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.

RNA expression profiling of renal allografts in a nonhuman primate identifies variation in NK and endothelial gene expression.

RNA transcript expression estimates are a promising method to study the mechanisms and classification of renal allograft rejections. Here we use the Nanostring platform to profile RNA expression in renal allografts in a nonhuman primate (NHP), the Cynomolgus monkey. We analyzed protocol and indication 278 archival renal allograft samples, both protocol and indication from 76 animals with diagnoses of chronic antibody-mediated rejection (CAMR), acute cellular rejection (TCMR), and MIXED (both CAMR and TCMR), plus normals and samples with no pathological rejection using a Cynomolgus-specific probe set of 67 genes. Analysis identified RNA expression heterogeneity of endothelial and NK genes within CAMR and TCMR, including the stages of CAMR. Three factors were partitioned into additional groups. One group with the longest allograft survival time is pure CAMR without NK or CD3. Three mixed groups show variation in NK and CD3. TCMR was split into 2 groups with variation in NK genes. Additional validation of the complete gene-set correlated many of the genes with diagnoses of CAMR, MIXED, and TCMR rejections and with Banff histologic criteria defined in human subjects. These NHP data demonstrate the utility of RNA expression profiling to identify additional heterogeneity of endothelial and NK RNA gene expressions.

RNA expression profiling of nonhuman primate renal allograft rejection identifies tolerance.

Tolerance induction to prevent allograft rejection is a long-standing clinical goal. However, convincing and dependable tolerance identification remains elusive. Hypothesizing that intragraft RNA expression is informative in both rejection and tolerance, we profile intrarenal allograft RNA expression in a mixed chimerism renal allograft model of cynomolgus monkeys and identify biologically significant tolerance. Analysis of 67 genes identified 3 dominant factors, each with a different pattern of gene expressions, relating to T cell-mediated rejection (TCMR), chronic antibody-mediated rejection (CAMR), or Tolerance. Clustering these 3 factors created 9 groups. One of the 9 clustered groups, the Tolerance cluster, showed the lowest probability of terminal rejection, the longest duration of allograft survival, and the lowest relative risk of terminal rejection. The Tolerance factor consists of a novel set of gene expressions including cytokine and immunoregulatory genes adding mechanistic insights into tolerance. The Tolerance factor could not be identified within current pathologic diagnostic categories. The TCMR and CAMR factors are dominant to the Tolerance factor, causing rejection even if the Tolerance factor is present. These 3 factors determine the probability of terminal rejection or tolerance. This novel a posteriori approach permits identification of pathways of rejection, including tolerance.

The Banff 2017 Kidney Meeting Report: Revised diagnostic criteria for chronic active T cell-mediated rejection, antibody-mediated rejection, and prospects for integrative endpoints for next-generation clinical trials.

The kidney sessions of the 2017 Banff Conference focused on 2 areas: clinical implications of inflammation in areas of interstitial fibrosis and tubular atrophy (i-IFTA) and its relationship to T cell-mediated rejection (TCMR), and the continued evolution of molecular diagnostics, particularly in the diagnosis of antibody-mediated rejection (ABMR). In confirmation of previous studies, it was independently demonstrated by 2 groups that i-IFTA is associated with reduced graft survival. Furthermore, these groups presented that i-IFTA, particularly when involving >25% of sclerotic cortex in association with tubulitis, is often a sequela of acute TCMR in association with underimmunosuppression. The classification was thus revised to include moderate i-IFTA plus moderate or severe tubulitis as diagnostic of chronic active TCMR. Other studies demonstrated that certain molecular classifiers improve diagnosis of ABMR beyond what is possible with histology, C4d, and detection of donor-specific antibodies (DSAs) and that both C4d and validated molecular assays can serve as potential alternatives and/or complements to DSAs in the diagnosis of ABMR. The Banff ABMR criteria are thus updated to include these alternatives. Finally, the present report paves the way for the Banff scheme to be part of an integrative approach for defining surrogate endpoints in next-generation clinical trials.

Molecular Assessment of Microcirculation Injury in Formalin-Fixed Human Cardiac Allograft Biopsies With Antibody-Mediated Rejection.

