Video: Gaining New Insights on Cellular Response Using Simple Plex

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Hello and welcome to Teach Me in 10. 

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In this discussion, 

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Dr. Jean Dunne shares valuable insights 

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gleaned from her research 

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measuring cytokines 

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to assess 

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cellular responses 

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in clinical and research settings. 

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And we'll be looking 

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at unlocking cellular response 

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with Simple Plex technology. 

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So without further ado, 

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let's get into the episode. 

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Hi Jean, 

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welcome to Teach Me in 10. 

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So as we only have 10 minutes, 

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we best get started. 

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And my first question for you has to be 

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can you briefly outline your research 

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focus: understanding 

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cellular response 

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and its implications 

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for disease-modifying drugs? 

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Hello, Lucy. 

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Yes, absolutely. 

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So we're interested 

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in the cellular response 

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in common viruses 

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and how they relate to diseases 

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such as multiple sclerosis, 

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which is a research focus of ours. 

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So currently, research 

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into viral association 

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in multiple 

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sclerosis has focused 

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on the Epstein–Barr virus, or EBV. 

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And this is a very common virus 

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which most people easily deal 

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with in childhood or adolescence. 

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It's called infectious mononucleosis 

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or kissing disease. 

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Okay, 

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so what happens 

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is that the virus is controlled 

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by immune cells 

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which patrol and deal with any exposures. 

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And a very large study, 

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so I think there were 10 million 

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samples included 

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from US 

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military personnel, 

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identified new adult infection 

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with Epstein–Barr 

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(EBV) 

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as a prerequisite 

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for subsequent development of MS. 

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So that links MS 

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causally 

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with Epstein–Barr disease. 

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And that study measured 

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antibody responses. 

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Now, we've carried out 

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a subsequent study that's been happily 

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published this year, in 2024, 

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and our study looked 

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at the cellular responses 

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to Epstein–Barr in patients with MS. 

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So, we looked at various 

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therapies 

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that are used to treat patients with MS 

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and we looked at the impact 

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of these therapies on the cytokine 

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responses of immune cells 

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that respond to Epstein–Barr. 

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The piece of Epstein–Barr 

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that we focused on is called 

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EBNA-1 

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or Epstein–Barr 

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nuclear-associated antigen 1. Perfect. 

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So then from that, 

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how have you developed 

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the in-house cytokine release 

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immunoassays? And how are these 

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aiding clinical researchers 

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in studying inflammatory cytokine response? 

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So we based the 

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assays on 

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tests that we routinely use 

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for latent TB. 

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It's called the interferon 

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gamma release assay. 

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And these assays 

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are commercially supplied in a kit 

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with TB antigens. Blood is added, 

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patient blood, it's incubated 

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overnight 

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and an ELISA is 

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used to measure interferon gamma. 

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So the in-house assays that we develop 

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use viral 

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peptides 

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or whole recombinant antigens. 

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Now, they can be from any source 

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but the ones obviously that we used in 

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the MS 

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studies are EBNA-1 

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from Epstein–Barr. 

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So these are added to whole blood 

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and cultured overnight 

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and then the 

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cytokines produced by the blood cells 

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are then measured in the supernatant. 

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And this is a very flexible assay system 

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and Ella provides 

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the great advantage of multiplex 

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cytokine measurement on a single sample. 

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I see. 

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So what were the main challenges 

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in creating these diagnostic tests 

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and what kind of key 

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lessons were learned? 

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Well, for 

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us, the main 

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challenge is optimizing the assay. 

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So that means establishing 

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the amount of antigen 

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that's required to elicit a response 

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and then, after that, deciding 

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on the appropriate cytokines to measure. 

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So it's important 

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to be able to identify a responder group 

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to establish the assay because 

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you have to have a positive 

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and perhaps a negative control. 

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And these can be healthy controls 

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or they may be part of your disease 

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cohort, as in the MS study. 

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So a key lesson for us 

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was to conduct a pilot study, 

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including reasonable numbers 

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of healthy controls, and 

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within the patient cohort 

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to identify subgroups. 

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For example, in the MS study 

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we didn't 

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initially include treatment 

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naive patients, 

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because they were fewer in numbers, 

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but eventually this cohort proved 

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to be essential for comparison. 

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They were our baseline comparator. 

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So another key lesson is to identify 

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the cytokine signature 

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of the cellular response 

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you wish to measure. 

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For T cell responses, 

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we used interferon gamma 

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and IL-2, eventually. 

