A robust multiplex immunofluorescence and digital pathology workflow for the characterisation of the tumour immune microenvironment

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usa-input-error-message selection-validation-message"> Name must be less than 100 characters </div> </div> <div class="action-panel-control-wrap existing-collections-controls"> <label for="collections-action-panel-add-to-existing" class="action-panel-label"> Choose a collection: </label> <select id="collections-action-panel-add-to-existing" class="action-panel-selector collections-action-add-to-existing" data-ga-category="save_share" data-ga-action="select_collection" data-ga-label="($('#collections-action-add-to-existing').val() === 'Favorites') ? 'Favorites' : 'non_favorites_collection'"> </select> <div class="collections-retry-load-on-error usa-input-error-message selection-validation-message"> Unable to load your collection due to an error<br> <a href="#">Please try again</a> </div> </div> </div> <div class="action-panel-actions"> <button class="action-panel-submit" type="submit" data-loading-label="Adding..." data-pinger-ignore data-ga-category="save_share" data-ga-action="collections" data-ga-label="add"> Add </button> <button class="action-panel-cancel" aria-label="Close 'Add to Collections' panel" ref="linksrc=close_collections_panel" aria-controls="collections-action-panel" aria-expanded="false" data-ga-category="save_share" data-ga-action="collections" data-ga-label="cancel"> Cancel </button> </div> </form> </div> </div> <div id="bibliography-action-panel" class="bibliography-action-panel action-panel in-progress-dots-panel" aria-hidden="true" data-bibliography-open-panel-enabled="false" data-bibliography-open-panel-url-hash="#open-bibliography-panel"> <div class="inner-wrap"> <h3 class="action-panel-heading"> Add to My Bibliography </h3> <form id="bibliography-action-panel-form" class="bibliography-action-panel-form action-panel-content action-form action-panel-smaller-selectors" data-add-to-bibliography-max-amount="100" data-add-to-bibliography-batch-size="10" data-bibliography-delegates-url="/list-bibliography-delegates/" data-add-to-bibliography-url="/add-to-bibliography/" data-get-article-ids-by-search-url="/get-article-ids-by-search/" data-mybib-root-url="https://www.ncbi.nlm.nih.gov/myncbi/collections/mybibliography/"> <input type="hidden" name="csrfmiddlewaretoken" value="vzx1PkxkxW3mADFiulVuICzoPksVVTUL6aiYSgjGxuDyXHSt3wdMJqzI04nANvio"> <div class="action-panel-control-wrap bibliographies-controls"> <div class="choice-group"> <ul class="bibliographies-action-add radio-group-items"> <li> <input name="bibliography" id="my-bibliography" class="my-bibliography" type="radio" checked/> <label for="my-bibliography">My Bibliography</label> </li> </ul> </div> </div> <div class="bibliographies-retry-load-on-error usa-input-error-message selection-validation-message"> Unable to load your delegates due to an error<br> <a href="#">Please try again</a> </div> <div class="action-panel-actions"> <button class="action-panel-submit" type="submit" data-loading-label="Adding..." data-pinger-ignore> Add </button> <button class="action-panel-cancel" aria-label="Close 'Add to bibliography' panel" ref="linksrc=close_bibliography_panel" aria-controls="bibliography-action-panel" aria-expanded="false" data-ga-category="save_share" data-ga-action="mybib" data-ga-label="cancel"> Cancel </button> </div> </form> </div> </div> <div id="saved-search-action-panel" class="saved-search-action-panel action-panel " aria-hidden="true" data-saved-search-open-panel-enabled="false" data-saved-search-open-panel-url-hash="#open-saved-search-panel"> <div class="inner-wrap"> <h2 class="action-panel-heading"> Your saved search </h2> <form id="saved-search-action-panel-form" class="saved-search-action-panel-form action-panel-content action-form" data-create-saved-search-url="/create-saved-search/" data-try-search-terms-url="/try-search-term/" data-saved-search-root-url="https://www.ncbi.nlm.nih.gov/myncbi/searches/"> <input type="hidden" name="csrfmiddlewaretoken" value="vzx1PkxkxW3mADFiulVuICzoPksVVTUL6aiYSgjGxuDyXHSt3wdMJqzI04nANvio"> <div class="action-panel-control-wrap"> <label for="saved-search-name" class="action-panel-label saved-search-name-label required-field-asterisk"> Name of saved search: </label> <input maxlength="200" type="text" name="saved-search-name" id="saved-search-name" class="saved-search-name" value="" required pattern="[^"&=<>\/]*" title="The following characters are not allowed in the Name field: "&=<>/"> </div> <div class="action-panel-control-wrap"> <label for="saved-search-term" class="action-panel-label required-field-asterisk"> Search terms: </label> <textarea name="saved-search-term" id="saved-search-term" class="saved-search-term" required></textarea> </div> <div class="test-search-term-wrap"> <a href="#" class="try-search-term">Test search terms</a> </div> <div class="choice-group action-panel-extra-margin-top"> <span class="action-panel-label" id="fieldset-label"> Would you like email updates of new search results? </span> <fieldset id="saved-search-alert" aria-describedby="fieldset-label"> <legend class="usa-sr-only">Saved Search Alert Radio Buttons</legend> <ul class="radio-group-items"> <li> <input type="radio" id="saved-search-alert-yes" class="saved-search-alert-yes" name="saved-search-alert" value="yes" checked> <label for="saved-search-alert-yes" class="action-panel-label">Yes</label> </li> <li> <input aria-label="No radio input" type="radio" id="saved-search-alert-no" class="saved-search-alert-no" name="saved-search-alert" value="no"> <label for="saved-search-alert-no" class="action-panel-label">No</label> </li> </ul> </fieldset> </div> <div class="alert-schedule-wrap"> <div class="action-panel-control-wrap"> <label class="action-panel-label"> Email: </label> <span aria-label="Email address" id="saved-search-email" class="action-panel-label"><span class="action-panel-label-bold"></span> (<a class="myncbi-account-settings" href="https://www.ncbi.nlm.nih.gov/account/settings/">change</a>)</span> </div> <div class="action-panel-control-wrap action-panel-extra-margin-top"> <label for="saved-search-frequency" class="action-panel-label"> Frequency: </label> <select id="saved-search-frequency" class="no-border-panel-selector saved-search-frequency"> <option value="monthly">Monthly</option> <option value="weekly">Weekly</option> <option value="daily">Daily</option> </select> </div> <div class="action-panel-control-wrap saved-search-monthly-additional"> <label for="saved-search-monthly-on-day" class="action-panel-label"> Which day? </label> <select id="saved-search-monthly-on-day" class="no-border-panel-selector"> <option value="Sunday">The first Sunday</option> <option value="Monday">The first Monday</option> <option value="Tuesday">The first Tuesday</option> <option value="Wednesday">The first Wednesday</option> <option value="Thursday">The first Thursday</option> <option value="Friday">The first Friday</option> <option value="Saturday">The first Saturday</option> <option value="day">The first day</option> <option value="weekday">The first weekday</option> </select> </div> <div class="action-panel-control-wrap saved-search-weekly-additional"> <label for="saved-search-weekly-on-day" class="action-panel-label"> Which day? </label> <select id="saved-search-weekly-on-day" class="no-border-panel-selector saved-search-weekly-on-day"> <option value="Sunday">Sunday</option> <option value="Monday">Monday</option> <option value="Tuesday">Tuesday</option> <option value="Wednesday">Wednesday</option> <option value="Thursday">Thursday</option> <option value="Friday">Friday</option> <option value="Saturday">Saturday</option> </select> </div> <div class="action-panel-control-wrap"> <label for="saved-search-report" class="action-panel-label"> Report format: </label> <select id="saved-search-report" class="no-border-panel-selector saved-search-report"> <option value="DocSum">Summary</option> <option value="DocSumText">Summary (text)</option> <option value="Abstract">Abstract</option> <option value="AbstractText">Abstract (text)</option> <option value="MEDLINE">PubMed</option> </select> </div> <div class="action-panel-control-wrap"> <label for="saved-search-amount" class="action-panel-label"> Send at most: </label> <select id="saved-search-amount" class="no-border-panel-selector saved-search-amount"> <option value="1">1 item</option> <option value="5" selected>5 