John C Tigges, BA, BS

I am the Director/Manager at the Flow Cytometry Core Facility and Center for Extracellular Vesicle Detection at Beth Israel Deaconess Medical Center in Boston, MA (https://research.bidmc.org/flow-cytometry-core/). I have extensive hands-on experience as well as demonstrated expertise in flow cytometry, microscopy, and flow cytometry-specific software programming and implementation. It is through this experience that I have been asked to participate in multiple training programs, task forces, education initiatives, collaborations, and consulting positions. I have been able to use my expertise to consult on multiple innovations and methodologies with researchers and industry partners. I have collaborated with software engineers at Beckman Coulter in the development of flow cytometry acquisition and analysis software Kaluza and WARP. Additionally, I have been involved in multiple groups focused on the development of new instrumentation and techniques to advance scientific technologies. I have been a key opinion leader with Beckman Coulter (BC) on the development of the Astrios seven laser, forty-nine parameter, and six-way sort logic cell sorter and subsequent scatter detection upgrade version, AstriosEQ. Furthermore, I was a KOL for the creation of the CytoFLEX SRT, a fully automated benchtop cell sorter. In addition, I have been involved with the BC CytoFLEX nano from its inception, designing the multi-scatter aspects of the instrument, to its commercial release. Consulting with BC, I was able to perform alpha- and beta- testing of the nano for use in a core facility. I was instrumental in the design and testing of Propel Labs one of a kind small particle detection system, NanoView. The AstriosEQ, CytoFLEX SRT, and NanoView provide the ability to detect and sort submicron particles. The nano allows for sub populating nanoparticles >40nm. The methodologies derived from the cutting-edge instrumentation were published in collaboration with Dr. Jennifer Jones, NIH/NCI, in the Journal of Extracellular Vesicles.

Recently, I have been working with Dr. David Melton using nano-flow cytometry to characterize EV populations involved in preeclampsia. This work has led to multiple K grants currently in review. Additionally, the EV core facility has been assisting Dr. Alec Schmaier develop a full profile of platelet EVs involved in Thrombosis and potential treatment modalities. Using the CytoFLEX nano and EV MACSPlex, we have been able to characterize many targets, and the work is highlighted in a manuscript in peer review. Moreover, using the MOSAIC spectral technology, I am collaborating with several BIDMC and MGH PIs to characterize multifluorescent signatures of cancer cells, and alveolar lung cells involved in asthmatic conditions. In particular, work with Dr. Analisa DiRuscio, incorporates both spectral analysis and nanoparticle analysis to understand the effects of loaded LNPs on tumor cell progression.

Throughout my career at BIDMC, I have collaborated with Dr. Ionita Ghiran to develop several techniques and devices to advance discovery. I have tested and validated magnetic levitation for the detection of malaria and sepsis using both microscopy and cell phone attachment. This project landed Dr. Ghiran a Bill and Melinda Gates Foundation award. In addition, my collaboration with Dr. Ghiran has contributed to understanding the function of EVs and EV isolation. Dr. Ghiran and I have developed methods to purify EV populations from biofluids using Top14 protein depletions and a custom Top10 protein depletion capture bead assay. This novel technique allows for the isolation of pure EVs for the detection of miRNA. miRNAs are detected via molecular beacons directly introduced to the EV populations, or by attachment of MBs to capture beads. We are currently designing a multiplex bead assay for the detection of MBs in plasma. Currently, we are working on a flow cytometry CBA method to characterize post-transcriptional modifications on miRNA from cancer

derived EVs. The patented methodology from Dr. Ghiran is currently being peer reviewed for publication. Moreover, we are collaborating with Alida Biosciences to create a Flow Cytometric assay to be commercially available. Finally, I am collaborating with Dr. Jonathan Burnie, HMS, on the full characterization of several viruses to build protocols and methodologies for Flow Virometry. Currently, our collaboration has led to several presentations, poster sessions at major conferences, and two pending manuscripts.

Throughout my career, I have been asked to consult on instruments designed by start-up companies such as Ferrologix, Lase Innovation, and AcouSort. Ferrologix is developing a ratcheting cytometry system for rare event analysis and EV phenotyping. Our collaboration will involve testing beta instrumentation for the detection of low abundance GFP transfected cells and EV samples labeled with tetraspanin fluorescent antibodies. Ferrologix has recently secured a SBIR grant with my assistance and support in regard to EV detection and current needs in the field of nano-FC. Lase Innovation uses laser barcoding technology to perform Cyclic Flow Cytometry to simplify high parameter immunophenotyping. In collaboration with members of the Cancer Center, we will be beta-testing the lase particles along with the new instrumentation to identify forty separate antibodies in a 4x10 cyclical format. Both commercially released and beta technologies have contributed to the advancement of cytometry and enhanced the capabilities of researchers in the BIDMC flow cytometry core facility. AcouSort is a Swedish based company developing an instrument capable of separating EVs from biofluids using a charge dependent methodology. Through beta-testing and consultation, AcouSort has been able to release the AcouPrep.

Currently, my expertise centers around setting up and describing a comprehensive methodology and standardization of EV analysis using nanoFC (nano-Flow Cytometry). NIST controls of different size ranges, fluorescent intensities, and materials are required to set up distribution curves. These are then used for instrument optimization and as a reference guide. Using these controls, FACS instruments can be primed for the detection, analysis and sorting of specific EV populations. This allows for cross platform comparison and the ability to monitor QC, QA. This work has been published through the EV Flow Cytometry Working Group’s publication of MIFLOWCytEV guidelines. Additionally, this work is ongoing through the ERCC2 subgroup that I am Co-Chairing for an EV benchmarking initiative to standardize nanoFC. This work has been presented at the NIH/ERCC Spring Meeting and is regularly updated to the ERCC steering committee. Upon completion, the developed materials and protocols will be published and distributed to all international ERCC members and made available for the broader EV research community. This work will allow for cross-institutional collaboration for EV studies. Furthermore, I specialize in describing the use of nanoparticles to optimize a flow cytometer for small particle detection. This work was published as a methodology in Methods of Molecular Biology. The development of proper setup and standardization of nanoFC has allowed me to participate in many educational talks, sessions, and manuscripts to teach EV research to an international audience. My contribution to the EV field has added another layer to my ability to teach and mentor researchers in cytometry methods and instrumentation. The standardization protocols developed in the Flow Cytometry Core have led to collaborative research efforts with the NIH/NCI, Northeastern University, University College Dublin, Trinity College Dublin, St. James Hospital, McGill University, Massachusetts General Hospital, Dana Farber Cancer Institute, University of Massachusetts Boston, and industry partners such as Beckman Coulter, PureTech, and BioLegend. Furthermore, the developed protocols for standardization, characterization, and sorting of EVs has been acknowledged through an international collaboration with UCD for a Precision Oncology Ireland grant. As a member of this prestigious team, BIDMC flow cytometry core will be a Center of Excellence for the discovery of biomarkers in cancer modalities. Finally, my work within the field of EV detection and characterization has been rewarded through an MLSC Research Infrastructure grant. The award has funded the development of a first of its kind nanoparticle core facility. The new facility is equipped with an orthogonal approach to nanoparticle detection incorporating microscopy, DLS, Raman Spectroscopy, Flow Cytometry, NTA, and MRPS. This award has been a culmination of my research efforts, KOL appointments, and consulting opportunities to promote nanoparticle science and nanomedicine.