RESUMEN
OBJECTIVES: The cardiac damage biomarkers cardiac troponin T (cTnT) and troponin I (cTnI) are used to identify patients with myocardial infarction (MI). To make the correct clinical decisions it is important to identify false positive results due to troponin assay interference. Often interferences are caused by high-molecular weight immunocomplexes called macrotroponin that may result in false troponin elevations because of delayed troponin clearance, or heterophilic antibodies that crosslink troponin assay antibodies and generate troponin-independent signals. DESIGN & METHODS: We describe and compare four methods for cTnI assay interference analysis using a protein G spin column method, gel filtration chromatography and two versions of a sucrose gradient ultracentrifugation for cTnI assay interference analysis on five patients with confirmed cTnI interference and one MI patient without cTnI interference from our troponin interference referral center. RESULTS: The protein G spin column method had a high between run variability but was still able to identify all five patients with cTnI interference. The sucrose gradient ultracentrifugation methods and the gel filtration method had simlar performancec and correctly identified the immunocomplexes that caused the cTnI interference. CONCLUSIONS: Our experience is that these methods are sufficient to safely confirm or exclude positive cTnI assay interference.
Asunto(s)
Infarto del Miocardio , Troponina I , Humanos , Troponina T , Infarto del Miocardio/diagnóstico , Anticuerpos , Anticuerpos Heterófilos , BiomarcadoresRESUMEN
BACKGROUND: Inappropriate and uncontrolled activation of the cascade systems in the blood is a driving force in adverse inflammatory and thrombotic reactions elicited by biomaterials, but limited data are available on the activation of the contact system by polymers and the present study was undertaken to investigate these mechanisms in established models. METHODS: Polymer particles were incubated in (1) EDTA-plasma (10 mM) to monitor the adsorption of 20 selected proteins; (2) lepirudin-anticoagulated plasma to evaluate contact system activation, monitored by the formation of complexes between the generated proteases factor[F]XIIa, FXIa and kallikrein and the serpins C1-inhibitor [C1INH] and antithrombin [AT]; (3) lepirudin-anticoagulated whole blood to determine cytokine release. RESULTS: Strong negative correlations were found between 10 cytokines and the ratio of deposited FXII/C1INH, generated FXIIa-C1INH complexes, and kallikrein-C1INH complexes. Formation of FXIIa-C1INH complexes correlated negatively with the amount of C3a and positively with deposited IgG. CONCLUSIONS: A reciprocal relationship was found between activation of the contact system and the complement system induced by the polymers studied here. The ratios of FXII/C1INH or C4/C4BP, adsorbed from EDTA-plasma are useful surrogate markers for cytokine release and inflammatory response to materials intended for blood contact.
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Materiales Biocompatibles , Activación de Complemento , Polímeros , Adsorción , Antitrombinas/metabolismo , Coagulación Sanguínea/efectos de los fármacos , Proteínas Sanguíneas/química , Proteínas Inactivadoras del Complemento 1/metabolismo , Proteína Inhibidora del Complemento C1 , Complemento C3a/análisis , Citocinas/metabolismo , Activación Enzimática , Factor XIIa/metabolismo , Factor XIa/metabolismo , Vidrio , Hirudinas/farmacología , Humanos , Interacciones Hidrofóbicas e Hidrofílicas , Inmunoglobulina G/análisis , Calicreínas/metabolismo , Tamaño de la Partícula , Poliestirenos , Cloruro de Polivinilo , Proteínas Recombinantes/farmacologíaRESUMEN
Inappropriate complement activation is often responsible for incompatibility reactions that occur when biomaterials are used. Complement activation is therefore a criterion included in legislation regarding biomaterials testing. However, no consensus is yet available regarding appropriate complement-activation-related test parameters. We examined protein adsorption in plasma and complement activation/cytokine release in whole blood incubated with well-characterized polymers. Strong correlations were found between the ratio of C4 to its inhibitor C4BP and generation of 10 (mainly pro-inflammatory) cytokines, including IL-17, IFN-γ, and IL-6. The levels of complement activation products correlated weakly (C3a) or not at all (C5a, sC5b-9), confirming their poor predictive values. We have demonstrated a direct correlation between downstream biological effects and the proteins initially adhering to an artificial surface after contact with blood. Consequently, we propose the C4/C4BP ratio as a robust, predictor of biocompatibility with superior specificity and sensitivity over the current gold standard.
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Materiales Biocompatibles/efectos adversos , Activación de Complemento/efectos de los fármacos , Inflamación/sangre , Inflamación/inducido químicamente , Polímeros/efectos adversos , Proteína de Unión al Complemento C4b/inmunología , Complejo de Ataque a Membrana del Sistema Complemento/inmunología , Proteínas del Sistema Complemento/inmunología , Humanos , Inflamación/inmunología , Interferón gamma/inmunología , Interleucina-17/sangre , Interleucina-17/inmunología , Interleucina-6/sangre , Interleucina-6/inmunologíaRESUMEN
The aim of this study was to create polymeric materials with known properties to study the preconditions for complement activation. Initially, 22 polymers were screened for complement activating capacity. Based on these results, six polymers (P1-P6) were characterized regarding physico-chemical parameters, for example, composition, surface area, pore size, and protein adsorption from human EDTA-plasma. P2, P4, and reference particles of polystyrene and polyvinyl chloride, were hydrophobic, bound low levels of protein and were poor complement activators. Their accessible surface was limited to protein adsorption in that they had pore diameters smaller than most plasma proteins. P1 and P3 were negatively charged and adsorbed IgG and C1q. A 10-fold difference in complement activation was attributed to the fact that P3 but not P1 bound high amounts of C1-inhibitor. The hydrophobic P5 and P6 were low complement activators. They selectively bound apolipoproteins AI and AIV (and vitronectin), which probably limited the binding of complement activators to the surface. We demonstrate the usefulness of the modus operandi to use a high-throughput procedure to synthesize a great number of novel substances, assay their physico-chemical properties with the aim to study the relationship between the initial protein coat on a surface and subsequent biological events. Data obtained from the six polymers characterized here, suggest that a complement-resistant surface should be hydrophobic, uncharged, and have a small available surface, accomplished by nanostructured topography. Additional attenuation of complement can be achieved by selective enrichment of inert proteins and inhibitors.
