High variability in Factor IX one-stage assay in samples spiked with nonacog beta pegol among different pairs of reagent/detection system.

INTRODUCTION: Haemophilia B (HB) is an X-linked hereditary bleeding disorder characterized by coagulation factor IX (FIX) deficiency. To improve the quality of life of patients and adherence to treatment, recombinant factor concentrates modified to extend their half-life have been developed, called extended half-life factors (EHL: extended half-life). Nonacog beta pegol (N9-GP) is a glycopegylated recombinant human FIX molecule that has a half-life of 93 h with a single dose and has shown a higher recovery percentage than other molecules. To diagnose and monitor the treatment of haemophiliac patients, FIX activity is determined with the one-stage clotting assay (OSA) and/or the chromogenic assay. The objective of this work, carried out in three centres, was to measure the recovery of N9-PG with 10 different activated partial thromboplastin time (APTT) reagents on three platforms, in samples spiked in vitro with N9-GP, at four different concentration levels. METHODS: It was measured the recovery of N9-GP with 10 different APTT reagents (polyphenol, ellagic acid, silice dioxide, colloidal silica as APTT activator on three platforms, in sample spiked in vitro with N9-GP. RESULTS: The results show heterogeneity in the activity of N9-GP measured by OSA with the different APTT reagents when the calibrations were performed with the specific calibrator of each coagulometer. A recovery percentage between 87% and 108% was obtained only with polyphenol and ellagic acid as activator in the three platforms evaluated. The other reagents studied overestimate or underestimate, with no clear profile. When a calibration curve was performed with a calibrator prepared from the N9-GP vial, all APTT reagents met the established recovery requirement. CONCLUSION: APTT reagents with polyphenol or ellagic acid as activator would be the only ones appropriate when using the commercially available OSA with specific calibrator to monitor patients treated with N9-GP.

The fibrinogen prothrombin time-derived method is not useful in patients anticoagulated with low molecular weight heparins or rivaroxaban.

Essentials Fibrinogen prothrombin time-derived (FIBPT-d) behavior in anticoagulated patients is under studied. FIBPT-d method overestimates fibrinogen in rivaroxaban and low molecular weight heparin samples. Unfractionated heparin and dabigatran samples showed similar bias to the control group. Rabbit brain and human recombinant thromboplastin behavior was different in rivaroxaban samples. SUMMARY: Background The fibrinogen prothrombin time-derived (FIBPT-d) method with photo-optical coagulometers is easy and economical. However, there are few reports on the behavior of this test on samples from patients anticoagulated with direct oral anticoagulants or low molecular weight heparin (LMWH). Objective To compare fibrinogen results obtained with the Clauss (FIB C) method and the FIBPT-d method with two thromboplastins in anticoagulated patients. Population The study population comprised 295 consecutive anticoagulated patients: 99 treated with vitamin K antagonists (VKAs), 49 treated with unfractionated heparin (UFH), 47 treated with LMWH, 50 treated with rivaroxaban, 50 treated with dabigatran, and 100 normal controls (NCs). Methods Dabigatran samples were analyzed by the use of FIB C with HemosIL Fibrinogen C or 100 NHI thrombin units mL-1 reagents; rabbit brain and human recombinant thromboplastins with HemosIL PTFibrinogen HS plus (HS) and Recombiplastin 2G (RP) were used for FIBPT-d method. Heparin and rivaroxaban levels were assessed with HemosIL Liq antiXa with specific calibrators; dabigatran levels were determined with the HemosIL Direct Thrombin Inhibitor Assay. All assays were performed on the ACL TOP platform in two laboratories. Percentage biases for the FIBPT-d method versus the FIB C method were calculated by the use of Bland-Altman plots. Results Positive biases of the FIBPT-d method versus the FIB C method with both thromboplastins were seen in NC samples (13.7% and 18.9% for HS and RP, respectively), but biases with HS in rivaroxaban and VKA patient samples were higher than that in NC samples, at 31.9% and 34.0%, respectively. LMWH patient samples showed higher bias than NC samples: 26.5% and 29.3.0% with HS and RP, respectively. UFH and dabigatran patient samples showed similar bias as NC samples. Conclusion The FIBPT-d method should not be used in anticoagulated patients, because the FIBPT-d mathematical algorithm has been validated only in normal subjects, so overestimation could occur in these patients.

