Abstract A042: cfDNA collection and purification improved and simplified: Inexpensive at-home device reduces large volumes of urine to a ucfdna containing stable filter
Authors: Taub Floyd E , Ray Lindsey , Barstis Elijah , Henry Charles , Arnold Kirsten 
Published: November 2024

Introduction: In order to reduce the numerous burdens venipuncture introduces, including the hard limit of sensitivity that the small sample size inflicts on all analytic methods seeking ultimate sensitivity, we have developed an inexpensive, use anywhere device, that can handle a full day's output of urine; it collects and stabilizes urine cfDNA on filters that can be transported by regular mail. Methods: Large volumes of urine are problematic for many reasons, including filter clogging. Thus, we tested many pre-filters as well as capture filters with various polyelectrolyte modifications, to capture the DNA. As degradation of cfDNA in urine is problematic, we tested methods of preserving the DNA as soon as it was voided and methods to ensure that the captured DNA was stable. We evaluated the stability of the captured DNA, the ability to elute it in the lab, and the ability to use it in downstream methods. Results - Collection: Extensive experimentation has revealed combination that can capture the cfDNA from a full void of urine without clogging. Since urine is abundant, high yield is not critical, however we observed recovery of spiked oligos of over 90%, using capture with the polyelectrolyte chitosan. An “instant dissolve” packet of additives and buffers begins stabilizing the DNA as soon as it hits the collection container. The home user then pushes the urine through an industrial syringe (which may be used as the collection device) and filter cartridge, discarding the flow through. They then cap the filter cartridge for standard mailing. Since DNase cannot bind to chitosan bound DNA, stability was anticipated; both cfDNA and spiked in oligo laggers are stable for at least four days. Multiple voids can be captured on a filter(s) during the day. Results - Elution and downstream: When the filter cartridge is received in the lab, the prefilter containing cells can be saved for gDNA and analysis of cells that may be important especially in urogenital cancer. The cfDNA capture filter is washed and eluted in under 15 min. From just 150 mls of urine, over 2 ug, vs the typical 10s of ng from other methods, is eluted. The released DNA is active in PCR, allowing the detection not only of alu and Line1 sequences but also beta-actin. In these experiments under 4 genome equivalents (the lowest amount used) detected single copy sequences. Conclusions: A cfDNA from urine device that allows at home collection, at any frequency, of any volume, in a stable easy to transport form has been created. The device allows easy transport at ambient temperature without biofluid concerns. The eluate is PCR ready with a minimum of steps and no equipment. Home collection will facilitate screening programs, monitoring of many diseases, and additional data collection in trials. For the first time daily output of cfDNA can be easily harvested and measured. Comprehensive total daily amounts and frequent longitudinal timepoints will complement current cross-sectional blood cfDNA testing and provide a more comprehensive picture. Urine also increases screening compliance.

Citation Format: Floyd E Taub, Lindsey Ray, Elijah Barstis, Charles Henry, Kirsten Arnold. cfDNA collection and purification improved and simplified: Inexpensive at-home device reduces large volumes of urine to a ucfdna containing stable filter [abstract]. In: Proceedings of the AACR Special Conference: Liquid Biopsy: From Discovery to Clinical Implementation; 2024 Nov 13-16; San Diego, CA. Philadelphia (PA): AACR; Clin Cancer Res 2024;30(21_Suppl):Abstract nr A042.