Accurate measurement of emicizumab in the presence of factor (F) VIII is required in patients with severe hemophilia A treated with emicizumab, as well as additional need for FVIII substitution or ...emicizumab prophylaxis in patients with acquired or moderate to mild hemophilia A. However, the presence of FVIII potentially biases the results.
To assess the impact of plasma FVIII activity on determined emicizumab levels and evaluate different strategies for correction for or preanalytical inhibition of FVIII.
Evaluated strategies comprised of the following: (1) calculation of actual emicizumab plasma levels based on measured FVIII activities and FVIII-affected emicizumab values, (2) preanalytical heat treatment (56 °C for 40 minutes), and (3) neutralization of FVIII activity using FVIII inhibitors. Emicizumab levels and FVIII activities were measured using a modified FVIII one-stage clotting assay and a chromogenic FVIII assay based on bovine factors, respectively.
Spiking experiments revealed a consistent linear association between FVIII activities and determined (FVIII-affected) emicizumab results at different emicizumab input levels (∼0.12 μg/mL per IU/dL of FVIII). This principally allowed for mathematical correction of measured emicizumab levels in the presence of FVIII. While a 40% to 50% activity loss of intrinsic plasma emicizumab through heat treatment was observed in patient samples, emicizumab spiked into FVIII-deficient plasma was not or only marginally affected. Application of inhibitor-based FVIII neutralization led to good agreement of results when compared with direct quantification of emicizumab by liquid chromatography-tandem mass spectrometry.
Inhibitor-based FVIII neutralization appears to be a feasible strategy for accurate measurement of plasma emicizumab levels in the presence of FVIII activity.
The endothelial cell-dependent PC (protein C) pathway is critically involved in the regulation of coagulation, anti-inflammatory, and cytoprotective signaling. Its reactivity shows high ...interindividual variability, and it contributes to prothrombotic disorders, such as the FVL (factor V Leiden) mutation.
Endothelial colony-forming cells (ECFCs) were isolated from heparinized peripheral blood from healthy individuals and FVL carriers. Confluent monolayers of ECFCs were overlaid with plasma, and thrombin formation was initiated by addition of tissue factor (1 pmol/L). Subsequently, thrombin and APC (activated PC) formation rates were measured over time using oligonucleotide-based enzyme capture assays. To induce downregulation of TM (thrombomodulin) expression, ECFCs were stimulated with IL-1β (interleukin 1β). In vivo APC response rates were monitored in study participants after infusion of low-dose rFVIIa (recombinant activated factor VII).
The median peak APC concentration was 1.12 nmol/L in experiments with IL-1β stimulated ECFCs and 3.66 nmol/L without IL-1β. Although thrombin formation rates were comparable, APC formation rates were significantly higher in FVL carriers (n=6) compared to noncarriers (n=5) as evidenced by a higher ratio between the area under the curve of APC generation to the area under the curve of thrombin generation (median 0.090 versus 0.031,
=0.017). These ex vivo results were correlated with an increased APC response to rFVIIa-induced thrombin formation in FVL carriers in vivo.
Patient-specific ex vivo modeling of the PC pathway was achieved using blood-derived ECFCs. The correlation between in and ex vivo APC response rates confirms that the autologous PC model accurately depicts the in vivo situation.
Short biotinylated oligodeoxynucleotides immobilized on streptavidin-coated magnetic beads allow for convenient and rapid purification of single-stranded oligodeoxynucleotides from crude asymmetric ...PCR mixtures, facilitating the selection of DNA aptamers.
Rosai‐Dorfman disease (RDD) is a rare disease which characterized by proliferation and overproduction of histiocytes in the lymph nodes appearing as lymphadenopathy, however, it may also occur in ...extranodal sites. The occurrence of unusual manifestations of the disease such as the appearance of the mass in an unusual area may increase the probability of misdiagnosis. Herein, we describe a case of RDD in an old woman with an unusual appearance of RDD in the leg that was successfully treated by thalidomide.
Bart syndrome is a rare genetic disorder characterized by aplasia cutis congenita, epidermolysis bullosa (EB), and nail abnormalities. We reported an unusual case of Bart syndrome associated with ...skeletal abnormalities and bilateral clubfoot.
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