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Thrombosis in extracorporeal membrane oxygenation (ECMO) circuits remains a frequent complication. We characterize the location, extent, structure, and clinical implications of thrombi in 53 ECMO circuits from 46 pediatric patients. The tubing, pump, and oxygenator were examined for visible thrombi. Representative samples of thrombi were collected for histologic, immunofluorescence, and immunohistochemical analysis. Thrombi were found in 81% of ECMO circuits. The most clinically significant were inflow oxygenator membrane surface thrombi (11% of circuits), arterial tubing thrombi (30%), and venous tubing (26%) or connector thrombi (26%). Oxygenator membrane surface thrombi resulted in rapidly increasing delta pressure across the oxygenator over 1-2 days, oxygenator failure, and circuit replacement. Oxygenator membrane surface thrombi were associated with intravascular venous thrombosis and bacterial infection before starting ECMO. Arterial cannula/tubing thrombi led in one case to aortic and mesenteric artery thrombosis followed by bowel infarction. In 11% of cases, venous tubing thrombi grew large enough to break off and embolize to the pump, resulting in increased hemolysis. Antifibrinolytic therapy during ECMO was associated with an increased risk of pump thromboembolism. Other less clinically significant thrombi included pump axle thrombi with thrombus fragments trapped in the oxygenator (45%), and deep oxygenator membrane thrombi (15%). Examination of ECMO circuits after removal is a useful quality improvement tool that can elucidate the cause of circuit problems, indicate patients at increased risk of thrombosis, and suggest areas for possible improvements.

Context.— Assessing direct oral anticoagulant (DOAC) drug levels by reliable laboratory assays is necessary in a number of clinical scenarios. Objective.— To evaluate the performance of DOAC-specific assays for various concentrations of dabigatran and rivaroxaban, assess the interlaboratory variability in measurement of these DOACs, and investigate the responsiveness of the routine clotting assays to various concentrations of these oral anticoagulants. Design.— College of American Pathologists proficiency testing survey data from 2013 to 2016 were summarized and analyzed. Results.— For dabigatran, the interlaboratory coefficient of variation (CV) of ecarin chromogenic assay was broad (ranging from 7.5% to 29.1%, 6.3% to 15.5%, and 6.8% to 9.0% for 100-ng/mL, 200-ng/mL, and 400-ng/mL targeted drug concentrations, respectively). The CV for diluted thrombin time for dabigatran was better overall (ranging from 11.6% to 17.2%, 9.3% to 12.3, and 7.1% to 11.2% for 100 ng/mL, 200 ng/mL, and 400 ng/mL, respectively). The rivaroxaban-calibrated anti-Xa assay CVs also showed variability (ranging from 11.5% to 22.2%, 7.2% to 10.9%, and 6.4% to 8.1% for 50-ng/mL, 200-ng/mL, and 400-ng/mL targeted drug concentrations, respectively). The prothrombin time (PT) and activated partial thromboplastin time (aPTT) showed variable dose- and reagent-dependent responsiveness to DOACs: PT was more responsive to rivaroxaban and aPTT to dabigatran. The undiluted thrombin time showed maximum prolongation across all 3 dabigatran concentrations, making it too sensitive for drug-level monitoring, but supporting its use as a qualitative screening assay. Conclusions.— DOAC-specific assays performed reasonably well. While PT and aPTT cannot be used safely to determine DOAC degree of anticoagulation, a normal thrombin time excludes the presence of dabigatran.

Extracorporeal membrane oxygenation (ECMO) can be lifesaving but suffers from high rates of bleeding and repeated transfusions. Current monitoring of blood cell damage during ECMO is limited to platelet counts, hematocrit, and plasma hemoglobin levels. Extracelluar vesicles (EV) are small cell fragments released when cells are activated/injured. The objective was to evaluate flow cytometric measurements of EV during ECMO as an indication of platelet, red cell, and endothelial activation/injury. Samples were collected from 55 patients (1 day to 19 years) during 58 ECMO runs. ECMO activated or injured blood cells, but the extent was highly variable and patient dependent. On average platelet activation was increased sevenfold during ECMO with up to 60-fold increased activation during the first 24 hours in some patients. EV associated with platelet and red-cell injury were increased eightfold on average but up to 200-fold in patients with disseminated intravascular coagulation, severe hemolysis, or massive transfusion. Approximately 9% of ECMO patients showed a red-cell and endothelial activation pattern that was associated with poor prognosis. Extracellular vesicles with autofluorescence similar to bilirubin appeared to come from monocytes processing hemoglobin. ECMO is associated with a highly variable, sustained increase in platelet, red-cell, and endothelial activation and injury that is a combination of circuit and transfusion related events, the patients underlying condition and possibly genetic influences on blood cell activation and injury. Extracellular vesicle measurements may improve our understanding of cellular activation and injury during ECMO as we work to improve the biocompatibility of these systems.

Hyperfibrinolytic bleeding occurs when fibrinolytic activity is in excess compared to fibrin formation at the site of a hemostatic clot, leading to clot dissolution and new or increased bleeding. Hyperfibrinolytic bleeding can occur systemically due to increased secretion or infusion of plasminogen activators, decreased fibrinolytic inhibitors, or reduced clearance of plasminogen activators. The most common causes of hyperfibrinolytic bleeding are iatrogenic—increased tissue plasminogen activator release during cardiopulmonary bypass for open heart surgery and infusion of thrombolytic agents to lyse a pathologic thrombus. Other acquired causes of systemic hyperfibrinolytic bleeding include cirrhosis/liver transplantation, transient release of plasminogen activators, and hyperfibrinolysis associated with the acute coagulopathy of severe trauma and disseminated intravascular coagulation. The primary diagnostic test for acute severe hyperfibrinolysis is increased lysis on whole blood viscoelastic testing. Localized fibrinolytic bleeding can occur during orthopedic surgery and in some cases of menorrhagia. Acquired causes of hyperfibrinolytic bleeding are more common than hereditary forms of the disorder, but hereditary hyperfibrinolysis should be kept in mind in patients with an unexplained lifelong bleeding disorder. The most common hereditary disorders of fibrinolysis are plasminogen activator inhibitor 1 deficiency, antiplasmin deficiency, Quebec platelet disorder, and persistently elevated plasminogen activator levels. Diagnosis of hereditary hyperfibrinolysis usually requires specialized testing for the specific fibrinolytic activator or inhibitor.

