Tissue biopsy is the gold standard for assessing allograft rejection and managing treatment in solid organ transplant. However, this approach requires an invasive procedure that is expensive, requires advanced planning, and is associated with increased risks of complications. It has been reported that the survival rate of a transplanted kidney is 10-13 years for living donors and 7-9 years for deceased donors, 75% of transplanted liver will survive at least 5 years, 12.5 years for heart, and 7.8 years for bilateral lung transplant and 4.8 years for a single lung.1  However, next generation sequencing (NGS) has provided a non-invasive method of early intervention to improve allograft survival by quantifying donor-derived cell-free DNA (dd-cfDNA) in solid organ transplant and monitor chimerism post-hematopoietic stem cell transplant (HSCT).

Cell-free DNA is degraded DNA that is released in circulation during cellular apoptosis in all individuals. In transplanted patients however, cfDNA is derived from recipient and donor organs, which serves as a diagnostic biomarker post-transplant. Whole blood sample is collected, and the plasma is used to quantify the percent of dd-cfDNA relative to the total amount of circulating cfDNA. Donor-derived-cfDNA has a half-life of 10 minutes to 2.5 hours thus detection of elevated levels of donor derived cfDNA has been correlated with active allograft injury. NGS assays uses cfDNA as a biomarker by specifically targeting hundreds of informative single nucleotide polymorphisms (SNPs) to discriminate between donor and recipient cfDNA to eliminate the need for prior genotyping of the recipient or donor. 2,3

Future advancements are incorporating NGS platforms in monitoring chimerism post-HSCT.  Short tandem repeat (STR) by polymerase chain reaction (PCR) is the gold standard in quantitative chimerism analysis to monitor engraftment of donor cells. Chimerism analysis is expressed as the ratio between recipient and donor cells with a target of complete chimerism, the detection of only donor genotype. However, the limitation of lower sensitivity of the STR-PCR assay presents a barrier for timely intervention. Recent developments in NGS technologies, using peripheral blood or bone marrow, present a highly sensitive application in chimerism analysis to aid in earlier treatment intervention.4

The use of NGS assays has revolutionized detection and surveillance of allograft injury post-solid organ transplant by providing a non-invasive and sensitive approach to extend the survival of transplanted organs. NGS also provides a promising technique in improving the patient’s quality of life post-HSCT. While NGS is rapidly advancing, approaches in standardizing procedures and quality management are required. A major obstacle in NGS standardization is the lack of an established proficiency testing system. However, efforts in formulating stringent guidelines to maintain quality during assay development and validation are being led by regulatory agencies.5




  1. Lori Kurtzman. How Long Do Transplanted Organs Last?. Wexner Medical Center.2020.
  2. Dengu F. Next-Generation Sequencing Methods to Detect Donor-Derived Cell-Free DNA After Transplantation. Transplantation Reviews. 2020;34.
  3. Jackson AM, Amato-Menker C, Bettinotti M. Cell-free DNA Diagnostic in Transplantation Utilizing Next Generation Sequencing, Human Immunology. 2021; 82:850-858.
  4. Tozzo P, Delicati A, Zambello R. Chimerism Monitoring Techniques after Hematopoietic Stem Cell Transplantation: An Overview of the Last 15 years of Innovation. Diagnostics. 2021;11:621.
  5. Endrullat C, Glokler J, Franke P. Standardization and Quality Management in Next-Generation Sequencing. Applied & Translational Genomics. 2016;10:2-9.

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