{"id":38100,"date":"2025-10-20T10:00:47","date_gmt":"2025-10-20T08:00:47","guid":{"rendered":"https:\/\/immunostep.com\/?p=38100"},"modified":"2025-10-17T10:37:49","modified_gmt":"2025-10-17T08:37:49","slug":"optimisation-of-multiplex-assays-for-cell-therapies","status":"publish","type":"post","link":"https:\/\/immunostep.com\/es\/2025\/10\/20\/optimisation-of-multiplex-assays-for-cell-therapies\/","title":{"rendered":"Optimisation of Multiplex Assays for Cell Therapies"},"content":{"rendered":"<p>The growing field of <a href=\"https:\/\/immunostep.com\/en\/applications\/car-t-cell-analysis\/\"><strong data-start=\"390\" data-end=\"417\">advanced cell therapies,<\/strong><\/a> including CAR-T and modified NK cells, demands comprehensive phenotypic and functional characterisation. In this context,<a href=\"https:\/\/immunostep.com\/en\/products\/multiplex-assays\/\"> multiplex assays <\/a>have become essential tools for obtaining <strong data-start=\"734\" data-end=\"759\">multidimensional data<\/strong> in a single analysis, enabling simultaneous assessment of functional, activation, and viability parameters.<\/p>\n<p>However, optimising these assays requires a robust strategy that correctly combines <strong data-start=\"955\" data-end=\"972\">marker panels<\/strong>, <strong data-start=\"974\" data-end=\"995\">calibration beads<\/strong>, and <strong data-start=\"1001\" data-end=\"1028\">compensation parameters<\/strong> to ensure both <strong data-start=\"1044\" data-end=\"1063\">reproducibility<\/strong> and <strong data-start=\"1068\" data-end=\"1083\">sensitivity<\/strong>.<\/p>\n<h3 data-start=\"1093\" data-end=\"1152\"><strong data-start=\"1096\" data-end=\"1152\">1. The Importance of Rational Multiplex Panel Design<\/strong><\/h3>\n<p data-start=\"1154\" data-end=\"1329\">The foundation of any effective multiplex assay lies in a <strong data-start=\"1212\" data-end=\"1244\">rational marker panel design<\/strong>. In the context of cell therapy development, the goal is to simultaneously assess:<\/p>\n<ul data-start=\"1331\" data-end=\"1574\">\n<li data-start=\"1331\" data-end=\"1393\">\n<p data-start=\"1333\" data-end=\"1393\">Expression of <strong data-start=\"1347\" data-end=\"1369\">activation markers<\/strong> (<em data-start=\"1371\" data-end=\"1390\">CD69, CD25, CD137<\/em>)<\/p>\n<\/li>\n<li data-start=\"1394\" data-end=\"1457\">\n<p data-start=\"1396\" data-end=\"1457\">Presence of <strong data-start=\"1408\" data-end=\"1432\">exhaustion molecules<\/strong> (<em data-start=\"1434\" data-end=\"1454\">PD-1, TIM-3, LAG-3<\/em>)<\/p>\n<\/li>\n<li data-start=\"1458\" data-end=\"1526\">\n<p data-start=\"1460\" data-end=\"1526\">Production of <strong data-start=\"1474\" data-end=\"1501\">intracellular cytokines<\/strong> (<em data-start=\"1503\" data-end=\"1523\">IFN-\u03b3, TNF-\u03b1, IL-2<\/em>)<\/p>\n<\/li>\n<li data-start=\"1527\" data-end=\"1574\">\n<p data-start=\"1529\" data-end=\"1574\">Cell <strong data-start=\"1534\" data-end=\"1565\">viability and proliferation<\/strong> status<\/p>\n<\/li>\n<\/ul>\n<p data-start=\"1576\" data-end=\"1949\">To achieve optimal resolution, fluorochromes with <strong data-start=\"1626\" data-end=\"1654\">minimal spectral overlap<\/strong> should be selected according to the laser configuration and dynamic range of the cytometer used. Combining <strong data-start=\"1762\" data-end=\"1786\">bright fluorochromes<\/strong> (such as PE or APC) for low-expression markers and <strong data-start=\"1838\" data-end=\"1862\">stable fluorochromes<\/strong> (FITC, PerCP-Cy5.5) for highly expressed markers enhances overall assay performance.