Molecular Microscope Diagnostic System in Patients after Kidney Transplantation-First Experience.

BACKGROUND: The diagnosis of graft rejection relies on the identification of donor-specific antibodies along with histological findings. Borderline changes are particularly challenging, representing non-rejection findings in up to 70% of cases. The analysis aimed to compare the results of histopathological conclusions with the findings from examination using a molecular microscope, which assesses gene expression (whole-genome microarray chip technology). METHODS: Molecular microscope examination (MMDx) was applied to twelve patients (six men and six women) who underwent either indication or protocol graft biopsy. RESULTS: The average age of patients was 46.6 years ± 4.2 (average follow-up from kidney transplantation was 6.1 months ± 1.2). MMDx examination was performed during indication biopsy in 11 patients and protocol biopsy in 1 patient. A total of 33% of the findings matched and 50% did not. Finally, we present a case of a patient with acute cellular rejection findings without clinical and laboratory correlation, where the use of MMDx significantly altered the treatment strategy. CONCLUSIONS: MMDx examination is suitable for complementing patients with ambiguous histological findings and a clinical picture not corresponding to biopsy results. The limitations of MMDx include cost and its inability to evaluate the potential recurrence of the underlying kidney disease in the graft.

Relationship between blood and tissue-based rejection-related transcripts in heart transplantation.

BACKGROUND: The purpose of the study is to investigate the relationship between blood and tissue-derived rejection-related transcripts from blood gene expression profiling (GEP) and molecular microscope in the setting of allograft rejection in heart transplant. METHODS: All heart transplant patients from August 2021 to May 2022 with both circulating blood GEP (AlloMap) and endomyocardial biopsy with molecular microscope diagnostic system (MMDx) within 4 weeks were included (N = 173 samples). We obtained individual blood GEP-based messenger RNA transcript expression levels of the 11 target genes from CareDx. Student's t-test was performed to compare blood GEP transcript expression levels between no rejection and rejection as assessed by MMDx. A Scatter plot with Spearman correlation analysis was performed to compare the relationship between transcript expression levels from AlloMap and MMDx, with and without allograft rejection. RESULTS: There were 52 samples (30.1%) with antibody-mediated rejection (ABMR) and 15 samples (8.7%) with T-cell-mediated rejection (TCMR), as assessed by MMDx. Expression of one of the blood ITGA4 (Integrin alpha 4) expression level was elevated in ABMR, compared to no ABMR (4,607.5 vs 4,217.5; p = 0.019). Most tissue rejection-associated transcript expression levels were elevated in ABMR, and tissue ROBO4 expression correlated with the blood ITGA4 expression with moderate or greater effect size in all samples (Spearman's R = 0.31; p < 0.001). There was also a positive correlation between blood ITGA4 and tissue ROBO4 expression in samples without ABMR (Spearman's R = 0.33; p < 0.001), but no correlation between blood ITGA4 and tissue ROBO4 expression in samples with ABMR (Spearman's R = 0.009; p = 0.513). CONCLUSIONS: Circulating blood ITGA4 expression is elevated in antibody-mediated rejection (AMR) and correlates with myocardial expression of ROBO4. The knowledge of individual transcript expression levels in blood and in tissue may provide insights into various disease processes in heart transplant patients. Taken together, the results of our study reveal an overlap between 2 objective post-heart transplant rejection surveillance methods, identify potential novel markers for ABMR, and reveal the need for a deeper understanding of molecular mechanisms underlying allograft rejection.

The utilization of molecular microscope in management of heart transplant recipients in the era of noninvasive monitoring.

INTRODUCTION: Monitoring for graft rejection is a fundamental tenet of post-transplant follow-up. In heart transplantation (HT) in particular, rejection has been traditionally assessed with endomyocardial biopsy (EMB). EMB has potential complications and noted limitations, including interobserver variability in interpretation. Additional tests, such as basic cardiac biomarkers, cardiac imaging, gene expression profiling (GEP) scores, donor-derived cell-free DNA (dd-cfDNA) and the novel molecular microscope diagnostic system (MMDx) have become critical tools in rejection surveillance beyond standard EMB. METHODS: This paper describes an illustrative case followed by a review of MMDx within the context of other noninvasive screening modalities for rejection. CONCLUSIONS: We suggest MMDx be used to assist with early detection of rejection in cases of discordance between EMB and other noninvasive studies.

Rejection! Or is it? Correlation among molecular microscope diagnostic system, histopathology and clinical judgement following heart transplantation.

