Clot Formation And Lysis Assay Research Articles

Delayed Plasma Clot Lysis in Adult Patients with Primary Immune Thrombocytopenia (ITP)

Introduction: Primary immune thrombocytopenia (ITP) is an orphan disease characterized by very low platelet counts. Patients have heterogeneous bleeding phenotypes, which are not only determined by platelet counts, also a paradoxically increased thrombotic risk has been observed.Aim: To investigate, whether the fibrinolysis inhibitors plasminogen activator inhibitor-1 (PAI-1) and α2-antiplasmin are associated with impaired plasma clot lysis in primary ITP patients in comparison to non-immunologic thrombocytopenic controls (TPC) and healthy controls (HC). Furthermore, associations with bleeding severity and previous thrombotic events were investigated.Methods: Patients from the Vienna ITP biobank (EC 1843/2016), a multi-centric study including adult patients with primary ITP were investigated and compared to age- and sex-matched control groups: TPC with thrombocytopenia after chemotherapy and HC. Informed consent was obtained from all individuals before study inclusion. A clot formation and lysis assay (CLA) was performed according to the recommendations of the ISTH SSC. Platelet poor plasma samples were measured in duplicates for each patient and control. PAI-1 (PAI-1 Actibind ELISA, Technoclone, Vienna, Austria) and α2-antiplasmin by the chromogenic STA Stachrom antiplasmin assay (Diagnostic Stago, Asnieres, France) were measured. Bleeding severity was measured using the ITP-specific ITP-ISTH BAT (Rodeghiero et al. 2013).Results: In total, 37 primary ITP patients, 18 TPC and 156 healthy controls were analyzed (Table 1). Primary ITP patients had a higher BMI than HC. Bleeding severity was higher and more ITP patients had a thrombosis history compared to HC, whereas there was no difference in comparison to TPC. PAI-1 activity was highest in ITP patients, with a statistically significant difference in comparison to HC. α2-antiplasmin activity was higher in ITP patients than in TPC, whereas there was no difference in comparison to HC.After adjustment for sex, age, BMI and fibrinogen, primary ITP patients had a reduced clot formation rate (V max) and significantly delayed plasma clot lysis compared to TPC and HC (Table 2). Also, the lag phase and time to peak absorbance (TTP) were prolonged with a significant difference in comparison to HC.To investigate outliers of PAI-1 and α2-antiplasmin, we calculated cut-offs at the 75 th percentile of healthy controls (PAI-1: ≥ 3.1 U/mL, α2-antiplasmin: ≥ 107.0 %). 14 (37.8 %) ITP patients had PAI-1 levels and 10 (27.0 %) ITP patients had α2-antiplasmin activity above the cut-off. ITP patients with high PAI-1 levels had mildly delayed clot lysis in comparison to those below with a significantly lower maximal lysis rate (mLR). ITP patients with α2-antiplasmin activity above the cut-off had a significantly shorter lag phase, faster V max and shorter TTP than patients below the cut-off, whereas there was no difference in clot lysis. No differences between ITP patients above or below the respective cut-offs of PAI-1 and α2-antiplasmin regarding their bleeding severity and thrombosis incidence were observed (Table 3).Conclusion: Primary ITP patients have a tendency towards increased PAI-1 activity, which is associated with considerably delayed plasma clot lysis. Albeit an association with the bleeding score could not be identified, this impaired lysis could be seen as a counter-regulation and at least contribute to the relatively mild bleeding tendency in patients with ITP. [Display omitted] DisclosuresPabinger: CSL Behring: Consultancy, Honoraria, Research Funding; NovoNordisk: Consultancy, Research Funding; Bayer: Consultancy, Honoraria; Takeda: Consultancy, Honoraria; Daiichi Sanchyo: Consultancy, Honoraria; Alexion: Consultancy, Honoraria; Novartis: Consultancy, Honoraria; Amgen: Consultancy, Honoraria; Pfizer: Consultancy, Honoraria.

