307.4 A novel multiplex digital PCR (mdPCR) assay for the detection of donor-derived cell-free DNA in pediatric transplant recipients
Monday March 27, 2023 from 10:00 to 11:00
Hill Country CD
Presenter

Lee Ann Baxter-Lowe, United States

Professor

Pathology and Laboratory Medicine

USC and CHLA

Abstract

A novel multiplex digital PCR (mdPCR) assay for the detection of donor-derived cell-free DNA in pediatric transplant recipients

Rebecca L. Edwards1, Johanna E. Takach2, Michael J. McAndrew 2, Jondavid Menteer3,4, Rachel M. Lestz3,5, Douglas Whitman2, Lee Ann Baxter-Lowe1,3.

1Department of Pathology & Laboratory Medicine, Children's Hospital Los Angeles, Los Angeles, CA, United States; 2Luminex Corporation, A DiaSorin Company, Austin, TX, United States; 3Keck School of Medicine, University of Southern California , Los Angeles, CA, United States; 4Division of Cardiology, Children's Hospital Los Angeles, Los Angeles, CA, United States; 5Division of Nephrology, Children's Hospital Los Angeles, Los Angeles, CA, United States

Introduction: Prolonging graft survival is especially important in pediatric solid organ transplantation because the patient's potential lifespan generally exceeds graft survival. Donor-derived cell-free DNA (DD-cfDNA) is increasingly used as a non-invasive tool for monitoring graft health. Barriers to taking full advantage of this biomarker in pediatric patients include relativity large blood volume requirements, long turnaround time, and high cost. Our goal is to develop a lower-cost DD-cfDNA test that is suited to smaller blood samples from small children, and that can provide same-day results at the transplant center.
Methods: This multiplex assay can use a panel of novel probes (Luminex, Austin, TX) to quantify up to 15 targets in a single reaction when used with the QuantStudio Absolute Q Digital PCR system (Thermo Fisher Scientific, San Jose, CA). This system utilizes a microfluidic array-based plate to separate each reaction into more than 20,000 partitions. An assay with up to 16 samples can be completed in less than 3 hrs. The amount of target present is calculated using Poisson statistics and data is analyzed with a custom R package. The performance characteristics of a prototype assay with 12 probes were determined using artificial mixtures of sheared human genomic DNA (3.3 ng to 33.3 ng). DD-cfDNA levels from blood collected from pediatric kidney and heart recipients was also quantified.
Results: This prototype assay achieved absolute quantification of DNA copies and could discriminate donor and recipient cell-free DNA. With a sample input of 10,000 human genome equivalents (hge), the limit of blank (LoB) for 7/12 optimized probes was 0 – 0.02% and the analytical limit of detection (LoD) was 0 – 0.03%. When testing 1,000 hge, for 7/12 optimized probes LoB was 0 – 0.18% and the analytical was LoD 0 – 0.30%. Intra- and inter-laboratory reproducibility was demonstrated. The assay was used to measure DD-cfDNA in samples from patients with stable graft function and those experiencing rejection. Results showed that high levels of DD-cfDNA were consistent with clinical observations and that same-day results are feasible. Factors that influenced assay performance included DNA input and probe design.
Conclusion: Our study demonstrates the advantages of this approach for DD-cfDNA detection, including absolute quantification, high sensitivity, ease of use, same-day results, and a relatively low cost. Further development of this method may substantially improve patient management, with potential to prolong patient and graft survival.

GoFARR Fund Project 8031000-00013350.


Lectures by Lee Ann Baxter-Lowe


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