Acquired hemophilia A in the setting of dual anticoagulation therapy and lupus anticoagulant: a case report.

BACKGROUND: Acquired hemophilia A is a disorder caused by autoantibodies against coagulation factor VIII that may present with severe bleeding. We report a rare case of acquired hemophilia A presenting with coexisting lupus anticoagulant. CASE PRESENTATION: An 81-year-old Caucasian female presented with large ecchymoses over the torso and extremities in the setting of an enoxaparin bridge to warfarin. Anticoagulation was held, but she continued to develop bruises with significant anemia and prolonged coagulation studies that failed to correct with mixing. Workup revealed factor VIII activity < 1% and a positive lupus anticoagulant. Initial testing for a factor VIII inhibitor was confounded by the presence of lupus anticoagulant, requiring a chromogenic Bethesda assay to confirm the presence of the inhibitor, establishing the diagnosis of acquired hemophilia A. The patient was initially treated with oral prednisone 80 mg daily and factor VIII inhibitor bypassing activity 25 units/kg twice daily before transitioning to susoctocog alfa 50 units/kg twice daily after placement of a tunneled line for outpatient rituximab infusions. On discharge, the patient's ecchymoses were resolving and factor VIII levels improved. Following completion of rituximab therapy, the patient's factor VIII activity normalized and factor VIII inhibitor was suppressed. CONCLUSIONS: Diagnosis of acquired hemophilia A can be confounded by other causes of abnormal coagulation studies and may require specialized testing, such as a chromogenic Bethesda assay, to confirm the presence of a factor VIII inhibitor.

Sex and Tamoxifen confound murine experimental studies in cardiovascular tissue engineering.

Tissue engineered vascular grafts hold promise for the creation of functional blood vessels from biodegradable scaffolds. Because the precise mechanisms regulating this process are still under investigation, inducible genetic mouse models are an important and widely used research tool. However, here we describe the importance of challenging the baseline assumption that tamoxifen is inert when used as a small molecule inducer in the context of cardiovascular tissue engineering. Employing a standard inferior vena cava vascular interposition graft model in C57BL/6 mice, we discovered differences in the immunologic response between control and tamoxifen-treated animals, including occlusion rate, macrophage infiltration and phenotype, the extent of foreign body giant cell development, and collagen deposition. Further, differences were noted between untreated males and females. Our findings demonstrate that the host-response to materials commonly used in cardiovascular tissue engineering is sex-specific and critically impacted by exposure to tamoxifen, necessitating careful model selection and interpretation of results.

Tandem phage-display for the identification of non-overlapping binding pairs of recombinant affinity reagents.

The 'sandwich' binding format, which uses two reagents that can bind simultaneously to a given analyte, is the gold standard in diagnostics and many biochemical techniques. One of the bottlenecks in creating a sandwich assay is identifying pairs of reagents that bind non-competitively to the target. To bridge this gap, we invented Megaprimer Shuffling for Tandem Affinity Reagents (MegaSTAR) to identify non-competitive binding pairs of recombinant affinity reagents through phage-display. The key innovation in MegaSTAR is the construction of a tandem library, in which two reagents are randomly-displayed on the phage surface. This is accomplished by using a pool of 300-nucleotide long 'megaprimers', which code for previously-selected reagents, to prime second strand synthesis of a single-stranded DNA template and generate millions of pair-wise combinations. The tandem library is then affinity selected to isolate pairs that both reagents contribute to binding the target. As a proof-of-concept, we used MegaSTAR to identify pairs of fibronectin type III monobodies for three human proteins. For each target, we could identify between five and fifteen unique pairs and successfully used a single pair in a sandwich assay. MegaSTAR is a versatile tool for generating sandwich ELISA-grade and bispecific reagents.