<\/p>\n<h3 data-start=\"1956\" data-end=\"2028\"><strong data-start=\"1959\" data-end=\"2028\">2. Calibration Beads: The Key to Inter-Experimental Comparability<\/strong><\/h3>\n<p data-start=\"2030\" data-end=\"2273\"><a class=\"decorated-link cursor-pointer\" href=\"https:\/\/immunostep.com\/en\/products\/calibration-beads\/\" target=\"_new\" rel=\"noopener\" data-start=\"2030\" data-end=\"2108\"><strong data-start=\"2031\" data-end=\"2052\">Calibration beads\u00a0<\/strong><\/a>are indispensable for converting fluorescence signals into <strong data-start=\"2168\" data-end=\"2203\">standardised quantitative units<\/strong>, ensuring comparability between experiments, days, and instruments.<\/p>\n<p data-start=\"2275\" data-end=\"2347\">Their use in multiplex assay optimisation serves two primary purposes:<\/p>\n<ol data-start=\"2348\" data-end=\"2610\">\n<li data-start=\"2348\" data-end=\"2468\">\n<p data-start=\"2351\" data-end=\"2468\"><strong data-start=\"2351\" data-end=\"2399\">Adjusting detector linearity and sensitivity<\/strong>, by establishing calibration curves for each fluorescence channel.<\/p>\n<\/li>\n<li data-start=\"2469\" data-end=\"2610\">\n<p data-start=\"2472\" data-end=\"2610\"><strong data-start=\"2472\" data-end=\"2516\">Controlling inter-instrument variability<\/strong>, ensuring that shifts in gain or optical alignment do not affect biological interpretation.<\/p>\n<\/li>\n<\/ol>\n<p data-start=\"2612\" data-end=\"2816\">When <strong data-start=\"2617\" data-end=\"2634\">control beads<\/strong> and <strong data-start=\"2639\" data-end=\"2661\">compensation beads<\/strong> are properly combined, the system achieves complete harmonisation, facilitating reliable comparison of multiple experiments under consistent conditions.<\/p>\n<h3 data-start=\"2823\" data-end=\"2908\"><strong data-start=\"2826\" data-end=\"2908\">3. Combining Markers and Calibration: Towards a Truly Multidimensional Reading<\/strong><\/h3>\n<p data-start=\"2910\" data-end=\"3115\">The real power of multiplex assays is realised when <strong data-start=\"2962\" data-end=\"2984\">functional markers<\/strong>, <strong data-start=\"2986\" data-end=\"3007\">calibration beads<\/strong>, and <strong data-start=\"3013\" data-end=\"3043\">advanced gating strategies<\/strong> are integrated to provide a comprehensive view of cellular behaviour.<\/p>\n<p data-start=\"3117\" data-end=\"3531\">By using multidimensional analysis software such as <a href=\"https:\/\/www.flowjo.com\/\">FlowJo<\/a>, <a href=\"https:\/\/cytobank.org\/\">Cytobank<\/a>, or <a href=\"https:\/\/www.sonybiotechnology.com\/global\/products\/software\/spectro-flo\/\">SpectroFlo<\/a>, researchers can integrate date on mean fluorescence intensity (MFI), subpopulation frequency, and functional correlations to generate unique immunophenotypic signatures for each treatment.<\/p>\n<p data-start=\"3533\" data-end=\"3594\">A well-optimised multiplex assay can simultaneously reveal:<\/p>\n<ul data-start=\"3595\" data-end=\"3780\">\n<li data-start=\"3595\" data-end=\"3662\">\n<p data-start=\"3597\" data-end=\"3662\">Changes in <strong data-start=\"3608\" data-end=\"3645\">activation or exhaustion profiles<\/strong> of CAR-T cells<\/p>\n<\/li>\n<li data-start=\"3663\" data-end=\"3713\">\n<p data-start=\"3665\" data-end=\"3713\"><strong data-start=\"3665\" data-end=\"3687\">Cytotoxic capacity<\/strong> against target antigens<\/p>\n<\/li>\n<li data-start=\"3714\" data-end=\"3780\"><strong data-start=\"3716\" data-end=\"3751\">Coordinated cytokine production<\/strong> within a single population<\/li>\n<\/ul>\n<p data-start=\"3782\" data-end=\"3902\">All this can be achieved in a single experimental acquisition, reducing both sample consumption and analysis time.