Precise diagnosis of antibody-mediated rejection (AMR) in cardiac allograft endomyocardial biopsies (EMBs) remains challenging. This study assessed molecular diagnostics in human EMBs with AMR. A set of 34 endothelial, natural killer cell and inflammatory genes was quantified in 106 formalin-fixed, paraffin-embedded EMBs classified according to 2013 International Society for Heart and Lung Transplantation (ISHLT) criteria. The gene set expression was compared between ISHLT diagnoses and correlated with donor-specific antibody (DSA), endothelial injury by electron microscopy (EM) and prognosis. Findings were validated in an independent set of 57 EMBs. In the training set (n = 106), AMR cases (n = 70) showed higher gene set expression than acute cellular rejection (ACR; n = 21, p < 0.001) and controls (n = 15, p < 0.0001). Anti-HLA DSA positivity was associated with higher gene set expression (p = 0.01). Endothelial injury by electron microscopy strongly correlated with gene set expression, specifically in AMR cases (r = 0.62, p = 0.002). Receiver operating characteristic curve analysis for diagnosing AMR showed greater accuracy with gene set expression (area under the curve [AUC] = 79.88) than with DSA (AUC = 70.47) and C4d (AUC = 70.71). In AMR patients (n = 17) with sequential biopsies, increasing gene set expression was associated with inferior prognosis (p = 0.034). These findings were confirmed in the validation set. In conclusion, biopsy-based molecular assessment of antibody-mediated microcirculation injury has the potential to improve diagnosis of AMR in human cardiac transplants.

Chronic Antibody-Mediated Rejection in Nonhuman Primate Renal Allografts: Validation of Human Histological and Molecular Phenotypes.

Molecular testing represents a promising adjunct for the diagnosis of antibody-mediated rejection (AMR). Here, we apply a novel gene expression platform in sequential formalin-fixed paraffin-embedded samples from nonhuman primate (NHP) renal transplants. We analyzed 34 previously described gene transcripts related to AMR in humans in 197 archival NHP samples, including 102 from recipients that developed chronic AMR, 80 from recipients without AMR, and 15 normal native nephrectomies. Three endothelial genes (VWF, DARC, and CAV1), derived from 10-fold cross-validation receiver operating characteristic curve analysis, demonstrated excellent discrimination between AMR and non-AMR samples (area under the curve = 0.92). This three-gene set correlated with classic features of AMR, including glomerulitis, capillaritis, glomerulopathy, C4d deposition, and DSAs (r = 0.39-0.63, p < 0.001). Principal component analysis confirmed the association between three-gene set expression and AMR and highlighted the ambiguity of v lesions and ptc lesions between AMR and T cell-mediated rejection (TCMR). Elevated three-gene set expression corresponded with the development of immunopathological evidence of rejection and often preceded it. Many recipients demonstrated mixed AMR and TCMR, suggesting that this represents the natural pattern of rejection. These data provide NHP animal model validation of recent updates to the Banff classification including the assessment of molecular markers for diagnosing AMR.

Banff Histopathological Consensus Criteria for Preimplantation Kidney Biopsies.

The Banff working group on preimplantation biopsy was established to develop consensus criteria (best practice guidelines) for the interpretation of preimplantation kidney biopsies. Digitally scanned slides were used (i) to evaluate interobserver variability of histopathologic findings, comparing frozen sections with formalin-fixed, paraffin-embedded tissue of wedge and needle core biopsies, and (ii) to correlate consensus histopathologic findings with graft outcome in a cohort of biopsies from international medical centers. Intraclass correlations (ICCs) and univariable and multivariable statistical analyses were performed. Good to fair reproducibility was observed in semiquantitative scores for percentage of glomerulosclerosis, arterial intimal fibrosis and interstitial fibrosis on frozen wedge biopsies. Evaluation of frozen wedge and core biopsies was comparable for number of glomeruli, but needle biopsies showed worse ICCs for glomerulosclerosis, interstitial fibrosis and tubular atrophy. A consensus evaluation form is provided to help standardize the reporting of histopathologic lesions in donor biopsies. It should be recognized that histologic parameters may not correlate with graft outcome in studies based on organs deemed to be acceptable after careful clinical assessment. Significant limitations remain in the assessment of implantation biopsies.

