ALBERT

Description: Background: Inherited thrombophilia (IT) has been described as a risk factor for venous thromboembolism (VTE) in children. So far the majority of studies performed in the field were either retrospective or prospective on small numbers of patients. Thus, the results are contradictory or inconclusive mainly due to lack of statistical power. The aim of this study was to better estimate the impact of IT on early VTE onset and recurrence in children as a prerequisite to develop primary and secondary treatment options. Methods: A systematic search of publications listed in the electronic databases (Pubmed, Medline, EMBASE, Web of Science, The Cochrane Library) up to August 2007 using key words in combination both as MeSH terms and text words, was conducted. Citations were screened by two independent group members and those meeting the inclusion criteria were retained. Articles were included if published after 1990, when pediatric VTE was started to be systematically investigated. Findings: Twenty case-control and 17 cohort studies from 13 countries met the inclusion criteria. In these studies 〉 70% of patients had at least one clinical risk factor. The summary odds ratios (OR) and 95% confidence intervals (CI) of included studies under a fixed-effects and random-effects model showed statistically significant associations between the IT traits investigated and VTE onset (table). For the rare event of VTE recurrence, 1227 patients (eight studies) were evaluated: at the present state due to high heterogeneity, a trend towards association with recurrent VTE was found for ≥2 IT traits in the fixed-effects model (0R/CI: 2.8/1.6–4.8). Interpretation: The present meta-analysis gives evidence that the detection of inherited thrombophilia is clincially meaningful in children with VTE and underlines the importance of a pediatric thrombophilia screening program. Summary of Data Risk Factors OR/CI:fixed model OR/CI:random model patients/controls 2470/4119 N/A FV G1691A 3.5/2.9–4.2 3.2/2.3–4.4 FII G20210A 2.2/1.5–3.3 2.2/1.5–3.4 Protein C defiiciency 9.8/5.9–16 9.9/6.1–16.1 Protein S deficiency 7.1/3.9–13.2 6.8/3.7–12.7 Antithrombin deficiency 7.9/3.8–16.6 7.3/3.4–15.3 Lipoprotein(a) 4.4/3,2–5.9 4/2.4–6.6 ≥ 2 risk factors 12.6/7.3–21.8 11.6/6.2–20.2

Description: The recurrence rate of thrombosis in children following a first thrombotic event ranges from 3% in neonates to 8% in older children. The relative importance of the factor II and factor FV mutations is unknown. We present a multicentre cohort study to assess the rate of symptomatic VTE recurrence per 1000 person-years in children heterozgous for the FII and FV mutations following a first VTE. Data were pooled to increase power for the secondary aims, e.g. time to recurrence, and predictors of recurrence. Between January 1994 and December 2006, 251 consecutively enrolled VTE patients aged newborn to ≤18 years (median 5.2 years: male n=141) carrying the FII (n=61) or FV mutation (n=190) were followed for a median of 58 (max 156) months. 128 of 251 VTE patients (51%) had at least one underlying medical condition at VTE onset, and 15 were heterozygous for both mutations. Children received acute anticoagulation (AC) with unfractionated heparin or low-molecular weight heparin, followed by AC with LMWH or warfarin for a three to 6 month period in 70% of cases. Of the 251 patients enrolled, 24 (9.5%: recurrence rate of 19.3 per 1000 person-years, 95% confidence interval (CI): 12.9–28.8) had recurrent VTE at a median (min-max) of 3.5 (0.1–120) months. Not including combined defects, the recurrence rate per 1000 person-years was 41.4 (95%CI: 22.3–77) for patients with the FII mutation, and 14 (95%CI: 8.3–23.6) for carriers of the FV mutation. Median (min-max) age at recurrence was 13.4 (0.1–17) years, 12 of 24 patients were male (50%), and in 21 of 24 children (87.5%) recurrence occurred after withdrawal of AC. When comparing FII with FV subjects, Cox regression analysis showed that the factor II mutation (HR/95%CI: 2.5/1.1–5.9; p=0.031) was associated more frequently with a second VTE. In addition, older age (〉 2 years) at first VTE onset (HR/95%CI: 1.1/1.01–1.14; p=0.025) independently influences the second VTE event [3.5 years (non-recurrence) vs. 12.7 years (recurrence); p〈 0.0001]. Among patients suffering from recurrent VTE, 58% occurred within the first six months following VTE onset. Time to recurrence (FII vs. FII and age at onset 〉/〈 2 ears) is shown in the Kaplan Meier analyses. The overall VTE recurrence rate of 9.5% is within the range recently reported in children. However, when comparing FII and FV carriers, the factor II G20210A variant is more often associated with a recurrent VTE. In addition, independently from the underlying gene mutation age 〉 2 years at first VTE increase the risk of a second symptomatic venous thrombosis. Figure Figure