Context.—The College of American Pathologists (CAP) developed proficiency testing for platelet function assays by using blood collected by the participant added to challenge tubes containing either saline (normal) or tirofiban (abnormal).Objective.—To analyze platelet function proficiency testing for Platelet Function Analyzer PFA-100, platelet aggregation, PlateletWorks, and PlateletMapping.Design.—Proficiency testing data from 2012–2016 were analyzed.Results.—For PFA-100, a total of 1200 laboratories participated; the coefficient variation (CV) of cartridge closure times was 22% (saline); 44,952 of 45,616 survey responses (99%) provided an interpretation, and 42,934 of 44,952 (96%) were correct. For optical platelet aggregation, 190 laboratories participated; the CV was 17% (saline), 7444 of 7813 survey responses (95%) provided an interpretation, and 7015 of 7444 (94%) were correct. For PlateletWorks, 60 laboratories participated; the CV was 3% to 11% (saline); 2412 of 2454 survey responses (98%) provided an interpretation, and 1207 of 1276 (95%) were correct for adenosine diphosphate (ADP) and 936 of 1136 (82%) for collagen. For PlateletMapping, 200 laboratories participated. For ADP, 1128 of 2697 survey responses (42%) provided an interpretation, but only 927 of 1128 (82%) were correct. For arachidonic acid, 1139 of 2604 survey responses (44%) provided an interpretation and 964 of 1139 (85%) were correct.Conclusions.—CAP is the first to provide proficiency testing for platelet aggregation, PlateletWorks, and PlateletMapping. Platelet aggregation, PFA-100, and PlateletWorks using ADP as an agonist performed well with more than 90% of laboratories providing an interpretation and a similar number providing correct results. PlateletWorks using collagen and PlateletMapping showed worse interpretive accuracy than the other methods.

Objectives To determine the relationship between baseline variations in the partial thromboplastin time (PTT) and the discordance between the PTT and anti-Xa heparin activity (anti-Xa) during heparin therapy. Methods The baseline PTT on heparin was determined using automated heparin neutralization with protamine (prPTT). The prPTT was used to calculate a baseline-corrected PTT on heparin to reduce discordance with anti-Xa measurements. Results The prPTT removed up to 1 U/mL of heparin, returning baseline values for normal, factor-deficient, and lupus inhibitor plasmas. A prolonged prPTT was seen in 97 (53%) of 182 samples from heparinized patients. The heparinized PTT was discordant compared with anti-Xa in 64 (35%) of 182 samples and 43 (67%) of 64 discordant samples, and 46% of concordant samples showed a prolonged prPTT. A baseline-corrected PTT reduced discordance with anti-Xa measurements by 64%. Conclusions PTT/anti-Xa discordance due to baseline PTT prolongation could be reduced using a baseline-corrected PTT.

Liver hemorrhage, the major cause of death in hepatic trauma, is notoriously difficult to control. We report on the use of high-intensity focused ultrasound (HIFU) to arrest the bleeding from incisions made in rabbit livers. A HIFU transducer, with a spherically curved aperture of 6.34 cm2 area, a focal length of 4 cm and a frequency of 3.3 MHz was used. In approximately 94% of the incisions, the hemorrhage was reduced to a slow oozing of blood in less than 2 min. The maximum temperature of liver tissue around the incision area, during HIFU application, was measured to be 86°C. The mechanism of hemostasis, confirmed by histological examination, appears to be coagulative necrosis of a volume of liver tissue around the incision. We believe that acoustic hemostasis, with the unique characteristic of “volume cauterization”, offers a novel method for the management of liver hemorrhage and, thus, has major clinical implications.

The prothrombin time (PT)3 and partial thromboplastin time (PTT) are useful assays for evaluation of coagulation factor levels and for monitoring anticoagulant therapy with warfarin and heparin, but they are of less value for predicting risk of thrombosis or bleeding in patients with acquired hemostatic defects. As new anticoagulant drugs become available and pathophysiologic mechanisms of abnormal hemostasis are unveiled, it is clear that new laboratory tests are needed to support drug monitoring, establish diagnosis, and develop therapeutic strategies. Over the past several years, a number of existing tests have been improved to offer additional information not available from PT/PTT, which include thromboelastography to evaluate fibrinolysis and target antifibrinolysis, thrombin generation assays (TGAs) for predicting bleeding and thrombotic risk, and anti-Xa and direct thrombin inhibitor assays to monitor direct oral anticoagulants. First-generation TGAs were performed in whole blood and plasma, but these were cumbersome and …

Chromogenic anti-Xa assays for unfractionated heparin monitoring (heparin activity) are susceptible to interference from hemolysis and icterus. The purpose of this study was to better understand the effect of hemolysis and icterus on anti-Xa heparin activity and to predict the magnitude of the error. Increasing levels of hemoglobin and unconjugated bilirubin were added to pooled normal plasma or buffer containing known levels of heparin. Increased plasma hemoglobin or bilirubin produced falsely increased residual factor Xa activity as measured by the absorbance change (OD/min) in the Stago heparin activity assay. This increased absorbance change slope resulted in falsely lower estimates of heparin activity. The falsely lower heparin activity measurement occurred even when heparin was not present, indicating it was not due to heparin neutralization. In a sample containing 0.62 ± 0.06 U/mL heparin and 228 mg/dL hemoglobin, the measured heparin activity was 0.41 ± 0.03 U/mL, underestimating heparin activity by 0.21 ± 0.07 U/mL. Interference occurred if plasma hemoglobin was above 70 mg/dL or bilirubin was above 16 mg/dL, which happened in 16%-26% of samples from pediatric patients on extracorporeal life support (ECLS). In conclusion, hemolysis and icterus were common in ECLS patients, leading to underestimates of unfractionated heparin activity and potentially higher doses of heparin than intended. The magnitude of the heparin activity measurement error could be predicted based on plasma hemoglobin and bilirubin levels until these levels exceeded the technical limits of the assay, ~230 mg/dL hemoglobin and 55 mg/dL bilirubin.

Reduced levels of coagulation factors are associated with an increased risk of bleeding. Therefore, it is not surprising that chronic elevation of some coagulation factors has been associated with an increased risk of venous thrombosis. The strongest association has been between chronic elevated factors VIII, IX, XI, and prothrombin and venous thrombosis. There is currently no known association between venous thrombotic risk and any of the other coagulation factors. Although elevations of some coagulation factors do have an increased risk of thrombosis, it is not clear that knowing the factor status of a patient with venous thrombosis has any effect on either initial antithrombotic therapy or therapy during recurrence, and therefore routine evaluation of factor levels in patients with venous thrombosis is not recommended.

Fibrinolytic testing separates into three groups: (1) screening tests such as euglobulin lysis time and percent clot lysis on thromboelastometry, (2) routine tests such as fibrinogen and D-dimer, and (3) esoteric tests typically sent to reference laboratories such as tissue plasminogen activator (t-PA) activity and antigen, plasminogen activator inhibitor 1 (PAI-1) activity and antigen, plasminogen activity and antiplasmin activity. Screening assays are typically used to detect severe fibrinolysis associated with active bleeding (surgery, trauma). D-dimer is most often used to rule out venous thromboembolism, whereas t-PA, PAI-1, and antiplasmin assays are used to diagnose hereditary fibrinolytic bleeding disorders.