items</option> <option value="10">10 items</option> <option value="20">20 items</option> <option value="50">50 items</option> <option value="100">100 items</option> <option value="200">200 items</option> </select> </div> <div> <input type="checkbox" id="saved-search-send-if-no-result" class="saved-search-send-if-no-result" name="saved-search-send-if-no-result"> <label for="saved-search-send-if-no-result" class="action-panel-label smaller-checkbox"> Send even when there aren't any new results </label> </div> <div class="action-panel-control-wrap option-text-in-email-wrap"> <label for="saved-search-email-text" class="action-panel-label"> Optional text in email: </label> <textarea name="saved-search-email-text" id="saved-search-email-text" class="saved-search-email-text"></textarea> </div> </div> <div class="action-panel-actions"> <button class="action-panel-submit" type="submit" data-loading-label="Saving..." data-ga-category="save_share" data-ga-action="alert" data-ga-label="save"> Save </button> <button class="action-panel-cancel" aria-label="Close 'Your saved search' panel" ref="linksrc=close_saved_search_panel" aria-controls="saved-search-action-panel" aria-expanded="false" data-ga-category="save_share" data-ga-action="alert" data-ga-label="cancel"> Cancel </button> </div> </form> </div> </div> <div id="citation-manager-action-panel" class="citation-manager-action-panel action-panel" aria-hidden="true"> <div class="inner-wrap"> <h2 class="action-panel-heading"> Create a file for external citation management software </h2> <form id="citation-manager-action-panel-form" class="action-panel-content action-form" action="/results-export-ids/" data-by-search-action="/results-export-search-data/" data-by-ids-action="/results-export-ids/" method="post" data-by-search-method="post" data-by-ids-method="post"> <input type="hidden" name="csrfmiddlewaretoken" value="vzx1PkxkxW3mADFiulVuICzoPksVVTUL6aiYSgjGxuDyXHSt3wdMJqzI04nANvio"> <input name="results-format" type="hidden" value="pubmed"/> <div class="action-panel-actions"> <button class="action-panel-submit" type="submit" data-loading-label="Sending..." data-ga-category="save_share" data-ga-action="citation_manager" data-ga-label="save"> Create file </button> <button class="action-panel-cancel" aria-label="Close 'Send citations to citation manager' panel" ref="linksrc=close_citation_manager_panel" aria-controls="citation-manager-action-panel" aria-expanded="false" data-ga-category="save_share" data-ga-action="citation_manager" data-ga-label="cancel"> Cancel </button> </div> </form> </div> </div> <div id="rss-action-panel" class="rss-action-panel action-panel " aria-hidden="true"> <div class="inner-wrap"> <h2 class="action-panel-heading"> Your RSS Feed </h2> <form id="rss-action-panel-form" class="rss-action-panel-form action-panel-content action-form" data-create-rss-feed-url="/create-rss-feed-url/" data-search-form-term-value=""> <input type="hidden" name="csrfmiddlewaretoken" value="vzx1PkxkxW3mADFiulVuICzoPksVVTUL6aiYSgjGxuDyXHSt3wdMJqzI04nANvio"> <div class="action-panel-control-wrap"> <label for="rss-name" class="action-panel-label required-field-asterisk"> Name of RSS Feed: </label> <input maxlength="200" placeholder="Name" type="text" name="rss-name" id="rss-name" class="rss-name" value='' required pattern="[^"&=<>\/]*" title="The following characters are not allowed in the Name field: "&=<>/"> </div> <div class="rss-limit-wrap"> <div class="action-panel-control-wrap action-panel-extra-margin-top"> <label for="rss-limit" class="action-panel-label"> Number of items displayed: </label> <select id="rss-limit" class="no-border-panel-selector rss-limit"> <option value="5">5</option> <option value="10">10</option> <option value="15" selected="selected">15</option> <option value="20">20</option> <option value="50">50</option> <option value="100">100</option> </select> </div> </div> <div class="action-panel-actions"> <button class="action-panel-submit" type="submit" data-loading-label="Creating..." data-ga-category="save_share" data-ga-action="alert" data-ga-label="save"> Create RSS </button> <button class="action-panel-cancel" aria-label="Close 'Your RSS' panel" ref="linksrc=close_rss_panel" aria-controls="rss-action-panel" aria-expanded="false" data-ga-category="save_share" data-ga-action="alert" data-ga-label="cancel"> Cancel </button> </div> <div class="action-panel-control-wrap rss-link-copy-wrap"> <label for="rss-link" class="usa-sr-only">RSS Link</label> <input placeholder="Your RSS Feed Link" type="text" name="rss-link" id="rss-link" class="rss-link" title="RSS Link"> <button type="button" disabled class="rss-link-copy-button disabled" data-ga-category="save_share" data-ga-action="rss" data-ga-label="copy"> Copy </button> </div> </form> </div> </div> </div> </div> <div class="article-page" id="article-page" data-article-pmid="32671911"> <aside class="page-sidebar"> <div class="inner-wrap"> <div class="full-text-links"> <div class="full-view"> <h3 class="title"> Full text links </h3> <div class="full-text-links-list"> <a class="link-item dialog-focus" 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collection: </label><input type="text" name="add-to-new-collection" id="collections-action-dialog-add-to-new" class="collections-action-add-to-new" pattern="[^"&=<>\/]*" title="The following characters are not allowed in the Name field: "&=<>/" maxlength="100" data-ga-category="collections_button" data-ga-action="create_collection" data-ga-label="non_favorties_collection"><div class="collections-new-name-too-long usa-input-error-message selection-validation-message"> Name must be less than 100 characters </div></div><div class="action-panel-control-wrap existing-collections-controls"><label for="collections-action-dialog-add-to-existing" class="action-panel-label"> Choose a collection: </label><select id="collections-action-dialog-add-to-existing" class="action-panel-selector collections-action-add-to-existing" data-ga-category="collections_button" data-ga-action="select_collection" data-ga-label="($('.collections-action-add-to-existing').val() === 'Favorites') ? 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Molecular oncology" data-pinger-ignore> Mol Oncol </button><div id="full-view-journal" class="journal-actions-dropdown dropdown dropdown-container" hidden aria-label="Dropdown menu for journal Molecular oncology"><div class="title">Actions</div><div class="content"><ul class="journal-actions-links"><li><a class="search-in-pubmed-link dropdown-block-link" href="/?term=%22Mol+Oncol%22%5Bjour%5D&sort=date&sort_order=desc" data-href="/?term=%22Mol+Oncol%22%5Bjour%5D&sort=date&sort_order=desc" ref="linksrc=search_in_pubmed_journal_name_link&journal_abbrev=Mol Oncol" data-ga-category="search" data-ga-action="journal_link" data-ga-label="Mol Oncol"> Search in PubMed </a></li><li><a class="search-in-nlm-catalog-link dropdown-block-link" ref ="linksrc=search_in_nlm_catalog_link" href="https://www.ncbi.nlm.nih.gov/nlmcatalog?term=%22Mol+Oncol%22%5BTitle+Abbreviation%5D" data-ga-category="search_catalog" data-ga-action="journal_link" data-ga-label="Mol Oncol"> Search in NLM Catalog </a></li><li><a class="add-to-search-link dropdown-block-link" ref ="linksrc=add_to_search_link" role="button" data-search-term='"Mol Oncol"[jour]' data-ga-category="search" data-ga-action="add_to_search" data-ga-label='"Mol Oncol"[jour]' href="#"> Add to Search </a></li></ul></div></div></div><span class="period">. </span><span class="cit">2020 Oct;14(10):2384-2402.