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Solving the Challenges of Pharmacodynamic and Biomarker Analysis by Flow Cytometry for Support of Multiple Sites

Posted on: January 20, 2023


The power of flow cytometry is that it allows you to really understand - from an immunological standpoint and a cellular stand standpoint – not just the efficacy of the drug, but also the impact the drug is having on the patient’s immune system, allowing us to answer questions like “Is this drug safe?” Flow Cytometry, in addition to monitoring wanted immune responses, it can help determine if you're augmenting the patient's immune cells in a negative fashion. It serves as an incredibly powerful tool. There are two major platforms that are utilized for measuring cellular immune responses; they are gold standard in the space at this point. The first is Flow Cytometry, the other is ELISpot. We offer both from the KCAS family. We have capabilities up to about 20 parameters and 18 colors/Biomarkers. The next generation of flow cytometers are spectral based and can go up to 60 biomarkers, but pose a challenge with regression of the data, interpretation of the data, and how to store the data. In my 29+ years of drug development rarely does the advancement in technology get ahead of the scientist and the physician.


All CROs face the same critical challenge of sample integrity when supporting Multiple Sites for Flow Cytometry.


To me the most critical step is getting the sample (in this case, the peripheral blood mononuclear cells or whole blood) to a sophisticated flow cytometry laboratory in a timely fashion and in a way that preserves the sample’s quality and integrity. You must ensure that they are stored at the right temperatures and handled properly along the way. KCAS’s team has decades of expertise in handling these samples properly, and we also have a lot of depth and knowledge around the preservation of those samples using such things as Cyto-Chex BCT tubes or SMART tubes.


We often get asked by clients if they should use PBMCs or whole blood. Similar to other CROs we recommend a feasibility study to assess this. What separates us from others is the multiple collection options we can present, how to design and test the collection of samples to provide high quality data in a cost-effective manner, 13+ years of development and validation flow cytometry assays for support of clinical trials, and 4 sites located globally [2 sites located in the United States (Kansas City and Philadelphia) and 2 sites located in Europe (Lyon and Milan)]. For instance, for immunophenotyping (IPT) the Cyto-Chex BCT tube can often preserve a whole blood sample for up to 7 days and for support of the T-cell, B-cell, and NK cell panel (TBNK panel) it can preserved for up to 14 days. This can allow for batching of samples and avoid having to test samples within 48 hours of collection, which is the standard expiration time for K2EDTA, ACD, and Heparin whole blood. If your panel is IPT then a logical starting point is a feasibility study using the Cyto-Chex BCT tube. Alternatively, if you are looking at intracellular staining the Cyto-Chex BCT tube is most likely not the best option for whole blood, but perhaps the SMART tube would be more suitable. We have shown stability of whole blood collected in SMART tubes to be stable at -80 for over 1 year. SMART tubes are an excellent alternative however the one disadvantage it requires sample processing at the clinical site and is not a direct blood draw. You first must use a standard venipunture tube and then transfer/process the sample into a smart tube. If you have performed multi-site studies supporting flow cytometry introduction of a new process can be a disaster if you don’t provide a detailed lab manual. Did I mention KCAS has a dedicated clinical supplies and kitting team that can provide detailed lab manuals and videos for support of flow cytometric clinical trials?


Unfortunately, a whole blood sample preserved may often not be feasible and you must invest in isolating PBMCs. We recommend collecting in CPT tubes and if a feasibility study shows up to 30 hours of stability prior to isolating, harvesting, and freezing them you should ship them to the bioanalytical lab. Should the feasibility study show instability <30 hours (we suggest at least 30 hours to allow for shipping from different time zones and allowing for time to accession the sample upon arrival) you will need to invest in training your clinical sites to isolate, harvest and freeze the cells on site. This can be challenging due to some sites not having proper equipment and proper training on how to collect PBMCs from a CPT tube. So long as they have a centrifuge KCAS has manual/video to train your clinical sites on collecting PBMCs from CPT tubes.


Additionally, we at KCAS help you design your Phase I trial with a proactive approach to help make it seamless to scale it to Phase II and Phase III trials. Meaning, often a Phase I trial is a single site, and you can design a sophisticated panel that requires PBMCs or whole blood collection but when you must expand to multiple sites you will have both logistical and technical issues if you do not proactively address the challenges with having multiple sites support the trial. The best way to address this challenge is to qualify each site by collecting samples from normal donors and have the flow cytometry lab test the samples flow cytometrically, but this is not always practical. An alternative is to implement some type of checks and balances as you on board each site, such as having the sites do a viability count prior to freezing the cells.


So where does this leave us with respect to how to support a multi-site flow cytometry assay?


The first question to address in my opinion is can I use PBMCs. For both sample integrity/quality and cost effectiveness collecting PBMCs is optimal. If PBMCs are an option, then you can determine if the site must isolate the PBMCs or if they can be overnight shipped to the flow cytometry lab and then isolated. If PBMCs are not an option and whole blood must be collected, can I preserve or stabilize it. For multiple site clinical trial flow cytometry assays we avoid fresh whole blood for numerous reasons, including delays in shipping that result in the sample expiring, the cost of having to test a single sample at a time, and the time lost having to manage testing one sample at a time instead of batching them. The good news is we at KCAS with four sites located globally, we got you covered.


If you have need to test your PBMCs or whole blood using Flow Cytometry let KCAS simplify the process by working with you to develop the best way to collect, ship and store your samples for support of your multi-site studies.


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