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BACKGROUND The Sysmex CN-6500 is a new haemostasis analyser with an integrated immunoassay module that performs chemiluminescence enzyme assay (CLEIA) in addition to coagulation, turbidimetric, chromogenic and platelet aggregation tests. AIMS To evaluate the analytical performance of the CN-6500 against the predicate device (Sysmex HISCL-800) for soluble thrombomodulin (TM), thrombin-antithrombin (TAT), tissue plasminogen activator/plasminogen activator inhibitor 1 complex (tPAI-C) and plasmin α2 plasmin inhibitor complex (PIC) assays. METHODS Imprecision was assessed by testing two levels of quality control plasmas 10 times on 5 separate days. Comparability was studied in 230 plasmas from normal donors (n = 30), patients with suspected disseminated intravascular coagulation (DIC, n = 100), sepsis (n = 20) or liver disease (n = 20), lipaemic (n = 20), haemolysed (n = 20) and icteric samples (n = 20). Limit of detection, limit of quantitation and linearity were determined by testing serial dilutions of normal plasma. Sample carryover was assessed by testing samples with high and low normal levels of the analytes concerned. RESULTS The CN-6500 performed 21 CLEIA tests per hour, while simultaneously performing coagulation tests. Acceptable between-run imprecision was obtained using commercial controls with normal and high activity for each analyte (%CV 0.99) across the measurement range. The lower limits of detection and quantitation were as follows: TM 0.1/

BACKGROUND The CN-6000 (Sysmex Corp.) is a new haemostasis analyser with blood coagulation, amidolytic, immuno-turbidometric and light transmission aggregometry (LTA) capabilities. Transmitted light is monitored at multiple wavelengths (340, 405, 575, 660, 800 nm), from an LED light source. AIMS To evaluate the performance of the CN-6000 against a predicate device. METHODS The CN-6000 was evaluated against the CS-5100 (Sysmex) for 14 different tests, using 880 samples from normal subjects, anticoagulated patients, critically ill patients, plasmas with high or low fibrinogen content or abnormal levels of interfering substances. Between-day assay imprecision was assessed using commercial QC materials (n = 10 replicates on each of 5 days). RESULTS Acceptable levels of imprecision were obtained for all assays. Agreement between the two analysers was excellent for all assays. Throughput was 35% higher using the CN-6000 (337 vs 250 tests per hour for PT, aPTT and fibrinogen). The CN-6000 also demonstrated improved clot detection in plasmas with high levels of interfering substances as demonstrated by a 29% reduction in "vote-outs" due to low light transmission (24 vs 34). CONCLUSIONS The CN-6000 demonstrated excellent comparability with the predicate instrument and acceptable levels of imprecision in all assays. Improvements in throughput and clot detection in the presence of interfering substances were also shown.

This guidance document was prepared on behalf of the International Council for Standardization in Haematology (ICSH), by the ADAMTS13 Assay Working Group, which comprises an international group of both clinical and laboratory experts. The document provides recommendations on best practice for the performance of ADAMTS13 assays in clinical laboratories. ADAMTS13 assays support the differential diagnosis of thrombotic microangiopathies and have utility in the management of thrombotic thrombocytopenic purpura (TTP). There are three types of assay: activity, antigen and autoantibody/inhibitor assays. Methods for activity assays differ in terms of sensitivity, specificity, precision and turnaround time. The most widely used assays involve VWF peptide substrates and either chromogenic ELISA or FRET techniques, although chemiluminescence assays and rapid screening tests have recently become available. Tests for autoantibodies and inhibitors allow confirmation of acquired, immune-mediated TTP, while antigen assays may be useful in congenital TTP and as prognostic markers. In this document, we have attempted to describe ADAMTS13 assays and the conditions that affect them, as well as: blood collection, sample processing, quality control, standardization and clinical utility; recognizing that laboratories in different parts of the world have varying levels of sophistication. The recommendations are based on expert opinion, published literature and good clinical laboratory practice.

INTRODUCTION: The XN-550 is a new, automated, compact, haematology analyser designed to generate a full blood count with a standard five-part white blood cell differential and an immature granulocyte count, as well as an optional reticulocyte and optical platelet (PLT) counts. The aim of the study was to evaluate the performance of the XN-550 and compare it to the established XN-20 system. METHODS: We evaluated the basic parameter and special measurement channels of the XN-550, using the XN-20 (which has a similar operating system), as a reference analyser. Precision, carry-over and throughput evaluations were performed. In addition, a total of 202 samples including normal controls and various pathological samples were studied for comparability. RESULTS: Good correlations with the reference analyser were obtained for all parameters except basophils. The XN-550 offers impedance and optical PLT counts and the latter showed a better correlation and less scatter than the impedance count and was comparable to the XN-20 fluorescent count at PLT counts ≤40×109/L. Precision was good, and no significant carry-over was detected. CONCLUSIONS: The XN-550 was simple and easy to use, while maintaining the good diagnostic sensitivity seen with high-range systems such as the XN-20, making this compact device suitable for near-patient services and smaller satellite laboratories.

Summary Introduction A new prothrombin time reagent (Revohem™ PT) based on recombinant human tissue factor produced by the silkworm-baculovirus expression system was tested. The aim of this study was to compare the performance of the new PT reagent with two widely used routine PT reagents. Methods All testing was performed on a Sysmex CS-5100 coagulometer. Revohem™ PT was tested for imprecision and stability using normal and abnormal lyophilized commercial control plasmas. Comparability was assessed with two widely used reagents: one containing recombinant human tissue factor (Reagent A) and the other a human placental thromboplastin (Reagent B) using a wide range of normal and abnormal plasmas and analyser-specific ISI values. Results Excellent between-day imprecision was obtained for Revohem™ PT (CV

Introduction Morphological assessment of the blood smear has been performed by conventional manual microscopy for many decades. Recently, rapid progress in digital imaging and information technology has led to the development of automated methods of digital morphological analysis of blood smears. Methods A panel of experts in laboratory hematology reviewed the literature on the use of digital imaging and other strategies for the morphological analysis of blood smears. The strengths and weaknesses of digital imaging were determined, and recommendations on improvement were proposed. Results By preclassifying cells using artificial intelligence algorithms, digital image analysis automates the blood smear review process and enables faster slide reviews. Digital image analyzers also allow remote networked laboratories to transfer images rapidly to a central laboratory for review, and facilitate a variety of essential work functions in laboratory hematology such as consultations, digital image archival, libraries, quality assurance, competency assessment, education, and training. Different instruments from several manufacturers are available, but there is a lack of standardization of staining methods, optical magnifications, color and display characteristics, hardware, software, and file formats. Conclusion In order to realize the full potential of Digital Morphology Hematology Analyzers, pre-analytic, analytic, and postanalytic parameters should be standardized. Manufacturers of new instruments should focus on improving the accuracy of cell preclassifications, and the automated recognition and classification of pathological cell types. Cutoffs for grading morphological abnormalities should depend on clinical significance. With all current devices, a skilled morphologist remains essential for cell reclassification and diagnostic interpretation of the blood smear.

