Performance and reference intervals of thrombin generation test: results from the Brazilian longitudinal study of adult health (ELSA-Brasil). A cross-sectional study

ABSTRACT BACKGROUND: The thrombin generation test (TGT) has shown promise for investigation of hemorrhagic and thrombotic diseases. However, despite its potential, it still needs standardization. Moreover, few studies have established reference values for TGT parameters. In Brazil, these values have not yet been established. OBJECTIVE: To determine TGT performance and reference intervals for TGT parameters in healthy individuals. DESIGN AND SETTING: Cross-sectional study conducted among participants in the Brazilian Longitudinal Study of Adult Health (Estudo Longitudinal de Saúde do Adulto, ELSA-Brasil). METHODS: The reference sample consisted of 620 healthy individuals. The calibrated automated thrombogram (CAT) method, under low and high tissue factor (TF) conditions, was used to assess thrombin generation. Test performance was analyzed using intra and interassay coefficients of variation (CV) and reference intervals were calculated using the nonparametric method proposed by the International Federation of Clinical Chemistry and the Clinical and Laboratory Standards Institute. RESULTS: The intraassay CV ranged from 1.4% to 2.2% and the interassay CV, 6.8% to 14.7%. The reference intervals for TGT parameters under low and high TF conditions were, respectively: lagtime: 3.0-10.3 and 1.4-3.7 min; endogenous thrombin potential (ETP): 1134.6-2517.9 and 1413.6-2658.0 nM.min; normalized ETP: 0.6-1.3 and 0.7-1.4; peak: 103.2-397.7 and 256.4-479.0 nM; normalized peak: 0.3-1.3 and 0.7-1.2; and time-to-peak: 5.6-16.0 and 3.4-6.7 min. These parameters were categorized relative to sex. Conclusion: TGT performance was adequate and the proposed reference intervals were similar to those of other studies. Our findings may be useful for consolidating the TGT, through contributing to its standardization and validation.

Hemostatic evaluation of rabbits envenomed with Bothrops alternatus treated with anti-bothropic serum, desmopressin and tranexamin acid / Avaliação hemostática de coelhos envenenados com Bothrops alternatus e tratados com soro antibotrópico, desmopressina e ácido tranexâmico

In Brazil, snakes from the Bothrops genus are responsible for thousands of accidents, and their venoms are mainly composed of proteolytic enzymes. Although the antibothropic serum produced by the Brazilian Institutes is remarkably efficient, more studies are necessary, especially in veterinary medicine. The venom contain enzymes and non-enzymatic proteins that interfere with hemostasis leading to hemorrhage or even thrombosis. Possible treatment associations with known bothropic antivenom were the reason for the development of the present study. The aim of this study was to evaluate hemostasis alterations caused by Bothrops alternatus venom in rabbits followed by treatments with anti-bothropic serum, tranexamic acid and desmopressin. Twenty New Zealand rabbits were distributed into five groups (n=4) that were experimentally envenomed with 150mcg/kg of B. alternatus venom via intramuscular injection and treated as follow: Group 1 (G1) was the positive control and received venom and PBS/BSA; Group 2 (G2) was treated with tranexamic acid; Group 3 (G3) with desmopressin; Group 4 (G4) with tranexamic acid and anti-bothropic serum; and Group 5 (G5) with anti-bothropic serum and desmopressin. Blood samples were collected before venom administration, and one, four, eight and 12 hours after, for Partial activated partial thromboplastin time, Prothrombin Time, Thrombin Time and fibrinogen evaluation. Thrombin generation (TG) test was carried out with a pool of samples from final times (8 and 12h). At the end of 12h, all animals were euthanized and necropsy was conducted. Samples from muscle tissue, heart, lungs and kidney were analyzed. Classic coagulation tests showed no significant differences amongst groups and times. However, TG indicated that the venom causes a hypocoagulability state, which was not reversed by proposed treatments. Histology showed muscle inflammation, hemorrhage and necrosis, as well as hemorrhage in other tissues with no differences amongst groups. B. alternatus envenomation causes hypocoagulability detected by TG assay, but not through classical coagulation tests. The use of tranexamic acid and desmopressin for hemostasis stabilization after inoculation of the venom did not show advantage in coagulation restoration.(AU)