</span> </div> <span class="citation-doi"> doi: 10.1002/1878-0261.12764. </span> <span class="secondary-date"> Epub 2020 Sep 1. </span> </div> <h1 class="heading-title"> A robust multiplex immunofluorescence and digital pathology workflow for the characterisation of the tumour immune microenvironment </h1> <div class="inline-authors"> <div class="authors"> <div class="authors-list"> <span class="authors-list-item "><a class="full-name" href="/?term=Viratham+Pulsawatdi+A&cauthor_id=32671911" ref="linksrc=author_name_link" data-ga-category="search" data-ga-action="author_link" data-ga-label="Amélie Viratham Pulsawatdi">Amélie Viratham Pulsawatdi</a><sup class="affiliation-links"><span class="author-sup-separator"> </span><a class="affiliation-link" title="Patrick G Johnston Centre for Cancer Research, Queen's University Belfast, Belfast, UK." href="#full-view-affiliation-1" ref="linksrc=author_aff"> 1 </a></sup><span class="comma">, </span></span><span class="authors-list-item "><a class="full-name" href="/?term=Craig+SG&cauthor_id=32671911" ref="linksrc=author_name_link" data-ga-category="search" data-ga-action="author_link" data-ga-label="Stephanie G Craig">Stephanie G Craig</a><sup class="affiliation-links"><span class="author-sup-separator"> </span><a class="affiliation-link" title="Patrick G Johnston Centre for Cancer Research, Queen's University Belfast, Belfast, UK." href="#full-view-affiliation-1" ref="linksrc=author_aff"> 1 </a></sup><span class="comma">, </span></span><span class="authors-list-item "><a class="full-name" href="/?term=Bingham+V&cauthor_id=32671911" ref="linksrc=author_name_link" data-ga-category="search" data-ga-action="author_link" data-ga-label="Victoria Bingham">Victoria Bingham</a><sup class="affiliation-links"><span class="author-sup-separator"> </span><a class="affiliation-link" title="Patrick G Johnston Centre for Cancer Research, Queen's University Belfast, Belfast, UK." href="#full-view-affiliation-1" ref="linksrc=author_aff"> 1 </a></sup><span class="comma">, </span></span><span class="authors-list-item "><a class="full-name" href="/?term=McCombe+K&cauthor_id=32671911" ref="linksrc=author_name_link" data-ga-category="search" data-ga-action="author_link" data-ga-label="Kris McCombe">Kris McCombe</a><sup class="affiliation-links"><span class="author-sup-separator"> </span><a class="affiliation-link" title="Patrick G Johnston Centre for Cancer Research, Queen's University Belfast, Belfast, UK." href="#full-view-affiliation-1" ref="linksrc=author_aff"> 1 </a></sup><span class="comma">, </span></span><span class="authors-list-item "><a class="full-name" href="/?term=Humphries+MP&cauthor_id=32671911" ref="linksrc=author_name_link" data-ga-category="search" data-ga-action="author_link" data-ga-label="Matthew P Humphries">Matthew P Humphries</a><sup class="affiliation-links"><span class="author-sup-separator"> </span><a class="affiliation-link" title="Patrick G Johnston Centre for Cancer Research, Queen's University Belfast, Belfast, UK." href="#full-view-affiliation-1" ref="linksrc=author_aff"> 1 </a></sup><span class="comma">, </span></span><span class="authors-list-item "><a class="full-name" href="/?term=Senevirathne+S&cauthor_id=32671911" ref="linksrc=author_name_link" data-ga-category="search" data-ga-action="author_link" data-ga-label="Seedevi Senevirathne">Seedevi Senevirathne</a><sup class="affiliation-links"><span class="author-sup-separator"> </span><a class="affiliation-link" title="Patrick G Johnston Centre for Cancer Research, Queen's University Belfast, Belfast, UK." href="#full-view-affiliation-1" ref="linksrc=author_aff"> 1 </a></sup><span class="comma">, </span></span><span class="authors-list-item "><a class="full-name" href="/?term=Richman+SD&cauthor_id=32671911" ref="linksrc=author_name_link" data-ga-category="search" data-ga-action="author_link" data-ga-label="Susan D Richman">Susan D Richman</a><sup class="affiliation-links"><span class="author-sup-separator"> </span><a class="affiliation-link" title="Leeds Institute of Medical Research at St James's, University of Leeds, Leeds, UK." href="#full-view-affiliation-2" ref="linksrc=author_aff"> 2 </a></sup><span class="comma">, </span></span><span class="authors-list-item "><a class="full-name" href="/?term=Quirke+P&cauthor_id=32671911" ref="linksrc=author_name_link" data-ga-category="search" data-ga-action="author_link" data-ga-label="Phil Quirke">Phil Quirke</a><sup class="affiliation-links"><span class="author-sup-separator"> </span><a class="affiliation-link" title="Leeds Institute of Medical Research at St James's, University of Leeds, Leeds, UK." href="#full-view-affiliation-2" ref="linksrc=author_aff"> 2 </a></sup><span class="comma">, </span></span><span class="authors-list-item "><a class="full-name" href="/?term=Campo+L&cauthor_id=32671911" ref="linksrc=author_name_link" data-ga-category="search" data-ga-action="author_link" data-ga-label="Leticia Campo">Leticia Campo</a><sup class="affiliation-links"><span class="author-sup-separator"> </span><a class="affiliation-link" title="CRUK/MRC Oxford Institute for Radiation Oncology, Oxford University, Oxford, UK." href="#full-view-affiliation-3" ref="linksrc=author_aff"> 3 </a></sup><span class="comma">, </span></span><span class="authors-list-item "><a class="full-name" href="/?term=Domingo+E&cauthor_id=32671911" ref="linksrc=author_name_link" data-ga-category="search" data-ga-action="author_link" data-ga-label="Enric Domingo">Enric Domingo</a><sup class="affiliation-links"><span class="author-sup-separator"> </span><a class="affiliation-link" title="CRUK/MRC Oxford Institute for Radiation Oncology, Oxford University, Oxford, UK." href="#full-view-affiliation-3" ref="linksrc=author_aff"> 3 </a></sup><span class="comma">, </span></span><span class="authors-list-item "><a class="full-name" href="/?term=Maughan+TS&cauthor_id=32671911" ref="linksrc=author_name_link" data-ga-category="search" data-ga-action="author_link" data-ga-label="Timothy S Maughan">Timothy S Maughan</a><sup class="affiliation-links"><span class="author-sup-separator"> </span><a class="affiliation-link" title="CRUK/MRC Oxford Institute for Radiation Oncology, Oxford University, Oxford, UK." href="#full-view-affiliation-3" ref="linksrc=author_aff"> 3 </a></sup><span class="comma">, </span></span><span class="authors-list-item "><a class="full-name" href="/?term=James+JA&cauthor_id=32671911" ref="linksrc=author_name_link" data-ga-category="search" data-ga-action="author_link" data-ga-label="Jacqueline A James">Jacqueline A James</a><sup class="affiliation-links"><span class="author-sup-separator"> </span><a class="affiliation-link" title="Patrick G Johnston Centre for Cancer Research, Queen's University Belfast, Belfast, UK." href="#full-view-affiliation-1" ref="linksrc=author_aff"> 1 </a><span class="author-sup-separator"> </span><a class="affiliation-link" title="Belfast Health and Social Care Trust, Belfast, UK." href="#full-view-affiliation-4" ref="linksrc=author_aff"> 4 </a></sup><span class="comma">, </span></span><span class="authors-list-item "><a class="full-name" href="/?term=Salto-Tellez+M&cauthor_id=32671911" ref="linksrc=author_name_link" data-ga-category="search" data-ga-action="author_link" data-ga-label="Manuel Salto-Tellez">Manuel Salto-Tellez</a><sup class="affiliation-links"><span class="author-sup-separator"> </span><a class="affiliation-link" title="Patrick G Johnston Centre for Cancer Research, Queen's University Belfast, Belfast, UK." href="#full-view-affiliation-1" ref="linksrc=author_aff"> 1 </a><span class="author-sup-separator"> </span><a class="affiliation-link" title="CRUK/MRC Oxford Institute for Radiation Oncology, Oxford University, Oxford, UK." href="#full-view-affiliation-3" ref="linksrc=author_aff"> 3 </a><span class="author-sup-separator"> </span><a class="affiliation-link" title="Belfast Health and Social Care Trust, Belfast, UK." href="#full-view-affiliation-4" ref="linksrc=author_aff"> 4 </a></sup></span> </div> </div> </div> <div class="short-article-details"> Affiliations <button class="more-details" id="toggle-authors" data-alt-text="Collapse" aria-controls="expanded-authors" aria-expanded="false" data-pinger-ignore data-fetch-url="/32671911/long-authors/" data-id-prefix=""> <span class="title"> Expand </span> </button> </div> <div class="extended-article-details" id="expanded-authors" aria-hidden="true"> <div class="expanded-authors" id="full-view-expanded-authors"> <div class="affiliations"> <h3 class="title"> Affiliations </h3> <ul class="item-list"> <li data-affiliation-id="full-view-affiliation-1" id="full-view-affiliation-1" ><sup class="key">1</sup> Patrick G Johnston Centre for Cancer Research, Queen's University Belfast, Belfast, UK.</li> <li data-affiliation-id="full-view-affiliation-2" id="full-view-affiliation-2" ><sup class="key">2</sup> Leeds Institute of Medical Research at St James's, University of Leeds, Leeds, UK.</li> <li data-affiliation-id="full-view-affiliation-3" id="full-view-affiliation-3" ><sup class="key">3</sup> CRUK/MRC Oxford Institute for Radiation Oncology, Oxford University, Oxford, UK.</li> <li data-affiliation-id="full-view-affiliation-4" id="full-view-affiliation-4" ><sup class="key">4</sup> Belfast Health and Social Care Trust, Belfast, UK.