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But when we conducted pilot studies, 

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we included a number of other  

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T cell-associated 

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and innate-associated cytokines, 

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and in an ideal world, 

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a broad range of cytokines 

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would be measured 

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but cost is a limiting factor, 

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I'm afraid, 

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in reality. Absolutely. 

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So what would you say 

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are the essential capabilities 

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you seek in immunoassay 

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platforms for diagnostic monitoring? 

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Well, reproducibility is essential. 

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We need a low coefficient of variation 

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for inter- and intra-assay comparisons 

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because we need to be able 

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to rely on the results 

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over a period of testing. A wide 

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dynamic range is a great advantage 

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and a wide range of sample types 

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such as plasma, serum, 

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stimulation supernatants 

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and perhaps cerebrospinal fluid. 

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Also, for our purposes, 

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a wide range of cytokines, chemokines 

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or other biomarkers to be available 

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for testing on that platform, 

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because that gives us 

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a great possibility 

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in what we look for. 

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Ease of use, with minimal 

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requirement for operator intervention 

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during testing, is obviously 

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a great advantage 

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and short 

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setup times and short 

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daily and monthly maintenance. 

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Another 

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capability would be 

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low volume of samples 

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because these are often 

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precious samples, 

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and quick time from initial sample 

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testing to results generation, 

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because we're anxious to gather results 

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and obviously good 

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engineering support and training. 

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Absolutely. 

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And then, from that, 

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how do patients benefit 

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from using the Simple Plex 

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platform on the Ella instrument 

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for cytokine measurement? 

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Well, all care is patient-centered 

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and it should be personalized. 

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And on the Ella platform, 

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the ease, 

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reproducibility and speed 

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of measurement of the cytokines 

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makes it possible to upscale 

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and translate these assays 

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which are essentially 

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a research assay, 

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but then they need to be translated 

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into use in the clinical laboratory. 

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So the Ella platform has enabled us 

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to develop 

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novel assays 

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to measure these individual responses. 

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We've been able to look at various 

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cytokine responses 

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in a dynamic system, 

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allowing us to choose the cytokines 

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that best represent the cellular processes 

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we want to measure. 

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And the initial studies 

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pave the way for things 

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like longitudinal studies, 

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looking at response to treatment 

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and possibly 

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predicting changes required in dosage 

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or treatment type. 

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Nice. 

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So what are the strengths 

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and weaknesses of the Ella 

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instrument, 

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particularly regarding its capacity 

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for measuring 

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multiple cytokines 

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on limited samples 

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and its high sensitivity 

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and reproducibility? 

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Well, as you say, 

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there are great advantages in being able 

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to measure 

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a range of cytokines 

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on a small, precious sample, 

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and this is especially true 

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when developing and optimizing assays 

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that we will translate 

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to the diagnostic and monitoring service. 

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So a single-step test 

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for a range of cytokines saves time 

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and human resources 

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and it produces 

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a huge amount of information 

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that we can 

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then analyze 

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to identify the most useful measures, 

270 

00:08:00,878 --> 00:08:02,187 

so the most appropriate 

271 

00:08:02,187 --> 00:08:04,101 

cytokines in our MS study. 

272 

00:08:05,141 --> 00:08:07,458 

So the sensitivity and reproducibility 

273 

00:08:07,458 --> 00:08:09,371 

are also essential in order 

274 

00:08:09,371 --> 00:08:11,486 

to be able to trust results. 

275 

00:08:11,486 --> 00:08:13,568 

You need to be able to trust that 

276 

00:08:13,568 --> 00:08:14,676 

if you test it today 

277 

00:08:14,676 --> 00:08:16,018 

and if you test it in 10 weeks, 

278 

00:08:16,018 --> 00:08:17,395 

it will be the same. 

279 

00:08:17,395 --> 00:08:18,301 

And also, 

280 

00:08:18,301 --> 00:08:19,141 

we need to trust them 

281 

00:08:19,141 --> 00:08:21,323 

because these results will change 

282 

00:08:21,323 --> 00:08:23,471 

as we do longitudinal studies 

283 

00:08:23,471 --> 00:08:25,553 

in each patient, 

284 

00:08:25,553 --> 00:08:27,903 

it may be change in response to disease 

285 

00:08:27,903 --> 00:08:30,891 

development or in response to treatment. 

