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.

Protein modelling to understand FGB mutations leading to congenital hypofibrinogenaemia.

INTRODUCTION: Congenital hypofibrinogenaemia is a quantitative fibrinogen disorder characterized by proportionally decreased levels of functional and antigenic fibrinogen. Mutations accounting for quantitative fibrinogen disorders are relatively frequent in the conserved COOH-terminal globular domains of the γ and Bß chains. The latter mutations are of particular interest since the Bß-chain is considered the rate-limiting chain in the hepatic production of the fibrinogen hexamer. AIM: The aim of this study was to study the molecular pattern of four patients with congenital hypofibrinogenaemia. METHODS: Four novel fibrinogen Bß-chain mutations leading to congenital hypofibrinogenaemia were identified in four women with heterogeneous symptoms. The human fibrinogen beta chain precursor protein sequence (P02675) was obtained from the UniProt database. The resulting models were analysed using swisspdbviewer 4.1.0. RESULTS: Three patients were heterozygous for different missense mutations located in the highly conserved ß nodule: c.882G>C:Arg294Ser (Arg264Ser), c.1298G>T:Trp433Leu (Trp403Leu) and c.1329C>G:Asn443Lys (Asn413Lys). Modelling analyses predicted major structural modifications likely to result in impaired fibrinogen secretion. One patient was heterozygous for an intron 7 donor splice mutation (c.1244 + 1G>A), leading to the complete abolishment of the donor site. CONCLUSIONS: Protein modelling of new causative mutations and comparison of molecular, biochemical and clinical data continue to yield valuable information on the development and course of fibrinogen disorders as well as on the choice of the most appropriate treatments.

[Myelodysplastic syndromes and autoimmunity]. / Syndromes myélodysplasiques et auto-immunité.

Myelodysplastic syndromes (MDS) are characterized by inefficient hematopoiesis and result in peripheral cytopenia. This heterogenous disease group arises from clonal disorders of hematopoietic stem cells. Several cohort studies and numerous case reports have highlighted a link between MDS and autoimmune disorders. Patients with MDS are more prone to develop particular systemic inflammatory diseases. On the other hand, patients suffering from autoimmune diseases are at higher risk to develop MDS. The scope of this article is to review the association between myelodysplasia and autoimmunity. It gives practical clues when to look for MDS in a patient suffering from autoimmunity and lists the main autoimmune diseases which may complicate the course of a MDS.

Bilateral periorbital ecchymoses. An often missed sign of amyloid purpura.

Immunoglobulin light chain (AL) amyloidosis is a systemic disease caused by a plasma cell clone synthesizing an unstable light chain, which forms amyloid fibrils. Deposition of amyloid fibrils affects primarily kidney, heart, nervous system, spleen, liver, gastrointestinal tract and the skin. Skin bleeding in these patients is called amyloid purpura. Classically, it occurs spontaneously and bilaterally in the periorbital region. Vessel wall fragility and damage by amyloid are the principal causes of periorbital and gastrointestinal bleeding. Additionally, coagulation factor inhibitory circulating paraprotein, hyperfibrinolysis, platelet dysfunction or isolated acquired factor X deficiency may contribute to even more severe, diffuse bleedings. Early diagnosis remains essential for improving prognosis of patients with AL amyloidosis. Although pictures of amyloid purpura have been often reported in the literature, the clinical diagnosis may be delayed. We report a case of cutaneous manifestation of AL amyloidosis diagnosed not until one year after the appearance of the first symptoms. Diagnostic work-up revealed that the patient suffered from multiple myeloma with secondary AL amyloidosis. Atraumatic ecchymoses at the face, particularly the eyelids as well as in the neck should raise the suspicion of AL amyloidosis.

My patient is thrombocytopenic! Is (s)he? Why? And what shall I do? A practical approach to thrombocytopenia.

