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Objectives Activated partial thromboplastin time (aPTT) is the primary test used to monitor intravenous (IV) direct thrombin inhibitors (DTIs) but has many limitations. The plasma diluted thrombin time (dTT) has shown better correlation with DTI levels than aPTT. This study compared dose-response curves for dTT and aPTT in pediatric patients receiving argatroban and bivalirudin. Methods A retrospective review of pediatric patients treated with argatroban (n = 45) or bivalirudin (n = 14) monitored with dTT and aPTT. Results The dTT assay was calibrated to report DTI concentrations in µg/mL for argatroban and bivalirudin with good analytic sensitivity and specificity. The dTT was fivefold more likely to show a stable dose-response slope than the aPTT (P Conclusions The dTT assay was more likely to show a stable dose response and have a stronger correlation with DTI dose than aPTT. Argatroban shows more variation in dose response than bivalirudin.

Extracorporeal membrane oxygenation (ECMO) can be life-saving, but suffers from thrombus formation in the circuit with associated risks of oxygenator occlusion, hemolysis and arterial embolism. The formation of thrombin is the key step to thrombus formation and two factors are needed for sustained thrombin generation, a coagulation activator to initiate the process and a procoagulant phospholipid surface for the coagulation system to assemble on.The purpose of this study was to use thrombin generation potential (TGP) and other assays to determine the specific coagulation activators and sources of procoagulant phospholipid that are present in ECMO patient plasma. Samples were collected from 60 patients on ECMO (age 1d-19y) followed evaluation of native and stimulated TGP, measurement of factor II levels and determination of procoagulant extracellular vesicle levels by flow cytometry.During ECMO, native (unstimulated) TGP was increased, followed by a decrease back towards normal after ECMO ended. The main activator of TGP in ECMO plasma was increased FXIa (100% of samples tested), while increased tissue factor activity was present in 7%. Procoagulant phospholipids were present in plasma from ECMO patients in the form of circulating platelet and red cell extracellular vesicles, which were increased 2 to 7-fold compared to normal. Procoagulant extracellular vesicle levels correlated with increased plasma native TGP activity.ECMO activates coagulation in plasma primarily through activation of the contact system and formation of activated factor XIa and generation of circulating procoagulant extracellular vesicles through platelet and red cell activation.

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.

Most pediatric patients show a decline in platelet counts while on extracorporeal life support (ECLS) and require multiple platelet transfusions. To better understand platelet loss during ECLS, this study estimated platelet loss rates due to diagnostic phlebotomy, platelet activation, bleeding and other causes.We collected data on 91 patients (1d-20y, 50 M, 41F). Platelet losses were estimated based on changes in platelet count, patient+circuit blood volume, and transfused platelet volumes. Platelet extracellular vesicles were measured by flow cytometry.Median platelet loss was 2.8 × 10Our study is the first to quantitate total, phlebotomy and activation related platelet loss during ECLS. Platelet activation accounts for ~32% of total platelet loss, while bleeding doubles the platelet loss rate. The etiology of the remaining platelet loss is unknown.

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.

To balance the risk of bleeding versus circuit thrombosis during extracorporeal life support (ECLS), it is important to monitor anticoagulants and hemostasis. We evaluated the prothrombin time (PT), partial thromboplastin time (PTT), activated clotting time (ACT), and antifactor Xa heparin activity (aXa) correlation with changes in coagulation factor and heparin levels using in vitro and in vivo samples. aXa correlated with heparin (r = 0.97) and antithrombin (r = 0.98) but was unaffected by other parameters. PT correlated with coagulation factors (r = 0.88) but was minimally affected by heparin or other parameters. When single parameters were changed, ACT was insensitive to

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.

