Anti-TRBC1 antibody-based flow cytometric detection of T-cell clonality: standardization of sample preparation and diagnostic implementation

Simple Summary The anti-TRBC1 antibody JOVI-1 has recently been identified as a flow cytometry marker potentially useful for assessment of T-cell clonality. The aim of this study was to optimize a flow cytometric method for routine use of anti-TRBC1 to assess T-cell clonality and validate it in a large series of normal and pathological samples. Our results showed that the best resolution to accurately identify TRBC1(+) cells was achieved by adding the CD3 antibody either simultaneously or after TRBC1. In addition, TRBC1(+)/TRBC1(-) ratios within different T alpha beta-cell subsets are provided as expected reference ranges for polyclonal T-cells. Based on the optimized approach here proposed, we detected monoclonal T alpha beta-cell populations with high specificity (96%) and a high analytical sensitivity/level of detection (<= 10(-4)), when clonal T-cells exhibited immunophenotypic aberrancies. These findings further support and extend previous observations about the utility of TRBC1 for the diagnostic screening and monitoring of clonal T alpha beta-cell populations.A single antibody (anti-TRBC1; JOVI-1 antibody clone) against one of the two mutually exclusive T-cell receptor beta-chain constant domains was identified as a potentially useful flow-cytometry (FCM) marker to assess T alpha beta-cell clonality. We optimized the TRBC1-FCM approach for detecting clonal T alpha beta-cells and validated the method in 211 normal, reactive and pathological samples. TRBC1 labeling significantly improved in the presence of CD3. Purified TRBC1(+) and TRBC1(-) monoclonal and polyclonal T alpha beta-cells rearranged TRBJ1 in 44/47 (94%) and TRBJ1+TRBJ2 in 48 of 48 (100%) populations, respectively, which confirmed the high specificity of this assay. Additionally, TRBC1(+)/TRBC1(-) ratios within different T alpha beta-cell subsets are provided as reference for polyclonal cells, among which a bimodal pattern of TRBC1-expression profile was found for all TCRV beta families, whereas highly-variable TRBC1(+)/TRBC1(-) ratios were observed in more mature vs. naive T alpha beta-cell subsets (vs. total T-cells). In 112/117 (96%) samples containing clonal T alpha beta-cells in which the approach was validated, monotypic expression of TRBC1 was confirmed. Dilutional experiments showed a level of detection for detecting clonal T alpha beta-cells of <= 10(-4) in seven out of eight pathological samples. These results support implementation of the optimized TRBC1-FCM approach as a fast, specific and accurate method for assessing T-cell clonality in diagnostic-FCM panels, and for minimal (residual) disease detection in mature T alpha beta(+) leukemia/lymphoma patients.Stemcel biology/Regenerative medicine (incl. bloodtransfusion

Pharmacological and nutritional targeting of voltage-gated sodium channels in the treatment of cancers

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Clinical, biochemical and molecular markers of injury before transplantation

*"' .• The use of organs from donors after circulatory death (DCD) has been recommended as one strategy to enlarge the donor pool and raise the transplant rate. However, DCD allografts had higher incidence of early post-transplant dysfunction. The general aim of this research project was to develop clinical and experimental strategies to reduce the incidence of early post-transplant dysfunction of kidney and liver allografts from DCD. First the ability of a clinical scoring system based on donor data for identifying DCD kidneys with high-risk of post-transplant dysfunction was evaluated using the Oxford and the UK National DCD kidney transplant cohorts. This works suggest that stratification of DCD kidneys before transplantation might allow early identification of kidneys in which lower graft function and survival could be expected if any additional therapeutic intervention is implemented. Second, as it has been suggested that hypothermic machine perfusion (HMP) may protect DCD kidneys from additional preservation injury and improve their outcome after transplantation, this work explored the benefit of HMP as preservation technique fo DCD kidneys in Oxford and discusses the potential of this technique for reducing the incidence of post-transplant dysfunction in DCD kidneys. The Oxford. Liver Group has provided evidence of the benefit of preservation with normothermic machine perfusion (NMP) on post-transplant function and survival of DCD liver allografts. In this work, the molecular mechanisms associated with this benefit were characterized using micro array technology. This analysis suggests that the beneficial effect ofNMP may be associated with the induction of the ischaemic preconditioning phenomenon and highlights a group of genes with potential for gene therapy. Finally, this works provides the "proof-of-concept" that the use of a non-mammalian viral vector for gene transfer of kidneys and livers during conventional cold preservation is feasible and is not associated with additional tissue injury.EThOS - Electronic Theses Online ServiceGBUnited Kingdo

The deceased donor score system in kidney transplants from deceased donors after cardiac death.

SUMMARY: A clinical score to identify kidneys from donors after cardiac death (DCD) with a high risk of dysfunction following transplantation could be a useful tool to guide the introduction of new algorithms for the preservation of these organs and improve their outcome after transplantation. We investigated whether the deceased donor score (DDS) system could identify DCD kidneys with higher risk of early post-transplant dysfunction. The DDS was validated in a cohort of 168 kidney transplants from donors after brain death (DBD) and then applied to a cohort of 56 kidney transplants from DCD. In the DBD cohort, the DDS grade predicted the incidence of delayed graft function (DGF) and levels of serum creatinine at 3 and 12 months post-transplant. Similarly, in the DCD cohort, the DDS grade correlated with DGF and also predicted the levels of serum creatinine at 3 and 12 months. Interestingly, the DDS identified a subgroup of marginal DCD kidneys in which minimization of cold ischemia time produced better early clinical outcome. These results highlight the impact of early interventions on clinical outcome of marginal DCD kidneys and open the possibility of using the DDS to identify those kidneys that may benefit most from therapeutic interventions before transplantation