An automated microfluidic platform for high-throughput newborn SCID screening

描述(申请人提供)：本次研究合作的总体目标是开发一种高通量数字微流控仪器，能够同时筛查数十名新生儿的严重联合免疫缺陷(SCID)。鉴于2010年1月21日，联邦新生儿和儿童遗传性疾病咨询委员会(ACHDNC)一致建议将严重联合免疫缺陷(SCID)纳入新生儿统一筛查小组，州筛查层面对这种技术平台的需求非常大。增加到统一新生儿筛查小组的条件成为各州采用筛查计划的强大动机，因此低成本、高通量的解决方案是面临严重预算危机的州的关键。除非能够实现免疫重建，否则SCID在生命的第一或第二年通常是致命的。众所周知，对于那些在3.5个月前接受这种治疗的人来说，骨髓移植的结果是94%的存活率。SCID婴儿在出生时或婴儿早期没有外在的身体异常，以提醒医生这种情况的存在，所以通常只有在严重感染发生后才能做出诊断。显然，新生儿筛查是对大多数患有这种疾病的婴儿进行早期诊断和最佳治疗的唯一希望。目前进行新生儿筛查的方法是从婴儿身上收集干燥的血迹，然后将其送到实验室进行分析。虽然过去威斯康星州试点新生儿SCID筛查计划的成本估计为每项测试5-6美元，但仍有许多与实施这项测试相关的前期成本。例如，TREC检测涉及许多DNA提取步骤，需要对人员进行高级培训。同样，高通量实时聚合酶链式反应检测需要昂贵的前期和维护成本。随后，我们坚信，用于新生儿筛查中SCID的DNA分析的数字微流控平台将以非常低的成本实现自动化。在这个项目中，将开发一个完整的TREC提取和检测数字微流控平台，没有移动部件，用于3 mm冲头干血斑。提取、纯化和浓缩DNA、对TREC执行随后的热循环PCR以及光学检测的所有步骤都将围绕ALL的核心数字微流控平台进行开发。我们将用大约100个正常的干血斑样本和大约10个白细胞减少的样本(代表SCID样本)来验证正常和SCID影响的斑点之间的区别。在完成这笔赠款中提出的目标后，我们相信我们将接近拥有一种完全自动化的高通量解决方案，该解决方案不仅可以用于州实验室的SCID筛查，而且还可以用于其他新生儿疾病的筛查。 与公共卫生相关：严重的联合免疫缺陷(更普遍地称为“泡泡病”)在生命的第一年或第二年普遍是致命的。骨髓移植可以为这种疾病带来挽救生命的免疫重建，对于在3.5个月前接受移植的婴儿来说，存活率为96%。这项提议的中心目标是开发一个完整的高通量、数字微流控DNA分析芯片实验室平台，这将导致一种低得多的、完全自动化的解决方案，用于筛查这种致命疾病的新生儿。这将降低公共卫生实验室进行这种疾病筛查的技术和资金障碍。

DESCRIPTION (provided by applicant): The overall goal of this research collaboration is to develop a high-throughput digital microfluidic instrument capable of screening dozens of newborns simultaneously for severe combined immunodeficiency (SCID). There is a significant need at the state-screening level for such a technology platform given that on January 21, 2010, a federal Advisory Committee on Heritable Disorders in Newborns and Children (ACHDNC), unanimously recommended that severe combined immunodeficiency (SCID) be added to the Uniform Newborn Screening Panel. Conditions added to the Uniform Newborn Screening Panel become a strong motivator for states to adopt screening programs, so a low-cost, high-throughput solution is a key for states facing critical budget crises. SCID is universally fatal in the first or second year of life unless immune reconstitution can be achieved. It is known that bone marrow transplantation results in a 94% survival rate for those given this treatment before 3.5 months of life. SCID infants have no outward physical abnormalities at birth or in early infancy to alert physicians that the condition exists, so the diagnosis is usually made only after serious infections develop. It is clear that newborn screening is the only hope for early diagnosis and optimal treatment for most infants with this condition. Newborn screening is currently performed by collecting dried bloodspots from infants and then sending them to a lab for analysis. While cost estimates from a past Wisconsin pilot newborn SCID screening program estimated $5-6 per test, there are still many upfront costs associated with implementing this test. For instance TREC detection involves many DNA extraction steps requiring advanced training of personnel. Likewise, high-throughput real-time PCR instrumentation requires costly upfront and maintenance costs. Subsequently, we strongly believe that a digital microfluidic platform for performing DNA analysis assays for SCID in newborn screening will enable walkaway automation at a very low cost. In this project, a complete TREC extraction and detection digital microfluidic platform with no moving parts will be developed for use with 3mm punch dried blood spots. All the steps for extracting, purifying, and concentrating DNA, performing subsequent thermocycled PCR for TRECs, and optical detection will be developed around ALL's core digital microfluidic platform. We will validate with about 100 normal dried blood spot samples and about 10 leukoreduced samples (representing SCID samples) to demonstrate discrimination between normal and SCID affected spots. Upon completion of the proposed aims in this grant, we believe we will be near to a having a completely automated high-throughput solution that can not only be leveraged for SCID screening in the state labs, but for other newborn disorders, as well. PUBLIC HEALTH RELEVANCE: Severe combined immunodeficiency (more popularly known as "bubble boy disease") is universally fatal in the first or second year of life. Bone marrow transplantation can result in life-saving immune reconstitution for this condition with a 96% survival rate for the infants that undergo transplantation before 3.5 months of life. The central goal of this proposal is to develop a complete high-throughput, digital microfluidic DNA analysis lab-on- a-chip platform that would lead to a significantly inexpensive and completely automated solution for screening newborns for this lethal condition. This would lower the technology and financial barrier for public health laboratories to undertake screening for this condition.