The XIIIth Banff Conference on Allograft Pathology: The Banff 2015 Heart Meeting Report: Improving Antibody-Mediated Rejection Diagnostics: Strengths, Unmet Needs, and Future Directions.

The 13th Banff Conference on Allograft Pathology was held in Vancouver, British Columbia, Canada from October 5 to 10, 2015. The cardiac session was devoted to current diagnostic issues in heart transplantation with a focus on antibody-mediated rejection (AMR) and small vessel arteriopathy. Specific topics included the strengths and limitations of the current rejection grading system, the central role of microvascular injury in AMR and approaches to semiquantitative assessment of histopathologic and immunophenotypic indicators, the role of AMR in the development of cardiac allograft vasculopathy, the important role of serologic antibody detection in the management of transplant recipients, and the potential application of new molecular approaches to the elucidation of the pathophysiology of AMR and potential for improving the current diagnostic system. Herein we summarize the key points from the presentations, the comprehensive, open and wide-ranging multidisciplinary discussion that was generated, and considerations for future endeavors.

The Banff 2015 Kidney Meeting Report: Current Challenges in Rejection Classification and Prospects for Adopting Molecular Pathology.

The XIII Banff meeting, held in conjunction the Canadian Society of Transplantation in Vancouver, Canada, reviewed the clinical impact of updates of C4d-negative antibody-mediated rejection (ABMR) from the 2013 meeting, reports from active Banff Working Groups, the relationships of donor-specific antibody tests (anti-HLA and non-HLA) with transplant histopathology, and questions of molecular transplant diagnostics. The use of transcriptome gene sets, their resultant diagnostic classifiers, or common key genes to supplement the diagnosis and classification of rejection requires further consensus agreement and validation in biopsies. Newly introduced concepts include the i-IFTA score, comprising inflammation within areas of fibrosis and atrophy and acceptance of transplant arteriolopathy within the descriptions of chronic active T cell-mediated rejection (TCMR) or chronic ABMR. The pattern of mixed TCMR and ABMR was increasingly recognized. This report also includes improved definitions of TCMR and ABMR in pancreas transplants with specification of vascular lesions and prospects for defining a vascularized composite allograft rejection classification. The goal of the Banff process is ongoing integration of advances in histologic, serologic, and molecular diagnostic techniques to produce a consensus-based reporting system that offers precise composite scores, accurate routine diagnostics, and applicability to next-generation clinical trials.

Long-term impact of CMV infection on allografts and on patient survival in renal transplant patients with protocol biopsies.

Cytomegalovirus (CMV) infection is a frequent complication of early posttransplantation. This study examines its impact on chronic allograft changes, long-term graft loss, and patient survival. We studied 594 patients who had protocol biopsies at 6 wk, and 3 and 6 mo posttransplantation. Chronic allograft changes were evaluated according to the updated Banff classification [interstitial fibrosis/tubular atrophy (IF/TA), vascular and glomerular lesions]. Follow-up data were available for up to 10 yr. CMV infection was diagnosed in 153 of 594 patients (26%) in the first year after transplantation, mostly within the first 3 mo. Graft survival was reduced in patients with CMV (P = 0.03) as well as the combined allograft/patient survival (P = 0.008). Prevalence of IF/TA at 6 wk after transplantation was already threefold higher in patients who experienced CMV infection later on compared with patients without CMV (P = 0.005). In multivariate analyses, CMV viremia or disease was not a significant factor for graft loss or death. In conclusion, patients with CMV infection posttransplantation show more chronic allograft changes early on, even before CMV infection, and development of IF/TA is not more prevalent in patients with CMV. Our data do not support a significant role of CMV in patient and graft outcomes.

Chemical Basis for Qualitative and Quantitative Differences Between ABO Blood Groups and Subgroups: Implications for Organ Transplantation.

