Cell-Free DNA in Pediatric Solid Organ Transplantation Using a New Detection Method of Separating Donor-Derived from Recipient Cell-Free DNA Evgenia Preka , a,b, * ,† Drew Ellershaw , c,† Natalie Chandler , c Helena Ahlfors , c Helen Spencer , d Lyn S. Chitty , e Matthew J. Fenton , d,‡ and Stephen D. Marks a,f,‡ BACKGROUND: The use of cell-free DNA (cfDNA) as a noninvasive biomarker to detect allograft damage is expanding rapidly. However, quantifying the low frac- tion of donor-derived cfDNA (ddcfDNA) is challenging and requires a highly sensitive technique. ddcfDNA detection through unique donor single nucleotide poly- morphisms (SNPs) is a recent new approach, however there are limited data in pediatric solid organ transplant (SOT) recipients. METHODS: We developed an assay using a combination of 61 SNPs to quantify the ddcfDNA accurately using a custom R script to model for both the patient and donor genotypes requiring only a single sample from the allograft recipient. Performance of the assay was vali- dated using genomic DNA (gDNA), cfDNA and donor samples where available. RESULTS: The R “genotype-free” method gave results comparable to when using the known donor genotype. applicable to both related and unrelated pairs and can reliably measure ddcfDNA (limit of blank, below 0.12%; limit of detection, above 0.25%; limit of quanti- fication 0.5% resulting in 84% accuracy). 159 pediatric SOT recipients (kidney, heart, and lung) were tested without the need for donor genotyping. Serial sampling was obtained from 82 patients. CONCLUSION: We have developed and validated a new as- say to measure the fraction of ddcfDNA in the plasma of pediatric SOT recipients. Our method can be applicable in any donor-recipient pair without the need for donor genotyping and can provide results in 48 h at a low cost. Additional prospective studies are required to demonstrate its clinical validity in a large cohort of pediatric SOT recipients. Introduction Performing a tissue biopsy remains the gold standard for monitoring allograft health in pediatric solid organ transplant (SOT) recipients. However, allograft biopsies are invasive, therefore posing risk, especially for young recipients requiring general anesthesia with a major complication rate of 1% in percutaneous renal trans- plant biopsies (1). Moreover, around 25% of allograft biopsies yield an inadequate specimen, and the detec- tion and management of subclinical rejection is complex and largely unknown. Furthermore, there is a lack of a reliable biomarker in pediatric cardiothoracic transplant recipients (equivalent to serum creatinine) to detect allo- graft function changes (2). Despite the fact that protocol biopsies are commonly used in kidney, heart, and lung a Department of Paediatric Nephrology, Great Ormond Street Hospital for Children NHS Foundation Trust, London, UK; b Paediatric Nephrology, University Hospital Southampton NHS Foundation Trust, Southampton, UK; c London North Genomic Laboratory Hub, Great Ormond Street Hospital for Children NHS Foundation Trust, London, UK; d Cardiothoracic Unit, Great Ormond Street Hospital for Children NHS Foundation Trust, London, UK; e North Thames Genomic Laboratory Hub, Great Ormond Street Hospital for Children NHS Foundation Trust and Genetics and Genomic Medicine, UCL Great Ormond Street Institute of Child Health; f University College London Great Ormond Street Institute of Child Health, NIHR Great Ormond Street Hospital Biomedical Research Centre, London, UK. * Address correspondence to this author at: Paediatric Nephrology, University Hospital Southampton NHS Foundation Trust, Southampton, UK. Fax þ44-2381205230; e-mail evgenia.preka@gmail.com † These authors have equally contributed to this work. ‡ These authors have equally contributed. Evgenia Preka (ORCID ID: 0000-0001-7130-0439)- Evgenia.preka@gmail.com Drew Ellershaw (ORCID ID: 0000-0002-8803-2490)- Drew.Ellershaw@gosh.nhs.uk Natalie Chandler (ORCID ID: 0000-0003-1396-0740)- Natalie.Chandler@gosh.nhs.uk Helena Ahlfors (ORCID ID: 0000-0002-8183-0895)- Helena.Ahlfors@gosh.nhs.uk Helen Spencer (ORCID ID: 0000-0003-2474-3290)- Helen.Spencer@gosh.nhs.uk Lyn S. Chitty (ORCID ID: 0000-0002-4857-7138)- l.chitty@ucl.ac.uk Matthew J. Fenton (ORCID ID: 0000-0001-9757-7954)- Matthew.Fenton@gosh.nhs.uk Stephen D. Marks (ORCID ID: 0000-0001-9850-8352)- Stephen.Marks@gosh.nhs.uk Received January 16, 2020; accepted July 6, 2020. DOI: 10.1093/clinchem/hvaa173 V C American Association for Clinical Chemistry 2020. All rights reserved. For permissions, please email: journals.permissions@oup.com. 1300 Clinical Chemistry 66:10 Molecular Diagnostics and Genetics 1300–1309 (2020) Downloaded from https://academic.oup.com/clinchem/article/66/10/1300/5901070 by guest on 27 April 2023