RESUMEN
BACKGROUND: Coagulation activation is a hallmark of cancer, and anticoagulants have shown tumour-inhibiting properties. However, recent trials have failed to demonstrate improved survival with low-molecular-weight heparin (LMWH) in cancer populations. This has raised the question of suboptimal adherence as a possible explanation for the lack of benefit. Still, there is no standardised method to directly monitor LMWH in patient plasma. Here, we directly determine LMWH levels in patients using the Heparin Red assay to objectively assess adherence and how this associates with the patient outcome in the RASTEN trial. METHODS: RASTEN is a multicentre, randomised phase III trial investigating if the addition of LMWH to standard therapy can improve survival in small-cell lung cancer. LMWH was measured in plasma (N = 258) by the Heparin Red assay and compared with the anti-factor Xa (anti-FXa) activity assay. RESULTS: Both methods could differentiate patients in the LMWH arm from the control arm and patients receiving therapeutic LMWH owing to thrombosis. Receiver Operating Characteristic (ROC) analysis yielded adherence rates of 85% for anti-FXa and 68% for Heparin Red. No survival benefits were found in the adherent subgroup compared with the control arm (hazard ratio [HR]: 1.26; 95% confidence interval [CI]: 0.95-1.67; P = 0.105 and HR: 1.19; 95% CI: 0.89-1.60; P = 0.248 for anti-FXa and Heparin Red, respectively). Heparin Red could define patients with high probability of adherence to LMWH treatment, which warrants prospective studies for further validation. Our finding that the LMWH-adherent subpopulation did not show improved survival excludes that the negative outcome of RASTEN was due to poor adherence.
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Anticoagulantes/administración & dosificación , Protocolos de Quimioterapia Combinada Antineoplásica/uso terapéutico , Coagulación Sanguínea/efectos de los fármacos , Enoxaparina/administración & dosificación , Neoplasias Pulmonares/tratamiento farmacológico , Cumplimiento de la Medicación , Carcinoma Pulmonar de Células Pequeñas/tratamiento farmacológico , Trombosis/tratamiento farmacológico , Anciano , Anticoagulantes/efectos adversos , Anticoagulantes/sangre , Protocolos de Quimioterapia Combinada Antineoplásica/efectos adversos , Pruebas de Coagulación Sanguínea , Monitoreo de Drogas , Enoxaparina/efectos adversos , Enoxaparina/sangre , Femenino , Humanos , Neoplasias Pulmonares/sangre , Neoplasias Pulmonares/mortalidad , Masculino , Persona de Mediana Edad , Factores de Riesgo , Carcinoma Pulmonar de Células Pequeñas/sangre , Carcinoma Pulmonar de Células Pequeñas/mortalidad , Suecia , Trombosis/sangre , Trombosis/mortalidad , Factores de Tiempo , Resultado del TratamientoRESUMEN
Haemophilia A and B diagnosis and disease severity classification are determined on the basis of results from factor VIII (FVIII) and factor FIX (FIX) activity assays, respectively. These assays are also used for potency labelling, postinfusion monitoring of factor replacement products and testing for FVIII/FIX inhibitors. This review focuses on activated partial thromboplastin time (APTT)-based one-stage assays (OSAs) and two-stage chromogenic substrate assays (CSAs). Currently, there is considerable inter-laboratory variability in the results obtained using OSAs, which can be intensified in a reagent-specific manner by the presence of the new modified recombinant factor replacement products that are entering the market. Furthermore, the use of CSAs, which tend to show less variability, especially with the new modified products, is recommended in a number of clinical scenarios. Clinical laboratories may, therefore, need to establish CSAs for routine use. In this review, we aim to improve understanding and help establish best practices by describing the methodology behind OSAs and CSAs and highlighting assay advantages and limitations. We argue that there can be value in offering both assay methodologies in clinical laboratories that contribute to the care of patients with haemophilia A or B. Educating both laboratory scientists and clinicians about the strengths and weaknesses of each type of assay will help to establish the necessary dialogue that is key to ensuring not only that the appropriate assays are used in the right clinical situations, but also that the results are interpreted correctly.
