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Essentials New VWF activity assays are increasingly used but information on their comparability is limited. This is an ISTH SSC-organized study (expert labs, 5 countries) to compare all available assays. VWF activity by six assays correlated well with each other. The new assays show improved characteristics - minor differences are noted. SUMMARY: Background Several new assays have become available to measure von Willebrand factor (VWF) activity. The new assays appear to have improved performance characteristics compared with the old reference standard, ristocetin cofactor activity (VWF:RCo), but information is limited about how they compare with VWF:RCo and each other. Methods The von Willebrand factor Subcommittee of the International Society for Thrombosis and Haemostasis (ISTH) Scientific and Standardization Committee (SSC) designed a collaborative study involving expert laboratories from several countries to compare available tests with each other and with VWF:RCo. Eight laboratories from five countries were provided with blinded samples from normal healthy individuals and well-characterized clinical cases. Laboratories measured VWF activity using all tests available to them; data from six laboratories, not affected by thawing during transportation, are included in this study. Results All tests correlated well with VWF:RCo activity (r-values ranged from 0.963 to 0.989). Slightly steeper regression lines for VWF:Ab and VWF:GPIbM were clinically insignificant. The new assays showed improved performance characteristics. Of the commercially available assays, the VWF:GPIbR using the AcuStar system was the most sensitive and could reliably detect VWF activity below 1 IU dL-1 . The lower limit of the measuring interval for the VWF:GPIbM and the VWF:GPIbR assays was in the 3-4 and 3-6 IU dL-1 range, respectively. Inter-laboratory variation was also improved for most new assays. Conclusion All VWF activity assays correlated well with each other and the VWF:RCo assay. The slight differences in characteristics found in the COMPASS-VWF study will assist the VWF community in interpreting and comparing activity results.
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INTRODUCTION: Rivaroxaban is non-inferior to warfarin for the treatment of venous thromboembolism, with regard to clinical efficacy and safety. The ex-vivo effects of warfarin versus therapeutic dose rivaroxaban on in-vivo markers of coagulation activation and thrombin generation remain undefined. The aim of this study was to compare the effects of warfarin and therapeutic dose rivaroxaban on ex-vivo thrombin generation (TG), and the in-vivo markers of coagulation activation, prothrombin fragment 1.2 (F1.2), thrombin-antithrombin complex (TAT), and D-dimer. METHODS: Eighty-five patients with venous thromboembolism were studied, 45 on warfarin, target INR 2.5 and 40 on rivaroxaban 20mg once daily. RESULTS: Anticoagulation was in therapeutic range in 71% (32/45) warfarin and 65% (26/40) rivaroxaban treated patients. 8 patients on warfarin and 9 patients on rivaroxaban had subtherapeutic INR and rivaroxaban levels respectively. Both rivaroxaban and warfarin reduced endogenous thrombin potential (ETP) and peak thrombin, and prolonged lag time and time to peak, compared to normal controls (p<0.0001). The lag time and time to peak TG were longer, and peak thrombin was lower in patients receiving rivaroxaban (p<0.0001) compared with warfarin, although warfarin-treated patients had lower ETP (p=0.0008). In-vivo coagulation activation markers were within the normal ranges in all rivaroxaban-treated patients (including those with levels considered to be subtherapeutic) and in 37/45 warfarin-treated patients who had an INR≥2.0. The warfarin-treated patients with subtherapeutic INRs exhibited slightly raised F1.2 and/or TAT. CONCLUSION: In conclusion, both rivaroxaban and warfarin provided effective anticoagulation, as assessed by inhibition of TG and makers of in-vivo coagulation activation.