No Brasil, as serpentes do gênero Bothrops são responsáveis por milhares de acidentes, e seus venenos são compostos principalmente de enzimas proteolíticas. Embora o soro antiofídico produzido pelos institutos brasileiros seja notavelmente eficiente, mais estudos são necessários, especialmente na medicina veterinária. O veneno contem enzimas e proteínas não-enzimáticas que interferem com a hemostasia levando a hemorragias ou trombose. A associação de outros tratamentos ao soro antibotrópico foi a razão para o desenvolvimento do presente estudo. O objetivo deste estudo foi avaliar as alterações da hemostasia causadas pelo veneno de Bothrops alternatus em coelhos, após tratamento com soro antibotrópico, ácido tranexâmico e desmopressina. Vinte coelhos da Nova Zelândia foram distribuídos em cinco grupos (n = 4) que foram submetidos a experimentos com 150mcg/kg de veneno de B. alternatus por injeção intramuscular. O Grupo 1 (G1) foi o controle positivo e recebeu veneno e PBS / BSA, enquanto o Grupo 2 (G2) foi tratado com ácido tranexâmico, o Grupo 3 (G3) com desmopressina, o Grupo 4 (G4) com ácido tranexâmico e soro antibotrópico, e o Grupo 5 (G5) com soro antibotrópico e desmopressina. As amostras de sangue foram coletadas antes da administração do veneno, e uma, quatro, oito e 12 horas após os tratamentos para realização de tempo de tromboplastina parcial ativada parcial (TTPa), tempo de protrombina (TP), tempo de trombina (TT) e mensuração de fibrinogênio. Para o ensaio de geração de trombina (TG) foi realizado com um pool de amostras nos tempos finais (8 e 12h). Ao final das 12h, todos os animais foram sacrificados e a necropsia foi realizada. Amostras de tecido muscular, coração, pulmões e rins foram analisadas. Os testes TTPa, TP, TT e fibrinogênio não mostraram diferenças significativas entre os grupos e os tempos. No entanto, o TG indicou que o veneno causa um estado de hipocoagulabilidade, que não foi revertido pelos tratamentos propostos. Na histologia, foram observadas inflamação muscular, hemorragia e necrose, além de hemorragia em outros tecidos, sem diferenças entre os grupos. O envenenamento por B. alternatus causa hipocoagulabilidade detectada mais precocemente pelo teste de geração de trombina. O uso de ácido tranexâmico e desmopressina para estabilização da hemostasia após a inoculação do veneno não mostrou vantagem na restauração da coagulação.(AU)

Thrombin generation and thromboelastometry tests in dogs with chronic kidney disease / Testes de geração de trombina e tromboelastometria em cães com doença renal crônica

Patients with chronic kidney disease (CKD) have paradoxical hemostatic potential because they have bleeding episodes but are also prone to thrombosis. Few studies have evaluated blood viscoelastic properties in dogs with kidney disease; on the other hand, hypercoagulability has been observed in these patients. It is also emphasized that the platelet function and its participation in this process have not yet been fully understood. The objective of this study was to evaluate and compare the Thrombin Generation Test (TGT) and also viscoelastic properties of the blood measured by thromboelastometry (TEM) in dogs with proteinuria in CKD. Twenty healthy dogs (Control Group) and 19 dogs with CKD in stage III or IV, classified according to International Renal Interest Society - IRIS, were selected, and the reference test of urine protein:creatinine ratio (UPCR) should be greater than one (CKD group). Blood samples for TEM, thrombin generation, Prothrombin Time (PT), activated Partial Thromboplastin Time (aPTT), and fibrinogen concentration was collected at a single time for both groups after inclusion criteria being confirmed. Statistical analysis was performed according to the distribution of variables at 5% significance level. Differences were observed between healthy dogs and those with proteinuria in CKD noted in TEM. The TGT was unable to differentiate between sick and healthy groups. However, when the nephropathy was stratified, increases in TTP and peak thrombin concentration by TGT were observed in females and dogs over 30 days of diagnosis of CKD. Both tests signaled a discrete state of hypercoagulability. In fact, TEM is more sensitive to detect hypercoagulability in dogs with CKD. However, the TGT has potential clinical application by allowing long-term sample storage.(AU)

