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

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.

Fig 1. myTAI_HEART clinical testing workflow schematic.

Fig 2. Electropherogram image of sheared gDNA, simulating cfDNA.

An Agilent 2100 Bioanalyzer instrument and high sensitivity DNA Kit were used to demonstrate the 164 bp peak corresponding to the median distribution of gDNA sheared by ultrasonication to the size range of cfDNA of apoptotic origin. FU, fluorescence units; bp, base pairs. Peaks at 35 and 10380 bp represent lower and upper internal kit standards.

Fig 3. Alu 115bp (ALU115) and 247bp (ALU247) PCR primer designs.

Forward and reverse primers of ALU115 are indicated by green text, ALU247 primers by orange text. Brackets indicate the size of fragments (140–200 bp) generated by enzymatic apoptotic cleavage as compared to the total length of the Alu element. ALU115 primers amplify apoptotic and longer DNA fragments, while ALU247 primers only amplify sequences longer than apoptotic DNA.

Fig 4. Equivalence of the “no donor genotype” algorithm for DF determination.

Samples passing QC used in creation of the “no donor genotype” algorithm are shown (N = 1128). Inset magnifies the 0–1% range. Line shows equality.

Fig 5. Effect of RT whole blood storage duration (0–24 hr) in Streck BCT tubes on myTAI_HEART cfDNA DF.

Significant drop in plasma cfDNA DF (0.2% +/-0.05% per day, p<0.01) was observed in four manufactured BCT whole samples (each at a unique starting DF) when plasma separation was delayed by 24 hrs at RT post-phlebotomy. DF is highly sensitive to cfDNA dilution by even very low levels of leukocyte lysis prior to plasma separation. See text for methodological detail.

Fig 6. DNA fragmentation assay linearity results.

(A) ALU115 (B) ALU247, see Table 8 for statistical data.

Fig 7. Interfering substance results at 25 ng/ml total cfDNA (TCF), DNA fragmentation assay, one-way analysis of Alu ratio with connecting letters report.

At this TCF concentration, small, but statistically significant differences compared to the unspiked controls, but not compared to diluting solvent controls, were seen for Sirolimus, EDTA, and bilirubin. These findings, in concert with those at lower and higher cfDNA concentrations, indicate lack of clinically relevant effects of these substances on the DNA Fragmentation Assay (see text).

Fig 8. Quantitative effects of leukocyte lysis on Alu ratio (A) and DF (B).

Fig 9. Bioanalyzer electropherograms of patient plasma cfDNA samples.

(A) Patient sample collected and processed per TAI protocol shows predominant singlet and doublet apoptotic cfDNA peaks at 186 bp and 362 bp, respectively, without larger fragments produced by cellular lysis. (B) Human sample procured and processed by a commercial vendor with delayed centrifugation (> 24hrs) shows a small peak at 178 bp (probably apoptotic) and a large, broad peak centered at 7822 bp, consistent with origin from leukocyte lysis. In both figures, sharp peaks at 35 bp and 10380 bp are internal kit markers.

Fig 10. TCF linearity 2–1,000 ng/mL.

The TCF assay is linear from 2–1,000 ng cfDNA/mL plasma. The adjusted linear fit equation is ng/mL (y) = -0.455701 + 1.2255499*Expected ng/mL(x).

Fig 11. Distribution of TCF concentration within asymptomatic healthy heart transplant recipients (264 samples from 106 subjects).

Fig 12. Distribution of TCF concentration in included (healthy) and excluded (potentially unhealthy) cohorts of the heart transplant recipient population.

Open circles represent the 264 “healthy” samples after exclusions; grey dots represent excluded samples from potentially “unhealthy” subjects. Samples are linearly arranged along the x-axis in order of increasing TCF (total cfDNA) concentration.

Fig 13. Limit of blank distribution, DF determination by quantitative genotyping.

LoB = 0.110% using the classical nonparametric approach applied to 757 samples (see text). Note that no estimated “system noise” has been subtracted from the LoB, yielding a pure LoB reference value for DF determination.

Fig 14. Linearity of DF in three blood lot reconstruction series.

Fig 15. Interfering substance testing, quantitative genotyping, 0.6% DF reconstruction.

Fig 16. DF (%) results, TECAN extractions 1 and 2, carryover/cross-contamination testing.

Relative sample positions were maintained from extraction through the entire myTAI_HEART workflow (extraction through qGT). Green positions = high DF samples; white positions = low DF samples.

Fig 17. Receiver operating characteristic (ROC) curve, ACR 0R versus ACR 1R+2R+3R.

Area under the Curve (AUC) was a robust 0.842. Using the DF cutoff of 0.32%, NPV for grade 2R or higher ACR, the intended use of the myTAI_HEART assay, was 100.00% for grade 2R or higher ACR, with 100.00% sensitivity and 75.48% specificity.