Comparative analysis of thrombin generation platforms for patients with coagulation factor deficiencies: A comprehensive assessment.

INTRODUCTION: Thrombin generation assays (TGAs) assess the overall functionality of the hemostatic system and thereby provide a reflection of the hemostatic capacity of patients with disorders in this system. Currently, four (semi-)automated TGA platforms are available: the Calibrated Automated Thrombogram, Nijmegen Hemostasis Assay, ST Genesia and Ceveron s100. In this study, we compared their performance for detecting patients with congenital single coagulation factor deficiencies. MATERIALS AND METHODS: Pooled patient samples, healthy control samples and normal pooled plasma were tested on all four platforms, using the available reagents that vary in tissue factor and phospholipid concentrations. The TGA parameters selected for analysis were peak height and thrombin potential. Results were normalized by using the calculated mean of healthy controls and a correction for between-run variation. Outcomes were presented as relative values, with the mean of healthy controls standardized to 100 %. RESULTS: Across all platforms and reagents used, thrombin potentials and peak heights of samples with coagulation factor deficiencies were lower than those of healthy controls. Reagents designed for bleeding tendencies yielded the lowest values on all platforms (relative median peak height 19-32 %, relative median thrombin potential 19-45 %). Samples representing more severe coagulation factor deficiencies generally exhibited lower relative peak heights and thrombin potentials. CONCLUSIONS: Thrombin generation assays prove effective in differentiating single coagulation factor deficient samples from healthy controls, with modest discrepancies observed between the platforms. Reagents designed for assessing bleeding tendencies, featuring the lowest tissue factor and phospholipid concentrations, emerged as the most suitable option for detecting coagulation factor deficiencies.

Decreased Thrombin Generation is Associated with Increased Thrombin Generation Biomarkers and Blood Cellular Indices in Pulmonary Embolism.

Pulmonary embolism (PE) is a heterogenous condition with variable clinical presentations. Thrombin generation potential (TGP) and biomarkers, and blood cellular indices can reflect the underlying pathophysiology and risk stratification of PE. This case-control study analyzed TGP in 209 PE patients from Loyola University, Pulmonary Embolism Response Team program compared to normal human plasma (NHP) controls. The present study evaluates TGP and biomarkers, and cellular indices in relation to PE severity, according to the European Society of Cardiology (ESC) guidelines. Statistical analysis including median with interquartile range (IQR), 2-tailed Wilcoxon Mann-Whitney test, Chi-square test, and Spearman Correlational analysis were performed. There were 209 patients with PE, with an almost equal distribution between sex, and a median age of 63 years. Significant downregulation in peak thrombin and endogenous thrombin potential (ETP), as well as upregulation in lag time, were observed in PE patients versus controls. Biomarker analysis revealed pronounced elevations, with D-dimer demonstrating the most significant increase. Blood cellular indices also rose in PE patients, correlating with disease severity. PE severity was associated with higher TGP and biomarker levels. Mortality rates differed significantly across risk categories and were highest in patients with elevated cellular indices. TGP and biomarkers are intricately linked to PE severity and can aid in risk stratification. Elevated cellular indices are associated with increased mortality, highlighting their potential as prognostic markers. These findings could enhance the precision of PE management strategies.

Whole blood storage duration alters fibrinogen levels and thrombin formation.

