In Dr Shets and Dr. Theins labs, blood samples were obtained from healthy volunteers and sickle cell patients to further studies on: 1. Platelet Activation in Sickle Cell Disease: Platelets were isolated from whole blood, to study in vitro platelet NLRP3 inflammasome activity. Whole blood was used to study platelet aggregation and ATP luminescence. 2. Tissue Damage and Release of Cell-Free DNA: Optimization of cell-free DNA extraction, quantitation and library preparation methodology. Whole genome sequencing (WGS) and whole genome bisulfite sequencing (WGBS), and quantitative polymerase chain reaction (qPCR), where the cell-free DNA extracted from healthy volunteers was used as an independent healthy control to compare with sickle cell disease patients who are in steady-state and crisis. 3. Neutrophil Activation and Extracellular Trap Formation: Blood from healthy volunteers and plasma from sickle cell patients were used for in vitro neutrophil functional studies (neutrophils extracellular traps formation). In separate experiments blood from both healthy donors and sickle cells patients were used to isolate low density granulocytes (LDGs) using density gradient centrifugation and flow cytometry. 4. Microparticles: Isolation of RBC pellet and plasma from blood samples of SCD patients and Healthy volunteers for microparticle isolation and investigation. 5. GBT 440 standardization assays: Blood samples from SCD patients and healthy controls were obtained to study in vitro the effects of GBT-440 at different doses on oxygen equilibrium curves. The findings from this study revealed that GBT-440 binds to the alpha chain of hemoglobin in a 1:1 ratio based on mass spectrometry findings. Moreover, further studies revealed that quantifying the percentage of hemoglobin modified by GBT 440 in a severely anemic patient with SCD by oxygen equilibrium curve measurement was more accurate than extrapolating from pharmacokinetic data. 6. Platelet Activation: Blood from healthy volunteers was used for in vitro platelet activation studies (thrombin generation and blood coagulation). In Dr. Tisdales lab, studies focused on developing genetic strategies aimed at correction of SCD through modification of autologous hematopoietic stem cells (HSCs) from the marrow. These furthered optimization of HSCs for gene therapy. 1. Collection of bone marrow HSCs is being optimized in an ongoing clinical trial testing lentiviral gene transfer to HSCs in patients with SCD. Therefore, bone marrow continues to be collected from volunteer patients to optimize cell processing and HSC enrichment. Twenty milliliters of BM from subjects with SCD (HbSS genotype) was collected in different anticoagulants (Heparin, ACD-A) and processed immediately (day 0) or stored at 4 degrees C and processed the following day (day 1). After isolation via Ficoll density gradient centrifugation, the mononuclear (MN) layer was stained with antibodies against inflammatory markers (CD36, CD35, CD11b, CD62L, CD62P), non-MN cells (GPA, CD66b, CD41/61), or processed for CD34+ selection using a magnetic microbead CD34+ selection kit and stained for CD34, CD45, and GPA expression. 2. Data were analyzed by conventional and imaging flow cytometry, the latter confirming post-CD34+ selection flow data and demonstrating antibody intensity as a characterization of HSC heterogeneity and progenitor lineage. In Dr Eatons lab, blood has been obtained from sickle cell patients to develop assays that quantify the rate of sickling and the effects of cell volume, hemoglobin concentration on sickling kinetics. 1. These approaches are being used to test the effects of anti-sickling drugs and to study the effects of human variation in iron availability, on the rate of sickling. Further studies are ongoing in this regard. Specifically, the effect of anti-sickling drugs are being evaluated in healthy controls and those with sickle cell trait. 2. Studies to standardize assays to measure oxygen equilibrium curves in the presence of antisickling agents are also underway. In Dr Fitzhughs lab, blood has been obtained from sickle cell patients and healthy controls to standardize cytokine measurements in SCD patients who underwent haploidentical hematopoietic stem cell transplantation. In particular, these measurements included assays for thrombospondin and platelet factor 4 which helped interpret plasma and serum biomarkers of successful and failed engraftment.