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Inhibidores de Factor de Coagulación Sanguínea/sangre , Factor IX/metabolismo , Factor VIII/metabolismo , Pruebas Hematológicas/métodos , Hemofilia A/sangre , Hemofilia B/sangre , Animales , HumanosRESUMEN
The treatment of patients with haemophilia A has remarkably improved over the years and the journey to a potential cure continues. Replacement therapy has been the cornerstone of treatment, and, despite major advances, the development of neutralizing antibodies, eg inhibitors, has thus far not been possible to avoid. How, and to what extent, the new non-factor-based options will modify treatment strategy and inhibitor risk is unclear and it is essential that every haemophilia treatment centre is linked to an educated and skilled laboratory performing the relevant inhibitor assays. The current review will focus on when to consider and how to perform inhibitor assay(s) in the management of patients with haemophilia A.
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Anticuerpos/inmunología , Hemofilia A/diagnóstico , Hemofilia A/inmunología , Laboratorios , Técnicas de Laboratorio Clínico , Hemofilia A/tratamiento farmacológico , Humanos , Control de CalidadRESUMEN
INTRODUCTION: Assay discrepancy in factor VIII activity between the one-stage and the chromogenic assays has been described in approximately one third of patients with non-severe haemophilia A. Whether assay discrepancy may also occur in patients with haemophilia B remains unknown. AIM: This study compared the results from the one-stage and the chromogenic assays in patients with haemophilia B. METHODS: Plasma samples from patients with haemophilia B attending the haemophilia centre in Malmö, Sweden, were collected after a wash-out period of more than 7 days and analysed with both assays. RESULTS: Fifty samples from 36 patients were analysed. No discrepancy was found in patients with severe haemophilia B. Among the 44 plasma samples from patients with non-severe disease, 15 showed a twofold or greater difference between the results of the two methods, with the chromogenic method presenting the higher value (mean FIX:Cone-stage 0.02 vs. FIX:Cchromo 0.06 IU mL-1 ). Of these 15 samples, 14 were from seven individuals from five families with the same mutated amino acid at the N-terminal cleaving site of the activation peptide (FIX: c.572G>A; p.Arg191His or FIX: c.571C>T; p.Arg191Cys). These mutations were not observed in any patients with non-discrepant results. The reported bleeding frequency for these patients was low and indicative of a mild bleeding phenotype. CONCLUSION: Our findings imply that assay discrepancy occurs for factor IX activity and that both type of assays are needed for a correct diagnosis and classification of haemophilia B. The underlying mechanism by which the mutation influences the assays remains to be determined.
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Pruebas de Coagulación Sanguínea/métodos , Compuestos Cromogénicos/metabolismo , Hemofilia B/sangre , Adolescente , Adulto , Anciano , Anciano de 80 o más Años , Factor IX/genética , Factor IX/metabolismo , Factor VIII/genética , Factor VIII/metabolismo , Femenino , Hemofilia B/genética , Humanos , Masculino , Persona de Mediana Edad , Mutación , Adulto JovenRESUMEN
INTRODUCTION: Apixaban is an oral direct factor Xa inhibitor developed for the prophylaxis and treatment of thromboembolic disorders. Laboratory monitoring is not necessary, but the effects on common coagulation reagents and assays constitute clinically valuable information. OBJECTIVES: To investigate the effects of apixaban on commonly used coagulation methods, and to evaluate anti-FXa assays for specific determination of the drug concentration. MATERIALS AND METHODS: Apixaban was added to plasma from healthy subjects in the concentration range 0-1000 µg L(-1) , and analyses were performed with different reagents for activated partial thromboplastin time (APTT), prothrombin time (PT), antithrombin, protein C, and protein S. A lupus anticoagulant assay and an APTT assay with varying phospholipid concentrations were used to study the phospholipid dependence. RESULTS: In general, apixaban showed fewer effects in vitro than have been shown for rivaroxaban, another direct FXa inhibitor. The concentration needed to double the APTT varied between 2200 and 4700 µg L(-1) , and the concentration needed to double the PT varied between 700 and 3900 µg L(-1) . The effects on antithrombin, protein C and protein S assays were dependent on the type of reagent. Apixaban did not cause false-positive lupus anticoagulant results. Chromogenic anti-FXa assays showed linear dose-response curves with apixaban. CONCLUSIONS: Therapeutic concentrations of apixaban variably affect different assay groups, and even different reagents within an assay group. The effects were much smaller than with rivaroxaban. The use of APTT and/or PT assays to screen the anticoagulant activity of apixaban cannot be recommended. A chromogenic anti-FXa assay can be used for reliable measurements of apixaban concentration.