Automated platelet counting history and current platelet parameters available, including MPV and reticulated platelets which aid in the diagnostic and management decisions of platelet disorders.

Reduced ADAMTS13 activity is seen in thrombotic thrombocytopenic purpura (TTP), and may lead to accumulation of prothrombotic ultra-large von Willebrand factor (ULVWF) multimers in vivo. ADAMTS13 activity and its relationship with VWF antigen (VWF:Ag) levels and platelet function in 'non-TTP related' TIA or ischaemic stroke has not been comprehensively studied.In this prospective pilot observational analytical case-control study, ADAMTS13 activity and VWF:Ag levels were quantified in platelet poor plasma in 53 patients in the early phase (≤ 4 weeks) and 34 of these patients in the late phase (≥ 3 months) after TIA or ischaemic stroke on aspirin. Data were compared with those from 22 controls not on aspirin. The impact of ADAMTS13 on platelet function in whole blood was quantified by measuring Collagen-ADP (C-ADP) and Collagen-Epinephrine closure times on a platelet function analyser (PFA-100(®)).Median ADAMTS13 activity was significantly reduced in the early phase (71.96% vs. 95.5%, P0.01) but not in the late phase after TIA or stroke compared with controls (86.3% vs. 95.5%, P=0.19). There was a significant inverse relationship between ADAMTS13 activity and VWF:Ag levels in the early phase (r=-0.31; P=0.024), but not in the late phase after TIA or stroke (P=0.74). There was a positive correlation between ADAMTS13 activity and C-ADP closure times in early phase patients only, likely mediated via VWF:Ag levels.ADAMTS13 activity is reduced and VWF:Ag expression is increased within 4 weeks of TIA or ischaemic stroke onset, and can promote enhanced platelet adhesion and aggregation in response to stimulation with collagen and ADP via VWF-mediated pathways. These data improve our understanding of the dynamic haemostatic and thrombotic profiles of ischaemic cerebrovascular disease (CVD) patients, and are important in view of the potential future role that ADAMTS13 may have to play as an anti-thrombotic agent in CVD.

Key points Finding an inherited complement abnormality in HSCT-associated TMA provides a rationale for the use of a complement inhibitor. Alternative complement inhibitors such as Coversin should be considered in patients who are resistant to eculizumab.

SummaryBackground Rituximab has been documented in the treatment of acute (≤ 3 days from admission), relapsed/refractory thrombotic thrombocytopenic purpura (TTP) and given as prophylaxis in selected cases to prevent acute relapse. The precise timing of rituximab in acute TTP has not been determined. Objective To perform a retrospective analysis of rituximab use in a large TTP referral center over an 8-year period. Patients/Methods We assessed response to treatment and outcome for all patients treated with rituximab, including 91 patients presenting with 104 episodes of acute TTP and 15 patients given rituximab as prophylaxis to prevent relapse. In the acute TTP group we assessed the benefit of giving early (≤ 3 days from admission) vs. later (> 3 days) rituximab. Results In acute de novo TTP, previously untreated with rituximab, rituximab was given ≤ 3 days from admission to 54 patients and > 3 days from admission to 32 patients. Earlier administration (≤ 3 days) was associated with faster attainment of remission (12 vs. 20 days, P

Introduction The gold standard for the determination of the erythrocyte sedimentation rate (ESR) is the Westergren method. Other methods to measure the ESR have become available. They range from modest modifications of the Westergren method to very different methodologies. The ICSH therefore established a Working Group to investigate these new approaches and compile recommendations for their validation and verification. Methods A panel of six experts in laboratory hematology examined the peer-reviewed literature and EQA surveys from over 6000 laboratories on four continents performing ESR testing. This information was used to create lists of ESR instrument manufacturers and their methods. Results Only 28% of laboratories surveyed used the unmodified Westergren method, while 72% of sites used modified or alternate methods. Results obtained with the new instruments could differ from results obtained with the Westergren method by up to 142%. Different non-Westergren methods showed differences from each other of up to 42%. The new methods were often significantly faster, safer, and less labor-intensive. They reduced costs and often used standard EDTA tubes, eliminating the need for a dedicated ESR tube. Conclusion Based on the consensus of the Working Group, recommendations for manufacturers for the validation of new ESR methods were developed. In addition, a list of recommendations for laboratories that are moving to modified or alternate methods was compiled, addressing instrument performance verification and communications of results to clinical users.

Accurate and reliable platelet counting is critical for the clinical management of platelet disorders, especially in thrombocytopenia. The platelet count is used to determine if the patient requires a platelet transfusion. As the prophylactic transfusion trigger is now set anywhere between 10-20 × 10(9)/L (depending upon the institution) it is therefore important that reliable and accurate counts are obtained in severely thrombocytopenic samples. The accuracy and precision of automated platelet counts is totally reliant upon optimal discrimination of platelets from other cells and interfering particles. However, clinicians often still rely upon counts that have been generated using so called "1-dimensional" cell size analysis systems, which not only fail to discriminate platelets from cell fragments of similar size but exclude large platelets from the final count. Also the current reference method for platelet counting (the manual phase count), upon which analysers are usually calibrated is highly imprecise, time consuming and unreliable. Thus there has been a demand for improvements in platelet counting technology in order to improve accuracy of counting in thrombocytopenia so that correct clinical decisions can be made. More recent developments including the introduction of "2-dimensional" optical counting and immunoplatelet counting within automated systems are significant advances. The availability of new technologies coupled with the recent development of a new candidate international reference method (flow cytometric immunocounting using the PLT/RBC ratio) should therefore improve the overall reliability of platelet counting especially in thrombocytopenia. In this review, the history and recent advances in platelet counting methodologies will be presented. The relative advantages and disadvantages of each technology will then be discussed along with their potential impact on improved accuracy of platelet counting.

