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
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 analytica...
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| creator | North, Paula E Ziegler, Emily Mahnke, Donna K Stamm, Karl D Thomm, Angela Daft, Paul Goetsch, Mary Liang, Huan Ling Baker, Maria Angeles Vepraskas, Adam Rosenau, Chris Dasgupta, Mahua Simpson, Pippa Mitchell, Michael E Tomita-Mitchell, Aoy |
| description | 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. |
| doi_str_mv | 10.1371/journal.pone.0227385 |
| format | Article |
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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.</description><identifier>ISSN: 1932-6203</identifier><identifier>EISSN: 1932-6203</identifier><identifier>DOI: 10.1371/journal.pone.0227385</identifier><identifier>PMID: 31929557</identifier><language>eng</language><publisher>United States: Public Library of Science</publisher><subject>Adolescent ; Adult ; Antibodies ; Apoptosis ; Biology and Life Sciences ; Biomarkers - blood ; Biopsy ; Blood ; Cell-Free Nucleic Acids - blood ; Child ; Child, Preschool ; Children ; Deoxyribonucleic acid ; DNA ; DNA fragmentation ; Female ; Fragmentation ; Genotypes ; Genotyping ; Graft Rejection ; Heart ; Heart Transplantation ; Humans ; Infant ; Infants ; Injury analysis ; Linearity ; Male ; Mathematical analysis ; Medicine and Health Sciences ; Multiplexing ; Next-generation sequencing ; Optimization ; Patients ; Pediatrics ; Quality assurance ; Rejection ; Research and Analysis Methods ; Sensitivity analysis ; Single-nucleotide polymorphism ; Tissue Donors ; Transplants - metabolism ; Vitamin E ; Young Adult</subject><ispartof>PloS one, 2020-01, Vol.15 (1), p.e0227385-e0227385</ispartof><rights>2020 North et al. This is an open access article distributed under the terms of the Creative Commons Attribution License: http://creativecommons.org/licenses/by/4.0/ (the “License”), which permits unrestricted use, distribution, and reproduction in any medium, provided the original author and source are credited. Notwithstanding the ProQuest Terms and Conditions, you may use this content in accordance with the terms of the License.</rights><rights>2020 North et al 2020 North et al</rights><lds50>peer_reviewed</lds50><oa>free_for_read</oa><woscitedreferencessubscribed>false</woscitedreferencessubscribed><citedby>FETCH-LOGICAL-c526t-396d00c626310a0af434d65d1e553bebaf8cae65b31b57ba2154f45300a593b63</citedby><cites>FETCH-LOGICAL-c526t-396d00c626310a0af434d65d1e553bebaf8cae65b31b57ba2154f45300a593b63</cites><orcidid>0000-0002-3996-1277 ; 0000-0002-7746-7528 ; 0000-0002-7844-7326</orcidid></display><links><openurl>$$Topenurl_article</openurl><openurlfulltext>$$Topenurlfull_article</openurlfulltext><thumbnail>$$Tsyndetics_thumb_exl</thumbnail><linktopdf>$$Uhttps://www.ncbi.nlm.nih.gov/pmc/articles/PMC6957190/pdf/$$EPDF$$P50$$Gpubmedcentral$$Hfree_for_read</linktopdf><linktohtml>$$Uhttps://www.ncbi.nlm.nih.gov/pmc/articles/PMC6957190/$$EHTML$$P50$$Gpubmedcentral$$Hfree_for_read</linktohtml><link.rule.ids>230,314,727,780,784,864,885,2100,2926,23865,27923,27924,53790,53792,79371,79372</link.rule.ids><backlink>$$Uhttps://www.ncbi.nlm.nih.gov/pubmed/31929557$$D View this record in MEDLINE/PubMed$$Hfree_for_read</backlink></links><search><contributor>Tchantchaleishvili, Vakhtang</contributor><creatorcontrib>North, Paula E</creatorcontrib><creatorcontrib>Ziegler, Emily</creatorcontrib><creatorcontrib>Mahnke, Donna K</creatorcontrib><creatorcontrib>Stamm, Karl D</creatorcontrib><creatorcontrib>Thomm, Angela</creatorcontrib><creatorcontrib>Daft, Paul</creatorcontrib><creatorcontrib>Goetsch, Mary</creatorcontrib><creatorcontrib>Liang, Huan Ling</creatorcontrib><creatorcontrib>Baker, Maria Angeles</creatorcontrib><creatorcontrib>Vepraskas, Adam</creatorcontrib><creatorcontrib>Rosenau, Chris</creatorcontrib><creatorcontrib>Dasgupta, Mahua</creatorcontrib><creatorcontrib>Simpson, Pippa</creatorcontrib><creatorcontrib>Mitchell, Michael E</creatorcontrib><creatorcontrib>Tomita-Mitchell, Aoy</creatorcontrib><title>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</title><title>PloS one</title><addtitle>PLoS One</addtitle><description>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.