Accuracy and consistency of anti-Xa activity measurement for determination of rivaroxaban plasma levels.

Essentials Accurate determination of anticoagulant plasma concentration is important in clinical practice. We studied the accuracy and consistency of anti-Xa assays for rivaroxaban in a multicentre study. In a range between 50 and 200 µg L-1 , anti-Xa activity correlated well with plasma concentrations. The clinical value might be limited by overestimation and intra- and inter-individual variation. SUMMARY: Background Determining the plasma level of direct oral anticoagulants reliably is important in the work-up of complex clinical situations. Objectives To study the accuracy and consistency of anti-Xa assays for rivaroxaban plasma concentration in a prospective, multicenter evaluation study employing different reagents and analytical platforms. Methods Rivaroxaban 20 mg was administered once daily to 20 healthy volunteers and blood samples were taken at peak and trough levels (clinicaltrials.gov NCT01710267). Anti-Xa activity was determined in 10 major laboratories using different reagents and analyzers; corresponding rivaroxaban plasma concentrations were measured by high-performance liquid chromatography-tandem mass spectrometry (HPLC-MS). Findings Overall Pearson's correlation coefficient of anti-Xa levels and HPLC-MS results was 0.99 for Biophen® Heparin (95% CI, 0.99, 0.99), Biophen® DiXaI (95% CI, 0.99, 0.99) and STA® anti-Xa liquid (95% CI, 0.99, 1.00). Correlation was lower in rivaroxaban concentrations below 50 µg L-1 and above 200 µg L-1 . The overall bias of the Bland-Altman difference plot was 14.7 µg L-1 for Biophen Heparin, 17.9 µg L-1 for Biophen DiXal and 19.0 µg L-1 for STA anti-Xa liquid. Agreement between laboratories was high at peak level but limited at trough level. Conclusions Anti-Xa activity correlated well with rivaroxaban plasma concentrations, especially in a range between 50 and 200 µg L-1 . However, anti-Xa assays systematically overestimated rivaroxaban concentration as compared with HPLC-MS, particularly at higher concentrations. This overestimation, coupled with an apparent interindividual variation, might affect the interpretation of results in some situations.

Impact of a product-specific reference standard for the measurement of a PEGylated rFVIII activity: the Swiss Multicentre Field Study.

INTRODUCTION: Measuring factor VIII (FVIII) activity can be challenging when it has been modified, such as when FVIII is pegylated to increase its circulating half-life. Use of a product-specific reference standard may help avoid this issue. AIM: Evaluate the impact of using a product-specific reference standard for measuring the FVIII activity of BAX 855 - a pegylated FVIII - in eight of Switzerland's main laboratories. METHODS: Factor VIII-deficient plasma, spiked with five different concentrations of BAX 855, plus a control FVIII sample, was sent to the participating laboratories. They measured FVIII activity by using either with a one-stage (OSA) or the chromogenic assay (CA) against their local or a product-specific reference standard. RESULTS: When using a local reference standard, there was an overestimation of BAX 855 activity compared to the target concentrations, both with the OSA and CA. The use of a product-specific reference standard reduced this effect: mean recovery ranged from 127.7% to 213.5% using the OSA with local reference standards, compared to 110% to 183.8% with a product-specific reference standard, and from 146.3% to 182.4% using the CA with local reference standards compared to 72.7% to 103.7% with a product-specific reference standard. CONCLUSION: In this in vitro study, the type of reference standard had a major impact on the measurement of BAX 855 activity. Evaluation was more accurate and precise when using a product-specific reference standard.

Impact of electronic reminders on venous thromboprophylaxis after admissions and transfers.