Over 150 mutations have been identified in the ADAMTS13 gene in patients with congenital thrombotic thrombocytopenic purpura (TTP). The majority of these (86%), lead to reduced (50%) secretion of mutant recombinant ADAMTS13. The mechanism by which this occurs has not been investigated in vitro. Two novel ADAMTS13 mutations (p.I143T and p.Y570C) identified in two congenital adolescence onset TTP patients were studied, to investigate their effects on ADAMTS13 secretion and subcellular localisation.HEK293T cells were transiently transfected with wild type or mutant ADAMTS13 cDNA. Immunofluorescence and confocal microscopy were used to study localisation within the endoplasmic reticulum (ER) and Golgi. The cell proteasome and lysosomes were inhibited in cells stably expressing ADAMTS13 to investigate degradation of ADAMTS13 by either organelle.Both mutations severely impaired secretion and both mutants localised within the ER and Golgi. Proteasome inhibition led to the intracellular accumulation of both mutants, suggesting proteasome degradation. Lysosome inhibition on the other hand did not lead to increased intracellular accumulation of the mutants.Proteasome degradation of these ADAMTS13 mutants contributed to their reduced secretion.

Rivaroxaban is established for the treatment and secondary prevention of venous thromboembolism, but whether it is useful in patients with antiphospholipid syndrome is uncertain.This randomised, controlled, open-label, phase 2/3, non-inferiority trial, done in two UK hospitals, included patients with antiphospholipid syndrome who were taking warfarin for previous venous thromboembolism, with a target international normalised ratio of 2·5. Patients were randomly assigned 1:1 to continue with warfarin or receive 20 mg oral rivaroxaban daily. Randomisation was done centrally, stratified by centre and patient type (with vs without systemic lupus erythematosus). The primary outcome was percentage change in endogenous thrombin potential (ETP) from randomisation to day 42, with non-inferiority set at less than 20% difference from warfarin in mean percentage change. Analysis was by modified intention to treat. Other thrombin generation parameters, thrombosis, and bleeding were also assessed. Treatment effect was measured as the ratio of rivaroxaban to warfarin for thrombin generation. This trial is registered with the ISRCTN registry, number ISRCTN68222801.Of 116 patients randomised between June 5, 2013, and Nov 11, 2014, 54 who received rivaroxaban and 56 who received warfarin were assessed. At day 42, ETP was higher in the rivaroxaban than in the warfarin group (geometric mean 1086 nmol/L per min, 95% CI 957-1233 vs 548, 484-621, treatment effect 2·0, 95% CI 1·7-2·4, p0·0001). Peak thrombin generation was lower in the rivaroxaban group (56 nmol/L, 95% CI 47-66 vs 86 nmol/L, 72-102, treatment effect 0·6, 95% CI 0·5-0·8, p=0·0006). No thrombosis or major bleeding were seen. Serious adverse events occurred in four patients in each group.ETP for rivaroxaban did not reach the non-inferiority threshold, but as there was no increase in thrombotic risk compared with standard-intensity warfarin, this drug could be an effective and safe alternative in patients with antiphospholipid syndrome and previous venous thromboembolism.Arthritis Research UK, Comprehensive Clinical Trials Unit at UCL, LUPUS UK, Bayer, National Institute for Health Research Biomedical Research Centre.

Activated protein C resistance (APCR) within a thrombin generation test (TGT) system is associated with an increased risk of venous thromboembolism (VTE). However, application of the TGT is restricted by the analytical platforms used to monitor thrombin generation. Using a routine coagulation analyser we have developed an automated chromogenic TGT that is sensitive to defects in the protein C pathway.The TGT was performed on a TOP500 analyser, in the presence and absence of Protac. The reaction was monitored using a substrate with slow kinetics for thrombin (S-2444). Results were expressed as the area under the curve normalised ratio (AUCnr). Assay results were compared with Coatest APCR (expressed as APC-ratio [CoAPCr]).Samples were obtained from 35 healthy subjects and 91 patients with previous history of VTE. Of these patients, 19, 17, and 9 had heterozygous factor V Leiden (FVL), antiphospholipid syndrome (APS), and protein C/protein S deficiencies (PC/PS) respectively.Inter-assay imprecision in the presence and absence of Protac were 20% and5% respectively. There was a significant difference between the AUCnr of normals (median [IQR]: 2.8 [2.4-4.7]) compared to: FVL (1.0 [0.7-1.2]); PC/PS (1.1 [0.9-1.2]); and APS (1.1 [0.8-1.4]); p0.001 for each comparison. No significant difference was seen between the AUCnr of normals and other VTE patients. The detection rate of AUCnr and CoAPCr were: 100% and 56% for FVL; 88% and 44% for PC/PS; and 64% and 45% for APS respectively.The automated TGT exhibited good sensitivity to defects in the protein C pathway.

Introduction Total hip/knee replacement surgery (THR/TKR respectively) is associated with an increased risk of venous thromboembolism. Dabigatran is recommended as a thromboprophylactic agent post orthopaedic surgery. The aim of this study was to assess the post-operative (Day-1 and Day-2) effect of prophylactic Dabigatran on: the thrombin generation (TG) assay; prothrombin fragment 1.2 (F1.2); thrombin-antithrombin complexes (TAT); D-dimer (D-D); and other coagulation parameters. Methods and samples Nineteen patients (12 THR, 7 TKR) who received 110 mg dabigatran 4 hours post-operatively, then 220 mg the following day, were recruited. Blood was collected: pre-operatively (Pre-); peri-operatively (Peri-); 19 hours after 110 mg dabigatran (Day-1); and 17 hours after 220 mg dabigatran (Day-2). The TG assay was measured using the Calibrated Automated Thrombogram and a low concentration of tissue factor. Other coagulation parameters measured included activated partial thromboplastin time (APTT), thrombin-time (TT), ecarin-clotting time (ECT) and Hemoclot tests. Results From Pre- to Peri-, ETP/peak-thrombin, F1.2, TAT and D-D increased significantly. From Peri- to Day-1 and Day-2: TAT reduced progressively; D-D increased; F1.2 did not change significantly; lag-time and time-to-peak prolonged; ETP/Peak-thrombin increased spuriously, due to Dabigatran interfering with the α-2 macroglobulin:thrombin complex in the TG assay. APTT, TT, ECT and Hemoclot increased progressively post-operatively; good correlations were seen between these tests. Conclusion The effect of dabigatran on the TG assay, showed a spurious increase in ETP and Peak-thrombin due to its interference with the TG assay. Dabigatran reduced TAT, but not F1.2, suggesting that thrombin was still being generated after surgery, but was blocked by Dabigatran.

