ALBERT

Description: Abstract 2988 Poster Board II-964 BACKGROUND: In pediatric venous thromboembolism (VTE), duration of hypercoagulability—which has implications for recurrence risk and duration of therapy–is not readily assessed despite a battery of laboratory assays for comprehensive thrombophilia testing. Global clotting assays offer the possibility to better understand duration of hypercoagulability on an individualized basis. OBJECTIVE: We sought to evaluate overall coagulability and fibrinolytic potential over time in children with acute VTE and to compare these findings with D-dimer and established thrombophilia traits. METHODS: The Clot Formation and Lysis (CloFAL) global assay was performed in platelet-poor plasma in 58 children enrolled in a single-institutional prospective inceptional cohort study of VTE at The Children's Hospital, Colorado, between March 2006 and June 2009. This spectrophotometric fibrin registration assay employing clotting activation with dilute tissue factor and phospholipid and fibrinolytic enhancement with tissue plasminogen activator has been previously shown analytically sensitive to physiologic and pathologic alterations in multiple components of the coagulation and fibrinolytic systems (Goldenberg et al., Thromb Res 2005). Hypercoagulability was defined by an area under the curve (AUC) of the CloFAL waveform that exceeded the upper limit of age-appropriate reference values established in healthy children (n=26) using the non-parametric method of Tukey, and hypofibrinolysis was similarly defined as a fibrinolytic index (FI, which relates to the slope of decline in absorbance over the period of 30 minutes following maximal amplitude of clot formation) that was below the lower limit of normal. Coagulation index (CI, measured as the AUC over the first 30 minutes of the assay, indexed to the pooled normal plasma standard) was also evaluated, and compared to established reference ranges. Analyses were grouped by period post-diagnosis, as follows: acute (0-1 month; n=10), subacute (1-3 months; n=12), early chronic (3-6 months; n=10), late chronic (≥1 year; n=32). Comprehensive testing for genetic and acquired thrombophilia states was performed in all subjects, along with serial assessment of D-dimer and automated euglobulin lysis time (ELT). RESULTS: Of the 58 children with VTE evaluated for global coagulative and fibrinolytic capacity, 25% underwent repeat testing in follow-up. A positive relationship with factor VIII activity (FVIII) was demonstrated for both CI and AUC (r=0.37, P=0.006 and r=0.52, P

Description: Important predictors of adverse outcomes of thrombosis in children, including postthrombotic syndrome (PTS), have recently been identified. Given this knowledge and the encouraging preliminary pediatric experience with systemic thrombolysis, we sought to retrospectively analyze our institutional experience with a thrombolytic regimen versus standard anticoagulation for acute, occlusive deep venous thrombosis (DVT) of the proximal lower extremities in children in whom plasma factor VIII activity and/or D-dimer concentration were elevated at diagnosis, from within a longitudinal pediatric cohort. Nine children who underwent the thrombolytic regimen and 13 who received standard anticoagulation alone were followed from time of diagnosis with serial clinical evaluation and standardized PTS outcome assessments conducted in uniform fashion. The thrombolytic regimen was associated with a markedly decreased odds of PTS at 18 to 24 months compared with standard anticoagulation alone, which persisted after adjustment for significant covariates of age and lag time to therapy (odds ratio [OR] = 0.018, 95% confidence interval [CI] = 〈 0.001-0.483; P = .02). Major bleeding developed in 1 child, clinically judged as not directly related to thrombolysis for DVT. These findings suggest that the use of a thrombolysis regimen may safely and substantially reduce the risk of PTS in children with occlusive lower-extremity acute DVT, providing the basis for a future clinical trial.

Description: BACKGROUND: The Clot Formation and Lysis (CloFAL) assay, modified from prior global assay methods by He et al., 1999, and Smith et al., 2003, involves activation of coagulation and fibrinolysis in platelet-poor plasma via addition of a physiologic reactant solution in a multi-well microassay plate. Continuous spectropohotometric data analysis allows measurement of kinetic absorbance changes of the plasma sample over three hours, which yields a unique clot formation and lysis curve. Using parameters of the curve, coagulation and fibrinolytic indices (CI and FI) are calculated relative to a simultaneously run pooled normal plasma standard. METHODS: Platelet-poor plasma obtained from pregnant women at term (n=24), neonatal cord blood (n=29), and healthy children (n=22) were analyzed using the CloFAL assay. Healthy adult (n=22) plasma samples, as well as those of individuals with factor deficiencies, were obtained commercially. Fibrinolytic alterations in vitro were also investigated. Intra-assay coefficients of variation (CVs) for normal controls ranged from 3–12% for all assay parameters, with inter-assay CVs of 5–15%. RESULTS: Representative CloFAL curves for healthy adults and children, pregnant women, and newborn infants are shown in Figure 1. Coagulation potential (measured by median CI) was significantly increased, while fibrinolytic capacity (measured by median FI) was markedly decreased, in pregnant women as compared to healthy adults (CI: 239% vs. 115%, FI: 59% vs. 95%; P

