Rejection Grade Research Articles

Deep learning-enabled assessment of cardiac allograft rejection from endomyocardial biopsies.

Endomyocardial biopsy (EMB) screening represents the standard of care for detecting allograft rejections after heart transplant. Manual interpretation of EMBs is affected by substantial interobserver and intraobserver variability, which often leads to inappropriate treatment with immunosuppressive drugs, unnecessary follow-up biopsies and poor transplant outcomes. Here we present a deep learning-based artificial intelligence (AI) system for automated assessment of gigapixel whole-slide images obtained from EMBs, which simultaneously addresses detection, subtyping and grading of allograft rejection. To assess model performance, we curated a large dataset from the United States, as well as independent test cohorts from Turkey and Switzerland, which includes large-scale variability across populations, sample preparations and slide scanning instrumentation. The model detects allograft rejection with an area under the receiver operating characteristic curve (AUC) of 0.962; assesses the cellular and antibody-mediated rejection type with AUCs of 0.958 and 0.874, respectively; detects Quilty B lesions, benign mimics of rejection, with an AUC of 0.939; and differentiates between low-grade and high-grade rejections with an AUC of 0.833. In a human reader study, the AI system showed non-inferior performance to conventional assessment and reduced interobserver variability and assessment time. This robust evaluation of cardiac allograft rejection paves the way for clinical trials to establish the efficacy of AI-assisted EMB assessment and its potential for improving heart transplant outcomes.

Electron Microscopy Reveals Evidence of Perinuclear Clustering of Mitochondria in Cardiac Biopsy-Proven Allograft Rejection.

Acute cellular rejection is a major complication in heart transplantation. We focus on the analysis of new ultrastructural findings in cardiac biopsy rejection based on mitochondrial intracellular organization. This study includes heart transplanted patients from a single center who were referred for endomyocardial biopsies as a scheduled routine screening. Participants were divided into two groups: patients transplanted without allograft rejection (Grade 0R), and patients with biopsy-proven allograft rejection (Grade ≥ 2R). Using electronic microscopy, we detected a significant increase in the volume density of mitochondria (p < 0.0001) and dense bodies (p < 0.01) in the rejection group. The most relevant finding was the presence of local accumulations of mitochondria close to the nuclear envelope, pressing and molding the morphology of this membrane in all rejection samples (100%). We identified this perinuclear clustering of mitochondria phenomenon in a 68 ± 27% of the total cardiac nucleus observed from rejection samples. We did not observe this phenomenon in any non-rejection samples, reflecting excellent sensitivity and specificity. We have identified a specific phenomenon affecting the architecture of the nuclear membrane—perinuclear clustering of mitochondria—in endomyocardial biopsies from patients with cardiac rejection. This ultrastructural approach might complement and improve the diagnosis of rejection.

Native T1 mapping detects both acute clinical rejection and graft dysfunction in pediatric heart transplant patients

BackgroundCardiovascular magnetic resonance (CMR) is emerging as an important tool for cardiac allograft assessment. Native T1 mapping may add value in identifying rejection and in assessing graft dysfunction and myocardial fibrosis burden. We hypothesized that CMR native T1 values and features of textural analysis of T1 maps would identify acute rejection, and in a secondary analysis, correlate with markers of graft dysfunction, and with fibrosis percentage from endomyocardial biopsy (EMB).MethodsFifty cases with simultaneous EMB, right heart catheterization, and 1.5 T CMR with breath-held T1 mapping via modified Look-Locker inversion recovery (MOLLI) in 8 short-axis slices and subsequent quantification of mean and peak native T1 values, were performed on 24 pediatric subjects. A single mid-ventricular slice was used for image texture analysis using nine gray-level co-occurrence matrix features. Digital quantification of Masson trichrome stained EMB samples established degree of fibrosis. Markers of graft dysfunction, including serum brain natriuretic peptide levels and hemodynamic measurements from echocardiography, catheterization, and CMR were collated. Subjects were divided into three groups based on degree of rejection: acute rejection requiring new therapy, mild rejection requiring increased ongoing therapy, and no rejection with no change in treatment. Statistical analysis included student’s t-test and linear regression.ResultsPeak and mean T1 values were significantly associated with acute rejection, with a monotonic trend observed with increased grade of rejection. Texture analysis demonstrated greater spatial heterogeneity in T1 values, as demonstrated by energy, entropy, and variance, in cases requiring treatment. Interestingly, 2 subjects who required increased therapy despite low grade EMB results had abnormal peak T1 values. Peak T1 values also correlated with increased BNP, right-sided filling pressures, and capillary wedge pressures. There was no difference in histopathological fibrosis percentage among the 3 groups; histopathological fibrosis did not correlate with T1 values or markers of graft dysfunction.ConclusionIn pediatric heart transplant patients, native T1 values identify acute rejection requiring treatment and may identify graft dysfunction. CMR shows promise as an important tool for evaluation of cardiac grafts in children, with T1 imaging outperforming biopsy findings in the assessment of rejection.