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Anticoagulantes/uso terapéutico , Inhibidores del Factor Xa/uso terapéutico , Relación Normalizada Internacional/métodos , Morfolinas/uso terapéutico , Tiofenos/uso terapéutico , Tromboembolia Venosa/tratamiento farmacológico , Adulto , Anciano , Coagulación Sanguínea/efectos de los fármacos , Femenino , Humanos , Masculino , Persona de Mediana Edad , Rivaroxabán , Warfarina/uso terapéuticoRESUMEN
BACKGROUND: Antiphospholipid antibodies may interfere with the anticoagulant activity of activated protein C (APC) to induce acquired APC resistance (APCr). AIMS: To investigate the frequency and characteristics of APCr by using recombinant human APC (rhAPC) and endogenous protein C activation in antiphospholipid syndrome (APS). METHODS: APCr was assessed in APS and non-APS venous thromboembolism (VTE) patients on warfarin and normal controls with rhAPC or Protac by thrombin generation. IgG anti-protein C and anti-protein S antibodies and avidity were assessed by ELISA. RESULTS: APS patients showed greater resistance to both rhAPC and Protac than non-APS patients and normal controls (median normalized endogenous thrombin potential inhibition): APS patients with rhAPC, 81.3% (95% confidence interval [CI] 75.2-88.3%; non-APS patients with rhAPC, 97.7% (95% CI 93.6-101.8%; APS patients with Protac, 66.0% (95% CI 59.5-72.6%); and non-APS patients with Protac, 80.7 (95% CI 74.2-87.2%). APS patients also had a higher frequency and higher levels of anti-protein C antibodies, with 60% (15/25) high-avidity antibodies. High-avidity anti-protein C antibodies were associated with greater APCr and with a severe thrombotic phenotype (defined as the development of recurrent VTE while patients were receiving therapeutic anticoagulation or both venous and arterial thrombosis). Twelve of 15 (80%) patients with high-avidity anti-protein C antibodies were classified as APS category I. CONCLUSION: Thrombotic APS patients showed greater APCr to both rhAPC and activation of endogenous protein C by Protac. High-avidity anti-protein C antibodies, associated with greater APCr, may provide a marker for a severe thrombotic phenotype in APS. However, in patients with category I APS, it remains to be established whether anti-protein C or anti-ß2 -glycoprotein I antibodies are responsible for APCr.
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Resistencia a la Proteína C Activada/inmunología , Anticuerpos Antifosfolípidos/sangre , Síndrome Antifosfolípido/complicaciones , Proteína C/inmunología , Tromboembolia Venosa/inmunología , Resistencia a la Proteína C Activada/sangre , Resistencia a la Proteína C Activada/diagnóstico , Resistencia a la Proteína C Activada/tratamiento farmacológico , Adulto , Anciano , Anticoagulantes/uso terapéutico , Síndrome Antifosfolípido/sangre , Síndrome Antifosfolípido/diagnóstico , Síndrome Antifosfolípido/tratamiento farmacológico , Síndrome Antifosfolípido/inmunología , Biomarcadores/sangre , Pruebas de Coagulación Sanguínea , Estudios de Casos y Controles , Estudios Transversales , Ensayo de Inmunoadsorción Enzimática , Femenino , Fibrinolíticos/uso terapéutico , Humanos , Péptidos y Proteínas de Señalización Intercelular , Masculino , Persona de Mediana Edad , Péptidos/uso terapéutico , Fenotipo , Proteína C/uso terapéutico , Proteínas Recombinantes/uso terapéutico , Índice de Severidad de la Enfermedad , Tromboembolia Venosa/sangre , Tromboembolia Venosa/diagnóstico , Tromboembolia Venosa/prevención & control , Warfarina/uso terapéuticoAsunto(s)
Anticoagulantes/uso terapéutico , Coagulación Sanguínea/efectos de los fármacos , Monitoreo de Drogas/métodos , Morfolinas/uso terapéutico , Tiempo de Protrombina , Tiofenos/uso terapéutico , Tromboplastina , Adulto , Anciano , Anticoagulantes/sangre , Anticoagulantes/farmacocinética , Femenino , Humanos , Masculino , Persona de Mediana Edad , Morfolinas/sangre , Morfolinas/farmacocinética , Valor Predictivo de las Pruebas , Reproducibilidad de los Resultados , Rivaroxabán , Tiofenos/sangre , Tiofenos/farmacocinéticaRESUMEN