BackgroundThe Sysmex XN haematology instrument performs automatic reflex testing, depending on sample results. A nucleated red blood cell (NRBC) count is provided on all samples. The instrument has a smaller footprint (34%) than previous Sysmex XE analysers.MethodsAn evaluation comparing all results to the Sysmex XE-2100 and manual microscopic differential and morphology (n=390) was performed followed by a workflow study of 1000 samples to compare speed of operation and number of blood films reviews required from both systems.ResultsThe new features on the instrument are: (1) white cell and NRBC channel, all samples include the NRBC count; (2) white cell precursor channel: false positive flags for blasts, abnormal lymphocytes and atypical lymphocytes are reduced significantly without a statistical increase of false negatives; (3) low white cell count mode: suggested setting of 9/l. An extended count is more precise and provides an accurate differential. Fluorescent platelet count is performed in a dedicated channel. If the red cell or platelet size histograms are abnormal or if the platelet count is low, then a fluorescent platelet count is automatically performed. Good correlation with the XE-2100 and manual differential was found and the improved results compared to the reference flow cytometric analysis for platelet counts, especially below 30×109/l (XE-2100, R2=0.500; XN, R2=0.875).ConclusionThe XN showed reduced sample turnaround time of 10% and reduced number of blood films for examination, 49% less than the XE-2100 without loss of sensitivity with more precise and accurate results on low cell counts.

Patients receiving warfarin are at increased risk of bleeding when their International Normalised Ratio (INR)4.5. Although not standardised above 4.5 the INR is measured in over-anticoagulated patients, consequently we have examined the reliability of INR results ≥4.5. We assessed: the relationship between different prothrombin time systems for INRs4.5; the relationships between the INR and levels of vitamin K-dependent coagulation factors (VKD-CF) and thrombin generation test (TGT) parameters; and the impact that variation in results would have on warfarin dosing.INRs were performed using a CoaguChek XS Plus point-of-care (POC) device (measuring range 0.6-8.0). For POC INRs ≥4.5, laboratory INRs were also measured using a recombinant tissue factor (rTF) and a rabbit brain (RBT) thromboplastin.There was good correlation between POC (INR ≥4.5,8.0) and Lab INRs (rTF n=154, rs=0.87, p0.0001; RBT n=102, rs=0.76, p0.0001); and significant correlations between each of the VKD-CF and the INR, the strongest being with FVII (POC INR rs=-0.53 p0.0001; Lab rTF-INR rs=-0.70 p0.0001). TGT peak thrombin and ETP also showed good correlations with INR values (R(2)0.71). Using POC and Lab rTF-INR, 109/154 (71%), or POC and Lab RBT-INR 75/102 (74%) results exhibited dosage concordance and/or were within 0.5 INR units. In the remaining patients variation in warfarin dosing was generally slight.Our data suggest that CoaguChek XS Plus INRs4.5 and8.0 are comparable to laboratory INRs (both methods) and it is probably unnecessary to perform laboratory INRs for clinical management of patients with INRs4.5 including those8.0.

related to the subsections of this guideline. The writing group produced the draft guideline, which was subsequently revised by consensus by members of the Haemostasis and Thrombosis Task Force of the BCSH. The guideline was then reviewed by a sounding board of British haematologists, the BCSH and the British Society for Haematology Committee and comments incorporated where appropriate. The ‘GRADE’ system was used to quote levels and grades of evidence, details of which can be found at http://www.bcshguidelines.com. The objective of this guideline is to provide healthcare professionals with clear, up-to-date, and practical guidance on the management of TTP and related thrombotic microangiopathies, defined by thrombocytopenia, microangiopathic haemolytic anaemia (MAHA) and small vessel thrombosis.

A persistently elevated level of factor VIII (FVIII) is an independent risk factor for venous thromboembolism (VTE). Although the pathophysiology of VTE is unclear, the involvement of thrombin generation (TG) has been postulated. Consequently this study was designed to (i) investigate the relationships between FVIII, Thrombin generation test (TGT) parameters and D-dimer in VTE patients, (ii) determine whether elevated levels of FVIII and increased TG in these patients are transient or sustained.After an initial period of anticoagulation had been completed 91 VTE patients and 52 healthy controls were recruited. FVIII levels were determined by one-stage clotting (FVIII:C) and chromogenic (FVIII:Ch) assays. The potential to generate thrombin was measured using the Calibrated Automated Thrombogram (CAT) and D-Dimer was by immuno-turbidometric assay.Patients' FVIII:C levels and FVIII:Ch, exhibited good agreement (rs=0.94; p0.0001), although FVIII:C exhibited a mean bias of -6%. FVIII:Ch show a significant correlation with TGT Peak Thrombin (rs=0.30; p=0.004) and Peak Thrombin was found to be significantly higher (p=0.04) in patients with FVIII200 iu/dL. Furthermore elevated levels of FVIII and increased thrombin generation parameters appeared to be consistent over time.Our data suggests that high FVIII leading to increased TG confers a significant risk of recurrent VTE and therefore we speculate that these patients may benefit from prolonged anticoagulation therapy.

The guideline writing group was selected to be representative of UK-based medical experts. MEDLINE was systematically searched for publications in English up to the Summer of 2010 using key words platelet, platelet function testing and platelet aggregometry. Relevant references generated from initial papers and published guidelines/reviews were also examined. Meeting abstracts were not included. The writing group produced the draft guideline, which was subsequently revised and agreed by consensus. Further comment was made by members of the Haemostasis and Thrombosis Task Force of the British Committee for Standards in Haematology. The guideline was then reviewed by a sounding board of approximately 40 UK haematologists, the British Committee for Standards in Haematology (BCSH) and the British Society for Haematology Committee and comments incorporated where appropriate. Criteria used to quote levels and grades of evidence are as outlined in appendix 7 of the Procedure for Guidelines Commissioned by the BCSH [http://www.bcshguidelines.com/BCSH_PROCESS/EVIDENCE_LEVELS_AND_GRADES_OF_RECOMMENDATION/43_GRADE.html]. The objective of this guideline is to provide healthcare professionals with clear guidance on platelet function testing in patients with suspected bleeding disorders. The guidance may not be appropriate to patients receiving antiplatelet therapy and in all cases individual patient circumstances may dictate an alternative approach.

