysis \u2013 but other sources of body fluids include urine, stool, saliva, pleural fluid, cerebral spinal fluid and peritoneal fluid or washings.<sup><a href=\"https:\/\/pubmed.ncbi.nlm.nih.gov\/31803629\/\" target=\"_blank\" rel=\"noopener\">2<\/a><\/sup><\/span><\/p>\n<h2><b><span>Liquid biopsy targets<\/span><\/b><\/h2>\n<p class=\"MsoNormal\"><span lang=\"EN-US\">The list of potential <\/span><span lang=\"EN-US\">tumor-derived <\/span><span lang=\"EN-US\">materials that can be analyzed in a liquid biopsy includes:<\/span><\/p>\n<ul>\n<li><b><span lang=\"EN-US\">Circulating tumor cells (CTCs)<\/span><\/b><sup><span lang=\"EN-US\"><a href=\"https:\/\/pubmed.ncbi.nlm.nih.gov\/32708837\/\" target=\"_blank\" rel=\"noopener\">3<\/a><\/span><\/sup><span lang=\"EN-US\"> \u2013 <\/span><span lang=\"EN-US\">intact cancer cells shed by both primary and metastatic tumors.<\/span><\/li>\n<li><b><span lang=\"EN-US\">Circulating cell-free DNA (<\/span><\/b><b><span lang=\"EN-US\">cfDNA)<\/span><\/b><sup><span lang=\"EN-US\"><a href=\"https:\/\/doi.org\/10.3389\/fcell.2021.639233\" target=\"_blank\" rel=\"noopener\">4<\/a> <\/span><\/sup><span lang=\"EN-US\">and <b>circulating tumor DNA (ctDNA)<\/b>\u2013fragments of DNA released from cells (cfDNA), and the tumor-derived DNA fraction (ctDNA).<\/span><\/li>\n<li><b><span lang=\"EN-US\">Extracellular vesicles (EVs)<\/span><\/b><sup><span lang=\"EN-US\"><a href=\"https:\/\/pubmed.ncbi.nlm.nih.gov\/30864674\/#:~:text=Tumor%E2%80%91derived%20extracellular%20vesicles%20(TD,a%20crosstalk%20between%20malignant%20cells.\" target=\"_blank\" rel=\"noopener\">5<\/a><\/span><\/sup><span lang=\"EN-US\">\u2013 small membrane-bound vesicles, including micro-vesicles and exosomes, which can carry cargo \u2013 such as nucleic acids and proteins \u2013 during tumor metastasis.<\/span><\/li>\n<li><b><span lang=\"EN-US\">Circulating cell-free RNA (cfRNA)<\/span><\/b><span lang=\"EN-US\"> \u2013 cancer-related messenger RNAs or non-coding RNAs, including microRNAs (miRNAs).<\/span><\/li>\n<\/ul>\n<div class=\"flex flex-column flex-align-items-center image-modal\"><img decoding=\"async\" class=\"img-responsive\" alt=\"A list of the the tumor-derived materials that liquid biopsy can detect.\" src=\"https:\/\/peymantaeidi.net\/stem-cell\/wp-content\/uploads\/2023\/02\/54533-tn-liquid-biopsy-2-jm-v21676901307272.jpg\" \/><\/div>\n<p><i>The tumor-derived materials that liquid biopsy can detect. Credit: Technology Networks<\/i><\/p>\n<h2><b><span>Liquid biopsy protocol and steps<\/span><\/b><\/h2>\n<p class=\"MsoNormal\"><span lang=\"EN-US\">Liquid biopsy protocols are focused on the rapid detection of clinically relevant information with high sensitivity, selectivity and specificity while remaining minimally invasive.<\/span><span lang=\"EN-US\"><\/span><\/p>\n<p class=\"MsoNormal\"><span lang=\"EN-US\">The <a href=\"https:\/\/www.technologynetworks.com\/diagnostics\/articles\/liquid-biopsy-reading-the-signs-of-disease-from-biofluid-365850\" target=\"_blank\" rel=\"noopener\">techniques selected for a liquid biopsy<\/a> will depend on the nature of the target.