Correlation between microvascular inflammation in endomyocardial biopsies and rejection transcripts, donor-specific antibodies and graft dysfunction in antibody-mediated rejection

The pathology-based diagnosis of antibody-mediated rejection (AMR) following heart transplantation relies on the 2013 Working Formulation in which microvascular inflammation (MVI) is considered as present or absent regardless of its extent. This work assessed the biological and clinical value of a semiquantitative evaluation of the extent of MVI in endomyocardial biopsies (EMB). We retrospectively graded the extent of MVI in 291 EMB from 291 patients according to a 4-point scale where MVI scores of 0, 1, 2 and 3 represented 0%, 1–10%, 11–50% and > 50% of the myocardial area, respectively. We analyzed the association between the MVI score and tissue rejection molecular activity assessed by micro-arrays or reverse transcriptase multiplex ligation-dependent probe amplification, current pathology classification (pAMR), circulating anti-HLA donor-specific antibodies (DSA) and graft dysfunction. Overall, 172 (59.1%), 33 (11.4%), 42 (14.4%) and 44 (15.1%) EMB were given MVI scores of 0, 1, 2 and 3, respectively. pAMR1(H+) and pAMR2/3 categories were found to be heterogenous in terms of MVI score. Acute cellular rejection grades did not influence the MVI score. In both molecular approaches, we observed a stepwise increase in the expression of AMR-related transcripts with increasing MVI scores, independently of the C4d or CD68 status ( Fig. 1 , P < 0.001). Both the frequency and mean fluorescence intensity of DSA gradually increased with the MVI score ( P < 0.001). Acute graft dysfunction was more frequent in MVI score 3 ( P < 0.001). The intensity of MVI in EMB, based on a semiquantitative evaluation of its extent, has biological and clinical importance.

Correlation Between Microvascular Inflammation in Endomyocardial Biopsies and Rejection Transcripts, Donor-specific Antibodies, and Graft Dysfunction in Antibody-mediated Rejection.

The pathology-based diagnosis of cardiac antibody-mediated rejection (AMR) relies on the 2013 International Society for Heart and Lung Transplantation Working Formulation, in which microvascular inflammation (MVI) is considered as present or absent regardless of its extent. This work assessed the biological and clinical value of a semiquantitative evaluation of the extent of MVI in endomyocardial biopsies (EMBs). We retrospectively graded the extent of MVI in 291 EMB from 291 patients according to a 4-point scale in which MVI scores of 0, 1, 2, and 3 represented 0%, 1%-10%, 11%-50%, and >50% of the myocardial area, respectively. We analyzed the association between the MVI score and tissue rejection molecular activity assessed by microarrays or reverse transcriptase multiplex ligation-dependent probe amplification, current pathology classification (pathologic AMR [pAMR]), anti-HLA donor-specific antibodies, and graft dysfunction. Overall, 172 (59.1%), 33 (11.4%), 42 (14.4%), and 44 (15.1%) EMB were given MVI scores of 0, 1, 2, and 3, respectively. pAMR1(H+) and pAMR2/3 categories were found to be heterogeneous in terms of MVI score. Acute cellular rejection grades did not influence the MVI score. In both molecular approaches, we observed a stepwise increase in the expression of AMR-related transcripts with increasing MVI scores, independent of the C4d or CD68 status (P < 0.001). Both the frequency and mean fluorescence intensity of donor-specific antibodies gradually increased with the MVI score (P < 0.001). Acute graft dysfunction was more frequent in MVI score 3 (P < 0.001). The intensity of MVI in EMB, based on a semiquantitative evaluation of its extent, has biological and clinical importance.

