Integrated System for FFPE Sample Preparation

FFPE 样品制备集成系统

• 批准号: 8833602
• 负责人: Gregory John Kellogg
• 金额: $ 16.36万
• 依托单位: COVARIS, INC.
• 依托单位国家: 美国
• 财政年份: 2014
• 资助国家: 美国
• 起止时间: 2014-09-26 至 2015-08-31
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/8833602
• 关键词: Acoustics; Adhesives; Animals; Automation; Benchmarking; Binding; Biocompatible Materials; Biological Assay; Biological Markers; Biopsy Specimen; Buffers; Caliber; Caring; Chemicals; Complex; Computers; Custom; DNA; DNA Sequence; DNA amplification; Development; Devices; Diagnosis; Digestion; Disease; Disease Progression; Endopeptidase K; Failure; Film; Fluorescence; Fluorescent Dyes; Formalin; Freezing; Frequencies; Future; Gases; Genetic; Genome; Geometry; Hair; Hand; Healthcare; Heating; Human; Individual; Lead; Length; Liquid substance; Malignant Neoplasms; Manuals; Mass Spectrum Analysis; Measures; Mechanics; Mediating; Methods; Metric; Microfluidics; Microscope; Modeling; Modification; Molds; Molecular; Monitor; Nucleic Acids; Organic solvent product; Outcome; Paraffin; Paraffin Embedding; Particle Size; Patients; Performance; Phase; Plastics; Polymerase; Positioning Attribute; Preparation; Process; Proteins; Proteome; Protocols documentation; Reading; Reagent; Recording of previous events; Research; Research Personnel; Residual state; Running; Sampling; Solid; Solutions; Source; Staging; Structure; Surface; System; Techniques; Technology; Testing; Time; Tissue Sample; Tissues; Transducers; Tumor Tissue; Ultrasonics; Ultraviolet Rays; Vacuum; Vacuum Pumps; Waxes; Work; anticancer research; base; cancer diagnosis; cancer genome; chemical bond; computerized; cost effective; design; genetic analysis; improved; instrument; instrumentation; magnetic beads; next generation sequencing; novel; pressure; prototype; public health relevance; scale up; seal; sound; tumor

DESCRIPTION (provided by applicant): Many samples taken from individuals and animals are stored by treating them with chemical, formalin, and then encasing them in paraffin or wax; these are called formalin fixed paraffin encased (FFPE) samples. Usually this storage is very good for preserving them for observation under microscopes, but it presents some problems in terms of analysis of their DNA. Genetic analyses are important because many of these preserved samples are biopsies of cancer tissue, and cancers are diseases brought on by changes in the DNA. By understanding the genetic content of these samples, we can look for specific DNA sequences that can be used to diagnose cancer. We can also use the information in these samples to determine what therapies might work best, and to measure how well the therapies applied are working. There are some instruments and kits of reagents available for getting at the DNA in FFPE samples, but they are usually complicated. Often they use dangerous chemicals. They are inconsistent in the quality of DNA extracted, which can sometimes end up broken into small pieces or bound with proteins that make them unusable for genetic analysis techniques like sequencing, where the order of the different components of DNA are read out. Often they just extract too little of the DNA. Adaptive Focused Acoustics (AFA) is an ultrasonic technology that focuses sound energy onto small volumes. It is used for many processes like breaking up tissue samples to extract proteins and DNA and grinding solids into uniformly-sized particles. Recently AFA was applied to FFPE samples in a process performed manually. The process removes the paraffin and breaks the tissue down to extract the DNA. It provides 2-3 times as much DNA as other methods, and that DNA is of higher quality. This project will develop a system that uses AFA for FFPE samples with a special instrument and a special plastic cartridge. A vacuum pump on the instrument will move the liquids in the cartridge, and AFA will remove the wax, extract the DNA, and mix the liquids and reagents in the cartridge. The DNA that AFA removes from samples will also be purified by binding to little magnetic beads, which are held in place by magnets on the machine. This makes the extracted DNA ready for use in other applications, like DNA sequencing. This instrument and cartridge will provide a reliable, reproducible, and labor-saving way to use FFPE samples that are very valuable and sometimes irreplaceable and that can be used for research or by doctors in diagnosing and treating patients. We will do this by breaking down the whole process into small pieces and developing those individually. The pieces are the extraction of DNA using AFA; chemical treatment to remove proteins and to break any chemical bonds between the DNA and itself or remaining protein contamination; and purifying the DNA. The performance of each step will be compared to the manual method. The ways we will measure the performance include determining just how much DNA there is by adding a fluorescent dye and measuring the fluorescence; measuring the length of the DNA molecules (which tells us if they are too broken up to be used for other applications); measuring the contamination by residual protein by seeing how much UV light is absorbed by the DNA solutions; and finally by using a DNA amplification method called quantitative PCR that only works well if the DNA is intact and not bound to other contaminants in the sample. We will also sequence a few samples that have been processed by the prototype. When all of the pieces have been developed individually, they will be put together. For the project, the cartridge to do this work will be mae using a computerized milling or cutting machine that can make high quality models of structures with features of size less than that of the diameter of a human hair. When the cartridge is made into a product it will be made from molded plastic. The instrument will be run by a computer and modified from an existing Covaris AFA instrument. If this project is successful, the next phase will be to solve manufacturing problems, including storing reagents on the cartridge, and scaling the cartridge up to many samples.