OBJECTIVES Anticoagulation with unfractionated heparin remains the most common therapy used to prevent circuit thrombosis during extracorporeal membrane oxygenation, but no consensus exists on the optimal method or targets for heparin monitoring. From 2015 to 2018, we switched from monitoring heparin during extracorporeal membrane oxygenation using activated clotting times to anti-Xa heparin activity assays. This study describes the transition from activated clotting time to anti-Xa heparin activity assay monitoring and the associated clinical changes. DESIGN Retrospective analysis at single institution. SETTING Referral Children's Hospital. PATIENTS A total of 145 pediatric patients over 152 extracorporeal membrane oxygenation runs using 206 extracorporeal membrane oxygenation circuits. INTERVENTIONS Anticoagulation protocol quality improvement. MEASUREMENTS AND MAIN RESULTS From 2015 to 2018, heparin monitoring during extracorporeal membrane oxygenation changed from hourly activated clotting time to anti-Xa heparin activity assay every 6 hours with an associated 75% reduction in the circuit changes per extracorporeal membrane oxygenation day. Over the 4 years, patients with an average anti-Xa heparin activity assay of at least 0.25 U/mL showed a 59% reduction in circuit changes per extracorporeal membrane oxygenation day compared with less than 0.15 U/mL. In addition to its association with reduced circuit changes, anti-Xa heparin activity assay monitoring was also associated with reduced heparin dose changes per day from 11 ± 4 to 2 ± 1 (p < 0.001), smaller heparin dose changes (less variation in dose), and reduced diagnostic phlebotomy volumes from 41 ± 6 to 25 ± 11 mL/day (p < 0.001). The number of patients with reported bleeding decreased from 69% using activated clotting time to 51% (p = 0.03). Transfusion rates did not change. CONCLUSIONS Over 4 years, we replaced the activated clotting time assay with the anti-Xa heparin activity assay for heparin monitoring during extracorporeal membrane oxygenation. Minimum anti-Xa heparin activity assay levels of 0.25 U/mL were associated with reduced circuit changes. Further studies are needed to determine the optimum anti-Xa heparin activity assay therapeutic range during extracorporeal membrane oxygenation.

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.

Microvesicles are fragments of cells released when the cells are activated, injured, or apoptotic. Analysis of microvesicle levels in blood has the potential to shed new light on the pathophysiology of many diseases. Flow cytometry is currently the only method that can simultaneously separate true lipid microvesicles from other microparticles in blood, determine the cell of origin and other microvesicle characteristics, and handle large numbers of clinical samples with a reasonable effort, but expanded use of flow cytometric measurement of microvesicle levels as a clinical and research tool requires improved, standardized assays. The goal of this review is to aid investigators in applying current best practices to microvesicle measurements. First pre-analytical factors are evaluated and data summarized for anticoagulant effects, sample transport and centrifugation. Next flow cytometer optimization is reviewed including interference from background in buffers and reagents, accurate microvesicle counting, swarm interference, and other types of coincidence errors, size calibration, and detection limits using light scattering, impedance and fluorescence. Finally current progress on method standardization is discussed and a summary of current best practices provided. © 2016 Clinical Cytometry Society.

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.

The presence of hemolysis or icterus can interfere with chromogenic anti-Xa assays for unfractionated heparin monitoring (heparin activity), resulting in falsely lower estimates of heparin activity. Dilution is a common method for mitigating interference in spectroscopic assays that measure a single analyte by endpoint analysis and has been proposed as a solution for hemolysis interference in heparin activity assays. Heparin activity assays measure heparin/antithrombin complex activity using a chromogenic rate of absorbance change method. Heparin activity is a complex equilibrium of heparin binding to multiple proteins, including antithrombin, heparin cofactor II, platelet factor 4, histidine-rich glycoprotein, vitronectin, fibronectin, and others; some enhance while others neutralize heparin activity. The objective of this study was to determine how dilution affects heparin activity with different starting levels of antithrombin. First samples were diluted without interference (no hemolysis). Samples with 1 U/mL heparin in pooled normal plasma (PNP) and 50% PNP (to simulate 50% antithrombin levels) were diluted 1:1 with PNP versus OK buffer. Dilution resulted in nonlinear changes. Samples with 100% antithrombin recovered 88% of expected activity with PNP versus 74% with OK buffer; samples with 50% antithrombin recovered 111% with PNP versus 77% with OK buffer. Samples were then spiked with varying concentrations of hemolysate and 1:1 dilutions with PNP demonstrated nonlinear recoveries ranging from 41% to 84%. We were unable to recover the original heparin activity with or without hemolysis due to the complex interaction of initial heparin, antithrombin, and other binding protein concentrations with diluent antithrombin and other diluent heparin binding protein levels. When PNP is used as the diluent, the recovery will be lower when the initial antithrombin level is normal due to the addition of other heparin binding proteins; when the initial antithrombin level is low, the recovery is higher due to the addition of antithrombin from the diluent plasma. The recovery is always lower when buffer is used due to dilution of sample antithrombin. Careful consideration should be taken in evaluating the utility of sample dilution to mitigate interference from hemolysis and icterus in heparin activity assays, particularly in patients with fluctuating levels of antithrombin, such as those on extracorporeal life support or with liver failure.