<span> <\/span>For instance, it may involve the isolation, enumeration and characterization of CTCs \u2013 or the isolation and molecular analysis of other tumor-derived materials. <\/span><\/p>\n<h3><b><span lang=\"EN-US\">CTCs<\/span><\/b><\/h3>\n<h4><span lang=\"EN-US\">1.<span>&nbsp;&nbsp;&nbsp; <\/span><\/span><b><span lang=\"EN-US\">Isolation<\/span><\/b><\/h4>\n<p class=\"MsoNormal\"><span lang=\"EN-US\">Over the last decade, several technologies have emerged that aim to <\/span><span lang=\"EN-US\">isolate and separate CTCs from the massive pool of circulating blood cells \u2013 and then count and characterize them<\/span><span lang=\"EN-US\">.<sup><a href=\"https:\/\/pubmed.ncbi.nlm.nih.gov\/32708837\/\" target=\"_blank\" rel=\"noopener\">3<\/a><\/sup><\/span><span lang=\"EN-US\"> Each of these approaches exploits a key difference that discriminates CTCs from normal blood cells (such as differential gene\/protein expression, morphology, volume or biophysical properties) to enable the sensitive selection and capture of these very rare cells. They include:<\/span><\/p>\n<p class=\"MsoNormal\"><b><span lang=\"EN-US\">Immune-based detection methods <\/span><\/b><span lang=\"EN-US\">\u2013 these are the most widely used approaches and use antibodies to selectively bind cell-surface antigens that discriminate CTCs from blood cells. These antibodies may be conjugated to magnetic nanoparticles<sup><a href=\"http:\/\/dx.doi.org\/10.20517\/2394-4722.2020.48\" target=\"_blank\" rel=\"noopener\">7<\/a><\/sup> or immobilized on the walls of microfluidic chips<sup><a href=\"https:\/\/www.ncbi.nlm.nih.gov\/pmc\/articles\/PMC6372907\/\" target=\"_blank\" rel=\"noopener\">8<\/a><\/sup> to isolate CTCs. A new generation of CTC antigen-based dual modality platforms combines immunomagnetic beads with microfluidics.<\/span><\/p>\n<ul>\n<li><b><span lang=\"EN-US\">Size-dependent isolation techniques<sup> <\/sup><\/span><\/b><span lang=\"EN-US\">\u2013 including filtration, microfluidics, centrifugation and inertial focusing.<\/span><\/li>\n<li><b><span lang=\"EN-US\">Direct imaging <\/span><\/b><span lang=\"EN-US\">\u2013 imaging-based detection methods<b> <\/b>use specific fluorescent tags to identify and count CTCs in blood. Each of these technologies is unique in its sample preparation, detection algorithms and fluorophores.<\/span><\/li>\n<li><b><span lang=\"EN-US\">Dielectrophoresis \u2013 <\/span><\/b><span lang=\"EN-US\">which relies on particles with different polarization that move differently under a non-uniform electric field.<\/span><\/li>\n<\/ul>\n<p class=\"MsoListParagraphCxSpMiddle\"><span lang=\"EN-US\">&nbsp;<\/span><\/p>\n<h4><span lang=\"EN-US\">2.<span>&nbsp;&nbsp;&nbsp; <\/span><\/span><b><span lang=\"EN-US\">Enumeration<\/span><\/b><\/h4>\n<p class=\"MsoNormal\"><span lang=\"EN-US\">The methods used to count the number of CTCs detected in a sample include impedance<sup><a href=\"https:\/\/pubmed.ncbi.nlm.nih.gov\/31767345\/\" target=\"_blank\" rel=\"noopener\">10<\/a><\/sup>, high-throughput imaging<sup><a href=\"https:\/\/pubmed.ncbi.nlm.nih.gov\/31605225\/\" target=\"_blank\" rel=\"noopener\">11<\/a><\/sup>, flow cytometry<sup><a href=\"https:\/\/pubmed.ncbi.nlm.nih.gov\/31194699\/\" target=\"_blank\" rel=\"noopener\">12<\/a><\/sup> and artificial intelligence.