Endoluminal devices such as metallic flow diversion (FD) and aneurysm bridging (AB) stents are used for treatment of intracranial aneurysms. Treatments are associated with thrombogenic events mandating the use of dual antiplatelet therapy in all cases. In the current in vitro study, we utilize a slow binding fluorogenic thrombin specific substrate to measure the thrombin generation potential of six devices: four FD devices (Pipeline™ Flex embolization device, Pipeline™ Flex embolization device with Shield Technology™, SILK+, FRED™) and two AB devices (Solitaire™ AB, LEO+). We show that the Pipeline™ Flex embolization device with Shield Technology™ has significantly lower peak thrombin and takes significantly longer time to achieve peak thrombin (time to peak) compared to the other three FD devices (p

Antibody-mediated rejection (AMR) is a major cause of kidney allograft loss. Currently, AMR diagnosis relies on biopsy which is an invasive procedure. A noninvasive biomarker of acute AMR could lead to early diagnosis and treatment of this condition and improve allograft outcome. Microvesicles are membrane-bound vesicles released from the cell surface after injury. We hypothesized that because AMR is associated with allograft endothelial injury and C4d deposition, plasma microvesicles positive for endothelial (CD144) marker and C4d are increased in this condition.We studied microvesicle concentration in the plasma of 95 kidney transplant patients with allograft dysfunction and compared with 23 healthy volunteers. Biopsy diagnosis and scoring was performed using Banff classification.In the 28 subjects with AMR, the density of C4d+/CD144+ microvesicles was on average 11-fold (P = 0.002) higher than transplant recipients with no AMR and 24-fold (P = 0.008) than healthy volunteers. Densities of C4d+ and C4d+/annexin V+ (C4d+/AVB+) microvesicles were also increased in AMR patients compared with no AMR and healthy subjects. C4d+/AVB+ microvesicles correlated with AMR biopsy severity. Nine patients with acute AMR that received treatment showed a mean 72% decrease (P = 0.01) in C4d+/CD144+ microvesicle concentration compared with pretreatment values.Quantification of plasma C4d+ microvesicles provides information about presence of AMR, its severity and response to treatment in transplant patients.

An international normalized ratio (INR) higher than 9 is associated with a high risk of bleeding, yet most studies have focused on outpatients with lower INR. We retrospectively analyzed diagnosis, bleeding, treatment, and mortality in 162 patients with INR higher than 9, including inpatients and outpatients with and without warfarin treatment. Patients without anticoagulant treatment with INR higher than 9 had a poor prognosis, 67% experienced bleeding and 74% died. Among outpatients receiving warfarin with INR higher than 9, 11% had bleeding, but none died. Among inpatients receiving warfarin, 35% had bleeding and 17% died. Factors associated with bleeding were older age, renal failure, and alcohol use. Withholding warfarin or giving vitamin K treatment was ineffective at reducing the INR within 24 hours, whereas plasma infusion immediately dropped the INR to 2.4 ± 0.9. Because of underlying disease, comorbidities, and medications, hospitalized patients with INR higher than 9 may not respond quickly to withholding warfarin or vitamin K treatment, and plasma infusion may be needed to reduce INR and the risk of bleeding within 24 hours.

Context.—Proper diagnosis and therapy of fibrinogen deficiency requires high-quality fibrinogen assays.Objective.—To assess the interlaboratory bias, precision, and grading of fibrinogen assays used by laboratories participating in the United States College of American Pathologists proficiency testing program in coagulation.Design.—Two identical vials of normal plasma were sent to more than 3500 laboratories. Participants measured fibrinogen levels using local methods.Results.—Fifty different fibrinogen methods were evaluated. All-method bias was 8.3% (range of method-specific biases, 0.0%–27.0%) and all-method coefficient of variation was 7.7% (range of method-specific coefficients of variation, 0.7%–25.8%). After controlling for reagent/instrument type, mean fibrinogen levels were 11.6% higher for prothrombin time–based reagents compared to Clauss (P < .001), and coefficient of variation was 46% lower for mechanical endpoint instruments compared to photo-optical. Most testing events (97.4%) could be reliably graded as pass or fail using a target range of ±20% from the method mean (total pass rate, 98.8%). Total fail rate was 3.0-fold lower for mechanical instruments compared to photo-optical (0.5% versus 1.5%, P = .001). Nonetheless many photo-optical methods had very high precision and very low fail rates.Conclusions.—Fibrinogen assays showed highly variable methodology and performance characteristics. Bias, precision, and grading were affected by the type of reagent or instrument used.

Use of a coagulation panel [prothrombin time (PT), partial thromboplastin time (PTT), thrombin time (TT) and fibrinogen], intended for evaluation of bleeding, tripled over 6 years, out of proportion to admissions, surgery, or transfusions. To determine whether the panels were ordered appropriately, we classified 28,737 sets of panel results into groups followed by chart reviews to determine typical patient histories. In 39% of panels, PT/PTT was normal. Prolonged PT occurred in 33% of results, due to liver failure (8%), warfarin (23%), and presumed vitamin K deficiency (69%). Prolonged PTT occurred in 34% of results and was primarily associated with long PT or lupus inhibitors. Prolonged PTT and TT (15% of panels) indicated heparin therapy. Fibrinogen was normal in 98% and low in 1.4%. Critical fibrinogen (below 100 mg/dl, 0.6% of panels) was associated with bleeding in 90% of patients. Only 8% of panel orders were clinically indicated based on patient history. Clinician interviews indicated many were unaware the panel included fibrinogen and TT. Interventions included an education program and an order form change. The education program had no effect on overall order volume or test selection. A later order form change made TT and fibrinogen a separate order. This reduced TT and fibrinogen testing by 90% without complaints or changes in blood transfusion statistics. We conclude that many coagulation test panel orders were not clinically indicated, that PT more often diagnosed vitamin K deficiency than bleeding risk, and that order-based restriction of testing was more effective than educational programs at introducing change in clinical test utilization.

We evaluated the accuracy and precision of von Willebrand disease (vWD) testing performed by up to 50 North American Specialty Coagulation Laboratories from 2004 through 2009, using proficiency samples from healthy subjects (n = 7) and patients with type 1 vWD (n = 7) or type 2 vWD (n = 3). We analyzed 2,212 submitted results. Precision was highest for von Willebrand factor (vWF) antigen assays (coefficient of variation, 14%), which were performed predominantly by latex immunoassays, and lowest for ristocetin cofactor assays (coefficient of variation, 28%), which were increasingly replaced by collagen binding and immunofunctional methods during the 6-year evaluation period. Overall interpretation error rates ranged from 3% for normal samples, 28% for type 1 vWD, and 60% for type 2 vWD. Type 2 vWD samples were correctly identified by all laboratories using collagen binding/antigen ratios but by only one third of laboratories using ristocetin cofactor/antigen or immunofunctional/antigen ratios. In 2009, only 27% (12/45) of laboratories performed vWF multimer analysis, with error rates ranging from 7% to 22%.

