The molecular phenotypes of injury, steatohepatitis, and fibrosis in liver transplant biopsies in the INTERLIVER study.

To extend previous molecular analyses of rejection in liver transplant biopsies in the INTERLIVER study (ClinicalTrials.gov #NCT03193151), the present study aimed to define the gene expression selective for parenchymal injury, fibrosis, and steatohepatitis. We analyzed genome-wide microarray measurements from 337 liver transplant biopsies from 13 centers. We examined expression of genes previously annotated as increased in injury and fibrosis using principal component analysis (PCA). PC1 reflected parenchymal injury and related inflammation in the early posttransplant period, slowly regressing over many months. PC2 separated early injury from late fibrosis. Positive PC3 identified a distinct mildly inflamed state correlating with histologic steatohepatitis. Injury PCs correlated with liver function and histologic abnormalities. A classifier trained on histologic steatohepatitis predicted histologic steatohepatitis with cross-validated AUC = 0.83, and was associated with pathways reflecting metabolic abnormalities distinct from fibrosis. PC2 predicted histologic fibrosis (AUC = 0.80), as did a molecular fibrosis classifier (AUC = 0.74). The fibrosis classifier correlated with matrix remodeling pathways with minimal overlap with those selective for steatohepatitis, although some biopsies had both. Genome-wide assessment of liver transplant biopsies can not only detect molecular changes induced by rejection but also those correlating with parenchymal injury, steatohepatitis, and fibrosis, offering potential insights into disease mechanisms for primary diseases.

Molecular diagnosis of ABMR with or without donor-specific antibody in kidney transplant biopsies: Differences in timing and intensity but similar mechanisms and outcomes.

We studied the clinical, histologic, and molecular features distinguishing DSA-negative from DSA-positive molecularly defined antibody-mediated rejection (mABMR). We analyzed mABMR biopsies with available DSA assessments from the INTERCOMEX study: 148 DSA-negative versus 248 DSA-positive, compared with 864 no rejection (excluding TCMR and Mixed). DSA-positivity varied with mABMR stage: early-stage (EABMR) 56%; fully developed (FABMR) 70%; and late-stage (LABMR) 58%. DSA-negative patients with mABMR were usually sensitized, 60% being HLA antibody-positive. Compared with DSA-positive mABMR, DSA-negative mABMR was more often C4d-negative; earlier by 1.5 years (average 2.4 vs. 3.9 years); and had lower ABMR activity and earlier stage in molecular and histology features. However, the top ABMR-associated transcripts were identical in DSA-negative versus DSA-positive mABMR, for example, NK-associated (e.g., KLRD1 and GZMB) and IFNG-inducible (e.g., PLA1A). Genome-wide class comparison between DSA-negative and DSA-positive mABMR showed no significant differences in transcript expression except those related to lower intensity and earlier time of DSA-negative ABMR. Three-year graft loss in DSA-negative mABMR was the same as DSA-positive mABMR, even after adjusting for ABMR stage. Thus, compared with DSA-positive mABMR, DSA-negative mABMR is on average earlier, less active, and more often C4d-negative but has similar graft loss, and genome-wide analysis suggests that it involves the same mechanisms. SUMMARY SENTENCE: In 398 kidney transplant biopsies with molecular antibody-mediated rejection, the 150 DSA-negative cases are earlier, less intense, and mostly C4d-negative, but use identical molecular mechanisms and have the same risk of graft loss as the 248 DSA-positive cases.

Evolving the surveillance and workup of heart transplant rejection: A real-world analysis of the Molecular Microscope Diagnostic System.

The Molecular Microscope Diagnostic System (MMDx) analyzes RNA transcripts of transplanted heart tissue to differentiate among T cell-mediated rejection (TCMR), antibody-mediated rejection (AMR), injury, and healthy tissue. However, little is known about its performance in relation to other modalities in a real-world heart transplant population. We evaluated whether MMDx performs in agreement with other validated modalities. Two hundred and twenty-eight corresponding endomyocardial biopsies (EMBx) and MMDx specimens from 135 adult heart transplant patients were retrospectively reviewed with correlating donor-derived cell-free DNA (dd-cfDNA). Rejection was classified on EMBx in 29 specimens (TCMR ≥ 2R and/or AMR ≥ 1), on MMDx in 56 specimens, and in 74 values with dd-cfDNA ≥0.20%. Despite moderate agreement between EMBx and MMDx (84% agreement, Cohen's kappa, 0.48, p < .001), systematic differences were observed (McNemar's test, p < .001) where MMDx classified 32 of 37 discordant cases as rejection. MMDx and dd-cfDNA demonstrated slight agreement (72% agreement, Cohen's kappa, 0.39, p < .001); however, systematic differences were also apparent where MMDx classified 12 of 50 discordant specimens as rejection when dd-cfDNA was <0.20% (McNemar's test, p < .001). Our findings provide insight on the performance of MMDx relative to other modalities in a heart transplant cohort and have implications on the surveillance and workup of allograft rejection in heart transplantation.