286 

00:08:31,461 --> 00:08:33,778 

A disadvantage is the in-house 

287 

00:08:33,778 --> 00:08:36,766 

assay should be translatable into an IVD 

288 

00:08:36,766 --> 00:08:38,646 

or CE-marked product 

289 

00:08:38,646 --> 00:08:39,921 

to enable us to use it 

290 

00:08:39,921 --> 00:08:42,204 

in the clinical diagnostics setting. 

291 

00:08:42,204 --> 00:08:43,043 

And this is something 

292 

00:08:43,043 --> 00:08:44,621 

that needs investment 

293 

00:08:44,621 --> 00:08:47,240 

of both time and resources. 

294 

00:08:47,240 --> 00:08:48,146 

I see. 

295 

00:08:48,146 --> 00:08:49,355 

So I'd love 

296 

00:08:49,355 --> 00:08:50,832 

if you could share your experience 

297 

00:08:50,832 --> 00:08:51,806 

in creating a novel 

298 

00:08:51,806 --> 00:08:53,283 

cytokine release assay 

299 

00:08:53,283 --> 00:08:54,122 

and the significance 

300 

00:08:54,122 --> 00:08:54,995 

of using a platform 

301 

00:08:54,995 --> 00:08:56,271 

that offers this fast, 

302 

00:08:56,271 --> 00:08:58,184 

hands-free operation. 

303 

00:08:58,184 --> 00:08:58,822 

Absolutely. 

304 

00:08:58,822 --> 00:09:01,911 

So, most people with multiple sclerosis 

305 

00:09:02,347 --> 00:09:05,201 

have high antibodies to a latent 

306 

00:09:05,201 --> 00:09:07,450 

particle of EBV called EBNA-1. 

307 

00:09:07,450 --> 00:09:08,155 

And, as I said, 

308 

00:09:08,155 --> 00:09:09,934 

previous studies have used this 

309 

00:09:09,934 --> 00:09:11,881 

antibody to demonstrate 

310 

00:09:11,881 --> 00:09:14,131 

Epstein–Barr reactivation. 

311 

00:09:14,131 --> 00:09:14,903 

However, when we looked 

312 

00:09:14,903 --> 00:09:16,145 

at these antibodies, 

313 

00:09:16,145 --> 00:09:16,649 

we showed that 

314 

00:09:16,649 --> 00:09:17,589 

they didn't change 

315 

00:09:17,589 --> 00:09:18,797 

in response to therapies 

316 

00:09:18,797 --> 00:09:20,174 

that are used for MS, 

317 

00:09:20,174 --> 00:09:21,349 

so antibody levels 

318 

00:09:21,349 --> 00:09:22,490 

can't be used to monitor 

319 

00:09:22,490 --> 00:09:25,243 

treatment responses in patients. 

320 

00:09:25,243 --> 00:09:26,451 

In contrast, 

321 

00:09:26,451 --> 00:09:27,492 

when we looked at the 

322 

00:09:27,492 --> 00:09:29,271 

when we developed the cellular assays 

323 

00:09:29,271 --> 00:09:31,219 

and we looked at the cellular responses 

324 

00:09:31,219 --> 00:09:32,226 

to EBNA-1 

325 

00:09:32,226 --> 00:09:34,643 

using interferon gamma and IL-2 

326 

00:09:34,643 --> 00:09:36,657 

multiplexes on the Ella, 

327 

00:09:36,657 --> 00:09:37,396 

we showed that 

328 

00:09:37,396 --> 00:09:39,242 

the majority of new patients 

329 

00:09:39,242 --> 00:09:41,827 

had a cellular response to EBNA-1 

330 

00:09:41,827 --> 00:09:44,479 

and these are the treatment naive cohort, 

331 

00:09:44,479 --> 00:09:46,527 

so they've never been treated. 

332 

00:09:46,527 --> 00:09:49,951 

And this response, this cytokine response 

333 

00:09:49,951 --> 00:09:51,160 

to EBNA-1 

334 

00:09:51,160 --> 00:09:53,208 

was significantly decreased in patients 

335 

00:09:53,208 --> 00:09:55,054 

treated with immunosuppressive 

336 

00:09:55,054 --> 00:09:57,505 

and anti-B cell therapies. 

337 

00:09:57,505 --> 00:09:58,445 

And these therapies 

338 

00:09:58,445 --> 00:10:00,023 

mostly work by suppressing 

339 

00:10:00,023 --> 00:10:01,836 

the inflammatory response or 

340 

00:10:01,836 --> 00:10:03,682 

by eliminating immune cells. 