OBJECTIVE: Clinical decision support has the potential to improve prevention of venous thromboembolism (VTE). The purpose of this prospective study was to analyze the effect of electronic reminders on thromboprophylaxis rates in wards to which patients were admitted and transferred. The latter was of particular interest since patient handoffs are considered to be critical safety issues. METHODS: The trial involved two study periods in the six departments of a university hospital, three of which were randomly assigned to the intervention group displaying reminders during the second period. At 6 h after admission or transfer, the algorithm checked for prophylaxis orders within 0-30 h of the patient's arrival, increasing the specificity of the displayed reminders. RESULTS: The significant impact of the reminders could be seen by prophylaxis orders placed 6-24 h after admission (increasing from 8.6% (223/2579) to 12% (307/2555); p<0.0001) and transfer (increasing from 2.4% (39/1616) to 3.7% (63/1682); p=0.034). In admission wards, the rate of thromboprophylaxis increased from 62.4% to 67.7% (p<0.0001), and in transfer wards it increased from 80.2% to 84.3% (p=0.0022). Overall, the rate of prophylaxis significantly increased in the intervention group from 69.2% to 74.3% (p<0.0001). No significant changes were observed in the control group. Postponing prophylaxis checks to 6 h after admissions and transfers reduced the number of reminders by 62% and thereby minimized the risk of alert fatigue. CONCLUSIONS: The reminders improved awareness of VTE prevention in both admission and transfer wards. This approach may contribute to better quality of care and safer patient handoffs.

Heparin-induced thrombocytopenia associated with thrombotic microangiopathy.

Some cases of thrombotic microangiopathy (TMA) are refractory to plasma exchange therapy (PE) with persistence or recurrence of thrombocytopenia. We report two patients suffering from TMA of different aetiologies (associated with disseminated malignancy, typical haemolytic uraemic syndrome) with recurrent or persistent thrombocytopenia despite adequate therapy including PE. Since both patients were exposed to unfractionated heparin, heparin-induced thrombocytopenia (HIT) was suspected as a cause. Pretest probabilities for HIT were intermediate. ELISA for PF4/heparin antibodies was strongly positive in both cases, and HIT was confirmed by heparin-induced platelet activation assay. Anticoagulation with lepirudin was initiated, with subsequent rapid increase of the platelet count. TMA might represent a predisposition for HIT. This could be due to TMA-related platelet activation with increased PF4 release. In TMA patients exposed to heparin and with refractory or rapidly recurrent thrombocytopenia HIT should always be considered as a possible cause.

Acquired thrombotic thrombocytopenic purpura. Development of an autoimmune response.

The von Willebrand factor (VWF)-cleaving metalloprotease, ADAMTS13 (adisintegrin and metalloprotease with thrombospondin type 1 motifs-13) is the only known target of the dysregulated immune response in acquired TTP. Autoantibodies to ADAMTS13 either neutralize its activity or accelerate its clearance, thereby causing a severe deficiency of ADAMTS13 in plasma. As a consequence, size regulation of VWF is impaired and the persistence of ultra-large VWF (ULVWF) multimers facilitates microvascular platelet aggregation causing microangiopathic haemolytic anaemia and ischaemic organ damage. Autoimmune TTP although a rare disease with an annual incidence of 1.72 cases has a mortality rate of 20% even with adequate therapy. We describe the mechanisms involved in ADAMTS13 autoimmunity with a focus on the role of B- and T-cells in the pathogenesis of this disorder. We discuss the potential translation of recent experimental findings into future therapeutic concepts for the treatment of acquired TTP.

Outcome of influenza infections in outpatients after allogeneic hematopoietic stem cell transplantation.

BACKGROUND: Influenza can cause significant morbidity and mortality in patients after hematopoietic stem cell transplantation (HSCT). The diagnostic methods and antiviral treatment have scarcely been investigated. METHODS: We retrospectively identified influenza-infected patients with upper or lower respiratory tract infection (RTI) diagnosed by culture and polymerase chain reaction (PCR) testing between November 2007 and April 2008. Treatment with oseltamivir 75 mg twice daily and serial nasal swabs were performed at the discretion of the treating physician. RESULTS: We identified 21 influenza infections in 19 patients: 19 with upper RTI and 2 with lower RTI. At diagnosis, all 21 samples were positive for PCR with a median influenza load of 5.9 log(10) copies/mL. Culture was positive in 14 (67%) patients. Influenza A virus was diagnosed in 8 (38%) episodes and influenza B virus in 13 (62%) episodes. Two patients were sequentially infected by influenza A, followed by B after 38 and 47 days, respectively. Eighteen (86%) patients were treated with oseltamivir for 11 days (median, interquartile range [IQR]: 8-14). No progression to lower RTI or mortality occurred. Shedding persisted for 12 days (median, IQR: 8-13). Absolute lymphocyte count at diagnosis correlated inversely with shedding of the virus (P<0.001). CONCLUSIONS: Oseltamivir is well tolerated and may reduce mortality of influenza virus-infected patients after HSCT. PCR may help to optimize diagnosis and to monitor treatment strategies.

