Human Specimen Collection to Support Basic and Clinical Research

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The overall scope and purpose of this research protocol is to support assay development and research conducted within the NHLBI and other divisions of the intramural research program, especially the Sickle Cell Branch. Biospecimens, typically include blood samples that are collected from subjects, including those with sickle cell trait and subjects with sickle cell disease. The various methods and assays developed by different collaborators are listed below. The Sickle Cell Branch is focused on developing a greater understanding of acute pathophysiology, genetics and vascular biology in Sickle Cell Disease. The following assays were developed in the sickle cell branch laboratories: 1. Platelet function assays: Platelets isolated from whole blood, to study in vitro platelet aggregation studies. Whole blood was used to study platelet aggregation and ATP luminescence. The protocol has provided samples to standardize the assay using healthy ethnic matched controls. 2. Cell-Free DNA and mitochondrial DNA assays: 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 (neutrophil extracellular trap formation). In separate experiments blood from both healthy donors and sickle cells patients are used to isolate low density granulocytes (LDGs) using density gradient centrifugation and flow cytometry. 4. Cell derived extracellular vesicles (EVs) in plasma: Assays to study plasma borne cell-derived EVs obtained from SCD patients and Healthy volunteers are being standardized for two newly established research protocols to study the impact of acute vaso-occlusive crisis and venous thrombosis on plasma EV numbers and coagulation profile. 5. Assays to quantify and study the isomerase function of protein disulfide isomerase (PDI) have been standardized using this protocol. The results of this assay will be used to support research conducted in a phase 2 study of a small molecule inhibitor of plasma PDI. The Cellular and Molecular Therapy Branch is focused on developing genetic strategies aimed at correction of SCD through modification of autologous hematopoietic stem cells (HSCs) from the marrow and on hematopoietic stem cell transplantation as a cure for SCD. The following assays are under development in the Cellular and Molecular Therapy Branch: 1. In vitro modification of autologous hematopoietic stem cells (HSCs) from the marrow 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. Data are analyzed by conventional and imaging flow cytometry, the latter confirming post-CD34+ selection flow data and demonstrating variations in antibody labeling intensity to characterize HSC heterogeneity and progenitor lineage. 2. In other studies, blood is obtained from sickle cell patients and healthy controls to standardize cytokine measurements in SCD patients and healthy controls and compare them with SCD patients who underwent haploidentical hematopoietic stem cell. Measurements include assays for thrombospondin and platelet factor 4 as serum biomarkers of engraftment. 3. Studying the life span of erythrocytes that are modified by either gene transfer or HSC transplantation is critical to understanding the impact of such therapies on anemia in SCD. Assays to study erythrocyte half-life using biotin labeling are currently supported by this protocol. 4. In collaboration with NHGRI, an assay to isolate reticulocytes from anticoagulated whole blood obtained from SCD patients and standardize single cell RNA sequencing of reticulocytes is being developed. This methodology has been successfully completed. The Laboratory of Chemical Physics is concerned with studying the thermodynamics, kinetics, and mechanism of fiber formation of hemoglobin S, and its relation to the pathophysiology and therapy of sickle cell disease. This protocol supports laboratory research by providing blood 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 on the rate of sickling. Specifically, the effect of anti-sickling drugs is 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 anti-sickling agents are also underway including the inter-individual variability of these assays.