Initiation of noninvasive surveillance for allograft rejection in heart transplant patients>1 year after transplant.

Gene expression profiling (GEP) and donor-derived, cell-free DNA (dd-cfDNA) measurement are alternative methods to endomyocardial biopsy (EMB) to monitor for rejection following heart transplantation. We aim to describe our use of GEP and dd-cfDNA in heart transplant recipients>1-year post-transplantation. This is a single-center, retrospective study in post-transplant recipients. For patients who were>1-year post-transplantation and deemed to be at elevated clinical risk for rejection, we collected both GEP and dd-cfDNA every 3 months. Baseline characteristics including GEP, dd-cfDNA levels, rejection episodes, and number of biopsies were obtained. Since July 2019, there were 18 patients being followed with GEP and dd-cfDNA who were>1-year post-transplantation. Nine EMBs had been performed in seven patients due to as follows; three due to elevated GEP ({greater than or equal to} 34), one due to elevated dd-cfDNA ({greater than or equal to} .20%), two due to elevations of both GEP and dd-cfDNA, two due to clinical rejection and one to follow up a post rejection episode. One of the two biopsies due to elevations of both GEP and dd-cfDNA showed acute cellular rejection grade 2R. None of the biopsies due to either an elevation in the GEP or dd-cfDNA revealed any significant rejection. In this study, the use of both GEP and dd-cfDNA led to an increased number of EMB in patients>1-year post-transplantation. Further studies are needed to validate these findings and evaluate long-term consequences of these diagnostic tests in this population.

Abstract 13830: Prediction of Cardiac Rejection Trajectories via Machine Learning Derived Features From Digital Endomyocardial Biopsy Images

Introduction: Cellular rejection occurs in 20-40% of transplant recipients with an increased risk of graft failure. Previously, we developed a model for predicting ISHLT rejection grades via the automated extraction of morphologic features in Endomyocardial biopsy (EMB) images. Considering the frequent discordance between conventional rejection grade and clinical rejection severity, in this work, we sought to identify morphologic features that predict the clinical trajectory of rejection events. Methods: Our study comprised 299 EMBs with grade and trajectory labels. Trajectory labels are based on the development of overt clinical signs of allograft injury as “clinically evident” or “clinically silent”. Morphologic features describing number, spatial arrangement of lymphocytes, and shape, orientation of interstitial fibers were computationally extracted. To identify the top features associated with clinically evident disease, the T-test method was applied across 500 iterations of 3-fold cross validation. In each iteration, a quadratic discriminant analysis model was trained with top 10 features on 2 folds of dataset using trajectory labels (M trj ) and was validated on one hold out test set. This model was trained using grade labels (M grd ) to predict “high” (2R + 3R) or “low” (0R + 1R). To assess feature importance, in each model, the frequency of every feature appearing in the classifier was measured through iterations. Results: The mean area under the receiver operating curve of M trj and M grd was 0.80±0.04 and 0.84±0.02 correspondingly. The top features for predicting grades differ substantially from those for predicting clinical trajectory (Figure 1). Conclusions: The additional features required to predict clinical trajectory vs. rejection grade may explain the discordance between conventional histology and rejection syndrome observed in clinical practice, and highlights the translational potential of computer-assisted histologic analysis of EMBs.

Abstract 11935: The Index of Microcirculatory Resistance to Predict Subsequent Acute Cellular Rejection and Cardiac Events After Heart Transplantation