Blood group ABH(O) carbohydrate antigens are carried by precursor structures denoted type I-IV chains, creating unique antigen epitopes that may differ in expression between circulating erythrocytes and vascular endothelial cells. Characterization of such differences is invaluable in many clinical settings including transplantation. Monoclonal antibodies were generated and epitope specificities were characterized against chemically synthesized type I-IV ABH and related glycans. Antigen expression was detected on endomyocardial biopsies (n = 50) and spleen (n = 11) by immunohistochemical staining and on erythrocytes by flow cytometry. On vascular endothelial cells of heart and spleen, only type II-based ABH antigens were expressed; type III/IV structures were not detected. Type II-based ABH were expressed on erythrocytes of all blood groups. Group A1 and A2 erythrocytes additionally expressed type III/IV precursors, whereas group B and O erythrocytes did not. Intensity of A/B antigen expression differed among group A1 , A2 , A1 B, A2 B and B erythrocytes. On group A2 erythrocytes, type III H structures were largely un-glycosylated with the terminal "A" sugar α-GalNAc. Together, these studies define qualitative and quantitative differences in ABH antigen expression between erythrocytes and vascular tissues. These expression profiles have important implications that must be considered in clinical settings of ABO-incompatible transplantation when interpreting anti-ABO antibodies measured by hemagglutination assays with reagent erythrocytes.

AST Cutting Edge of Transplantation 2013 Meeting Report: a comprehensive look at B cells and antibodies in transplantation.

Antibody-mediated rejection (ABMR) represents a significant clinical challenge for solid organ transplantation. Mechanistic understanding of ABMR is incomplete and diagnostic accuracy for ABMR is limited, and as a result, targeted treatment remains elusive and new treatment modalities are difficult to validate. Three hundred twenty-six participants from 15 countries met for the first Cutting Edge of Transplantation (CEOT) symposium organized by the American Society of Transplantation (AST) in Chandler, Arizona, February 14-16, 2013. During the 3-day interactive symposium, presentations, moderated poster sessions and round table discussions addressed cutting edge knowledge of B and plasma cell biology, mechanisms of antibody-mediated tissue injury, advances and limitations in ABMR diagnostics, as well as current and potential new treatment options for ABMR. The outcome of the meeting identified the following unmet needs for: (a) improved understanding of the regulation of B cell maturation and antibody response to enable targeted therapies; (b) more precise diagnostics of ABMR, including molecular pathology, risk stratification by sensitive antibody testing and monitoring of treatment effects; and (c) innovative multicenter trial designs that enhance observational power, in particular, in assessing synergistic multimodality therapies with reduced toxicities.

Cardiovascular MRI predicts 5-year adverse clinical outcome in heart transplant recipients.

Imaging recommendations for the follow-up of heart transplant recipients (HTRs) lack evidence justifying their prognostic value. Cardiovascular magnetic resonance imaging (CMRI) can characterize heart structure and function and has prognostic value in many myocardial diseases. We hypothesized that CMRI evaluation of cardiac allografts would predict adverse events. We performed CMRI on 60 HTRs evaluating biventricular size, function and myocardial scar. We performed survival analysis to identify independent predictors of cardiovascular (CV) death or hospitalization. Participants had a mean age of 51 ± 14 years, mean graft age of 3.5 years (±4) and 75% are male. Median follow-up time was 4.9 years with 22 CV hospitalizations and 7 CV deaths. A multivariable survival analysis of imaging and clinical variables identified myocardial scar (hazard ratio [HR] of 10.7, p = 0.005), right ventricular end- diastolic volume index (RVEDVI; 1.1/mL/m(2) , p = 0.001), graft age (HR = 1.2/year, p = 0.004) and previous allograft rejection (HR = 4.4, p = 0.006) as predictive of time to CV death or hospitalization. CMRI-derived myocardial scar and RVEDVI are independently associated with CV outcomes in HTRs.

Banff Initiative for Quality Assurance in Transplantation (BIFQUIT): reproducibility of polyomavirus immunohistochemistry in kidney allografts.