</li> </ul> </div> </div> </div> <ul class="identifiers" id="full-view-identifiers"> <li> <span class="identifier pubmed"> <span class="id-label"> PMID: </span> <strong class="current-id" title="PubMed ID">32671911</strong> </span> </li> <li> <span class="identifier pmc"> <span class="id-label"> PMCID: </span> <a class="id-link" target="_blank" rel="noopener" ref="linksrc=article_id_link&article_id=PMC7530793&id_type=PMC" href="https://pmc.ncbi.nlm.nih.gov/articles/PMC7530793/" data-ga-category="full_text" data-ga-action="PMCID" > PMC7530793 </a> </span> </li> <li> <span class="identifier doi"> <span class="id-label"> DOI: </span> <a class="id-link" target="_blank" rel="noopener" ref="linksrc=article_id_link&article_id=10.1002/1878-0261.12764&id_type=DOI" href="https://doi.org/10.1002/1878-0261.12764" data-ga-category="full_text" data-ga-action="DOI" > 10.1002/1878-0261.12764 </a> </span> </li> </ul> <div class="in-clipboard-label " hidden> Item in Clipboard </div> </div> <div class="short-view" id="short-view-heading"> <h1 class="heading-title"> A robust multiplex immunofluorescence and digital pathology workflow for the characterisation of the tumour immune microenvironment </h1> <div class="short-citation"> <span class="authors-list"> <span class="authors-list-item "><span class="full-name">Amélie Viratham Pulsawatdi</span><span class="citation-separator"> et al.</span></span> </span> <span class="citation-journal"> Mol Oncol<span class="citation-separator">.</span> </span> <span class="date"> <span class="citation-year">2020 Oct</span><span class="citation-separator">.</span> </span> </div> <div class="more-article-details-section with-display-settings"> <button class="more-article-details" id="toggle-article-details" data-alt-text="Hide details" ref="linksrc=toggle_article_details" aria-controls="full-article-details" aria-expanded="false" data-fetch-url="/32671911/long-authors/" data-id-prefix=""> <span class="title">Show details</span> </button> <div class="display-options" data-format-key="format" data-default-format-value="abstract" data-current-format-value="abstract" role="region"> <button class="trigger usa-button" title="Change format" data-ga-category="display_options" 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oncology" data-pinger-ignore> Mol Oncol </button><div id="short-view-journal" class="journal-actions-dropdown dropdown dropdown-container" hidden aria-label="Dropdown menu for journal Molecular oncology"><div class="title">Actions</div><div class="content"><ul class="journal-actions-links"><li><a class="search-in-pubmed-link dropdown-block-link" href="/?term=%22Mol+Oncol%22%5Bjour%5D&sort=date&sort_order=desc" data-href="/?term=%22Mol+Oncol%22%5Bjour%5D&sort=date&sort_order=desc" ref="linksrc=search_in_pubmed_journal_name_link&journal_abbrev=Mol Oncol" data-ga-category="search" data-ga-action="journal_link" data-ga-label="Mol Oncol"> Search in PubMed </a></li><li><a class="search-in-nlm-catalog-link dropdown-block-link" ref ="linksrc=search_in_nlm_catalog_link" href="https://www.ncbi.nlm.nih.gov/nlmcatalog?term=%22Mol+Oncol%22%5BTitle+Abbreviation%5D" data-ga-category="search_catalog" data-ga-action="journal_link" data-ga-label="Mol Oncol"> Search in NLM Catalog </a></li><li><a class="add-to-search-link dropdown-block-link" ref ="linksrc=add_to_search_link" role="button" data-search-term='"Mol Oncol"[jour]' data-ga-category="search" data-ga-action="add_to_search" data-ga-label='"Mol Oncol"[jour]' href="#"> Add to Search </a></li></ul></div></div></div><span class="period">. </span><span class="cit">2020 Oct;14(10):2384-2402.</span> </div> <span class="citation-doi"> doi: 10.1002/1878-0261.12764. </span> <span class="secondary-date"> Epub 2020 Sep 1. </span> </div> <div class="expanded-authors" id="short-view-expanded-authors"> <div class="authors"> <h3 class="title"> Authors </h3> <div class="authors-list"> <span class="authors-list-item "><a class="full-name" href="/?term=Viratham+Pulsawatdi+A&cauthor_id=32671911" ref="linksrc=author_name_link" data-ga-category="search" data-ga-action="author_link" data-ga-label="Amélie Viratham Pulsawatdi">Amélie Viratham Pulsawatdi</a><sup class="affiliation-links"><span class="author-sup-separator"> </span><a class="affiliation-link" title="Patrick G Johnston Centre for Cancer Research, Queen's University Belfast, Belfast, UK." href="#short-view-affiliation-1" ref="linksrc=author_aff"> 1 </a></sup><span class="comma">, </span></span><span class="authors-list-item "><a class="full-name" href="/?term=Craig+SG&cauthor_id=32671911" ref="linksrc=author_name_link" data-ga-category="search" data-ga-action="author_link" data-ga-label="Stephanie G Craig">Stephanie G Craig</a><sup class="affiliation-links"><span class="author-sup-separator"> </span><a class="affiliation-link" title="Patrick G Johnston Centre for Cancer Research, Queen's University Belfast, Belfast, UK." href="#short-view-affiliation-1" ref="linksrc=author_aff"> 1 </a></sup><span class="comma">, </span></span><span class="authors-list-item "><a class="full-name" href="/?term=Bingham+V&cauthor_id=32671911" ref="linksrc=author_name_link" data-ga-category="search" data-ga-action="author_link" data-ga-label="Victoria Bingham">Victoria Bingham</a><sup class="affiliation-links"><span class="author-sup-separator"> </span><a class="affiliation-link" title="Patrick G Johnston Centre for Cancer Research, Queen's University Belfast, Belfast, UK." href="#short-view-affiliation-1" ref="linksrc=author_aff"> 1 </a></sup><span class="comma">, </span></span><span class="authors-list-item "><a class="full-name" href="/?term=McCombe+K&cauthor_id=32671911" ref="linksrc=author_name_link" data-ga-category="search" data-ga-action="author_link" data-ga-label="Kris McCombe">Kris McCombe</a><sup class="affiliation-links"><span class="author-sup-separator"> </span><a class="affiliation-link" title="Patrick G Johnston Centre for Cancer Research, Queen's University Belfast, Belfast, UK." href="#short-view-affiliation-1" ref="linksrc=author_aff"> 1 </a></sup><span class="comma">, </span></span><span class="authors-list-item "><a class="full-name" href="/?term=Humphries+MP&cauthor_id=32671911" ref="linksrc=author_name_link" data-ga-category="search" data-ga-action="author_link" data-ga-label="Matthew P Humphries">Matthew P Humphries</a><sup class="affiliation-links"><span class="author-sup-separator"> </span><a class="affiliation-link" title="Patrick G Johnston Centre for Cancer Research, Queen's University Belfast, Belfast, UK." href="#short-view-affiliation-1" ref="linksrc=author_aff"> 1 </a></sup><span class="comma">, </span></span><span class="authors-list-item "><a class="full-name" href="/?term=Senevirathne+S&cauthor_id=32671911" ref="linksrc=author_name_link" data-ga-category="search" data-ga-action="author_link" data-ga-label="Seedevi Senevirathne">Seedevi Senevirathne</a><sup class="affiliation-links"><span class="author-sup-separator"> </span><a class="affiliation-link" title="Patrick G Johnston Centre for Cancer Research, Queen's University Belfast, Belfast, UK." href="#short-view-affiliation-1" ref="linksrc=author_aff"> 1 </a></sup><span class="comma">, </span></span><span class="authors-list-item "><a class="full-name" href="/?term=Richman+SD&cauthor_id=32671911" ref="linksrc=author_name_link" data-ga-category="search" data-ga-action="author_link" data-ga-label="Susan D Richman">Susan D Richman</a><sup class="affiliation-links"><span class="author-sup-separator"> </span><a class="affiliation-link" title="Leeds Institute of Medical Research at St James's, University of Leeds, Leeds, UK." href="#short-view-affiliation-2" ref="linksrc=author_aff"> 2 </a></sup><span class="comma">, </span></span><span class="authors-list-item "><a class="full-name" href="/?term=Quirke+P&cauthor_id=32671911" ref="linksrc=author_name_link" data-ga-category="search" data-ga-action="author_link" data-ga-label="Phil Quirke">Phil Quirke</a><sup class="affiliation-links"><span class="author-sup-separator"> </span><a class="affiliation-link" title="Leeds Institute of Medical Research at St James's, University of Leeds, Leeds, UK." href="#short-view-affiliation-2" ref="linksrc=author_aff"> 2 </a></sup><span class="comma">, </span></span><span class="authors-list-item "><a class="full-name" href="/?term=Campo+L&cauthor_id=32671911" ref="linksrc=author_name_link" data-ga-category="search" data-ga-action="author_link" data-ga-label="Leticia Campo">Leticia Campo</a><sup class="affiliation-links"><span class="author-sup-separator"> </span><a class="affiliation-link" title="CRUK/MRC Oxford Institute for Radiation Oncology, Oxford