Pregnancy is associated with significant haemostatic changes, with a progressive rise in most clotting factors. There is limited data on the changes of factor XIII (FXIII) level during pregnancy. This study assesses changes in FXIII activity during normal pregnancy and establish FXIII reference range during each trimester of pregnancy and immediate postnatal period. This is a cross sectional study of 376 women with normal uneventful pregnancies. Plasma FXIII activity was measured during first (weeks 0-12, n = 116), second (weeks 13-28, n = 132), third trimester (weeks 29-42, n = 128) and postnatal (day 0-3; n = 30). Samples were also collected from non-pregnant women (n = 25) as a control group. FXIII was assayed on CS-5100 analyser using chromogenic reagent. The mean ± SD FXIII activity was 112 ± 29 IU dL(-1) during first trimester, 96 ± 26 IU dL(-1) during second trimester, 83 ± 21 IU dL(-1) during third trimester, 90 ± 19 IU dL(-1) during postnatal period, and 113 ± 26 IU dL(-1) in the control. The reference range was calculated during the first (55-169 IU dL(-1)), second (45-147 IU dL(-1)), third trimester (42-125 IU dL(-1)) and postnatal period (61-137 IU dL(-1)). There was a significant reduction in the mean FXIII activity during the second and third trimester compared to the first trimester and control group (P < 0.0001). During the immediate postnatal period, the mean FXIII activity was not statistically different compared to the third and second trimester levels but was significantly lower compared to the first trimester (P < 0.0001) level and the control group (P = 0.0002). This study establishes the reference range for FXIII activity during the three trimesters of normal pregnancy and immediate postnatal period. Women have a significantly decreased level of FXIII activity during a normal uneventful pregnancy.
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Factor XIII/metabolismo , Trimestres del Embarazo/sangre , Adulto , Estudios Transversales , Femenino , Humanos , Persona de Mediana Edad , Embarazo , Resultado del Embarazo , Valores de Referencia , Factores de Riesgo , Adulto JovenRESUMEN
INTRODUCTION: The investigation of platelet function by aggregometry requires specialist equipment and is labour intensive. We have developed an automated platelet aggregation method on a routine coagulation analyser. METHODS: We used a CS-2000i (Sysmex) with prototype software to perform aggregation in platelet-rich plasma (PRP), using the following agonists: ADP (0.5-10 µm), epinephrine (0.5-10 µm), collagen (0.5-10 mg/µL), ristocetin (0.75-1.25 mg/mL) and arachidonic acid (0.12-1.0 mm). Platelet agonists were from Hyphen Biomed, and an AggRAM aggregometer (Helena Biosciences) was used as the reference instrument. RESULTS: CS-2000i reaction cuvette stirrer speed was found to influence reaction sensitivity and was optimized to 800 rpm. There were no clinically significant changes in aggregation response when the PRP platelet count was 150-480 x 10(9) /L, but below this there were changes in the maximum amplitude (MA) and slope (rate). Dose response with each of the agonists was comparable between CS-2000i and an AggRAM aggregometer and normal subjects receiving antiplatelet drugs. Aggregation imprecision was similar on both the CS-2000i and AggRAM systems, with a cv for 2-5 µm ADP MA and slope varying between 3-12%. CONCLUSION: Our preliminary studies indicated that optimal sensitivity using the CS-2000i was obtained with a reaction cuvette stirrer speed of 800 rpm and a PRP platelet count of 200-300 x 10(9) /L; aggregation with a PRP count <100 x 10(9) /L showed poor sensitivity. Imprecision and detection of antiplatelet drug effects was similar between the CS-2000i and AggRAM. These data demonstrate that CS-2000i is comparable to a stand-alone aggregometer, although CS-2000i has the advantages of walk-away technology and also required a smaller sample volume than the AggRAM (44% less).