Rebound Hypercoagulability Following a Conventional Course of Anticoagulation and the Risk of Recurrent Venous Thromboembolism in Young Patients: First Analysis of the Kids-DOTT Multicenter Trial-Derived Biobank

Background:Knowledge of prognostic factors for recurrence in adult and pediatric VTE is limited. Elevated plasma levels of D-dimer (a marker of coagulation activation) following a 6-month course of anticoagulation in adults with unprovoked VTE are associated with a heightened risk of VTE recurrence, warranting prolonged therapy. In patients with provoked VTE, and in young VTE patients more generally, prognostic factors remain largely undefined. Recently, using a global assay of clot formation and lysis on plasma samples collected serially over time, we observed a phenomenon of rebound hypercoagulability at 3 months post-diagnosis, relative to 4-6 weeks post-diagnosis, in a group of children with provoked VTE who were enrolled in a single-institution prospective inception cohort study.Objectives:The objectives of this work were:(1) toevaluate the prevalence of relative rebound hypercoagulability at 3 months status-post acute VTE in patients <21 years old; and (2) to investigate an association of relative rebound hypercoagulability with recurrent VTE.Methods:We combined clinical data and banked plasma biospecimens from an ongoing NHLBI-sponsored multinational multicenter trial of VTE treatment in patients <21 years old (the Kids-DOTT trial) with those from a single-institutional prospective cohort study of pediatric VTE at Johns Hopkins All Children's Hospital (St. Petersburg, FL, USA). The clot formation and lysis (CloFAL) spectrophotometric assay was performed as previously described, on banked plasma samples from 4-6 weeks and 3 months post-diagnosis of acute VTE, with heparinase pre-treatment of samples prior to assay. Those few patients (n=3) who received agents other than low molecular weight heparin at these time points were excluded. Assay measurements included maximal amplitude of the CloFAL waveform (MA), time to maximal amplitude (T1), and area under the curve at 60 minutes, indexed to that of the pooled normal plasma standard (AUC60). For each patient, the change in each CloFAL parameter between the 3 months and the 4-6 week post-VTE time points was then calculated. We used descriptive statistics to summarize data on patient characteristics VTE presentation, treatment, and outcomes. To explore prognostic cutoffs for percent change in each parameter receiver operating characteristics curves were generated, and inter-assay coefficients of variation of the assay were also considered. Thresholds of 50% increase in MA or AUC60, or 50% decrease in T1, were ultimately selected as candidate phenotypes of relative rebound hypercoagulability. An association between relative rebound hypercoagulability and VTE recurrence was determined via univariate logistic regression, using odds ratios (OR) and 95% confidence intervals (CI), with two-sided alpha=0.05. The blind was maintained in the Kids-DOTT trial throughout data transfer and analysis.Results: The final study population consisted of 88 patients, with a median age of 12.6 years (range, 0.01-20.8 years). VTE was classified as provoked in 98% of cases. Patient and VTE characteristics at baseline and outcomes during follow-up are shown in Table 1. Median follow up time was 52 weeks (range 9-123 weeks). Relative rebound hypercoagulability was found in 13% of the study population at 3 months post-diagnosis. Recurrent VTE developed in 10% of patients. Logistic regression analysis revealed that change in CloFAL AUC60 and T1 were not prognostic of VTE recurrence. However, relative rebound hypercoagulability as measured by change in MA was associated with a statistically significant, 4-fold increase in the risk of recurrent VTE (OR=4.47, 95%CI=1.05-19.0; P=0.04).Conclusions:Relative rebound hypercoagulability, as measured by change in MA in the CloFAL assay, developed in 13% of a multicenter study population of patients <21 years of age with predominantly-provoked VTE, and was associated with a 4-fold increased risk of recurrent VTE. Future work should seek to investigate mechanisms of relative rebound hypercoagulability following a conventional course of anticoagulation in young VTE patients, and to substantiate findings of its prognostic significance for VTE recurrence, via a future multicenter validation cohort.Acknowledgment:The Kids-DOTT trial is supported via dual U01 awards from NIH NHLBI (Clinical Coordinating Center U01, Goldenberg, PI; Data Coordinating Center U01, Hiatt, PI). [Display omitted] DisclosuresNo relevant conflicts of interest to declare.

In Children with Provoked Venous Thromboembolism, Increasing Plasma Coagulability during the First 3 Months Postdiagnosis is Prognostic of Recurrence.

