Search

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.

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

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.

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.

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.

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.

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.

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 (

The importance of testing for anticardiolipin antibodies (aCL) in the diagnosis of antiphospholipid syndrome (APS) in patients with thrombosis has recently been challenged (ISTH SSC meeting, Boston 2002). We have analyzed the antiphospholipid serology of 123 patients with persistent antiphospholipid antibodies (aPL) attending our hematology department. The cohort was tested for anti-beta(2)-glycoprotein I (beta(2)-GPI) antibodies and aCL of IgG and IgM class and for lupus anticoagulant (LA). Ninety-six of these patients fulfilled Sapporo clinical criteria for APS and 70 of these patients had venous and/or arterial thrombosis. Patients with LA plus anti-beta(2)-GPI antibodies had significantly higher levels of IgG aCL and anti-beta(2)-GPI antibodies than those exhibiting positivity for only LA or anti-beta(2)-GPI antibodies (P0.05). Patients with aCL IgG levels over 60 GPLU were found in all cases to be positive for LA and anti-beta(2)-GPI antibodies; 25.2% (31/123) of all patients and 26.04% (25/96) of patients fulfilling Sapporo clinical criteria for APS were positive for aCL only. The mean IgG aCL level in the Sapporo clinical criteria positive patients who had aCL only was 11.5 GPLU (normal5 GPLU). These data indicate that omission of aCL testing from the clinical investigation of APS could lead to a failure to diagnose the syndrome in a proportion of patients.

Objective: To identify patients with poor tissue factor pathway inhibitor (TFPI) response to heparin and observe any association with increased risk of excessive coagulation activation, morbidity, or mortality. Design: Prospective, observational cohort study. Setting: University hospital. Participants: Patients (n = 96) undergoing cardiopulmonary bypass for various types of surgery. Interventions: None. Measurements and Main Results: TFPI antigen and activity were determined in patients before and after heparin administration, before cardiopulmonary bypass for cardiac surgery. The clinical progress of each patient was recorded. Median levels of TFPI activity were 0.98 U/mL (interquartile range, 0.83 to 1.14 U/mL) preheparin and 2.34 U/mL (2.18 to 2.54 U/mL) postheparin (p < 0.0001), representing a median 2.3-(2.1- to 2.8-) fold increase. Median TFPI antigen levels were 92.4 ng/mL (73.0 to 119.5 ng/mL) preheparin and 422.9 ng/mL (398.7 to 501.6 ng/mL) postheparin (p < 0.0001), representing a median 4.6-fold (3.6- to 6.2-fold) increase. Two patients had low (

Summary Background The true frequency of deep-vein thrombosis (DVT) during long-haul air travel is unknown. We sought to determine the frequency of DVT in the lower limb during longhaul economy-class air travel and the efficacy of graduated elastic compression stockings in its prevention. Methods We recruited 89 male and 142 female passengers over 50 years of age with no history of thromboembolic problems. Passengers were randomly allocated to one of two groups: one group wore class-I below-knee graduated elastic compression stockings, the other group did not. All the passengers made journeys lasting more than 8 h per flight (median total duration 24 h), returning to the UK within 6 weeks. Duplex ultrasonography was used to assess the deep veins before and after travel. Blood samples were analysed for two specific common gene mutations, factor V Leiden (FVL) and prothrombin G20210A (PGM), which predispose to venous thromboembolism. A sensitive Ddimer assay was used to screen for the development of recent thrombosis. Findings 12/116 passengers (10%; 95% CI 4·8–16·0%) developed symptomless DVT in the calf (five men, seven women). None of these passengers wore elastic compression stockings, and two were heterozygous for FVL. Four further patients who wore elastic compression stockings, had varicose veins and developed superficial thrombophlebitis. One of these passengers was heterozygous for both FVL and PGM. None of the passengers who wore class-I compression stockings developed DVT (95% CI 0–3·2%). Interpretation We conclude that symptomless DVT might occur in up to 10% of long-haul airline travellers. Wearing of elastic compression stockings during long-haul air travel is associated with a reduction in symptomless DVT.

Antiplatelet drugs are used in a wide range of disorders, either as sole agents or as adjuncts to other therapies. Aspirin has been shown to be clinically effective in a number of ischaemic conditions and has been in use for many years. The newer agents, ticlopidine and clopidogrel (which are thought to inhibit ADP-mediated platelet reactions) are also effective and may prove to be superior to aspirin in certain indications. However, ticlopidine in particular has a different spectrum of side-effects, which may eventually limit its widespread use. The Gp IIb/IIIa antagonists have been most extensively investigated in the acute coronary syndromes, and shown to significantly improve outcome. Most of these studies have utilized agents which need to be given parenterally, and subsequently oral compounds are currently being developed. A number of other antiplatelet drugs such as prostacyclin and its analogues, as well as thromboxane inhibitors have been studied over the years, but overall they have failed to demonstrate any real clinical advantage.