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Automatización de Laboratorios/normas , Plaquetas/efectos de los fármacos , Agregación Plaquetaria/efectos de los fármacos , Adenosina Difosfato/farmacología , Automatización de Laboratorios/instrumentación , Células Cultivadas , Colágeno/farmacología , Epinefrina/farmacología , Humanos , Pruebas de Función Plaquetaria , Ristocetina/farmacología , Sensibilidad y EspecificidadRESUMEN
von Willebrand disease (VWD) is caused by a quantitative and/or qualitative deficiency of the von Willebrand factor (VWF). The laboratory diagnosis of VWD is dependent on the measurement of VWF antigen (VWF:Ag) and ristocetin cofactor activity (VWF:RCo). The aim of this study was to undertake a two-centre evaluation of two new automated VWF:Ag and VWF:RCo assays systems from Instrumentation Laboratory (Bedford, USA). Using the two new analytical systems that operated with different detection principles: immunoturbidimetric (TOP500 analyser) and chemiluminescent (AcuStar analyser), VWF:Ag and VWF:RCo levels were determined in samples from 171 healthy normal subjects, 80 VWD patients (16 type 1, 58 type 2 and 6 type 3) and 7 acquired von Willebrand syndrome patients. With commercial lyophilized normal and pathological plasmas VWF: Ag and VWF:RCo assays performed on both analysers exhibited low levels of inter-assay imprecision (AcuStar: CV% range 3.3-6.9; TOP500: CV% range 2.6-6.3). Samples from normal healthy subjects (range: VWF:Ag 44.6-173.9 IU dL(-1) ; VWF:RCo 43.1-191.5 IU dL(-1)) and patients (range: VWF:Ag <0.3-115.1 IU dL(-1) ; VWF:RCo <0.5-57.2 IU dL(-1)) showed a good correlation between the two VWF:Ag and VWF:RCo methods (rs = 0.92 and 0.82 respectively), with only a few inconsistent cases among the patients' samples evaluated. The chemiluminescent assays had a lower limit of detection for both VWF:Ag and VWF:RCo compared to immunoturbidimetric tests (0.3 IU dL(-1) vs. 2.2 IU dL(-1) and 0.5 IU dL(-1) vs. 4.4 IU dL(-1) respectively). The TOP500 and AcuStar VWF:Ag and VWF:RCo assays were precise and compare well between centres, making these systems suitable for the diagnosis of VWD in non-specialized and reference laboratories.
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Pruebas de Coagulación Sanguínea/métodos , Ristocetina/metabolismo , Factor de von Willebrand/metabolismo , Pruebas de Coagulación Sanguínea/instrumentación , Humanos , Reproducibilidad de los Resultados , Ristocetina/sangre , Sensibilidad y Especificidad , Enfermedades de von Willebrand/sangre , Enfermedades de von Willebrand/diagnóstico , Factor de von Willebrand/inmunologíaRESUMEN
INTRODUCTION: The Clinical and Laboratory Standards Institute (CLSI) has produced a guideline detailing how to determine the activated partial thromboplastin time's (APTT) sensitivity to clotting factor deficiencies, by mixing normal and deficient plasmas. Using the guideline, we determined the factor sensitivity of two APTT reagents. METHODS: APTTs were performed using Actin FS and Actin FSL on a Sysmex CS-5100 analyser. The quality of factor-deficient and reference plasmas from three commercial sources was assessed by assaying each of the clotting factors within the plasmas and by performing thrombin generation tests (TGT). RESULTS: Testing samples from 50 normal healthy subjects gave a two-standard deviation range of 21.8-29.2 s for Actin FS and 23.5-29.3 s for Actin FSL. The upper limits of these ranges were subsequently used to determine APTT factor sensitivity. Assay of factor levels within the deficient plasmas demonstrated that they were specifically deficient in a single factor, with most other factors in the range 50-150 iu/dL (Technoclone factor VII-deficient plasma has 26 iu/dL factor IX). APTTs performed on mixtures of normal and deficient plasmas gave diverse sensitivity to factor deficiencies dependent on the sources of deficient plasma. TGT studies on the deficient plasmas revealed that the potential to generate thrombin was not solely associated with the levels of their component clotting factors. CONCLUSION: Determination of APTT factor sensitivity in accordance with the CLSI guideline can give inconsistent and misleading results.