Immunohistochemistry (IHC) is the gold standard for diagnosing (positive vs. negative) polyomavirus BK (BKV) nephropathy and has the potential for disease staging based on staining intensity and quantification of infected cells. This multicenter trial evaluated the reproducibility of BKV IHC among 81 pathologists at 60 institutions. Participants stained tissue microarray slides and scored them for staining intensity and percentage of positive nuclei. Staining protocol details and evaluation scores were collected online. Slides were returned for centralized panel re-evaluation and kappa statistics were calculated. Individual assessment of staining intensity and percentage was more reproducible than combined scoring. Inter-institutional reproducibility was moderate for staining intensity (κ = 0.49) and percentage (κ = 0.42), fair for combined (κ = 0.25) and best for simple positive/negative scoring (κ = 0.78). Inter-observer reproducibility was substantial for intensity (κ = 0.74), percentage (κ = 0.66), positive/negative (κ = 0.78) and moderate for combined scoring (κ = 0.43). Inter-laboratory reproducibility was fair for intensity (κ = 0.37), percentage (κ = 0.40) and combined (κ = 0.24), but substantial for positive/negative scoring (κ = 0.67). BKV RNA copies/cell correlated with staining intensity (r = 0.56) and percentage (r = 0.62). These results indicate that BKV IHC is reproducible between observers but scoring should be simplified to a single-feature schema. Standardization of tissue processing and staining protocols would further improve inter-laboratory reproducibility.

Banff fibrosis study: multicenter visual assessment and computerized analysis of interstitial fibrosis in kidney biopsies.

Increasing interstitial fibrosis (IF) in native and kidney transplant biopsies is associated with functional decline and serves as a clinical trial end point. A Banff 2009 Conference survey revealed a range in IF assessment practices. Observers from multiple centers were asked to assess 30 renal biopsies with a range of IF and quantitate IF using two approaches on trichrome, Periodic acid-Schiff (PAS) and computer-assisted quantification of collagen III immunohistochemistry (C-IHC) slides, as well as assessing percent of cortical tubular atrophy% (TA%) and Banff total cortical inflammation score (ti-score). C-IHC using whole slide scans was performed. C-IHC assessment showed a higher correlation with organ function (r = -0.48) than did visual assessments (r = -0.32--0.42); computerized and visual C-IHC assessment also correlated (r = 0.64-0.66). Visual assessment of trichrome and C-IHC showed better correlations with organ function and C-IHC, than PAS, TA% and ti-score. However, visual assessment of IF, independent of approach, was variable among observers, and differences in correlations with organ function were not statistically significant among C-IHC image analysis and visual assessment methods. C-IHC image analysis correlated among three centers (r > 0.90, p < 0.0001, between all centers). Given the difficulty of visual IF assessment standardization, C-IHC image could potentially accomplish standardized IF assessment in multicenter settings.

Banff 2013 meeting report: inclusion of c4d-negative antibody-mediated rejection and antibody-associated arterial lesions.

The 12th Banff Conference on Allograft Pathology was held in Comandatuba, Brazil, from August 19-23, 2013, and was preceded by a 2-day Latin American Symposium on Transplant Immunobiology and Immunopathology. The meeting was highlighted by the presentation of the findings of several working groups formed at the 2009 and 2011 Banff meetings to: (1) establish consensus criteria for diagnosing antibody-mediated rejection (ABMR) in the presence and absence of detectable C4d deposition; (2) develop consensus definitions and thresholds for glomerulitis (g score) and chronic glomerulopathy (cg score), associated with improved inter-observer agreement and correlation with clinical, molecular and serological data; (3) determine whether isolated lesions of intimal arteritis ("isolated v") represent acute rejection similar to intimal arteritis in the presence of tubulointerstitial inflammation; (4) compare different methodologies for evaluating interstitial fibrosis and for performing/evaluating implantation biopsies of renal allografts with regard to reproducibility and prediction of subsequent graft function; and (5) define clinically and prognostically significant morphologic criteria for subclassifying polyoma virus nephropathy. The key outcome of the 2013 conference is defining criteria for diagnosis of C4d-negative ABMR and respective modification of the Banff classification. In addition, three new Banff Working Groups were initiated.

Banff initiative for quality assurance in transplantation (BIFQUIT): reproducibility of C4d immunohistochemistry in kidney allografts.