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

Background: The pharmacokinetic properties of enoxaparin may lead to supratherapeutic antifactor Xa (anti-Xa) levels and increased bleeding when standard treatment doses are used in patients with morbid obesity. Objective: To evaluate the dose of enoxaparin needed to achieve therapeutic anti-Xa levels in a prospective, masked observational cohort of heterogeneous inpatients with morbid obesity and to determine whether patients with morbid obesity treated with 1 mg/kg of enoxaparin are at increased risk of supratherapeutic levels and bleeding events compared to patients receiving lower doses. Methods: Hospitalized patients with a body mass index ≥40 kg/m2 or actual body weight ≥140 kg and prescribed treatment doses of enoxaparin >60 mg per day were enrolled and consented to phlebotomy for determination of anti-Xa levels. Results: Forty-one patients were included for data analysis. The dose of enoxaparin that resulted in therapeutic and supratherapeutic anti-Xa levels at steady state was 0.83 mg/kg and 0.98 mg/kg (−0.11; 95% confidence interval [CI] −0.20 to −0.01, P = .02), respectively. Enoxaparin dose as mg/kg of actual body weight was an independent predictor of having a supratherapeutic anti-Xa level. Patients with doses Conclusion: Patients with morbid obesity required less than the recommended 1 mg/kg enoxaparin dose to achieve therapeutic peak anti-Xa levels; therefore, initiation with lower dosages is prudent and anti-Xa monitoring should guide dosage adjustments.

Objectives: Thromboelastography Platelet Mapping (TEGPM) is an assay designed to detect platelet inhibition due to aspirin or clopidogrel-like drugs. The purpose of this study was to evaluate potential causes of error in the design or operation of the assay. Methods: We evaluated percent inhibition of platelets due to aspirin or clopidogrel using TEGPM, which measures clot viscoelastic maximum amplitude (MA) after activation with adenosine diphosphate (ADP) or arachidonic acid (AA) and subtraction of MA due to fibrin (MAFibrin). Results: MAFibrin measured in heparinized blood showed an unstable increasing pattern in 28% of samples (16 of 58). The platelet aggregation inhibitor eptifibatide corrected increasing MAFibrin in 14 of 16 cases, while the thrombin inhibitor argatroban corrected increasing MAFibrin in six of 16 cases, suggesting that unanticipated platelet activation/ aggregation was a more important cause of unstable rising MAFibrin than uninhibited thrombin. The unstable increased MAFibrin falsely increased percent ADP inhibition on average from 19% to 38% and percent AA inhibition from 29% to 58%. Heparinized samples showed platelet clumping and had procoagulant platelet microvesicle levels double those in citrate anticoagulant. Conclusions: Unanticipated platelet activation/aggregation occurring in the heparinized TEGPM samples lead to erroneous percent inhibition results.