Prognostic factors for venous thromboembolism (VTE) recurrence following provoked VTE are largely unknown. Using the Clot Formation and Lysis (CloFAL) assay, single institutional research has shown overall improvement in acute hypercoagulability during the first 3 months postpediatric VTE, yet a rise in plasma coagulability in a subgroup of patients. We sought to define the incidence of rise in coagulability during the first 3 months post-provoked VTE, to investigate its relationship with elevated D-dimer, and to test the hypothesis that a marked rise in coagulability is independently prognostic of VTE recurrence. CloFAL and D-dimer assays were performed on plasma at 4 to 6 weeks and 3 months post-VTE in the Johns Hopkins pediatric VTE cohort and National Institutes of Health-sponsored Kids-DOTT trial. Associations of VTE recurrence with D-dimer and CloFAL assay measures were evaluated via logistic regression. Eighty-seven patients were included. Median follow-up was 1 year. Complete veno-occlusion was determined in 12% at 6 weeks. During the first 3 months post-VTE, a marked rise in coagulability was observed by CloFAL assay in 17% of patients, while D-dimer was elevated in 21%. Recurrent VTE occurred in 10% of patients. CloFAL assay, but not D-dimer, was associated with recurrence (odds ratio [OR] 5.87, 95% confidence interval [95% CI], 1.34-25.8]). After adjustment for veno-occlusion, patients with a marked rise in coagulability by CloFAL assay had a 10-fold increased risk of recurrent VTE (OR 10.33 [95% CI, 1.83-58.19]). Future work should seek to elucidate the mechanisms underlying a rise in plasma coagulability following provoked VTE and to substantiate its prognostic utility for recurrent VTE.

A Rise in Plasma Coagulability during the First 3 Months Following Provoked Venous Thromboembolism Is Associated with the Development of Post-Thrombotic Syndrome in Children and Young Adults: Analysis of the Kids-DOTT Multicenter Trial-Derived Biobank

Background:Knowledge of prognostic factors for the development of post-thrombotic syndrome (PTS) is limited, particularly in the pediatric population. Elevated plasma levels of D-dimer and FVIII, both markers of coagulation activation and inflammation, are associated with an increased risk of adverse thrombosis outcomes, including PTS, in children. Similarly, increased levels of inflammatory cytokines, such as interleukin-6 (IL-6), have been associated with venous endothelial dysfunction and fibrosis, suggesting a potential role in the development of PTS. Given the strong interplay between inflammation and coagulation, the aims of this study were to: (1)Investigate a hypothesized association between increased levels of plasma cytokines during the first 3 months post-diagnosis of deep venous thrombosis (DVT) with the development of PTS in patients &lt;21 years old; and (2) to investigate an association between increased plasma coagulability during the first 3 months post-DVT diagnosis with the development of PTS. Methods:We used banked plasma biospecimens and masked clinical data from an ongoing NHLBI-sponsored multinational multicenter trial of VTE treatment in patients &lt;21 years old (the Kids-DOTT trial, NCT00687882). All patients with an extremity DVT who underwent PTS assessment at 1 year were included in this analysis. The Clot Formation and Lysis (CloFAL) spectrophotometric assay was performed, as previously described, on banked plasma samples obtained at 6 weeks and 3 months post-VTE diagnosis. All samples were pre-treated with heparinase prior to assay. Assay measurements included maximal amplitude of the CloFAL waveform (MA), time to maximal amplitude (T1), and area under the curve at 60 minutes, indexed to that of the pooled normal plasma standard (AUC60). Levels of IL-6, IL-8, IL-12/IL-23p40, IL-17, vascular endothelial growth factor (VEGF), tumor necrosis factor (TNF)-α, interferon (IFN)-γ and C-reactive protein (CRP) were measured via V-Plex kit (Meso Scale Discovery, Rockville, MD). For each patient, the change in each laboratory parameter between the 3 months and 6 weeks post-VTE time points was calculated. The diagnosis of PTS was made at the one year follow-up assessment using the Manco-Johnson Instrument. Descriptive statistics were used to summarize data on patient and VTE characteristics as well as PTS outcome. Putative prognostic factors for PTS (age group, complete thrombus occlusion, CloFAL parameters, and inflammatory cytokines) were evaluated via univariate logistic regression, using odds ratios (OR) and 95% confidence intervals (CI). Variables with P-values &lt;0.1 in univariate analyses were pre-specified for inclusion in an adjusted (multivariate) regression analysis. The blind was maintained in the Kids-DOTT trial throughout data transfer and analysis. Results: A total of 80 patients were included in the present analysis. Median age was 12.7 years (range 0.04 - 20.8 years). Patient and VTE characteristics at baseline, as well as PTS outcomes, are summarized in Table 1. PTS developed in 34% of patients by the 1 year post-VTE follow up visit. Univariate logistic regression analysis identified change in CloFAL AUC60 (OR=1.46, 95%CI=1.02-2.09; P=0.036) and complete thrombus occlusion at 6 weeks post-VTE (OR=3.29, 95%CI=0.93-11.6; P=0.064) as putative prognostic factors for PTS (Table 2). After adjustment in multivariate regression, change in CloFAL AUC60 remained the only possibly-significant (P=0.06) independent prognostic factor for PTS. Specifically, for each 1 A.U. increase in CloFAL AUC from the 6 weeks to 3 months post-VTE diagnosis, the odds of PTS increased by more than 40% (OR=1.43, 95%CI=0.99-2.08; Table 3). Changes in none of the measured inflammatory cytokines were prognostic of PTS. Conclusions: PTS developed in 34% of patients &lt;21 years old enrolled in enrolled in an ongoing multicenter trial of provoked DVT treatment. A rise in plasma coagulability during the first 3 months post-DVT diagnosis, as measured by increase in AUC60 in the CloFAL assay is a candidate prognostic factor for PTS in patients &lt;21 years old with provoked DVT. Future work should seek to investigate the mechanisms of increased plasma coagulability following a course of anticoagulation therapy in young VTE patients, and to substantiate findings of its potential prognostic significance for the development of PTS. Disclosures Everett: ImmunArray: Consultancy, Patents &amp; Royalties. Goldenberg:NIH: Other: research support and salary support.