Elevated levels of circulating procoagulants like tissue factor may increase the risk of systemic coagulation activation, thrombin generation, and consumptive coagulopathy. I measured procoagulant activity in plasma by using a clot-based assay that incorporated normal plasma to replace missing factors, corn trypsin inhibitor to block contact activation, factor VIIa to improve sensitivity to tissue factor activity, and anti–tissue factor antibodies to measure tissue factor–specific activity. Procoagulant activity was evaluated in 58 trauma patients. Trauma patients without coagulopathy (n = 50) showed 5-fold higher procoagulant activity than did control subjects (P < .001), whereas trauma patients with coagulopathy (n = 8) showed 10-fold higher activity than control subjects (P < .001) and 2-fold higher activity than trauma patients without coagulopathy (P = .03). In control subjects, tissue factor activity was below the detection limit of the assay. Tissue factor activity was 3- to 4-fold higher in trauma patients with coagulopathy vs patients without coagulopathy (P = .002). Trauma patients with coagulopathy have increased circulating tissue factor activity. In a number of clinical disorders, including shock, trauma, and sepsis, systemic coagulation activation can occur, leading to varying degrees of coagulation factor consumption and vascular fibrin deposition. Systemic exposure or release into the vascular system of substances that activate the coagulation system (procoagulants) may have a role in consumptive coagulopathies. Normal blood from healthy patients contains little procoagulant activity. 1 If contact activation is blocked in normal blood, little if any thrombin generation occurs. 2 In disease states, though, blood may contain several different forms of procoagulant that can help initiate thrombin generation, including soluble, cell, and microparticle-bound tissue factor; cancer-derived procoagulants; procoagulant phospholipids; activated factor XIa; and others. 3-6 Acute coagulopathy of trauma (ACT) has been described that can have adverse effects on patient outcome. 7-9 This trauma coagulopathy seems to arise from a combination of tissue trauma and shock, exacerbated by hemodilution, hypothermia, acidemia, and inflammation. ACT is associated with activation of the coagulation and fibrinolytic

BACKGROUND: Evaluation of hemostasis in bleeding patients requires both accuracy and speed. STUDY DESIGN AND METHODS: As an alternative to point-of-care testing, we developed an emergency hemorrhage panel (EHP: prothrombin time [PT], fibrinogen, platelet count, hematocrit) for use in making transfusion decisions on bleeding patients with a goal of less than 20-minute turnaround time (TAT) when performed in the clinical laboratory on automated instruments. Because point-of-care samples are not checked for clotting or hemolysis, we evaluated their effect on automated testing. RESULTS: TAT was reduced by moving the sample immediately to testing and shortening centrifugation times. Clotting in samples was rare (1.1%) and shortened the PT by only 0.7 seconds. It lowered fibrinogen on average 18%, but resulted in only one of 2300 samples changing from normal to low fibrinogen. Hemolysis had no clinically significant effect on the PT or fibrinogen. Therefore, hemolysis checks were eliminated and clot checks minimized. Initially TAT averaged 15 ± 4 minutes (range, 8-30 min), but 9% of samples exceeded the 20-minute goal due to low fibrinogens that slowed testing. A revised fibrinogen assay with expanded calibration range resulted in a TAT of 14 ± 3 minutes (range, 6-28 min) with only 2% of samples exceeding the 20-minute goal. By limiting EHPs to patients that were actively bleeding, EHPs accounted for only 8 of 243 coagulation samples per day. CONCLUSION: Limiting EHPs to bleeding patients and modifications to the process and assays used for hemostasis testing lead to TATs of less than 20 minutes for critical testing in the clinical laboratory.

BACKGROUND: Trauma patients are at risk of developing an acute coagulopathy of trauma (ACT) related to tissue injury, shock, and hemodilution. ACT is incompletely understood, but is similar to disseminated intravascular coagulation (DIC) and is associated with poor outcome. STUDY DESIGN AND METHODS: Thrombin generation assays were used to evaluate plasma hemostasis in 42 trauma patients, 25 normal subjects, and 45 patients on warfarin and in laboratory-prepared factor reduced plasma. RESULTS: Prolonged prothrombin time (PT), more than 18 seconds, or an international normalized ratio of greater than 1.5 was present in 15 trauma patients indicating possible ACT. Native thrombin generation (no activator added, contact activation blocked) showed that Trauma with ACT patients had lag times 68% shorter and peak thrombin generation threefold higher than normal patients indicating the presence of circulating procoagulants capable of initiating coagulation systemically. Trauma patients had lower platelet counts and fibrinogen and Factor (F)II levels putting them at increased risk of bleeding. In laboratory-prepared isolated factor-reduced samples and in patients with vitamin K–dependent factor deficiency due to warfarin, thrombin generation decreased in direct proportion to FII levels. In contrast, in diluted plasma and in trauma patients with reduced factor levels, thrombin generation was increased and associated with slower inhibition of thrombin generation (prolonged termination time) and decreased antithrombin levels (43% of normal in Trauma with ACT). CONCLUSIONS: Thrombin generation studies indicate that Trauma with ACT patients show dysregulated hemostasis characterized by excessive non–wound-related thrombin generation due to a combination of circulating procoagulants capable of activating coagulation systemically and reduced inhibitor levels allowing systemic thrombin generation to continue once started.

Background: Ionized calcium (iCa) is measured frequently in hospitalized patients, and hypocalcemia is frequently found, seemingly supporting the practice. Methods: We retrieved the results of 58 040 iCa tests and records of intravenous (IV) and oral calcium supplementation from laboratory and hospital information systems and evaluated them for frequency of testing, frequency of hypocalcemia, and effects of calcium supplementation. Results: Serial and daily iCa testing was common and responsible for a substantial fraction of all iCa tests ordered. Half of all patients tested had iCa values below the reference interval. IV, but not oral, calcium therapy increased mean iCa concentrations, but the effect of calcium administration was small compared with the spontaneous increase in iCa that occurred in similar patients who received no calcium treatment. A retrospective analysis suggested that a low total calcium (tCa) concentration ( Conclusions: Testing for iCa and IV calcium supplementation were significantly reduced with a reflexive calcium testing strategy that provided iCa testing only to patients with low tCa. Adverse clinical outcomes possibly associated with hypocalcemia did not increase.

