The selection criteria in the present case report study were noncomplicated heart transplantation (HTx) and postoperative course with later EMB confirming acute cellular rejection (ACR) or antibody-mediated rejection (AMR) at the time of blood collection. The patients were subjected to standard clinical practice. The timeline for performing EMBs is presented in Table I. Patients who failed to appear at their scheduled blood draw appointment were not included in the study. Histology, immunohistochemistry and EMB were performed according standard protocols and guidelines (13-15).

Patient 1 (male; age, 63 years) underwent orthotopic HTx (OHT) in March 2016 at the Cardio Center of the Institute for Clinical and Experimental Medicine (Prague, Czech Republic) due to dilated cardiomyopathy. Standard immunosuppression was applied. The patient received induction with rabbit antithymocyte globulin (rATG; Fresenius). The maintenance therapy consisted of tacrolimus (Astellas Pharma Europe B.V., Leiderdorp), mycophenolate mofetil (Salutas Pharma) and corticosteroids (Zentiva a.s.). The patient was postoperatively treated for 10 days with Tamiflu (F. Hoffmann-La Roche Ag) due to a positive test for influenza AB in a donor. In the first EMB, there were histological findings without evidence of cellular or antibody-mediated rejection. Echocardiography showed good heart graft function and insignificant pericardial effusion, without progression. Cardiopulmonary compensation during hospitalization was without heart rhythm disturbance. Donor 1 (male; age, 36 years) had suffered from epilepsy since childhood and had suffered brain death due to post-hypoxic brain damage after an epileptic seizure.

Patient 2 (female; age, 65 years) underwent OHT in October 2017 at the Cardio Center of the Institute for Clinical and Experimental Medicine (Prague, Czech Republic) due to congenital cardiac malformation. The patient had been affected by an atrial septal defect and underwent surgical closure in 1959 and reoperation (surgical closure with bovine pericardial patch and tricuspid valve plastic surgery) in 2011. In 2014, the patient had been hospitalized multiple times due to right-sided heart failure. In 2016, treatment with sildenafil (Mylan) was started because of the detection of significant pulmonary arterial hypertension. In March 2017, the patient was added to the waiting list for HTx. The patient suffered from comorbidities such as permanent atrial fibrillation, diabetes mellitus and dyslipidemia. Up to the 7th postoperative day, the patient was supported with inotropic milrinone for right-sided decompensation due to problematic borderline diuresis with high furosemide (FSM; Zentiva a.s.) support/preoperatively administered FSM at a dose of 1 g per day for right-sided failure, without the need for an elimination method. The patient received the same induction with rATG (Fresenius) and maintenance therapy as patient 1. Corticoid-induced deterioration of diabetes was compensated for by insulin therapy. The first three EMBs showed histological findings without evidence of cellular or antibody-mediated rejection. Echocardiography showed a good heart graft with maximum moderate tissue relaxation with inversion recovery and mild right ventricular dysfunction and treated arterial hypertension. Donor 2 (female; age, 50 years) had suffered from hypertension, hypertensive encephalopathy and hypothyroidism, and brain death had occurred as a result of intracerebral hemorrhage.

Aortic tissues for genomic DNA extraction were collected during OHT from donors and recipients from March 2016 to October 2017 at the Institute for Clinical and Experimental Medicine (Prague, Czech Republic). The screening of 48 single-nucleotide polymorphisms (SNPs; minor allele frequency range 0.4-0.5; Table SI) for identification of the homozygous status was performed using microfluidic chips (Fluidigm; cat. no. 48.48 IFC) on a BioMark^™ system (Fluidigm). The protocol was performed according to manufacturer's recommendations stated in the Fluidigm SNP Genotyping User Guide (PN68000098 REV.18, Appendix C-SNP Type^™ Assays for SNP Genotyping on the Dynamic Array^™ IFCs).

Blood samples (10 ml) were collected (before their corresponding biopsy) in EDTA-containing tubes on the 10th day and at the end of the 1st, 6th and 12th month after OHT (from April 2016 to October 2018 at the Cardio Center of the Institute for Clinical and Experimental Medicine, Prague, Czech Republic), at the times when EMB was performed. Plasma was separated (centrifugation at 1,500 x g, 15 min, room temperature) within 30 min of blood collection and stored at -80˚C in RNase-/DNase-free tubes. cfDNA was extracted from 1 ml of plasma using a Plasma/Serum Cell-Free Circulating DNA Purification MIDI kit (cat. no. 55600; Norgen Biotek). DNA was eluted in a final volume of 45 µl. No artificial spike was used.