Os pacientes com doença renal crônica (DRC) apresentam um potencial hemostático paradoxal, pois apresentam episódios de sangramento, mas também são propensos à trombose. Poucos estudos avaliaram as propriedades viscoelásticas sanguíneas em cães com doenças renais, entretanto, a hipercoagulabilidade já foi observada nestes pacientes. Ressalta-se ainda que a função plaquetária e sua participação neste processo ainda não foram totalmente esclarecidas. O objetivo foi avaliar e comparar o teste de geração de trombina (TGT) e as propriedades viscoelásticas sanguíneas medidas pela tromboelastometria (TEM) em cães com DRC proteinúrica. Foram selecionados 20 cães saudáveis (grupo controle) e 19 cães com DRC em estágios III ou IV classificados segundo o IRIS e a relação proteína/creatinina urinária maior que um (grupo DRC). As amostras de sangue para a realização da tromboelastometria (TEM), geração de trombina, tempo de protrombina (TP), tempo de tromboplastina parcial ativada (TTPA) e concentração de fibrinogênio foram colhidas em momento único para ambos os grupos após os critérios de inclusão confirmados. A análise estatística foi realizada de acordo com a distribuição das variáveis, ao nível de 5% de significância. Foi observada diferença entre os cães saudáveis e os com DRC proteinúrica observados na TEM. O teste de geração de trombina não foi capaz de diferenciar os grupos doente e saudável. Entretanto, quando os nefropatas foram analisados de forma estratificada, foram observados aumentos do ETP e da concentração máxima de trombina (peak) pelo TGT em fêmeas e em cães com mais de 30 dias de diagnóstico da DRC. Ambos os testes sinalizando para um discreto estado de hipercoagulabiliade. A tromboelastometria é mais sensível para detectar a hipercoagulabilidade em cães com DRC. Entretanto, o teste de geração de trombina tem melhor aplicabilidade por permitir o armazenamento da amostra em longo prazo.(AU)

Standardization and evaluation of the performance of the thrombin generation test under hypo- and hypercoagulability conditions

ABSTRACT Background: In order to standardize a thrombin generation() protocol, we analyzed the analytical variables and sensitivity of this test to hypo/hypercoagulability states. Methods: The effect of the tissue factor concentration and the intra- and interassay precision were analyzed. To evaluate the hypercoagulability status, the plasma of women under an oral contraceptive was tested, while plasma from hemophilia A patients at 1, 3 and 7 days after recombinant FVIII infusion, and lyophilized plasma deficient in FVII or FVIII were used for the evaluation of hypocoagulability. Results: The intra-assay coefficient of variation was <10% with 1 and 5 pM of low and high TF. The oral contraceptive users showed increased thrombin generation in comparison to non-users, which was more pronounced with low TF (endogenous thrombin potential ETP) p = 0.0009; peak p = 0.0009; lagtime p = 0.0008). In relation to the FVIII-deficient plasma, a higher TG was observed as FVIII levels were increased and a better discrimination was obtained for different concentrations of FVIII with low TF (ETP p < 0.0001; peak p < 0.0001; lagtime p = 0.0004). Using low TF, plasma from hemophilia A patients showed higher TG values after 1 day of recombinant FVIII infusion vs after 3 days (ETP p < 0.0001; peak p < 0.0001; lagtime p = 0.0407), while the lowest values were observed after 7 days. With FVII-deficient plasma, thrombin generation was lower than normal plasma and a more pronounced difference was observed with high TF compared to low TF (ETP p < 0.0001; peak p < 0.0001; lagtime p < 0.0001). Conclusion: Under our conditions the thrombin generation test seems to be sensitive to evaluation of hyper/hypocoagulability states. Standardization of the thrombin generation test may have an application in the evaluation of bleeding and thrombotic disorders.