Coagulative function assessed preoperatively via the clot formation and lysis assay as a prognostic factor for blood loss in adolescents undergoing posterior spinal fusion for idiopathic scoliosis.

Posterior spinal fusion for adolescent idiopathic scoliosis is a complex surgery often associated with clinically significant blood loss leading to perioperative blood transfusion. Knowledge of risk factors for blood loss and transfusion stems mostly from retrospective studies. We sought to prospectively investigate putative prognostic factors for intraoperative blood loss and perioperative blood transfusion in adolescent idiopathic scoliosis patients undergoing posterior spine fusion, including clinical characteristics, surgical factors, and preoperative assessment of overall coagulative and fibrinolytic functions in plasma using the clot formation and lysis (CloFAL) assay. Following Internal Review Board approval, adolescents 10 to <21years old with idiopathic scoliosis undergoing posterior spine fusion were enrolled preoperatively in a single-institutional prospective cohort and biobanking study. Clinical data were collected on patient characteristics, surgical approach, perioperative management, intraoperative estimated blood loss, and blood transfusion through hospital discharge. Coagulative and fibrinolytic functions in plasma were measured on preoperative samples by CloFAL assay (Coagulation Index and modified Fibrinolytic Index). Univariate linear regression and multivariable linear regression were performed to identify predictors of weight-indexed intraoperative estimated blood loss EBL (EBL/kg). The final study population included 74 patients. Median age was 14.8years (SD=2.2). After adjustment for other putative prognostic factors via multivariable linear regression, coagulative function as determined preoperatively by CloFAL Coagulation Index was an independent predictor of intraoperative (EBL)/kg. Specifically, each 10% increase in CloFAL CI was associated with 3% decrease in the geometric mean of EBL/kg (OR 0.97, 95%CI 0.94-0.99, P=.01). In adolescents undergoing posterior spinal fusion for idiopathic scoliosis, increased coagulative function measured preoperatively using the CloFAL assay is independently associated with decreased intraoperative blood loss. Future studies should expand upon these investigations of plasma coagulative and fibrinolytic capacities in combination with clinical factors, to guide precise preventive strategies against blood loss and blood transfusion in this patient population.

Monitoring hypercoagulability and hypofibrinolysis following acute venous Thromboembolism in Children: Application of the CloFAL assay in a prospective inception cohort study