Donor genetic variants as risk factors for thrombosis after liver transplantation: A genome-wide association study.

Thrombosis after liver transplantation substantially impairs graft- and patient survival. Inevitably, heritable disorders of coagulation originating in the donor liver are transmitted by transplantation. We hypothesized that genetic variants in donor thrombophilia genes are associated with increased risk of posttransplant thrombosis. We genotyped 775 donors for adult recipients and 310 donors for pediatric recipients transplanted between 1993 and 2018. We determined the association between known donor thrombophilia gene variants and recipient posttransplant thrombosis. In addition, we performed a genome-wide association study (GWAS) and meta-analyzed 1085 liver transplantations. In our donor cohort, known thrombosis risk loci were not associated with posttransplant thrombosis, suggesting that it is unnecessary to exclude liver donors based on thrombosis-susceptible polymorphisms. By performing a meta-GWAS from children and adults, we identified 280 variants in 55 loci at suggestive genetic significance threshold. Downstream prioritization strategies identified biologically plausible candidate genes, among which were AK4 (rs11208611-T, p = 4.22 × 10-05 ) which encodes a protein that regulates cellular ATP levels and concurrent activation of AMPK and mTOR, and RGS5 (rs10917696-C, p = 2.62 × 10-05 ) which is involved in vascular development. We provide evidence that common genetic variants in the donor, but not previously known thrombophilia-related variants, are associated with increased risk of thrombosis after liver transplantation.

Discovering novel injury features in kidney transplant biopsies associated with TCMR and donor aging.

We previously characterized the molecular changes in acute kidney injury (AKI) and chronic kidney disease (CKD) in kidney transplant biopsies, but parenchymal changes selective for specific types of injury could be missed by such analyses. The present study searched for injury changes beyond AKI and CKD related to specific scenarios, including correlations with donor age. We defined injury using previously defined gene sets and classifiers and used principal component analysis to discover new injury dimensions. As expected, Dimension 1 distinguished normal vs. injury, and Dimension 2 separated early AKI from late CKD, correlating with time posttransplant. However, Dimension 3 was novel, distinguishing a set of genes related to epithelial polarity (e.g., PARD3) that were increased in early AKI and decreased in T cell-mediated rejection (TCMR) but not in antibody-mediated rejection. Dimension 3 was increased in kidneys from older donors and was particularly important in survival of early kidneys. Thus high Dimension 3 scores emerge as a previously unknown element in the kidney response-to-injury that affects epithelial polarity genes and is increased in AKI but depressed in TCMR, indicating that in addition to general injury elements, certain injury elements are selective for specific pathologic mechanisms. (ClinicalTrials.gov NCT01299168).

Molecular phenotyping of rejection-related changes in mucosal biopsies from lung transplants.

Diagnosing lung transplant rejection currently depends on histologic assessment of transbronchial biopsies (TBB) with limited reproducibility and considerable risk of complications. Mucosal biopsies are safer but not histologically interpretable. Microarray-based diagnostic systems for TBBs and other transplants suggest such systems could assess mucosal biopsies as well. We studied 243 mucosal biopsies from the third bronchial bifurcation (3BMBs) collected from seven centers and classified them using unsupervised machine learning algorithms. Using the expression of a set of rejection-associated transcripts annotated in kidneys and validated in hearts and lung transplant TBBs, the algorithms identified and scored major rejection and injury-related phenotypes in 3BMBs without need for labeled training data. No rejection or injury, rejection, late inflammation, and recent injury phenotypes were thus scored in new 3BMBs. The rejection phenotype correlated with IFNG-inducible transcripts, the hallmarks of rejection. Progressive atrophy-related changes reflected by the late inflammation phenotype in 3BMBs suggest widespread time-dependent airway deterioration, which was especially pronounced after two years posttransplant. Thus molecular assessment of 3BMBs can detect rejection in a previously unusable biopsy format with potential utility in patients with severe lung dysfunction where TBB is not possible and provide unique insights into airway deterioration. ClinicalTrials.gov NCT02812290.

