A reliable method for avoiding false negative results with Luminex single antigen beads; evidence of the prozone effect.

Luminex single antigen bead (SAB) assays have become an essential tool in monitoring the status of antibody to the Human Leucocyte Antigen (HLA) of patients both before and after transplantation. In addition SAB data is used to aid risk stratification to assess immunological risk of humoral rejection in solid organ transplantation (CTAG/BTAG guidelines) [1]. Increasingly laboratories are reporting false negative results at high antibody titre due to a prozone effect. Here we report a case study where the prozone effect led to a false negative antibody result that could have resulted in adverse outcome. We describe a method to reliably remove the prozone effect through heat inactivation and the addition of Ethylenediaminetetraacetic acid (EDTA) to the Luminex wash buffer.

A single-centre comparison of the clinical outcomes at 6 months of renal transplant recipients administered Adoport® or Prograf® preparations of tacrolimus.

BACKGROUND: The use of generic formulations of immunosuppressive drugs in place of brand name drugs offers considerable cost savings. Brand name tacrolimus (Prograf®) came off patent in April 2008. However, published evidence supporting therapeutic equivalence of generic formulations of tacrolimus in solid organ transplantation is lacking. The South West Transplant Centre switched from administering Prograf® to a generic formulation (Adoport®) for de novo transplant recipients in November 2010. This study sought to compare the clinical outcomes of renal transplant recipients administered Prograf® with those receiving Adoport®. METHODS: Data regarding patient characteristics and clinical outcomes were collected retrospectively for all patients undergoing renal transplantation at the South West Transplant Centre between 8 November 2009 and 8 November 2011 to whom tacrolimus was prescribed. RESULTS: A total of 48 patients received Prograf® and 51 received Adoport®. At 6 months, no statistically significant differences were identified in the rates of patient survival, graft survival, acute allograft rejection, delayed graft function, calcineurin inhibitor toxicity or cytomegalovirus infection occurring within the two groups. CONCLUSIONS: This is the first study to compare the clinical outcomes of patients receiving Adoport® with those receiving brand name tacrolimus. We report comparable clinical outcomes at 6 months in patients receiving either Prograf® or Adoport® from the time of renal transplantation. These early outcome data therefore support the use of Adoport® in place of Prograf® as a potential cost-saving measure.

Activation of NF-kappaB and MAP kinase cascades by hypothermic stress in endothelial cells.

Signal transduction pathways and transcription factors are likely to be important mediators of stress responses to ischaemia and reperfusion injury following renal transplantation. We have investigated the activation of the transcription factor nuclear factor kappaB (NF-kappaB) and the mitogen activated protein kinases (MAPK), p44/42 (ERK 1/2), p38 and c-Jun N-terminal kinase (JNK) during cold stress at 4 degrees C. Human umbilical vein endothelial cells (HUVECs) were subjected to 72 h of hypothermia in a renal preservation solution. NF-kappaB activation was assessed by electromobility shift assays and MAPK activation by immunoblotting. Cell viability and apoptosis was assessed. Hypothermia activated the NF-kappaB complex, ERK 1/2 and p38 MAPK pathway. There was a 6-fold increase in NF-kappaB in the nucleus within minutes of hypothermia, correlating with p38 (p = 0.01) and ERK 1/2 activation (p = 0.03). A significant relationship was found between ERK 1/2, p38 and NF-kappaB throughout the 72 h time course (p = 0.01). In contrast, hypothermia had no effect on JNK phosphorylation. Inhibition of MAPK with an MEK inhibitor (PD098059) blocked the activation of NF-kappaB but a specific p38 inhibitor (SB203580) had no effect on NF-kappaB. Increased lactate production after 48 h indicated a switch towards anaerobic metabolism during prolonged hypothermia. Endothelial cells had a high viability and no DNA fragmentation throughout the experiment. Activation of stress pathways during organ procurement may be important in the quality of stored grafts.