Background
Diagnosing and monitoring acute cellular rejection (ACR) is a major objective in the surveillance of heart-transplanted patients. The aim of this study was to evaluate the value of global longitudinal strain (GLS), measured by two-dimensional speckle-tracking echocardiography, as a noninvasive tool for graft function monitoring in relation to ACR.
Methods
The study population consisted of all heart-transplanted patients who underwent biopsy and corresponding echocardiography at one institution from 2011 to 2013 ( n = 64). ACR was classified according to the International Society of Heart and Lung Transplantation (0R–3R). Changes in graft function were serially evaluated before, during, and in the resolving period after ACR.
Results
No sign of rejection was seen in 268 biopsies (52.7%), minimal rejection (1R) in 202 biopsies (39.7%), and moderate rejection (2R) in 39 biopsies (7.7%); no patients had severe (3R) rejection. A significant difference in GLS was observed comparing the groups with 0R (−15.5%; 95% confidence interval, −16.2% to −14.2%), 1R (−15.3%; 95% confidence interval, −16.0% to −14.6%), and 2R (−13.8%; 95% confidence interval, −14.6% to −12.9%) rejection ( P < .0001). GLS remained significantly reduced in the 2R group despite the exclusion of patients with impaired systolic function (ejection fraction < 50%), allograft vasculopathy, and late rejection (>2 years) after transplantation. In the serial assessment, GLS was decreasing significantly at the time of moderate 2R rejection and improved significantly in the resolving period. The traditional diastolic Doppler parameters, E-wave deceleration time and isovolumetric relaxation time, were unaffected by rejections, whereas the E/A and E/e′ ratios were significantly higher in the 2R group ( P = .004 and P = .01) compared with the 0R and 1R groups.
Conclusions
GLS is significantly reduced during moderate (2R) ACR and improves significantly in the resolving period. The present results provide encouraging evidence to consider the routine use of GLS as a marker of graft function involvement during ACR.
Acute cellular rejection (ACR) is an inflammatory response, seen most frequently within 3 to 6 months after heart transplantation (HTX). Only approximately one-third of patients remain free of ACR episodes during the first year after HTX. ACR is associated with the development of cardiac allograft vasculopathy (CAV) and poor outcomes.
Standard surveillance of ACR after HTX is done through endomyocardial biopsies (EMB) performed either because of clinically suspected rejection or as part of a routine program. In some cases, arrhythmias or sudden drop in left ventricular (LV) ejection fraction (LVEF) leads to suspected rejection. However, our clinical experience shows that LVEF is often within the normal range during ACR, suggesting that LVEF is an inappropriate parameter to detect impaired myocardial function during ACR. Echocardiographic assessments with diastolic indices, fractional shortening (FS), and myocardial performance index have all been associated with ACR but have not found a significant role in rejection monitoring. Thus, reliable methods for graft function surveillance during ACR are lacking. It is well known that the subendocardial longitudinally oriented fibers of the myocardium are very sensitive to ischemia and fibrosis. Two-dimensional (2D) speckle-tracking echocardiography (STE) is a new echocardiographic modality for evaluation of longitudinal myocardial deformation. There are limited data on the serial changes in longitudinal function during and after ACR.
Thus, the aim of the present study was to evaluate the impact of ACR on global longitudinal strain (GLS) serially, using 2D STE during and after ACR.
Methods
Study Population
All HTX patients followed at our center from 2011 to 2013 were consecutively included in our study ( Figure 1 A). At each hospitalization during follow-up, all patients underwent comprehensive echocardiographic examinations. We retrospectively reviewed all biopsies performed within follow-up at the right-censoring date, July 1, 2013. Only echocardiographic examinations with corresponding biopsies were included in the analysis. We excluded examinations within the first month after HTX to minimize bias by pretransplantation ischemic injury.