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Tiempo de Tromboplastina Parcial/normas , Factores de Coagulación Sanguínea/metabolismo , Pruebas de Coagulación Sanguínea/métodos , Pruebas de Coagulación Sanguínea/normas , Humanos , Tiempo de Tromboplastina Parcial/métodos , Guías de Práctica Clínica como Asunto , Juego de Reactivos para Diagnóstico , Valores de Referencia , Sensibilidad y EspecificidadRESUMEN
The ristocetin cofactor assay (VWF:RCo) is the reference method for assessing von Willebrand factor (VWF) activity in the diagnosis of von Willebrand's Disease (VWD). However, the assay suffers from poor reproducibility and sensitivity at low levels of VWF and is labour intensive. We have undertaken an evaluation of a new immunoturbidimetric VWF activity (VWF:Ac) assay (INNOVANCE(®) VWF Ac. Siemens Healthcare Diagnostics, Marburg, Germany) relative to an established platelet-based VWF:RCo method. Samples from 50 healthy normal subjects, 80 patients with VWD and 50 samples that exhibited 'HIL' (i.e. Haemolysis, Icterus or Lipaemia) were studied. VWF:Ac, VWF:RCo and VWF:Ag were performed on a CS-analyser (Sysmex UK Ltd, Milton Keynes, UK), all reagents were from Siemens Healthcare Diagnostics. The VWF:Ac assay, gave low intra- and inter-assay imprecision (over a 31-day period, n = 200 replicate readings) using commercial normal (Mean 96.2 IU dL(-1), CV < 3.0%) and pathological (Mean 36.1 IU dL(-1), CV < 3.5%) control plasmas. The normal and clinical samples exhibited good correlation between VWF:RCo (range 3-753 IU dL(-1)) and VWF:Ac (rs = 0.97, P < 0.0001), with a mean bias of 5.6 IU dL(-1). Ratios of VWF:Ac and VWF:RCo to VWF:Ag in the VWD samples were comparable, although VWF:Ac had a superior lower level of detection to that of VWF:RCo (3% and 5% respectively). A subset (n = 97) of VWD and HIL samples were analysed for VWF:Ac at two different dilutions to assess the effect on relative potency, no significant difference was observed (P = 0.111). The INNOVANCE(®) VWF Ac assay was shown to be reliable and precise.
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Ensayo de Inmunoadsorción Enzimática/métodos , Enfermedades de von Willebrand/diagnóstico , Factor de von Willebrand/análisis , Anticuerpos Monoclonales , Humanos , Receptores de GABA-B/metabolismo , Reproducibilidad de los ResultadosRESUMEN
INTRODUCTION: 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. PATIENTS AND METHODS: 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. RESULTS: Patients' FVIII:C levels and FVIII:Ch, exhibited good agreement (rs=0.94; p<0.0001), 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 FVIII>200 iu/dL. Furthermore elevated levels of FVIII and increased thrombin generation parameters appeared to be consistent over time. CONCLUSION: 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.
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Factor VIII/análisis , Trombina/análisis , Tromboembolia Venosa/sangre , Tromboembolia Venosa/epidemiología , Adulto , Anciano , Anticoagulantes/uso terapéutico , Biomarcadores/sangre , Estudios de Cohortes , Femenino , Humanos , Masculino , Persona de Mediana Edad , Prevalencia , Estudios Prospectivos , Medición de Riesgo , Factores de Riesgo , Reino Unido/epidemiología , Tromboembolia Venosa/tratamiento farmacológico , Adulto JovenRESUMEN
INTRODUCTION: 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 INRs >4.5; 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. METHODS: 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. RESULTS: There was good correlation between POC (INR ≥4.5, <8.0) and Lab INRs (rTF n=154, rs=0.87, p<0.0001; RBT n=102, rs=0.76, p<0.0001); and significant correlations between each of the VKD-CF and the INR, the strongest being with FVII (POC INR rs=-0.53 p<0.0001; Lab rTF-INR rs=-0.70 p<0.0001). 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. CONCLUSIONS: Our data suggest that CoaguChek XS Plus INRs >4.5 and <8.0 are comparable to laboratory INRs (both methods) and it is probably unnecessary to perform laboratory INRs for clinical management of patients with INRs >4.5 including those >8.0.
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Anticoagulantes/administración & dosificación , Relación Normalizada Internacional/métodos , Sistemas de Atención de Punto , Tiempo de Protrombina/métodos , Warfarina/administración & dosificación , Adulto , Anciano , Anciano de 80 o más Años , Animales , Factores de Coagulación Sanguínea/metabolismo , Femenino , Humanos , Masculino , Persona de Mediana Edad , Conejos , Trombina/metabolismo , Adulto JovenRESUMEN
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 (â¼20 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, <10-50 IU dL(-1)). The CS-2000i results exhibited no clinically significant variation between batches (mean cv = 7%). The automated VWF:RCo assay offers a more sensitive, reproducible, robust and less laborious alternative to standard aggregometry.