INTRODUCTION: Whole blood resuscitation for hemorrhagic shock in trauma represents an opportunity to correct coagulopathy in trauma while also supplying red blood cells. The production of microvesicles in stored whole blood and their effect on its hemostatic parameters have not been described in previous literature. We hypothesized that microvesicles in aged stored whole blood are procoagulant and increase thrombin production via phosphatidylserine. METHODS: Whole blood was obtained from male C57BL/6 male mice and stored in anticoagulant solution for up to 10 days. At intervals, stored whole blood underwent examination with rotational thromboelastography, and platelet-poor plasma was prepared for analysis of thrombin generation. Microvesicles were prepared from 10-day-old whole blood aliquots and added to fresh whole blood or platelet-poor plasma to assess changes in coagulation and thrombin generation. Microvesicles were treated with recombinant mouse lactadherin prior to addition to plasma to inhibit phosphatidylserine's role in thrombin generation. RESULTS: Aged murine whole blood had decreased fibrin clot formation compared with fresh samples with decreased plasma fibrinogen levels. Thrombin generation in plasma from aged blood increased over time of storage. The addition of microvesicles to fresh plasma resulted in increased thrombin generation compared with controls. When phosphatidylserine on microvesicles was blocked with lactadherin, there was no difference in the endogenous thrombin potential, but the generation of thrombin was blunted with lower peak thrombin levels. CONCLUSION: Cold storage of murine whole blood results in decreased fibrinogen levels and fibrin clot formation. Aged whole blood demonstrates increased thrombin generation, and this is due in part to microvesicle production in stored whole blood. One mechanism by which microvesicles are procoagulant is by phosphatidylserine expression on their membranes.

Hemostatic imbalance induced by tamoxifen in estrogen receptor-positive breast cancer patients: An observational study.

BACKGROUND: Estrogen receptor (ER)-positive (ER+) breast cancer accounts for approximately 75% of all breast cancers. Tamoxifen, a selective estrogen receptor modulator, is the standard adjuvant treatment. Although better tolerated than aromatase inhibitors, tamoxifen increases the risk of venous thromboembolism (VTE) 1.4-fold. AIM: To assess the hemostatic imbalance induced by tamoxifen in adjuvant treatment of ER+ breast cancer. METHOD: Twenty-five patients in remission from ER+ breast cancer under tamoxifen were included. One hundred and thirty one age- and BMI-matched healthy controls were included to establish reference ranges of thrombin generation assay (TGA) parameters. TGA was performed in the absence and presence of exogenous activated protein C (APC) to calculate the normalized APC sensitivity ratio (nAPCsr), a marker of APC resistance. RESULTS: All TG parameters except the endogenous thrombin potential (ETP) (-APC) were significantly impacted by tamoxifen (p < 0.001). In absence of APC, regardless of TGA parameters, at least 50% of results were outside the reference ranges except for ETP, which was above the upper reference limit in only two individuals. The most impacted parameter was the Peak Height with 52% (-APC) and 80% (+APC) of results above the upper reference range limit, respectively. The nAPCsr was significantly higher in tamoxifen users (mean ± standard deviation = 3.18 ± 0.91) compared to the control group (2.19 ± 0.92, p < 0.0001). CONCLUSION: This observational study showed that patients in remission from ER+ breast cancer taking tamoxifen had altered thrombin generation, as well as an acquired APC resistance. Moreover, this is the first study using the validated ETP-based APC resistance assay in tamoxifen-treated patients.

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.

Produccion de Trombina y Tromboplastina tisular para el diagnostico de alteraciones hemorragicas y tromboticas / Production of Thrombim and Tromboplastim for diagnosis of hemorrhagic and thombotic disorders

Se produjo trombina a partir de plasma humano y tromboplastina de cerebro de conejo, se realizo el control de calidad de la trombina mediante (TT) y tiempo de Protrombina (PT) respectivamente, en muestras normales y patologicas. Se obtuvo una trombina de muy alta actividad y un rendimiento del reactivo suficiente para la determinacion de 2500 pruebas de TT. Asimismo se obtuvo tromboplastina de buena calidad con un rendimiento del reactivo suficiente para 240 pruebas a partir de 6,7 g de cerebro.

