Device for measuring blood concentrations of unbound bilirubin in neonates

描述（由申请方提供）：本项目的总体目标是开发一种用于测量未结合未结合胆红素（Bf）浓度的试验，以比现有方法更准确地诊断新生儿胆红素诱导的神经毒性。虽然超过70%的新生儿有血胆红素水平升高，但对大多数婴儿来说，这种高胆红素血症是良性的。然而，对于某些婴儿，胆红素的影响可能是神经毒性的，导致从可逆性听力缺陷到更严重和可能致命的核黄疸神经后遗症的缺陷。特别是极低出生体重（ELBW）婴儿的结局非常差，其中约25-30%死亡，另外25-30%有严重的神经功能缺损（例如脑瘫）。推测这是由于胆红素神经毒性所致。目前通过监测总血清胆红素（TSB）评估新生儿胆红素神经毒性，高水平会导致光疗和/或换血。然而，最近的研究，特别是我们自己在ELBW婴儿中的一项新研究表明，使用TSB不会检测到潜在的胆红素毒性（高假阴性），并且使用基于TSB水平的光疗不会改善这些婴儿中非常差的结局。相反，对TSB水平极高（e 25 mg/dL）（换血阈值）的足月婴儿的研究最近发现，在多达80%的这些婴儿中（高假阳性），换血（本身与约2%的死亡率相关）可能是不必要的。这意味着由于高TSB而不必要地接受光疗治疗的婴儿的百分比可能更大，因为光疗治疗的阈值更低。TSB作为诊断失败的原因是胆红素是高度不溶性的，并且在血液中绝大多数与白蛋白结合，通常只有约10-4处于未结合或“游离”状态（Bf）。然而，只有TSB的Bf组分可以穿过血脑屏障并引起核黄疸。TSB不能解释改变白蛋白-白蛋白结合的许多因素，因此不能解释Bf。为了改善结果并减少不必要的治疗，需要一种准确且简单易用的Bf测量方法。该项目的具体目标是，在第一阶段，开发一种简单的一次性检测盒，其中包含我们最近开发的荧光标记的突变脂肪酸结合蛋白（FABP），可以通过结合Bf后荧光的变化来准确定量Bf。这些探针将使用近红外（NIR）荧光，从而允许测量5 μ L未稀释全血中的Bf。在第二阶段，我们将开发一种比率荧光仪器， 测量样品盒上的Bf，并将在不到1分钟的时间内显示Bf浓度。

DESCRIPTION (provided by applicant): The overall goal of this project is to develop a test for measuring the concentration of unbound unconjugated bilirubin (Bf) to more accurately diagnose than current methods, bilirubin-induced neurotoxicity in newborns. Although more than 70% of newborns have elevated blood bilirubin levels, for most infants this hyperbilirubinemia is benign. However, for some infants bilirubin effects can be neurotoxic, resulting in deficits ranging from reversible hearing defects to the more severe and possibly fatal neurological sequelae of kernicterus. In particular very poor outcomes occur in extremely low birth weight (ELBW) infants where approximately 25-30% die and an additional 25-30% have severe neurologic deficits (e.g. cerebral palsy). This is presumed to be due to bilirubin neurotoxicity. Bilirubin neurotoxicity in neonates is currently assessed by monitoring total serum bilirubin (TSB) and high levels cause phototherapy and/or exchange transfusion to be initiated. However, recent studies and in particular a new study of our own in ELBW infants demonstrates that using TSB will not detect the underlying bilirubin toxicity (high false negativity) and using phototherap based on TSB levels will not improve the very poor outcomes in these infants. Conversely, studies in term infants presenting with extremely high TSB levels (e 25 mg/dL), the threshold for exchange transfusion, have recently found that the exchange transfusion (itself associated with ~ 2% mortality) may have been unnecessary in as many as 80% of these infants (high false positivity). This implies that the percent of infants unnecessarily treated with phototherapy because of high TSB may be even larger because the threshold for phototherapy treatment is lower. The reason for the failure of TSB as a diagnostic is that bilirubin is highly insoluble and n blood the vast majority is bound to albumin, normally only about 10-4 is in the unbound or "free" state (Bf). However, only the Bf fraction of TSB can cross the blood brain barrier and cause kernicterus. TSB cannot account for the many factors that change bilirubin-albumin binding and therefore Bf. What is needed to improve outcomes and reduce unnecessary therapy, is an accurate and simple to use method for measuring Bf. The specific Aims of the project are, in Phase I, to develop a simple disposable cartridge containing our recently developed fluorescently labeled mutant fatty acid binding proteins (FABPs) that allow accurate quantitation of Bf by a change in fluorescence upon binding Bf. These probes will use near infrared (NIR) fluorescence and thereby allowing measurements of Bf in 5 �L of undiluted whole blood applied to the cartridge. In Phase II we will develop a ratio fluorescence instrument that is optimized for measuring Bf on sample cartridge and will display the Bf concentration in less than 1 minute.