PHOTOCHEMICAL DECONTAMINATION OF PLATELET CONCENTRATES

治疗性血小板输注目前估计为5-7百万 单位，并预计将增加扩大使用器官 移植和癌症强化化疗。 的健康风险 与血小板输注相关的重要性主要是因为 血液中的病毒传染源传播的可能性 尽管输血前的血液制品测试。 的目的 建议开发一种技术来抑制B型肝炎病毒（HBV）， 人类非甲非乙型肝炎病毒（NANBV）和巨细胞病毒（CMV） 保存血小板功能的血小板浓缩物（PC）。 该项目的信息将普遍适用于未来 血液制品中其他病毒灭活的研究， 这些过程对细胞和血浆成分的影响，以及 最终预防输血相关的病毒性疾病。 发展了一种光化学去污技术 利用8-甲氧基补骨脂素（8-MOP）和长波紫外 紫外线照射（UVA），以杀灭PC中的广谱病毒， 保存体外血小板功能。 本项目谋求 优化PCD技术的灭活效率， PC中的细胞外和细胞内病毒，以开发体外测定 量化病毒灭活的效率， 用于病毒灭活研究的模型，以证明有效性 使用体外和体内PCD处理对PC进行去污 测定，并确定PCD处理的血小板的体内存活 在灵长类动物中。[1]的文件。 光化学失活动力学 细胞外和细胞内人CN 4V和鼠CMV在PC中的应用 体外细胞培养系统和鼠感染性模型将被 测定了 细胞外和细胞外的光化学失活动力学 细胞内鸭嗜肝DNA病毒（DHBV），一种人HBV的模型， 使用鸭肝细胞和鸡蛋感染性模型在PC中测定。 猫鼻气管炎病毒（FeRTV）将用作 细胞内病毒，因为高水平的感染性可以实现在 PC与此代理红细胞、淋巴细胞和血浆的作用 将确定脂质污染对PC中病毒灭活的影响。 在 除8-MOP外，还可使用其它的庚烯衍生物[4，5 ′，8-三甲基庚烯 (TMP)和4 ′-氨甲基-4，5 ′，8-三甲基庚烯（AMT）]将被 研究优化PCD技术。[2]的文件。 细胞内 将测量光加合物的形成以评估 前病毒DNA/RNA交联和聚合酶链反应（PCR） 将使用扩增测定来量化抑制效率 细胞核和病毒DNA转录。[3]。 确定后 基于病毒灭活研究的最佳灭活条件， 这些条件对PCD处理的PC的体外功能的影响将 被衡量。 将通过测量以下指标评估血小板功能： 产量、胞外pH、形态评分、聚集反应、 分泌，细胞外乳酸脱氢酶，血栓素B-2 生产、致密体含量和颗粒含量。[4]的文件。 人类PC， 加入已知量的HBV和NANBV， 各种PCD条件，将被输入黑猩猩， 评估肝炎的发作。[5]。体内回收率， PCD处理的食蟹猴PC的存活和止血功能将 在正常和血小板减少的食蟹猴中测量。

Therapeutic platelet transfusion is currently estimated at 5-7 million units annually, and is expected to increase with expanded use of organ transplantation and intensive cancer chemotherapy. The health risks associated with platelet transfusion are significant primarily because of the potential for transmission of blood-home viral infectious agents despite pretransfusion blood product testing. The objective of this proposal is to develop a technique to inactivate hepatitis B virus(HBV), hepatitis non-A, non-B virus(NANBV), and cytomegalovirus(CMV) in human platelet concentrates(PC) with preservation of platelet function. Information from this project will be generally applicable to future studies of the inactivation of other viruses in blood products, to the effects of these processes on cellular and plasma components, and ultimately to the prevention of transfusion-associated viral disease. A photochemical decontamination(PCD) technique has been developed utilizing 8-methoxypsoralen (8-MOP) and long wavelength ultraviolet irradiation (UVA) to inactivate a wide spectrum of viruses in PC with preservation of in vitro platelet function. This project seeks to optimize the efficiency of the PCD technique for inactivation of extracellular and intracellular viruses in PC, to develop in vitro assays to quantify the efficiency of viral inactivation, to develop animal models for study of viral inactivation, to demonstrate the effectiveness of the PCD treatment for decontamination of PC using in vitro and in vivo assays, and to determine the in vivo survival of PCD treated platelets in primates. [1]. The photochemical inactivation kinetics of extracellular and intracellular human CN4V and murine CMV in PC using in vitro cell culture systems and a murine infectivity model will be measured. The photochemical inactivation kinetics of extracellular and intracellular duck hepadna virus(DHBV), a model for human HBV, will be determined in PC using duck hepatocyte and egg infectivity models. Feline rhinotracheitis virus(FeRTV) will be used as a model of intracellular virus, since high levels of infectivity can be achieved in PC with this agent. The effects of red cell, lymphocyte, and plasma lipid contamination on viral inactivation in PC will be determined. In addition to 8-MOP, other psoralen derivatives [4, 5', 8-trimethylpsoralen (TMP) and 4'-aminomethyl-4, 5', 8-trimethylpsoralen (AMT)] will be studied to optimize the PCD technique. [2]. The degree of intracellular photoadduct formation will be measured to assess the efficiency of proviral DNA/RNA crosslinking, and the polymerase chain reaction (PCR) amplification assay will be used to quantify the efficiency of inhibition of nuclear and viral DNA transcription. [3]. After determination of optimal inactivation conditions based on viral inactivation studies, the effects of these conditions on in vitro function of PCD treated PC will be measured. Platelet function will be assessed by measuring: platelet yield, extracellular pH, morphology score, aggregation response, secretion, extracellular lactate dehydrogenase, thromboxane B-2 production, dense body content and granule content. [4]. Human PC, to which known amounts of HBV and NANBV have been added, treated under various PCD conditions, will be transfused into chimpanzees followed by evaluation for the onset of hepatitis. [5]. The in vivo recovery, survival and hemostatic function of PCD treated cynomolgus PC will be measured in normal and thrombocytopenic cynomolgus monkeys.