BACKGROUND: Clinical studies conducted to elucidate the systemic response to cardiopulmonary bypass (CPB) did not differentiate possible effect of different types of cardiac surgical pathologies and operations on outcomes and have typically combined different procedures. We hypothesized that valve surgery induces more prominent systemic reaction compared to isolated on-pump CABG. METHODS: Twenty-seven patients undergoing primary on-pump CABG (Group 1, n = 14) or valve surgery with or without CABG (Group 2, n = 13) were prospectively enrolled. Heparin-bonded circuits were used in all patients. Cardiotomy suction was only used in Group 2. Clinical and laboratory markers were evaluated. RESULTS: Clinical measurements, including chest tube output, blood transfusion requirement, inotropic support requirement, and duration of ICU stay were not significantly different. Thrombin generation (PF-1.2) was significantly higher in Group 2 (p = 0.001). tPA was also significantly higher in Group 2 at 15 and 60 minutes on CPB (p < 0.01). Group 2 had significantly higher inflammatory response shown by elevation of IL6 (p = 0.005). Neuronal injury markers, S100beta and NSE, were significantly higher at the termination of CPB in Group 2 (p < 0.01). At no point of time course for any marker, Group 1 had significantly higher response compared to Group 2. CONCLUSIONS: Valve surgery induced more prominent systemic response than CABG. The possible explanations include the difference in baseline disease pathophysiology, and/or difference associated with the procedures such as open systems and use of cardiotomy suction. Future clinical studies assessing systemic response to CPB and therapies to blunt these need consider and account for these observed differences.

SummaryActivated partial thromboplastin time (aPTT) monitoring of direct thrombin inhibitors (DTIs) is vulnerable to interference from many sources. If the baseline aPTT is prolonged, as occurs with lupus inhibitors, alternative methods are required to monitor DTI levels. We compared the plasma diluted thrombin time (1:4 dilution of patient plasma with normal plasma) and the aPTT in patient samples spiked with argatroban, bivalirudin, or lepirudin at three concentration levels. Each drug was spiked into five samples with lupus inhibitors, five samples with deficient vitamin K-dependent factors, three samples with elevated D-dimers, and eight samples with normal baseline aPTT values. The aPTT overestimated the spiked DTI concentration in all samples with lupus inhibitors, low levels of vitamin K-dependent factors, and elevated D-dimers. In samples with normal baseline aPTTs, the aPTT failed to correctly estimate the spiked drug concentration in four of 24 samples spiked with argatroban, seven of 24 spiked with lepirudin, and three of 24 spiked with bivalirudin. The plasma diluted thrombin time was not affected by lupus inhibitors, low vitamin K-dependent factor levels or elevated D-dimer levels and correctly estimated the spiked drug level in 63 of 63 samples spiked with argatroban, 63 of 63 samples spiked with bivalirudin, and 62 of 63 samples spiked with lepirudin. In conclusion, the plasma diluted thrombin time appears to be a viable alternative to the aPTT for monitoring DTI levels, especially in patients with lupus inhibitors or low levels of vitamin K-dependent factors.

Summary. Background: Associations between common F7 haplotypes, plasma factor VII (FVII) levels, and cardiovascular risk have recently been reported in population studies involving predominantly European men. Methods: We assessed associations between F7 haplotypes and cardiovascular risk in two US population-based studies: a case–control study of these alleles related to a decreased risk of arterial thrombotic outcomes such as myocardial infarction (MI) in young-to-middle-aged women (n = 671), and a cohort study of cardiovascular disease risk factors in young women (n = 1040). Results: The high-expression F7 haplotype B (containing the promoter variant allele −402A) was associated with an increased FVII level among controls, but not with MI risk. Women carrying a≥1 copy of the low FVII expression level haplotype C (containing the −401T/−323del/−122C and Gln353 alleles) had decreased FVII levels and decreased risk of MI (odds ratio 0.54, 95% CI 0.31–0.93) compared with women homozygous for the most common haplotype A. Haplotype C was also associated with a decreased body mass index (BMI) and an increased high-density lipoprotein (HDL) cholesterol level, but not with MI risk after adjustment for these metabolic risk factors. In a cohort study composed of young US women, individuals homozygous for haplotype C had a lower BMI and lower systolic blood pressure, but the association between the F7 haplotype and HDL cholesterol was not confirmed. Conclusion: Common FVII haplotypes may contribute to the risk of MI in women, but the mechanisms appear complex. The association between F7 haplotypes and MI susceptibility may be mediated in part through an influence on atherogenic risk factors such as BMI.

Mixing studies for prolonged prothrombin time (PT)/activated partial thromboplastin time (aPTT) are used to estimate whether the prolongation is due to an inhibitor or factor deficiency. We propose a new method of mixing study interpretation based on estimation of average factor level changes. Factor level vs. PT/aPTT curves were prepared for single factor, vitamin K-dependent factor, and all factor deficiencies. These curves were used to predict the factor level in the sample and the correction needed to differentiate deficiencies from inhibitors. We compared this estimated factor correction (EFC) method to normal range, percentage correction, and Rosner index. For a given factor level, multiple factor deficiencies prolonged the PT/aPTT more than single factor deficiency, necessitating different thresholds for defining correction on mixing studies. The EFC method was superior to other the correction methods, correctly identifying 38 of 39 known inhibitors, single and multiple factor deficiencies, and correctly identifying inhibitor vs. deficiency in 50 of 59 patient samples. In 99 adult patient mixing studies over 18 months, 30% showed deficiency only, 30% inhibitor only, whereas 40% showed evidence of both. The EFC method for PT/aPTT mixing study interpretation was more accurate than the comparison methods at determining deficiency versus inhibitor.

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 μg/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.

The U.S. College of American Pathologists (CAP) has conducted a focused study of the proficiency testing for von Willebrand disease (vWD) analysis from 2003 to 2005. This report summarizes the findings regarding the accuracy and precision of the various assays at different analyte levels, as well as the influence of the reference material used to construct the assay standard curve. The results show that testing of von Willebrand factor (vWF):antigen (vWF:Ag) and ristocetin cofactor activity (vWF:RCo) is reasonably accurate, with all-method mean values falling within 3.2 and 5.6%, respectively, of the International Society on Thrombosis and Haemostasis Secondary Coagulation Standard (lot 2) assigned values. vWF:Ag measurements are reasonably precise (all-method coefficients of variation [CVs] = 10.7 to 15.1%), even at lower levels of vWF. The highest precision was observed for immunoturbidometric assays (CVs, 6.3 to 9.7%). vWF:RCo measurements are less precise (all-method CVs, 23.3 to 30.9%). The reference materials used in the standard curves for immunoturbidometric vWF:Ag assays appear to have accurately assigned vWF values for the majority of commercial suppliers.