<\/p>\n<h3 data-start=\"3909\" data-end=\"3963\"><strong data-start=\"3912\" data-end=\"3963\">4. Best Practices for Experimental Optimisation<\/strong><\/h3>\n<p data-start=\"3965\" data-end=\"4065\">To ensure the highest levels of reproducibility and sensitivity in multiplex assays, we recommend:<\/p>\n<ul data-start=\"4067\" data-end=\"4406\">\n<li data-start=\"4067\" data-end=\"4153\">\n<p data-start=\"4069\" data-end=\"4153\"><strong data-start=\"4069\" data-end=\"4095\">Validate each antibody<\/strong> individually before integrating it into the full panel.<\/p>\n<\/li>\n<li data-start=\"4154\" data-end=\"4226\">\n<p data-start=\"4156\" data-end=\"4226\">Use <strong data-start=\"4160\" data-end=\"4183\">daily control beads<\/strong> to adjust PMT voltage and gain settings.<\/p>\n<\/li>\n<li data-start=\"4227\" data-end=\"4321\">\n<p data-start=\"4229\" data-end=\"4321\">Implement <strong data-start=\"4239\" data-end=\"4271\">FMO (Fluorescence Minus One)<\/strong> controls to correctly define gating boundaries.<\/p>\n<\/li>\n<li data-start=\"4322\" data-end=\"4406\">\n<p data-start=\"4324\" data-end=\"4406\">Record all <strong data-start=\"4335\" data-end=\"4358\">instrument settings<\/strong> and <strong data-start=\"4363\" data-end=\"4390\">compensation parameters<\/strong> for each run.<\/p>\n<\/li>\n<\/ul>\n<p data-start=\"4408\" data-end=\"4542\">These practices not only ensure analytical reliability but also enhance traceability in <strong data-start=\"4496\" data-end=\"4503\">GMP<\/strong> or <strong data-start=\"4507\" data-end=\"4539\">quality control environments<\/strong>.<\/p>\n<h3 data-start=\"4549\" data-end=\"4566\"><strong data-start=\"4552\" data-end=\"4566\">Conclusion<\/strong><\/h3>\n<p data-start=\"4568\" data-end=\"5024\">The optimisation of multiplex assays for cell therapies is a critical step towards the <strong data-start=\"4655\" data-end=\"4721\">comprehensive characterisation of advanced cell-based products<\/strong>. The intelligent integration of <strong data-start=\"4754\" data-end=\"4765\">markers<\/strong>, <strong data-start=\"4767\" data-end=\"4784\">fluorochromes<\/strong>, and <strong data-start=\"4790\" data-end=\"4811\">calibration beads<\/strong> provides a truly <strong data-start=\"4829\" data-end=\"4857\">multidimensional insight<\/strong> into cellular states, driving both basic research and <a href=\"https:\/\/immunostep.com\/en\/quality-control-solutions\/\"><strong data-start=\"4913\" data-end=\"4932\">quality control<\/strong> <\/a>inadvances therapy manufacturing.<\/p>\n","protected":false},"excerpt":{"rendered":"<p>The growing field of advanced cell therapies, including CAR-T and modified NK cells, demands comprehensive phenotypic and functional characterisation. In this context, multiplex assays have become essential tools for obtaining multidimensional data in a single analysis, enabling simultaneous assessment of functional, activation, and viability parameters. However, optimising these assays requires a robust strategy that correctly [&hellip;]<\/p>\n","protected":false},"author":225,"featured_media":38101,"comment_status":"closed","ping_status":"closed","sticky":false,"template":"","format":"standard","meta":{"footnotes":"","_links_to":"","_links_to_target":""},"categories":[2033],"tags":[2031,2349,2154,2287,2117,2159,2215],"class_list":["post-38100","post","type-post","status-publish","format-standard","has-post-thumbnail","hentry","category-immunology","tag-car-t","tag-celltherapies","tag-immune-cells","tag-immune-resistance","tag-immunology","tag-therapy","tag-treatments"],"yoast_head":"<!-- This site is optimized with the Yoast SEO plugin v23.6 - https:\/\/yoast.com\/wordpress\/plugins\/seo\/ -->\n<title>Optimisation of Multiplex Assays for Cell Therapies | Immunostep Biotech<\/title>\n<meta name=\"description\" content=\"At Immunostep we have been innovating since 2001 to develop comprehensive products that contribute to improve.\" 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