Thrombotic thrombocytopenic purpura (TTP) is an acute prothrombotic disorder. Human immunodeficiency virus (HIV) is an identified precipitant. This study reviewed 30 episodes of HIV-associated TTP in 24 patients from the South-East England Apheresis units, over the last 10 years. All patients were heterosexual Black Africans. First presentation of TTP revealed a new diagnosis of HIV in eight patients. TTP relapse occurred on six occasions (in four patients) as a result of non-adherence to highly active antiretroviral therapy (HAART). Prompt initiation/re-initiation of HAART in parallel with plasma exchange (PEX)±steroid led to prompt remission. Adjunct immunomodulatory agents (e.g. Rituximab) were required in 10% of cases. Once-daily HAART regimens are recommended, being compatible with PEX requirement, maximizing drug exposure between PEX. High viral loads (>500,000 copies/ml) require more PEX to remission. ADAMTS13 activity was reduced (

The Antiphospholipid Syndrome (APS) Clinical Research Task Force (CRTF) was one of six Task Forces developed by the 13th International Congress on Antiphospholipid Antibodies (aPL) organization committee with the purpose of: a) evaluating the limitations of APS clinical research and developing guidelines for researchers to help improve the quality of APS research; and b) prioritizing the ideas for a well-designed multicenter clinical trial and discussing the pragmatics of getting such a trial done. Following a systematic working algorithm, the Task Force identified five major issues that impede APS clinical research and the ability to develop evidence-based recommendations for the management of aPL-positive patients: (1) aPL detection has been based on partially or non-standardized tests, and clinical (and basic) APS research studies have included patients with heterogeneous aPL profiles with different clinical event risks; (2) clinical (and basic) APS research studies have included a heterogeneous group of patients with different aPL-related manifestations (some controversial); (3) thrombosis and/or pregnancy risk stratification and quantification are rarely incorporated in APS clinical research; (4) most APS clinical studies include patients with single positive aPL results and/or low-titer aPL ELISA results; furthermore, study designs are mostly retrospective and not population based, with limited number of prospective and/or controlled population studies; and (5) lack of the understanding the particular mechanisms of aPL-mediated clinical events limits the optimal clinical study design. The Task Force recommended that there is an urgent need for a truly international collaborative approach to design and conduct well-designed prospective large-scale multi-center clinical trials of patients with persistent and clinically significant aPL profiles. An international collaborative meeting to formulate a good research question using ‘FINER’ (Feasible; Interesting; Novel; Ethical; and Relevant) criteria took place in November 2010.

The antiphospholipid syndrome (APS) is defined by the presence of thrombosis and/or pregnancy morbidity in combination with the persistent presence of circulating antiphospholipid antibodies: lupus anticoagulant, anticardiolipin antibodies and/or anti-β2-glycoprotein I antibodies in medium to high titers. The management of thrombosis in patients with APS is a subject of controversy. This set of recommendations is the result of an effort to produce guidelines for therapy within a group of specialist physicians in Cardiology, Neurology, Hematology, Rheumatology and Internal Medicine, with a clinical and research focus on APS.

Summary Introduction: Performance of the Celltac F haematology analyser (MEK-8222) which provides 22 parameters, including a 5-part differential, was compared with the Sysmex XE-2100. Methods: 242 EDTA samples were investigated. Differential results from both instruments were compared with the reference microscopic count. Flagging performance was compared with cell morphology seen in the blood films. Results: Precision met or exceeded manufacturer’s specifications, carryover was minimal (≤1.37%) and linearity was excellent (R ≥ 0.99). Results were stable for at least 8 h at room temperature and for 24 h at 4 °C. Comparisons were excellent for white blood cells, red cell count, Hb, HCT and platelets (R ≥ 0.98). All other red cell and platelet parameters showed good correlation with the XE-2100 (R ≥ 0.93) except for mean cell haemoglobin concentration. The differential was comparable to the XE-2100 for neutrophils, lymphocytes and eosinophils and acceptable for monocytes. Correlation of automated differentials with manual reference counts and the efficiency of flagging of blasts, immature granulocytes and platelet clumps were similar for both instruments. Celltac F demonstrated better efficiency for atypical lymphocyte and platelet clumps. Conclusion: The Celltac F shows broadly comparable analytical performance to the XE-2100 for the parameters assessed. The Celltac F is recommendable for medium-sized laboratories or as a back-up instrument in larger laboratories.

Activation of the complement cascade is an important mechanism for antiphospholipid antibody-mediated thrombosis. We examined the effects of rEV576 (coversin), a recombinant protein inhibitor of complement factor 5 activation, on antiphospholipid antibody-mediated tissue factor up-regulation and thrombosis. Groups of C57BL/6J mice ( n = 5) received either IgG from a patient with antiphospholipid syndrome (APS) or control IgG from normal human serum (NHS). Each of these groups of mice had IgG administration preceded by either rEV576, or phosphate buffer control. For each of the four treatment groups, the size of induced thrombus, tissue factor activity in carotid homogenates, anticardiolipin and anti-β2glycoprotein I (anti-β2GPI) levels were measured 72 h after the first injection. Mice treated with IgG-APS had significantly higher titers of anticardiolipin antibodies and anti-β2GPI at thrombus induction compared with those treated with IgG-NHS. The IgG-APS/phosphate buffer treatment induced significantly larger thrombi and tissue factor activity compared with other groups. Mice treated with IgG-APS/rEV576 had significantly smaller thrombi and reduced tissue factor activity than those treated with IgG-APS/phosphate buffer. The data confirm involvement of complement activation in antiphospholipid antibody-mediated thrombogenesis and suggest that complement inhibition might ameliorate this effect.