Discrepancy analysis comparing molecular and histology diagnoses in kidney transplant biopsies.

Discrepancy analysis comparing two diagnostic platforms offers potential insights into both without assuming either is always correct. Having optimized the Molecular Microscope Diagnostic System (MMDx) in renal transplant biopsies, we studied discrepancies within MMDx (reports and sign-out comments) and between MMDx and histology. Interpathologist discrepancies have been documented previously and were not assessed. Discrepancy cases were classified as "clear" (eg, antibody-mediated rejection [ABMR] vs T cell-mediated rejection [TCMR]), "boundary" (eg, ABMR vs possible ABMR), or "mixed" (eg, Mixed vs ABMR). MMDx report scores showed 99% correlations; sign-out interpretations showed 7% variation between observers, all located around boundaries. Histology disagreed with MMDx in 37% of biopsies, including 315 clear discrepancies, all with implications for therapy. Discrepancies were distributed widely in all histology diagnoses but increased in some scenarios; for example, histology TCMR contained 14% MMDx ABMR and 20% MMDx no rejection. MMDx usually gave unambiguous diagnoses in cases with ambiguous histology, for example, borderline and transplant glomerulopathy. Histology lesions or features associated with more frequent discrepancies (eg, tubulitis, arteritis, and polyomavirus nephropathy) were not associated with increased MMDx uncertainty, indicating that MMDx can clarify biopsies with histologic ambiguity. The patterns of histology-MMDx discrepancies highlight specific histology diagnoses in which MMDx assessment should be considered for guiding therapy.

The molecular diagnosis of rejection in liver transplant biopsies: First results of the INTERLIVER study.

Molecular diagnosis of rejection is emerging in kidney, heart, and lung transplant biopsies and could offer insights for liver transplant biopsies. We measured gene expression by microarrays in 235 liver transplant biopsies from 10 centers. Unsupervised archetypal analysis based on expression of previously annotated rejection-related transcripts identified 4 groups: normal "R1normal " (N = 129), T cell-mediated rejection (TCMR) "R2TCMR " (N = 37), early injury "R3injury " (N = 61), and fibrosis "R4late " (N = 8). Groups differed in median time posttransplant, for example, R3injury 99 days vs R4late 3117 days. R2TCMR biopsies expressed typical TCMR-related transcripts, for example, intense IFNG-induced effects. R3injury displayed increased expression of parenchymal injury transcripts (eg, hypoxia-inducible factor EGLN1). R4late biopsies showed immunoglobulin transcripts and injury-related transcripts. R2TCMR correlated with histologic rejection although with many discrepancies, and R4late with fibrosis. R2TCMR , R3injury , and R4late correlated with liver function abnormalities. Supervised classifiers trained on histologic rejection showed less agreement with histology than unsupervised R2TCMR scores. No confirmed cases of clinical antibody-mediated rejection (ABMR) were present in the population, and strategies that previously revealed ABMR in kidney and heart transplants failed to reveal a liver ABMR phenotype. In conclusion, molecular analysis of liver transplant biopsies detects rejection, has the potential to resolve ambiguities, and could assist with immunosuppressive management.

T cell exhaustion is associated with antigen abundance and promotes transplant acceptance.

Exhaustion of T cells limits their ability to clear chronic infections or eradicate tumors. Here, in the context of transplant, we investigated whether T cell exhaustion occurs and has a role in determining transplant outcome. A peptide/MHC tetramer-based approach was used to track exhausted CD8+ T cells in a male-to-female skin transplant model. Transplant of large whole-tail skins, but not small tail skins (0.8 cm × 0.8 cm), led to exhaustion of anti-male tetramer+ CD8+ T cells and subsequently the acceptance of skin grafts. To study CD4+ T cell exhaustion, we used the TCR-transgenic B6 TEa cells that recognize a major transplant antigen I-Eα from Balb/c mice. TEa cells were adoptively transferred either into B6 recipients that received Balb/c donor skins or into CB6F1 mice that contained an excessive amount of I-Eα antigen. Adoptively transferred TEa cells in skin-graft recipients were not exhausted. By contrast, virtually all adoptively transferred TEa cells were exhausted in CB6F1 mice. Those exhausted TEa cells lost ability to reject Balb/c skins upon further transfer into lymphopenic B6.Rag1-/- mice. Hence, T cell exhaustion develops in the presence of abundant antigen and promotes transplant acceptance. These findings are essential for better understanding the nature of transplant tolerance.

