Digital droplet PCR for rapid quantification of donor DNA in the circulation of transplant recipients as a potential universal biomarker of graft injury

J Beck, S Bierau, S Balzer, R Andag… - Clinical …, 2013 - academic.oup.com
J Beck, S Bierau, S Balzer, R Andag, P Kanzow, J Schmitz, J Gaedcke, O Moerer, JE Slotta…
Clinical chemistry, 2013academic.oup.com
BACKGROUND Cell-free DNA (cfDNA) from grafts in the circulation of transplant recipients
is a potential biomarker of rejection. Its usefulness was investigated after heart
transplantation during the maintenance phase by use of microarrays and massive parallel
sequencing of donor and recipient DNA. Disadvantages of these methods are high costs,
long turnaround times, and need for donor DNA. Therefore, we sought to develop a rapid
and cost-effective method using digital droplet PCR (ddPCR). METHODS Plasma samples …
BACKGROUND
Cell-free DNA (cfDNA) from grafts in the circulation of transplant recipients is a potential biomarker of rejection. Its usefulness was investigated after heart transplantation during the maintenance phase by use of microarrays and massive parallel sequencing of donor and recipient DNA. Disadvantages of these methods are high costs, long turnaround times, and need for donor DNA. Therefore, we sought to develop a rapid and cost-effective method using digital droplet PCR (ddPCR).
METHODS
Plasma samples were collected from stable recipients after liver (LTx, n = 10), kidney (KTx, n = 9), and heart (HTx, n = 8) transplantation as well as from 7 additional patients directly after LTx. Known single-nucleotide polymorphisms were selected for high minor allelic frequencies, of which 41 hydrolysis probe assays were established. Plasma cfDNA was preamplified, followed by conventional real-time PCR to define informative (heterologous) SNPs, which were then used for quantification (percentage) of graft-derived cfDNA (GcfDNA) using ddPCR.
RESULTS
Mean recovery was 94% (SD, 13%) with an imprecision of 4%–14% with the use of controls with 2% minor allele. GcfDNA in stable patients was <6.8% (LTx), <2.5% (KTx), and <3.4% (HTx). On the day of LTx, GcfDNA was approximately 90% and by day 10 it was <15% in complication-free LTx recipients. In 2 patients with biopsy-proven rejection, GcfDNA increased to >60%, whereas in 1 patient with cholestasis no increase was found.
CONCLUSIONS
A novel, cost-effective, rapid technique was developed to quantify GcfDNA in transplant recipients. This technique embodies a promising, potentially universal biomarker for early detection of rejection, which could enable more effective therapeutic interventions.
Oxford University Press