Detection of C4d is crucial for diagnosing antibody-mediated-rejection. We conducted a multicenter trial to assess the reproducibility for C4d immunohistochemistry on paraffin tissue. Unstained slides from a tissue microarray (TMA) comprising 44 kidney allograft specimens representing a full analytical spectrum for C4d were distributed to 73 institutions. Participants stained TMA slides using local protocols and evaluated their slides following the Banff C4d schema. Local staining details and evaluation scores were collected online. Stained slides were returned for centralized panel re-evaluation. Kappa statistics were used to determine reproducibility. Poor interinstitutional reproducibility was observed (kappa 0.17), which was equally due to limitations in interobserver (kappa 0.44) and interlaboratory reproducibility (kappa 0.46). Depending on the cut-off, reproducibility could be improved by omitting C4d grading and only considering ± calls. Heat-induced epitope recovery (pH 6-7, 20-30 min, citrate buffer) with polyclonal antibody incubation (<1:80, >40 min) appeared as best practice. The BIFQUIT trial results indicated that C4d staining on paraffin sections varies considerably between laboratories. Refinement of the current Banff C4d scoring schema and harmonization of tissue processing and staining protocols is necessary to achieve acceptable reproducibility.

Molecular diagnosis of T cell-mediated rejection in human kidney transplant biopsies.

Histologic diagnosis of T cell-mediated rejection is flawed by subjective assessments, nonspecific lesions and arbitrary rules. This study developed a molecular test for T cell-mediated rejection. We used microarray results from 403 kidney transplant biopsies to derive a classifier assigning T cell-mediated rejection scores to all biopsies, and compared these with histologic assessments. The score correlated with histologic lesions of T cell-mediated rejection (infiltrate, tubulitis). The accuracy of the classifier for the histology diagnoses was 89%. Very high and low molecular scores corresponded with unanimity among three pathologists on the presence or absence of T cell-mediated rejection, respectively. The molecular score had low sensitivity (50%) and positive predictive value (62%) for the histology diagnoses. However, histology showed similar disagreement between pathologists--only 45-56% sensitivity of one pathologist with diagnoses of T cell-mediated rejection by another. Discrepancies between molecular scores and histology were mostly when histology was ambiguous ("borderline") or unreliable, e.g. in cases with scarring or inflammation induced by tissue injury. Vasculitis (isolated v-lesion TCMR) was particularly discrepant, with most cases exhibiting low TCMR scores. We propose new rules to integrate molecular tests and histology into a precision diagnostic system that can reduce errors, ambiguity and interpathologist disagreement.

Molecular diagnosis of antibody-mediated rejection in human kidney transplants.

Antibody-mediated rejection is the major cause of kidney transplant failure, but the histology-based diagnostic system misses most cases due to its requirement for C4d positivity. We hypothesized that gene expression data could be used to test biopsies for the presence of antibody-mediated rejection. To develop a molecular test, we prospectively assigned diagnoses, including C4d-negative antibody-mediated rejection, to 403 indication biopsies from 315 patients, based on histology (microcirculation lesions) and donor-specific HLA antibody. We then used microarray data to develop classifiers that assigned antibody-mediated rejection scores to each biopsy. The transcripts distinguishing antibody-mediated rejection from other conditions were mostly expressed in endothelial cells or NK cells, or were IFNG-inducible. The scores correlated with the presence of microcirculation lesions and donor-specific antibody. Of 45 biopsies with scores>0.5, 39 had been diagnosed as antibody-mediated rejection on the basis of histology and donor-specific antibody. High scores were also associated with unanimity among pathologists that antibody-mediated rejection was present. The molecular score also strongly predicted future graft loss in Cox regression analysis. We conclude that microarray assessment of gene expression can assign a probability of ABMR to transplant biopsies without knowledge of HLA antibody status, histology, or C4d staining, and predicts future failure.

Precision diagnostics in transplantation: from bench to bedside.

The Canadian and American Societies of Transplantation held a symposium on February 22, 2012 in Quebec City focused on discovery, validation and translation of new diagnostic tools into clinical transplantation. The symposium focused on antibody testing, transplantation pathology, molecular diagnostics and laboratory support for the incompatible patient. There is an unmet need for more precise diagnostic approaches in transplantation. Significant potential for increasing the diagnostic precision in transplantation was recognized through the integration of conventional histopathology, molecular technologies and sensitive antibody testing into one enhanced diagnostic system.