[Thrombotic microangiopathies]. / Thrombotische Mikroangiopathien.

Thrombotic microangiopathies are disorders of microvascular occlusion and are characterized by thrombocytopenia and microangiopathic haemolytic anaemia. Varying additional signs and symptoms of organ ischaemia may be present. Thrombotic microangiopathies are pathophysiologically heterogenous disorders and include primarily thrombotic thrombocytopenic purpura and haemolytic uraemic syndrome. Besides this, they may occur in association with certain diseases or drugs, or after allogeneic haematopoietic stem cell transplantation.

Measurement of von Willebrand factor cleaving protease (ADAMTS-13): results of an international collaborative study involving 11 methods testing the same set of coded plasmas.

BACKGROUND: ADAMTS-13 is a von Willebrand factor (VFW)-cleaving protease. Its congenital or acquired deficiency is associated with thrombotic thrombocytopenic purpura (TTP) and more rarely with the hemolytic uremic syndrome. We report on a survey evaluating 11 methods for ADAMTS-13 measurement performed in different labs. DESIGN: Two plasmas, one normal and one from a patient with familial TTP, were mixed at the co-ordinating center to prepare 6 plasmas with 0%, 10%, 20%, 40%, 80% and 100% ADAMTS-13 levels. Each plasma was aliquoted and assembled into sets of 60 (coded from 1 to 60), each containing 10 copies of the original 6 plasmas. Plasmas were frozen and shipped in dry ice to 10 labs with a common frozen reference plasma. Laboratories were asked to measure ADAMTS-13 with their methods. Results were sent to the coordinating center for statistical analysis. RESULTS: Of the 10 methods performed under static conditions 9 were quantitative and one was semiquantitative. One method performed under flow conditions evaluated the extent of cleavage of endothelial cell-derived ultralarge VWF string-like structures and expressed results as deficient, normal, or borderline. Linearity (expected-vs-observed levels), assessed as the squared correlation coefficient, ranged from 0.98 to 0.39. Reproducibility, expressed as the coefficient of variation for repeated measurements, ranged from < 10% to 83%. The majority of methods were able to discriminate between different ADAMTS-13 levels. The majority were able to detect the plasma with 0% level and some of them to discriminate between 0% and 10%. Overall the best performance was observed for three methods measuring cleaved VWF by ristocetin cofactor, collagen binding, and immunoblotting of degraded multimers of VWF substrate, respectively. The poor interlaboratory agreement of results was hardly affected by the use of the common standard. The method performed under flow conditions identified the plasmas with 0%, 10%, 20% and 40% activity as deficient in 7, 5, 1 and 3 of the 10 replicate measurements. The plasmas with 80% and 100% were identified as normal in all of the 10 replicate measurements. CONCLUSIONS: The survey shows varied performance, but supports an optimistic view about the reliability of current methods for ADAMTS-13.

Measurement of von Willebrand factor-cleaving protease (ADAMTS-13) activity in plasma: a multicenter comparison of different assay methods.

A severely deficient ADAMTS-13 activity (<5%) is a key laboratory finding confirming the diagnosis of thrombotic thrombocytopenic purpura (TTP), whereas a mildly or moderately decreased activity is found in various other conditions. Laboratory tests for ADAMTS-13 activity must reliably identify a severe deficiency and detect inhibitory antibodies against ADAMTS-13. We carried out a multicenter comparison of different assays for ADAMTS-13 activity in plasma, including the quantitative immunoblotting of degraded von Willebrand factor (VWF) substrate, the residual collagen binding activity and ristocetin cofactor activity of degraded VWF, and an immunoradiometric assay. The main goal was to investigate whether all assays concordantly identified severe ADAMTS-13 deficiency and detected inhibitory antibodies. ADAMTS-13 activity was determined by five laboratories in 30 plasma samples of patients with hereditary and acquired TTP and other conditions. ADAMTS-13 activity values of the samples ranged from <3% to > 100%. Concerning the identification of a severe ADAMTS-13 deficiency, good interassay and interlaboratory agreement was observed with only one false-negative and two false-positive results by two laboratories using a collagen binding assay. For samples with normal or mildly to moderately reduced ADAMTS-13 activity, results were less concordant. There was good agreement for the detection of strong inhibitors. We conclude that all assays investigated are useful as a screening test in suspected TTP. Further assay improvement is needed, however.