Endotoxin is a powerful trigger of systemic inflammation. Since cardiac surgery exposes patients to endotoxemia, this study was set up to define the relationship between preoperative endogenous endotoxin immune status, gut perfusion, and outcome following cardiac valve replacement surgery.Observational study.University hospital.Fifty-nine consecutive patients undergoing cardiac valve replacement.Blood was assayed for IgG and IgM endotoxin core antibody (EndoCAb) levels preoperatively, immediately postoperatively, and at 4 h and 24 h postoperatively. Intraoperative gut mucosal perfusion was assessed using gastric tonometry. Complications were assessed for groups above and below the median EndoCAb value of a healthy population (100 median units micro/mL). Of the 59 patients, 12 developed at least one of a set of predefined complications. Of these 12, all had preoperative levels of IgM EndoCAb below 100 MU/mL (p0.025). Eleven had IgG EndoCAb levels below 100 MU/mL (0.05p0.1). There was no relationship between the fall in gastric intramucosal pH and exposure to endotoxin as implied by the fall in unbound IgM EndoCAb levels, although the specificity of tonometry for predicting complications could be improved by considering the patient's preoperative EndoCAb status.Preoperative EndoCAb levels were related to poor outcome following cardiac surgery and may be used to improve the specificity of GI tonometry in predicting postoperative complications.

Summary XR5118, a diketopiperazine-based low molecular weight inhibitor of plasminogen activator inhibitor-1 (PAI-1) activity, was studied ex vivo and in vivo in the rat to determine whether inhibition of PAI-1 activity resulted in increased fibrinolysis and protection against thrombus formation. XR5118 reversed the inhibitory effects of human PAI-1 against tissue-type plasminogen activator (tPA), in an vitro amidolytic assay (S2251) with an IC50 value of 3.5 μM±0.19 μM (n=7). This activity was confirmed in in vitro fibrinolysis assays against both human and rat PAI-1 and, following intravenous administration to rats, XR5118 (1–5mg/kg) dose-dependently increased clot lysis in an ex vivo dilute blood clot lysis time (DBCLT) assay. At 5 mg/kg, XR5118 increased clot lysis by 41±1.6% (n=39, P

Recently methods have become available to assay the haemostasis proteins tissue factor pathway inhibitor (TFPI) and activated factor VII (FVIIa). These assays are primarily used in research and in some studies patients may be receiving heparin therapy. We investigated the effect of heparin and its neutralisation by protamine sulphate, hexadimethrine bromide (polybrene) and triethylaminoethyl (TEAE) cellulose, on functional assays for TFPI, FVIIa and also factor VII (FVII). In the clotting assay for FVIIa using truncated recombinant tissue factor, heparin had little effect up to IU/ml, but concentrations higher than this grossly prolonged the clotting time. Protamine and polybrene neutralisation of heparin resulted in some prolongation of the clotting time despite adequate heparin neutralisation, and in addition, in the absence of heparin each of these substances themselves affected the clotting time. TEAE neutralisation of heparin appeared to be effective in the FVIIa assay, although in the absence of heparin we observed a 5% decrease in the clotting time. In the amidolytic substrate assays for TFPI and FVII, heparin with or without neutralisation resulted in only small changes in the optical density at 405nm and hence plasma levels of these factors were not significantly changed.

Flow Cytometry has now been successfully incorporated into automated blood cell counters to allow routine reporting of five cell differentials of up to 100 samples per hour. Sophisticated flagging of abnormal red cell, platelet and white cell parameters will reduce film inspection to under 10% of the total peripheral blood counts. Using the flow cytometer a routine reticulocyte count could also be incorporated into the standard print out. Reticulocyte RNA is stained with thiazole orange and according to staining intensity reticulocyte maturation indexes could be produced which may enable prediction of recovery from aplastic events. As well as the standard lymphocyte count, lymphocyte subset analysis particularly of T4 and T8 counts can also be incorporated into a routine report. This will become an increasing necessity for monitoring chronic viral diseases such as HIV positive conditions. Further subset analysis of the T8 lymphocyte populations including division of the T8 population into cytotoxic killer cells/suppressor cells can also be performed by using specific labelled monoclonal antibodies. For diagnostic investigation of haemotological malignancies in both peripheral blood and bone marrow samples several additional fluorescent monoclonal antibodies can be used in the flow cytometer as appropriate. This will also allow monitoring of response to intensive treatment. For the investigation of thrombocytopenia or neutropenia cell bound associated immunoglobulin can be quantified by a labelling index and specific cell fluorescence. Thus immune meditated thrombocytopenia and neutropenia can be rapidly diagnosed and the response to treatment monitored. All these applications should be possible by the end of the decade with a single automated blood cell counter incorporating versatile flow cytometry.

The initial problem is to clinically recognize a bleeding tendency and then to define the defect by specific laboratory tests before the apropriate prophylactic or therapeutic measures can be instigated