This study provides the first estimates of microparticle numbers in platelet-rich plasma (PRP) from normal individuals, closer to in-vivo levels, using higher-resolution flow cytometry. We measured platelet (CD41+) and annexin V+ microparticles in fresh and frozen aliquots of PRP, platelet-poor plasma, platelet-free plasma (PFP), and microparticles isolated by high-speed centrifugation. PRP from healthy individuals contained 730,000/μl total microparticles based on light-scattering measurements. A median of 27,000/μl microparticles in PRP were of platelet origin and 120,000/μl annexin V+, and of these, 24,000/μl were dual-positive procoagulant platelet microparticles. Double centrifugation of PRP removed 99% of platelets, but also 80% of annexin V+ CD41+, 93% of annexin V+ CD41-, and 58% of annexin V- CD41+ microparticles. Loss of microparticles with centrifugation varied from individual to individual. Microparticle counts after isolation by centrifugation and double washing were not significantly different than counts in the original PFP sample, but lower than in PRP. Freeze-thawing of PFP had no effect on platelet microparticle counts, but slightly increased annexin V+, CD41- counts. Freeze-thawing of isolated washed microparticles resulted in a 30-50% increase in annexin V+ microparticles. PRP contains large numbers of cellular microparticles, including platelet and annexin V+ microparticles, which are lost to varying degrees when PRP is double centrifuged to remove platelets. Microparticles remaining in PFP can be recovered by high-speed centrifugation without loss compared to the original PFP sample. Freeze-thawing has variable effects on microparticle counts depending on the sample preparation used.

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.

Cardiopulmonary bypass (CPB) is a unique clinical scenario that results in widespread activation of the hemostatic system. However, surgery also results in normal increases in coagulation activation, platelet activation, and fibrinolysis that are associated with normal wound hemostasis. Conventional CPB interferes with normal hemostasis by diluting hemostatic cells and proteins, through reinfusion of shed blood, and through activation on the bypass circuit surface of multiple systems including platelets, the kallikrein-kinin system, and fibrinolysis. CPB activation of the kallikrein-kinin system increases activated factor XIIa, kallikrein, bradykinin, and tissue plasminogen activator levels, but has little effect on thrombin generation. Increased tissue plasminogen activator and circulating fibrin result in increased plasmin generation, which removes hemostatic fibrin. The nonendothelial surface of the bypass circuit, along with circulating thrombin and plasmin, lead to platelet activation, platelet receptor loss, and reduced platelet response to wounds. In this review, we highlight the major mechanisms responsible for CPB-induced activation of the hemostatic system and examine some of the markers described in the literature. Additionally, strategies used to reduce this activation are discussed, including limiting cardiotomy suction, increasing circuit biocompatibility, antithrombin supplementation, and antifibrinolytic use. Determining which patients will most benefit from specific therapies will ultimately require investigation into genetic phenotypes of coagulation protein expression. Until that time, however, a combination of approaches to reduce the hemostatic activation from CPB seems warranted.

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%.

Summary. Background: Microparticle size measurements are often calibrated on flow cytometers using polystyrene microspheres that forward scatter more light vs. particle diameter than cellular microparticles. Methods: We compared theoretical with measured forward angle light scattering on the LSRII, FC500 and Apogee A40 using polystyrene and silica microspheres vs. synthetic lipid vesicles and platelets, then compared plasma microparticle counts using different calibration strategies. Results: Polystyrene and silica microspheres with higher refractive indices forward scattered more light with a wavelength of 488 nm for a given size microparticle than did lipid vesicles or platelets. The LSRII and FC500 did not count many, and were unable to separate by size, polystyrene microspheres

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.