Introduction: Single center data suggest that the index of microcirculatory resistance (IMR) measured early after heart transplantation predicts subsequent acute cellular rejection. Hypothesis: To validate whether IMR measured early after transplantation can predict subsequent acute cellular rejection and long-term outcome in a large multicenter cohort. Methods: From 5 international heart transplantation cohorts, 237 patients who underwent IMR measurement early after transplantation were enrolled. The primary outcome was biopsy-proven acute cellular rejection of grade 2 or 3 within 1 year after transplantation. A key secondary outcome was major adverse cardiac events (MACE, the composite of death, re-transplantation, myocardial infarction, stroke, graft dysfunction, and readmission due to a cardiac cause) at 10 years. Results: IMR was measured at a median of 7 weeks (interquartile range: 3 to 10 weeks) post-transplantation. Patients with acute cellular rejection grade 2 or 3 at the time of IMR assessment were excluded. At 1 year, the incidence of acute cellular rejection was 14.0%. IMR was associated proportionally with the risk of acute cellular rejection (per increase of 1-unit IMR; adjusted hazard ratio [aHR], 1.04; 95% confidence interval [CI], 1.01-1.06; P=0.002). The incidence of acute cellular rejection in patients with IMR ≥ 18 was 23.8%, while the incidence of acute cellular rejection in those with IMR &lt; 18 was 5.5% (aHR, 4.09; 95% CI, 1.67-10.0; P=0.002). At 10 years, MACE occurred in 86 (36.3%) patients. IMR was significantly associated with the risk of MACE (per increase of 1-unit IMR; aHR, 1.03; 95% CI, 1.01-1.05; P=0.002). Conclusions: IMR measured early after heart transplantation is associated with subsequent acute cellular rejection at 1 year and clinical events at 10 years. Early IMR measurement after transplantation identifies patients at higher risk and may guide personalized post-transplantation management.

Synergistic Effect of DBP with CTAB on Flotation Separation of Quartz from Collophane

Collophane is difficult to upgrade by reverse flotation of quartz with amine collector alone due to its low grade, complex structure, fine dissemination grain size, etc. This investigation was conducted to explore the synergistic effect of dibutyl phthalate (DBP) as a surfactant with cetyltrimethyl ammonium bromide (CTAB) as the collector on the separation of quartz from collophane by means of micro-flotation tests, surface tension and aggregate size measurements, and froth water mass fraction/recovery characterization. It was found that DBP reduced the surface tension of the reagent solution and enhanced the collision probability between bubbles and quartz particles by increasing the size of aggregates through increased hydrophobic interaction between the quartz particles and DBP droplets. The addition of DBP reduced the entrainment of fine collophane particles as a result of improved defoaming and increased the flotation recovery of quartz without resulting in any flotation of collophane at dosages lower than 200 mg/L. Flotation test results with the binary artificial mineral mixture showed that DBP improved the P2O5 recovery, SiO2 rejection, and P2O5 grade by up to 7%, 12%, and 1%, respectively.

The Positive Impact of Donor Bone Marrow Cells Transplantation into Immunoprivileged Compartments on the Survival of Vascularized Skin Allografts

Using the vascularized skin allograft (VSA) model, we compared the tolerogenic effects of different allogeneic bone marrow transplantation (BMT) delivery routes into immunoprivileged compartments under a 7-day protocol immunosuppressive therapy. Twenty-eight fully MHC mismatched VSA transplants were performed between ACI (RT1a) donors and Lewis (RT11) recipients in four groups of seven animals each, under a 7-day protocol of alfa/beta TCRmAb/CsA (alpha/beta-TCR monoclonal antibodies/Cyclosporine A therapy). Donor bone marrow cells (BMC) (100 × 106 cells) were injected into three different immunoprivileged compartments: Group 1: Control, without cellular supportive therapy, Group 2: Intracapsular BMT, Group 3: Intragonadal BMT, Group 4: Intrathecal BMT. In Group 2, BMC were transplanted under the kidney capsule. In Group 3, BMC were transplanted into the right testis between tunica albuginea and seminiferous tubules, and in Group 4, cells were injected intrathecally. The assessment included: skin evaluation for signs and grade of rejection and immunohistochemistry for donor cells engraftment into host lymphoid compartments. Donor-specific chimerism for MHC class I (RT1a) antigens and the presence of CD4+/CD25+ T cells were assessed in the peripheral blood of recipients. The most extended allograft survival, 50–78 days, was observed in Group 4 after intrathecal BMT. The T cells CD4+/CD25+ in the peripheral blood were higher after intrathecal BMC injection than other experimental groups at each post-transplant time point. Transplantation of BMC into immunoprivileged compartments delayed rejection of fully mismatched VSA and induction of robust, donor-specific chimerism.

Coronary Microcirculatory Dysfunction and Acute Cellular Rejection After Heart Transplantation.