Thrombin generation assays for global evaluation of the hemostatic system: perspectives and limitations

Abstract The existing techniques to evaluate hemostasis in clinical laboratories are not sensitive enough to detect hypercoagulable and mild hypocoagulable states. Under different experimental conditions, the thrombin generation test may meet these requirements. This technique evaluates the overall balance between procoagulant and anticoagulant forces and has provided new insights in our understanding of the coagulation cascade, as well as of the diagnosis of hypocoagulability and hypercoagulability conditions. Thrombin generated in the thrombin generation test can be quantified as platelet-rich or platelet-poor plasma using the calibrated automated thrombogram method, which monitors the cleavage of a fluorogenic substrate that is simultaneously compared to the known thrombin activity in a non-clotting plasma sample. The calibrated automated thrombogram method is an open system, in which different antibodies, proteins, enzymes and peptides can be introduced to answer specific questions regarding hemostatic processes. The thrombin generation test has great clinical potential, such as in monitoring patients taking anticoagulants and antiplatelet drugs, screening for genetic or acquired thrombotic disorders, and evaluating bleeding risk control in patients with hemophilia using bypass agents or replacement therapy. Different to conventional coagulation tests, the thrombin generation test can be used for an overall evaluation of hemostasis, the results of which can then be used to evaluate specific characteristics of hemostasis, such as prothrombin time, activated partial thromboplastin time, and levels of fibrinogen and other coagulation factors. The introduction of this method will contribute to a better understanding and evaluation of overall hemostatic processes; however, this method still requires standardization and clinical validation.

Benefits of Thromboelastography and Thrombin Generation Assay for Bleeding Prediction in Patients With Thrombocytopenia or Hematologic Malignancies

BACKGROUND: Thromboelastography (TEG) provides comprehensive information on the whole blood clot formation phases, whereas thrombin generation assay (TGA) reveals the endogenous thrombin levels in plasma. We investigated the potential significance of TEG and TGA parameters for prediction of clinical bleeding in hematologic patients on the basis of the patient's platelet levels. METHODS: TEG and TGA were performed in 126 patients with thrombocytopenia or hematologic malignancies. The bleeding tendencies were stratified on the basis of the World Health Organization bleeding grade. RESULTS: Maximum amplitude (MA) and clot formation in TEG and endogenous thrombin potential (ETP) in TGA showed significant associations with high bleeding grades (P=0.001 and P=0.011, respectively). In patients with platelet counts ≤10×10⁹/L, low MA values were strongly associated with a high bleeding risk. For bleeding prediction, the area under the curve (AUC) of MA (0.857) and ETP (0.809) in patients with severe thrombocytopenia tended to be higher than that of platelets (0.740) in all patients. Patients with platelet counts ≤10×10⁹/L displayed the highest AUC of the combined MA and ETP (0.929). CONCLUSIONS: Both TEG and TGA were considered to be good predictors of clinical bleeding in patients with severe thrombocytopenia. Combination of the ETP and MA values resulted in a more sensitive bleeding risk prediction in those with severe thrombocytopenia.

Activation of the Intrinsic Coagulation Pathway in Patients With Chronic Urticaria

PURPOSE: Although coagulation activation has been reported in chronic urticaria, data pertaining to detailed changes in coagulation factors and global coagulation status are lacking. The current study evaluated global coagulation status in patients with chronic urticaria using thrombin generation assay (TGA) and the levels of individual coagulation factors. METHODS: Patients with chronic urticaria (n=57) and 20 healthy controls were enrolled. TGA was performed under stimulation with 2 concentrations of tissue factor (TF). Coagulation factors and conventional coagulation assays were also analyzed. RESULTS: Although patients with chronic urticaria showed prolonged activated partial thromboplastin time, prothrombin time did not differ significantly between patients and controls. In both 1 pM and 5 pM TF-stimulated TGA, peak thrombin and endogenous thrombin potential (ETP) levels were markedly decreased in patients with chronic urticaria. As expected, intrinsic coagulation factors (VIII, IX, and XII), as well as coagulation factors of the common pathway (II, V, and X), were consistently decreased. Additionally, D-dimer was significantly increased in patients as compared to controls. In multivariate regression analysis, the presence of chronic urticaria was the only significant independent contributor to the low ETP value. CONCLUSIONS: Chronic urticaria is characterized by in vivo coagulation activation through the intrinsic coagulation pathway, which can be measured with sensitivity using TGA.