Plasminogen activator inhibitor-1 activity and 4G/5G polymorphism in hemodialysis.

INTRODUCTION: Chronic insufficiency alters homeostasis, in part due to endothelial inflammation. Plasminogen activator inhibitor-1 (PAI-1) is increased in renal disease, contributing to vascular damage. We assessed PAI-1 activity and PAI-1 4G/5G polymorphism in hemodialysis (HD) subjects and any association between thrombotic vascular access (VA) events and PAI-1 polymorphism. METHODS: Prospective, observational study in 36 HD patients: mean age: 66.6 +/- 12.5 yr, males n=26 (72%), time on HD: 28.71 +/- 22.45 months. Vascular accesses: 10 polytetrafluoroethylene grafts (PTFEG), 22 arteriovenous fistulae (AVF), four dual lumen catheters (CAT). Control group (CG): 40 subjects; mean age: 60.0 +/- 15 yrs, males n=30 (75%). Group A (GA): thrombotic events (n=12), and group B (GB): No events (n=24). Groups were no different according to age (69.2 +/- 9.12 vs. 65.3 +/- 14.5 yrs), gender (males: 7; 58.3% vs. 18; 81.8%), time on HD (26.1 +/- 14.7 vs. 30.1 +/- 38.7 months), causes of renal failure. Time to follow-up for access thrombosis: 12 months. RESULTS: PAI-1 levels in HD: 7.21 +/- 2.13 vs. CG: 0.42 +/- 0.27 U/ml (p<0.0001). PAI-1 4G/5G polymorphic variant distribution in HD: 5G/5G: 6 (17%), 4G/5G: 23 (64%); 4G/4G: 7 (19%) and in CG: 5G/5G: 14 (35%); 4G/5G: 18 (45%); 4G/4G: 8 (20%). C-reactive protein (CRP) in HD: 24.5 +/- 15.2 mg/L vs. in CG 2.3 +/- 0.2 mg/L (p<0.0001). PAI-1 4G/5G variants: GA: 5G/5G: 3; 4G/5G: 8; 4G/4G: 1; GB: 5G/5G: 3; 4G/5G: 15; 4G/4G: 6. Thrombosis occurred in 8/10 patients (80%) with PTFEG, 3/22 (9%) in AVF, and 1/4 (25%) in CAT. Among the eight PTFEG patients with thrombosis, seven were PAI 4G/5G. CONCLUSIONS: PAI-1 levels were elevated in HD patients, independent of their polymorphic variants, 4G/5G being the most prevalent variant. Our data suggest that in patients with PTFEG the 4G/5G variant might be associated with an increased thrombosis risk.

Factor XIII en transplante de médula ósea / Factor XIII in Bone Marrow Transplantation