Although individual thrombophilia tests are frequently performed in children with venous thromboembolism (VTE), global assays provide the opportunity to fill the gap in knowledge regarding their net impact on overall coagulative (and in some cases fibrinolytic) function. We first evaluated analytic sensitivity of the Clot Formation and Lysis (CloFAL) global assay to hypercoagulability and alterations in fibrinolysis, and then characterized changes in plasma coagulative and fibrinolytic capacities over time in children with acute VTE. In plasma ex vivo and in vitro experiments, the CloFAL assay area-under-the-curve (AUC) was analytically sensitive to hypercoagulable states, and its modified fibrinolytic index (FI2) was sensitive to both hyper- and hypofibrinolytic conditions. Clinical data and plasma samples for assay were collected during follow-up of 50 children enrolled in a prospective inception cohort study of VTE from May 2006 to June 2010. Follow-up periods were designated as follows: acute (<1month post-event), sub-acute (1–3months), early chronic (3–12months), and late chronic (>12months). Since most children were sampled at fewer than three pre-defined follow-up periods, study population findings were grouped by timepoint. AUC was significantly increased, and FI2 significantly decreased, in the acute period of VTE when compared to healthy controls, indicating hypercoagulability and hypofibrinolysis, respectively. One-third of patients were hypercoagulable, and 23% were hypofibrinolytic, in the late chronic phase. AUC and FI2 were strongly correlated with functional fibrinogen levels. These findings indicate the utility of the CloFAL assay in monitoring plasma coagulative and fibrinolytic capacities in children with VTE. Studies of its potential role in outcome prediction are ongoing.

Utility of Clot Formation and Lysis Assay to Monitor Global Coagulation State of Patients with Severe Acute Pancreatitis

Systemic inflammation affects hemostasis during severe acute pancreatitis (SAP). A hypercoagulable state occurs more frequently in SAP, which is not fully detected by traditional coagulation testing. The aim of this study was to evaluate the contribution of clot formation and lysis (CloFAL) assay to improve monitoring of global coagulation in patients with SAP. Twenty-five patients with SAP who were treated from December 2009 to April 2011 were studied. Plasma was collected at the time of admission, and CloFAL was measured using the CloFAL analyzer. The parameters evaluated include coagulation time (CT), fibrinolysis time (FT), and maximum amplitude (MA), from which the accelerating coagulation extent (ACE, MA/CT), accelerating fibrinolytic extent (AFE, MA/FT), and balance level exponent (BLE, ACE/AFE) were calculated. In addition, laboratory values for the traditional coagulation testing were measured. Values were compared to a control group of 20 healthy subjects. The MA, FT, ACE, and BLE values of the CloFAL assay were significantly increased in the SAP group compared to the control group (p\0.05 for all measurements). For the traditional coagulation testing, fibrinogen, plasminogen, and D-dimer levels were higher in patients in the SAP group compared to the control group (p\0.05). Our findings using the CloFAL analyzer indicate that the hypercoagulable state was due to increased fibrin generation and invariable fibrinolysis in patients with SAP. CloFAL assay is a simple and useful global coagulation assay to monitor hypercoagulable states during SAP.

Use of Global Assays to Predict Bleeding Severity In Congenital Factor VII Deficiency.

AbstractAbstract 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 (<1- 60 U/dL); bleeding score: 1.1 (0-3.2). FVII:C varied indirectly with PT (r= -0.76, P<0.001) and time to maximal amplitude in the CloFAL assay (r= -0.51, P=0.01), and directly with CloFAL coagulation index (r= 0.47, P=0.03). FVII:C was also negatively correlated with maximal velocity (Vmax) of plasmin generation (r= -0.67, P<0.001). Bleeding manifestations were severe (bleeding score >/= 3) in all three homozygotes/compound heterozygotes; moderate in 1/21 heterozygotes; mild in 13/21; and absent in the remaining 7 heterozygotes. Among heterozygotes, while there was no significant correlation between bleeding score and FVII:C or PT, there was a significant negative correlation with CloFAL area under the curve (AUC); i.e., as bleeding score increased, AUC decreased. CloFAL AUC explained 19% of variability in bleeding score among heterozygotes (P=0.047). Considering all subjects, CloFAL AUC was significantly higher among those without, versus with, a prior history of bleeding (P=0.046) (Table 1). CloFAL AUC also demonstrated a significant negative correlation with bleeding score (P=0.02) (Table 2). STP values did not correlate with history of bleed or bleeding score. Conclusion:These findings indicate that the CloFAL assay is unique in explaining considerable phenotypic variability in congenital FVII deficiency. Larger prospective studies are warranted to evaluate the capacity of the CloFAL assay to predict bleeding severity in this disease and guide prophylactic replacement therapy during episodes of hemostatic stress.Table 1:Correlation of CloFAL AUC with Bleeding HistoryBleeding HistoryMean AUC (% PNP)No History of Bleed (n=7)152.43P-value: 0.046History of Bleed (n=17)111.73r value: −0.42Table 2:Correlation of CloFAL AUC with Bleeding ScoreBleeding ScoreMean AUC (% PNP)Asymptomatic (0) (n=7)152.43Mild (1) (n=13)116.05Moderate (2) (n=1)90.2P-value: 0.02Severe (3) (n=3)100.2r value: −0.49 Disclosures:Grunzke:NHF/Baxter Clinical Fellowship Award 2008–2010: Research Funding.

