Función renal y nivel plasmático de dabigatrán en el valle, medido por un ensayo de trombina diluida / Renal function and plasma dabigatran level measured at trough by diluted thrombin time assay

El dabigatrán etexilato (inhibidor directo de trombina) es eficaz en la prevención tromboembólica en pacientes con fibrilación auricular. No requiere control rutinario de laboratorio, pero dada su eliminación renal, sería importante medirlo ante el deterioro de la función renal. Los objetivos del trabajo fueron verificar la calidad analítica del ensayo tiempo de trombina diluido para medición de la concentración plasmática (cc) de dabigatrán, correlacionar las cc con las pruebas básicas de coagulación tiempo de protrombina (TP) y tiempo de tromboplastina parcial activada (APTT) y evaluarlas de acuerdo al clearance de creatinina (CLCr). Se utilizaron muestras de plasma de 40 pacientes que recibían dabigatrán 150 (n = 19) o 110 (n = 21) mg/12 horas, colectadas 10-14 horas después de la última toma. Los ensayos de trombina diluida HemosIL DTI para la medición de dabigatrán, TP y APTT (IL), fueron realizados en coagulómetros fotoópticos ACL TOP 300 y 500 (IL). El DTI presentó coeficiente de variación intraensayo < 5.4% e interensayo < 6.0%, rango de linealidad 0-493 ng/ml; cc medidas en pacientes: mediana 83 (4-945) ng/ml. Individuos con CLCr en tercil inferior (< 46.1 ml/min) presentaron cc significativamente más elevadas, 308 (49-945), que los de tercilos medio, 72 (12-190), y superior, 60 (4-118) ng/ml. Las correlaciones cc vs. APTT o TP fueron moderadas, r2 = 0.59, -0.66, p < 0.0001, respectivamente. La prueba ensayada permitió cuantificar el nivel de dabigatrán plasmático tanto en pacientes con función renal normal como deteriorada, representando una herramienta útil en situaciones clínicas como deterioro de la función renal, pre cirugía o emergencias.

Dabigatran etexilate (direct thrombin inhibitor) is effective in preventing embolic stroke in patients with atrial fibrillation. It does not require laboratory control, but given the high renal elimination, its measurement in plasma is important in renal failure. The objectives of the study were to verify the analytical quality of the diluted thrombin time assay for measurement of dabigatran plasma concentration (cc), correlate cc with classic coagulation assays, prothrombin time (PT) and activated partial thromboplastin time (APTT), and evaluate them according to the creatinine clearance (CLCr). Forty plasma samples of patients (34 consecutive and 6 suspected of drug accumulation) receiving dabigatran at 150 (n = 19) or 110 (n = 21) mg/12 hours were collected. Blood samples were drawn at 10-14 hours of the last intake. Dabigatran concentration was determined by diluted thrombin time (HemosIl DTI, Instrumentation Laboratory (IL). PT and APTT (IL) were performed on two fotooptical coagulometers, ACL TOP 300 and 500 (IL). DTI presented intra-assay coefficient of variation < 5.4% and inter-assay < 6%, linearity range 0-493 ng/ml. Patients´ cc: median 83 (4-945) ng/ml. Individuals with CLCr in the lowest tertile (22.6-46.1 ml/min) showed significantly higher median cc: 308 (49-945), compared to the average 72 (12-190) and highest tertile, 60 (4-118) ng/ml. Correlation between cc and APTT or PT were moderate, r2 = 0.59 and -0.66, p < 0.0001, respectively. DTI test allowed us to quantify plasma dabigatran levels, both in patients with normal or altered renal function, representing a useful tool in clinical situations such as renal failure, pre surgery or emergencies.

Dabigatran en plasma: tiempos de trombina diluido, Ecarin y veneno de víbora de Russell diluido / Plasma dabigatran: diluted thrombin time, Ecarin and diluted Russell Viper Venom Time / Dabigatrana em plasma: tempos de trombina diluído, Ecarin e veneno de víbora de Russell diluído

