Assessing the anticoagulant effect of dabigatran in children: an in vitro study.

OBJECTIVES: Age-related changes in the hemostatic system result in variation in response to anticoagulants and coagulation assays over childhood. This study used in vitro methods to determine i) optimum coagulation assays for dabigatran in children and ii) anticoagulant effect of dabigatran across pediatric age groups. MATERIALS AND METHODS: Pooled plasma samples from healthy children aged 0 to <1, 1 to <5, 5 to <10, 10 to <17 years and adults were spiked with increasing concentrations of dabigatran and the effect was assessed in five coagulation assays. The samples were also assessed for overall hemostasis potential using a fibrin clot formation and lysis assay. RESULTS: In all five coagulation assays, there were no differences in responses to dabigatran over all pediatric age groups. The international normalized ratio was the least sensitive measure. Activated partial thromboplastin time showed moderate sensitivity and a nonlinear response curve. Thrombin time (TT), dilute TT (dTT) and ecarin clotting time were linearly correlated with dabigatran concentrations; however, the ecarin time and TT were overly sensitive. In the overall hemostasis potential assay, increasing dabigatran concentrations delayed the initiation of clot formation and reduced the time to 50% clot lysis. The responses to initiation of clot formation and clot lysis were consistent across all pediatric groups and comparable to responses in adults. CONCLUSION: The dTT is the most suitable assay for measuring dabigatran concentrations in children. Fibrin clot generation and lysis assay responses to dabigatran in children over all ages were consistent and comparable to those of adults.

Dabigatran assessment in patients with acute complications using routine coagulation assays.

Complications while on dabigatran therapy, particularly bleeding and thrombosis, are occurring, and require laboratory assessment. The utility of routine coagulation assays has been previously evaluated in stable patients, but not those with acute complications. The purpose of this study was to determine how to employ routine coagulation assays to assess dabigatran in patients with acute complications. Seventeen patients on dabigatran presenting with various complications were evaluated. In addition, plasma samples with various fibrinogen levels were spiked in vitro with dabigatran ranging from low trough levels to the highest supratherapeutic concentrations reported (5000 ng/ml). INR, partial thromboplastin time (PTT), thrombin time (TT, Diagnostica Stago reagent), and fibrinogen were assayed and results compared to that of the Hemoclot Thrombin Inhibitors assay. Interference in the Clauss fibrinogen assay was assessed using a variety of commercial reagents. The majority of patients on dabigatran with acute complications demonstrated a significant negative bias in PTT results compared to normal plasma. TT remained highly sensitive to the presence of dabigatran (at least 10 ng/ml) under all circumstances investigated. There was wide variation in the sensitivity of commercial fibrinogen assays to dabigatran, with some even showing interference in the therapeutic range but this could be mitigated. The PTT is unreliable as a method for assessment of dabigatran in patients with acute complications. The TT assay is a simple and reliable alternative, particularly when combined with a fibrinogen level.

Specimen requirements for the haemostasis laboratory.

Sample integrity is one of the most important details to consider for the production of quality results in the laboratory. Many factors have the potential to adversely affect the sample: intrinsic patient characteristics (caused by the underlying malady and/or treatment, incorrect patient preparation, etc.), difficult or incorrectly performed collection of sample, correct timing of sample collection relative to drug administration, incorrect processing and transport within the laboratory-just to name a few. This chapter outlines standard common requirements with explanations as a basis for those limitations, and practical laboratory advice to attain and maintain dependable samples.

Fibrinogen.

Fibrinogen is the final essential building block of the clotting process. Thus, all of the preliminary "cause and effect" events in the clotting cascade rely on the work of this molecule to measure their success. The most commonly used laboratory method for measuring fibrinogen is the Clauss fibrinogen assay. The Clauss fibrinogen assay is a quantitative, clot-based, functional assay. The assay measures the ability of fibrinogen to form fibrin clot after being exposed to a high concentration of purified thrombin. Plasma samples are pre-diluted which minimize assay interference from substances like heparin and fibrinogen degradation products. In brief, the diluted plasma is incubated at 37°C prior to the addition of the pre-warmed (37°C) thrombin reagent. From the exact moment of the addition of thrombin, the time to clot is measured. The clotting time in seconds is interpolated from a standard curve made using various dilutions of assayed standard plasma. The following chapter includes detailed information on the Clauss fibrinogen assay. Other fibrinogen assays used include fibrinogen levels derived from prothrombin time assays and antigenic methods. Fibrinogen measurements using the prothrombin time and antigenic based assays are described in brief.

D-dimer and fibrinogen/fibrin degradation products.

Although clinical requests for D-dimer are generally in the minority of assays in the routine clinical laboratory, they are an important aspect-especially if the laboratory supports an active emergency room and hematology service. Throughout the literature, D-dimer assays have been used for many purposes in the research setting; however it is generally the negative predictive value of the assay that is the most common piece of information being utilized from the standpoint of a clinician. Research or clinical needs will dictate the type of assay required-a qualitative, semiquantitative, or quantitative D-dimer assay may be appropriate for a particular purpose. Commonalities and differences between these assay types are outlined here, as well as universal concerns regarding standardization of D-dimer assay results.

Phosphorylcholine-coated circuits improve preservation of platelet count and reduce expression of proinflammatory cytokines in CABG: a prospective randomized trial.