Data were analyzed in two groups: (1) To assess LV function during ACR, we performed a cross-sectional analysis of all biopsies and corresponding echocardiographic examinations performed within 24 hours of the biopsies. Biopsies were divided into three groups (0R, 1R, and 2R) according to the International Society of Heart and Lung Transplantation classification. (2) Furthermore, we serially investigated changes in graft function before and during subclinical ACR episodes. Baseline was the latest biopsy without rejection, and follow-up was at the time of 2R ACR, detected by routine biopsy without clinically suspected ACR. Additionally, we investigated changes in graft function in the resolving period after ACR. Baseline in this analysis was at the time of 2R ACR (clinical or subclinical), the first follow-up visit was <1 month after 2R ACR, and the second follow-up visit was >3 months after 2R ACR ( Figure 1 B). We only investigated cases with decreasing rejection grades during follow-up. Not all patients had second follow-up visits, mainly because of new ACR episodes within the 3 months of follow-up or no clinical indication for additional biopsies >3 months after ACR.
EMB
Biopsies were performed according to standard local hospital procedure using the internal jugular or femoral vein. Patients underwent routine biopsies the first 2 years after HTX. Biopsies were scheduled weekly during the first 6 weeks, every 2 weeks until 3 months, every month until 6 months, and every 2 months for the rest of the first postoperative year. Biopsies were performed every 3 months between years 1 and 2. Subsequently, biopsies were performed only when rejection was clinically suspected. Biopsy samples were routinely examined for histologic signs of antibody-mediated rejection. Biopsy samples were examined for C4D and CD68 deposits only in patients with clinical signs or symptoms.
An experienced cardiac pathologist, blinded to the results of echocardiography and coronary angiography, analyzed all biopsies. ACR was histopathologically graded according to guidelines of the International Society of Heart and Lung Transplantation. ACR ≥ 2R were treated with 1 g intravenous methylprednisolone for 3 days. We calculated the number of previous ≥2R rejections for each examination.
Echocardiography
We used a commercially available ultrasound system (Vivid 9; GE Vingmed Ultrasound AS, Horten, Norway) with a 3.5-MHz phased-array transducer (M5S). Echocardiography was performed within 24 hours of the biopsy and always before medical treatment of rejection. The observer was blinded to clinical status, biopsy analysis, and vasculopathy status.
From a parasternal view, M-mode measurement included septal and posterior wall thickness and end-diastolic and end-systolic diameters of the left and right ventricles.
From an apical view, 2D LVEF measurements were based on end-systolic and end-diastolic LV volumes, using the biplane method of disks.
Peak systolic mitral annular velocities (S′) were estimated in EchoPAC (GE Vingmed Ultrasound AS) from tissue velocity images as the averages of septal, lateral, anterior, and posterior mitral annular velocities. Likewise, tissue tracking was estimated as an average of basal septal, lateral, anterior, and posterior movement. GLS was obtained from frame-by-frame tracking of speckle patterns throughout the left-sided myocardium in standard 2D cine loops. The speckle area of interest was manually adjusted for optimal tracking results. Segments with unacceptably low tracking quality due to poor image acquisition or artifacts were excluded. EchoPAC calculated GLS as the average longitudinal systolic strain of 17 myocardial segments at the time of peak value during systole. A higher negative value of strain indicates a higher magnitude of strain. EchoPAC allowed the calculation of GLS only when tracking quality was adequate in at least five of six segments in each view. In case of GLS not being reported because of the exclusion of only one projection (two or more segments with inadequate tracking), an average of the remaining segments was calculated and used as a measure for the projection. Subsequently, GLS was manually calculated as an average of all three projections.
A single investigator (T.S.C.), blinded to clinical data, analyzed data offline, using dedicated software (EchoPAC PC SW-Only, 112) and stored them digitally.
Angiography and CAV
Angiography was performed annually to detect CAV. An experienced cardiologist reviewed all angiograms and compared them with previous angiograms. All examinations were analyzed blinded to patient clinical status, echocardiography, and biopsies. CAV was classified according to International Society of Heart and Lung Transplantation guidelines.