Objective: Heparin and other oxygenator coatings have been used in attempts to reduce hemostatic activation during cardiopulmonary bypass (CPB). This study evaluated whether an oxygenator coated with poly 2-methoxyethylacrylate (PMEA) (X-coating; Terumo Corporation, Tokyo, Japan) would cause less activation of coagulation and fibrinolytic systems during CPB in children than a noncoated oxygenator. Design: Observational study. Setting: University-affiliated children’s hospital. Patients: Twenty-six patients, 3 months to 5 years old, who underwent congenital heart surgery for repair of a ventricular septal defect, atrial septal defect, or both. Interventions: Patients were divided into 2 age-matched groups based on the type of oxygenator used: a noncoated oxygenator (group NC) versus a PMEA-coated oxygenator (group C). Measurements and Main Results: Blood samples for coagulation and fibrinolytic markers were compared before, during, and after CPB. Despite increases in thrombin generation markers (F1.2 and TAT) at certain times during CPB in group C compared to group NC, a comparison over all times during CPB were not statistically different between groups. Overall D-dimer concentrations during CPB were elevated in group C compared to group NC ( p = 0.02). Active tPA and active PAI-1 were not different between groups during or after CPB. Group C had higher platelet counts (181,000 ± 29,000) during CPB than group NC (155,000 ± 57,000, p = 0.04) but not postoperatively. Twelve hours postoperatively, chest tube outputs were 8.8 ± 3 mL/kg in group C and 19.1 ± 12 mL/kg in group NC ( p = 0.003). The corresponding outputs 24 hours after surgery were 12.4 ± 3 mL/kg and 24 ± 11 mL/kg, respectively ( p = 0.005). Conclusions: Except for a somewhat higher platelet count during CPB, there was no indication that PMEA coating resulted in less activation of coagulation and fibrinolytic systems. The lower postoperative chest tube output observed after CPB with PMEA-coated oxygenators needs to be studied further.

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.

Microvesicles are fragments of cells released when the cells are activated, injured, or apoptotic. Analysis of microvesicle levels in blood has the potential to shed new light on the pathophysiology of many diseases. Flow cytometry is currently the only method that can simultaneously separate true lipid microvesicles from other microparticles in blood, determine the cell of origin and other microvesicle characteristics, and handle large numbers of clinical samples with a reasonable effort, but expanded use of flow cytometric measurement of microvesicle levels as a clinical and research tool requires improved, standardized assays. The goal of this review is to aid investigators in applying current best practices to microvesicle measurements. First pre-analytical factors are evaluated and data summarized for anticoagulant effects, sample transport and centrifugation. Next flow cytometer optimization is reviewed including interference from background in buffers and reagents, accurate microvesicle counting, swarm interference, and other types of coincidence errors, size calibration, and detection limits using light scattering, impedance and fluorescence. Finally current progress on method standardization is discussed and a summary of current best practices provided. © 2016 Clinical Cytometry Society.

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.

The purpose of this study was to determine quantitatively the effects of consumption, hemodilution, and blood loss on coagulation and fibrinolytic factor levels during cardiopulmonary bypass.A combination of measured levels of prothrombin, antithrombin, fibrinogen, plasminogen, and antiplasmin along with their activation markers F1.2, thrombin-antithrombin complex, fibrinopeptide A, plasmin-antiplasmin complex, and D-dimer were used with a computer model of each patient's vascular and hemostatic systems to estimate the cardiopulmonary bypass-associated loss of each factor because of hemodilution, blood loss, and consumption.University hospital.Nine patients undergoing coronary artery bypass graft surgery.None.At baseline, it was estimated that on average 2%, 3%, and 25%, respectively, of the baseline liver secretion of plasminogen, prothrombin,and fibrinogen were consumed by activation of these proteins. During cardiopulmonary bypass, thrombin and plasmin generation were increased, whereas fibrin generation was decreased because of heparin. Compared with baseline, hemodilution during cardiopulmonary bypass resulted in an average 35% +/- 7% decrease in the concentration of coagulation and fibrinolytic proteins, whereas blood loss was responsible for an average 6% +/- 5% decrease in these proteins. Blood loss varied substantially among patients, resulting in1% to 14% decreases in hemostatic protein levels. On average, consumption because of activation resulted in less than a 1% drop in the concentration of coagulation and fibrinolytic factors during cardiopulmonary bypass.Hemodilution is the primary cause of the drop in coagulation and fibrinolytic proteins during routine cardiopulmonary bypass, followed by blood loss, whereas consumption accounts for less than a 1% drop in most patients.