Imaging the dynamics and microstructure of fibrin clot polymerization in cardiac surgical patients using spectrally encoded confocal microscopy.

During cardiac surgery with cardiopulmonary bypass (CPB), altered hemostatic balance may disrupt fibrin assembly, predisposing patients to perioperative hemorrhage. We investigated the utility of a novel device termed spectrally-encoded confocal microscopy (SECM) for assessing fibrin clot polymerization following heparin and protamine administration in CPB patients. SECM is a novel, high-speed optical approach to visualize and quantify fibrin clot formation in three dimensions with high spatial resolution (1.0 µm) over a volumetric field-of-view (165 × 4000 × 36 µm). The measurement sensitivity of SECM was first determined using plasma samples from normal subjects spiked with heparin and protamine. Next, SECM was performed in plasma samples from patients on CPB to quantify the extent to which fibrin clot dynamics and microstructure were altered by CPB exposure. In spiked samples, prolonged fibrin time (4.4 ± 1.8 to 49.3 ± 16.8 min, p < 0.001) and diminished fibrin network density (0.079 ± 0.010 to 0.001 ± 0.002 A.U, p < 0.001) with increasing heparin concentration were reported by SECM. Furthermore, fibrin network density was not restored to baseline levels in protamine-treated samples. In CPB patients, SECM reported lower fibrin network density in protaminized samples (0.055 ± 0.01 A.U. [Arbitrary units]) vs baseline values (0.066 ± 0.009 A.U.) (p = 0.03) despite comparable fibrin time (baseline = 6.0 ± 1.3, protamine = 6.4 ± 1.6 min, p = 0.5). In these patients, additional metrics including fibrin heterogeneity, length and straightness were quantified. Note, SECM revealed that following protamine administration with CPB exposure, fibrin clots were more heterogeneous (baseline = 0.11 ± 0.02 A.U, protamine = 0.08 ± 0.01 A.U, p = 0.008) with straighter fibers (baseline = 0.918 ± 0.003A.U, protamine = 0.928 ± 0.0006A.U. p < 0.001). By providing the capability to rapidly visualize and quantify fibrin clot microstructure, SECM could furnish a new approach for assessing clot stability and hemostasis in cardiac surgical patients.

Recommendations for clinical laboratory testing for antithrombin deficiency; Communication from the SSC of the ISTH.

Hereditary deficiency of antithrombin, a natural anticoagulant, causes a thrombophilia with a high risk for venous thromboembolism. Guidance for laboratory testing to diagnose antithrombin deficiency include the use of an activity assay for initial testing, performing an antigen test and activity-to-antigen ratio when the activity level is low, using pediatric reference ranges until the age of 6 months, excluding acquired causes of low antithrombin (e.g. liver dysfunction, proteinuria, heparin, disseminated intravascular coagulation, thrombosis, surgery) or falsely normal/elevated results (e.g. argatroban, bivalirudin, dabigatran in factor IIa-based assays; rivaroxaban, apixaban, edoxaban, but not betrixaban in Xa-based assays). Molecular testing, if available, may help determine the risk for thrombosis as this might vary among the different mutations. Moreover, it will identify mutations that can be missed by traditional activity assays. Strategies for interpreting laboratory test results are provided.

Platelet aggregation response in immune thrombocytopenia patients treated with romiplostim.