Description: BACKGROUND: The significance of the lupus anticoagulant (LA) and other antiphospholipid antibodies (APA) in children with thromboembolism (TE) has not been as clearly defined in children as among adults. OBJECTIVE: To characterize TE presentation and prothrombotic risk factors among children with TE who are positive for the LA using the dilute Russell Viper Venom Time (dRVVT) as a screening test. METHODS: Children with acute TE were consecutively enrolled in a prospective inceptional cohort study and a compehensive APA testing battery was serially employed. APA testing included StaClot-LA, as well as ELISA for IgG and IgM binding to prothrombin, protein C, protein S, cardiolipin and β2GP1. RESULTS: 34 children who were dRVVT positive within 3 months of acute thrombosis were evaluated with the entire APA battery on 1 to 5 occasions over 3 months to 4 years. Multiple APA were detected in 20 (59% of) study participants (5 tests, n=1; 4 tests, n=2; 3 tests, n=6; 2 tests, n=11; 1 test, n=14). Findings were: dRVVT (100%), anti-prothrombin (50%), StaClot (18%), anti-protein S and ACA (12% each), anti-β2GP1 (6%) and anti-protein C (3%). TE presentation and prothrombotic risk factors were compared between these 20 children with multiple APA and 14 children who were dRVVT positive only (Tables 1 and 2). Children with LA-associated TE who had multiple APA were more likely to present with parenchymal sites of thrombosis (p=0.03) and appeared less likely to have otherwise-idiopathic TE (i.e., no additional prothrombotic risk factors identified), although the latter trend was not statistically significant. CONCLUSIONS: The majority of children with acute TE and a positive dRVVT in whom a comprehensive battery of APA is performed demonstrate multiple APA, among which anti-prothrombin antibodies are the most common. When compared to children positive for dRVVT only, children with LA-associated thrombosis who have multiple APA are particularly predisposed to parenchymal thrombotic events and may be more likely to experience otherwise-idiopathic TE. Future investigations will evaluate the risk of recurrent TE in children with thrombosis relative to findings on serial comprehensive APA testing. Table 1. Thrombus Sites*, by APA Group. APA group Limb DVT Extensive PE CSVT Parenchymal thrombosis Arterial thrombosis Total * Some patients had 〉1 site. Abbreviations: DVT=deep venous thrombosis; PE=pulmonary embolism; CSVT=cerebral sinovenous thrombosis. Arterial thrombosis includes ischemic arterial stroke. Multiple APA 8 (40%) 6 (30%) 2 (10%) 6 (30%) 3 (15%) 20 (100%) dRVVT only 7 (50%) 4 (29%) 0 (0%) 0 (0%) 3 (21%) 14 (100%) Table 2. Prothrombotic Risk Factors, by APA Group. APA Group Infection Surgery/Trauma SLE Other* None Total * Includes anatomic anomaly, obesity, sedentary condition, oral contraceptive use, genetic thrombophilia, central venous catheter, and cardiac disease. Abbreviation: SLE=systemic lupus erythematosus. Multiple APA 4 (20%) 3 (15%) 2 (10%) 4 (20%) 7 (35%) 20 (100%) dRVVT only 2 (14%) 1 (7%) 0 (0%) 9 (64%) 2 (14%) 14 (100%)

Description: BACKGROUND: Antiphospholipid antibodies (APA) that persist ≥12 weeks in adults with acute thrombosis are predictive of thrombus recurrence. The significance of APA in children with thrombosis is unclear. This study was developed to examine the relationship of a persistently positive lupus anticoagulant (≥ 12 weeks), assessed by the dilute Russell Viper Venom Time (dRVVT), to adverse thrombus outcomes including progression or recurrence. In addition, the relationship of a persistently positive dRVVT to the prevalence and titer of several other APA in children with thrombosis were examined. METHODS: Data from a consented prospective inceptional cohort study of pediatric thrombosis and thrombophilia (COMIRB 05–0339) were extracted for this analytic project. Eligibility included age ≤ 21 years at presentation, objectively confirmed thrombus, dRVVT within 4 weeks of presentation and repeated ≥ 12 weeks later. Patients with a persistently positive dRVVT (ratio of Screen/Confirm ≥1.2) were classified as having antiphospholipid syndrome (APS positive), and those with a negative dRVVT were classified as APS negative. Twelve or more weeks from presentation, patient plasmas were tested in ELISA assays for the presence and titer of IgM and IgG antibodies directed against protein C (PC), protein S (PS), prothrombin (II), β-2-Glycoprotein I (B2GPI), and cardiolipin (ACA). A euglobulin clot lysis assay (ELT) and hexagonal (II) phase phospholipid assay (STACLOT LA, Diagnostica Stago, Inc.) were also performed. RESULTS: The cohort included 122 cases with thrombosis. Of these, 35 failed eligibility due to transient dRVVT positivity or lack of blood sampling within the specified time period. Of the remaining 87 patients, 43 were APS positive and 44 were APS negative. APS positivity was associated with longer duration of therapy (p