Introduction The purpose of this study was to estimate, in patients undergoing cardiopulmonary bypass (CPB), the in vivo rates of tissue plasminogen activator (tPA) and plasminogen activator inhibitor 1 (PAI-1) secretion, plasmin generation, fibrin degradation, and plasmin inhibition by aprotinin versus antiplasmin. Materials and methods Estimates of in vivo rates were based on measured levels of tPA, PAI-1, antiplasmin, plasmin–antiplasmin complex (PAP), total aprotinin, plasmin–aprotinin complex and D-dimer, combined with a computer model of each patient's vascular system that continuously accounted for secretion, clearance, hemodilution, blood loss and transfusion. Plasmin regulation was studied in nine control patients undergoing CPB without aprotinin versus six patients treated with aprotinin. Results In controls, plasmin–antiplasmin levels rose from a baseline of 3.0±0.9 to a peak of 8.1±2.7 nmol/L after CPB due to an average 44-fold rise in the plasmin generation rate. This rise in plasmin generation during CPB lead to increased fibrin degradation causing D-dimer levels to increase from a baseline of 1.2±0.6 to a peak of 9.7±4.4 nmol/L due to an average 74-fold rise in the D-dimer generation rate. During CPB in the aprotinin group, plasmin–antiplasmin levels dropped, plasmin–aprotinin complex levels rose, while D-dimer levels remained unchanged from baseline. Compared to controls, the aprotinin group showed similar rates of plasmin generation during CPB, but an 11-fold faster plasmin inhibition rate and a 10-fold lower D-dimer generation rate. Conclusions The rise in plasmin generation and fibrin degradation that occurs during standard CPB is suppressed by the addition of aprotinin, which returns the patient to near baseline fibrin degradation rates during CPB.

P-selectin is a marker of platelet activation. Previous studies have reported elevated P-selectin in patients with congestive heart failure (CHF) and depression as separate disorders. We examined if comorbid depression had an effect on platelet activation in CHF patients. Soluble (s)P-selectin was measured in 108 CHF patients; 24 with comorbid depression. There were no significant differences in age, cardiac parameters or (s)P-selectin levels between CHF-only patients and patients with comorbid depression. Our data show no group differences in P-selectin values, which suggests that comorbid depression has no additive effect on platelet activation in CHF patient.

Numerous studies have demonstrated that increased C-reactive protein (CRP) levels predict coronary heart disease, stroke, peripheral vascular disease, and diabetes, and are associated with features of the metabolic syndrome. Only three previous studies have investigated the heritability of CRP levels, primarily in samples of Caucasian families. The purpose of the present study was to estimate the magnitude of genetic influences on CRP levels, and to examine potential associations between variation in the APOE gene and CRP levels, using a sample of 562 individual Japanese Americans from 68 extended kindreds. In general, correlation coefficients between first-degree relatives for CRP were approximately 0.2, and spouse correlations did not differ from zero, consistent with genetic influences. Heritability estimates were approximately 0.3 (p < 0.01), even with adjustment for factors known to influence CRP levels. A significant relationship was seen between unadjusted CRP levels and APOE genotypes (p = 0.02), with the highest mean CRP level among epsilon2 carriers (1.20 mg/L), and nearly the same mean levels among epsilon3/epsilon3 subjects and epsilon4 carriers (0.72 and 0.74 mg/L, respectively). However, this relationship was diminished with adjustment for covariates (p = 0.07). These results demonstrate the presence of both genetic and environmental effects on CRP levels among Asian Americans, and additional studies are needed to determine if the APOE gene contributes to these genetic influences.

We studied the relationship between sustained submaximal exercise, increased tissue plasminogen activator (t-PA) levels and decreased hepatic clearance of t-PA. Six healthy male volunteers exercised for 35 min while receiving constant rate infusions of either saline or two different doses of recombinant t-PA for 90 min (40 min before, 35 min during and 15 min after exercise). Liver blood flow was estimated simultaneously by constant rate indocyanine green infusion. Since t-PA is cleared rapidly by the liver in direct proportion to liver blood flow, it was expected that a significant decrease in liver blood flow during sustained submaximal exercise would be associated with a proportional increase in plasma t-PA. During submaximal exercise with a saline (placebo) infusion, steady-state t-PA antigen increased from a resting baseline of 6.3 +/- 3.1 to 15.1 +/- 5.1 ng/ml; with a 20 microg/min t-PA infusion, t-PA antigen increased from 33 +/- 12 to 84 +/- 25 ng/ml during exercise; and with a 40 microg/min t-PA infusion, t-PA antigen increased from 77 +/- 38 to 166 +/- 42 ng/ml during exercise. During submaximal exercise, liver blood flow fell on average 71, 68 and 70%, respectively, during the three procedures, while calculated t-PA clearance decreased on average 59, 59 and 53%. t-PA concentration versus time curves, displayed in proportional units, were similar. The comparable relative increases in endogenous and exogenous t-PA with simultaneous proportional decreases in liver blood flow suggests that diminished hepatic t-PA clearance is the major cause of increased t-PA concentration and blood fibrinolytic activity enhancement during sustained submaximal exercise.

A rapidly growing body of evidence demonstrates important associations between the metabolic syndrome, characterized by a cluster of risk factors or phenotypes that include dyslipidemia, central obesity, hypertension, and hyperinsulinemia, and both cardiovascular disease and type 2 diabetes. The purpose of the present study was to characterize the metabolic syndrome in a sample of 432 individuals from 68 Japanese-American families, using factor analysis of quantitative phenotypes, and to estimate the heritability of these independent factors. Using nine characteristic phenotypes that included LDL particle size and C-reactive protein (CRP), factor analysis identified three multivariate factors interpreted as lipids, body fat/insulin/glucose/CRP, and blood pressure, explaining 65% of the variance. Heritability analysis revealed significant genetic effects on all of the factors: lipids (h2 = 0.52, P < 0.001), body fat/insulin/glucose/CRP (h2 = 0.27, P = 0.016), and blood pressure (h2 = 0.25, P = 0.026). This analysis shows that independent, multivariate factors of the metabolic syndrome are heritable, demonstrating genetic influences on the underlying pathophysiological mechanisms of the syndrome.

Background: The purpose of this study was to evaluate the relative importance of systemic hypercoagulability, pre-existing and acquired risk factors, and specific injury patterns in the development of venous thromboembolism (VTE) after injury. Methods: Injured patients with an Injury Severity Score ≥ 15 were followed with lower extremity venous duplex ultrasonography, prothrombin fragment 1 + 2, and quantitative D-dimer levels at 1 and 3 days and then weekly until discharge. Results: Among 101 patients with a mean Injury Severity Score of 27.3 ± 10.5 followed for 12.4 ± 8.7 days, 28 (27.7%) developed a lower extremity thrombosis, 2 (1.9%) sustained a pulmonary embolism, and 1 (0.9%) had a symptomatic upper extremity thrombosis. Although admission fragment 1 + 2 and D-dimer levels were elevated in 81.4% and 100% of patients, respectively, mean levels were not significantly different in those with or without VTE. VTE was more common (p 40 years, immobilization for > 3 days, spine fractures, and lower extremity fractures. However, only obesity (p = 0.004) and immobilization > 3 days (p = 0.05) were independent predictors of VTE in a multivariate analysis. Conclusion: Although elevated in seriously injured patients, neither markers of activated coagulation nor specific injury patterns are predictive of VTE. Associations with immobilization and obesity suggest that VTE after injury is a systemic hypercoagulable disorder with local manifestations of thrombosis related to lower extremity stasis.

