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Non-Invasive Assay Improves Surveillance of Solid-Organ Transplants

By LabMedica International staff writers
Posted on 28 Oct 2016
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Image: The Access Array microfluidic system generates next-generation sequencing (NGS) (Photo courtesy of Fluidigm).
Image: The Access Array microfluidic system generates next-generation sequencing (NGS) (Photo courtesy of Fluidigm).
Patients who have received a solid organ transplant require lifelong immunosuppressive therapy. The threat of transplant rejection due to insufficient drug therapy must be balanced against increased risks of infections and cancer from excessive immunosuppression.

A significant unmet need exists for non-invasive diagnostic tools to monitor transplant recipients, especially for early detection of active injury and rejection. A new non-invasive test has been developed that measures donor-derived cell-free DNA (dd-cfDNA) in plasma that has the potential to reduce complications and rejection, improving outcomes in transplant recipients.

Scientists working at CareDx, Inc, (Brisbane, CA, USA) and their colleagues collected blood samples healthy, non-transplant volunteers and transplant patients. CfDNA was extracted from thawed plasma using the Circulating Nucleic Acid kit and concentrated by centrifugal vacuum concentration. For the dd-cfDNA assay to be applicable to different transplant recipients without requiring separate genotyping of either donor or recipient, single nucleotide polymorphisms (SNPs) were selected to ensure that the same SNP panel could be used for individuals with different ancestral heritages.

The dd-cfDNA assay is based on targeted amplification of DNA regions harboring 266 SNPs and the measurement by next generation sequencing (NGS) of each allele contribution at each SNP position. Preamplified material was further amplified using 48 limited complexity multiplexes (1 to 11 targets per reaction) on the Access Array microfluidic system. Index sequences and Illumina sequencing adapters were added to each sample DNA by polymerase chain reaction (PCR), and the sample was qualified and quantified by capillary electrophoresis.

The team reported that data are presented from a multi-center heart transplantation study showing that dd-cfDNA was, on average, three-fold higher in patients experiencing acute rejection than in stable transplant recipients without acute rejection. A decrease in dd-cfDNA levels upon successful anti-rejection treatment was also observed. The assay quantified the fraction of dd-cfDNA in both unrelated and related donor-recipient pairs. The dd-cfDNA assay can reliably measure dd-cfDNA (limit of blank, 0.10%; limit of detection, 0.16%; limit of quantification, 0.20%) across the linear quantifiable range (0.2% to 16%) with across-run CVs of 6.8%. Precision was also evaluated for independently processed clinical sample replicates and is similar to across-run precision.

Marica Grskovic, PhD, Associate Director, R&D, CareDx, Inc, and lead investigator of the study said, “dd-cfDNA is an emerging biomarker of transplanted organ injury, and the availability of a clinical-grade, analytically validated assay is critical for advancement of this biomarker toward improving the outcomes of transplant patients. These results show promise in using cfDNA not only to detect rejection, but also to monitor response to treatment. The ongoing measurement of cfDNA may allow clinicians to better personalize care, adjust immunosuppression regimens, and improve the long-term outcomes of transplant recipients.” The study was published on October 7, 2016, in The Journal of Molecular Diagnostics.

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