PURPOSE: Little is known about clinical decision making among discordant findings concerning for rejection with endomyocardial biopsy (EMBx) and Molecular Microscope Diagnostic System (MMDx) in patients following heart transplantation. METHODS: Two hundred and twenty-eight corresponding EMBx and MMDx specimens from 135 adult heart transplant patients were retrospectively reviewed. Rejection was classified as t-cell mediated rejection ≥2R and/or antibody mediated rejection ≥1. Clinical decision making among concordant and discordant cases of EMBx and MMDx results were reviewed. RESULTS: Patient characteristics were comparable between concordant and discordant patient groups (median age 60 yrs., 76% male, and 71% White). A total of 167/228 specimens (73%) were concordant for no rejection with 98% agreement in clinical decision making and 25/228 (11%) concordant for rejection with 64% agreement in clinical decision making. Among the 36/228 (16%) discordant samples, clinical decision-making agreed on treatment for rejection in five of the MMDx samples and three of the EMBx samples. CONCLUSIONS: MMDx can be an additional tool to diagnose rejection not detected by the traditional EMBx and influence clinical decision making in guiding appropriate treatment. Ongoing investigation into the clinical utility of MMDx is warranted to determine the significance of discordant findings among diagnostic modalities when assessing for rejection.

Kidney Allograft Monitoring by Combining Donor-Derived Cell-Free DNA and Molecular Gene Expression: A Clinical Management Perspective.

Donor-derived cell-free DNA (dd-cfDNA) may safely assess kidney allograft rejection. Molecular Microscope (MMDx®) gene expression may offer increased precision to histology. This single-center retrospective study monitored kidney transplant recipients for rejection at specified time intervals by utilizing creatinine (SCr), proteinuria, donor-specific antibodies (DSAs), and dd-cfDNA. A clinically indicated biopsy sample was sent for histopathology and MMDx®. Patients were categorized into rejection (Rej) and non-rejection (NRej) groups, and further grouped according to antibody-mediated rejection (ABMR) subtypes. Rej and NRej groups included 52 and 37 biopsies, respectively. Median follow-up duration was 506 days. DSAs were positive in 53% and 22% of patients in both groups, respectively (p = 0.01). Among these groups, pre- and post-intervention median SCr, proteinuria, and dd-cfDNA at 1 month, 2 months, and at the last follow-up revealed significant difference for dd-cfDNA (all p = 0.01), however, no difference was found for SCr and proteinuria (p > 0.05). The AUC was 0.80 (95% CI: 0.69-0.91), with an optimal dd-cfDNA criterion of 2.2%. Compared to histology, MMDx® was more likely to diagnose ABMR (79% vs. 100%) with either C4d positivity or negativity and/or DSA positivity or negativity. Hence, a pre- and post-intervention allograft monitoring protocol in combination with dd-cfDNA, MMDx®, and histology has aided in early diagnosis and timely individualized intervention.

Bradycardia in Recent Heart Transplant: Will the Microscope Illuminate the True Answer?CME.

Transplant recipients are at risk of developing rejection that may cause significant morbidity and mortality following transplantation The clinical presentation of rejection may be atypical, leading to difficulties in diagnosis and management especially in cases with a nondiagnostic biopsy specimen. The emergence of artificial intelligence may aid in clinical decision making when traditional techniques are inconclusive.

Recent Advancements in the Assessment of Renal Transplant Dysfunction with an Emphasis on Microarray Molecular Diagnostics.

Conventional assessment of renal transplant rejection and injury through use of histology, C4d staining, and HLA antibody testing, has been the standard approach to transplant management. By many measures, these methods of conventional assessment may be considered flawed, particularly with the subjective nature of histologic diagnoses. The Alberta Transplant Applied Genomics Center has developed the Molecular Microscope diagnostic system, which uses microarrays to measure gene expression. These data are analyzed using classifiers (weighted equations) that compare the tested biopsy to a proprietary reference set of biopsies to provide objective measures of the status of the renal transplant.

Revealing Higher Order Protein Structure Using Mass Spectrometry.

The development of rapid, sensitive, and accurate mass spectrometric methods for measuring peptides, proteins, and even intact protein assemblies has made mass spectrometry (MS) an extraordinarily enabling tool for structural biology. Here, we provide a personal perspective of the increasingly useful role that mass spectrometric techniques are exerting during the elucidation of higher order protein structures. Areas covered in this brief perspective include MS as an enabling tool for the high resolution structural biologist, for compositional analysis of endogenous protein complexes, for stoichiometry determination, as well as for integrated approaches for the structural elucidation of protein complexes. We conclude with a vision for the future role of MS-based techniques in the development of a multi-scale molecular microscope. Graphical Abstract ᅟ.