Discovery and cross-validation of peripheral blood and renal biopsy gene expression signatures from ethnically diverse kidney transplant populations.

We determined peripheral blood (PB) and biopsy (Bx) RNA expression signatures in a Brazilian and US cohort of kidney transplant patients. Phenotypes assigned by precise histology were: acute rejection (AR), interstitial fibrosis/tubular atrophy/chronic rejection (CR), excellent functioning transplants (TX), and glomerulonephritis recurrence (GN). Samples were analyzed on microarrays and profiles from each cohort were cross-validated on the other cohort with similar phenotypes. We discovered signatures for each tissue: (1) AR vs TX, (2) CR vs TX, and (3) GN vs TX using the Random Forests algorithm. We validated biopsies signatures of AR vs TX (area under the curve [AUC] 0.97) and CR vs TX (AUC 0.87). We also validated both PB and Bx signatures of AR vs TX and CR vs TX with varying degrees of accuracy. Several biological pathways were shared between AR and CR, suggesting similar rejection mechanisms in these 2 clinical phenotypes. Thus, we identified gene expression signatures for AR and CR in transplant patients and validated them in independent cohorts of significantly different racial/ethnic backgrounds. These results reveal that there are strong unifying immune mechanisms driving transplant diseases and identified in the signatures discovered in each cohort, suggesting that molecular diagnostics across populations are feasible despite ethnic and environmental differences.

Generating automated kidney transplant biopsy reports combining molecular measurements with ensembles of machine learning classifiers.

We previously reported a system for assessing rejection in kidney transplant biopsies using microarray-based gene expression data, the Molecular Microscope® Diagnostic System (MMDx). The present study was designed to optimize the accuracy and stability of MMDx diagnoses by replacing single machine learning classifiers with ensembles of diverse classifier methods. We also examined the use of automated report sign-outs and the agreement between multiple human interpreters of the molecular results. Ensembles generated diagnoses that were both more accurate than the best individual classifiers, and nearly as stable as the best, consistent with expectations from the machine learning literature. Human experts had ≈93% agreement (balanced accuracy) signing out the reports, and random forest-based automated sign-outs showed similar levels of agreement with the human experts (92% and 94% for predicting the expert MMDx sign-outs for T cell-mediated (TCMR) and antibody-mediated rejection (ABMR), respectively). In most cases disagreements, whether between experts or between experts and automated sign-outs, were in biopsies near diagnostic thresholds. Considerable disagreement with histology persisted. The balanced accuracies of MMDx sign-outs for histology diagnoses of TCMR and ABMR were 73% and 78%, respectively. Disagreement with histology is largely due to the known noise in histology assessments (ClinicalTrials.gov NCT01299168).

The impact of donor and recipient common clinical and genetic variation on estimated glomerular filtration rate in a European renal transplant population.

Genetic variation across the human leukocyte antigen loci is known to influence renal-transplant outcome. However, the impact of genetic variation beyond the human leukocyte antigen loci is less clear. We tested the association of common genetic variation and clinical characteristics, from both the donor and recipient, with posttransplant eGFR at different time-points, out to 5 years posttransplantation. We conducted GWAS meta-analyses across 10 844 donors and recipients from five European ancestry cohorts. We also analyzed the impact of polygenic risk scores (PRS), calculated using genetic variants associated with nontransplant eGFR, on posttransplant eGFR. PRS calculated using the recipient genotype alone, as well as combined donor and recipient genotypes were significantly associated with eGFR at 1-year posttransplant. Thirty-two percent of the variability in eGFR at 1-year posttransplant was explained by our model containing clinical covariates (including weights for death/graft-failure), principal components and combined donor-recipient PRS, with 0.3% contributed by the PRS. No individual genetic variant was significantly associated with eGFR posttransplant in the GWAS. This is the first study to examine PRS, composed of variants that impact kidney function in the general population, in a posttransplant context. Despite PRS being a significant predictor of eGFR posttransplant, the effect size of common genetic factors is limited compared to clinical variables.

Banff Lung Report: Current knowledge and future research perspectives for diagnosis and treatment of pulmonary antibody-mediated rejection (AMR).

The Lung session of the 2017 14th Banff Foundation for Allograft Pathology Conference, Barcelona focused on the multiple aspects of antibody-mediated rejection (AMR) in lung transplantation. Multidimensional approaches for AMR diagnosis, including classification, histological and immunohistochemical analysis, and donor- specific antibody (DSA) characterization with their current strengths and limitations were reviewed in view of recent research. The group also discussed the role of tissue gene expression analysis in the context of unmet needs in lung transplantation. The current best practice for monitoring of AMR and the therapeutic approach are summarized and highlighted in this report. The working group reached consensus of the major gaps in current knowledge and focused on the unanswered questions regarding pulmonary AMR. An important outcome of the meeting was agreement on the need for future collaborative research projects to address these gaps in the field of lung transplantation.