描述（由申请人提供）：许多从个人和动物身上采集的样本都是通过用化学品、福尔马林处理，然后用石蜡或蜡包裹来储存的；这些样品称为福尔马林固定石蜡包裹 (FFPE) 样品。通常这种存储方式非常适合保存它们以便在显微镜下观察，但它在 DNA 分析方面带来了一些问题。遗传分析很重要，因为许多保存的样本都是癌症组织的活组织检查，而癌症是由 DNA 变化引起的疾病。通过了解这些样本的遗传内容，我们可以寻找可用于诊断癌症的特定 DNA 序列。我们还可以使用这些样本中的信息来确定哪些疗法可能最有效，并衡量所采用的疗法的效果如何。 有一些仪器和试剂盒可用于获取 FFPE 样品中的 DNA，但它们通常很复杂。他们经常使用危险化学品。它们提取的 DNA 质量不一致，有时最终会破碎成小片段或与蛋白质结合，使其无法用于测序等遗传分析技术，测序是读出 DNA 不同成分的顺序。通常他们提取的 DNA 太少。 自适应聚焦声学 (AFA) 是一种超声波技术，可将声能聚焦到小体积上。它用于许多过程，例如分解组织样本以提取蛋白质和 DNA，以及将固体研磨成大小均匀的颗粒。最近，AFA 被应用于手动执行的 FFPE 样品。该过程去除石蜡并分解组织以提取 DNA。它提供的 DNA 数量是其他方法的 2-3 倍，并且 DNA 质量更高。 该项目将开发一个系统，使用 AFA 处理 FFPE 样品，并配有特殊仪器和特殊塑料盒。仪器上的真空泵将移动卡盒中的液体，AFA 将去除蜡，提取 DNA，并将卡盒中的液体和试剂混合。 AFA 从样本中去除的 DNA 也将通过与小磁珠结合来纯化，这些磁珠由机器上的磁铁固定到位。这使得提取的 DNA 可以用于其他应用，例如 DNA 测序。该仪器和试剂盒将为使用 FFPE 样品提供可靠、可重复且省力的方式，这些样品非常有价值，有时是不可替代的，可用于研究或供医生诊断和治疗患者。 我们将通过将整个过程分解为小块并单独开发来实现这一点。这些片段是使用 AFA 提取的 DNA；化学处理以去除蛋白质并破坏 DNA 与其自身之间的任何化学键或剩余的蛋白质污染物；并纯化DNA。每个步骤的性能将与手动方法进行比较。我们衡量性能的方法包括通过添加荧光染料并测量荧光来确定 DNA 的含量；测量 DNA 分子的长度（这可以告诉我们它们是否过于破碎而无法用于其他应用）；通过观察 DNA 溶液吸收了多少紫外线来测量残留蛋白质的污染；最后，使用称为定量 PCR 的 DNA 扩增方法，该方法只有在 DNA 完整且不与样品中的其他污染物结合的情况下才能正常工作。我们还将对原型机处理过的一些样本进行测序。当所有部件都单独开发后，它们将被组合在一起。 对于该项目，完成这项工作的墨盒将使用计算机铣床或切割机，该机器可以制作高质量的结构模型，其尺寸特征小于人类头发的直径。当墨盒被制成产品时，它将由模制塑料制成。该仪器将由计算机运行，并由现有的 Covaris AFA 仪器进行修改。 如果该项目成功，下一阶段将解决制造问题，包括将试剂存储在卡盒上，以及将卡盒扩大到容纳许多样品。