Thrombin generation increasing with age and decreasing with use of heparin indicated by calibrated automated thrombogram conducted in Chinese / 生物医学与环境科学（英文）

<p><b>OBJECTIVE</b>Calibrated Automated Thrombogram(CAT) is a test to monitor the generation of thrombin. It can be described by four parameters: lag time, peak thrombin, endogenous thrombin potential (ETP) and time to peak (ttPeak). This study aims to determine the normal ranges of CAT parameters in Chinese, and evaluate whether thrombin generation is correlated with the concentration of heparin/low molecular weight heparin.</p><p><b>METHODS</b>Plasma from 120 healthy subjects were collected to determine the normal rangea of CAT parameters in Chinese. Normal plasma pool (NPP, n=25) spiked with different concentrations of heparin or enoxaparin were used to detecte CAT parameters. The overall and age specific normal ranges of CAT parameters were calculated using descriptive statistics method with mean±2SD. The correlation between CAT parameters and age or concentrations of heparin, enoxaparin were analyzed with linear regression model.</p><p><b>RESULTS</b>The normal ranges for lag time, peak thrombin, ETP, ttPeak in the subjects were 3.648±2.465 min, 367.39±151.93 nmol/L, 2277±1030 nmol/L•min and 6.372±4.280 min respectively. Age was linearly correlated with lag time (r=-0.6583, P<0.0001), peak thrombin (r=0.4863, P<0.0001), ETP (r=0.3608, P<0.0014) and ttPeak (r=-0.6313, P<0.0001). The values of ETP/peak ratio were linearly correlated with concentrations of heparin.</p><p><b>CONCLUSION</b>The normal ranges of four CAT parameters for Chinese were determined. CAT parameters are associated with age. ETP/peak ratio could be used to monitor the process of anticoagulation therapy.</p>

Efecto de los anticuerpos antifosfolipídicos en la formación y degradación de la malla de fibrina en pacientes con aborto recurrente / Effect of antiphospholipid antibodies on the formation and lysis of fibrin network in patients with recurrent miscarriage

En el presente trabajo se estudió el proceso de formación y disolución de la malla de fibrina y la generación de plasmina en un grupo de pacientes con aborto recurrente (AR) debido a la presencia de anticuerpos antifosfolipídicos (N= 10), mujeres con AR sin el síndrome antifosfolipídico (SAF) (N= 6) y se comparó con un grupo de mujeres sanas (N= 8). Del grupo de pacientes estudiadas con SAF, nueve fueron positivas para anticuerpos anticardiolipina (aCL), cinco para la anti-b2-glicoproteína I (anti-b2GPI), cuatro para ambos anticuerpos, una para anticuerpos antiprotrombina (aPT) y anticoagulante lúpico (AL). El proceso de formación de la fibrina y su disolución fue estudiado por turbidimetría y la generación de plasmina mediante sustrato cromogénico S2251. Las curvas de polimerización de la(s) paciente(s) con AR sin SAF y AL presentaron un incremento en la pendiente y turbidez final, comparado con las del grupo control de mujeres sanas. La velocidad de disolución del coágulo fue mayor en la paciente con AL (21 ± 0) 10-4 DDO/seg y en las AR sin SAF (19,6 ± 5,7) 10-4 DDO/seg, comparado con el grupo control (14,5 ± 2,8) 10-4 DDO/seg. La generación de plasmina estuvo incrementada solamente en las AR sin SAF (85 ± 24%) comparado con 52 ± 3% en el grupo control, p= 0,005. Los cambios observados en el proceso de polimerización y fibrinólisis de la(s) paciente(s) con AR sin SAF y AL pudieran estar relacionados con el incremento en los niveles de fibrinógeno, mientras que los de la generación de plasmina con la entidad mórbida.