Interrelación entre el tiempo de tromboplastina parcial activada (TTPA), la generación de trombina y el sistema antigénico ABO / Interrelation between activated partial thromboplastin time (APTT), thrombin generation and ABO antigenic system

No disponible

Marcadores de generacion de trombina en insuficiencia renal de niños con sindrome uremico hemolitico epidemico / Markers of thrombin generation in kidney failure of children with epidemic hemolytic uremic syndrome

Se presentan aquí los niveles de los marcadores tempranos de activación de la coagulación y del TNF-alpha en 12 niños con la forma epidémica del síndrome urémico hemolítico, de 16 meses de edad, (12-18) (mediana y rango) Todos los pacientes se recuperaron de las enfermedad dentro de las 2 a 4 semanas de evolución. Se tomaron cuatro muestras de sangre: al ingreso al hospital, luego en la primera y segunda semana y en la remisión. Las determinaciones del F1+2 TAT y TNF-alpha se realizaron por técnicas de ELISA comerciales, mientras que el factor von Willebrand se determinó por el método de Laurell. Los valores de F1+2 y del TAT al ingreso fueron 7.8 nM (3.7-12.3) y 22.7 ng/ml (8-76) respectivamente. Además, se encontró correlación significativa entre los niveles de F1+2 vs creatinina sérica, r:0.47 p < 0.001; F1+2 vs úrea sérica, r:0.66 p < 0.001; TAT vs creatinina sérica, r:0.77 p < 0.001; TAT vs urea , r:0.59 p <0.001. La mediana del FvW al ingreso en 11/12 niños fue de 260 por ciento (170-420). Los niveles del FvW se correlacionaron con los del F1+2; r:0.77 p < 0.001 y con los del TAT, r:0.41 p < 0.01. Los valores de estos marcadores séricos tendieron a normalizarse con la mejoría de la enfermedad. Se encontró una correlación negativa entre el recuento plaquetario y los niveles del F1+2, r:-0.64 p<0.001. Los niveles de TNF-alpha estuvieron aumentados en 5 niños, 22.2 pg/ml (17.2-53.7). Los resultados sugieren que estas anormalidades pueden ser atribuidas a un estímulo común sobre células endoteliales.

Marcadores de generacion de trombina en insuficiencia renal de niños con sindrome uremico hemolitico epidemico / Markers of thrombin generation in kidney failure of children with epidemic hemolytic uremic syndrome

Se presentan aquí los niveles de los marcadores tempranos de activación de la coagulación y del TNF-alpha en 12 niños con la forma epidémica del síndrome urémico hemolítico, de 16 meses de edad, (12-18) (mediana y rango) Todos los pacientes se recuperaron de las enfermedad dentro de las 2 a 4 semanas de evolución. Se tomaron cuatro muestras de sangre: al ingreso al hospital, luego en la primera y segunda semana y en la remisión. Las determinaciones del F1+2 TAT y TNF-alpha se realizaron por técnicas de ELISA comerciales, mientras que el factor von Willebrand se determinó por el método de Laurell. Los valores de F1+2 y del TAT al ingreso fueron 7.8 nM (3.7-12.3) y 22.7 ng/ml (8-76) respectivamente. Además, se encontró correlación significativa entre los niveles de F1+2 vs creatinina sérica, r:0.47 p < 0.001; F1+2 vs úrea sérica, r:0.66 p < 0.001; TAT vs creatinina sérica, r:0.77 p < 0.001; TAT vs urea , r:0.59 p <0.001. La mediana del FvW al ingreso en 11/12 niños fue de 260 por ciento (170-420). Los niveles del FvW se correlacionaron con los del F1+2; r:0.77 p < 0.001 y con los del TAT, r:0.41 p < 0.01. Los valores de estos marcadores séricos tendieron a normalizarse con la mejoría de la enfermedad. Se encontró una correlación negativa entre el recuento plaquetario y los niveles del F1+2, r:-0.64 p<0.001. Los niveles de TNF-alpha estuvieron aumentados en 5 niños, 22.2 pg/ml (17.2-53.7). Los resultados sugieren que estas anormalidades pueden ser atribuidas a un estímulo común sobre células endoteliales. (AU)