341 

00:10:03,682 --> 00:10:06,670 

So they're two types of treatment. 

342 

00:10:06,905 --> 00:10:07,879 

And a different 

343 

00:10:07,879 --> 00:10:09,389 

therapy, natalizumab or 

344 

00:10:09,389 --> 00:10:10,833 

Tysabri, works 

345 

00:10:10,833 --> 00:10:12,579 

by preventing the cells 

346 

00:10:12,579 --> 00:10:14,694 

from crossing the blood–brain barrier. 

347 

00:10:14,694 --> 00:10:16,842 

Okay, so in that case, 

348 

00:10:16,842 --> 00:10:18,622 

all of the MS patients were shown 

349 

00:10:18,622 --> 00:10:20,334 

to have a high interferon gamma 

350 

00:10:20,334 --> 00:10:22,919 

and IL-2 response to EBNA-1 

351 

00:10:22,919 --> 00:10:23,792 

and this indicated 

352 

00:10:23,792 --> 00:10:25,101 

that the cells were held 

353 

00:10:25,101 --> 00:10:26,511 

in the peripheral compartment, 

354 

00:10:26,511 --> 00:10:28,626 

so they're held in the bloodstream, 

355 

00:10:28,626 --> 00:10:30,439 

and they don't cross over the blood–brain 

356 

00:10:30,439 --> 00:10:31,949 

barrier to create damage 

357 

00:10:31,949 --> 00:10:34,232 

in the central nervous system. 

358 

00:10:34,232 --> 00:10:35,004 

And these are all 

359 

00:10:35,004 --> 00:10:37,522 

actually really novel findings that, 

360 

00:10:37,522 --> 00:10:38,328 

you know, 

361 

00:10:38,328 --> 00:10:39,369 

are enabled 

362 

00:10:39,369 --> 00:10:40,376 

by the 

363 

00:10:40,376 --> 00:10:42,189 

use of the Ella platform. 

364 

00:10:42,189 --> 00:10:43,062 

So we 

365 

00:10:43,062 --> 00:10:44,371 

were able to measure 

366 

00:10:44,371 --> 00:10:47,090 

a range of cytokines in the pilot study. 

367 

00:10:47,090 --> 00:10:49,239 

The supernatants were small, around 

368 

00:10:49,239 --> 00:10:50,749 

200 microliters. 

369 

00:10:50,749 --> 00:10:52,327 

So the panel design 

370 

00:10:52,327 --> 00:10:54,442 

capability and multiplexing, 

371 

00:10:54,442 --> 00:10:55,013 

together 

372 

00:10:55,013 --> 00:10:56,154 

with the range of available 

373 

00:10:56,154 --> 00:10:57,632 

cytokines, was really useful 

374 

00:10:57,632 --> 00:10:59,512 

for the initial pilot. 

375 

00:10:59,512 --> 00:11:00,653 

And the results identified 

376 

00:11:00,653 --> 00:11:01,962 

the relevant cytokines 

377 

00:11:01,962 --> 00:11:03,339 

that we went on to test in 

378 

00:11:03,339 --> 00:11:04,648 

the full cohort. 

379 

00:11:04,648 --> 00:11:07,334 

So the short preparation time 

380 

00:11:07,334 --> 00:11:08,609 

and the reliability 

381 

00:11:08,609 --> 00:11:10,624 

and the speed of results – for us, 

382 

00:11:10,624 --> 00:11:12,537 

we were very happy to get quick results – 

383 

00:11:13,847 --> 00:11:15,895 

you know,  makes the Ella 

384 

00:11:15,895 --> 00:11:16,835 

a great platform, 

385 

00:11:16,835 --> 00:11:17,607 

most especially 

386 

00:11:17,607 --> 00:11:21,031 

for the multiplex parameters. In general, 

387 

00:11:21,031 --> 00:11:22,978 

there were very few assay failures, 

388 

00:11:22,978 --> 00:11:23,750 

and there was very low 

389 

00:11:23,750 --> 00:11:25,462 

inter- and intra-assay 

390 

00:11:25,462 --> 00:11:27,577 

coefficient of variability. 

391 

00:11:27,577 --> 00:11:29,256 

In our hands, less than 2% 

392 

00:11:29,256 --> 00:11:31,270 

for interferon gamma and IL-2, 

393 

00:11:31,270 --> 00:11:32,110 

which is great. Wow. 