Comparative sensitivity of commonly used thromboplastins to ex vivo therapeutic rivaroxaban levels

von Willebrand's disease (VWD) is regarded as the most common congenital bleeding disorder, and although not available in all laboratories von Willebrand factor (VWF) activity is most frequently assessed as ristocetin cofactor (VWF:RCo). This test can be technically challenging, is subject to poor sensitivity (∼20 IU dL(-1) VWF:RCo) and has a high degree of inter- and intra-assay imprecision [coefficient of variation (cv) > 25%]. We studied an automated assay using a combined fixed platelet/ristocetin reagent (BC von Willebrand reagent, Siemens Healthcare Diagnostics) on the CS-2000i analyser (Sysmex UK Ltd). Initially inter- and intra-assay imprecision was assessed. The automated method showed good day-to-day reproducibility and linearity of standard curves. This technique, also gave low intra- and inter-assay imprecision using commercial normal (cv < 4.5%) and pathological (cv < 8.1%) control plasmas. We then compared automated technique results from 30 healthy normal subjects and 39 VWD patients to those obtained using standard aggregometry (Bio/Data, PAP4) with lyophilised fixed platelets (Helena BioSciences) and ristocetin (American Biochemical and Pharmaceutical Ltd). The automated method had a sensitivity limit of approximately 10 IU dL(-1) vs. 20 IU dL(-1) for aggregometry. Samples giving results within the aggregometry measurable range (n = 50) exhibited good correlation with the automated technique (median 70 IU dL(-1), range 7-184 IU dL(-1); and 64 IU dL(-1), 6-138 IU dL(-1) respectively; R(2) = 0.85). We subsequently compared 3 different batches of BC von Willebrand reagent, using a second group of normal subjects and VWD patients (n = 35, 55-139 IU dL(-1) and n = 30

P>Total hip/knee replacement surgeries are associated with an increased risk of venous thromboembolism and post-operative thromboprophylaxis has become standard treatment. This study aimed to: (i) assess the impact of hip/knee replacement surgery on ex vivo thrombin generation (TG), prothrombin fragments 1 + 2 (F1 + 2), thrombin-antithrombin complexes (TAT) and D-dimer; (ii) compare the anticoagulant effects of dalteparin and rivaroxaban on TG 24 h after surgery. Haemostatic variables were assessed in plasma samples of 51 patients taken pre-operatively, peri-operatively, and 24 h post-operatively. Prophylaxis, once a day, with dalteparin or rivaroxaban, starting 6-8 h post-operatively, was administered in 25 (14 knee/11 hip) and 26 patients (13 knee/13 hip) respectively. TG, F1 + 2, TAT and D-dimer increased during surgery. Dalteparin patients showed a variable TG response 24 h after surgery: conversely, the effect of rivaroxaban on TG was consistent across individuals. Good correlation was seen between rivaroxaban levels and TG-lag-time (rs = 0 center dot 46, P = 0 center dot 01); TG-time-to-Peak (rs = 0 center dot 53, P = 0 center dot 005); TG-peak-thrombin (rs = -0 center dot 59, P = 0 center dot 001); and TG-velocity-index-rate (rs = -0 center dot 61, P = 0 center dot 0009). Patients who received rivaroxaban showed a greater decrease of TG, F1 + 2 and TAT (but not D-dimer) than those on dalteparin. TG increases during hip/knee replacement surgery. Rivaroxaban inhibits TG more than dalteparin at 24 h after surgery.

Summary. Background: Increasingly, patients with acute, idiopathic, antibody mediated thrombotic thrombocytopenic purpura (TTP) are being treated with rituximab to achieve a durable remission, however, there is the potential that it is removed by plasma exchange (PEX). Objectives: To look at the pharmacokinetics and pharmacodynamics of rituximab in patients with acute idiopathic TTP undergoing PEX. Patients and methods: Patients who received rituximab for acute idiopathic TTP (group 1, n = 30) and a control group (group 2, n = 3) of TTP patients in remission receiving rituximab electively as maintenance were included. Rituximab levels were measured before/after each infusion, before/after PEX and in follow-up. ADAMTS-13 activity, anti-ADAMTS-13 IgG and CD19% were measured to assess response. Results: The median number of PEX to remission after rituximab was 10 (range 4–25). In group 1 there was no significant incremental rise in the peak serum rituximab level until dose 4. Trough levels were lower in patients who had had PEX since their last rituximab infusion. In the control group, there was an incremental rise in the peak serum rituximab level and all patients had detectable trough levels. The median fall in rituximab per PEX was 65%. All patients achieved CD19 < 1%. In group 1, the median time to undetectable rituximab was 5 months (range 0–12 months) and to B cell return was 7 months (range 3–24 months). ADAMTS-13 increased and anti-ADAMTS-13 fell after therapy. There were three deaths and two relapses in group 1. Relapse was not temporally related to B cell return.

Background Octapharma PPGmbH has recently modified its manufacturing process for solvent/detergent-treated plasma to incorporate a prion reduction step, in which a 3 log reduction has been demonstrated. The current study was undertaken to assess the impact of this procedure on haemostatic variables in the new product OctaplasLG in comparison with standard Octaplas. Methods Production batches of standard Octaplas (n = 4) and OctaplasLG (n = 16) were assessed for levels of coagulation factors, physiological protease inhibitors, markers of activation and procoagulant microparticles. Global haemostasis was assessed by a thrombin generation test (TGT) and rotational thromboelastometry (ROTEM). Results Mean levels of factors: II, V, VII, IX, X, XI, XII and XIII, VWF:Ag, antithrombin, protein C and free protein S were all > 75 u/dl. ADAMTS-13 activity levels were normal. Factor VIII and VWF:RCo were > 55 u/dl. TGT and ROTEM were similar in both preparations, and microparticles were present at negligible levels. Two units of OctaplasLG had slightly elevated levels of Prothrombin Fragments 1 + 2, but D-Dimer and thrombin-antithrombin complexes were normal in all batches. Conclusion These studies indicate that the affinity chromatography procedure used in OctaplasLG does not appear to adversely affect the proven haemostatic quality of Octaplas, while offering a selective reduction in the concentration of pathological prion proteins.