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 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: Abstract 3477 Poster Board III-414 Background Congenital afibrinogenemia (CA) is a rare bleeding disorder. While thrombosis is a recognized complication of dysfibrinogemia, the risk for thrombosis in CA has not been well studied. Life-threatening thrombosis with onset shortly after puberty has developed in 2 of 3 patients with CA followed through the coagulation program at The Children's Hospital, Colorado. Patient 1 suffered recurrent peripheral arterial thrombosis following cryoprecipitate administration during adolescence, massive myocardial infarction (MI) at age 19 years and death from spontaneous intracranial hemorrhage at age 20 years. Patient 2 has had superficial thrombophlebitis following intravenous (IV) infusion of fibrinogen concentrate, as well as bilateral pulmonary embolism with infarction 1 month following last infusion, without further recurrence over the course of 6 months of judicious use of anticoagulation and fibrinogen replacement. Patient 3 is 14 years old and receives fibrinogen replacement episodically for bleeding events, without thrombotic complications to date. Objective The object of this study was to investigate overall thrombin and plasmin generation potential and overall coagulative and fibrinolytic potential in CA, in order to generate hypotheses regarding pathophysiology of bleeding and thrombotic complications. Methods Plasma was collected from 2 CA patients at asymptomatic baseline states and following fibrinogen concentrate replacement. Plasma was isolated from whole blood within 1 hour, via double centrifugation at 2500 x g at 4°C x 15 minutes, and stored at -70°C until time of assay. Standard assays included fibrinogen activity by Clauss clotting assay and thrombin-antithrombin complexes (TAT) by ELISA (Siemens, Marburg, GE). Measurement of overall coagulative and fibrinolytic capacity in plasma was performed by Clot Formation and Lysis (CloFAL) global assay, as previously described (Goldenberg et al., Haemophilia, 2008). Simultaneous Thrombin and Plasmin generation assay (STP) was performed by fluorometric method with the same reagents as used in the CloFAL assay (dilute tissue factor, phospholipid, tissue plasminogen activator), as previously reported (Grunzke et al., J Thromb Haemost 2009 (abstract)). Results TAT was elevated to twice the upper limit of normal (9.4 mcg/L) in patient 1 six months following MI. In patient 2, TAT increased progressively from age 15 (2.9 mcg/L) to 20 years (13.7 mcg/L) while STP showed progressive shortening of time to peak thrombin generation by 60% over these 5 years (Figure 1). Baseline plasmin generation was nearly undetectable in both patients studied. As shown in Table I, TAT increased significantly at peak post infusion of fibrinogen concentrate in both patients evaluated during half-life and recovery studies. As shown in Figure 2, panels A and B, coagulative capacity by CloFAL assay and velocity of plasmin generation by STP showed dose dependence to fibrinogen following IV replacement with concentrate in patient 3. Discussion These data suggest that baseline and post infusion thrombin generation may be increased in post pubertal patients with CA. Furthermore, given undetectable plasmin generation in the absence of fibrinogen, the hemostatic balance in CA is simultaneously hemorrhagic and prothrombotic. These preliminary findings call for further prospective evaluation in other identified patients with CA. Disclosures: Grunzke: National Hemophilia Foundation/Baxter Clinical Fellowship Award: Research Funding. Manco-Johnson:CSL Behring: Honoraria, Research Funding.

Description: Abstract 3668 Background: Congenital FVII deficiency, the most common of the autosomal recessive rare coagulation disorders (RCDs), is characterized by genotypic variability as well as phenotypic heterogeneity ranging from absent to severe hemorrhage, and rarely thrombosis. Prothrombin time (PT) and FVII activity assays do not reliably predict bleeding phenotype. Global assays of coagulation and fibrinolysis may better characterize overall hemostatic balance and could aid in the assessment of hemorrhagic and thrombotic risk in RCDs. Objective: We sought to investigate whether a global assay might better predict bleeding phenotype in FVII deficiency. Methods: Four North American centers enrolled 24 known FVII deficient subjects (21 heterozygotes (FVII:C 20–60 U/dl) and 3 homozygotes/compound heterozygotes (FVII:C 〈 .01U/dL)) at asymptomatic baseline in a collaborative cross-sectional study. Venipuncture was performed for determination of baseline factor VII activity (FVII:C) and prothrombin time (PT) in plasma. The Clot Formation and Lysis (CloFAL) global assay and the Simultaneous Thrombin and Plasmin generation (STP) assay were performed centrally at the University of Colorado by previously described methods (Goldenberg et. al, Haemophilia 2008 and Grunzke et al., J Thromb Haemost 2009 (abstract)). Bleeding history data were obtained retrospectively and locally using a standardized case report form, in order to assign each subject a bleeding score (0-3.2) as previously described by Mariani et al. (Thromb Haemost, 2005), but slightly modified as follows: 0 (asymptomatic); 1 (mild): 1–2 symptoms excluding hemarthrosis, CNS and GI bleeding; 2 (moderate): 〉/=3 symptoms excluding hemarthrosis, CNS or GI bleeding; 3 (severe): hemarthrosis, CNS or GI bleeding. Within each score, decimals indicated the # of symptoms. Results: Median values (ranges) for age, FVII:C, and bleeding score were as follows: age, 12 years (3-36 years); FVII:C, 39 U/dL (

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.