Solving the riddle of a thrombocytopenic patient is a difficult and fascinating task. The spectrum of possible aetiologies is wide, ranging from an in vitro artefact to severe treatment-resistant thrombocytopenic bleeding conditions, or even life-threatening prothrombotic states. Moreover, thrombocytopenia by itself does not protect from thrombosis and sometimes a patient with a low platelet count requires concomitant antithrombotic treatment as well. In order to identify and treat the cause and the effects of the thrombocytopenia, you have to put together several pieces of information, solving a unique jig-jaw puzzle. The present work is not a textbook article about thrombocytopenia, rather a collection of differential diagnostic thoughts, treatment concepts, and some basic knowledge, that you can retrieve when facing your next thrombocytopenic patient. Enjoy reading it, but most importantly enjoy taking care of patients with a low platelet count. I bet the present work will assist you in this challenging and rewarding clinical task.

Rivaroxaban: Quantification by anti-FXa assay and influence on coagulation tests: a study in 9 Swiss laboratories.

INTRODUCTION: Rivaroxaban (RXA) is licensed for prophylaxis of venous thromboembolism after major orthopaedic surgery of the lower limbs. Currently, no test to quantify RXA in plasma has been validated in an inter-laboratory setting. Our study had three aims: to assess i) the feasibility of RXA quantification with a commercial anti-FXa assay, ii) its accuracy and precision in an inter-laboratory setting, and iii) the influence of 10mg of RXA on routine coagulation tests. METHODS: The same chromogenic anti-FXa assay (Hyphen BioMed) was used in all participating laboratories. RXA calibrators and sets of blinded probes (aim ii.) were prepared in vitro by spiking normal plasma. The precise RXA content was assessed by high-pressure liquid chromatography-tandem mass spectrometry. For ex-vivo studies (aim iii), plasma samples from 20 healthy volunteers taken before and 2 - 3hours after ingestion of 10mg of RXA were analyzed by participating laboratories. RESULTS: RXA can be assayed chromogenically. Among the participating laboratories, the mean accuracy and the mean coefficient of variation for precision of RXA quantification were 7.0% and 8.8%, respectively. Mean RXA concentration was 114±43µg/L .RXA significantly altered prothrombin time, activated partial thromboplastin time, factor analysis for intrinsic and extrinsic factors. Determinations of thrombin time, fibrinogen, FXIII and D-Dimer levels were not affected. CONCLUSIONS: RXA plasma levels can be quantified accurately and precisely by a chromogenic anti-FXa assay on different coagulometers in different laboratories. Ingestion of 10mg RXA results in significant alterations of both PT- and aPTT-based coagulation assays.

Laboratory-based ROTEM(®) analysis: implementing pneumatic tube transport and real-time graphic transmission.

INTRODUCTION: ROTEM(®) is considered a helpful point-of-care device to monitor blood coagulation. Centrally performed analysis is desirable but rapid transport of blood samples and real-time transmission of graphic results are an important prerequisite. The effect of sample transport through a pneumatic tube system on ROTEM(®) results is unknown. The aims of the present work were (i) to determine the influence of blood sample transport through a pneumatic tube system on ROTEM(®) parameters compared to manual transportation, and (ii) to verify whether graphic results can be transmitted on line via virtual network computing using local area network to the physician in charge of the patient. METHODS: Single centre study with 30 normal volunteers. Two whole blood samples were transferred to the central haematology laboratory by either normal transport or pneumatic delivery. EXTEM, INTEM, FIBTEM and APTEM were analysed in parallel with two ROTEM(®) devices and compared. Connection between central laboratory, emergency and operating rooms was established using local area network. RESULTS: All collected ROTEM(®) parameters were within normal limits. No statistically significant differences between normal transport and pneumatic delivery were observed. Real-time transmission of the original ROTEM(®) curves using local area network is feasible and easy to establish. CONCLUSION: At our institution, transport of blood samples by pneumatic delivery does not influence ROTEM(®) parameters. Blood samples can be analysed centrally, and results transmitted live via virtual network computing to emergency or operating rooms. Prior to analyse blood samples centrally, the type of sample transport should be tested to exclude in vitro blood activation by local pneumatic transport system.