Objectives.—To review the state of the art relating to elevated hemostatic factor levels as a potential risk factor for thrombosis, as reflected by the medical literature and the consensus opinion of recognized experts in the field, and to make recommendations for the use of specific measurements of hemostatic factor levels in the assessment of thrombotic risk in individual patients. Data Sources.—Review of the medical literature, primarily from the last 10 years. Data Extraction and Synthesis.—After an initial assessment of the literature, key points were identified. Experts were assigned to do an in-depth review of the literature and to prepare a summary of their findings and recommendations. A draft manuscript was prepared and circulated to every participant in the College of American Pathologists Conference XXXVI: Diagnostic Issues in Thrombophilia prior to the conference. Each of the key points and associated recommendations was then presented for discussion at the conference. Recommendations were accepted if a consensus of the 27 experts attending the conference was reached. The results of the discussion were used to revise the manuscript into its final form. Conclusions.—Consensus was reached on 8 recommendations concerning the use of hemostatic factor levels in the assessment of thrombotic risk in individual patients. Detailed discussion of the rationale for each of these recommendations is presented in the article. This is an evolving area of research. While routine use of factor level measurements is not recommended, improvements in assay methodology and further clinical studies may change these recommendations in the future.

Thrombotic adverse outcomes contribute to significant morbidity and mortality after major invasive surgery. Surgery creates an acute phase or ‘stress’ response, that causes the release of prothrombotic proteins and thrombocytosis. Some patients exhibit an exaggerated response. In cardiothoracic surgery the same acute phase response contributes to a variety of adverse outcomes from abnormal bleeding to coronary artery thrombosis and stroke. A number of genetic variants or polymorphisms have been elucidated that have bearing on peri-operative risk and adverse outcomes. These polymorphisms have been correlated with venous thrombosis, accelerated atherosclerosis, unstable angina and acute myocardial infarction (MI) in the non-surgical setting. The contribution of these polymorphisms to adverse outcomes after surgery is being investigated. By understanding some key polymorphisms and potentially testing for them in the future, we may be better able to target expensive pharmacological therapies as well as predicting or preventing certain adverse prothrombotic outcomes.

Hemolytic uremic syndrome (HUS) usually occurs after infection with Shiga toxin-producing bacteria. Thrombotic thrombocytopenic purpura, a disorder with similar clinical manifestations, is associated with deficient activity of a circulating metalloprotease that cleaves von Willebrand factor at the Tyr842-Met843 peptide bond in a shear stress-dependent manner. We analyzed von Willebrand factor-cleaving metalloprotease activity and the status of von Willebrand factor in 16 children who developed HUS after Escherichia coli O157:H7 infection and in 29 infected children who did not develop this complication. Von Willebrand factor-cleaving metalloprotease activity was normal in all subjects, but von Willebrand factor size was decreased in the plasma of each of 16 patients with HUS. The decrease in circulating von Willebrand factor size correlated with the severity of thrombocytopenia and was proportional to an increase in von Willebrand factor proteolytic fragments in plasma. Immunohistochemical studies of the kidneys in four additional patients who died of HUS demonstrated glomerular thrombi in three patients, and arterial and arteriolar thrombi in one patient. The glomerular thrombi contained fibrin but little or no von Willebrand factor. A decrease in large von Willebrand factor multimers, presumably caused by enhanced proteolysis from abnormal shear stress in the microcirculation, is common in HUS.

Reduced factor XIIIA levels and decreased clot strength have been associated with increased bleeding after cardiopulmonary bypass (CPB). The purpose of this study was to evaluate the relationship between hemostatic factors, including factor XIIIA, and clot strength before, during and after CPB. Factor XIIIA antigen, platelet counts, fibrinogen, factor V activity, tissue plasminogen activator and clot strength (by thromboelastograph) were measured at baseline, after 45 min of CPB, at the end of CPB and 4 h post-operatively in 34 patients. Baseline factor XIIIA antigen was 5.2 +/- 1.4 mg/l. On average, factor XIIIA levels dropped to 64% and clot strength to 77% of baseline values after 45 min on CPB and remained below baseline during the immediate post-operative period. Clot strength was significantly correlated (r = 0.81) with platelet count and fibrinogen but not plasma factor XIIIA levels. Addition of 10 mg/l recombinant factor XIII[a2] significantly increased clot strength. Postoperative bleeding at 2 h was inversely correlated with platelet count, factor XIIIA antigen and clot strength measured at the end of CPB. Maintenance of adequate platelet counts and factor XIIIA levels at the end of CPB may play a role in maintaining clot strength and reducing blood loss.

Objective: To determine the effects of hemofiltration on serum aprotinin levels during cardiopulmonary bypass (CPB) surgery. Design: Prospective, randomized study. Setting: University of Washington Medical Center, single institution. Participants: Patients undergoing cardiac surgery without contraindications to aprotinin administration. Interventions: Patients were randomized to full-Hammersmith and half-Hammersmith dosing regimens of aprotinin and were further randomized to hemofiltration or no hemofiltration. Measurements and Main Results: Serum aprotinin levels were studied before CPB, 60 and 120 minutes into CPB, and at the end of CPB before protamine administration. Each group experienced a decrease in serum aprotinin levels with the institution of CPB, attributable to hemodilution and redistribution of aprotinin outside of the vascular compartment. During CPB, aprotinin levels declined further, but no significant difference was observed between patients who received hemofiltration and those who did not. Hematocrit values were significantly higher at the end of CPB in the hemofiltration groups. Patients receiving half-Hammersmith dosing regimens maintained aprotinin levels throughout CPB, which have been shown to inhibit plasmin but were lower than levels previously shown to inhibit kallikrein. Conclusions: Hemofiltration during CPB did not significantly alter serum aprotinin levels in patients receiving half-Hammersmith and full-Hammersmith dosing regimens of aprotinin. Copyright © 2000 by W.B. Saunders Company

A 5 µg/kg bolus of tissue plasminogen activator (t-PA) was infused into 11 healthy subjects followed by measurement of t-PA activity and antigen, PAI-1 activity and antigen, t-PA/PAI-1 complex, plasmin/antiplasmin (PAP) complex and D-dimer over 4 h. Infusion of t-PA resulted in a rise in PAP levels in all subjects from a baseline of 2.4 ± 1.1 nmol/l to a peak of 5.1 ± 2.3 nmol/l, but had no effect on plasminogen, antiplasmin or D-dimer levels. Using a kinetic model of plasminogen activation in vivo, the second-order rate constant for t-PA binding to plasminogen was estimated to be 3,100 ± 1,300 mol–1l·s–1, 200–500 times slower than t-PA accelerated by fibrin. The half-life of PAP was 4.5 ± 1.6 h. In healthy subjects, the PAP level in plasma represents the average rate of plasminogen activation over the last 2–8 h.