Acute cellular rejection is a major determinant of mortality and retransplantation after heart transplantation. We sought to evaluate the prognostic implications of coronary microcirculatory dysfunction assessed by index of microcirculatory resistance (IMR) for the risk of acute cellular rejection after heart transplantation. The present study prospectively enrolled 154 heart transplant recipients who underwent scheduled coronary angiography and invasive coronary physiological assessment 1 month after transplantation. IMR is microcirculatory resistance under maximal hyperemia. By measuring hyperemic mean transit time using 3 injections (4 mL each) of room-temperature saline under maximal hyperemia, IMR was calculated as hyperemic distal coronary pressure×hyperemic mean transit time. The primary end point was biopsy-proven acute cellular rejection of grade ≥2R during 2 years of follow-up after transplantation and was compared by using multivariable Cox proportional hazards regression according to IMR. The incremental prognostic value of IMR, in addition to the model with clinical factors, was evaluated by comparison of C-index, net reclassification index, and integrated discrimination index. The mean age of recipients was 51.2±13.1 years (81.2% male), and the cumulative incidence of acute cellular rejection was 19.0% at 2 years. Patients with acute cellular rejection had significantly higher IMR values at 1 month than those without acute cellular rejection (23.1±8.6 versus 16.8±11.1, P=0.002). IMR was significantly associated with the risk of acute cellular rejection (per 5-U increase: adjusted hazard ratio, 1.18 [95% CI, 1.04-1.34], P=0.011) and the optimal cutoff value of IMR to predict acute cellular rejection was 15. Patients with IMR≥15 showed significantly higher risk of acute cellular rejection than those with IMR<15 (34.4% versus 3.8%; adjusted hazard ratio, 15.3 [95% CI 3.6-65.7], P<0.001). Addition of IMR to clinical variables showed significantly higher discriminant and reclassification ability for risk of acute cellular rejection (C-index 0.87 versus 0.74, P<0.001; net reclassification index 1.05, P<0.001; integrated discrimination index 0.20, P<0.001). Coronary microcirculatory dysfunction assessed by IMR measured early after heart transplantation showed significant association with the risk of acute cellular rejection. In addition to surveillance endomyocardial biopsy, early stratification using IMR could be a clinically useful tool to identify patients at higher risk of future acute cellular rejection after heart transplantation. Registration: URL: https://www.clinicaltrials.gov; Unique identifier: NCT02798731.

Face transplantation: A longitudinal histological study focusing on chronic active and mucosal rejection in a series with long-term follow-up

The 2007 Banff working classification of skin-containing Tissue Allograft Pathology addressed only acute T cell-mediated rejection in skin. We report the longitudinal long-term histological follow-up of six face transplant recipients, focusing on chronic and mucosal rejection. We identified three patterns suggestive of chronic rejection (lichen planus-like, vitiligo-like and scleroderma-like). Four patients presented lichen planus-like and vitiligo-like chronic rejection at 52±17months posttransplant with severe concomitant acute T cell-mediated rejection. After lichen planus-like rejection, two patients developed scleroderma-like alterations. Graft vasculopathy with C4d deposits and de novo DSA led to subsequent graft loss in one patient. Chronic active rejection was frequent and similar patterns were noted in mucosae. Concordance between 124 paired skin and mucosal biopsies acute rejection grades was low (κ=0.2, p=.005) but most grade 0/I mucosal rejections were associated with grade 0/I skin rejections. We defined discordant (grade≥II mucosal rejection and grade 0/I skin rejection) (n=55 [70%]) and concordant (grade≥II rejection in both biopsies) groups. Mucosal biopsies of the discordant group displayed lower intra-epithelial GranzymeB/FoxP3 ratios suggesting a less aggressive phenotype (p=.08). The grading system for acute rejection in mucosa may require phenotyping. Whether discordant infiltrates reflect a latent allo-immune reaction leading to chronic rejection remains an open question.