Los objetivos del trabajo fueron verificar la calidad analítica del ensayo tiempo de trombina diluido (DTI) para medición de la concentración plasmática (cc) de dabigatran comparando dos coagulómetros de detección foto-óptica, comparar los resultados con el tiempo de Ecarin (ECT) y correlacionar las cc con las pruebas básicas de coagulación Tiempo de protrombina (TP), APTT y Tiempo de trombina (TT), y tiempo de veneno de víbora de Russell con fosfolípidos concentrados (DRVVTC). Se tomaron 43 muestras de plasma en el valle (10-14 h de la última toma) de 40 pacientes que recibían dabigatran. DTI y ECT presentaron (%) repetitividad <5,4% y <7,5%, CV interensayo <6% y <9%, respectivamente, en el protocolo EP15A2, aceptables para un Error Total permitido (TEa) <15%. Las cc medidas en pacientes fueron: mediana 83 (4-945) ng/mL. La comparación de equipos ACL TOP 300 y 500 dio resultados equivalentes por procedimiento alternativo de comparación de métodos. La comparación ECT vs. DTI fue satisfactoria por regresión de Deming (pendiente 1,143, ordenada al origen -19,33). Las correlaciones de cc vs. APTT, TP y DRVVTC fueron moderadas y no lineales tendiendo a plateau a cc>350 ng/mL, r2 0,59, 0,66 y 0,59, respectivamente. El TT fue extremadamente sensible: >120 s a cc 50 ng/mL. DTI presentó un buen desempeño analítico y permitió cuantificar dabigatran plasmático a cc bajas y altas en ambos equipos utilizados. ECT presentó resultados comparables a DTI. Se verifica una correlación moderada entre cc de dabigatran y las pruebas clásicas y DRVVTC, pudiendo ser estimadores de cc a partir de los 50 ng/mL.

The aims of the study were to verify the analytical performance of Dilute Thrombin Time (DTI) test to measure plasma dabigatran concentration (cc) in two photo-optical coagulometers, compare Ecarin clotting Time (ECT) and DTI results, and correlate cc with classical coagulation tests: prothrombin time (PT), APTT, thrombin time (TT) and diluted Russell Viper Venom Time tests with high phospholipid concentration (DRVVTC). Forty three plasma samples from 40 patients taking dabigatran were drown at through (10-14 hs.since last dose). DTI and ECT showed repetitivity (%) <5.4% and <7.5%, interassay CV <6% and <9%, respectively, following EP15A2 protocol, acceptable considering a Allowed Total Error (TEa)<15%. Patients` cc: median 83 (4-945) ng/mL. The comparison between ACL TOP 300 and 500 coagulometers showed equivalent results by using the alternative method comparison test. ECT vs. DTI: acceptable by Deming`s regression (slope 1.143, Y insert -19.33). cc vs. APTT, TP and DRVVTC: nonlinear and moderate correlations with plateau reached at cc >350 ng/mL, r2 0.59, 0.66 y 0.59, respectively. TT is extremely prolonged at cc >50 ng/mL. In conclusion: DTI showed a good analytical performance in both coagulometers. ECT showed comparable results to DTI. We verified that dabigatran cc presented moderate correlations with PT, APTT and DRVVTC, and that these tests could only qualitative estimate cc >50 ng/mL.

Os objetivos do trabalho foram verificar a qualidade analítica do ensaio tempo de trombina diluído (DTI) para medição da concentração plasmática (cc) de dabigatrana, comparando dois coagulômetros de detecção foto-óptica, comparar os resultados com o tempo de Ecarina (ECT) e correlacionar as cc com os testes básicos de coagulação Tempo de protrombina (TP), APTT e Tempo de trombina (TT), e tempo de veneno de víbora de Russell com fosfolipídios concentrados (DRVVTC). Foram tomadas 43 amostras de plasma no vale (10-14 h. da última toma) de 40 pacientes que recebiam dabigatrana. DTI e ECT apresentaram (%) repetitividade <5,4% e <7,5%, CV interensaio <6% e <9%, respectivamente, no protocolo EP15A2, aceitáveis para um Erro Total permitido (TEa) <15%. Cc medidas em pacientes: mediana 83 (4-945) ng/mL. Comparação de equipamentos ACL TOP 300 e 500: resultados equivalentes por procedimento alternativo de comparação de métodos. Comparação ECT vs. DTI: satisfatória por regressão de Deming (pendente 1,143, ordenada à origem -19,33). Correlações cc vs. APTT, TP e DRVVTC: moderadas e não lineares tendendo a plateau a cc>350 ng/mL, r2 0,59; 0,66 e 0,59, respectivamente. O TT é extremamente sensível: >120 s a cc 50 ng/mL. DTI apresentou um bom desempenho analítico e permitiu quantificar dabigatrana plasmática a cc baixas e altas em ambos os equipamentos utilizados. ECT apresentou resultados comparáveis com DTI. Verifica-se uma correlação moderada entre cc de dabigatrana e os testes clássicos e DRVVTC, podendo ser estimadores de cc a partir dos 50 ng/mL.