University, Oxford, UK." href="#short-view-affiliation-3" ref="linksrc=author_aff"> 3 </a></sup><span class="comma">, </span></span><span class="authors-list-item "><a class="full-name" href="/?term=Domingo+E&cauthor_id=32671911" ref="linksrc=author_name_link" data-ga-category="search" data-ga-action="author_link" data-ga-label="Enric Domingo">Enric Domingo</a><sup class="affiliation-links"><span class="author-sup-separator"> </span><a class="affiliation-link" title="CRUK/MRC Oxford Institute for Radiation Oncology, Oxford University, Oxford, UK." href="#short-view-affiliation-3" ref="linksrc=author_aff"> 3 </a></sup><span class="comma">, </span></span><span class="authors-list-item "><a class="full-name" href="/?term=Maughan+TS&cauthor_id=32671911" ref="linksrc=author_name_link" data-ga-category="search" data-ga-action="author_link" data-ga-label="Timothy S Maughan">Timothy S Maughan</a><sup class="affiliation-links"><span class="author-sup-separator"> </span><a class="affiliation-link" title="CRUK/MRC Oxford Institute for Radiation Oncology, Oxford University, Oxford, UK." href="#short-view-affiliation-3" ref="linksrc=author_aff"> 3 </a></sup><span class="comma">, </span></span><span class="authors-list-item "><a class="full-name" href="/?term=James+JA&cauthor_id=32671911" ref="linksrc=author_name_link" data-ga-category="search" data-ga-action="author_link" data-ga-label="Jacqueline A James">Jacqueline A James</a><sup class="affiliation-links"><span class="author-sup-separator"> </span><a class="affiliation-link" title="Patrick G Johnston Centre for Cancer Research, Queen's University Belfast, Belfast, UK." href="#short-view-affiliation-1" ref="linksrc=author_aff"> 1 </a><span class="author-sup-separator"> </span><a class="affiliation-link" title="Belfast Health and Social Care Trust, Belfast, UK." href="#short-view-affiliation-4" ref="linksrc=author_aff"> 4 </a></sup><span class="comma">, </span></span><span class="authors-list-item "><a class="full-name" href="/?term=Salto-Tellez+M&cauthor_id=32671911" ref="linksrc=author_name_link" data-ga-category="search" data-ga-action="author_link" data-ga-label="Manuel Salto-Tellez">Manuel Salto-Tellez</a><sup class="affiliation-links"><span class="author-sup-separator"> </span><a class="affiliation-link" title="Patrick G Johnston Centre for Cancer Research, Queen's University Belfast, Belfast, UK." href="#short-view-affiliation-1" ref="linksrc=author_aff"> 1 </a><span class="author-sup-separator"> </span><a class="affiliation-link" title="CRUK/MRC Oxford Institute for Radiation Oncology, Oxford University, Oxford, UK." href="#short-view-affiliation-3" ref="linksrc=author_aff"> 3 </a><span class="author-sup-separator"> </span><a class="affiliation-link" title="Belfast Health and Social Care Trust, Belfast, UK." href="#short-view-affiliation-4" ref="linksrc=author_aff"> 4 </a></sup></span> </div> </div> <div class="affiliations"> <h3 class="title"> Affiliations </h3> <ul class="item-list"> <li data-affiliation-id="short-view-affiliation-1" id="short-view-affiliation-1" ><sup class="key">1</sup> Patrick G Johnston Centre for Cancer Research, Queen's University Belfast, Belfast, UK.</li> <li data-affiliation-id="short-view-affiliation-2" id="short-view-affiliation-2" ><sup class="key">2</sup> Leeds Institute of Medical Research at St James's, University of Leeds, Leeds, UK.</li> <li data-affiliation-id="short-view-affiliation-3" id="short-view-affiliation-3" ><sup class="key">3</sup> CRUK/MRC Oxford Institute for Radiation Oncology, Oxford University, Oxford, UK.</li> <li data-affiliation-id="short-view-affiliation-4" id="short-view-affiliation-4" ><sup class="key">4</sup> Belfast Health and Social Care Trust, Belfast, UK.</li> </ul> </div> </div> <ul class="identifiers" id="short-view-identifiers"> <li> <span class="identifier pubmed"> <span class="id-label"> PMID: </span> <strong class="current-id" title="PubMed ID">32671911</strong> </span> </li> <li> <span class="identifier pmc"> <span class="id-label"> PMCID: </span> <a class="id-link" target="_blank" rel="noopener" ref="linksrc=article_id_link&article_id=PMC7530793&id_type=PMC" href="https://pmc.ncbi.nlm.nih.gov/articles/PMC7530793/" data-ga-category="full_text" data-ga-action="PMCID" > PMC7530793 </a> </span> </li> <li> <span class="identifier doi"> <span class="id-label"> DOI: </span> <a class="id-link" target="_blank" rel="noopener" ref="linksrc=article_id_link&article_id=10.1002/1878-0261.12764&id_type=DOI" href="https://doi.org/10.1002/1878-0261.12764" data-ga-category="full_text" data-ga-action="DOI" > 10.1002/1878-0261.12764 </a> </span> </li> </ul> <div class="in-clipboard-label " hidden> Item in Clipboard </div> </div> </div> </header> <div class="actions-buttons inline"><div class="inner-wrap"><button class="full-text-links-button full-text-links-dialog-trigger" title="Open dialog with full text links" ref="linksrc=full_text_links"> Full text links </button><button class="citation-button citation-dialog-trigger" aria-label="Open dialog with citation text in different styles" data-ga-category="save_share" data-ga-action="cite" data-ga-label="open" data-all-citations-url="/32671911/citations/" data-citation-style="nlm" data-pubmed-format-link="/32671911/export/"><span class="button-label">Cite</span></button><button class="more-actions-button more-actions-dialog-trigger" title="Open dialog with more actions to take" ref="linksrc=more_actions_btn"></button><div class="display-options" data-format-key="format" data-default-format-value="abstract" data-current-format-value="abstract" role="region"><button class="trigger usa-button" title="Change format" data-ga-category="display_options" data-ga-action="display_options_dialog"><span class="button-label">Display options</span></button><div class="dropdown" hidden><div class="title"> Display options </div><div class="content"><div class="format-container"><span class="option-label"> Format </span><select name="format" data-initial-value="abstract" aria-label="Display format" id="inline-actions-display-format"><option value="abstract">Abstract</option><option value="pubmed">PubMed</option><option value="pmid">PMID</option></select></div></div></div></div></div></div> <div class="abstract" id="abstract"> <h2 class="title"> Abstract </h2> <div class="abstract-content selected" id="eng-abstract"> <p> Multiplex immunofluorescence is a powerful tool for the simultaneous detection of tissue-based biomarkers, revolutionising traditional immunohistochemistry. The Opal methodology allows up to eight biomarkers to be measured concomitantly without cross-reactivity, permitting identification of different cell populations within the tumour microenvironment. In this study, we aimed to validate a multiplex immunofluorescence workflow in two complementary multiplex panels and evaluate the tumour immune microenvironment in colorectal cancer (CRC) formalin-fixed paraffin-embedded tissue. We stained CRC and tonsil samples using Opal multiplex immunofluorescence on a Leica BOND RX immunostainer. We then acquired images on an Akoya Vectra Polaris and performed multispectral unmixing using inform. Antibody panels were validated on tissue microarray sections containing cores from six normal tissue types, using qupath for image analysis. Comparisons between chromogenic immunohistochemistry and multiplex immunofluorescence on consecutive sections from the same tissue microarray showed significant correlation (r<sub>s</sub> > 0.9, P-value < 0.0001), validating both panels. We identified many factors that influenced the quality of the acquired fluorescent images, including biomarker co-expression, staining order, Opal-antibody pairing, sample thickness, multispectral unmixing and biomarker detection order during image analysis. Overall, we report the optimisation and validation of a multiplex immunofluorescence process, from staining to image analysis, ensuring assay robustness. Our multiplex immunofluorescence protocols permit the accurate detection of multiple immune markers in various tissue types, using a workflow that enables rapid processing of samples, above and beyond previous workflows. </p> </div> <p> <strong class="sub-title"> Keywords: </strong> image analysis; multiplex immunofluorescence; opal methodology. </p> </div> <p class="copyright" id="copyright"> © 2020 The Authors. Published by FEBS Press and John Wiley & Sons Ltd. </p> <p class="disclaimer" id="disclaimer"> <a href="/disclaimer/" target="_blank" data-ga-category="literature_resources" data-ga-action="disclaimer_link" data-ga-label="under_abstract">PubMed Disclaimer</a> </p> <div class="conflict-of-interest" id="conflict-of-interest"> <h2 class="title"> Conflict of interest statement </h2> <div class="statement"> <p xmlns:xlink="http://www.w3.org/1999/xlink" xmlns:mml="http://www.w3.org/1998/Math/MathML" xmlns:p1="http://pubmed.gov/pub-one">Prof Phil Quirke has research funding with Roche, GeneFirst and Amgen, previous research funding from Halio, consultancy with Nordlai‐Adlyte and advisory boards with Merck, Amgen and Roche. Prof Manuel Salto‐Tellez is a senior scientific advisor to Philips Computational Pathology and Sonrai Analytics, and has received honoraria from Roche, AstraZeneca, Merck and GSK. These declarations of interest have no relationship with the submitted publication. All other authors declare no competing interests.