Statistical Analysis
Continuous data conforming to a normal distribution are presented as mean ± SD, and categorical data are presented as absolute values with percentages. A mixed model was used comparing continuous variables in the assessment of myocardial function during and after ACR, because of an unequal number of observations between patients. We used analysis of variance comparing continuous variables in patient characteristics and histograms and Q-Q plots to check for normality. To calculate sensitivity and specificity, patients were randomly divided into two approximately equally sized groups; a derivation group that was used to calculate the optimal cutoff values and a test group in which the cutoff values were used to determine sensitivity and specificity. In the test of variability, we used intraclass correlation coefficients (ICCs) and the absolute difference divided by the mean of the repeated observations and expressed as a percent. P values < .05 were considered statistically significant. We used a standard statistical software package (Stata/IC 11; StataCorp LP, College Station, TX).
Results
From January 1, 2011, to July 1, 2013, 64 patients (64.1% men) underwent a total of 585 EMBs. However, 76 EMBs were performed within the first month after transplantation and therefore excluded. We included a total of 509 EMBs and corresponding echocardiographic examinations ( Figure 1 A). Table 1 shows baseline characteristics.
| Variable | Value |
|---|---|
| Men | 41 (64.1%) |
| Donor age (y) | 42.4 ± 14.2 (10–65) |
| Diabetes | 8 (12.5%) |
| Hypertension | 51 (79.6%) |
| Hypercholesterolemia | 61 (96%) |
| Stroke/claudication | 4 (6.2%) |
| Vasculopathy | 19 (30%) |
| Reason for transplantation | |
| Cardiomyopathy | 41 (64.1%) |
| Ischemic heart disease | 16 (25.0%) |
| Congenital heart disease | 3 (4.7%) |
| Others | 4 (6.3%) |
Cross-Sectional Analysis
The biopsies were divided into three groups according to the grade of ACR. No sign of ACR was seen in 268 biopsies (52.7%), minimal ACR (1R) in 202 biopsies (39.7%), and moderate ACR (2R) in 39 biopsies (7.7%). The pathologist detected no episodes of severe (3R) ACR or humeral rejection. Table 2 displays demographics of the three groups (0R, 1R, and 2R). Graft age, number previous of ≥2R rejections, and former percutaneous coronary intervention in the transplanted heart were significantly higher in the 2R group.
| Variable | R0 ( n = 268) | R1 ( n = 202) | R2 ( n = 39) | ANOVA P |
|---|---|---|---|---|
| Men | 184 (68.7%) | 131 (64.8%) | 25 (64.1%) | .64 |
| Age (y) | 46 ± 14 | 46 ± 13 | 44 ± 13 | .70 |
| Donor age (y) | 46 ± 12 | 43 ± 13 | 44 ± 14 | .009 ∗ |