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Pruebas de Coagulación Sanguínea/métodos , Enfermedades de von Willebrand/diagnóstico , Factor de von Willebrand/análisis , Humanos , Reproducibilidad de los Resultados , Sensibilidad y Especificidad , Enfermedades de von Willebrand/sangreRESUMEN
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.
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Proteínas Sanguíneas/análisis , Hemostáticos/análisis , Plasma/química , Priones , Cromatografía de Afinidad/métodos , HumanosRESUMEN
BACKGROUND: The clot solubility test is the most widely used method for detection of factor (F)XIII deficiency. However, it will only detect severe deficiencies; consequently mild deficiencies and heterozygous states are probably under diagnosed. OBJECTIVE: As an alternative first-line screening test, we assessed an automated quantitative ammonia release assay (QARA). PATIENTS/METHODS: Inter-assay imprecision was evaluated with commercial normal and pathological control plasmas (10 replicates on each of 5 days). Using the QARA and other commercial assays a comparative assessment of congenital (FXIII range < 1-70 u dL(-1), n = 9) and acquired (n = 43) deficiencies was made. We also investigated the prevalence of acquired deficiencies in hospitalized patients using residual samples from adult patients (n = 1004) and from a paediatric intensive care unit (ICU, n = 56). RESULTS: Assay imprecision was acceptably low (normal control: mean 86.6 u dL(-1); cv = 2.0%; pathological control: mean 27.5 u dL(-1); cv = 3.8%). Using an iodoacetamide blanking procedure, the QARA results (FXIII range < 1-70 u dL(-1)) exhibited close agreement with those from an immuno-turbidometric FXIII A-subunit (FXIII-A) method. There was also good correlation (R(2) ≥ 0.89) between the QARA (range 20-180 u dL(-1)), a second chromogenic assay, the FXIII-A and FXIII A+B-subunit ELISA. We found that 21% of samples from adult patients had FXIII levels < 70 u dL(-1) (mean normal ± 2 SD 73-161 u dL(-1)) with 6% < 50 u dL(-1). Within the paediatric ICU samples, 52% were < 70 u dL(-1), with 21% < 50 u dL(-1). CONCLUSIONS: Our data demonstrates that the automated assay is sensitive, highly reproducible and the results from clinical samples suggest that acquired FXIII deficiency is a relatively common phenomenon in hospital patients after surgery and in ICU.
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Química Clínica/métodos , Deficiencia del Factor XIII/sangre , Deficiencia del Factor XIII/diagnóstico , Factor XIII/análisis , Adulto , Automatización , Niño , Estudios de Cohortes , Heterocigoto , Humanos , Sistema Inmunológico , Unidades de Cuidados Intensivos , Nefelometría y Turbidimetría/métodos , Prevalencia , Reproducibilidad de los Resultados , Cicatrización de HeridasRESUMEN
BACKGROUND: We have previously shown that fresh-frozen plasma (FFP) contains red blood cell-derived procoagulant microparticles (MPs) that are removable by 0.2 microm filtration. Given the limitations of current methods for accurately sizing MPs, we have applied the novel approach of dynamic light scattering (DLS) to characterize the size distributions of these MPs within FFP. METHODS: Fresh-frozen plasma was prepared from blood Group A and O donations (n = 10 of each) after an overnight hold of whole blood at 4 degrees C. On the day of analysis, plasma was thawed to 37 degrees C and daughter aliquots were studied pre- and post-filtration (0.2 microm filtration device, Ceveron MFU-500, Technoclone). MP size and dispersity was assessed using a Zetasizer Nano S (Malvern Instruments Ltd), which employs a 173 degrees backscatter detector and an N5 Submicron Particle Size Analyser (Beckman Coulter) using multi-angle measurements (30.1 degrees , 62.6 degrees and 90 degrees ). The analysers presented MP size distribution graphically as intensity plots, mean size, standard deviation and polydispersity index. RESULTS: Of the instruments used, only the N5 utilizing a 30.1 degrees angle of measurement could detect MPs of the expected size distribution and demonstrate their removal by filtration. MPs (range of mean particle diameters: pre, 101-464 nm; post, 21-182 nm filtration) were significantly smaller post-filtration (P < 0.0001), but polydispersity index (median: pre, 0.746, post, 0.769) exhibited no significant change. There was no significant difference between the size of MPs from blood Group O (pre, 247 nm) and Group A (pre, 289 nm) samples (P = 0.44). CONCLUSION: Our data demonstrates that DLS offers a novel approach to assessing MP size and distribution, a technique that could be easily adopted as a means of assessing MPs within either FFP or other blood products.
