Towards a consensus-based classification of childhood arterial ischemic stroke.

Background and purposeThe implementation of uniform nomenclature and classification in adult arterial ischemic stroke (AIS) has been critical for defining outcomes and recurrence risks according to etiology and in developing risk-stratified treatments. In contrast, current classification and nomenclature in childhood AIS are often overlapping or contradictory. Our purpose was to develop a comprehensive consensus-based classification system for childhood AIS.MethodsUsing a modified-Delphi method, members of the International Pediatric Stroke Study (IPSS) developed the Childhood AIS Standardized Classification And Diagnostic Evaluation (CASCADE) criteria. Two groups of pediatric stroke specialists from the IPSS classified 7 test cases using 2 methods each: (1) classification typical of the individual clinician's current clinical practice; and (2) classification based on the CASCADE criteria. Group 1 underwent in-person training in the utilization of the CASCADE criteria. Group 2 classified the same cases via an online survey, including definitions but without training. Inter-rater reliability (IRR) was assessed via multi-rater unweighted κ-statistic.ResultsIn Group 1 (with training), IRR was improved using CASCADE criteria (κ=0.78, 95% CI=[0.49, 0.94]), compared with typical clinical practice (κ=0.40, 95% CI=[0.11, 0.60]). In Group 2 (without training), IRR was lower than among trained raters (κ=0.61, 95% CI=[0.29, 0.77]), but higher than current practice (κ=0.23, 95% CI=[0.03, 0.36]).ConclusionsA new, consensus-based classification system for childhood AIS, the CASCADE criteria, can be used to classify cases with good IRR. These preliminary findings suggest that the CASCADE criteria may be particularity useful in the setting of prospective multicenter studies in childhood-onset AIS, where standardized training of investigators is feasible

Pharmacogenetics of warfarin in a paediatric population: time in therapeutic range, initial and stable dosing and adverse effects

Warfarin is used in paediatric populations, but dosing algorithms incorporating pharmacogenetic data have not been developed for children. Previous studies have produced estimates of the effect of polymorphisms in Cytochrome P450 2C9 (CYP2C9) and vitamin K epoxide reductase complex subunit 1 (VKORC1) on stable warfarin dosing, but data on time in therapeutic range, initial dosing and adverse effects are limited. Participants (n=97) were recruited, and routine clinical data and salivary DNA samples were collected from all participants and analysed for CYP2C9*2, *3 and VKORC1-1639 polymorphisms.VKORC1 -1639 was associated with a greater proportion of the first 6 months’ treatment time spent within the target International Normalised Ratio (INR) range, accounting for an additional 9.5% of the variance in the proportion of time. CYP2C9*2 was associated with a greater likelihood of INR values exceeding the target range during the initiation of treatment (odds ratio (OR; per additional copy) 4.18, 95% confidence interval (CI) 1.42, 12.34). CYP2C9*2 and VKORC1-1639 were associated with a lower dose requirement, and accounted for almost 12% of the variance in stable dose. VKORC1-1639 was associated with an increased likelihood of mild bleeding complications (OR (heterozygotes vs homozygotes) 4.53, 95% CI 1.59, 12.93). These data show novel associations between VKORC1-1639 and CYP2C9*2 and INR values in children taking warfarin, as well as replicating previous findings with regard to stable dose requirements. The development of pharmacogenomic dosing algorithms for children using warfarin has the potential to improve clinical care in this population

Assay of the von Willebrand factor (VWF) propeptide to identify patients with type 1 von Willebrand disease with decreased VWF survival

Type 1 von Willebrand disease (VWD) is characterized by a partial quantitative deficiency of von Willebrand factor (VWF). Few VWF gene mutations have been identified that cause dominant type 1 VWD. The decreased survival of VWF in plasma has recently been identified as a novel mechanism for type 1 VWD. We report 4 families with moderately severe type 1 VWD characterized by low plasma VWF:Ag and FVIII:C levels, proportionately low VWF:RCo, and dominant inheritance. A decreased survival of VWF in affected individuals was identified with VWF half-lives of 1 to 3 hours, whereas the half-life of VWF propeptide (VWFpp) was normal. DNA sequencing revealed a single (heterozygous) VWF mutation in affected individuals, S2179F in 2 families, and W1144G in 2 families, neither of which has been previously reported. We show that the ratio of steady-state plasma VWFpp to VWF:Ag can be used to identify patients with a shortened VWF half-life. An increased ratio distinguished affected from unaffected individuals in all families. A significantly increased VWFpp/VWF:Ag ratio together with reduced VWF:Ag may indicate the presence of a true genetic defect and decreased VWF survival phenotype. This phenotype may require an altered clinical therapeutic approach, and we propose to refer to this phenotype as type-1C VWD

The Effect of Factor VIII Deficiencies and Replacement and Bypass Therapies on Thrombus Formation under Venous Flow Conditions in Microfluidic and Computational Models