The experiment was performed according the protocol of Beck et al (16) with slight modifications. Preamplification of the cfDNA was conducted using the NEBNext Ultra II DNA Library Prep Kit (New England Biolabs) with a final elution volume of 21 µl. Quantification of cfDNA after this step was performed using a Qubit 4 fluorimeter (Thermo Fisher Scientific, Inc.).

The total cfDNA concentration was determined using a droplet PCR assay. PCR oligonucleotides and fluorophore-conjugated hydrolysis probes were purchased from GeneriBiotech. ddPCR was performed using a QX200 Droplet DPCR System (Bio-Rad Laboratories, Inc.) in a volume of 20 µl containing 10 µl 2x ddPCR Supermix for probes (no dUTP) (Bio-Rad Laboratories, Inc.), 7 µl of preamplified DNA, 1.8 µl of primer mix (forward primer: 5'-TAGGCCATAATACTCTTGA-3'; reverse primer: 5'-ACTGGCATTCTAACTAGA-3'), 0.5 µl of FAM-labeled probe (5'-TTGACATTTGGCCATTTTATAGGTCCA-3', and 0.5 µl of HEX-labeled probe (5'-TGACATTTGGGCATTTTATAGGTCCA-3'), according to the manufacturer's instructions. The thermocycling conditions were 95˚C for 10 min and 40 cycles of 94˚C for 30 sec and 57˚C for 1 min, followed by one final step at 98˚C for 10 min. Sample analysis of each experiment was performed using QuantaSoft v1.7 SW (Bio-Rad Laboratories, Inc.). Rare event detection settings were used to calculate the positive droplet concentration. The fluorescence threshold was set manually based on validation data obtained from homozygous variants of both versions, the temperature gradient and the negativity of no-template controls. All reactions were performed in duplicate. To account for the random distribution of target DNA into partitions, Poisson's statistical model was applied and the absolute quantity was calculated. Fractional abundance (%ddcfDNA) was calculated as the ratio of donor absolute quantity [copies (cp) per 1 ml] to absolute quantity of cfDNA (donor absolute quantity plus recipient absolute quantity).

The ddPCR concentration results were converted to a plasma-equivalent concentration (cp/ml) using the following equation:

where C_plasma is the concentration of the target within the plasma in cp/ml; C_ddPCR is the concentration of the target within the PCR in cp/µl; V_elution is the volume of eluent used during the cfDNA extraction step in µl; V_cfDNA is the volume of extracted cfDNA used in the PCR in µl; V_MM is the volume of all other PCR components in µl; and V_plasma is the volume of plasma used for cfDNA extraction in ml.

For comparing the quantification of ddcfDNA, two digital PCR systems were used, namely the QX200 digital droplet PCR system (Bio-Rad Laboratories) and the Qiacuity One dPCR system (5plex; Qiagen GmbH). The measurements were conducted on samples of patient 2. The first method used for Bio-Rad is provided in the Methods section. For the second measurement, Qiacuity was used, which involved a volume of 40 µl with 10 µl of 4X Probe PCR Master Mix (Qiagen GmbH), 8.8 µl of preamplified DNA, 4 µl of 10X primer-probe mix (0.8 µM forward primer: 5'-TAGGCCATAATACTCTTGA-3'; 0.8 µM reverse primer: 5'-ACTGGCATTCTAACTAGA-3'; 0.4 µM FAM-labeled probe (5'-TTGACATTTGGCCATTTTATAGGTCCA-3') and 4 µl of 10X primer-probe mix (0.8 µM forward primer: 5'-TAGGCCATAATACTCTTGA-3'; 0.8 µM reverse primer: 5'-ACTGGCATTCTAACTAGA-3'; 0.4 µM HEX-labeled probe (5'-TGACATTTGGGCATTTTATAGGTCCA-3') and 13.2 µl of RNAse free water, following the manufacturer's instructions. The thermocycling conditions were 95˚C for 2 min and 40 cycles of 95˚C for 15 sec and 60˚C for 30 min. Quiacuity Nanoplates 26k (Qiagen GmbH) with up to 26,000 partitions per well were used to conduct the measurements. The ddcfDNA absolute quantity and %ddcfDNA were calculated, and the Bland-Altman analysis was performed using GraphPad Prism 5 software, version 5.03 (GraphPad; Dotmatics) (17).