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Micropartículas Derivadas de Células/química , Luz , Tamaño de la Partícula , Plasma/química , Dispersión de Radiación , HumanosRESUMEN
Autoantibodies to ADAMTS13 (a disintegrin-like and metalloprotease with thrombospondin type I motif, member 13) play an important role in the development of microthrombosis in thrombotic thrombocytopenic purpura (TTP). In severe cases of antiphospholipid syndrome (APS), microthrombosis can occur similar to that seen in TTP, suggesting possible mutual pathogenic factors. However, the role of ADAMTS13 in APS is unknown. We hypothesised that aberrations in ADAMTS13 may occur in APS and evaluated ADAMTS13 and von Willebrand factor (VWF) in 68 patients with antiphospholipid antibodies (aPA) including 52 with APS. Thirty-three (49%) had IgG anti-ADAMTS13 with 12 of these patients having reduced ADAMTS13 activity, suggesting neutralising antibodies. Low ADAMTS13 activity (median 34%) was demonstrated in 22/68 (33%), all with normal ADAMTS13 antigen levels consistent with dysfunctional ADAMTS13. Reduced ADAMTS13 activity was not secondary to elevated von Willebrand factor (VWF), or increased VWF secretion (normal VWF propeptide), although a reduced VWF clearance was noted in APS. Analysis found no associations between the ADAMTS13 abnormalities and any aPA profile or thrombotic/obstetric complications, although this study was not adequately powered to address clinical associations. Nevertheless, these findings highlight that ADAMTS13 autoantibodies and ADAMTS13 dysfunction can occur in APS, and although the clinical significance remains undetermined, ADAMTS13 dysfunction may be contributory to thrombogenesis in autoimmune conditions other than TTP.
Asunto(s)
Proteínas ADAM/inmunología , Síndrome Antifosfolípido/inmunología , Autoanticuerpos/sangre , Factor de von Willebrand/metabolismo , Proteínas ADAM/sangre , Proteínas ADAM/fisiología , Proteína ADAMTS13 , Adulto , Anciano , Femenino , Semivida , Humanos , Inmunoglobulina G/sangre , Masculino , Persona de Mediana EdadRESUMEN
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 degrees 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 r(s)> or = 0.22, P< or = 0.003; peak thrombin r(s)> or = 0.48, P< or = 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.
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Coagulación Sanguínea/fisiología , Factor VIII/análisis , Plasma/fisiología , Humanos , Plasma/química , Control de Calidad , TromboelastografíaRESUMEN
BACKGROUND: We have previously demonstrated that clot formation in fresh-frozen plasma (FFP) is influenced by the presence of microparticles (MP). In this study, the cellular source(s), properties and influence of MPs on clot formation within FFP were further characterized. METHODS: Fresh-frozen plasma was prepared after an overnight hold of whole blood at 4 degrees C. We examined the effect of a 0.2 microm filtration device designed to remove cellular MPs on thrombin generation test (TGT) and Thrombelastography (TEG(R)) as well as clotting factors and physiological inhibitors: prothrombin time (PT); activated partial thromboplastin time (APTT), fibrinogen (Fg), factor VIII (FVIII), von Willebrand factor antigen (VWF:Ag), antithrombin III (AT-III) and protein C (PC). MPs were measured using a functional assay and also by flow cytometry. RESULTS: Microparticle levels by functional assay were reduced by filtration (pre- 5.11 vs. post- 4.43 nmol/l phosphatidylserine equivalent, P < 0.0001). Flow cytometry showed that the most numerous MPs were derived from red blood cells, with ~87% binding annexin V, most of which (94%) were removed by filtration. MP removal had minimal effect on the PT, APTT, Fg, VWF:Ag, AT-III or PC or FVIII, but a major effect on TGT (endogenous thrombin potential: pre- 1722 vs. post- 990 nM thrombin, P < 0.0001; peak thrombin: pre- 91 vs. post- 44 nM thrombin, P < 0.0001), which in turn reflected the changes seen in TEG(R), where post-filtration clots started forming more slowly and the rate of clot formation was reduced. CONCLUSION: These data suggest that MPs contribute towards clot formation in FFP.