Description: Among risk factors for developing thromboembolism (VTE) in children with acute lymphoblastic leukemia were Escherichia coli asparaginase, concomitant steroid use, presence of central venous lines, and thrombophilic abnormalities. Developing a predictive model for determining children at increased risk would be beneficial in targeting interventional studies to high-risk groups (HRGs). Predictive variables were incorporated into a risk assessment model, which was evaluated in 456 children and then validated in 339 patients. VTE risk by score was no greater than 2.5 for low-risk group (LRG) and greater than 2.5 for HRG. VTE rates at 3.5 months (validation cohorts) were 2.5% in LRG and 64.7% in HRG. In multivariate analysis adjusted for age, duration of asparaginase administration, enoxaparin prophylaxis, and T-immunophenotype, the HRG was significantly associated with VTE compared with the LRG (hazard/95% confidence interval [CI], 8.22/1.85-36.53). Model specificity was 96.2% and sensitivity was 63.2%. As secondary objective we investigated the use of enoxaparin for VTE prophylaxis in the HRG. HRG patients without enoxaparin prophylaxis showed a significantly reduced thrombosis-free survival compared with children on low-molecular-weight heparin (LMWH). On the basis of the high specificity, the model may identify children with leukemia at risk of VTE. LMWH may help prevent VTE in the HRG; this warrants assessment in larger cooperative clinical trials.

Description: Diagnosis of platelet function disorders is based on the nature of bleeding symptoms, a positive family history and abnormal laboratory tests. However, currently available methods are time and labour consuming and lack sensitivity and specificity. In the present study we evaluated the performance of the Cone and Plate (let) analyzer (CPA) for screening patients with a suspected platelet function disorder and for monitoring therapy. Consecutive patients presenting with spontaneous or post surgical bleeding were studied. All patients underwent primary and secondary hemostasis evaluation including CPA testing. In this test a volume of 200μL of citrated whole blood is placed onto polystyrene wells and submitted to flow (shear rate of 1800 sec. −1) by a Teflon cone for 2 minutes. The resulting platelet deposition is then read by an image analyzer and expressed as % of the well surface covered ( SC, %) with adhered platelets and as the average size (AS, μm2)of platelet aggregates. Patients with a platelet function disorder were further tested with a modified CPA test in which blood samples are pre-incubated (1 min.) with a sub-optimal concentration of an agonist prior to subjecting the sample to the regular CPA test. Response of platelets to the agonist leads to agglutination and micro aggregates formation in the suspension phase of the test tube with consequent decline in platelet adhesion. 165 consecutive bleeding patients were evaluated and compared to 54 healthy controls. We diagnosed 51 patients as suffering from a platelet function disorder, of whom 8 patients (GTA – 7, BSS – 1) were excluded. The remaining 43 patients presented complete unresponsiveness to epinephrine in repeated platelet aggregation tests while all other coagulation, liver and renal function tests were normal and similar to healthy controls. In the CPA test these 43 patients had a significantly lower SC values compared to the other patients or to healthy controls. To further explore the CPA’s diagnostic capability 6 patients were compared to 16 randomly chosen controls using the modified CPA test. Whereas SC declined significantly by 73% in healthy controls after pre-incubation with epinephrine a non significant decline of only 15% was noted in patients with platelet adrenergic receptor dysfunction (table). Seventeen patients with platelet adrenergic receptor dysfunction underwent surgery. Following pre-surgical administration of DDAVP SC values followed closely and the VWF:RCo levels whereas the response to epinephrine in routine aggregometry did not change. All patients underwent successful operations without excessive bleeding. Our data suggest that patients with adrenergic receptor dysfunction suffer from a combined aggregation and adhesion platelet defects. The CPA test was found useful for screening both adhesion and aggregation of platelet disorders including adrenergic receptor dysfunction as well as for monitoring DDAVP therapy in these patients. Adrenergic receptor dysfunction No disorder Healthy controls * max. response to epinephrine in aggregometry ** 1 min. incubation with epinephrine N 43 114 54 Bleeding time (min.) 5.6±2.5 5.5±2.3 6.7±3.1 Aggregomatry * 23% 82% 81% SC% 6.2±2.4 11.5±3.4 12.3±3.0 Modifioed CPA-pre 6.5±2.2 9.4±1.8 Modified CPA-post** 5.4±1.3 2.5±0.8