Schistocytic anaemia, severe thrombocytopenia, and renal dysfunction: thrombotic microangiopathy due to severe acquired ADAMTS-13 deficiency. Case 2.

We present the case of a woman (age: 64 years) with acute thrombotic microangiopathy due to severe acquired ADAMTS-13 (von Willebrand factor-cleaving protease) deficiency. She was successfully treated with plasma exchange therapy and glucocorticosteroids. She relapsed seven months later, and splenectomy led to lasting remission. Pathomechanisms of thrombotic thrombocytopenic purpura, especially the role of ADAMTS-13, are discussed and therapeutic measures outlined.

Von Willebrand factor-cleaving protease (ADAMTS-13) activity in thrombotic microangiopathies: diagnostic experience 2001/2002 of a single research laboratory.

BACKGROUND: Severe deficiency of von Willebrand factor-cleaving protease (ADAMTS-13) activity (<5% of normal) is specific for classical thrombotic thrombocytopenic purpura (TTP), a disorder presenting with thrombocytopenia, microangiopathic haemolytic anaemia and often with organ dysfunction such as neurological symptoms, renal failure, and fever. A certain, though according to several case series, variable percentage of patients with clinically diagnosed TTP and most patients with other forms of thrombotic icroangiopathies (TMA) do not show severe ADAMTS-13 deficiency. METHODS: We determined ADAMTS-13 activity in 508 plasma samples of 309 patients referred to our laboratory in 2001 and 2002. Plasma samples with ADAMTS-13 activity <5% were additionally tested for the presence of inhibitory antibodies. Patients were assigned to ten predefined clinical categories according to information provided in the referral letter (TMA not specified; TMA associated with neoplasia or chemotherapy; TMA following haematopoietic stem cell transplantation; TMA with additional disorder; idiopathic TTP; haemolytic-uraemic syndrome (HUS) not specified; HUS with diarrhoea prodrome; atypical HUS; other haematological disorder; no clinical information available). RESULTS: We detected 50 (16%) patients with severe ADAMTS-13 deficiency. Forty-four (88%) of these patients had been classified as idiopathic TTP, 2 as neoplasia- or chemotherapy-associated, and 4 as non-specified TMA. Among the patients labelled as acute idiopathic TTP, the prevalence of severe ADAMTS-13 deficiency was 63% (44/70). Inhibitory antibodies were found in 31 (62%) patients with ADAMTS-13 activity <5%. Of the 44 patients with acute idiopathic TTP, at initial presentation or at relapse, with ADAMTS-13 activity <5%, 11 were identified to have (probable) constitutional severe ADAMTS-13 deficiency. CONCLUSION: Severe ADAMTS-13 deficiency is found in about 60% of patients diagnosed with idiopathic TTP but in none of 111 diagnosed with HUS. Plasma ADAMTS-13 activity <5%, however, does not identify all patients clinically diagnosed with TTP. Detection of inhibitory antibodies against ADAMTS-13 helps to differentiate between acquired and constitutional forms of TTP, which may be important for treatment strategies.

Quantification and facilitated comparison of von Willebrand factor multimer patterns by densitometry.

The analysis of von Willebrand factor (vWF) multimers is an important laboratory tool for distinguishing among the numerous subtypes of von Willebrand disease (vWD). Comparability and reproducibility of this method are insufficient; standardization and external references are pending. Interlaboratory comparison of results therefore may be difficult. We applied densitometry to obtain a reproducible quantification of vWF multimer patterns in healthy donors, patients with vWD variants, and factor VIII/vWF concentrates to improve the reproducibility and comparability of vWF multimer analysis. Multimers were separated and visualized luminographically on x-ray films. Films were scanned and evaluated by densitometry. The variation inherent in vWF multimer analysis and the range of the normal could be quantified. In vWD variants and factor VIII/vWF concentrates, densitometry could quantify and visualize alterations of vWF multimer patterns and facilitate their comparison. Densitometry permits a precise quantitative comparison of sample patterns to a reference plasma. It could be a valuable tool offering reproducible quantification and additional visualization of normal and pathologic vWF multimer patterns, facilitating their comparison and contributing to a standardization of vWF multimer analysis.