</p> </div> </div> <div class="figures" id="figures"> <h2 class="title"> Figures </h2> <div class="figures-list" id="slides-container" itemscope itemtype="http://schema.org/ImageGallery"> <figure class="figure-item " itemscope itemtype="http://schema.org/ImageObject" itemprop="associatedMedia" data-slide-index="0" data-label-slug="fig-1"> <a class="figure-link" href="https://cdn.ncbi.nlm.nih.gov/pmc/blobs/a4a9/7530793/26552dce20d4/MOL2-14-2384-g001.jpg" itemprop="contentUrl" aria-describedby="figure-caption-0" role="button" data-image-width="709" data-image-height="708" data-image-alt="Fig. 1" data-pmc-id="PMC7530793" data-figure-id="mol212764-fig-0001"> <img class="figure-thumb" itemprop="thumbnail" id="article-image-0" src="https://cdn.ncbi.nlm.nih.gov/pmc/blobs/a4a9/7530793/45d3345c6a43/MOL2-14-2384-g001.gif" alt="Fig. 1" /> </a> <meta itemprop="width" itemtype="http://schema.org/ImageObject" content="709"> <meta itemprop="height" itemtype="http://schema.org/ImageObject" content="708"> <div class="figure-caption figure-caption-text" itemtype="http://schema.org/ImageObject" itemprop="caption" aria-hidden="true"> <div class="caption-wrap"> <strong class="figure-label"> <p> Fig. 1 </p> </strong> <div class="figure-caption-contents"><p> Epitope stability as a function… </p></div> </div> </div> <div class="figure-caption-medium figure-caption-text" aria-hidden="true"> <strong class="figure-label"> <p> Fig. 1 </p> </strong> <div class="figure-caption-contents"><p> Epitope stability as a function of antibody positioning in MP1. Line graphs depicting… </p></div> </div> <figcaption id="figure-caption-0" class="figure-caption-full figure-caption-text" itemtype="http://schema.org/ImageObject" itemprop="description"> <strong class="figure-label"> Fig. 1 </strong> <div class="figure-caption-contents">Epitope stability as a function of antibody positioning in MP1. Line graphs depicting how biomarker detection in low (left) and high (right) immune expressing CRC tissue changes according to the number of HIER cycles preceding antibody application, that is the position of an antibody in a multiplex. Fifteen chromogenic singleplexes were performed on each of the two CRC cases (<i>n</i> = 30). The corresponding images are shown below each graph: position 1 (left), position 3 (middle) and position 5 (right). Images were scanned at 40× magnification and are displayed at 20× magnification (scale bar = 50 µm).</div> </figcaption> </figure> <figure class="figure-item " itemscope itemtype="http://schema.org/ImageObject" itemprop="associatedMedia" data-slide-index="1" data-label-slug="fig-2"> <a class="figure-link" href="https://cdn.ncbi.nlm.nih.gov/pmc/blobs/a4a9/7530793/5bbe4b540d5e/MOL2-14-2384-g002.jpg" itemprop="contentUrl" aria-describedby="figure-caption-1" role="button" data-image-width="709" data-image-height="897" data-image-alt="Fig. 2" data-pmc-id="PMC7530793" data-figure-id="mol212764-fig-0002"> <img class="figure-thumb" itemprop="thumbnail" id="article-image-1" src="https://cdn.ncbi.nlm.nih.gov/pmc/blobs/a4a9/7530793/ec5b8522e364/MOL2-14-2384-g002.gif" alt="Fig. 2" /> </a> <meta itemprop="width" itemtype="http://schema.org/ImageObject" content="709"> <meta itemprop="height" itemtype="http://schema.org/ImageObject" content="897"> <div class="figure-caption figure-caption-text" itemtype="http://schema.org/ImageObject" itemprop="caption" aria-hidden="true"> <div class="caption-wrap"> <strong class="figure-label"> <p> Fig. 2 </p> </strong> <div class="figure-caption-contents"><p> Fluorescent singleplex development and comparison… </p></div> </div> </div> <div class="figure-caption-medium figure-caption-text" aria-hidden="true"> <strong class="figure-label"> <p> Fig. 2 </p> </strong> <div class="figure-caption-contents"><p> Fluorescent singleplex development and comparison to chromogenic singleplex. (A) MP1 Opal‐antibody pairings based… </p></div> </div> <figcaption id="figure-caption-1" class="figure-caption-full figure-caption-text" itemtype="http://schema.org/ImageObject" itemprop="description"> <strong class="figure-label"> Fig. 2 </strong> <div class="figure-caption-contents">Fluorescent singleplex development and comparison to chromogenic singleplex. (A) MP1 Opal‐antibody pairings based on the position, colocalisation and expected abundance of the biomarkers (columns 1–5). Columns 6–8 present the spectral properties of the fluorophores and the last column the fluorophores that qualify for MOTiF scanning. (B) Fluorescent singleplexes (<i>n</i> = 10) were compared to the chromogenic singleplexes (<i>n</i> = 10) previously performed on the same low and high immune expressing CRC tissue. Bar graph demonstrating the percentage of cell positivity for each biomarker in low and high immune expressing CRC tissue when stained with DAB (brown) versus Opal (multicolour) reagents. Statistical significance was determined by the Mann–Whitney <i>U</i>‐test. Difference of > 10% in cell positivity in four cases, marked with an asterisk. (C) ROI of two of these cases is displayed. For each case, the chromogenic stain is seen on the left and fluorescent stain on the right, with the original image above and with cell detection applied below. The CD3 stains are viewed at 20× magnification (scale bar = 50 µm), and the CK stains are seen at 10× magnification (scale bar = 100 µm). In the chromogenic CD3 stain, there are more positive cells (in brown) than there are in the fluorescent CD3 stain (in yellow), which is reflective of the different immune expression profiles that exist between nonserial sections. In the chromogenic CK stain, some DAB‐positive cells are not detected by <sc>qupath</sc> as being a negative cell (blue) nor a positive cell (red). However, all cells are identified in the fluorescent CK stain as being either negative (grey) or positive (green).</div> </figcaption> </figure> <figure class="figure-item " itemscope itemtype="http://schema.org/ImageObject" itemprop="associatedMedia" data-slide-index="2" data-label-slug="fig-3"> <a class="figure-link" href="https://cdn.ncbi.nlm.nih.gov/pmc/blobs/a4a9/7530793/889de5d1afba/MOL2-14-2384-g003.jpg" itemprop="contentUrl" aria-describedby="figure-caption-2" role="button" data-image-width="709" data-image-height="822" data-image-alt="Fig. 3" data-pmc-id="PMC7530793" data-figure-id="mol212764-fig-0003"> <img class="figure-thumb" itemprop="thumbnail" id="article-image-2" src="https://cdn.ncbi.nlm.nih.gov/pmc/blobs/a4a9/7530793/b7623811bfab/MOL2-14-2384-g003.gif" alt="Fig. 3" /> </a> <meta itemprop="width" itemtype="http://schema.org/ImageObject" content="709"> <meta itemprop="height" itemtype="http://schema.org/ImageObject" content="822"> <div class="figure-caption figure-caption-text" itemtype="http://schema.org/ImageObject" itemprop="caption" aria-hidden="true"> <div class="caption-wrap"> <strong class="figure-label"> <p> Fig. 3 </p> </strong> <div class="figure-caption-contents"><p> Overview of MP1 optimisation. (A)… </p></div> </div> </div> <div class="figure-caption-medium figure-caption-text" aria-hidden="true"> <strong class="figure-label"> <p> Fig. 3 </p> </strong> <div class="figure-caption-contents"><p> Overview of MP1 optimisation. (A) Example images viewed in <sc> inform </sc> as simulated DAB… </p></div> </div> <figcaption id="figure-caption-2" class="figure-caption-full figure-caption-text" itemtype="http://schema.org/ImageObject" itemprop="description"> <strong class="figure-label"> Fig. 3 </strong> <div class="figure-caption-contents">Overview of MP1 optimisation. (A) Example images viewed in <sc>inform</sc> as simulated DAB