Integrated System for FFPE Sample Preparation

Period of Performance: 07/01/2016 - 06/30/2017

$1.23MM

Phase 2 SBIR

Recipient Firm

Covaris, Inc.
WOBURN, MA 01801
Principal Investigator

Abstract

Summary Many samples taken from individuals and animals are stored by treating them with a 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 someproblems in terms of analysis of their DNA. Genetic analyses are important because many of these preservedsamples are biopsies of cancer tissue, and cancers are diseases brought on by changes in the DNA. Byunderstanding the genetic content of these samples, we can look for specific DNA sequences that can be usedto diagnose cancer. We can also use the information in these samples to determine what therapies might workbest, 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, butthey are usually complicated. Often they use dangerous chemicals. They are inconsistent in the quality ofDNA extracted, which can sometimes end up broken into small pieces or bound with proteins that make themunusable for genetic analysis techniques like sequencing, where the order of the different components of DNAare read out. Often they just extract too little of the DNA. And they require significant hands-on time. Adaptive Focused Acoustics (AFA) is an ultrasonic technology that focuses sound energy onto smallvolumes. It is used for many processes like breaking up tissue samples to extract proteins and DNA andgrinding solids into uniformly-sized particles. Recently AFA was applied to FFPE samples in a processperformed manually. The process removes the paraffin and breaks the tissue down to extract the DNA. Itprovides 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 specialplastic cartridge. Air pressure on the instrument will move the liquids in the cartridge, and AFA will remove thewax, extract the DNA, and mix the liquids and reagents in the cartridge. The DNA that AFA removes fromsamples will also be purified by binding to little magnetic beads, which are held in place by magnets on themachine. This makes the extracted DNA ready for use in other applications, like DNA sequencing. Thisinstrument and cartridge will provide a reliable, reproducible, and labor-saving way to use FFPE samples thatare very valuable and sometimes irreplaceable and that can be used for research or by doctors in diagnosingand 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 anychemical bonds between the DNA and itself or remaining protein contamination; and purifying the DNA. Theperformance of each step will be compared to the manual method. The ways we will measure theperformance include determining just how much DNA there is by adding a fluorescent dye and measuring thefluorescence; measuring the length of the DNA molecules (which tells us if they are too broken up to be usedfor other applications); measuring the contamination by residual protein by seeing how much UV light isabsorbed by the DNA solutions; and finally by using a DNA amplification method called quantitative PCR thatonly works well if the DNA is intact and not bound to other contaminants in the sample. We will also sequencea few samples that have been processed by the prototype. When all of the pieces have been developedindividually, they will be put together. For the project, the cartridge to do this work will be made using a computerized milling or cutting machinethat can make high quality models of structures with features of size less than that of the diameter of a humanhair. When the cartridge is made into a product it will be made from molded plastic. The instrument will be runby 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 storingreagents on the cartridge, and scaling the cartridge up to many samples.