Screening for lupus anticoagulant: improving the performance of the lupus-sensitive PTT-LA.

INTRODUCTION: The aim of the present work was to verify whether calculating a ratio between clotting times obtained with the sensitive PTT-LA and a less sensitive activated partial thromboplastin time (aPTT)-reagent may represent a valuable aPTT-based screening strategy for lupus anticoagulants (LA). METHODS: For the pilot study, plasma samples from normal subjects (n = 15) and from patients with LA (n = 10), therapeutic anticoagulation with vitamin K-antagonists (VKA) (n = 15) or unfractionated heparin (n = 15), coagulation factors deficiency (n = 16), and inhibitory antibodies against factor VIII or IX (n = 11) were studied. For the evaluation study, 1553 consecutive plasma samples from nonanticoagulated patients investigated for LA between January 2005 and December 2007 at our institution were studied. Following screening strategies were employed: Pathromtin-SL (aPTT-SL), PTT-LA (aPTT-LA), ratio aPTT-LA/aPTT-SL (aPTT-ratio), and Russell's viper venom (RVV) based LA-Check. LA positive samples were identified by mixing studies and diluted RVV confirmation test (LA-Check/LA-Sure). RESULTS: Pilot study: All screening strategies had a 100% sensitivity, and the aPTT-ratio reached the highest specificity (82%; 95%CI: 74-90%). Within the evaluation study, following sensitivities for LA screening were observed: aPTT-SL 59.0% (95%CI: 57-61%), aPTT-LA 82.1% (95%CI: 80-84%), aPTT-ratio 92.3% (95%CI: 91-94), and LA-Check 83.3% (95%CI: 82-85%). CONCLUSION: Calculating a ratio between the LA-sensitive PTT-LA and the less sensitive Pathromtin-SL improves the performance of the PTT-LA itself and represents a simple and sensitive aPTT-based integrated strategy for LA screening.

Variability of anti-PF4/heparin antibody results obtained by the rapid testing system ID-H/PF4-PaGIA.

BACKGROUND: Recent studies have shown that a low clinical pretest probability may be adequate for excluding heparin-induced thrombocytopenia. However, for patients with intermediate or high pretest probability, laboratory testing is essential for confirming or refuting the diagnosis. Rapid assessment of anti-PF4/heparin-antibodies may assist clinical decision-making. OBJECTIVES: To evaluate the performance of rapid ID-H/PF4-PaGIA. In particular, we verified reproducibility of results between plasma and serum specimens, between fresh and frozen samples, and between different ID-H/PF4-polymer lots (polystyrene beads coated with heparin/PF4-complexes). PATIENTS/METHODS: The samples studied were 1376 plasma and 914 corresponding serum samples from patients investigated for suspected heparin-induced thrombocytopenia between January 2000 and October 2008. Anti-PF4/heparin-antibodies were assessed by ID-H/PF4-PaGIA, commercially available ELISAs and heparin-induced platelet aggregation test. RESULTS: Among 914 paired plasma/serum samples we noted discordant results (negative vs. low-titre positive) in nine instances (1%; 95%CI, 0.4-1.6%). Overall, agreement between titres assessed in plasma vs. serum was highly significant (Spearman correlation coefficient, 0.975; P < 0.0001). Forty-seven samples tested both fresh and after freezing/thawing showed a good agreement, with one discordant positive/negative result (Spearman correlation coefficient, 0.970; P < 0.0001). Among 1376 plasma samples we noted a strikingly variable incidence of false negative results (none - 82%; 95%CI, 66-98%), depending on the employed ID-H/PF4-polymer lot. Faulty lots can be recognized by titrating commercial positive controls and stored samples of HIT-patients. CONCLUSION: Laboratories performing the assay should implement stringent internal quality controls in order to recognize potentially faulty ID-H/PF4-polymer lots, thus avoiding false negative results.