The thrombopoietin receptor agonist romiplostim is used for the long-term treatment of chronic immune thrombocytopenia (ITP). ITP patients have an increased thrombotic risk, which could be exacerbated if romiplostim increased platelet hyperreactivity or caused spontaneous platelet aggregation. To investigate this possibility, this study examined platelet function in romiplostim-treated ITP patients and healthy subjects. Light transmission platelet aggregometry utilizing arachidonic acid, collagen, epinephrine, ristocetin, ADP, and saline (to assess spontaneous aggregation) was performed for each subject. In addition, the ADP AC50 (ADP concentration that induced half-maximal aggregation) was determined for each patient as a sensitive measurement of altered platelet reactivity. Fifteen ITP patients and 7 healthy subjects entered the study. All ITP patients had active disease and were receiving weekly romiplostim as the sole ITP-directed therapy. Platelet aggregation in response to the strong agonists arachidonic acid, collagen, and ristocetin was not significantly different between ITP patients and healthy subjects (P = 0.2442, P = 0.0548, and P = 0.0879, respectively). Platelet aggregation in response to weak agonists was significantly reduced in ITP patients compared with that in healthy subjects: median (range) aggregation to ADP, 45% (15-84%) versus 89% (70-95%) (P = 0.0010), and epinephrine, 21% (1.6-90%) versus 88% (79-94%) (P = 0.0085). The median AC50 of ADP was threefold higher in ITP patients versus that in healthy subjects (6.3 µM vs 2.1 µM) (P = 0.0049). Significant spontaneous aggregation was not observed in any patient. Platelets from romiplostim-treated ITP patients do not show evidence for spontaneous aggregation or hyperreactivity, but instead have a modestly reduced aggregation response to ADP and epinephrine.

Deep Hypothermic Circulatory Arrest in a Patient With Cold-Induced Urticaria.

Acquired cold urticaria is a condition characterized by the onset of pruritic hives, swelling, and occasional severe systemic reactions, during the rewarming phase, after cold exposure. Pulmonary thromboendarterectomy (PTE) is a cardiac surgical procedure where organized thrombus is excised from the pulmonary vasculature in order to improve pulmonary blood flow and relieve pressure on the right ventricle. A PTE requires institution of deep hypothermic circulatory arrest (DHCA) in order to reduce blood flow from collateral vessels during thrombus excision. We describe a case of PTE performed under DHCA in a patient with ACU.

Guidance on the emergent reversal of oral thrombin and factor Xa inhibitors.

The new oral anticoagulants dabigatran, rivaroxaban and apixaban have advantages over warfarin which include no need for laboratory monitoring, less drug-drug interactions and less food-drug interactions. However, there is no established antidote for patients who are bleeding or require emergent surgery and there is a paucity of evidence to guide the clinical care during these situations. Members of thrombosis and anticoagulation groups participating in the Thrombosis and Hemostasis Summit of North America formulated expert opinion guidance for reversing the anticoagulant effect of the new oral anticoagulants and suggest: routine supportive care, activated charcoal if drug ingestion was within a couple of hours, and hemodialysis if feasible for dabigatran. Also, the pros and cons of the possible use of four factor prothrombin complex concentrate are discussed.

Mannose-binding lectin and its associated proteases (MASPs) mediate coagulation and its deficiency is a risk factor in developing complications from infection, including disseminated intravascular coagulation.

The first line of host defense is the innate immune system that includes coagulation factors and pattern recognition molecules, one of which is mannose-binding lectin (MBL). Previous studies have demonstrated that MBL deficiency increases susceptibility to infection. Several mechanisms are associated with increased susceptibility to infection, including reduced opsonophagocytic killing and reduced lectin complement pathway activation. In this study, we demonstrate that MBL and MBL-associated serine protease (MASP)-1/3 together mediate coagulation factor-like activities, including thrombin-like activity. MBL and/or MASP-1/3 deficient hosts demonstrate in vivo evidence that MBL and MASP-1/3 are involved with hemostasis following injury. Staphylococcus aureus infected MBL null mice developed disseminated intravascular coagulation (DIC), which was associated with elevated blood IL-6 levels (but not TNF-α and multi-organ inflammatory responses). Infected MBL null mice also develop liver injury. These findings suggest that MBL deficiency may manifest into DIC and organ failure during infectious diseases.

Warfarin-induced skin necrosis.

Warfarin-induced skin necrosis is a rare complication of anticoagulant therapy with a high associated morbidity and mortality requiring immediate drug cessation. Cutaneous findings include petechiae that progress to ecchymoses and hemorrhagic bullae. Characteristic dermatopathological findings are diffuse dermal microthrombi with endothelial cell damage and red cell extravasation with progression to full-thickness coagulative necrosis. The lesions of warfarin-induced skin necrosis may be difficult to differentiate from mimickers, but skin biopsy in conjunction with careful consideration of the clinical history, including time of onset, cutaneous distribution of the lesions, and laboratory findings, are essential for prompt diagnosis and patient treatment. Herein, we review the clinical and histologic features helpful for differentiating warfarin-induced skin necrosis and report a case illustrative of the diagnostic difficulty that may at times be encountered in clinical practice.