<div><p>Clinical evidence suggests that individuals with factor VIII (FVIII) deficiency (hemophilia A) are protected against venous thrombosis, but treatment with recombinant proteins can increase their risk for thrombosis. In this study we examined the dynamics of thrombus formation in individuals with hemophilia A and their response to replacement and bypass therapies under venous flow conditions. Fibrin and platelet accumulation were measured in microfluidic flow assays on a TF-rich surface at a shear rate of 100 s⁻¹. Thrombin generation was calculated with a computational spatial-temporal model of thrombus formation. Mild FVIII deficiencies (5–30% normal levels) could support fibrin fiber formation, while severe (<1%) and moderate (1–5%) deficiencies could not. Based on these experimental observations, computational calculations estimate an average thrombin concentration of ∼10 nM is necessary to support fibrin formation under flow. There was no difference in fibrin formation between severe and moderate deficiencies, but platelet aggregate size was significantly larger for moderate deficiencies. Computational calculations estimate that the local thrombin concentration in moderate deficiencies is high enough to induce platelet activation (>1 nM), but too low to support fibrin formation (<10 nM). In the absence of platelets, fibrin formation was not supported even at normal FVIII levels, suggesting platelet adhesion is necessary for fibrin formation. Individuals treated by replacement therapy, recombinant FVIII, showed normalized fibrin formation. Individuals treated with bypass therapy, recombinant FVIIa, had a reduced lag time in fibrin formation, as well as elevated fibrin accumulation compared to healthy controls. Treatment of rFVIIa, but not rFVIII, resulted in significant changes in fibrin dynamics that could lead to a prothrombotic state.</p></div

Thrombin generation under flow in FVIII deficiencies.

<p>The average thrombin concentration within a thrombus was calculated using a spatial-temporal computational model of thrombus formation on 2.3 fmol TF/cm². (A) Average thrombin concentration as a function of time for FVIII levels of 1, 5, 10, 20, and 100%. The dynamics of thrombin generation were quantified by three metrics: (B) Maximum thrombin concentration, which is the thrombin concentration at the end of the 5 min. simulation. (C) The lag time, which is the time to 1 nM thrombin. (D) The velocity, which is the slope of the average thrombin curve from the lag time to the end of the simulation. Each data point (•) represents a single simulation. The lines are extrapolations between simulation data points.</p

Thrombi formed under flow on collagen-TF surfaces from individuals with FVIII deficiencies.

<p>Recalcified whole blood was perfused over glass slides coated with 2.3/cm² and type 1 fibrillar collagen at 100 s⁻¹ for 5 min. Representative images of platelets (<i>blue</i>, anti-CD41) and fibrin(ogen) (<i>green</i>, Alexa488-fibrinogen) accumulation for a normal control (A) and hemophilia samples with plasma FVIII levels of 11.1% (B), 3.1% (C), and 0.4% (D) at 5 min. Scale bar = 25 µm. Scanning electron micrographs of thrombi from the same individuals; (E) 100% 11.1% (F), 3.1% (G), and 0.4% (H). Platelet aggregates are immersed in a fibrin mesh for the control (E) and form a starburst like pattern for mild hemophilia samples (F). The fibers on the surface in (G) and (H) are collagen fibers. Scale bar = 25 µm.</p

Fibrin deposition dynamics in response to rFVIII treatment.

<p>Four patients (1–4) with severe hemophilia where treated with rFVIII (see <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0078732#s2" target="_blank">Methods</a> for doses). Recalcified whole blood was perfused over glass slides coated with 2.3 fmol TF/cm² and type 1 fibrillar collagen at 100 s⁻¹ for 5 min before and 30 min after treatment with rFVIII. Platelets (<i>blue</i>, anti-CD41), fibrin(ogen) (<i>green</i>, Alexa488-fibrinogen) and their overlay immediately before (A–C) and 30 min. after (D–F) rFVIII injection. (G) Transient fibrin density pre-treatment (•) and post-treatment (○) in comparison to a normal control (◊). The dynamics of fibrin deposition before (black bars) and after (white bars) was characterized by (H) maximum fibrin density, (I) the lag time, and (J) the velocity. Error bars represent standard deviations of n = 3. Lines indicate comparisons between pairs according to the Mann-Whitney U-test.</p

Fibrin deposition dynamics in response to rFVIIa treatment.

<p>Two patients (5–6) with severe FVIII deficiency with high inhibitor titer were treated with 90 µg/mL rFVIIa. Recalcified whole blood was perfused over glass slides coated with 2.3 fmol TF/cm² and type 1 fibrillar collagen at 100 s⁻¹ for 5 min before and 30 min after treatment with rFVIIa. Platelets (<i>blue</i>, anti-CD41), fibrin(ogen) (<i>green</i>, Alexa488-fibrinogen) and their overlay immediately before (A–C) and 30 min. after (D–F) rFVIIa injection. (G) Transient fibrin density pre-treatment (•) and post-treatment (○) in comparison to a normal control (◊). The dynamics of fibrin deposition before (black bars) and after (white bars) was characterized by (H) maximum fibrin density, (I) the lag time, and (J) the velocity. Error bars represent standard deviations of n = 3. Lines indicate comparisons between pairs according to the Mann-Whitney U-test.</p