Ultrasound Imaging of Treatment‐Needed Cardiac Rejection with CD4‐Targeted Nanobubbles

AbstractAcute cardiac allograft rejection (ACAR) is one of the main reasons for mortality after heart transplantation. The timely diagnosis of treatment‐needed ACAR is of great importance in the clinic. Molecular imaging may make accurate diagnoses at the early stage to visualize the activity of specific pathological processes. The amount of CD4+‐lymphocyte infiltration is a crucial index in ACAR grading. CD4‐targeted nanobubbles (NBCD4) are fabricated to assess the infiltrating CD4+ lymphocytes, while isotype control nanobubbles (NBIso) are also fabricated. Animal models with ACAR grades from 0R to 3R are established. Ultrasound molecular imaging (USMI) is performed to distinguish different rejection grades with NBCD4. In the grade 2R and 3R groups, the USMI signals of NBCD4 are significantly higher than those of NBIso. The signals of NBCD4 in the 2R group are significantly lower than those in the 3R group and significantly higher than those in the 1R and 0R groups. Moreover, the signals of NBCD4 are strongly correlated with the rejection grades and the number of infiltrating CD4+ lymphocytes. As asymptomatic rejection with 2R or higher grades should be seriously treated, USMI with NBCD4 may be a new approach to timely diagnosis of treatment‐needed ACAR.

Calcineurin Inhibitor–Based Maintenance Immunosuppression in Lung Transplant Recipients: Optimal Serum Levels for Managing Acute Rejection and Renal Function

Although effective for curtailing alloimmune responses, calcineurin inhibitors (CNIs) have an adverse-effect profile that includes nephrotoxicity. In lung transplant (LTx) recipients, the optimal serum levels of the CNI tacrolimus necessary to control alloimmune responses and minimize nephrotoxicity are unknown. This retrospective, single-center study reviewed tacrolimus whole blood trough levels (BTLs), grades of acute cellular rejection (ACR), acute rejection scores, and creatinine clearance (CrCl) obtained in LTx recipients within the first year after their transplant procedure. Comparisons were made between the first 90 days post LTx (when tacrolimus BTLs were maintained >10 µg/L) and the remainder of the post-LTX year (when BTLs were <10 µg/L). Despite tacrolimus mean BTLs being higher during the first 90 days post LTx compared with the remainder of the first post-LTx year (10.4 ± 0.3 µg/L vs 9.5 ± 0.3 µg/L, P < .0001) there was no association with lower grades of ACR (P=.24). The intensity of ACR (as determined by acute rejection scores) did not correlate with tacrolimus mean BTLs at any time during the first posttransplant year (P=.79). During the first 90 days post LTx there was a significant decline in CrCl and a correlation between increasing tacrolimus mean BTLs and declining CrCl (r=-0.26, P=.03); a correlation that was not observed during the remainder of the year (r=-0.09, P=.52). In LTx recipients, maintaining BTLs of the CNI tacrolimus >10µg/L did not result in superior control of acute rejection responses but was associated with declining renal function.

Clinical Features and Outcomes of Combined Pulmonary Fibrosis and Emphysema After Lung Transplantation

Combined pulmonary fibrosis and emphysema (CPFE) is recognized as a characteristic syndrome of smoking-related interstitial lung disease that has a worse prognosis than idiopathic pulmonary fibrosis (IPF). However, outcomes after lung transplantation for CPFE have not been reported. The aim of this study is to describe the clinical features and outcomes of CPFE after lung transplantation. What are the clinical features and outcomes of CPFE after lung transplantation? This is a single-center retrospective cohort study of patients with CPFE and IPF who underwent lung transplantation at our center between January 2011 and December 2016. We defined CPFE as≥10%emphysema in the upper lung fields combined with fibrosis on high-resolution CT scan. We characterized the clinical features of patients with CPFE and compared their outcomes after lung transplantation with those with IPF. Twenty-seven of 172 (16%) patients with IPF met criteria for CPFE. Severe pulmonary hypertension was present in 16 of 27 (59%) patients with CPFE. On logistic regression analysis, CPFE was significantly associated with primary graft dysfunction (PGD) grade 3 (OR, 3.14; 95%CI, 1.18-8.37; P= .02). On competing risk regression analysis, CPFE was associated with acute cellular rejection (ACR) grade≥ A2, and chronic lung allograft dysfunction (CLAD) (hazard ratio [HR], 1.89; 95%CI, 1.10-3.25; P= .02; HR, 1.96; 95%CI, 1.02-3.77; P= .04, respectively). Five-year survival was 79.0%for the CPFE group and 75.4%for the IPF group (log-rank P= .684). After transplantation, patients with CPFE were more likely to develop PGD, ACR, and CLAD compared with those with IPF. However, survival was not significantly different between the two groups.

An automated computational image analysis pipeline for histological grading of cardiac allograft rejection.