Afibrinogenemia: a Disorder of Paradoxical Bleeding and Clotting.

Abstract 3477Poster Board III-414 BackgroundCongenital 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. ObjectiveThe 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. MethodsPlasma 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)). ResultsTAT 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.Table 1Patient 2: Time after fibrinogen concentrate (hrs)Fibrinogen (mg/dL)TAT (mcg/L)Patient 3: Time after fibrinogen concentrate (hrs)Fibrinogen (mg/dL)TAT (mcg/L)Pre-infusion<54.5Pre-infusion19.21.50.584.527.01.5285.52.11.378.83.048119.11.62447.02.29657.01.74835.71.87226.52.214413.02.2 DiscussionThese 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. [Display omitted] [Display omitted] Disclosures:Grunzke:National Hemophilia Foundation/Baxter Clinical Fellowship Award: Research Funding. Manco-Johnson:CSL Behring: Honoraria, Research Funding.

Evidence of Increased Plasma Coagulative Capacity by CloFAL Assay among Pediatric Factor V Leiden and Prothrombin G20210A Heterozygotes with, Versus without, a First-or Second-Degree Family History of Venous Thromboembolism

BACKGROUND: The factor V (FV) Leiden and prothrombin (PT) G20210A polymorphisms in heterozygous state are present in 5% and 1–2% of Caucasians, respectively, and confer approximately 5-fold and 2-fold increases in the risk of incident venous thromboembolism (VTE). While some families who carry these genetic thrombophilia traits exhibit a prothrombotic phenotype, others have no (or only a limited) history of VTE. The ability to discern which individuals with personal and familial carriage of these genetic thrombophilias possess a clinically meaningful increase in VTE risk remains elusive, and (particularly among children) is perhaps best informed presently by family history of VTE.OBJECTIVE AND HYPOTHESES: We sought to evaluate overall plasma coagulative capacity in FV Leiden and PT G20210A heterozygotes using the Clot Formation and Lysis (CloFAL) assay, a global turbidimetric plasma assay of tissue-factor induced fibrin clot formation and tissue-type plasminogen activator enhanced fibrinolysis. We hypothesized that children heterozygous for either thrombophilia would not uniformly demonstrate hypercoagulability, but that coagulative capacity would be increased among heterozygotes who have a family history of VTE.PATIENTS AND METHODS: Children aged birth to 18 years (inclusive) enrolled in prospective inceptional cohort study of thrombosis/thrombophilia/stroke were included in the analysis if they were found to be heterozygous for FV Leiden or PT G20210A upon comprehensive thrombophilia testing and had undergone CloFAL assay testing on a research basis. Data on personal and family history of thrombotic events, thrombophilia testing, and CloFAL assay findings were analyzed. Intergroup comparisons of continuous data were performed by Mann-Whitney U test and proportions were compared between groups using chi-square or Fisher's exact test, as appropriate.RESULTS: Characteristics of the study population are shown in Table 1. Approximately 70% of patients were evaluated for a family history of VTE (with/without known thrombophilia) and nearly 50% had personal histories of VTE or arterial ischemic stroke (AIS)/recurrent transient ischemic attack (TIA); those evaluated for events were significantly older than those without events, and this difference was statistically significant among those with a positive family history fo VTE. Hypercoagulability was shown in 50% of patients and hypofibrinolysis in 13% using the CloFAL assay. Plasma coagulative capacity and maximal amplitude (MA) of the CloFAL waveform were significantly increased in patients with, versus without, family history of VTE (coagulation index, CI: 102% vs. 72% of the adult normal pooled plasma standard, respectively, p=0.04; MA: 0.415 vs. 0.322, p=0.02), and were not explained by age differences between groups. However, in this relatively small study population, the proportion of CloFAL CI results that exceeded the upper limit of normal values did not significantly differ between those with, versus without, family history of VTE. Pediatric FV Leiden or PT G20210A heterozygotes with positive family history of VTE were more likely to have multi-trait (>1) thrombophilia, in which case a trend toward increased plasma coagulability was demonstrated (CI: 139% [multi-trait] vs. 86% [isolated trait]; p=0.07); superimposed thrombophilias in this group most often consisted of elevated factor VIII activity and Lp(a) concentration.CONCLUSIONS: The present findings using the CloFAL global assay indicate that, while pediatric FV Leiden or PT G20210A heterozygotes do not uniformly exhibit hypercoagulability, plasma coagulative capacity is nevertheless significantly increased among heterozgyotes who have a family history of VTE, which may relate to the presence of superimposed thrombophilias.Table 1. Summary characteristics of the study population.N32Median age at evaluation (range)9.5 y (1–18 y)Personal history of events*14 y (1–18 y)No personal history of events*8 y (2–18 y)FV Leiden heterozygote (n)26**PT G20210A heterozygote (n)8**Personal history of VTE (n)11Personal history of AIS/recurrent TIA4Family history (1st/2nd degree) of VTE71%Multi-trait (>1) thrombophilia45%Acquired thrombophilia24%Hypercoagulability by CloFAL assay50%Hypofibrinolysis by CloFAL assay13%*VTE, AIS, or recurrent TIA**Two patients were dual heterozygotes.