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Coagulación Sanguínea/efectos de los fármacos , Inhibidores del Factor Xa/química , Factor Xa/química , Pirazoles/química , Piridonas/química , Administración Oral , Calibración , Reacciones Falso Positivas , Voluntarios Sanos , Humanos , Relación Normalizada Internacional , Inhibidor de Coagulación del Lupus/química , Morfolinas/química , Tiempo de Tromboplastina Parcial , Fosfolípidos/química , Proteína C/química , Proteína S/química , Tiempo de Protrombina , Reproducibilidad de los Resultados , Rivaroxabán , Tiofenos/químicaRESUMEN
BACKGROUND: The risk of venous tromboembolism (VTE) in women taking combined oral contraceptives (COCs) is attributed to changes in coagulation and fibrinolysis. The impact of the COCs may be greater in women with preexisting thrombophilic defects. Nevertheless most women who suffer from venous thrombosis do not have any of the well known hereditary or acquired risk factors. A simple and sensitive marker of"thrombogenicity" has not been identified. OBJECTIVES: To investigate the effects of two different monophasic combined oral contraceptives (COCs) on the plasma concentrations of activated protein C-inhibitor of protein C ( APC-PCI) and on comparable hemostatic factors in fertile women. METHOD: Forty four healthy nulliparous women with regular menstrual periods were included and randomly assigned to start with a monophasic preparation containing 30µg ethinylestradiol and 150µg levonogestrel (LNG/EE) or a preparation containing 30µg ethinylestradiol and 150 ug desogestrel (DG/EE). After a wash out period of two months, treatment with the alternate preparation was initiated and continued for two more cycles. RESULTS: The plasma concentration of the APC-PCI complex and thrombin-antithrombin complex (TAT) increased during treatment with the two COCs. During DG/EE treatment the APC-PCI complex increased significantly more than during LNG/EE (p<0,01).The plasma concentration of D-dimer did not increase during OC treatment. CONCLUSION: The APC-PCI complex concentration, which serves as a marker for thrombin generation and indicates hypercoagulability, was increased during COC treatment compared to baseline. The method is a sufficiently sensitive marker to detect even small differences in the activation of coagulation.
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Anticonceptivos Orales Combinados/efectos adversos , Desogestrel/efectos adversos , Etinilestradiol/efectos adversos , Levonorgestrel/efectos adversos , Inhibidor de Proteína C , Proteína C , Trombofilia/inducido químicamente , Adulto , Antitrombina III , Coagulación Sanguínea/efectos de los fármacos , Anticonceptivos Orales Combinados/farmacología , Desogestrel/farmacología , Etinilestradiol/farmacología , Femenino , Productos de Degradación de Fibrina-Fibrinógeno/análisis , Humanos , Levonorgestrel/farmacología , Péptido Hidrolasas/sangre , Proteína C/análisis , Inhibidor de Proteína C/sangre , Trombofilia/diagnóstico , Adulto JovenAsunto(s)
Servicios de Planificación Familiar/métodos , Púrpura Trombocitopénica Trombótica/terapia , Adulto , Femenino , Humanos , Embarazo , Complicaciones Hematológicas del Embarazo/diagnóstico , Complicaciones Hematológicas del Embarazo/terapia , Púrpura Trombocitopénica Trombótica/diagnóstico , Resultado del TratamientoRESUMEN
INTRODUCTION: Rivaroxaban is an oral direct factor Xa inhibitor developed for prophylaxis and treatment of thromboembolic disorders. Laboratory monitoring is not necessary but the dose-dependent effects on common reagents and assay procedures are largely unknown. OBJECTIVES: To investigate the effect of rivaroxaban on commonly used coagulation assays. MATERIALS AND METHODS: Rivaroxaban was added to plasma from healthy subjects in the concentration range 0-1000 µg L(-1) and analyzed using different reagents for activated partial thromboplastin time (APTT), prothrombin time (PT), antithrombin, fibrinogen and activated protein C (APC) resistance assays. RESULTS: At an expected peak concentration of rivaroxaban in clinical use, the APTTs were almost invariably prolonged but at lower concentrations the effect was weak. The concentration needed to double the APTT varied between 389 ± 106 and 617 ± 149 µg L(-1) for different reagents. The PT assays showed a marked degree of difference. In general, the Quick PT type assays were more sensitive compared with the Owren type PT assays. The results from antithrombin assays were dependent on the type of reagent, with the Xa-based assay being sensitive for rivaroxaban with an estimated increase of 0.09 IU mL(-1) per 100 µg L(-1) rivaroxaban. There were only minor effects on fibrinogen assays based on thrombin reagents. The APTT-based assay for APC resistance is affected in a dose-dependent manner whereas an assay based on the activation of coagulation at the prothrombinase level was unaffected. CONCLUSIONS: Different assays, and even different reagents within an assay group, display variable effects by therapeutic concentrations of rivaroxaban.