AimAllograft rejection is a serious concern in heart transplant medicine. Though endomyocardial biopsy with histological grading is the diagnostic standard for rejection, poor inter-pathologist agreement creates significant clinical uncertainty. The aim of this investigation is to demonstrate that cellular rejection grades generated via computational histological analysis are on-par with those provided by expert pathologistsMethods and resultsThe study cohort consisted of 2472 endomyocardial biopsy slides originating from three major US transplant centres. The ‘Computer-Assisted Cardiac Histologic Evaluation (CACHE)-Grader’ pipeline was trained using an interpretable, biologically inspired, ‘hand-crafted’ feature extraction approach. From a menu of 154 quantitative histological features relating the density and orientation of lymphocytes, myocytes, and stroma, a model was developed to reproduce the 4-grade clinical standard for cellular rejection diagnosis. CACHE-grader interpretations were compared with independent pathologists and the ‘grade of record’, testing for non-inferiority (δ = 6%). Study pathologists achieved a 60.7% agreement [95% confidence interval (CI): 55.2–66.0%] with the grade of record, and pair-wise agreement among all human graders was 61.5% (95% CI: 57.0–65.8%). The CACHE-Grader met the threshold for non-inferiority, achieving a 65.9% agreement (95% CI: 63.4–68.3%) with the grade of record and a 62.6% agreement (95% CI: 60.3–64.8%) with all human graders. The CACHE-Grader demonstrated nearly identical performance in internal and external validation sets (66.1% vs. 65.8%), resilience to inter-centre variations in tissue processing/digitization, and superior sensitivity for high-grade rejection (74.4% vs. 39.5%, P < 0.001).ConclusionThese results show that the CACHE-grader pipeline, derived using intuitive morphological features, can provide expert-quality rejection grading, performing within the range of inter-grader variability seen among human pathologists.

Donor-derived cell-free DNA accurately detects acute rejection in lung transplant patients, a multicenter cohort study

Acute rejection, which includes antibody-mediated rejection and acute cellular rejection, is a risk factor for lung allograft loss. Lung transplant patients often undergo surveillance transbronchial biopsies to detect and treat acute rejection before irreversible chronic rejection develops. Limitations of this approach include its invasiveness and high interobserver variability. We tested the performance of percent donor-derived cell-free DNA (%ddcfDNA), a non-invasive blood test, to detect acute rejection. This multicenter cohort study monitored 148 lung transplant subjects over a median of 19.6 months. We collected serial plasma samples contemporaneously with TBBx to measure %ddcfDNA. Clinical data was collected to adjudicate for acute rejection. The primary analysis consisted of computing the area-under-the-receiver-operating-characteristic-curve of %ddcfDNA to detect acute rejection. Secondary analysis determined %ddcfDNA rule-out thresholds for acute rejection. ddcfDNA levels were high after transplant surgery and decayed logarithmically. With acute rejection, ddcfDNA levels rose six-fold higher than controls. ddcfDNA levels also correlated with severity of lung function decline and histological grading of rejection. %ddcfDNA area-under-the-receiver-operating-characteristic-curve for acute rejection, AMR, and ACR were 0.89, 0.93, and 0.83, respectively. ddcfDNA levels of <0.5% and <1.0% showed a negative predictive value of 96% and 90% for acute rejection, respectively. Histopathology detected one-third of episodes with ddcfDNA levels ≥1.0%, even though >90% of these events were coincident to clinical complications missed by histopathology. This study demonstrates that %ddcfDNA reliably detects acute rejection and other clinical complications potentially missed by histopathology, lending support to its use as a non-invasive marker of allograft injury.

The Investigation of Endomyocardial Biopsy Results, Plasma pro-BNP Levels and Non-invasive Parameters for Diagnosing of Acute Rejection in Patients Who Undergo Cardiac Transplantation

