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.

"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.

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.

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.

Collagen response and glycoprotein VI function decline progressively as canine platelets age in vivo.

Clinical and experimental observations suggest that platelet function deteriorates quickly with cell age. However, efforts to define age-dependent alterations have detected only modest biochemical changes occurring late in the cell life span. In this report, we demonstrate two significant alterations of the collagen response occurring during in vivo aging of canine platelets: a progressive increase in the EC50 for collagen types I, III and V and the emergence of a population of aged platelets which are refractory to collagen. Experiments with convulxin, a specific agonist for the collagen receptor glycoprotein VI (GPVI), also demonstrate an age-dependent decline in activation and the appearance of a non-reactive, aged population as observed with native collagens. Our studies indicate that canine platelets have two distinct binding levels for FITC-labeled convulxin and that the higher binding level disappears upon cell aging. During these studies one dog (#428) was identified whose platelets not only failed to demonstrate an age-dependent decrease in convulxin reactivity but also maintained a high convulxin-binding ability throughout their otherwise normal life span. Transfusion of biotinylated platelets from control dogs into dog #428 showed that the expected changes in collagen response and GPVI function did not occur in the transfused platelets. These observations demonstrate that the canine platelet response towards collagen is strongly dependent upon cell-age and suggest that this functional decline is at least partly due to an extrinsic-mediated alteration, possibly proteolytic, of GPVI.

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.

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.

Qualitative platelet defect and thrombohaemorrhagic complications in a patient with polycythaemia vera. Case 10.

We describe a 67-year-old patient with polycythaemia vera and pathological functional platelet studies. He not only suffered a transient ischaemic attack despite taking of antiplatelet agents, but also showed bleeding diathesis with cerebral bleeding and spontaneous suffusions. Platelet function studies and clinical findings improved after phlebotomy and cytoreductive treatment with hydroxyurea. Thrombosis and haemorrhage account predominantly for morbidity and mortality in patients with polycythaemia vera. The pathophysiological mechanisms to explain thrombosis and bleeding in patients with myeloproliferative disorders including polycythaemia vera were intensively studied. However, up to now no clear correlation of laboratory findings in relation to clinical history of thrombosis and bleeding was demonstrated. In this report the most important pathophysiological mechanisms and therapy with antiplatelet agents are discussed.

Acquired von Willebrand syndrome in a myeloproliferative disorder. Case 6.

We present a woman (age: 57 years) with an excessive bleeding episode under acetylsalicylic acid after bone marrow puncture due to an acquired von Willebrand syndrome (avWS) in the context of a myeloproliferative disorder. The laboratory features showed a high platelet concentration and a qualitative defect of von Willebrand factor (vWF) with a low normal vWF ristocetin cofactor activity, a normal vWF antigen and a decrease of the larger vWF multimers in plasma. The exact mechanism of avWS is still incompletely resolved. Myeloproliferative diseases are one of several underlying disorders that may cause avWS. The diagnosis of the underlying disease is important because its treatment may lead to an improvement of the vWF abnormality. For symptomatic treatment of bleeding, desmopressin, vWF concentrate infusion, intravenous immunoglobulin and/or fibrinolysis inhibitors can be tried.

[Oral anticoagulation with vitamin K antagonists]. / Die orale Antikoagulation mit Vitamin K-Antagonisten.

Coumarins are broadly employed in clinical practice and should therefore belong to the group of 'well known' drugs. The present review will deal with some concepts of anticoagulant therapy: the role of vitamin K in the synthesis of functional coagulation factors, some clinically relevant pharmacokinetic aspects of coumarins, the management of oral anticoagulant therapy with special emphasis on the laboratory monitoring, and the most frequent complication, bleeding.

[2-year-old boy with recurrent mucocutaneous hemorrhage--Glanzmann thrombasthenia. Role of the fibrinogen receptor (glycoprotein IIb-IIIa) in thrombocyte function]. / Zweijähriger Knabe mit rezidivierenden mukokutanen Blutungen--Thrombasthenie Glanzmann. Die Rolle des Fibrinogenrezeptors (Glycoprotein IIb-IIIa) in der Thrombozytenfunktion.

We report the clinical history of a patient suffering from a mucocutaneous bleeding diathesis since his birth. Discussing this case of Glanzmann's thrombasthenia we present essential aspects of clinical approach, diagnostic procedures and management of patients with platelet dysfunctions. Furthermore, we summarize the current understanding of platelet involvement in the haemostatic process and we discuss the role of the fibrinogen receptor glycoprotein IIb-IIIa.

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.