To better understand the regulation of tissue plasminogen activator (t-PA) and plasminogen activator inhibitor 1 (PAI-1) during liver transplantation, we used a computer model of the human circulatory system to simultaneously evaluate the effect of t-PA secretion, t-PA inhibition by PAI-1, hepatic clearance of t-PA, blood loss, transfusion and hemodynamics on t-PA and PAI-1 levels during liver transplantation in three patients that differed in severity of liver disease, blood loss and anhepatic changes in t-PA. Higher preoperative t-PA levels were primarily related to underlying liver disease and reduced hepatic clearance. During the anhepatic stage, when hepatic t-PA clearance was eliminated: (1) the expected rise in t-PA was modulated by the extent of bleeding, which acted as an alternate t-PA clearance mechanism; and (2) the ratio of t-PA:PAI-1 was increased due both to lower t-PA clearance and reduced PAI-1 secretion. Recirculation of the new liver was associated with renewed clearance of t-PA, an acute phase increase in PAI-1 and a drop in the t-PA:PAI-1 ratio. Understanding fibrinolytic regulation required simultaneous analysis of t-PA secretion, inhibition and clearance. Anhepatic t-PA levels could be predicted based on preoperative liver function and surgical blood loss, which acted as an alternate t-PA clearance mechanism.

Using human whole blood samples with and without contrast agent (CA), we evaluated the effect of exposures to focused, continuous wave (CW) 1.1-MHz ultrasound for durations of 10 ms to 1 s at spatial average intensities of 560 to 2360 W/cm2. Cavitation was monitored with a passive cavitation detector and hemolysis was determined with spectroscopy. In whole blood alone, no significant cavitation, heating or hemolysis was detected at any exposure condition. Conversely, cavitation and hemolysis, but not heating, were detected in whole blood with CA. A CA concentration as low as 0.28 microL CA per mL whole blood at an intensity of 2360 W/cm2 for 1 s resulted in measurable cavitation and a 6-fold increase in hemolysis compared to shams. Cavitation and hemolysis increased proportional to the concentration of CA and duration of exposure. In samples containing 4.2 microL CA per mL whole blood exposed for 1 s, a threshold was seen at 1750 W/cm2 where cavitation and hemolysis increased 10-fold compared to exposures at lower intensities. HIFU exposure of whole blood containing CA leads to significant hemolysis in vitro and may lead to clinically significant hemolysis in vivo.

Background To evaluate the effect of plasminogen activator inhibitor type 1 (PAI-1) levels on the clearance of total tissue plasminogen activator (TPA) antigen, we studied the clearance of active TPA and TPA/PAI-1 complex in subjects with low (181±109 pmol/L; n=7) and high (1166±322 pmol/L; n=4) baseline active PAI-1. Methods and Results A 5-μg/kg bolus of TPA was infused over a 15-second period followed by measurement of TPA activity, TPA antigen, TPA/PAI-1, TPA/C1 inhibitor, PAI-1 activity, and PAI-1 antigen over a 4-hour period. α-Phase clearance of total TPA antigen was faster in subjects with low PAI-1 (t 1/2 of 3.5±0.7 minutes) versus high PAI-1 (t 1/2 of 5.3±0.9 minutes) ( P =.006). Clearance of all factors was best fit by a two-compartment pharmacokinetic model based on a computer-simulated human circulatory system. The average hepatic clearance fraction in the two-compartment model was greater for active TPA (89±10%, t 1/2 of 2.4±0.3 minutes) than for TPA/PAI-1 complex (48±17%, t 1/2 of 5.0±1.8 minutes) ( P =.0006). Conclusions Plasma clearance of active TPA was faster than clearance of TPA/PAI-1 complex. High levels of active PAI-1 converted more TPA into TPA/PAI-1 complex, effectively slowing the clearance of total TPA antigen and explaining in part why high levels of PAI-1 activity are associated with increases in total TPA antigen.

Use of age-adjusted d-dimer levels has been recommended for evaluating patients with suspected pulmonary embolism. The authors of this commentary caution that clinicians must understand important d...

New anticoagulants, like the orally available direct thrombin inhibitor (DTI) dabigatran etexilate, have recently been introduced into the market for venous thromboembolic prophylaxis and for stroke prevention in atrial fibrillation. While dabigatran has been approved for use without the need for routine therapeutic monitoring, there are clinical scenarios in which monitoring can help guide clinical management. We report herein the application of a recently described plasma-diluted thrombin time (DTI assay) used to monitor intravenous DTI as a useful and easily implemented method to monitor oral DTIs.

A computer simulation of the circulatory system was used to kinetically model secretion, inhibition, and clearance of tissue plasminogen activator (t-PA) during three different processes that increase active t-PA levels: epinephrine infusion, exercise, and endurance training. Infusion of epinephrine stimulated an increase in t-PA secretion that was proportional to the plasma epinephrine concentration. In addition, epinephrine infusion increased hepatic blood flow and t-PA clearance, thus slowing the increase of plasma t-PA levels. During exercise, t-PA levels increased due both to increased t-PA secretion and to decreased clearance secondary to reduced hepatic blood flow. The increase in t-PA secretion during exercise was directly proportional to the epinephrine concentration in blood with the same ratio of t-PA secretion to epinephrine as found during epinephrine infusion, suggesting that increased plasma epinephrine during exercise was the primary stimulus for t-PA secretion. Lastly, the simulation predicted that 6 months of endurance training produced a decrease in resting plasminogen activator inhibitor type 1 (PAI-1) secretion, resulting in less t-PA inhibition and an overall increase in active t-PA after training. Accurate analysis of the regulation of active t-PA levels in blood required simultaneous modeling of t-PA and PAI-1 secretion, hepatic clearance, and inhibition of t-PA by PAI-1.

We compared plasma with whole blood (WB) international normalized ratio (INR) and fibrinogen using the same instrument and reagents. WBINRs were 50% higher than plasma INRs. After increasing the WB sample volume 40% and adjusting the International Sensitivity Index, WBINRs were similar to plasma INRs [adjusted WBINR = 0.99(plasma INR) – 0.02; r2 = 0.98; n = 155], but the average difference in WB vs plasma INR was 4-fold higher than duplicate plasma INRs. Variation in hematocrit was a major determinant of the accuracy of the WBINR, with increased error at high INRs. The WB fibrinogen assay was highly dependent on the sample hematocrit (r2 = 0.83), even after the sample volume was adjusted. Accurate WB fibrinogen measurements required a mathematical hematocrit correction. We conclude that WBINR and fibrinogen assays can be performed on point-of-care or automated analyzers, but sample volume must be adjusted to account for hematocrit. Accuracy is limited by variations in hematocrit with worsening accuracy for samples with high INRs or low fibrinogen levels.