R35 Equipment Supplement

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ABSTRACT Sepsis, burns and trauma are the most catastrophic and intractable challenges affecting our adult population. Trauma, sepsis, and critical illness are diseases of the bone marrow, and we have now demonstrated that many of the long-term complications and dismal clinical outcomes are secondary to ‘pathologic activation’ of myeloid populations. We were the first to report the expansion of MDSC subsets in sepsis, and have made significant contributions to our understanding of these cell populations. Studies we have conducted by this funding instrument have demonstrated that our definitions of MDSC phenotypes over-simplify and limit efforts to prognose and treat MDSC-mediated disease. We recognize that our ongoing studies require a more granular assessment of the function and phenotype of individual stem cell as well as myeloid and lymphoid cell populations over time in patients with differential outcomes after sepsis, trauma and burns. To achieve that goal, we require an increase in our capability to resolve and isolate individual MDSC subtypes. We currently are using a Bio-Rad S3 fluorescently-activated cell sorter that is seven years old, and has 2015 technology. This includes just two lasers, four colors and two side stream splitting. Our experience with the S3 has shown that we are limited to approximately 20,000 drops/second and 1000 events/second making sorts a day-long procedure. More problematic, Bio-Rad has informed us that they are discontinuing support for the S3 including parts. In its stead, we are requesting support for the purchase of a Becton-Dickinson FACSMelody® BRV 9 Color 4 way flow cytometer package including software, training and a one year maintenance contract. The advantages of the FACSMelody® over the existing instrument are significant: three versus two lasers, nine colors instead of four colors and four versus two side streams to simultaneously isolate at least two and likely three much higher enriched subsets of MDSCs. Also important is sorting speed. Using the current instrument, whole blood sorts take three to four hours per sample, and these times can be reduced five-fold with the new instrument. This not only increases throughput, but improves the viability of the isolated cells, a key component of improved rigor and reproducibility. We envision analyzing samples from over 75 subjects annually (150-200 samples) based on the proposed goals. Additional institutional resources guaranteed by the Department of Surgery/College of Medicine will the service contract (~$25k annually) for the life of the grant award and the costs of additional reagents required for the higher specificity (see attached letters). Through combined support from NIGMS and the Department of Surgery and College of Medicine, the programmatic goals can be achieved with high rigor and reproducibility.