Device for measuring blood concentrations of unbound bilirubin in neonates

描述（由申请人提供）：该项目的总体目标是开发一种检测未结合非共轭胆红素（Bf）浓度的方法，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，对于一些婴儿，胆红素的作用可能是神经毒性的，导致从可逆性听力缺陷到更严重甚至可能致命的核黄疸神经系统后遗症的缺陷。特别是极低出生体重（ELBW）婴儿的预后非常差，其中约25-30%死亡，另外25-30%有严重的神经功能缺陷（如脑瘫）。这被认为是由于胆红素神经毒性。胆红素对新生儿的神经毒性目前是通过监测血清总胆红素（TSB）来评估的，高水平的胆红素会导致光疗和/或交换输血。然而，最近的研究，特别是我们自己在ELBW婴儿中进行的一项新研究表明，使用TSB不能检测到潜在的胆红素毒性（高假阴性），并且基于TSB水平使用光疗也不能改善这些婴儿非常糟糕的结果。相反，对TSB水平极高（约25毫克/分升）的足月婴儿（换血阈值）的研究最近发现，多达80%的婴儿可能没有必要进行换血（本身与约2%的死亡率相关）（高假阳性）。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，因为光疗治疗的门槛较低。TSB不能作为诊断的原因是胆红素是高度不溶性的，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部分才能穿过血脑屏障，引起核毒性。TSB不能解释改变胆红素-白蛋白结合的许多因素，因此Bf。改善预后和减少不必要的治疗所需要的是一种准确和简单的测量Bf的方法。该项目的具体目标是，在第一阶段，开发一种简单的一次性药筒，其中包含我们最近开发的荧光标记突变脂肪酸结合蛋白（FABPs），该蛋白可以通过结合Bf时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未稀释的全血中的Bf。在第二阶段，我们将开发一种比例荧光仪器，优化为

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.