IHC images (‘pseudo‐DAB’) to show the bleed through issues encountered during MP1 protocol optimisation, in CRC tissue (left) and tonsil tissue (right). Images are displayed at 10× magnification (scale bar = 100 µm) and magnified fields of view (in the black boxes) at 20× magnification (scale bar = 50 µm). Top row: CD20 crosstalk in the CD8 channel. Bottom row: CK crosstalk in the CD8 and CD4 channels. The CK crossover into CD8 is more evident since CD8 was positioned directly after CK in this early MP1 protocol (staining order CD20 > CD3 > CK > CD8 > CD4). (B) Example images of a CRC core stained with a more developed MP1 protocol, following resolution of the bleed through issues. Magnified images of the same core (within the black box) highlight the clean staining obtained for each marker. Images are viewed at 10× magnification (scale bar = 200 µm) and magnified fields of view at 20× magnification (scale bar = 100 µm). All signal intensity counts are within the acceptable range of 20–25 except for CD20 (43.6), but this was resolved in the final protocol. (C) The optimised MP1 protocol with the spectrum of the MOTiF Opals used below. No overlap is seen between the emission peaks. Obtaining the final optimised MP1 protocol required the development of nine protocols and the use of <i>n</i> = 40 tissue sections.</div> </figcaption> </figure> <figure class="figure-item " itemscope itemtype="http://schema.org/ImageObject" itemprop="associatedMedia" data-slide-index="3" data-label-slug="fig-4"> <a class="figure-link" href="https://cdn.ncbi.nlm.nih.gov/pmc/blobs/a4a9/7530793/5e0116644f52/MOL2-14-2384-g004.jpg" itemprop="contentUrl" aria-describedby="figure-caption-3" role="button" data-image-width="709" data-image-height="510" data-image-alt="Fig. 4" data-pmc-id="PMC7530793" data-figure-id="mol212764-fig-0004"> <img class="figure-thumb" itemprop="thumbnail" id="article-image-3" src="https://cdn.ncbi.nlm.nih.gov/pmc/blobs/a4a9/7530793/19e4a552fa8d/MOL2-14-2384-g004.gif" alt="Fig. 4" /> </a> <meta itemprop="width" itemtype="http://schema.org/ImageObject" content="709"> <meta itemprop="height" itemtype="http://schema.org/ImageObject" content="510"> <div class="figure-caption figure-caption-text" itemtype="http://schema.org/ImageObject" itemprop="caption" aria-hidden="true"> <div class="caption-wrap"> <strong class="figure-label"> <p> Fig. 4 </p> </strong> <div class="figure-caption-contents"><p> Factors considered for DIA. (A)… </p></div> </div> </div> <div class="figure-caption-medium figure-caption-text" aria-hidden="true"> <strong class="figure-label"> <p> Fig. 4 </p> </strong> <div class="figure-caption-contents"><p> Factors considered for DIA. (A) A full‐face CRC section was scanned at 20×… </p></div> </div> <figcaption id="figure-caption-3" class="figure-caption-full figure-caption-text" itemtype="http://schema.org/ImageObject" itemprop="description"> <strong class="figure-label"> Fig. 4 </strong> <div class="figure-caption-contents">Factors considered for DIA. (A) A full‐face CRC section was scanned at 20× and 40× magnification. The same region, viewed at 40× magnification (scale bar = 20 µm), was selected on each scan. The only notable difference is the higher resolution of the 40× scan. (B) Full‐face CRC sections stained with MP1 (<i>n</i> = 5) were scanned using individual and batch exposure times. The scans of one of these samples are shown here at 1.5× magnification (scale bar = 500 µm). Spearman's rank correlation determined that no difference exists in their staining intensities. (C) Two serial full‐face CRC sections were stained, one for MP1 (top row) and one for MP2 (bottom row). The difference in their staining intensities is seen in the left column: CK (green) and CD4 (cyan), which are present in both panels, are more intense in MP2 than in MP1. This originates from the difference in their paraffin section thickness, seen in the right column via the autofluorescence channel: Autofluorescence in MP1 is greater than in MP2 on account of the MP1 section being thicker than the MP2 section. Images are viewed at 5× magnification (scale bar = 250 µm). (D) A colonic core before (top row) and after (bottom row) spectral unmixing in <sc>inform</sc>. The epithelium is expected to stain green for CK (Opal 480) and not yellow for CD3 (Opal 520). Other MP1 markers seen here are CD20 in red (Opal 570), CD4 in cyan (Opal 620) and CD8 in magenta (Opal 690). Images are viewed at 10× magnification (scale bar = 100 µm).</div> </figcaption> </figure> <figure class="figure-item " itemscope itemtype="http://schema.org/ImageObject" itemprop="associatedMedia" data-slide-index="4" data-label-slug="fig-5"> <a class="figure-link" href="https://cdn.ncbi.nlm.nih.gov/pmc/blobs/a4a9/7530793/a591c53b319e/MOL2-14-2384-g005.jpg" itemprop="contentUrl" aria-describedby="figure-caption-4" role="button" data-image-width="709" data-image-height="526" data-image-alt="Fig. 5" data-pmc-id="PMC7530793" data-figure-id="mol212764-fig-0005"> <img class="figure-thumb" itemprop="thumbnail" id="article-image-4" src="https://cdn.ncbi.nlm.nih.gov/pmc/blobs/a4a9/7530793/63b040fc7e9e/MOL2-14-2384-g005.gif" alt="Fig. 5" /> </a> <meta itemprop="width" itemtype="http://schema.org/ImageObject" content="709"> <meta itemprop="height" itemtype="http://schema.org/ImageObject" content="526"> <div class="figure-caption figure-caption-text" itemtype="http://schema.org/ImageObject" itemprop="caption" aria-hidden="true"> <div class="caption-wrap"> <strong class="figure-label"> <p> Fig. 5 </p> </strong> <div class="figure-caption-contents"><p> Overview of mIF validation. (A)… </p></div> </div> </div> <div class="figure-caption-medium figure-caption-text" aria-hidden="true"> <strong class="figure-label"> <p> Fig. 5 </p> </strong> <div class="figure-caption-contents"><p> Overview of mIF validation. (A) Nine consecutive sections were cut from a TMA… </p></div> </div> <figcaption id="figure-caption-4" class="figure-caption-full figure-caption-text" itemtype="http://schema.org/ImageObject" itemprop="description"> <strong class="figure-label"> Fig. 5 </strong> <div class="figure-caption-contents">Overview of mIF validation. (A) Nine consecutive sections were cut from a TMA block, of which seven were stained for single antibody DAB IHC and two for mIF. Section 4 stained for MP1 and section 5 stained for MP2. Three right images depict overviews of the TMA layout, a chromogenic TMA and a multiplexed TMA, with tissue annotation and cell detection applied in the latter two. The TMA consists of six normal human tissue types, plus sheep lung tissue for orientation. (B) MP1 validation results. Spearman's rank correlation graphs (top) and Bland–Altman plots (bottom) illustrating the relationship and agreement between DAB detection (from TMAs 1, 2, 3, 6, 7) and singleplex IF detection (from TMA 4), for each biomarker in MP1. The same cores (<i>n</i> = 17) across all the TMAs were used for analysis of the biomarkers. All correlations are strong (<i>r</i> <sub>s</sub> > 0.9, <i>P</i> < 0.0001) except for CD8, and all biases are within the limits of agreement.</div> </figcaption> </figure> <figure class="figure-item " itemscope itemtype="http://schema.org/ImageObject" itemprop="associatedMedia" data-slide-index="5" data-label-slug="fig-6"> <a class="figure-link" href="https://cdn.ncbi.nlm.nih.gov/pmc/blobs/a4a9/7530793/5199047e6112/MOL2-14-2384-g006.jpg" itemprop="contentUrl" aria-describedby="figure-caption-5" role="button" data-image-width="709" data-image-height="487" data-image-alt="Fig. 6" data-pmc-id="PMC7530793" data-figure-id="mol212764-fig-0006"> <img class="figure-thumb" itemprop="thumbnail" id="article-image-5" src="https://cdn.ncbi.nlm.nih.gov/pmc/blobs/a4a9/7530793/8e25bfa23e78/MOL2-14-2384-g006.gif" alt="Fig. 6" /> </a> <meta itemprop="width" itemtype="http://schema.org/ImageObject" content="709"> <meta itemprop="height" itemtype="http://schema.org/ImageObject" content="487"> <div class="figure-caption figure-caption-text" itemtype="http://schema.org/ImageObject" itemprop="caption" aria-hidden="true"> <div class="caption-wrap"> <strong class="figure-label"> <p> Fig. 6 </p> </strong> <div class="figure-caption-contents"><p> Importance of the detection order… </p></div> </div> </div> <div class="figure-caption-medium figure-caption-text" aria-hidden="true"> <strong class="figure-label"> <p> Fig. 6 </p> </strong> <div class="figure-caption-contents"><p> Importance of the detection order in digital assessment