Description: Objective To determine the importance of antithrombin [AT] deficiency as risk factor or predictor for fatal/non-fatal recurrent venous thromboembolism (VTE) in children. Methods In the present cohort of 874 consecutively enrolled pediatric patients with VTE aged newborn to

Description: Abstract 3169 Background: Adapted from the adult definition, pediatric antiphospholipid syndrome [APS] has been defined as one or more arterial or venous thrombosis associated with persistent antiphospholipid antibodies, i.e. IgM or IgG anticardiolipin antibodies [ACL: cut-off 〉 99th age-dependent percentile] or the presence of lupus anticoagulants confirmed in at least one follow-up visit more than 8 to 12 weeks apart. Antiphospholipid antibodies play an important role in the development of pediatric thromboembolism [TE] with arterial TE or stroke being more often associated with primary APS compared to deep venous thrombosis [DVT], which is observed predominantly in children with secondary APS. However, results of single studies on the risk thromboembolism onset associated with APS have been contradictory or inconclusive, mainly due to lack of statistical power. The aim of this study was to estimate the impact of APS on risk of childhood arterial and venous TE via meta-analysis of published observational studies. Methods: A systematic search of electronic databases (Medline, EMBASE, OVID, Web of Science, The Cochrane Library) for studies published from 1970 to 2010 was conducted using key words in combination both as MeSH terms and text words. Citations were independently screened by two authors and those meeting the a priori defined inclusion criteria were retained. Data on year of publication, study design, laboratory methodologies, country of origin, number of patients/controls, ethnicity, type and location of TE were abstracted. Publication bias indicator and heterogeneity across studies were evaluated, and summary odds ratios (ORs) and 95% confidence intervals (CIs) were calculated using both fixed-effects and random-effects models. Results: Eight of 319 (DVT) and seven out of 185 (arterial TE) references found met inclusion criteria: In total 1403 patients and 1904 population-based controls aged neonate to 18 years were enrolled. No significant heterogeneity was discerned across studies, and no publication bias was detected. In addition, regression analysis did not reveal statistical significant differences between locations of TE, age at first disease onset, study country, or publication year. Thus, data from arterial and venous TE were analyzed together. A statistically significant association with a first TE onset was demonstrated with a cumulative summary ORs/CIs (fixed-effects model) of 5.9/3.6-9.7. Conclusions: The present meta-analysis indicates that APS serves as a clinical meaningful risk factor for a first symptomatic TE in children. However, the impact of APS upon outcome and recurrence risk needs to be further investigated. Disclosures: No relevant conflicts of interest to declare.