<\/span><\/p>\n<h4><span lang=\"EN-US\">3.<span>&nbsp;&nbsp;&nbsp; <\/span><\/span><b><span lang=\"EN-US\">Characterization<\/span><\/b><\/h4>\n<p class=\"MsoNormal\"><span lang=\"EN-US\">Techniques used to characterize CTCs include immunostaining, fluorescent <i>in situ<\/i> hybridization (FISH), sequencing, quantitative reverse-transcription polymerase chain reaction (qRT-PCR), expression analysis and cell culture.<sup><a href=\"https:\/\/pubmed.ncbi.nlm.nih.gov\/32708837\/\" target=\"_blank\" rel=\"noopener\">3<\/a><\/sup><b> <\/b><\/span><\/p>\n<h3><b><span>EVs<\/span><\/b><\/h3>\n<p class=\"MsoNormal\"><span lang=\"EN-US\">The clinical application of EVs detected by liquid biopsy has so far been limited by the lack of simple, efficient procedures to obtain EVs with high purity.<sup><a href=\"https:\/\/www.hindawi.com\/journals\/bmri\/2021\/6611244\/\" target=\"_blank\" rel=\"noopener\">13<\/a><\/sup> Some of the major strategies used to isolate these extracellular vesicles include ultracentrifugation, polymer precipitation, ultrafiltration, size-exclusion chromatography, affinity isolation and microfluidics-based techniques.<sup><a href=\"https:\/\/www.hindawi.com\/journals\/bmri\/2021\/6611244\/\" target=\"_blank\" rel=\"noopener\">13<\/a><\/sup> Each of these approaches has its advantages and disadvantages.<\/span><\/p>\n<h3><b><span>cfDNA\/ctDNA<\/span><\/b><\/h3>\n<p class=\"MsoNormal\"><span>Several methods have been used to quantify and detect tumor mutations in cfDNA\/ctDNA<sup><a href=\"https:\/\/doi.org\/10.1186\/s13045-022-01351-y\" target=\"_blank\" rel=\"noopener\">14<\/a><\/sup> \u2013 including droplet digital PCR<sup><a href=\"https:\/\/pubmed.ncbi.nlm.nih.gov\/34049769\/\" target=\"_blank\" rel=\"noopener\">15<\/a><\/sup>, beads emulsion, amplification and magnetics (BEAMing)<sup><a href=\"https:\/\/pubmed.ncbi.nlm.nih.gov\/28945877\/\" target=\"_blank\" rel=\"noopener\">16<\/a><\/sup>, next-generation sequencing-based approaches<sup><a href=\"https:\/\/pubmed.ncbi.nlm.nih.gov\/34575039\/\" target=\"_blank\" rel=\"noopener\">17<\/a> <\/sup>(whole exome or whole genome sequencing), cancer personalized profiling by deep sequencing (CAPP-seq)<sup><a href=\"https:\/\/pubmed.ncbi.nlm.nih.gov\/28945877\/\" target=\"_blank\" rel=\"noopener\">16<\/a><\/sup> and tagged-amplicon deep sequencing (TAm-Seq).<sup><a href=\"https:\/\/pubmed.ncbi.nlm.nih.gov\/29547634\/\" target=\"_blank\" rel=\"noopener\">18<\/a><\/sup> The profiling of epigenetic features of ctDNA, such as methylation signatures<sup><a href=\"https:\/\/doi.org\/10.1186\/s13148-022-01337-0\" target=\"_blank\" rel=\"noopener\">19<\/a><\/sup>, may provide information about the tissue of origin. <\/span><\/p>\n<h3><b><span>cfRNA<\/span><\/b><\/h3>\n<p class=\"MsoNormal\"><span>Profiling circulating cell-free (cfRNA) <\/span><span lang=\"EN-US\">offers unique opportunities to detect cancer, predict the tumor of origin, and determine the molecular subtype.<sup><a href=\"https:\/\/doi.org\/10.1038\/s41467-021-22444-1\" target=\"_blank\" rel=\"noopener\">20<\/a><\/sup> &nbsp;But technical and biological variability, coupled with a lack of standardized protocols, have so far hindered the clinical development of RNA-based liquid biopsies.