An automated microfluidic platform for high-throughput newborn SCID screening

描述（由申请人提供）：本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）添加到统一新生儿筛查小组中，因此在国家筛查层面对这样一个技术平台有很大的需求。新生儿统一筛查小组增加的条件成为各州采用筛查项目的强大动力，因此低成本、高通量的解决方案是面临严重预算危机的各州的关键。除非能够实现免疫重建，否则SCID在生命的第一年或第二年普遍是致命的。众所周知，在3.5个月前接受骨髓移植治疗的患者存活率为94%。SCID婴儿在出生时或婴儿期早期没有外在的身体异常来提醒医生这种疾病的存在，因此通常只有在发生严重感染后才能做出诊断。很明显，新生儿筛查是早期诊断和最佳治疗的唯一希望，对于大多数患有这种疾病的婴儿。目前进行新生儿筛查的方法是从婴儿身上收集干血点，然后送到实验室进行分析。虽然过去威斯康星州新生儿SCID筛查试点项目的成本估计为每次测试5-6美元，但实施该测试仍有许多前期成本。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PCR仪器需要昂贵的前期和维护成本。随后，我们坚信，用于新生儿SCID筛查的DNA分析的数字微流控平台将以非常低的成本实现自动化。在本项目中，将开发一个完整的无运动部件的TREC提取和检测数字微流控平台，用于3mm穿孔干血点。提取、纯化和浓缩DNA，对trec进行后续的热循环PCR和光学检测的所有步骤将围绕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个白化样本（代表SCID样本）进行验证，以证明正常和SCID影响斑点之间的区别。在完成这项拨款的目标后，我们相信我们将接近于拥有一个完全自动化的高通量解决方案，不仅可以用于国家实验室的SCID筛查，还可以用于其他新生儿疾病。

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.