Influence of factor IX on overall plasma coagulability and fibrinolytic potential as measured by global assay: monitoring in haemophilia B

We sought to determine the influence of factor IX (FIX) deficiency upon overall coagulative and fibrinolytic capacities in plasma using the clot formation and lysis (CloFAL) assay, and to investigate the role of this global assay as an adjunctive monitoring tool in haemophilia B. CloFAL assay parameters were measured in vitro in platelet-poor plasma in relation to FIX activity and antigen (FIX:Ag), and were determined ex vivo among FIX-deficient patients (n = 41) in comparison to healthy individuals (n = 48). Supplementation of FIX-deficient plasma with FIX in vitro demonstrated a non-linear concentration dependence of FIX upon overall plasma coagulability. Ex vivo, coagulability was significantly decreased in FIX-deficient vs. healthy subjects among adults [median coagulation index (CI): 4% vs. 104% respectively; P < 0.001] and children (median CI: 9% vs. 63%; P < 0.001). Fibrinolytic capacity was increased in adult FIX-deficient vs. healthy subjects (median fibrinolytic index: 216% vs. 125%, respectively, P < 0.001), and was supported by a trend in shortened euglobulin lysis time (ELT). Severe haemophilia B patients showed heterogeneity in aberrant CloFAL assay waveforms, influenced partly by FIX:Ag levels. Patients with relatively preserved FIX:Ag (i.e. dysfunctional FIX) exhibited a shorter time to maximal amplitude in clot formation than those with type I deficiency. During patient treatment monitoring, markedly hypocoagulable CloFAL assay waveforms normalized following 100% correction with infused FIX. The CloFAL global assay detects FIX deficiency, demonstrates differences in coagulability between dysfunctional FIX and type I deficiency, and appears useful as an adjunctive test to routine FIX measurement in monitoring haemophilia B treatment.

Influence of Factor IX on Overall Plasma Coagulability and Fibrinolytic Potential in Hemophilia B as Measured by Global Assay.