Prevalence of heparin/platelet factor 4 antibodies before and after cardiac surgery.

BACKGROUND: The clinical significance of heparin/platelet factor 4 (PF4) antibodies in subjects undergoing cardiac surgery has not been systematically studied. We prospectively investigated whether the presence of heparin/PF4 antibodies would predict clinical thrombosis in this population. METHODS: In 299 patients scheduled for cardiac surgery between October 2003 and March 2005, the heparin/PF4 antibodies and platelet count were measured immediately prior to, and 5 days after, surgery. The patients were followed up at 30 days for thrombotic complications. RESULTS: The prevalence of the heparin/PF4 antibodies was 4.3% (13 of 299) prior to surgery and increased more than fivefold to 22.4% (62 of 277) postoperatively (p < 0.0001). Thromboembolic events occurred in 8.8% of patients with negative antibody and in 6.3% of patients with positive antibody (p = 0.77). Of the 62 patients with positive heparin/PF4 antibodies postoperatively, 22 (35.5%) were treated with a nonheparin anticoagulant. There was a trend toward higher rates of thromboembolic events in subjects who were thrombocytopenic compared with those who were not (17.1% and 6.7%, respectively, p = 0.06), regardless of antibody status. Two out of 8 patients (25%) with both thrombocytopenia and a positive antibody (clinical heparin-induced thrombocytopenia [HIT]) suffered a thromboembolic event, compared with 17 of 222 (7.7%) without clinical HIT (p = 0.13). CONCLUSIONS: The high prevalence of antibodies to the heparin/PF4 complex after cardiac surgery and the low rate of thromboembolic complications in this population suggest that the antibody alone does not confer an increased risk of thrombotic complications. Monitoring for thrombocytopenia is recommended.

Effect of fondaparinux on coagulation assays: results of College of American Pathologists proficiency testing.

CONTEXT: Fondaparinux, a factor Xa inhibitor, is approved for thromboprophylaxis after orthopedic surgery and for treatment of venous thromboembolism. It may also be efficacious, safe, and cost-effective for other patients; thus, more widespread use of fondaparinux is likely. The effect of fondaparinux on coagulation testing needs to be thoroughly examined. OBJECTIVE: To report the effects of fondaparinux on coagulation tests (prothrombin time, activated partial thromboplastin time, fibrinogen, antithrombin, factor VIII, thrombin time, anti-factor Xa) across diverse methodologies. DESIGN: Samples with different concentrations of fondaparinux (0, 0.4, 0.8, and 2.0 microg/mL) were sent to laboratories participating in the College of American Pathologists Comprehensive Coagulation proficiency survey (N = 898). Laboratory-specific methods were used to assay coagulation parameters. RESULTS: Prophylactic or therapeutic fondaparinux prolonged the prothrombin time by approximately 1 second and the activated partial thromboplastin time by 4 to 5 seconds, and reduced factor VIII from 119% to 107% and 102%, respectively. Supratherapeutic fondaparinux reduced factor VIII to 85%. The activated partial thromboplastin time was prolonged in 19%, 29%, and 52% of laboratories with prophylactic, therapeutic, and supratherapeutic fondaparinux levels, respectively. Fibrinogen, antithrombin, and thrombin time assays did not show clinically significant changes. When measuring fondaparinux concentration using an anti-factor Xa assay, the most accurate results were obtained when fondaparinux was used as the calibrator. CONCLUSIONS: Fondaparinux, even in prophylactic doses, slightly prolongs the prothrombin time and activated partial thromboplastin time and can interfere with factor VIII assays, but it has no clinically relevant effect on fibrinogen, antithrombin, or thrombin time. A fondaparinux standard curve should be used for reporting fondaparinux levels using an anti-factor Xa assay.