Description: Abstract 3154 Poster Board III-91 Background. The development of alloantibodies that inhibit the coagulant activity of factor VIII (FVIII) is currently the most challenging complication of treatment in persons with hemophilia. Among other factors known to influence inhibitor development, several reports in the literature claimed for a different rate of inhibitor development in hemophilia A (HA) patients after plasma derived (pd-) or recombinant (r-) FVIII administration. Aim of this study was to compare the incident rate of inhibitors in HA patients treated with pd- or r-FVIII through systematic appraisal of the literature. Methods Studies reporting data about inhibitor rate in previously untreated patients (PUPs) with severe (〈 0.01 UI/mL) or severe-moderate HA were searched in the following electronic databases: Medline, EMBASE, OVID, Web of Science, The Cochrane Library. Details about study and patient characteristics were abstracted. To avoid double counting of patients included in more than one report of the same authors/working groups, patient recruitment periods and catchment areas were evaluated and authors were contacted for clarification if needed. If any of the required data could not be found in the published report, the corresponding author was contacted to provide the missing data of interest. High responder (HR) inhibitors were defined as 〉=5 BU/mL. Inhibitors were defined as transient when spontaneously disappearing within 6 months without the need to change treatment regimen. Firstly, the incident rate of inhibitor was recalculated for each study as the number of new inhibitor cases during the observation period divided by the number of HA patients initially inhibitor-free. Secondly, the recalculated rates were pooled for pd- and r- treated cohorts with the random effect model of Laird and Mosteller for single-cohort studies. Thirdly, a summary rate ratio (RR) was calculated for the subset of studies reporting parallel cohorts of patients treated with pd- or r-FVIII concentrates using fixed-effects and random-effects models. Sensitivity analysis, meta-regression and multivariate ANOVA were used to investigate the effect of covariates. Heterogeneity across studies and publication bias were evaluated. Results Twenty-four trials were included (19 prospective), 21 of which reporting details on HR inhibitors for a total of 2113 patients (1170 treated exclusively with pd-, 943 with r-FVIII; 1143 were severe), median age at enrolment was 9.6 months. The total number of inhibitors was 389 of which 135 in patients treated with pd- and 254 in patients treated with r-FVIII. HR inhibitors were 256 (103 for pd- and 153 for r-FVIII). Non-transient inhibitors were 162 (59 for pd- and 103 for r-FVIII). Inhibitor testing was from every 5 exposure days to every 2 year. Pooled incident rate (95% CI) was in all trials 14.7 (10.7 to 19.9) for pd- and 26.6 (22.6 to 31.0) for r-; for prospective trials 9.5 (5.7 to 15.3) for pd- and 22.4 (17.1 to 28.3) for r-; for HR inhibitors 8.5 (4.8 to 14.6) for pd- and 15.4 (12.2 to 19.3) for r-; for non-transient inhibitors 12.7 (7.3 to 21.1) for pd- and 18.9 (14.3 to 24.6) for r-. Six non concurrent cohort studies including 1259 HA patients met the inclusion criteria for RR calculation. Compared to pd-FVIII a statistically significant association with inhibitor development was demonstrated for r-FVIII, with summary RR ranging (95% CI) for HR inhibitors of 1.7 (C.I. 1.3 to 2.7), p〈 0.001, I2 = 0%, Harbord-Egger bias indicator p=0.07, fixed effect model; for all inhibitor patients of 2.0 (1.5 to 2.6), p〈 0.001, I2 = 41.6%, Harbord-Egger bias indicator p=0.06, random effect model. In the complete study set, testing frequency and study period correlated with rate of inhibitors development at meta-regression. At multivariate ANOVA testing frequency and study period were the strongest determinants of inhibitor development, and type of concentrate lost its statistical significance in the complete model. Conclusions This systematic review suggests that a lower inhibitor rate is found in patients with severe HA with the use of pd-FVIII, but also underscores the critical role of study related characteristics in the evaluation of the true effect of source of factor VIII. Future randomized and prospective follow-up studies are warranted. Disclosures No relevant conflicts of interest to declare.

Description: Background: Severe hemophilia A and B patients with inhibitors experience serious musculoskeletal hemorrhage as well as high risk of limb and life threatening bleeds. However, lack of effect of FVIII or FIX substitution therapy and short functional half-life of by-passing agents, leave these patients with very limited bleed preventive treatment options. ALN-AT3 (Alnylam Pharmaceuticals, Cambridge, MA, USA), a subcutaneously administered investigational RNAi therapeutic targeting reduction of antithrombin for potential treatment of hemophilia is currently in phase 1 clinical development in hemophilia A and B patients without inhibitors. Initial data from that ongoing study in 12 patients suggest an AT KD dependent correction of thrombin generation. This study aims to assess changes in peak thrombin generation in samples from patients with severe hemophilia A and B with inhibitors following in vitro reduction of antithrombin. Materials and methods: Citrated plasma samples were obtained from patients with severe hemophilia A and B with high responding inhibitors. Samples were spiked in vitro with isotype specific control IgG or a monoclonal antibody (Haemtech Inc, Essex Junction, VT, USA) targeting antithrombin knockdown of 50% and 90%. Dynamic formation of thrombin was measured by calibrated automated thrombin generation using 1pM tissue factor PPP reagent and 4μM phospholipid (Thrombinoscope, Maastricht, The Nederlands). The primary effect measure was peak thrombin (nM). Data were tested by a 1-way ANOVA and p