Asunto(s)
Anticoagulantes/administración & dosificación , Pruebas de Coagulación Sanguínea , Coagulación Sanguínea/efectos de los fármacos , Inhibidores del Factor Xa , Morfolinas/administración & dosificación , Tiofenos/administración & dosificación , Resistencia a la Proteína C Activada/sangre , Administración Oral , Adulto , Antitrombinas/metabolismo , Relación Dosis-Respuesta a Droga , Femenino , Fibrinógeno/metabolismo , Humanos , Masculino , Tiempo de Tromboplastina Parcial , Valor Predictivo de las Pruebas , Tiempo de Protrombina , Reproducibilidad de los Resultados , RivaroxabánRESUMEN
INTRODUCTION: D-dimer assays are now widely used as the first-line test in the diagnostic algorithm of suspected deep vein thrombosis (DVT). The aim of this study was to evaluate the performance of two relatively new quantitative D-Dimer assays (Innovance and AxSYM) by comparison with a clinical gold standard. PATIENTS AND METHODS: 311 samples from outpatients with clinical suspicion of DVT, included in a prospective management study, was analysed (prevalence of DVT 23%). The diagnostic workup included estimation of pre-test probability, D-dimer determination, objective imaging as well as 3 month clinical follow up of negative patients. RESULTS: No significant differences were seen in sensitivity and negative predictive values between Innovance, AxSYM and the reference assays. The area under the ROC curve was slightly lower for the AxSYM assay and the correlation to the reference assays was only moderate (r < 0.8) whereas the agreement with the Vidas assay was near excellent (kappa = 0.8). The Innovance assay reached the highest AUC, showed a strong correlation with the reference assays (r > or = 0.9) and a good agreement with the Vidas assay (kappa = 0.76). In combination with a low pre-test probability score the Innovance assay reached a NPV of 100% (95% CI, 92-100) and the AxSYM assay 98% (95% CI, 87-100). CONCLUSION: The Innovance and AxSYM assays show an overall good and comparable performance for the exclusion of DVT when compared to the established assays. Our results for the AxSYM assay indicate that the optimal cut-off value needs to be further evaluated.
Asunto(s)
Productos de Degradación de Fibrina-Fibrinógeno/metabolismo , Juego de Reactivos para Diagnóstico/normas , Trombosis de la Vena/sangre , Trombosis de la Vena/diagnóstico , Adolescente , Adulto , Anciano , Anciano de 80 o más Años , Pruebas de Coagulación Sanguínea , Técnicas de Laboratorio Clínico , Femenino , Humanos , Masculino , Persona de Mediana Edad , Valor Predictivo de las Pruebas , Probabilidad , Estudios Prospectivos , Reproducibilidad de los ResultadosRESUMEN
OBJECTIVES: To investigate the reliability of a combined strategy of clinical assessment score followed by a local D-dimer test to exclude deep vein thrombosis. For comparison D-dimer was analysed post hoc and batchwise at a coagulation laboratory. DESIGN: Prospective multicenter management study. SETTING: Seven hospitals in southern Sweden. SUBJECTS: 357 patients with a suspected first episode of deep vein thrombosis (DVT) were prospectively recruited and pre-test probability score (Wells score) was estimated by the emergency physician. If categorized as low pre-test probability, D-dimer was analysed and if negative, DVT was considered to be ruled out. The primary outcome was recurrent venous thromboembolism (VTE) during 3 months of follow up. RESULTS: Prevalence of DVT was 23.5% (84/357). A low pre-test probability and a negative D-dimer result at inclusion was found in 31% (110/357) of the patients of whom one (0.9%, [95% CI 0.02-4.96]) had a VTE at follow up. Sensitivity, specificity, negative predictive value and negative likelihood ratio for our local D-dimer test in the low probability group were 85.7%, 74.5%, 98.2%, and 0,19 respectively compared to 85.6%, 67,6%, 97.9% and 0,23 using batchwise analysis at a coagulation laboratory. CONCLUSION: Pre-test probability score and D-dimer safely rule out DVT in about 30% of outpatients with a suspected first episode of DVT. One out of 110 patients was diagnosed with DVT during follow up. No significant difference in diagnostic performance was seen between local D-dimer test and the post hoc batch analysis with the same reagent in the low probability group.