Adequação de um sistema de hemodiálise em eqüinos / Adequacy of hemodialysis system in horses

Realizou-se a adequação da técnica de hemodiálise com eqüinos, distribuídos em quatro grupos experimentais de seis animais cada. Os animais do grupo I foram submetidos a cateterismo central unilateral (grupo-controle); os do grupo II foram submetidos a cateterismo central unilateral com cateter duplo-lúmen e a uma sessão de hemodiálise de seis horas; os do grupo III a cateterismo central unilateral com cateter duplo-lúmen e duas sessões de hemodiálise de seis horas e os do grupo IV a cateterismo central bilateral com cateter monolúmen e a uma sessão de hemodiálise de seis horas. Empregaram-se xilazina 10 por cento (0,4mg/kg) e acepromazina 2 por cento (0,008mg/kg) via intravenosa para sedação. Foram utilizados dois hemodialisadores conectados em série, do tipo fibras ocas, baixo fluxo, membrana de polissulfona e área de 1,8m². O fluxo sangüíneo médio foi de 319,18±97,41ml/minuto, associado a um fluxo de dialisato de 500ml/min. A anticoagulação foi feita com heparina sódica em 100UI/kg para primming, repetida na dose de 53,86±18,61UI/kg/hora. Foram avaliados: tempo de coagulação, tempo de protrombina, tempo de tromboplastina parcial ativada e contagem plaquetária, e verificado trombocitopenia nos grupos dialisados. O melhor acesso vascular foi proporcionado pelo cateterismo unilateral com cateter lúmen-duplo (Grupos II e III), podendo a técnica de hemodiálise ser empregada na espécie eqüina, com dialisadores de alta eficiência, em sessão de seis horas de diálise.

Hemodialysis adequacy was studied in four groups with six horses in each: the treatments: group I animals were submitted to unilateral central venous catheter (control group); group II animals were submitted to unilateral central venous double lumen catheter and one six-hour session of hemodialysis; group III horses were submitted to unilateral central venous double lumen catheter and to two six-hour session of hemodialysis, and group IV horses were submitted to bilateral central venous mono lumen catheter and to one six-hour session of hemodialysis. Xilazine 10 percent (0.4mg/kg) and acepromazine 2 percent (0.008 mg/kg) were iv administrated for sedation. Two hollow fiber, 1.8m² low flux polyssulfone hemodialysis apparatus were used in a connected serie. The mean blood flux was 319.18±97.41ml/min with a dialisate flux of 500ml/min. Anticoagulation was performed with sodium heparin, 100UI/kg for priming at the dose of 53.86±18.61UI/kg/h. Anticoagulation monitoring was performed by clotting time, protrombin time, tromboplastin activated time, and platelet number. Decrease in platelet number was detected in groups submitted to dialysis. The best vascular access was performed with double lumen catheter and the hemodialysis may be used in equine practice, with high performance dialyze used in six- hour session.

Partial characterization and anticoagulant activity of a heterofucan from the brown seaweed Padina gymnospora

The brown algae Padina gymnospora contain different fucans. Powdered algae were submitted to proteolysis with the proteolytic enzyme maxataze. The first extract of the algae was constituted of polysaccharides contaminated with lipids, phenols, etc. Fractionation of the fucans with increasing concentrations of acetone produced fractions with different proportions of fucose, xylose, uronic acid, galactose, and sulfate. One of the fractions, precipitated with 50 percent acetone (v/v), contained an 18-kDa heterofucan (PF1), which was further purified by gel-permeation chromatography on Sephadex G-75 using 0.2 M acetic acid as eluent and characterized by agarose gel electrophoresis in 0.05 M 1,3 diaminopropane/acetate buffer at pH 9.0, methylation and nuclear magnetic resonance spectroscopy. Structural analysis indicates that this fucan has a central core consisting mainly of 3-ß-D-glucuronic acid 1-> or 4-ß-D-glucuronic acid 1 ->, substituted at C-2 with alpha-L-fucose or ß-D-xylose. Sulfate groups were only detected at C-3 of 4-alpha-L-fucose 1-> units. The anticoagulant activity of the PF1 (only 2.5-fold lesser than low molecular weight heparin) estimated by activated partial thromboplastin time was completely abolished upon desulfation by solvolysis in dimethyl sulfoxide, indicating that 3-O-sulfation at C-3 of 4-alpha-L-fucose 1-> units is responsible for the anticoagulant activity of the polymer.