SNP screening identified 10 (patient 1) and 7 (patient 2) different homozygous variants suitable for the detection of donor DNA in paired samples (Table SI). All selected SNP primers (for sequences see Table SI) were validated before measurement to eliminate false-positive droplets. Only variant rs521861 (within the MYO5B gene) passed validation for using the ddPCR method. Target sequences C/G within rs521861 were analyzed in plasma cfDNA of the patients.

In patient 1, OHT was performed without any complications and the postoperative course was favorable. Echocardiographic parameters were normal except for mild dysfunction of the dilated right ventricle during the first days after Tx. Increased wall thickness was detected and was treated with intravenous (IV) pulse dose steroids. Other events were not observed during follow-up. EMB examination confirmed mild grades of ACR (1R/2) in the 1st month and (1R/1A) in the 6th month after OHT. The maximal ddcfDNA level of 270 cp/ml was detected in the 1st month after Tx. A decline in ddcfDNA to 80 cp/ml was then detected in the 6th month after OHT and 53 cp/ml (time without rejection; Fig. 1A). At the time of conclusion of the present study (October 2023), patient 1 is free of any cardiac problems and does not have any coronary graft disease.

In patient 2, the OHT was performed without any complications. Right-sided heart decompensation was treated with inotropic drugs (milrinone) until the 6th postoperative day. Echocardiography showed dilatation and dysfunction of the right ventricle and secondary tricuspid regurgitation, while all other parameters were normal. At one month after HTx, the right ventricle had a standard size and only mild dysfunction, but the wall thickness was detected to increase. EMBs were without any histological signs of acute cellular or humoral rejection at this point. In the 2nd month after transplantation, an oral pulse dose of steroids was indicated due to mild acute cellular rejection (grade 1R/2). Echocardiographic parameters were normal. EMB examination confirmed moderate-grade ACR (2R/3A) in the 3rd month after OHT, which was treated with 1,000 mg/day of IV methylprednisolone for 3 days. EMB examination at the end of the 6th month after OHT confirmed AMR grade 1+. Due to the histological findings of AMR, complement-dependent cytotoxicity (CDC) crossmatch and human leukocyte antigen antibody (HLA; Luminex^® bead assay; Luminex Corp.) testing were added; this was performed according to standard procedures. The specificity of HLA antibodies was defined by LABScreen Mixed and Single Antigen class I and class II beads (One Lambda Inc.). CDC crossmatch was negative. Only non-significant positivity of anti-HLA class II antibodies (anti-DR 13, anti-DR 14 and anti-DR 17; One Lambda Inc.) was detected. In view of the above, the tacrolimus dose was increased and a higher target tacrolimus level was set. Echocardiography showed only a mild thickening of the interventricular septum (14 mm) without deterioration of ventricular function. The patient was treated with Metroprolol succinate (Astra Zeneca Spa), Perindoprilum Argininum/Amlodipinum (Servier Industries Ltd.) and FSM. This was the last time when a rejection episode was detected. Corresponding to the EMB results, a slight increase of ddcfDNA 1,416 cp/ml was identified early in the sample collected 1 month after OHT, without serious rejection. The highest value of ddcfDNA at 1,846 cp/ml was detected 6 months after transplantation when AMR grade 1+ was also detected. Subsequently, there was a decline of ddcfDNA to 777 cp/ml in the rejection-free time (Fig. 1B). At the time of conclusion of the present study (October 2023), patient 2 is in a stable clinical condition, but is classified as New York Heart Association functional class III.

After transplantation, each patient is monitored and treated until death in our institute. Regular medical follow-ups are scheduled every 6 months to assess the status and function of the graft.

The ddcfDNA measurements obtained using two different digital PCR instruments were compared. There was a similar trend in the %ddcfDNA and in the plasma quantity of ddcfDNA (Table II and Fig. 2). The slight differences in results may have been influenced by the different PCR reagents, efficiency and specificity of the instruments.