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Productos de Degradación de Fibrina-Fibrinógeno/análisis , Trombosis de la Vena/diagnóstico , Adulto , Anciano , Anciano de 80 o más Años , Estudios de Cohortes , Medicina de Emergencia , Femenino , Humanos , Masculino , Persona de Mediana Edad , Valor Predictivo de las Pruebas , Estudios Prospectivos , Trombosis de la Vena/sangreRESUMEN
BACKGROUND: Exposure in a pig house causes airway inflammation and bronchial hyper-responsiveness which are not influenced by anti-asthma drugs, including a beta(2)-agonist (salmeterol). OBJECTIVES: We hypothesized that a glucocorticoid or a cyclo-oxygenase-inhibitor synergistically interacts with salmeterol offering a protection against dust-induced increased bronchial responsiveness and airway inflammation. As data did not confirm previous results a retrospective analysis of pooled data on dust-induced bronchial hyper-responsiveness from four other studies was performed. DESIGN: Fluticasone or ibuprofen was administered for 1 week and salmeterol or placebo was inhaled 1 h prior to a 3-h exposure in a pig barn in a double-blind, placebo-controlled, cross-over design (2-3 weeks apart) in 12 healthy subjects. Lung function, bronchial responsiveness to methacholine and inflammatory markers were evaluated before and after exposure. Pre- and postexposure bronchial responsiveness in nontreated subjects was retrospectively evaluated from four previous studies. SUBJECTS: Twelve healthy, nonatopic nonsmokers. RESULTS: Salmeterol partially protected against bronchial hyper-responsiveness but did not influence inflammatory markers. Fluticasone and ibuprofen did not add to these effects. The retrospective analysis showed that PD(20)FEV(1) after exposure in a pig barn is almost totally independent of pre-exposure PD(20)FEV(1)-level; all subjects end up at the same low postexposure PD(20)FEV(1). CONCLUSION: Contradictory to our previous results, salmeterol offered partial protection against enhanced bronchial responsiveness induced by exposure in a pig barn. This effect was not modified by fluticasone or ibuprofen. Our data clearly demonstrate that interventions altering bronchial responsiveness must be compared between groups with similar prechallenge bronchial responsiveness or in a cross-over design.
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Agonistas Adrenérgicos beta/uso terapéutico , Albuterol/análogos & derivados , Androstadienos/uso terapéutico , Antiinflamatorios no Esteroideos/uso terapéutico , Broncodilatadores/uso terapéutico , Polvo , Ibuprofeno/uso terapéutico , Adulto , Enfermedades de los Trabajadores Agrícolas/tratamiento farmacológico , Albuterol/uso terapéutico , Animales , Pruebas de Provocación Bronquial/métodos , Método Doble Ciego , Interacciones Farmacológicas , Femenino , Fluticasona , Volumen Espiratorio Forzado/efectos de los fármacos , Humanos , Masculino , Cloruro de Metacolina , Persona de Mediana Edad , Exposición Profesional/efectos adversos , Estudios Retrospectivos , Xinafoato de Salmeterol , Porcinos , Capacidad Vital/efectos de los fármacosRESUMEN
The concentration of the complex between activated protein C and the protein C inhibitor reflects the degree of activation of blood coagulation. A sandwich method has been devised that measures the complex concentration in blood plasma. A key feature of the method is that the catching monoclonal antibody recognizes a complex-dependent neoepitope in PCI, which is a prerequisite, since the concentration of uncomplexed PCI is approximately 10(4)-fold higher than that of the complex. In patients with atherosclerotic disease, those with aortic aneurysms exhibit a three-fold increase in complex concentration compared to that of normal subjects.