<\/span><sup><span><a href=\"https:\/\/doi.org\/10.1186\/s40364-022-00409-w\" target=\"_blank\" rel=\"noopener\">21<\/a><\/span><\/sup><span><\/span><\/p>\n<h2><b><span>References<\/span><\/b><\/h2>\n<ol>\n<li>Lone SN, Nisar S, Masoodi T, et al. Liquid biopsy: a step closer to transform diagnosis, prognosis and future of cancer treatments. <i>Mol. Cancer<\/i>. 2022;21(1):79. doi:<a href=\"https:\/\/doi.org\/10.1186\/s12943-022-01543-7\" target=\"_blank\" rel=\"noopener\">10.1186\/s12943-022-01543-7<\/a><\/li>\n<li>Satyal U, Srivastava A and Abbosh P. Urine Biopsy-Liquid Gold for Molecular Detection and Surveillance of Bladder Cancer. <i>Front Oncol.<\/i> 2019;19(9):1266. doi: <a href=\"https:\/\/pubmed.ncbi.nlm.nih.gov\/31803629\/\" target=\"_blank\" rel=\"noopener\">10.3389\/fonc.2019.01266<\/a>.<\/li>\n<li>Habli Z, AlChamaa W, Saab R, Kadara H, Khraiche ML. Circulating Tumor Cell Detection Technologies and Clinical Utility: Challenges and Opportunities. <i>Cancers (Basel)<\/i>. 2020;12(7):1930. doi:<a href=\"https:\/\/doi.org\/10.3390\/cancers12071930\">10.3390\/cancers12071930<\/a><\/li>\n<li>Yan Y, Guo Q, Wang F, et al. 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Current challenges and best practices for cell-free long RNA biomarker discovery. <i>Biomarker Research<\/i>. 2022;10(1):62. doi:<a href=\"https:\/\/doi.org\/10.1186\/s40364-022-00409-w\" target=\"_blank\" rel=\"noopener\">10.1186\/s40364-022-00409-w<\/a><\/li>\n<\/ol>\n<\/div>\n","protected":false},"excerpt":{"rendered":"<p>Listen with Speechify Register for FREE to listen to this article Listen Now Thank you.<\/p>\n","protected":false},"author":1,"featured_media":41228,"comment_status":"open","ping_status":"open","sticky":false,"template":"","format":"standard","meta":{"footnotes":""},"categories":[1],"tags":[],"_links":{"self":[{"href":"https:\/\/peymantaeidi.net\/stem-cell\/wp-json\/wp\/v2\/posts\/41226"}],"collection":[{"href":"https:\/\/peymantaeidi.net\/stem-cell\/wp-json\/wp\/v2\/posts"}],"about":[{"href":"https:\/\/peymantaeidi.net\/stem-cell\/wp-json\/wp\/v2\/types\/post"}],"author":[{"embeddable":true,"href":"https:\/\/peymantaeidi.net\/stem-cell\/wp-json\/wp\/v2\/users\/1"}],"replies":[{"embeddable":true,"href":"https:\/\/peymantaeidi.net\/stem-cell\/wp-json\/wp\/v2\/comments?post=41226"}],"version-history":[{"count":3,"href":"https:\/\/peymantaeidi.net\/stem-cell\/wp-json\/wp\/v2\/posts\/41226\/revisions"}],"predecessor-version":[{"id":41231,"href":"https:\/\/peymantaeidi.net\/stem-cell\/wp-json\/wp\/v2\/posts\/41226\/revisions\/41231"}],"wp:featuredmedia":[{"embeddable":true,"href":"https:\/\/peymantaeidi.net\/stem-cell\/wp-json\/wp\/v2\/media\/41228"}],"wp:attachment":[{"href":"https:\/\/peymantaeidi.net\/stem-cell\/wp-json\/wp\/v2\/media?parent=41226"}],"wp:term":[{"taxonomy":"category","embeddable":true,"href":"https:\/\/peymantaeidi.net\/stem-cell\/wp-json\/wp\/v2\/categories?post=41226"},{"taxonomy":"post_tag","embeddable":true,"href":"https:\/\/peymantaeidi.net\/stem-cell\/wp-json\/wp\/v2\/tags?post=41226"}],"curies":[{"name":"wp","href":"https:\/\/api.w.org\/{rel}","templated":true}]}}