"HIT on Trousseau" double trouble: acquired coagulopathy with femoral artery thrombosis.

Trousseau Syndrome is a paraneoplastic procoagulant phenomenon. Heparin-induced thrombocytopenia (HIT) is a rare complication of anticoagulation with heparin. To our knowledge, the coincidence of the two has not been reported so far. We report a case of an acute thrombosis of the left femoral artery and distal leg arteries in a patient with an otherwise normal cardiovascular status. Endovascular revascularization attempts using mechanical rotational thrombectomy catheter, aspiration and local thrombolysis were unsuccessful. Progressive coagulation along the intra-arterial catheter was seen. Surgical thrombectomy of the femoral-pedal axis was successful, but the patient developed an immune-mediated HIT postoperatively. An adenocarcinoma of the colon was the likely cause for the initial arterial thrombosis, and probably adversely affected endovascular revascularization attempts. Subsequent HIT with microvascular thrombosis worsened ischemic damage leading to a below knee-amputation, despite patent large vessels. Compared to venous thrombosis, arterial thrombosis is a rare manifestation of Trousseau syndrome. The coincidence of it with HIT is even rarer. There may be a causal relationship between the two.

Thrombotic thrombocytopenic purpura.

This overview summarizes the history of thrombotic thrombocytopenic purpura (TTP) from its initial recognition in 1924 as a most often fatal disease to the discovery in 1997 of ADAMTS-13 deficiency as a major risk factor for acute disease manifestation. The cloning of the metalloprotease, ADAMTS-13, an essential regulator of the extremely adhesive unusually large von Willebrand factor (VWF) multimers secreted by endothelial cells, as well as ADAMTS-13 structure and function are reviewed. The complex, initially devised assays for ADAMTS-13 activity and the possible limitations of static in vitro assays are described. A new, simple assay using a recombinant 73-amino acid VWF peptide as substrate will hopefully be useful. Hereditary TTP caused by homozygous or double heterozygous ADAMTS-13 mutations and the nature of the mutations so far identified are discussed. Recognition of this condition by clinicians is of utmost importance, because it can be easily treated and--if untreated--frequently results in death. Acquired TTP is often but not always associated with severe, autoantibody-mediated ADAMTS-13 deficiency. The pathogenesis of cases without severe deficiency of the VWF-cleaving protease remains unknown, affected patients cannot be distinguished clinically from those with severely decreased ADAMTS-13 activity. Survivors of acute TTP, especially those with autoantibody-induced ADAMTS-13 deficiency, are at a high risk for relapse, as are patients with hereditary TTP. Patients with thrombotic microangiopathies (TMA) associated with hematopoietic stem cell transplantation, neo-plasia and several drugs, usually have normal or only moderately reduced ADAMTS-13 activity, with the exception of ticlopidine-induced TMA. Diarrhea-positive-hemolytic uremic syndrome (D+ HUS), mainly occurring in children is due to enterohemorrhagic Escherichia coli infection, and cases with atypical, D- HUS may be associated with factor H abnormalities. Treatment of acquired idiopathic TTP involves plasma exchange with fresh frozen plasma (FFP), and probably immunosuppression with corticosteroids is indicated. We believe that, at present, patients without severe acquired ADAMTS-13 deficiency should be treated with plasma exchange as well, until better strategies become available. Constitutional TTP can be treated by simple FFP infusion that rapidly reverses acute disease and--given prophylactically every 2-3 weeks--prevents relapses. There remains a large research agenda to improve diagnosis of TMA, gain further insight into the pathophysiology of the various TMA and to improve and possibly tailor the management of affected patients.

Menorrhagia caused by severe hereditary factor VII deficiency. Case 1.

This report describes the clinical history and laboratory findings of three sisters with severe inherited factor VII deficiency. We present current knowledge about factor VII structure and function, and discuss clinical presentation as well as management options for patients affected by factor VII deficiency.