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Aneurisma de la Aorta/diagnóstico , Inmunoensayo/métodos , Complejos Multiproteicos/análisis , Inhibidor de Proteína C/metabolismo , Proteína C/metabolismo , Biomarcadores , Humanos , Inhibidor de Proteína C/química , Inhibidor de Proteína C/inmunologíaAsunto(s)
Factor V/genética , Complejos Multiproteicos/sangre , Inhibidor de Proteína C/sangre , Tromboembolia/sangre , Trombosis de la Vena/sangre , Resistencia a la Proteína C Activada/complicaciones , Resistencia a la Proteína C Activada/genética , Área Bajo la Curva , Productos de Degradación de Fibrina-Fibrinógeno/análisis , Genotipo , Heterocigoto , Humanos , Mutación Puntual , Proteína C/metabolismo , Inhibidor de Proteína C/metabolismo , Curva ROC , Factores de Riesgo , Tromboembolia/etiología , Tromboembolia/genética , Trombosis de la Vena/etiología , Trombosis de la Vena/genéticaRESUMEN
Our newly devised immunofluorometric sandwich assay for measuring plasma concentrations of activated protein C (APC) in complex with protein C inhibitor (PCI) was compared with testing for conventional markers of myocardial damage CKMB (creatine kinase MB), TNI (troponin I) and hypercoagulability (D-dimer, TAT) in 76 patients with suspected myocardial infarction (MI). APC-PCI complex levels in samples drawn on admission did not correlate with CKMB in the simultaneously drawn sample but correlated closely with maximal CKMB, which reflects MI size (r = 0.52). The areas under the receiver operating characteristics (ROC) curves calculated for the APC-PCI complex results obtained upon admission did not differ significantly from the corresponding values for CKMB, TNI or TAT. Our results show that in patients at risk for MI, the APC-PCI concentration is a sensitive and independent marker that can identify a subgroup of MI patients with normal CKMB but an increased APC-PCI level upon admission. It remains to be determined whether these patients would benefit from early intensive anticoagulant treatment.
Asunto(s)
Infarto del Miocardio/sangre , Inhibidor de Proteína C/sangre , Proteína C/metabolismo , Adulto , Anciano , Anciano de 80 o más Años , Antitrombina III , Enfermedad Coronaria/sangre , Creatina Quinasa/sangre , Femenino , Productos de Degradación de Fibrina-Fibrinógeno/análisis , Fluoroinmunoensayo , Humanos , Isoenzimas/sangre , Masculino , Persona de Mediana Edad , Péptido Hidrolasas/sangre , Proteína C/análisis , Curva ROC , Troponina I/sangreRESUMEN
Activated protein C (APC) is a serine proteinase that regulates blood coagulation. In plasma it is inhibited mainly by the protein C inhibitor (PCI). The plasma concentrations of APC-PCI complex is increased in hypercoagulative states such as deep venous thrombosis. Formation of the APC-PCI complex induces a drastic conformational change in PCI that exposes new epitopes (neoepitopes) on the molecule. We have devised a simple immunofluorometric sandwich assay for measurements of the concentrations of APC-PCI complex, employing as the catcher, a monoclonal antibody that has a high affinity (K(D) = 4 x 10(-11) M) for a complexation-specific neoepitope that is expressed on PCI. A monoclonal antibody against protein C is employed as the tracer. The method gives a linear dose-response curve (0.06-50 microg/l), has a low detection limit (0.06 microg/l) and no crossreactivity with native PCI at physiologic plasma concentrations. We have now determined the concentration of the APC-PCI complex in healthy individuals.
Asunto(s)
Inhibidor de Proteína C/sangre , Proteína C/análisis , Calibración , Reacciones Cruzadas , Ensayo de Inmunoadsorción Enzimática/métodos , Ensayo de Inmunoadsorción Enzimática/normas , Epítopos/inmunología , Epítopos/metabolismo , Fluoroinmunoensayo/métodos , Fluoroinmunoensayo/normas , Humanos , Unión Proteica/inmunología , Proteína C/inmunología , Proteína C/metabolismo , Inhibidor de Proteína C/inmunología , Inhibidor de Proteína C/metabolismo , Conformación Proteica , Estándares de Referencia , Sensibilidad y EspecificidadRESUMEN
A first clinical evaluation has been made of the performance of a newly devised immunofluorometric assay for measuring plasma concentrations of activated protein C (APC) in complex with protein C inhibitor (PCI). The method was compared with testing for other markers of hypercoagulability in a case-control study comprising 123 patients with clinical suspicion of deep vein thrombosis (DVT). The diagnosis was confirmed by ascending phlebography, and the thrombotic burden estimated with a newly developed scoring system. Receiver operating characteristics (ROC) curves calculated to demonstrate the discriminatory capacity of the methods, showed the area under the curves (AUCs) to be similar for the APC-PCI and D-dimer methods. However, in contrast to the D-dimer method, the APC-PCI method measures a well-defined analyte, a prerequisite for reliable comparisons of future clinical studies. The APC-PCI method appears to be particularly useful as a marker for detection of recently developed proximal thrombi.