BACKGROUND: The interaction of blood with foreign artificial surfaces during cardiopulmonary bypass (CPB) has been recognized as a major stimulus in evoking a systemic inflammatory and metabolic response. Phosphorylcholine (PC) is a new-generation coating material designed to ameliorate biocompatibility and thereby to reduce the detrimental interactions of CPB. We studied the effects of PC-coated perfusion circuits on platelet function and the humoral and cellular response to CPB. METHODS: Thirty patients undergoing coronary artery bypass grafting were randomized to PC-coated (PC group, n = 15) and noncoated (control group, n = 15) circuit groups. Clinical data, total blood loss, and pre- and postoperative platelet counts were recorded and IL-6 and TNF-alpha, CD41a, CD42b, and CD62p were measured at induction of anesthesia, after the initiation of CPB and at termination of CPB. RESULTS: There was a significantly improved preservation of platelet count following CPB in the PC group (p = 0.028), which was sustained over a period of 72 hours. The use of PC-coated circuits further resulted in a significant attenuation of TNF-alpha and IL-6 expression (p < 0.05 and p < 0.01); however, we were unable to detect any differences in clinical outcomes. CONCLUSIONS: Despite similar clinical outcome, the obvious reduction of cytokine expression and improved preservation of platelet count suggest superior biocompatibility of PC-coated circuits.

Platelet dysfunction in asphyxiated newborn piglets resuscitated with 21% and 100% oxygen.

Hemostatic disturbances are common in asphyxiated newborns after resuscitation. We compared platelet function in hypoxic newborn piglets reoxygenated with 21% or 100% oxygen. Piglets (1-3 d, 1.5-2.1 kg) were anesthetized and acutely instrumented for hemodynamic monitoring. After stabilization, normocapnic hypoxia was induced with an inspired oxygen concentration of 10-15% for 2 h. Piglets were then resuscitated for 1 h with 21% or 100% oxygen, followed by 3 h with 21% oxygen. Platelet counts and collagen (2, 5, and 10 microg/mL)-stimulated whole blood aggregation were studied before hypoxia and at 4 h of post-hypoxia/reoxygenation. Platelet function was studied using transmission electron microscopy and by measuring plasma thromboxane B2 (TxB2) and matrix metalloproteinase (MMP)-2 and -9 levels. Control piglets were sham-operated without hypoxia/reoxygenation. The hypoxemic (PaO2 33 mm Hg) piglets developed hypotension with metabolic acidosis (pH 7.02-7.05). Upon reoxygenation, piglets recovered and blood gases gradually normalized. At 4 h reoxygenation, platelet aggregation ex vivo was impaired as evidenced by a rightward-downward shifting of the concentration-response curves. Electron microscopy showed features of platelet activation. Plasma MMP-9 but not MMP-2 activity significantly increased. Resuscitation with 100% but not 21% oxygen increased plasma TxB2 levels. Platelet counts decreased after hypoxia/reoxygenation but were not different between groups during the experiment. Resuscitation of hypoxic newborn piglets caused platelet activation with significant deterioration of platelet aggregation ex vivo and increased plasma MMP-9 levels. High oxygen concentrations may aggravate the activation of prostaglandin-thromboxane mechanistic pathway.

Stability of heparin and physical compatibility of heparin/antibiotic solutions in concentrations appropriate for antibiotic lock therapy.

OBJECTIVES: The purpose of this study was to determine the biological stability of heparin and to test for physical compatibility in heparin/antibiotic solutions in concentrations suitable for antibiotic lock therapy. METHODS: Solutions were prepared with heparin 5000 U/mL or heparin 10 U/mL and cefazolin 10 mg/mL, ampicillin 10 mg/mL, or piperacillin 40 mg/mL. Solutions of vancomycin 2.5 mg/mL with heparin 5000 U/mL and vancomycin 2 mg/mL with heparin 10 U/mL were also prepared. The ability of each solution to elevate the activated partial thromboplastin time (APTT) of pooled normal plasma and the physical compatibility of the solutions were assessed for 14 days. RESULTS: The APTT levels never varied by more than 16.4% from baseline. Physical incompatibility never occurred before day 14 in any of the solutions. CONCLUSIONS: Mixing of antibiotics in the concentrations chosen for the study had no clinically significant effect on biological heparin activity, and all solutions were physically compatible for at least 14 days.

Pyridoxine as a template for the design of antiplatelet agents.

The B(6) vitamers have been shown to display beneficial therapeutic effects in cardiovascular related disorders. The design of novel antiplatelet agents using pyridoxine as a template has led to the discovery of a class of novel cardio- and cerebro-protective agents. The present study describes the synthesis of several of these derivatives along with the antiplatelet and antiischemic activity of derivative 16.

Coronary artery bypass surgery with heparin-coated perfusion circuits and low-dose heparinization.

OBJECTIVE: To evaluate the safety and efficacy of heparin-coated perfusion circuits with low-dose heparinization and centrifugal pumping compared with the standard method during coronary artery bypass grafting. DESIGN: Prospective, randomized, single-blind clinical trial. SETTING: A primary care institution. PATIENTS: Ninety patients who underwent first-time elective coronary artery bypass grafting were eligible for the study. After giving informed consent, they were randomly assigned to 1 of 3 groups (30/group). INTERVENTIONS: Perfusion on regular uncoated bypass equipment with a roller pump and full-dose heparinization (300 IU/kg bolus, activated clotting time [ACT] > 400 s) (group 1), on a heparin-coated oxygenator with a centrifugal pump and full-dose heparinization (group 2) and on fully heparin-coated bypass equipment with a centrifugal pump and low-dose heparinization (100 IU/kg bolus, ACT of 180-400 s) (group 3). Standard coronary artery bypass grafting was performed. OUTCOME MEASURES: Postoperative bleeding, transfusion requirements and clinical outcomes. RESULTS: There were no complications related to the study protocol. Study groups were similar in terms of postoperative bleeding, transfusion requirements and clinical outcomes. CONCLUSIONS: Heparin-coated cardiopulmonary bypass with low-dose heparinization and centrifugal pumping is a safe practice but showed no advantages over the use of regular uncoated bypass circuits for coronary bypass surgery.