394 

00:11:32,110 --> 00:11:34,493 

Yeah, it is very impressive. Incredible. 

395 

00:11:34,493 --> 00:11:36,071 

And then, finally, as we're 

396 

00:11:36,071 --> 00:11:37,112 

almost out of time, 

397 

00:11:37,112 --> 00:11:38,656 

what potential long-term 

398 

00:11:38,656 --> 00:11:39,697 

research advantages 

399 

00:11:39,697 --> 00:11:40,670 

come with employing 

400 

00:11:40,670 --> 00:11:41,677 

fast and efficient 

401 

00:11:41,677 --> 00:11:43,893 

analytical techniques like this? 

402 

00:11:43,893 --> 00:11:46,512 

Well, the great advantage of the 

403 

00:11:46,512 --> 00:11:48,996 

Ella multiplex is that we can quickly 

404 

00:11:48,996 --> 00:11:50,037 

and reproducibly, 

405 

00:11:50,037 --> 00:11:50,708 

and that's important, 

406 

00:11:50,708 --> 00:11:53,461 

test a range of biomarkers, 

407 

00:11:53,461 --> 00:11:55,643 

including cytokines and chemokines. 

408 

00:11:55,643 --> 00:11:57,490 

And these precious primary samples 

409 

00:11:57,490 --> 00:11:59,000 

are in supernatants. 

410 

00:11:59,000 --> 00:12:00,041 

So as I said, 

411 

00:12:00,041 --> 00:12:01,451 

we've used these cellular assays 

412 

00:12:01,451 --> 00:12:02,592 

in the multiple sclerosis 

413 

00:12:02,592 --> 00:12:03,835 

study where we looked at EBNA-1 

414 

00:12:03,835 --> 00:12:06,856 

and the changes following treatments. 

415 

00:12:06,890 --> 00:12:09,072 

But this work will lead 

416 

00:12:09,072 --> 00:12:10,582 

onto longitudinal studies 

417 

00:12:10,582 --> 00:12:12,127 

where we'll follow patients 

418 

00:12:12,127 --> 00:12:13,570 

through disease flares 

419 

00:12:13,570 --> 00:12:15,249 

and treatment changes, 

420 

00:12:15,249 --> 00:12:16,122 

and the multiplex 

421 

00:12:16,122 --> 00:12:17,129 

capability of 

422 

00:12:17,129 --> 00:12:18,539 

Ella will allow us to test 

423 

00:12:18,539 --> 00:12:20,117 

large numbers of samples 

424 

00:12:20,117 --> 00:12:21,493 

easily and quickly. 

425 

00:12:21,493 --> 00:12:24,045 

We only use 5 ml of blood, 

426 

00:12:24,045 --> 00:12:25,992 

and this is much easier to take compared 

427 

00:12:25,992 --> 00:12:28,073 

to the cerebrospinal fluid 

428 

00:12:28,073 --> 00:12:29,550 

which requires a lumbar puncture 

429 

00:12:29,550 --> 00:12:31,363 

and an in-hospital procedure, 

430 

00:12:31,363 --> 00:12:32,605 

and that's the current 

431 

00:12:32,605 --> 00:12:34,553 

diagnostic biomarker 

432 

00:12:34,553 --> 00:12:36,567 

in multiple sclerosis. 

433 

00:12:36,567 --> 00:12:39,387 

So in other studies, you know, to expand 

434 

00:12:39,387 --> 00:12:41,133 

it, we've used SARS-CoV-2 

435 

00:12:41,133 --> 00:12:42,442 

peptides and antigens 

436 

00:12:42,442 --> 00:12:42,912 

to measure 

437 

00:12:42,912 --> 00:12:44,423 

cellular responses 

438 

00:12:44,423 --> 00:12:47,713 

pre- and post-vaccination in patients 

439 

00:12:48,418 --> 00:12:49,693 

that are immunosuppressed. 

440 

00:12:49,693 --> 00:12:51,338 

So these include patients 

441 

00:12:51,338 --> 00:12:51,775 

on 

442 

00:12:51,775 --> 00:12:53,218 

immunosuppressive therapies 

443 

00:12:53,218 --> 00:12:55,535 

and those with inborn errors of immunity, 

444 

00:12:55,535 --> 00:12:57,918 

so babies that are born with 

445 

00:13:00,772 --> 00:13:01,813 

defects in their immune 

446 

00:13:01,813 --> 00:13:04,800 

system. And these are an important group 

447 

00:13:04,901 --> 00:13:06,983 

because they may need individualized 

448 

00:13:06,983 --> 00:13:08,560 

vaccination regimens 

449 

00:13:08,560 --> 00:13:09,735 

to ensure protection. 