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Proteínas Sanguíneas/química , Proteínas del Sistema Complemento/metabolismo , Ensayo de Materiales/métodos , Polímeros/química , Adsorción , Western Blotting , Activación de Complemento , Reactivos de Enlaces Cruzados/química , Electroforesis en Gel de Poliacrilamida , Endotoxinas/metabolismo , Citometría de Flujo , Hirudinas/metabolismo , Humanos , Concentración de Iones de Hidrógeno , Microscopía Electrónica de Rastreo , PorosidadRESUMEN
Blood incompatibility reactions caused by surfaces often involve platelet activation and subsequent platelet-initiated activation of the coagulation and complement cascades. The goal of this study was to immobilize apyrase on a biomaterial surface in order to develop an enzymatically active surface that would have the capacity to inhibit platelet activation by degrading ADP. We were able to immobilize apyrase on a polystyrene surface with preservation of the enzymatic activity. We then analyzed the hemocompatibility of the apyrase surface and of control surfaces by incubation with platelet-rich plasma (PRP) or whole blood. Monitoring of markers of platelet, coagulation, and complement activation and staining of the surfaces revealed decreased levels of platelet and coagulation activation parameters on the apyrase surface. The formation of antithrombin-thrombin and antithrombin-factor XIa complexes and the extent of platelet consumption were significantly lower on the apyrase surface than on any of the control surfaces. No significant differences were seen in complement activation (C3a levels). Staining of the apyrase surface revealed low platelet adherence and no formation of granulocyte-platelet complexes. These results demonstrate that it is possible to create an antithrombotic surface targeting the ADP amplification of platelet activation by immobilizing apyrase.
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Antitrombinas/farmacología , Apirasa/metabolismo , Apirasa/farmacología , Enzimas Inmovilizadas/metabolismo , Activación Plaquetaria/efectos de los fármacos , Animales , Antitrombinas/química , Apirasa/química , Materiales Biocompatibles/química , Materiales Biocompatibles/metabolismo , Coagulación Sanguínea/efectos de los fármacos , Activación de Complemento/efectos de los fármacos , Enzimas Inmovilizadas/química , Humanos , Ensayo de Materiales , Propiedades de SuperficieRESUMEN
The aim of this study was to evaluate a new approach to inhibit complement activation triggered by biomaterial surfaces in contact with blood. In order to inhibit complement activation initiated by the classical pathway (CP), we used streptococcal M protein-derived peptides that specifically bind human C4BP, an inhibitor of the CP. The peptides were used to coat polystyrene microtiter wells which served as a model biomaterial. The ability of coated peptides to bind C4BP and to attenuate complement activation via the CP (monitored as generation of fluid-phase C3a and binding of fragments of C3 and C4 to the surface) was investigated using diluted normal human serum, where complement activation by the AP is minimal, as well as serum from a patient lacking alternative pathway activation. Complement activation (all parameters) was significantly decreased in serum incubated in well surfaces coated with peptides. Total inhibition of complement activation was obtained at peptide coating concentrations as low as 1-5 microg/mL. Successful use of Streptococcus-derived peptides shows that it is feasible to control complement activation at a model biomaterial surface by capturing autologous complement regulatory molecules from plasma.
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Antígenos Bacterianos/química , Proteínas de la Membrana Bacteriana Externa/química , Materiales Biocompatibles/química , Proteínas Portadoras/química , Modelos Biológicos , Fragmentos de Péptidos/química , Proteína de Unión al Complemento C4b , Antígenos de Histocompatibilidad/metabolismo , Humanos , Unión Proteica , Propiedades de SuperficieRESUMEN
Heparin has traditionally been a widely used anticoagulant in blood research, but has been shown to be inappropriate for work with the complement system because of its complement-interacting properties. In this work, we have compared the effects of heparin with those of the specific thrombin inhibitor hirudin on complement and blood cells in vitro. Whole blood collected in the presence of hirudin (50 microg/mL) or heparin (1 IU/mL) was incubated in the slide chamber model. The plasma was analyzed for complement activation markers C3a and sC5b-9, and the polyvinylchloride test slides were stained for adhering cells. The integrity of the complement system was tested by incubating serum and hirudin-treated plasma in the presence of various activating agents. In contrast to heparin, the addition of hirudin generally preserved the complement reactivity, and complement activation in hirudin plasma closely resembled that in normal serum. Importantly, immunochemical staining of surface-bound cells demonstrated the inducible expression of tissue factor on bound monocytes from hirudin-treated blood, an effect that was completely abolished in heparin-treated blood. Our results indicate that hirudin as an anticoagulant produces more physiological conditions than heparin, making hirudin well-suited for in vitro studies, especially those addressing the regulation of cellular processes.