Aim: Heart failure is a disease with high mortality and morbidity, reducing the patient's quality of life. Each year 10% of heart failure patients progress to end-stage heart failure. Cardiac transplantation is the gold standard treatment method in these patients, however acute rejection is the most important factor affecting the success of this treatment. In this study, it was aimed to evaluate endomyocardial biopsy results, serum pro-BNP and non-invasive parameters in patients with acute rejection following cardiac transplantation.Patients and Methods: Twenty patients who underwent cardiac transplantation in our center were included in the study. The patients were divided into two groups, namely acute rejection (n: 10) and without rejection (n: 10). Echocardiography, electrocardiography (ECG), endomyocardial biopsy results, serum reactive proteins (CRP), sedimentation rate and serum pro-BNP levels, were evaluated among the patients and compared between the groups.Results: Endomyocardial biopsies obtained from patients with acute rejection revealed grade 1 rejection in 6 (60%) patients, grade 2 in 3 (30%) patients and grade 3 rejection in 1 (10%) patients. CRP and sedimentation rate were found to be similar between the groups (p&amp;gt; 0.05). High pro-BNP levels were found in patients with rejection (4843.20 ± 6690.10 pg / ml) when compared to the control group (496.30± 216.20 pg / ml) (p: 0.001). In addition, higher pro-BNP levels were detected with progressing of rejection grade (p: 0.03). The highest pro-BNP level was found in a patient with Grade-3 rejection (15211 pg / ml, p: 0.000).Conclusion: Our results show that serum pro-BNP levels are associated with acute rejection. In addition, higher pro-BNP levels were found to be associated with advanced rejection levels.

De Novo Human Leukocyte Antigen Allosensitization in Heartmate 3 Versus Heartmate II Left Ventricular Assist Device Recipients.

Left ventricular assist devices (LVADs) are associated with the development of antihuman leukocyte antigen (HLA) antibodies, which can create a challenge for future transplantation in these patients. The differential effects of Heartmate 3 (HM3) versus Heartmate II (HMII) on de novo HLA allosensitization remain unknown. Patients who underwent HMII or HM3 implantation and had no prior HLA antibodies by solid-phase assay (Luminex) testing were included in this study. Complement-dependent cytotoxicity (CDC) panel reactive antibody (PRA) levels and Luminex antibody profiles were followed until cardiac transplantation, device explantation, or death. Electronic medical records were reviewed to examine posttransplant outcomes. Thirty-eight HM3 and 34 HMII patients with complete data were followed for 1.5 ± 1.1 years on device support. HM3 and HMII groups had similar age at implant, female gender, ischemic heart failure etiology, bridge strategy at implant, as well as intraoperative and postoperative transfusion requirements. 39.5% of HM3 and 47.1% of HMII patients developed detectable HLA antibodies by Luminex testing (p = 0.516). Development of high-level (mean fluorescence intensity >10,000) antibodies was significantly lower in HM3 than HMII patients (5.3 vs. 20.6%, p = 0.049). CDC PRA testing showed fewer HM3 patients with a positive result (PRA > 0%) than HMII patients (39.4 vs. 70.0%, p = 0.015). Among transplanted patients, those who had developed de novo sensitization on LVAD support showed a trend toward incidence of moderate to severe grade rejection compared with unsensitized patients (23.8 vs. 4.8%, p = 0.078). HM3 is associated with lower risk of de novo HLA sensitization compared with HMII.

Identification of cell-free DNA methylation patterns unique to the human left ventricle as a potential indicator of acute cellular rejection.

Increased levels of donor-derived cell-free DNA (dd-cfDNA) in recipient plasma have been associated with rejection after transplantation. DNA sequence differences have been used to distinguish between donor and recipient, but epigenetic differences could also potentially identify dd-cfDNA. This pilot study aimed to identify ventricle-specific differentially methylated regions of DNA (DMRs) that could be detected in cfDNA. We identified 24 ventricle-specific DMRs and chose two for further study, one on chromosome 9 and one on chromosome 12. The specificity of both DMRs for the left ventricle was confirmed using genomic DNA from multiple human tissues. Serial matched samples of myocardium (n=33) and plasma (n=24) were collected from stable adult heart transplant recipients undergoing routine endomyocardial biopsy for rejection surveillance. Plasma DMR levels increased with biopsy-proven rejection grade for individual patients. Mean cellular apoptosis in biopsy samples increased significantly with rejection severity (2.4%, 4.4% and 10.0% for ACR 0R, 1R, and 2R, respectively) but did not show a consistent relationship with DMR levels. We identified multiple DNA methylation patterns unique to the human ventricle and conclude that epigenetic differences in cfDNA populations represent a promising alternative strategy for the non-invasive detection of rejection.