Cell-free DNA donor fraction analysis in pediatric and adult heart transplant patients by multiplexed allele-specific quantitative PCR: Validation of a rapid and highly sensitive clinical test for stratification of rejection probability

Autor: Emily Ziegler, Chris Rosenau, Adam Vepraskas, Michael E. Mitchell, Maria Angeles Baker, Angela Thomm, Huan Ling Liang, Mary Goetsch, Karl Stamm, Aoy Tomita-Mitchell, Paul G. Daft, Mahua Dasgupta, Paula E. North, Pippa Simpson, Donna K. Mahnke
Rok vydání: 2019
Předmět:
0301 basic medicine
Oncology
Graft Rejection
Male
Cardiovascular Procedures
Physiology
Biopsy
Transplants
DNA fragmentation
030204 cardiovascular system & hematology
Biochemistry
White Blood Cells
0302 clinical medicine
Animal Cells
Genotype
Medicine and Health Sciences
Child
Multidisciplinary
medicine.diagnostic_test
Genomics
Cardiac Transplantation
Tissue Donors
3. Good health
Transplant rejection
Body Fluids
Nucleic acids
Real-time polymerase chain reaction
Blood
Cell-free fetal DNA
Child
Preschool
Medicine
Female
Anatomy
Cellular Types
Cell-Free Nucleic Acids
Research Article
Adult
medicine.medical_specialty
Genotyping
Adolescent
Science
Immune Cells
Immunology
Surgical and Invasive Medical Procedures
Research and Analysis Methods
Blood Plasma
03 medical and health sciences
Young Adult
Alu Elements
Internal medicine
medicine
Genetics
SNP
Humans
Repeated Sequences
Molecular Biology Techniques
Molecular Biology
Transplantation
Blood Cells
business.industry
Infant
Biology and Life Sciences
Organ Transplantation
DNA
Cell Biology
medicine.disease
030104 developmental biology
Heart Transplantation
business
Biomarkers
Zdroj: PLoS ONE
PLoS ONE, Vol 15, Iss 1, p e0227385 (2020)
ISSN: 1932-6203
Popis: Lifelong noninvasive rejection monitoring in heart transplant patients is a critical clinical need historically poorly met in adults and unavailable for children and infants. Cell-free DNA (cfDNA) donor-specific fraction (DF), a direct marker of selective donor organ injury, is a promising analytical target. Methodological differences in sample processing and DF determination profoundly affect quality and sensitivity of cfDNA analyses, requiring specialized optimization for low cfDNA levels typical of transplant patients. Using next-generation sequencing, we previously correlated elevated DF with acute cellular and antibody-mediated rejection (ACR and AMR) in pediatric and adult heart transplant patients. However, next-generation sequencing is limited by cost, TAT, and sensitivity, leading us to clinically validate a rapid, highly sensitive, quantitative genotyping test, myTAIHEART®, addressing these limitations. To assure pre-analytical quality and consider interrelated cfDNA measures, plasma preparation was optimized and total cfDNA (TCF) concentration, DNA fragmentation, and DF quantification were validated in parallel for integration into myTAIHEART reporting. Analytical validations employed individual and reconstructed mixtures of human blood-derived genomic DNA (gDNA), cfDNA, and gDNA sheared to apoptotic length. Precision, linearity, and limits of blank/detection/quantification were established for TCF concentration, DNA fragmentation ratio, and DF determinations. For DF, multiplexed high-fidelity amplification followed by quantitative genotyping of 94 SNP targets was applied to 1168 samples to evaluate donor options in staged simulations, demonstrating DF call equivalency with/without donor genotype. Clinical validation studies using 158 matched endomyocardial biopsy-plasma pairs from 76 pediatric and adult heart transplant recipients selected a DF cutoff (0.32%) producing 100% NPV for ≥2R ACR. This supports the assay’s conservative intended use of stratifying low versus increased probability of ≥2R ACR. myTAIHEART is clinically validated for heart transplant recipients ≥2 months old and ≥8 days post-transplant, expanding opportunity for noninvasive transplant rejection assessment to infants and children and to all recipients >1 week post-transplant.
Databáze: OpenAIRE
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