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Técnica del Anticuerpo Fluorescente Directa , Inhibidor de Proteína C/análisis , Proteína C/análisis , Trombofilia/sangre , Trombosis de la Vena/diagnóstico , Resistencia a la Proteína C Activada/sangre , Resistencia a la Proteína C Activada/genética , Anticuerpos Monoclonales/inmunología , Antitrombina III/análisis , Área Bajo la Curva , Biomarcadores , Biotinilación , Proteína C-Reactiva/análisis , Estudios de Casos y Controles , Epítopos/inmunología , Factor V/análisis , Femenino , Productos de Degradación de Fibrina-Fibrinógeno/análisis , Humanos , Sustancias Macromoleculares , Masculino , Fragmentos de Péptidos/análisis , Péptido Hidrolasas/análisis , Flebografía , Proteína C/antagonistas & inhibidores , Proteína C/química , Proteína C/inmunología , Inhibidor de Proteína C/inmunología , Inhibidor de Proteína C/farmacología , Conformación Proteica , Protrombina/análisis , Protrombina/genética , Curva ROC , Sensibilidad y Especificidad , Índice de Severidad de la Enfermedad , Estreptavidina/análisis , Trombofilia/genética , Trombosis de la Vena/sangre , Trombosis de la Vena/diagnóstico por imagenRESUMEN
Protein C inhibitor, a serine proteinase inhibitor (serpin), is the physiologically most important inhibitor of activated protein C. We have made a monoclonal antibody (M36) that binds with equally high affinity to an epitope present in activated protein C-protein C inhibitor complexes and cleaved loop-inserted protein C inhibitor. Insertion of a synthetic N-acetylated tetradecapeptide (corresponding to residues P1-P14 of the reactive center loop) into beta-sheet A of the uncleaved inhibitor also exposed the epitope. The antibody had no apparent affinity for native uncleaved inhibitor or for the free peptide. Synthetic P1-P14 analogues, with Arg P13 or Ala P9 substituted to the residues found in mouse protein C inhibitor (Thr and Ile, respectively), were also inserted in beta-sheet A. The Arg P13/Thr substitution led to a greatly impaired reactivity with the antibody, whereas the Ala P9/Ile mutation resulted in a modest loss of reactivity with the antibody. These results indicate that complex formation leads to insertion of the reactive center loop in beta-sheet A from Arg P14 and presumably beyond Ala P9. Moreover, to the best of our knowledge, this is the first instance where the neoepitope of a complexation-specific monoclonal antibody has been localized to the loop-inserted part of beta-sheet A, the part of the serpin where the complexation-induced conformational change is most conspicuous.
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Epítopos/química , Epítopos/metabolismo , Inhibidor de Proteína C/química , Inhibidor de Proteína C/metabolismo , Proteína C/química , Proteína C/metabolismo , Animales , Anticuerpos Monoclonales/biosíntesis , Anticuerpos Monoclonales/química , Anticuerpos Monoclonales/metabolismo , Especificidad de Anticuerpos , Sitios de Unión , Simulación por Computador , Activación Enzimática , Epítopos/inmunología , Humanos , Ratones , Modelos Moleculares , Fragmentos de Péptidos/química , Fragmentos de Péptidos/metabolismo , Proteína C/inmunología , Inhibidor de Proteína C/biosíntesis , Inhibidor de Proteína C/inmunología , Conformación Proteica , Estructura Secundaria de Proteína , Relación Estructura-ActividadRESUMEN
An entirely new type of blood gas analyser has made its way into the marketplace, to be used, for example, in emergency rooms, intensive care units, ambulances, and bedside with quarantined patients in infectious diseases units. The instruments reviewed here employ new miniaturised analysis circuitry, integrated into the cassette on which the blood sample is applied. These instruments are designed for use by care-givers without specific laboratory training. Four point-of-care blood gas analysers are tested: OPTI 1 (AVL), I-STAT (HP), IRMA (Infiniti) och ABL 70 (Radiometer).