of MP1. (A) Example images… </p></div> </div> <figcaption id="figure-caption-5" class="figure-caption-full figure-caption-text" itemtype="http://schema.org/ImageObject" itemprop="description"> <strong class="figure-label"> Fig. 6 </strong> <div class="figure-caption-contents">Importance of the detection order in digital assessment of MP1. (A) Example images of tonsil core 3 stained with MP1 protocol, seen at 10× magnification (scale bar = 100 µm). Top row shows the original stains: a composite image followed by an image for each individual marker. Middle row illustrates the same composite image to which six different detection orders were applied. From left to right, the accuracy of the detection orders increases. From one order to the next, the biomarker that has been modified is underlined. Bottom row presents the phenotypes identified by each detection order as a percentage of the classified cells. Pie chart colours are in accordance with cell classification colours above. Unclassified cells are indicated in grey. The final detection order (CK > CD4 > CD3 > CD20 > CD8) best represents the original stain visually. (B) Scatter graphs showing the correlation between each multiplex detection order and the singleplex detection of MP1 TMA 4, for each biomarker. The same cores (<i>n</i> = 17) of MP1 TMA 4 were used for analysis of the biomarkers. Statistical significance was measured by Spearman's rank correlation coefficient. The final detection order (purple) best matches the singleplex results quantitatively (<i>r</i> <sub>s</sub> > 0.9, <i>P</i> < 0.0001 for all the biomarkers).</div> </figcaption> </figure> <figure class="figure-item tail" itemscope itemtype="http://schema.org/ImageObject" itemprop="associatedMedia" data-slide-index="6" data-label-slug="fig-7"> <a class="figure-link" href="https://cdn.ncbi.nlm.nih.gov/pmc/blobs/a4a9/7530793/ec076d7e38dc/MOL2-14-2384-g007.jpg" itemprop="contentUrl" aria-describedby="figure-caption-6" role="button" data-image-width="709" data-image-height="408" data-image-alt="Fig. 7" data-pmc-id="PMC7530793" data-figure-id="mol212764-fig-0007"> <img class="figure-thumb" itemprop="thumbnail" id="article-image-6" src="https://cdn.ncbi.nlm.nih.gov/pmc/blobs/a4a9/7530793/ed1b35d43bce/MOL2-14-2384-g007.gif" alt="Fig. 7" /> </a> <meta itemprop="width" itemtype="http://schema.org/ImageObject" content="709"> <meta itemprop="height" itemtype="http://schema.org/ImageObject" content="408"> <div class="figure-caption figure-caption-text" itemtype="http://schema.org/ImageObject" itemprop="caption" aria-hidden="true"> <div class="caption-wrap"> <strong class="figure-label"> <p> Fig. 7 </p> </strong> <div class="figure-caption-contents"><p> Cell classification in MP1 using… </p></div> </div> </div> <div class="figure-caption-medium figure-caption-text" aria-hidden="true"> <strong class="figure-label"> <p> Fig. 7 </p> </strong> <div class="figure-caption-contents"><p> Cell classification in MP1 using a bespoke script. (A) Decision tree visually representing… </p></div> </div> <figcaption id="figure-caption-6" class="figure-caption-full figure-caption-text" itemtype="http://schema.org/ImageObject" itemprop="description"> <strong class="figure-label"> Fig. 7 </strong> <div class="figure-caption-contents">Cell classification in MP1 using a bespoke script. (A) Decision tree visually representing the coding instructions in the script. Unexpected classes are indicated within the black boxes. The original script did not contain these unexpected phenotypes, as opposed to the updated script. TIL, tumour‐infiltrating lymphocyte. (B) Differences in cell classification were only observed in the lymphoid tissue cores (lymph node, spleen and tonsil) (<i>n</i> = 9) when comparing the original script (script 1) and the updated script (script 2). Only a percentage of CD4<sup>+</sup> cells that were found in hot immune regions are reclassified by script 2 as CD4<sup>+</sup>/CD8<sup>+</sup> cells. (C) Example displaying the reclassification of these CD4<sup>+</sup> cells (cyan) to CD4<sup>+</sup>/CD8<sup>+</sup> cells (orange). Colours are in accordance with the colour‐coded phenotypes of the decision tree. Unclassified cells are indicated in grey. The core shown is of lymph node 3, seen here at 10× magnification (scale bar = 100 µm).</div> </figcaption> </figure> </div> <div class="see-all actions-bar"> <button class="show-more" aria-controls="slides-container" data-ga-category="figure" data-ga-action="show_all"> All figures (7) </button> </div>  <div class="pswp figures-dialog" id="figures-dialog" tabindex="-1" role="dialog" aria-hidden="true" data-image-gallery-uid="article-figures" aria-label="Figures Dialog">  <div class="pswp__bg"></div>  <div class="pswp__scroll-wrap">  <div class="pswp__container"> <div class="pswp__item"></div> <div class="pswp__item"></div> <div class="pswp__item"></div> </div>  <div class="pswp__ui pswp__ui--hidden"> <div class="pswp__top-bar">  <div class="pswp__counter"></div> <button class="pswp__button pswp__button--close" title="Close (Esc)"></button> <button class="pswp__button pswp__button--share" title="Share"></button> <button class="pswp__button pswp__button--fs" title="Toggle fullscreen"></button> <button class="pswp__button pswp__button--zoom" title="Zoom in/out"></button>   <div class="pswp__preloader"> <div class="pswp__preloader__icn"> <div class="pswp__preloader__cut"> <div class="pswp__preloader__donut"></div> </div> </div> </div> </div> <div class="pswp__share-modal pswp__share-modal--hidden pswp__single-tap"> <div class="pswp__share-tooltip"></div> </div> <button class="pswp__button pswp__button--arrow--left" title="Previous (arrow left)"> </button> <button class="pswp__button pswp__button--arrow--right" title="Next (arrow right)"> </button> <div class="pswp__caption"> <div class="pswp__caption__center"></div> </div> </div> </div> </div> <div class="figure-dialog-caption"> <div class="figure-caption-link-container"> <a class="figure-caption-link" href="#">See this image and copyright information in PMC</a> </div> <div class="figure-caption-center" data-expand-title="click to show full caption" data-collapse-title="click to hide full caption"> </div> <button class="figure-caption-toggle-btn" aria-label="show full caption" data-expand-text="show full caption" data-collapse-text="hide full caption">  </button> </div> </div> <div class="similar-articles" id="similar"> <h2 class="title"> Similar articles </h2> <ul class="articles-list" id="similar-articles-list"> <li class="full-docsum"> <div class="docsum-content" style="display:inline;"> <a class="docsum-title" href="/35664673/" ref="article_id=35664673&linksrc=similar_articles_link&ordinalpos=1" data-ga-category="similar_article" data-ga-action="35664673" data-ga-label=""> Validation of an Accurate Automated Multiplex Immunofluorescence Method for Immuno-Profiling Melanoma. </a> <div class="docsum-citation full-citation"> <span class="docsum-authors full-authors">Yaseen Z, Gide TN, Conway JW, Potter AJ, Quek C, Hong AM, Long GV, Scolyer RA, Wilmott JS.</span> <span class="docsum-authors short-authors">Yaseen Z, et al.</span> <span class="docsum-journal-citation full-journal-citation">Front Mol Biosci. 2022 May 19;9:810858. doi: 10.3389/fmolb.2022.810858. eCollection 2022.</span> <span class="docsum-journal-citation short-journal-citation">Front Mol Biosci. 2022.</span> <span class="citation-part">PMID: <span class="docsum-pmid">35664673</span></span> <span class="free-resources spaced-citation-item citation-part">Free PMC article.</span> </div> </div> </li> <li class="full-docsum"> <div class="docsum-content" style="display:inline;"> <a class="docsum-title" href="/31783023/" ref="article_id=31783023&linksrc=similar_articles_link&ordinalpos=2" data-ga-category="similar_article" data-ga-action="31783023" data-ga-label=""> Multiplex immunofluorescence staining and image analysis assay for diffuse large B cell lymphoma. </a> <div class="docsum-citation full-citation"> <span class="docsum-authors full-authors">Lee CW, Ren YJ, Marella M, Wang M, Hartke J, Couto SS.</span> <span class="docsum-authors short-authors">Lee CW, et al.</span> <span class="docsum-journal-citation full-journal-citation">J Immunol Methods. 2020 Mar;478:112714. doi: 10.1016/j.jim.2019.112714. 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A robust multiplex immunofluorescence and digital pathology workflow for the characterisation of the tumour immune microenvironment - PubMed