Fig 1.
myTAIHEART 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.
Table 1.
Summary statistics across 94 targets for minor allele frequency (MAF) as presented in the GnomAD [77] 2019 database.
Table 2.
Subgroups from the 1000 genomes project [78] providing further distinction in the African/African American population.
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.
Table 3.
Passing-Bablock linear regression results, Method 1 (with donor genotype) algorithm vs Method 2 (without donor genotype) algorithm.
Fig 5.
Effect of RT whole blood storage duration (0–24 hr) in Streck BCT tubes on myTAIHEART 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.
Table 4.
Precision results, ALU115 qPCR.
Table 5.
Precision results, ALU247 qPCR.
Table 6.
Limit of detection results, ALU115 and ALU247.
Table 7.
Limit of quantitation results, myTAIHEART DNA fragmentation assay.
Fig 6.
DNA fragmentation assay linearity results.
(A) ALU115 (B) ALU247, see Table 8 for statistical data.
Table 8.
Linearity fit values, myTAIHEART DNA fragmentation assay.
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.
Table 9.
TCF concentration: Precision averages, standard deviations, and %CV.
Table 10.
TCF quantification, limit of detection calculation.
Table 11.
Limit of quantitation results, TCF.
Fig 10.
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.
Table 12.
Limit of detection calculations, quantitative genotyping (DF).
Table 13.
%CV of DF measurement for reconstructed samples at three DF levels.
Table 14.
Precision of DF determination across days, operators, reagent lots, and equipment lines for three reconstructed samples (low, medium, and high DF level).
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 myTAIHEART 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 myTAIHEART assay, was 100.00% for grade 2R or higher ACR, with 100.00% sensitivity and 75.48% specificity.