| Time since transplantation (d) | 467 ± 782 | 578 ± 942 | 1,106 ± 1,506 | .0003 ∗ |
| Weight (kg) | 80 ± 19 | 81 ± 18 | 78 ± 17 | .74 |
| Systolic blood pressure (mm Hg) | 136 ± 16 | 136 ± 16 | 134 ± 17 | .67 |
| Diastolic blood pressure (mm Hg) | 83 ± 12 | 85 ± 12 | 85 ± 14 | .44 |
| Heart rate (beats/min) | 87 ± 12 | 88 ± 13 | 91 ± 12 | .18 |
| Diabetes | 42 (16.6%) | 27 (13.7%) | 3 (7.7%) | .31 |
| Hypertension | 195 (77.1%) | 166 (84.7%) | 31 (79,5%) | .13 |
| Hypercholesterolemia | 243 (96.0%) | 194 (99.0%) | 39 (100%) | .08 |
| Present smoker | 34 (17.3%) | 17 (11.0%) | 6 (17.6%) | .22 |
| Vasculopathy | 57 (22.1%) | 41 (20.5%) | 14 (35.9%) | .11 |
| Former PCI treatment | 21 (8.1%) | 16 (8.0%) | 10 (25.6%) | .0015 ∗ |
| Medications | ||||
| Prednisolone | 215 (84.6%) | 169 (86.2%) | 32 (87.2%) | .84 |
| Cyclosporin | 62 (24.5%) | 34 (17.3%) | 9 (23.1%) | .18 |
| Tacrolimus | 184 (72.7%) | 157 (80.1%) | 29 (74.4%) | .19 |
| Mycophenolate | 250 (92.9%) | 185 (93.9%) | 33 (84.6%) | .20 |
| Everolimus | 65 (25.7%) | 61 (31.1%) | 16 (41.0%) | .11 |
| Statins | 236 (93.3%) | 181 (92.3%) | 38 (97.4%) | .51 |
| ACE inhibitors/ATII receptor blockers | 157 (62.5%) | 122 (62.9%) | 18 (48.6%) | .24 |
| Furosemide or bumetanide | 62 (24.5%) | 67 (34.2%) | 16 (41.0%) | .0228 ∗ |
| Thiazide | 33 (13.0%) | 12 (6.1%) | 0 (0%) | .0048 ∗ |
| Calcium channel blockers | 83 (32.4%) | 65 (33.7%) | 9 (24.3%) | .52 |
| β-blockers | 3 (1.2%) | 4 (2.0%) | 0 (0%) | .56 |
| Aspirin | 81 (32.0%) | 67 (35.2%) | 19 (48.7%) | .12 |
| Biochemistry | ||||
| Creatinine (μmol/L) | 118.4 ± 67.8 | 115.5 ± 58.7 | 116.9 ± 70.5 | .90 |
| Hemoglobin (mmol/L) | 7.8 ± 1.0 | 7.8 ± 0.9 | 7.8 ± 1.0 | .97 |
| Total cholesterol (mmol/:) | 4.9 ± 1.5 | 5.2 ± 1.7 | 5.0 ± 1.9 | .24 |
| s-tacrolimus (μg/L) | 7.7 ± 2.7 | 8.3 ± 3.4 | 8.1 ± 5.3 | .29 |
| s-everolimus (μg/L) | 4.0 ± 2.1 | 4.1 ± 3.3 | 5.2 ± 3.8 | .54 |
| s-ciclosporine (μg/L) | 184.6 ± 95.5 | 140.8 ± 51.3 | 156.1 ± 61.6 | .0448 ∗ |
Table 3 shows the various echocardiographic parameters. FS was unaffected in the 1R biopsy group (36.9%; 95% confidence interval [CI], 35.6%–38.2%) compared with the 0R biopsy group (36.9%; 95% CI 35.7%–38.2%) but was significantly lower in the 2R biopsy group (34.1%; 95% CI 32.1%–36.0%) ( P = .0039). LVEF was significantly lower in the biopsy group with 2R ACR (0R: 63.5% [95% CI, 62.0%–64.9%]; 1R: 63.8% [95% CI, 62.3%–65.3%]; and 2R: 60.5% [95% CI, 58.5%–62.5%]; P = .0003). LVEF remained unaffected during ACRs within the first 2 years after HTX. LVEFs were <50% in only 23 patients (0R, n = 9; 1R, n = 9; 2R, n = 5), and FS was <26% in only 20 patients (0R, n = 7; 1R, n = 7; 2R, n = 6).