Asunto(s)
Coagulación Sanguínea , Micropartículas Derivadas de Células/metabolismo , Plasma/metabolismo , Pruebas de Coagulación Sanguínea/métodos , Transfusión de Componentes Sanguíneos/métodos , Citometría de Flujo/métodos , HumanosRESUMEN
OBJECTIVE: The risk of stroke in patients with recently symptomatic carotid stenosis is considerably higher than in patients with asymptomatic stenosis. In the present study it was hypothesised that excessive platelet activation might partly contribute to this difference. METHODS: A full blood count was done and whole blood flow cytometry used to measure platelet surface expression of CD62P, CD63, and PAC1 binding and the percentage of leucocyte-platelet complexes in patients with acute (0-21 days, n = 19) and convalescent (79-365 days) symptomatic (n = 16) and asymptomatic (n = 16) severe (> or =70%) carotid stenosis. Most patients were treated with aspirin (37.5-300 mg daily) although alternative antithrombotic regimens were more commonly used in the symptomatic group. RESULTS: The mean platelet count was higher in patients with acute and convalescent symptomatic compared with asymptomatic carotid stenosis. There were no significant differences in the median percentage expression of CD62P and CD63, or PAC1 binding between the acute or convalescent symptomatic and asymptomatic patients. The median percentages of neutrophil-platelet (p = 0.004), monocyte-platelet (p = 0.046), and lymphocyte-platelet complexes (p = 0.02) were higher in acute symptomatic than in asymptomatic patients. In patients on aspirin monotherapy, the percentages of neutrophil-platelet and monocyte-platelet complexes (p = 0.03) were higher in acute symptomatic (n = 11) than asymptomatic patients (n = 14). In the convalescent phase, the median percentages of all leucocyte-platelet complexes in the symptomatic group dropped to levels similar to those found in the asymptomatic group. CONCLUSION: Increased platelet count and leucocyte-platelet complex formation may contribute to the early excess risk of stroke in patients with recently symptomatic carotid stenosis.
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Estenosis Carotídea/complicaciones , Estenosis Carotídea/fisiopatología , Activación Plaquetaria , Accidente Cerebrovascular/etiología , Accidente Cerebrovascular/fisiopatología , Enfermedad Aguda , Anciano , Aspirina/uso terapéutico , Femenino , Fibrinolíticos/uso terapéutico , Citometría de Flujo , Humanos , Leucocitos/fisiología , Masculino , Recuento de Plaquetas , Índice de Severidad de la EnfermedadRESUMEN
Replacement of normal levels of von Willebrand factor-cleaving protease (VWF:CP, ADAMTS13) activity from infused plasma is important in plasma exchange (PEX) for the treatment of thrombotic thrombocytopenic purpura (TTP) patients. We have studied the VWF:CP activity, VWF multimer distribution, VWF:Ag, protein S (PS) activity and free PS antigen levels in fresh frozen plasma (FFP), cryosupernatant (CSP) and virally inactivated components treated with methylene blue/light (MB) or solvent detergent (SD) processes. VWF:CP activity was normal in all components tested and was retained following overnight storage at room temperature. CSP and SD plasma contained reduced levels of the highest molecular weight VWF multimers. Protein S activity was reduced below the normal range in SD plasma, but within the normal range for the other components tested. Virally inactivated SD- and MB-treated plasma may be an effective alternative to FFP and CSP in PEX for TTP. Reduced PS activity in SD plasma may predispose to venous thromboembolism, especially if infused in large volumes.