Thrombotic thrombocytopenic purpura (TTP) in children is rare and is often thought to be due to congenital ADAMTS13 deficiency. We report seven new cases of noncongenital TTP in children and adolescents and perform a review of the literature where ADAMTS13 assays have been performed in paediatric acquired TTP. All new cases were female and the median age was 13 years. Presenting clinical features included bruising/petechiae or bleeding, fever, neurological, and renal impairment. Median Hb and platelet counts on admission were 66 g/l and 10 x 10(9)/l respectively. Two cases had raised Troponin T levels and one had an abnormal ECG. All cases had ADAMTS13 activity less than 5% and an inhibitor to ADAMTS13. The median number of plasma exchange to remission was 22.5. Six cases received rituximab. Three achieved normal ADAMTS13 activity and remain in remission. Two had persistently low ADAMTS13 activity with high anti-ADAMTS13 IgG levels and one of these relapsed. One had moderately reduced levels of ADAMTS13 in remission with no inhibitor, however, a fall in ADAMTS13 activity and increase in anti-ADAMTS13 IgG heralded clinical relapse. The literature review identified 12 acquired cases showing low ADAMTS13 activity and inhibition of ADAMTS13 (in 95%). In children and adolescents TTP may be due to acquired deficiency of ADAMTS13, associated with an inhibitor/Anti-ADAMTS13 IgG antibodies. Treatment of acquired disease requires PEX and usually immunosuppressive treatment. Rituximab appears to be an effective adjunctive treatment modality. Blood Coagul Fibrinolysis 21:245-250 (C) 2010 Wolters Kluwer Health vertical bar Lippincott Williams & Wilkins.

The widespread use of central neuraxial block (CNB) and the prevalence of anticoagulation for different indications have led to an inevitable overlap between the two. The most serious complication of CNB in anticoagulated patients is the risk of spinal/epidural haematoma. Performing CNB in these patients is a complex decision that should take into account the twin risks of bleeding and venous/arterial thrombosis if anticoagulation therapies were to be stopped. Various guidelines have been issued to achieve normal haemostasis and thus allow safe administration of CNB. However, the evidence base for many such recommendations is weak, relying mainly on case reports, small studies and pharmacokinetics of the drugs. Given these limitations it is crucial to fully assess individual risk factors and understand anticoagulant pharmacokinetics in order to appropriately set time intervals for catheter insertion/removal. This paper will review traditional and newer anticoagulation/antiplatelet therapies with a view to improving the management of anticoagulated patients undergoing CNB.

Atypical haemolytic uraemic syndrome (aHUS) is associated with a poor prognosis with regard to survival at presentation, recovery of renal function and transplantation. It is now established that aHUS is a disease of complement dysregulation with mutations in the genes encoding both complement regulators and activators, and autoantibodies against the complement regulator factor H. Identification of the underlying molecular abnormality in an individual patient can now help to guide their future management. In these guidelines we make recommendations for the investigation and management of aHUS patients both at presentation and in the long-term. We particularly address the role of renal transplantation alone and combined liver-kidney transplantation.

Summary Thrombotic microangiopathies are a relatively rare group of congenital and inherited disorders caused by defects in processing the ultra large forms of von Willibrand factor which pathologically give rise to platelet rich microthrombi in the micro arterial circulation leading to end organ damage particularly in the brain, heart and kidneys. Identification of the ADAMTS 13 gene has led to the definition of congenital deficiency of its activity or failure of activity due to the development of an inhibitory IgG antibody. The idiopathic autoimmune form of the disease is the most common. There are various subgroups of acquired TTP associated with HIV infection, pregnancy, pancreatitis, associated with bone marrow transplantation, various disseminated malignancies and certain drugs, particularly Clopidogrel. Diagnostic assays are now becoming widely available to identify ADAMTS 13 activity and also acquired antibodies to the enzyme. Mainline treatment is associated with daily plasma exchange with associated other immunosuppressant treatments particularly steroids and recently the use of Rituximab, a monoclonal anti-CD20 antibody. Despite improvement in treatment modalities there is still significant mortality of 10–20%, particularly if there is a delay in initiating plasma exchange. Relapse also occurs in 20–50% of patients although this may be improved by Rituximab therapy.

Evidence for cardiac involvement in thrombotic thrombocytopenic purpura (TTP) is uncommonly described.We retrospectively reviewed 41 patients assessing troponin T as a marker for cardiac involvement in acute TTP with clinical symptoms, electrocardiograms (ECG) and echocardiograms. A histopathological review of five patients who died of acute TTP was also undertaken.In 54% (22/41) of patients, troponin T wasor=0.05microg L(-1) (normal range 0-0.01 microg L(-1)). Half (12/22) had cardiac symptoms and 8/22 with a raised troponin T reported chest pain. ECG changes were present in 62% of patients with a raised troponin T. Median anti-ADAMTS 13 IgG antibody was significantly higher (P=0.018) in patients with troponin Tor=0.05 microg L(-1) (58.5% (range 17-162%), compared with patients with troponin T0.05 microg L(-1) (35%, range 9-134%). Patients who died had higher troponin T levels (median 0.305 microg L(-1)) and raised anti-ADAMTS 13 IgG (median 66.5%). On admission, there were no deaths in those with troponin Tor=0.04microg L(-1). Histology confirmed widespread myocardial microvascular thrombi.Clinical symptoms, ECG changes and echocardiograms are poor predictors of cardiac disease in acute TTP. Troponin T is specific for cardiac muscle and a sensitive marker of myocardial damage. In TTP patients, raised levels (or=0.05 microg L(-1)) signify myocardial necrosis associated with microvascular thrombi. Mortality and acute morbidity was associated with higher admission troponin T and raised IgG antibody (67%) to ADAMTS 13.

The decision to provide antiretroviral therapy to HIV-positive patients mainly depends on the CD4(+) T-cell count, with therapy indicated at a cut-off value of

Thrombotic thrombocytopenic purpura (TTP) may be idiopathic or secondary. We report seven TTP cases precipitated by pancreatitis. The patients were admitted with acute pancreatitis and at that time had no clinical or laboratory features of TTP. The median time to develop TTP after pancreatitis was 3 d. The patients had moderately reduced ADAMTS13 activity (mean activity 49%; normal range 66-126%) with no evidence of anti-ADAMTS13 inhibitory autoantibodies. The median number of plasma exchanges to remission was 10 (range 7-14) and no additional treatment with immunosuppression was required to maintain remission. There have been no relapses to date.

Automation of differentials is desirable for economic and time-saving reasons. Over the last 20 years, automated imaging processes have started to be introduced where stained blood films are scanned by a computer-driven microscope and leucocytes classified; however, early methods were slow and had difficulty in classifying abnormal cells. More recently the CellaVision DM96 (CellaVision AB, Lund, Sweden) has been introduced with added features such as continuous loading of slides and a faster throughput than previous instruments. The accuracy of CellaVision DM96 has been evaluated by comparing results to reference manual differentials. Results from different operators using the DM96 were compared with their own manual differential and to a 400-cell reference manual differential. Precision of the instrument was compared to the manual differential. The preclassification accuracy of the DM96 was 89.2%. Precision was similar to that of the 100-cell manual differential. The DM96 was faster than the manual method, even after reclassification by a laboratory scientist of any cells wrongly categorized by the instrument. The DM96 accuracy in morphological classification of leucocytes and red blood cells; depends upon both blood pathology and experience of the laboratory scientist using the instrument. For some cell types and operators, DM96 accuracy was better than the individual's 100 cell manual differential.