BACKGROUND: The Clot Formation and Lysis (CloFAL) global assay is a turbidimetric plasma assay utilizing coagluation activation by lipidated tissue factor and fibrinolytic enhancement by tissue plasminogen activator, and has recently been shown to be sensitive for factor VIII (FVIII) deficiency (Goldenberg et al., Thromb Res 2005; Goldenberg et al., Haemophilia 2006, in press). Deficiency of factor IX (FIX) is often clinically less severe than that of FVIII. We sought to evaluate the analytical sensitivity of the CloFAL assay for FIX deficiency, both in vitro and ex vivo.METHODS: The influence of FIX activity upon the CloFAL assay was examined in vitro in FIX-deficient plasma by mixing study with pooled normal plasma, addition of plasma-derived FIX concentrate, and repletion with recombinant FIX. The analytical sensitivity of the CloFAL assay for FIX deficiency was then studied clinically via comparison of healthy individuals to FIX-deficient adults (n=25) and children (n=19), of whom greater than 1/3 had mild hemophilia B (FIX activity >/= 5.0 U/dL). FIX-deficient subjects had not received exogenous FIX within the prior 96 hours and had no evidence of active hepatitis. Healthy adult and pediatric groups (n=25 each) were without chronic illness or family history of bleeding or thrombosis. None of the study subjects were taking medications that affect hemostasis or had recent active bleeding or acute infection.RESULTS: Alteration of FIX activity in vitro exerted considerable influence upon the CloFAL assay (Figure 1). Ex vivo, the CloFAL assay coagulation index (CI), a measure of the area under the clotting curve, was significantly decreased in FIX-deficient versus healthy subjects among adults and children alike (median CI, adults: 10% vs. 94%, respectively; median CI, children: 10% vs. 63%, respectively; P < 0.0001 for each), and correlated significantly with FIX activity in both age groups (r = 0.80 and r = 0.77; P < 0.0001 for each). In addition, the CloFAL assay fibrinolytic index (FI) was increased in FIX-deficient versus healthy subjects (median FI, adults: 228% vs. 109%, respectively, p<0.0001; median FI, children: 276% vs. 202%, respectively; p=0.7), although this difference was not statistically significant in children. Interestingly, severe hemophilia B patients (n=8; FIX activity < 1.0 U/dL) showed considerable heterogeneity in CloFAL assay waveforms (Figure 2). Nevertheless, the assay uniformly discriminated these patients from healthy controls.CONLUSION: This work demonstrates that the CloFAL global assay, which is highly influenced by FVIII activity, is also analytically sensitive to FIX deficiency. [Display omitted] [Display omitted]

Influence of factor VIII on overall coagulability and fibrinolytic potential of haemophilic plasma as measured by global assay: monitoring in haemophilia A

The objectives of the present study were to evaluate the analytical sensitivity of the recently developed Clot Formation and Lysis (CloFAL) global assay for factor VIII (FVIII) deficiency, both in vitro and ex vivo, to determine whether this global assay is influenced by FVIII inhibitors, and to investigate the coagulative response to FVIII replacement in haemophilia A patients using the CloFAL assay in comparison with FVIII activity. Among adults and children alike, the CloFAL assay coagulation index (CI) was significantly decreased in FVIII-deficient vs. healthy subjects (adults median CI: 2% vs. 94% respectively; children median CI: 3% vs. 63%; P < 0.001 for each), and correlated significantly with activated partial thromboplastin time-based FVIII activity across all individuals (r = 0.78; P < 0.001). The CloFAL assay was analytically sensitive to deficient FVIII activity and also influenced by the presence of von Willebrand factor. Severe haemophilia A patients without inhibitory antibodies to FVIII showed considerable heterogeneity in CloFAL assay waveforms, despite a uniformly diminished CI of 0-1%. During FVIII infusion half-life studies in patients with severe haemophilia A, the CloFAL assay demonstrated a marked rise in coagulability 30 min following infusion, with progressive decrease in coagulability towards baseline over the ensuing 48-h period. In each case, the profile of coagulative response to FVIII infusion as determined by CloFAL assay CI differed qualitatively from that measured by FVIII activity. These findings indicate that the CloFAL assay may be useful as an adjunctive test to FVIII activity measurements in the therapeutic monitoring of haemophilia A.

Evaluation of Physiologic and Pathologic Alterations of Coagulation and Fibrinolysis Using a New Global Assay.

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<0.001 for each). By contrast, CI was decreased, and FI notably increased, in neonatal cords versus children, although the former comparison did not achieve statistical significance (CI: 58% vs. 69%, P=0.09; FI: 210% vs. 142%, P<0.001). The influence of deficiencies of coagulation factors and fibrinolytic regulators upon CloFAL parameters was also investigated. The greatest impact upon CI occurred with severe deficiency of fibrinogen or factors II, V, VII, VIII, IX, or X. Furthermore, CI was sensitive to deficiencies of factor VIII and fibrinogen in a concentration-dependent manner. In addition, FI was increased by PAI-1 deficiency and by inhibition of thrombin-activatable fibrinolysis inhibitor (TAFI) activation, and was zero in the setting of aminocaproic acid treatment. Further studies revealed that the influence of heparin concentrations of up to 2 U/mL in plasma was completely reversible by heparinase treatment of samples prior to assay.CONCLUSION: These results indicate that the CLoFAL assay is reproducible and analytically sensitive to known physiologic and pathologic alterations in coagulation and fibrinolysis. The application of this global assay to patients with a variety of disorders of thrombosis and hemostasis is currently ongoing. [Display omitted]