The present work was intended to study the process of fibrin formation and lysis and plasmin generation in a group of patients with recurrent miscarriage (RM), due to the presence of antiphospholipid antibodies (N= 10); as well as in women with RM without the antiphospholipid syndrome (APS) (N= 6), compared with those of a group of healthy women (N= 8). In the group of patients with APS, nine were positive for antibodies against cardiolipin (aCL), five for anti-b2-glycoprotein I (anti-b2GPI), four for both antibodies, and one for antibodies against prothrombin (aPT) and lupus anticoagulant (LA). Fibrin formation and lysis was followed by turbidity and plasmin generation using chromogenic substrate S2251. The polymerization curves from RM patients without APS and the LA patient showed an increased slope and maximum turbidity compared to those of the control group. The speed of lysis was higher in the LA patient (21 ± 0) 10-4 DOD/seg and the RM patients without APS (19.6 ± 5.7) 10-4 DDO/seg, compared to that of the control group (14.5 ± 2.8) 10-4 DDO/seg. Plasmin generation increased only in RM patients without APS (85 ± 24%) against the control group (52 ± 3%), p= 0.005. The changes observed in the fibrin polymerization and lysis process of women with RM without APS and LA seem to be related to their higher fibrinogen levels, while the increased plasmin generation was related to the patients´ morbidity.

Effect of estrogen-progestin hormonal replacement therapy on blood coagulation and fibrinolysis in postmenopausal women

OBJECTIVE: To evaluate antithrombin III (AT), thrombin (Fragment 1+2 [F1+2] and thrombin-antithrombin [TAT]) generation markers, as well as other coagulation parameters, such as prothrombin time, partial activated thromboplastin time, thrombin time, fibrinogen, euglobulin lysis time, and platelet count, in postmenopausal women after hormonal therapy. STUDY DESIGN: Forty-five patients who received either 0.625 mg/day unopposed oral conjugated equine estrogen (CEE), 0.625 mg/day oral CEE plus medroxyprogesterone acetate (MP), or 50 µg/day transdermal 17beta-estradiol plus MP, were included. Tests were performed before (T0) and after 3 (T3), 6 (T6) and 12 (T12) months of treatment. AT was determined by an amidolytic method, whereas F1+2 and TAT complex were measured by ELISA. RESULTS: There was a significant reduction in the AT level of patients who received oral CEE plus MP at T3. There was no AT reduction in patients taking either oral CEE alone or transdermal 17beta-estradiol plus MP. F1+2 increased in all patients, but it reached statistical significance only in patients receiving transdermal 17beta-estradiol MP at T3. CONCLUSIONS: The CEE associated with MP treatment may reduce AT levels, whereas unopposed CEE or transdermal 17beta-estradiol plus MP does not change AT. These changes might not be clinically relevant in the general population; however, hormonal replacement therapy may increase the risk of thrombosis in women with congenital or acquired thrombophilia.

OBJETIVO: Avaliar os marcadores antitrombina III (AT), fragmento 1 + 2 da trombina (F1+2) e complexo trombina-antitrombina (TAT), bem como outros parâmetros da coagulação, como tempo de pró-trombina, tempo parcial de tromboplastina ativado, tempo de trombina, fibrinogênio e tempo de lise da euglobulina em mulheres na pós-menopausa após terapia hormonal. DESENHO DO ESTUDO: Foram incluídas 45 voluntárias que receberam estrogênios conjugados eqüinos (ECE) 0,625 mg/dia, isoladamente ou associado ao acetato de medroxiprogesterona (AMP) ou usaram o 17beta-estradiol (50 µg/dia) transdérmico com AMP. Os exames foram realizados antes do tratamento (T0) e após três (T3), seis (T6) e doze (T12) meses após o início do tratamento. AT foi avaliada pelo método amidolítico, enquanto que o F1+2 e o complexo TAT por ELISA. RESULTADOS: Houve redução significante nos níveis de AT em pacientes que receberam ECE associado ao AMP no T3. Não houve redução na AT em mulheres que usaram ECE isoladamente ou aquelas com 17beta-estradiol transdérmico e AMP. O F1+2 aumentou em todos os grupos, mas apenas o grupo com 17beta-estradiol transdérmico e AMP apresentou diferença significante durante o T3. CONCLUSÕES: A associação de ECE e AMP pode reduzir os níveis de AT, enquanto ECE isoladamente ou 17beta-estradiol transdérmico com AMP não modificam-o acentuadamente. Essas alterações poderiam ser mais relevantes clinicamente na análise populacional. Todavia, a terapia de reposição hormonal aumentaria o risco de trombose em mulheres com trombofilia prévia congênita ou adquirida.