Background Factor VIII (FVIII) levels are used as a quality marker of fresh-frozen plasma (FFP); however, other clotting factors are not routinely measured. Methods We assessed additional haemostatic parameters and the dynamics of coagulation using Thrombelastography (TEG®) and a thrombin generation test (TGT). FFP was prepared on the day of donation (Day 0) or after overnight hold at 4°C (Day 1). Results Factor VIII in Day 1 FFP was 18% lower than in Day 0. TEG® parameters in Day 1 FFP were consistent with increased coagulability and did not correlate with altered levels of clotting factors, but were consistent with the increased levels of microparticles seen in the Day 1 samples. TGT studies exhibited increased lag time, time to peak and reduced peak thrombin generation, but no change in endogenous thrombin potential (ETP) on Day 1. There was a weak association between FVIII level and both ETP and peak thrombin (ETP rs≥ 0·22, P≤ 0·003; peak thrombin rs≥ 0·48, P≤ 0·0001), which was influenced by ABO group, with the lowest levels in group O. Conclusion We conclude that levels of FVIII do not predict the haemostatic potential of FFP and that there may be a role for alternative technologies in monitoring the quality of FFP.

The SmartCheck INR (Unipath, Bedford, England) is a point-of-care device for professional and patient self-monitoring of oral anticoagulant therapy. It measures the international normalized ratio (INR) and assesses test strip integrity, temperature, and sample quality. No significant differences were found in SmartCheck INR results from 3 different instruments or in 3 test-strip lots. Within run imprecision was 0.89% and 6.36% for low and high control samples, respectively (mean INR, 0.99 and 4.08, respectively). Comparability was assessed in 68 patients receiving warfarin by using PTHS Plus (Instrumentation Laboratory, Lexington, MA) and Innovin (Dade Behring, Marburg, Germany) thromboplastins. Good correlations were observed between the methods (r = 0.89 and r = 0.90, respectively) with no significant differences in means: SmartCheck INR, 2.82; Innovin, 2.75; PTHS Plus, 2.74. Clinical agreement with laboratory methods was 88% for Innovin and 97% for PTHS Plus. The SmartCheck INR was easy to use with no mechanical failures during the evaluation and a low test-strip failure rate of 4.7%. The SmartCheck INR provides accurate and reproducible results and is suitable for routine monitoring of oral anticoagulant therapy in the majority of patients.

Background: Thrombotic thrombocytopenic purpura (TTP) is most commonly associated with deficiency or inhibition of von Willebrand factor-cleaving protease (ADAMTS-13) activity. ADAMTS-13 mutations and polymorphisms have been reported in childhood congenital TTP, but their significance in adult onset TTP remains unclear. Objectives: We sought to identify common ADAMTS-13 mutations in adults with late onset TTP and to investigate whether they may predispose acute clinical episodes of the disorder in adulthood. Patients/Methods/Results: We detected a missense mutation (C3178T) in exon 24 of ADAMTS-13 in 6/53 (11.3%) adult onset TTP patients, but no normal controls (n = 100). Three of the patients had pregnancy-associated TTP; three had chronic relapsing acute idiopathic TTP. C3178T encodes an arginine to tryptophan (R1060W) substitution in the TSP1-7 domain of ADAMTS-13. In vitro expression of mutant and wild-type ADAMTS-13 demonstrated that R1060W caused severe intracellular retention of ADAMTS-13 (

Summary The four main procedures for platelet counting are: manual phase contrast microscopy, impedance, optical light scatter/fluorescence and flow cytometry. Early methods to enumerate platelets were inaccurate and irreproducible. The manual count is still recognized as the gold standard or reference method, and until very recently the calibration of platelet counts by the manufacturers of automated cell counters and quality control material was performed by this method. However, it is time-consuming and results in high levels of imprecision. The introduction of automated full blood counters using impedance technology resulted in a dramatic improvement in precision. However, impedance counts still have limitations as cell size analysis cannot discriminate platelets from other similar-sized particles. More recently, light scatter or fluorescence methods have been introduced for automated platelet counting, but there are still occasional cases where an accurate platelet count remains a challenge. Thus, there has been interest in the development of an improved reference procedure to enable optimization of automated platelet counting. This method utilizes monoclonal antibodies to platelet cell surface antigens conjugated to a suitable fluorophore. This permits the possible implementation of a new reference method to calibrate cell counters, assign values to calibrators, and to obtain a direct platelet count on a variety of pathological samples. In future, analysers may introduce additional platelet parameters; a reliable method to quantify immature or reticulated platelets would be useful.

Thrombotic thrombocytopenic purpura (TTP) has been linked to a severe deficiency in ADAMTS13 (a disintegrin and metalloprotease with thrombospondin type 1 motif, member 13) activity. Since the identification of ADAMTS13, and its target cleavage sequence in von Willebrand factor (VWF), several novel ADAMTS13 activity, antigen and autoantibody assays have been developed. Our aim was to evaluate the potential use of these novel assays. ADAMTS13 activity and inhibitors were measured by overnight incubation of patient plasma with pure VWF followed by multimer or collagen binding analysis. ADAMTS13 activity (Rapid peptide assay), antigen and immunoglobulin G anti-ADAMTS13 were measured by enzyme-linked immunosorbent assay. 118 samples from seven TTP patients (six adult idiopathic, one congenital) were studied longitudinally during episodes of TTP, their treatment and prophylaxis. ADAMTS13 antigen levels varied considerably between patients and sample times, but in new cases of acute TTP, rapid assays of ADAMTS13 antigen, on serial samples, maybe helpful in confirming the diagnosis. The rapid peptide ADAMTS13 activity assay showed good concordance of results with the older activity assay methods. The change in ADAMTS13 activity mirrored the autoantibody level and in 5/6 acquired TTP cases, a fall in antibody appeared to predict a rise in ADAMTS13 activity, potentially allowing modification of patient management based on autoantibody levels.