Human Specimen Collection to Support Basic and Clinical Research

这项研究方案支持在NHLBI和内部研究计划的其他部门内进行的分析开发和研究。生物标本来自受试者、具有镰状细胞特征的受试者和患有镰状细胞病的受试者。 镰刀细胞科致力于加深对镰刀细胞病的急性病理生理学、遗传学和血管生物学的了解。在镰状细胞分支实验室开发了以下分析方法： 1.镰状细胞病中的血小板活化：从全血中分离血小板，进行体外血小板聚集研究。采用全血检测血小板聚集功能和ATP发光特性。该方案提供了样本，以使用健康的种族匹配对照来标准化检测。 2.无细胞DNA和线粒体DNA检测：无细胞DNA提取、定量和文库制备方法的优化。全基因组测序(WGS)和全基因组亚硫酸盐测序(WGBS)，以及定量聚合酶链式反应(QPCR)，其中从健康志愿者中提取的无细胞DNA作为独立的健康对照，与处于稳定状态和危象的镰状细胞病患者进行比较。 3.中性粒细胞活化与细胞外陷阱的形成：采用健康志愿者的血液和镰状细胞患者的血浆进行体外中性粒细胞功能研究(中性粒细胞外陷阱的形成)。在不同的实验中，来自健康捐献者和镰刀细胞患者的血液被用来使用密度梯度离心法和流式细胞术分离低密度粒细胞(LDGs)。 4.血浆中细胞来源的细胞外小泡(EVS)：从SCD患者和健康志愿者获得的血浆来源的细胞来源EV的分析正在标准化，用于研究急性血管闭塞危象和静脉血栓形成对血浆EV数量和凝血谱的影响的两项新的研究方案。 5.用该方法对蛋白质二硫键异构酶(PDI)异构酶功能的定量和研究进行了标准化。这项试验的结果将用于支持在血浆PDI小分子抑制剂第二阶段研究中进行的研究。 细胞和分子治疗科专注于开发旨在通过修改来自骨髓的自体造血干细胞(HSCs)来纠正SCD的遗传策略，并致力于通过造血干细胞移植来治愈SCD。细胞和分子治疗处正在开发以下检测方法： 1.在一项正在进行的临床试验中，对来自骨髓的自体造血干细胞(HSCs)进行体外修饰，以测试慢病毒基因转移到SCD患者的HSCs。因此，继续从志愿者患者身上收集骨髓，以优化细胞处理和HSC浓缩。通过常规和成像流式细胞术分析数据，后者证实了CD34选择后的流程数据，并展示了抗体标记强度的变化，以表征HSC的异质性和祖细胞谱系。 2.在其他研究中，从镰状细胞患者和健康对照组采集血液，以标准化SCD患者和健康对照组的细胞因子测定，并将其与接受单倍体相合造血干细胞治疗的SCD患者进行比较。测量包括作为植入的血清生物标记物的凝血酶反应蛋白和血小板因子4的检测。 3.研究通过基因转移或HSC移植修饰的红细胞的寿命，对于了解这类疗法对SCD贫血的影响至关重要。研究红细胞半衰期的分析目前正在开发中，试点研究的进行得到了该方案的支持。 4.与NHGRI合作，正在开发一种从SCD患者抗凝全血中分离网织红细胞的方法，并对网织红细胞的单细胞RNA测序进行标准化。 化学物理实验室致力于研究S血红蛋白纤维形成的热力学、动力学和机理，及其与镰状细胞病的病理生理和治疗的关系。该协议通过提供镰状细胞患者的血液来支持实验室研究，以开发量化镰状细胞比率以及细胞体积、血红蛋白浓度对镰状细胞动力学的影响的分析方法。 1.这些方法正被用来测试抗镰刀药的效果，并研究人类变异对镰刀率的影响。具体地说，正在评估抗病药物在健康对照组和具有镰状细胞特征的人中的效果。 2.在有抗镰刀剂存在的情况下测量氧平衡曲线的标准化分析也在进行中，包括这些分析的个体间变异性。

This research protocol support assay development and research conducted within the NHLBI and other divisions of the intramural research program. Biospecimens are collected from subjects, those with sickle cell trait and subjects with sickle cell disease. 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 Activation in Sickle Cell Disease: 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 are currently under development and conduct of pilot studies are 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. 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.