| Variable | 0R ( n = 268) | 1R ( n = 202) | 2R ( n = 39) | P |
|---|---|---|---|---|
| Parasternal M-mode | ||||
| LA diameter (cm) | 4.2 (4.0 to 4.4) | 4.3 (4.1 to 4.5) | 4.3 (4.1 to 4.5) | .41 |
| RV diameter (cm) | 2.9 (2.8 to 3.1) | 2.9 (2.8 to 3.1) | 2.9 (2.7 to 3.1) | .86 |
| IVS thickness (mm) | 10.7 (10.3 to 11.1) | 10.8 (10.4 to 11.2) | 10.7 (10.2 to 11.3) | .65 |
| PW thickness (mm) | 10.5 (10.2 to 10.8) | 10.8 (10.5 to 11.1) | 10.7 (10.3 to 11.2) | .0462 ∗ |
| FS (%) | 36.9 (35.7 to 38.2) | 36.9 (35.6 to 38.2) | 34.1 (32.1 to 36.0) | .0039 ∗ |
| LV EDD (cm) | 4.6 (4.5 to 4.7) | 4.6 (4.5 to 4.7) | 4.7 (4.6 to 4.8) | .24 |
| LV ESD (cm) | 2.9 (2.8 to 3.0) | 2.9 (2.8 to 3.0) | 3.1 (3.0 to 3.2) | .0009 ∗ |
| LV mass (g) | 177.2 (165.8 to 188.6) | 180.8 (169.3 to 192.4) | 183.3 (169.7 to 196.9) | .21 |
| Apical | ||||
| LVEF Simpson’s biplane (%) | 63.5 (62.0 to 64.9) | 63.8 (62.3 to 65.3) | 60.5 (58.5 to 62.5) | .0003 ∗ |
| LV EDV (mL) | 98.4 (92.3 to 104.3) | 98.0 (92.1 to 104.0) | 98.5 (91.6 to 105.4) | .93 |
| LV ESV (mL) | 36.7 (33.4 to 40.1) | 36.2 (32.8 to 39.6) | 40.0 (35.9 to 44.0) | .0182 ∗ |
| LA volume (ml) | 71.2 (63.9 to 78.4) | 71.3 (63.9 to 78.6) | 76.4 (67.9 to 84.8) | .10 |
| TAPSE (cm) | 1.6 (1.5 to 1.7) | 1.5 (1.4 to 1.6) | 1.5 (1.4 to 1.6) | .0019 ∗ |
| Diastole | ||||
| E/A ratio | 1.9 (1.7 to 2.1) | 2.0 (1.8 to 2.2) | 2.2 (2.0 to 2.4) | .0035 ∗ |
| E-wave deceleration time (msec) | 166.8 (160.9 to 172.6) | 164.9 (158.9 to 171.0) | 162.2 (153.5 to 170.8) | .42 |
| IVRT (msec) | 66.8 (64.6 to 69.0) | 67.8 (65.5 to 70.1) | 67.3 (63.6 to 70.9) | .61 |
| E/e′ ratio | 9.3 (8.5 to 10.1) | 9.4 (8.6 to 10.2) | 10.8 (9.6 to 12.0) | .0132 ∗ |
| Tissue Doppler | ||||
| S′ mean (cm/sec) | 6.0 (5.7 to 6.3) | 6.0 (5.7 to 6.3) | 5.6 (5.2 to 6.0) | .0227 ∗ |
| TT mean (mm) | 9.4 (8.9 to 9.9) | 9.2 (8.7 to 9.7) | 9.1 (8.4 to 9.7) | .25 |
| 2D STE | ||||
| GLS (%) | −15.5 (14.8 to 16.2) | −15.3 (14.6 to 16.0) | −13.8 (12.9 to 14.6) | <.0001 ∗ |
| Subanalysis | ||||
| GLS (graft age < 2 y) (%) | −15.8 (15.0 to 16.5) | −15.5 (14.7 to 16.3) | −14.2 (13.2 to 15.2) | .0002 ∗ |
| LVEF (graft age < 2 y) (%) | 64.4 (62.9 to 65.9) | 64.3 (62.8 to 65.9) | 63.8 (61.7 to 65.9) | .79 |
| GLS (patients without vasculopathy) (%) | −16.1 (15.4-16.9) | −15.9 (15.2 to 16.7) | −14.1 (13.1 to 15.1) | <.0001 ∗ |
| LVEF (patients without vasculopathy) (%) | 64.3 (62.8 to 65.9) | 64.6 (63.0 to 66.2) | 60.6 (58.3 to 62.9) | .0002 ∗ |
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