Circulating exosomal miR-92b: Its role for cancer immunoediting and clinical value for prediction of posttransplant hepatocellular carcinoma recurrence.

A recurrence of hepatocellular carcinoma (HCC) after living donor liver transplantation (LDLT) is one of the major concerns reflecting the higher mortality of HCC. This study aimed to explore the impact of circulating exosomes on HCC development and recurrence. One-shot transfusion of hepatoma serum to naïve rats induced liver cancer development with gradual elevation of alpha-fetoprotein (AFP), but exosome-free hepatoma serum failed to induce AFP elevation. The microarray analysis revealed miR-92b as one of the highly expressing microribonucleic acids in hepatoma serum exosomes. Overexpression of miR-92b enhanced the migration ability of liver cancer cell lines with active release of exosomal miR-92b. The hepatoma-derived exosomal miR-92b transferred to natural killer (NK) cells, resulting in the downregulation of CD69 and NK cell-mediated cytotoxicity. Furthermore, higher expression of miR-92b in serum exosomes was confirmed in HCC patients before LDLT, and its value at 1 month after LDLT was maintained at a higher level in the patients with posttransplant HCC recurrence. In summary, we demonstrated the impact of circulating exosomes on liver cancer development, partly through the suppression of CD69 on NK cells by hepatoma-derived exosomal miR-92b. The value of circulating exosomal miR-92b may predict the risk of posttransplant HCC recurrence.

Mechanistic Sharing Between NK Cells in ABMR and Effector T Cells in TCMR.

Human organ allograft rejection depends on effector lymphocytes: NK cells in antibody-mediated rejection (ABMR) and effector T cells in T cell-mediated rejection (TCMR). We hypothesized that NK cell CD16a stimulation and CD8 T cell TCR/CD3 stimulation represent highly similar effector systems, and should lead to shared molecular changes between ABMR and TCMR. We studied similarity between soluble proteins and the transcripts induced in CD16a stimulated NK cells and TCR/CD3-stimulated T cells in vitro. Of 30 soluble mediators tested, CD16a-activated NK cells and CD3/TCR activated T cells produced the same limited set of five mediators-CCL3, CCL4, CSF2, IFNG, and TNF-and failed to produce 25 others. Many transcripts increased in stimulated NK cells were also increased in CD3-stimulated CD8 T cells (FDR < 0.05), including IFNG, CSF2, CCL3, CCL4, and XCL1. We hypothesized that shared transcripts not produced by other cell types should be expressed both in ABMR and TCMR kidney transplant biopsies. CD160, XCL1, TNFRSF9, and IFNG were selective for TCR/CD3-activated T cells and CD16a-NK cells and all were strongly increased in ABMR and TCMR. The molecules such as CD160 and XCL1 shared between NK cells in ABMR and effector T cells in TCMR may hold insights into important rejection mechanisms.

The Effect of Cortex/Medulla Proportions on Molecular Diagnoses in Kidney Transplant Biopsies: Rejection and Injury Can Be Assessed in Medulla.

Histologic assessment of kidney transplant biopsies relies on cortex rather than medulla, but for microarray studies, the proportion cortex in a biopsy is typically unknown and could affect the molecular readings. The present study aimed to develop a molecular estimate of proportion cortex in biopsies and examine its effect on molecular diagnoses. Microarrays from 26 kidney transplant biopsies divided into cortex and medulla components and processed separately showed that many of the most significant differences were in glomerular genes (e.g. NPHS2, NPHS1, CLIC5, PTPRO, PLA2R1, PLCE1, PODXL, and REN). Using NPHS2 (podocin) to estimate proportion cortex, we examined whether proportion cortex influenced molecular assessment in the molecular microscope diagnostic system. In 1190 unselected kidney transplant indication biopsies (Clinicaltrials.govNCT01299168), only 11% had <50% cortex. Molecular scores for antibody-mediated rejection, T cell-mediated rejection, and injury were independent of proportion cortex. Rejection was diagnosed in many biopsies that were mostly or all medulla. Agreement in molecular diagnoses in paired cortex/medulla samples (23/26) was similar to biological replicates (32/37). We conclude that NPHS2 expression can estimate proportion cortex; that proportion cortex has little influence on molecular diagnosis of rejection; and that, although histology cannot assess medulla, rejection does occur in medulla as well as cortex.