Description: Background Previous results from our laboratory demonstrate an epitope specific response to high doses of FVIII within anti-C2 antibodies that was independent of antibody titer. In addition, for a panel of monoclonal antibodies (MAbs) directed against all FVIII domains the kinetics of inhibition influenced response to combinations of FVIII and recombinant factor VII (rFVIIa). The influence of inhibitor kinetics on response to treatment has also been demonstrated in inhibitor patient plasmas. The Bethesda assay detects the inhibitory capacity of anti-factor VIII (FVIII) antibodies to neutralize FVIII after 2 hours of incubation with pooled normal plasma (PNP). In this assay patient antibody is added to PNP as compared to the clinical scenario where recombinant FVIII (rFVIII) or plasma derived FVIII containing von Willebrand Factor (pdFVIII/VWF) is infused into the patient where antibody is already present. In this case the infused product is immediately available to interact with both VWF and anti-FVIII antibodies as compared to the Bethesda assay where VWF is already bound to FVIII when the antibody is added. Methods In this study we investigated the inhibitory kinetics of a panel of 20 anti-FVIII MAbs (Table) with known epitope specificity and inhibitory activity in a standard Bethesda assay. Inhibitor plasma consisted of a single MAb added to FVIII deficient plasma at 5 µg/ml. rFVIII and pd-FVIII/VWF were added to each inhibitor plasma and samples were sequentially removed at intervals between 5 and 90 minutes. FVIII activity was measured by a one-stage aPTT based assay. Results Of the MAb panel, 2 anti-A2 MAbs, 1D4 and 4A4, and the anti-A3 MAb F147 had full neutralization of both rFVIII and pd-FVIII/VWF. All 3 of these MAbs exhibit high inhibitory titers in the Bethesda assay. The majority of the other MAbs had improved neutralization kinetics and thus higher residual FVIII activity with pd-FVIII/VWF when compared to rFVIII. The figure below shows the residual FVIII activity at 15 minutes following the addition of rFVIII or pdFVIII/VWF into the inhibitor plasmas. Similar patterns were seen at the other time points. Three MAbs from the panel, two anti-A2 (4C7 and B157) and one anti-C2 (2-117), had significant inhibition of FVIII activity when rFVIII was added to the inhibitor plasma. This was not demonstrated in the standard Bethesda assay or when pd-FVIII/VWF was added to the inhibitor plasma. This demonstrates that the order of binding of VWF and anti-FVIII antibodies may be clinically relevant for a subset of FVIII epitopes. Conclusion The Bethesda assay in isolation neither predicts inhibitory kinetics nor defines response to various FVIII sources. FVIII source dependent neutralization kinetics and epitope mapping may be applied as additional tools for tailoring therapy in patients with inhibitors. Disclosures: No relevant conflicts of interest to declare.

Description: Background: Venous thromboembolism [TE] is a multifactorial disease and antithrombin [ATD]-, protein C [PCD]- or protein S [PSD]-deficiency constitutes a major risk factor. Since screening for thrombophilia is controversial, individuals at high risk for recurrence who benefit from screening need to be identified. Primary study objective was to determinethe individual recurrence risk in children compared to adults with TE with respect to their thrombophilia status. Methods: In 142 consecutively enrolled TE patients (children n=85; adults n=57) with ATD, PCD or PSD after exclusion of six children with purpura fulminans due to homozygous PCD or PSD we calculated i) the cumulative recurrence rates (CRR) at 1, 5 and 10 year following the first TE onset and, ii) in addition, the absolute recurrence risk (ARR) per 100 patient years (%). Results: At first TE onset in univariate analysis a higher rate of unprovoked TE was found in children, whereas adults presented with a higher rate of a positive TE family history and a higher rate of recurrence: 40 out of 136 patients showed a second TE after withdrawal of anticoagulation (AC) or insufficient AC [n=6]. Two events in children were fatal [pulmonary embolism]. The overall CRR at 1, 5 and 10 years was 10.9%, 20.4% and 29.2% with total ARR [95% CIs] of 5.3 [3.4-7.8] in adults compared to 2.1 [1.0-5.3] in children [p=0.004]. Whereas the ARR was no different between adults and children in ATD patients [5.1 versus 4.7; p=0.85] and in symptomatic PCD subjects [3.9 versus 1.6; p=0.17], adults with PSD showed a higher ARR compared to children [6.3 versus 0.1; p=0.001]. Positive family TE history did not predict recurrence. Conclusion: Given the high ARR of 5.3% in adults and 2.1% in children we suggest screening for ATD, PCD and PSD in adult and pediatric TE patients. The high rate of ARR should be taken into account when initiating future therapeutic trials. Duration and intensity of AC should be carefully reevaluated in patients with ATD, PCD or PSD. Disclosures Young: Baxter, Grifols: Consultancy, Honoraria. Nowak-Gottl:Bayer, LFB: Membership on an entity's Board of Directors or advisory committees.