</description><subject>Adolescent</subject><subject>Adult</subject><subject>Antibodies</subject><subject>Apoptosis</subject><subject>Biology and Life Sciences</subject><subject>Biomarkers - blood</subject><subject>Biopsy</subject><subject>Blood</subject><subject>Cell-Free Nucleic Acids - blood</subject><subject>Child</subject><subject>Child, Preschool</subject><subject>Children</subject><subject>Deoxyribonucleic acid</subject><subject>DNA</subject><subject>DNA fragmentation</subject><subject>Female</subject><subject>Fragmentation</subject><subject>Genotypes</subject><subject>Genotyping</subject><subject>Graft Rejection</subject><subject>Heart</subject><subject>Heart Transplantation</subject><subject>Humans</subject><subject>Infant</subject><subject>Infants</subject><subject>Injury analysis</subject><subject>Linearity</subject><subject>Male</subject><subject>Mathematical analysis</subject><subject>Medicine and Health Sciences</subject><subject>Multiplexing</subject><subject>Next-generation sequencing</subject><subject>Optimization</subject><subject>Patients</subject><subject>Pediatrics</subject><subject>Quality assurance</subject><subject>Rejection</subject><subject>Research and Analysis Methods</subject><subject>Sensitivity analysis</subject><subject>Single-nucleotide polymorphism</subject><subject>Tissue Donors</subject><subject>Transplants - 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Academic</collection><collection>PubMed Central (Full Participant titles)</collection><collection>DOAJ Directory of Open Access Journals</collection><jtitle>PloS one</jtitle></facets><delivery><delcategory>Remote Search Resource</delcategory><fulltext>fulltext</fulltext></delivery><addata><au>North, Paula E</au><au>Ziegler, Emily</au><au>Mahnke, Donna K</au><au>Stamm, Karl D</au><au>Thomm, Angela</au><au>Daft, Paul</au><au>Goetsch, Mary</au><au>Liang, Huan Ling</au><au>Baker, Maria Angeles</au><au>Vepraskas, Adam</au><au>Rosenau, Chris</au><au>Dasgupta, Mahua</au><au>Simpson, Pippa</au><au>Mitchell, Michael E</au><au>Tomita-Mitchell, Aoy</au><au>Tchantchaleishvili, Vakhtang</au><format>journal</format><genre>article</genre><ristype>JOUR</ristype><atitle>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</atitle><jtitle>PloS one</jtitle><addtitle>PLoS One</addtitle><date>2020-01-01</date><risdate>2020</risdate><volume>15</volume><issue>1</issue><spage>e0227385</spage><epage>e0227385</epage><pages>e0227385-e0227385</pages><issn>1932-6203</issn><eissn>1932-6203</eissn><abstract>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.</abstract><cop>United States</cop><pub>Public Library of Science</pub><pmid>31929557</pmid><doi>10.1371/journal.pone.0227385</doi><orcidid>https://orcid.org/0000-0002-3996-1277</orcidid><orcidid>https://orcid.org/0000-0002-7746-7528</orcidid><orcidid>https://orcid.org/0000-0002-7844-7326</orcidid><oa>free_for_read</oa></addata></record> |
| fulltext | fulltext |
| identifier | ISSN: 1932-6203 |
| ispartof | PloS one, 2020-01, Vol.15 (1), p.e0227385-e0227385 |
| issn | 1932-6203 1932-6203 |
| language | eng |
| recordid | cdi_plos_journals_2337055336 |
| source | MEDLINE; DOAJ Directory of Open Access Journals; Public Library of Science (PLoS); EZB-FREE-00999 freely available EZB journals; PubMed Central; Free Full-Text Journals in Chemistry |
| subjects | Adolescent Adult Antibodies Apoptosis Biology and Life Sciences Biomarkers - blood Biopsy Blood Cell-Free Nucleic Acids - blood Child Child, Preschool Children Deoxyribonucleic acid DNA DNA fragmentation Female Fragmentation Genotypes Genotyping Graft Rejection Heart Heart Transplantation Humans Infant Infants Injury analysis Linearity Male Mathematical analysis Medicine and Health Sciences Multiplexing Next-generation sequencing Optimization Patients Pediatrics Quality assurance Rejection Research and Analysis Methods Sensitivity analysis Single-nucleotide polymorphism Tissue Donors Transplants - metabolism Vitamin E Young Adult |
| title | 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 |
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