Hyalinosis Lesions in Renal Transplant Biopsies: Time-Dependent Complexity of Interpretation.

Because calcineurin inhibitor (CNI) immunosuppressive drugs induce arteriolar hyalinosis (ah) in kidney transplants, ah lesions can potentially provide information about drug exposure. We studied the relationship of ah lesions to findings and outcomes in 562 indication biopsies taken 3 days to 35 years after transplant. Prevalence of ah lesions increased with time of biopsy after transplant (TxBx). The ah scores correlated with arterial intimal thickening and atrophy-fibrosis but, unlike atrophy-fibrosis, did not increase until after 500 days because of a background of ah1 lesions in early biopsies reflecting donor aging. Correlation of ah scores with other features varied with TxBx-in early biopsies, donor age and related changes, and in very late biopsies, chronic antibody-mediated rejection and glomerulonephritis and associated lesions. After correction for TxBx, ah0 in intermediate time periods was associated with increased risk of T cell-mediated rejection and graft loss, probably because of underimmunosuppression and nonadherence. Thus, ah lesions in indication biopsies have multiple associations: donor age (early, usually ah1), chronic glomerular diseases (late, often ah2/3), and adequate exposure to CNIs at intermediate times. This threefold TxBx-dependent complexity must be considered when interpreting indication biopsies: ah lesions often indicate adequate CNI exposure, not toxicity, and unexpected ah0 should increase vigilance for nonadherence and underimmunosuppression.

Orthogonal Comparison of Molecular Signatures of Kidney Transplants With Subclinical and Clinical Acute Rejection: Equivalent Performance Is Agnostic to Both Technology and Platform.

We performed orthogonal technology comparisons of concurrent peripheral blood and biopsy tissue samples from 69 kidney transplant recipients who underwent comprehensive algorithm-driven clinical phenotyping. The sample cohort included patients with normal protocol biopsies and stable transplant (sTx) function (n = 25), subclinical acute rejection (subAR, n = 23), and clinical acute rejection (cAR, n = 21). Comparisons between microarray and RNA sequencing (RNA-seq) signatures were performed and demonstrated a strong correlation between the blood and tissue compartments for both technology platforms. A number of shared differentially expressed genes and pathways between subAR and cAR in both platforms strongly suggest that these two clinical phenotypes form a continuum of alloimmune activation. SubAR is associated with fewer or less expressed genes than cAR in blood, whereas in biopsy tissues, this clinical phenotype demonstrates a more robust molecular signature for both platforms. The discovery work done in this study confirms a clear ability to detect gene expression profiles for sTx, subAR, and cAR in both blood and biopsy tissue, yielding equivalent predictive performance that is agnostic to both technology and platform. Our data also provide strong biological insights into the molecular mechanisms underlying these signatures, underscoring their logistical potential as molecular diagnostics to improve clinical outcomes following kidney transplantation.

Real Time Central Assessment of Kidney Transplant Indication Biopsies by Microarrays: The INTERCOMEX Study.

The authors conducted a prospective trial to assess the feasibility of real time central molecular assessment of kidney transplant biopsy samples from 10 North American or European centers. Biopsy samples taken 1 day to 34 years posttransplantation were stabilized in RNAlater, sent via courier overnight at ambient temperature to the central laboratory, and processed (29 h workflow) using microarrays to assess T cell- and antibody-mediated rejection (TCMR and ABMR, respectively). Of 538 biopsy samples submitted, 519 (96%) were sufficient for microarray analysis (average length, 3 mm). Automated reports were generated without knowledge of histology and HLA antibody, with diagnoses assigned based on Molecular Microscope Diagnostic System (MMDx) classifier algorithms and signed out by one observer. Agreement between MMDx and histology (balanced accuracy) was 77% for TCMR, 77% for ABMR, and 76% for no rejection. A classification tree derived to provide automated sign-outs predicted the observer sign-outs with >90% accuracy. In 451 biopsy samples where feedback was obtained, clinicians indicated that MMDx more frequently agreed with clinical judgment (87%) than did histology (80%) (p = 0.0042). In 81% of feedback forms, clinicians reported that MMDx increased confidence in management compared with conventional assessment alone. The authors conclude that real time central molecular assessment is feasible and offers a useful new dimension in biopsy interpretation. ClinicalTrials.gov NCT#01299168.