DSE

Dobutamine infusion began at 5 μg/kg/min and was increased every 3 min to 10, 20, 40, and 50 μg/kg/min, with the addition of atropine as necessary to reach the predefined target heart rate ([220 − age] × 0.85). Two-dimensional image sequences were obtained from the parasternal long-axis and short-axis and apical two-chamber and four-chamber views examined at baseline, low dose, peak dose, and recovery. Before the angiographic data became available, regional wall motion abnormalities on digitally acquired cine loops were graded by two experienced observers as normal, hyperkinetic, hypokinetic, akinetic, or dyskinetic (scores 1–4) using the 17-segment model of the American Society of Echocardiography. Wall motion score index (WMSI) was calculated as the sum of the scores divided by the number of analyzed segments (1 = normal). Any deterioration of WMSI or persistence of baseline wall motion abnormality under dobutamine was considered abnormal.

Coronary Flow Measurement and CFR Determination

Coronary flow measurements were performed with the use of the 3V2c transducer of the Acuson Sequoia and the 4V1c transducer of the SC 2000, with second-harmonic capability. Harmonic 3.5-MHz and 4.3-MHz frequencies were chosen for visualization of left anterior descending coronary artery flow. For color Doppler coronary flow mapping, velocity ranges of ± 20 cm/sec were selected. Coronary flow in the middle to distal left anterior descending coronary artery was searched over the epicardial part of the distal anterior wall using a modified, foreshortened view obtained by slightly sliding the transducer upward and medially from an apical two-chamber view. The color Doppler frequency was set at 2.5 MHz, and the color priority was adjusted as needed. Blood flow velocity was measured using pulsed-wave Doppler, which displayed a characteristic biphasic flow pattern, with larger diastolic and smaller systolic components, from a sample volume of 3 mm placed on the color Doppler signal ( Figure 1 ). Peak systolic and peak diastolic velocities were recorded at baseline and under induced hyperemia by 0.56 mg/kg dipyridamole infusion over 4 min. If still negative, an additional 0.28 mg/kg over 2 min after 4 min of no dose was administered, as recommended by consensus statements. For each parameter, the three highest Doppler recordings were averaged. CFR was calculated as the ratio of hyperemic to baseline peak diastolic velocity. The previously defined standard cut point of ≤2 for this ratio was used to define abnormal CFR.

Foreshortened apical two-chamber view with the display of coronary flow in the distal left anterior descending coronary artery ( arrows ) by color ( top ) and pulsed-wave ( bottom ) Doppler at baseline and during hyperemia. D , Diastolic; S , systolic.

CA and Endomyocardial Biopsies

CA was performed annually and whenever necessary on the basis of clinical findings. Cine angiograms of each major coronary artery and its side branches in two orthogonal planes were obtained using the Judkins technique and biplane cine fluoroscopy. Two interventional cardiologists performed visual qualitative assessment of the angiograms blinded to echocardiographic data. According to International Society for Heart and Lung Transplantation recommendations, coronary abnormalities were defined as follows: CAV grade 0, no detectable lesion; CAV grade 1, mild (left main <50%, or primary vessel with maximum lesion <70%, or any branch stenosis <70% [including diffuse narrowing]); CAV grade 2, moderate (left main <50%, a single primary vessel ≥70%, or isolated branch stenosis ≥70% in branches of two systems); and CAV grade 3, severe (left main ≥50%, or two or more primary vessels ≥70% stenosis, or isolated branch stenosis ≥70% in all three systems). Diagnostic endomyocardial biopsies were performed annually after the first year simultaneously with CA. Additional biopsies were also performed in high-risk patients or whenever rejection was suspected on the basis of clinical or echocardiographic findings. Rejection diagnosis was made according to the revised International Society for Heart and Lung Transplantation cardiac biopsy grading.

Primary Outcome and Follow-Up

The primary aim of this study was to evaluate the predictive value of noninvasive CFR assessment and DSE in the determination of subsequent progression of CAV. For this purpose, we used CFR and dobutamine stress echocardiographic measurements at each yearly follow-up visit, as well as CA. Patients were followed for 5 years after transplantation. Assessment of CFR, DSE, and CA was performed annually and standard transthoracic echocardiography every 3 months. Patients who survived ≥1 year after transplantation participated in the study.

Statistical Analysis

Continuous variables are expressed as mean ± SD. Differences between the groups were assessed using Kruskal-Wallis and Mann-Whitney U tests. Bonferroni’s correction was used in post hoc analyses. The potential confounding effect of different parameters on CFR was tested using binary logistic regression analysis. Differences between the two dependent groups were analyzed using McNemar’s test. Noninvasive test performance for detecting CAV was calculated by considering whether CAV was present at any point over the duration of the study. In analyses of sensitivity, specificity, positive predictive value, and negative predictive value, noninvasive methods were censored after CA positivity, and to avoid double counting, positive results in CFR and dobutamine stress echocardiographic measurements were also excluded in subsequent follow-up once they became positive, if angiography had not shown positive results yet. This design is presented in Figure 2 . After determining the sensitivities and specificities of CFR and DSE at each time point separately, we calculated these values for CFR and DSE in combination in a serial design. This means that a result should be regarded as positive if both tests have positive findings. Sensitivity and specificity calculations were done for this combination according to the addition rules of probability:sensitiviy _combined = sensitivity _CFR × sensitivity _DSE , and specificity _combined = specificity _CFR + specificity _DSE − (specificity _CFR × specificty _DSE ).

Design of the study and results for each patient during the follow-up period. Gray shadowing indicates the test results that were not taken into consideration because they became positive after the angiographic evidence of CAV. Red bars indicate excluded noninvasive test results in subsequent follow-up once they became positive, to avoid double counting when calculating test positivity per patient for the overall accuracy of noninvasive tests to predict angiographic CAV.

Missing values were excluded, and analyses were performed only with available data. There were two patients with only one CFR value missing, one patient with only one dobutamine stress echocardiographic value missing, and one patient with the first-year assessments missing. P values < .05, on the basis of two-sided tests, were considered statistically significant. SPSS version 15.0 (SSPS, Inc, Chicago, IL) was used for the analyses in this study.