450 

00:13:09,735 --> 00:13:11,683 

They won't always be protected 

451 

00:13:11,683 --> 00:13:14,469 

by the same regime 

452 

00:13:14,469 --> 00:13:17,457 

as other cohorts. 

453 

00:13:17,692 --> 00:13:20,008 

And we're currently extending the studies 

454 

00:13:20,008 --> 00:13:23,298 

from the SARS-CoV-2 vaccination 

455 

00:13:23,466 --> 00:13:25,010 

to look at other common vaccine 

456 

00:13:25,010 --> 00:13:28,032 

responses in immunosuppressed patients. 

457 

00:13:28,032 --> 00:13:31,557 

So that's been the potential 

458 

00:13:31,859 --> 00:13:34,881 

long-term research advantages for us. 

459 

00:13:35,116 --> 00:13:35,955 

Perfect. 

460 

00:13:35,955 --> 00:13:36,123 

Well, 

461 

00:13:36,123 --> 00:13:38,036 

thank you so much for joining us here 

462 

00:13:38,036 --> 00:13:39,043 

in this episode. 

463 

00:13:39,043 --> 00:13:40,923 

It's been absolutely wonderful 

464 

00:13:40,923 --> 00:13:43,206 

speaking with you. Well, thank you. 

465 

00:13:43,206 --> 00:13:44,180 

It's been lovely 

466 

00:13:44,180 --> 00:13:47,201 

to have your support and care. 

467 

00:13:47,470 --> 00:13:48,511 

Thanks so much 

468 

00:13:48,511 --> 00:13:49,719 

for joining us in this episode, 

469 

00:13:49,719 --> 00:13:50,693 

because this episode 

470 

00:13:50,693 --> 00:13:52,170 

has been brought to you by Bio-Techne. 

471 

00:13:52,170 --> 00:13:54,721 

Bio-Techne is a global developer 

472 

00:13:54,721 --> 00:13:56,568 

manufacturer and supplier 

473 

00:13:56,568 --> 00:13:57,575 

of high quality 

474 

00:13:57,575 --> 00:13:59,421 

reagents, analytical instruments 

475 

00:13:59,421 --> 00:14:01,469 

and precision diagnostics. 

476 

00:14:01,469 --> 00:14:02,778 

Whether you are at the cutting edge 

477 

00:14:02,778 --> 00:14:03,819 

of academic research, 

478 

00:14:03,819 --> 00:14:05,431 

translating basic discoveries 

479 

00:14:05,431 --> 00:14:06,706 

to therapeutic leads 

480 

00:14:06,706 --> 00:14:07,915 

or at a facility 

481 

00:14:07,915 --> 00:14:09,392 

that requires the highest level 

482 

00:14:09,392 --> 00:14:10,836 

of diagnostic testing, 

483 

00:14:10,836 --> 00:14:11,608 

their award-winning 

484 

00:14:11,608 --> 00:14:13,152 

tools and solutions 

485 

00:14:13,152 --> 00:14:14,092 

empower scientists 

486 

00:14:14,092 --> 00:14:15,200 

and clinicians 

487 

00:14:15,200 --> 00:14:16,778 

to achieve reproducible 

488 

00:14:16,778 --> 00:14:18,591 

and consistent results. 

489 

00:14:18,591 --> 00:14:19,698 

Trusting Bio-techne 

490 

00:14:19,698 --> 00:14:21,041 

means choosing confidence 

491 

00:14:21,041 --> 00:14:22,082 

that every solution 

492 

00:14:22,082 --> 00:14:22,686 

you use 

493 

00:14:22,686 --> 00:14:24,163 

will help move you towards 

494 

00:14:24,163 --> 00:14:25,775 

better answers. 

495 

00:14:25,775 --> 00:14:27,353 

We've left some further resources below 

496 

00:14:27,353 --> 00:14:28,461 

should you like to learn more. 

497 

00:14:28,461 --> 00:14:29,300 

But thanks again 

498 

00:14:29,300 --> 00:14:30,576 

for watching this episode 

499 

00:14:30,576 --> 00:14:32,892 

and I hope we'll see you again very soon.