The evaluation of clotting time in bovine thrombin, reptilase©, and thrombin-like fraction of Crotalus durissus terrificus venom using bovine, equine, ovine, bubaline and human cryoprecipitates

The objective of this study was to evaluate the effects of the thrombin-like fraction of Crotalus durissus terrificus venom, Reptilase©, and bovine thrombin of fibrinogen polls on bovine, equine, ovine, bubaline and human cryoprecipitates. The authors also made a comparative study between animal and human cryoprecipitates to see if there was any possibility of future use in medicine. Fibrinogen levels in cryoprecipitate were studied using 48 blood samples obtained as follows: 12 samples from humans, 9 from bovine, 10 from equine, 10 from ovine and 7 from bubaline. The results obtained showed average levels of 375.50 mg per cent for humans, 218.33 mg per cent for bovine, 240.80 mg per cent for equine, 267.70 mg per cent for ovine and 664.00 mg per cent for bubaline. Upon the formation of pools of human and animals fibrinogens, the following results were obtained: 435 mg per cent for humans, 444 mg per cent for bovine, 337 mg per cent por equine, 390 mg per cent for ovine and 530 mg per cent for bubaline. Statistical analysis (using the analysis of variance for entirely randomized experiment for the calculation of F statistics) demonstrated that the bubaline fibrinogen level was higher than that of human, and both were higher than those of ovine, equine, and bovine. Clotting times were determined using different dilutions of bovine thrombin, thrombin-like fraction of Crotalus durissus terrificus venom, and Reptilase©. Comparing these clotting times, results for human and bovine were found to be very similar, whereas using equine, ovine and bubaline the results above a dilution of 1:3 were markedly different. The results obtained permitted the following conclusions to be drawn show that: 1) bovine thrombin presented better interactivity with fibrinogen extracted both from human and bovine cryoprecipitates; 2) there was similar behavior when bovine thrombin was substituted for Reptilase© and for the thrombin-like fraction of Crotalus durissus terrificus venom; 3) cryoprecipitate from bovine can, in special circumstances, substitute human cryoprecipitate in medical practice; 4) human and bovine cryoprecipitates can be used with both Reptilase© and Crotalus durissus terrificus fractions using a dilution up to 1:5; 5) the use of bovine cryoprecipitate can be recomended using either bovine thrombin, Reptilase©, or thrombin-like fraction of Crotalus durissus terrificus venom.

Avaliação da coagulação sanguínea em gestantes da maternidade escola Assis Chateaubriand - MEAC / Blood coagulation on pregnant women in the maternidade escola Assis Chateaubriand - MEAC

Com o objetivo de avaliar a coagulação sanguinea em gestantes da maternidade Escol a ASsis Chateaumbriand (MEAC), no periodo de 25 de janeiro a 25 de maio de 1992, foi analisado o plasma de 22 gestantes e 30 mulheres não grávidas que serviu como grupo de controle. Ficou evidenciado que os testes hemostáticos (tempo de protrombina, tempo parcial de de tromboplastina e tempo de trombina) resultaram em um aumento da coagulação com a diminuição da média dos tempos de coagulação, quando comparado ao grupo controle. O número de plaquetas dominuiu, com média de 175.545/mm3 na 26a semana para 148.727/mm3 na 36a semana de gestação. A concentração do fibrinogênio aumentou consideravelmente de 310mg/dl para 465,4mg/dl. Diante deste resultado, ficou evidente que o estudo é concordante com a literatura, onde as mudanças hemostáticas durante a gravidez levam a um estado de hipercoagulabilidade.

Alteraciones hemostaticas en diabetes mellitus idiopatica. / Hemostatic changes in idiopathic diabetes mellitus

En 22 pacientes diabeticos tipo II, 20 pacientes diabeticos tipo I y 22 sujetos normales se realizaron pruebas de hemostasia:Tiempo de trombina (TT), niveles de fibrinogeno, porcentaje de autoagregacion plaquetaria (AA) tiempo total de agregacion plaquetaria (TAP) usando epinefrina como agente indutor y tiempo de retardo de aparicion de la segunda onda de agregacion plaquetaria (RAP); se determinaron ademas niveles de hemoglobina glucosada (HbAlc) y glucemia de ayuno (Glu).Los niveles de fibrinogeno fueron mayores en los diabeticos que en los sujetos normales (p< 005) y proporcionales al tiempo de evolucion de la diabetes y a la Glu (p< 01).Los parametros de agregacion plaquetaria en los diabeticos mostraram hiperagregabilidad, juzgada por incremento en AA y acortamiento de TAP y RAP. Los niveles de HbA1c fueron mayores en ambos grupos de diabeticos. Se discute la "hiperfibrinogenemia" encontrada en los diabeticos y se propone a la posible glucosilacion de la molecula del fibrinogeno como responsable de algunas de las alteraciones encontradas