La subunidad A del Factor XIII (FXIII), parte activa del FXIII, es sintetizada casi exclusivamente por megacariocitos y precursores de monocitos-macrófagos. Determinándose la actividad del FXIII en pacientes sometidos a transplante de médula ósea autólogo (TAMO) y alegeneico (TMO). En 52 pacientes, 30 TAMO y 22 TMO, la actividad del FXIII fue estudiada en días fijos (basal, mitad de régimen condicionante, día 0, día +7, día +15 y día +30). Un descenso del FXIII fue encontrado en todos los casos, cuando se comparó con los niveles basales, alcalzando el nadir de la actividad al día +7. La caída del FXIII fué significativamente más pronunciada en TMO que en TAMO. Se demostró correlación entre la actividad del FXIII y el tiempo de recuperación hematopoyética. Siete de los 52 ptes. (13, 4 porciento) tuvieron niveles de actividad del FXIII inferiores al 10 porciento, sólo 3 sufrieron sangrados asociados. En TMO, los niveles de PAI estaban significativamente aumentados en los días +7, +15 y +30. En conclusión, la actividad del FXIII está siempre desminuída post-trasplante y el restablecimiento de los niveles basales se relaciona directamente con la velocidad de reconstitución hematopoyética. Diferencias en los tiempos de "engraftment" podrían explicar las diferencias encontradas entre TAMO y TMO. Finalmente, la ausencia de sangrado en ptes. con actividad del FXIII por debajo de niveles hemostáticos(<10 porciento) podría deberse a hipofibrinolisis concominante asociada a aumento del PAI

Factor XIII en transplante de médula ósea / Factor XIII in Bone Marrow Transplantation

La subunidad A del Factor XIII (FXIII), parte activa del FXIII, es sintetizada casi exclusivamente por megacariocitos y precursores de monocitos-macrófagos. Determinándose la actividad del FXIII en pacientes sometidos a transplante de médula ósea autólogo (TAMO) y alegeneico (TMO). En 52 pacientes, 30 TAMO y 22 TMO, la actividad del FXIII fue estudiada en días fijos (basal, mitad de régimen condicionante, día 0, día +7, día +15 y día +30). Un descenso del FXIII fue encontrado en todos los casos, cuando se comparó con los niveles basales, alcalzando el nadir de la actividad al día +7. La caída del FXIII fué significativamente más pronunciada en TMO que en TAMO. Se demostró correlación entre la actividad del FXIII y el tiempo de recuperación hematopoyética. Siete de los 52 ptes. (13, 4 porciento) tuvieron niveles de actividad del FXIII inferiores al 10 porciento, sólo 3 sufrieron sangrados asociados. En TMO, los niveles de PAI estaban significativamente aumentados en los días +7, +15 y +30. En conclusión, la actividad del FXIII está siempre desminuída post-trasplante y el restablecimiento de los niveles basales se relaciona directamente con la velocidad de reconstitución hematopoyética. Diferencias en los tiempos de "engraftment" podrían explicar las diferencias encontradas entre TAMO y TMO. Finalmente, la ausencia de sangrado en ptes. con actividad del FXIII por debajo de niveles hemostáticos(<10 porciento) podría deberse a hipofibrinolisis concominante asociada a aumento del PAI (AU)

[Heparin cofactor II, a thrombin inhibitor with a still not clarified physiologic role]. / Cofactor II de la heparina (HCII), un inhibidor de trombina cuyo rol fisiológico no está completamente esclarecido.

Heparin Cofactor II (HCII) is a glycoprotein in human plasma which inactivates thrombin rapidly in the presence of dermatan sulfate. Inhibition occurs by formation of a stable equimolar complex between HCII and thrombin. HCII association with thrombotic events has not always been observed, thus decreased HCII does not appear to be a strong risk factor for thromboembolic events. Reduced HCII levels have been detected in different clinical conditions, such as hepatic failure, disseminated intravascular coagulation, thalasemina, sickle cell anemia. Increased physiological levels have been found in pregnant women and oral contraception. In our laboratory, we measured HCII plasmatic levels in the normal Buenos Aires city population and in patients under different clinical conditions, such as sepsis, diabetis, burns, oral anticoagulation and in patients treated with heparin, hyperhomcysteinemia in whom septic and diabetic patients showed decreased values. HCII thrombin inhibition possibly takes place in extravascular sites where dermatan sulfate is present. HCII activity would be important in the regulation of wound healing, inflammation, or neuronal development.

Cofactor II de la heparina (HCII), un inhibidor de trombina cuyo rol fisiológico no está completamente esclarecido. / [Heparin cofactor II, a thrombin inhibitor with a still not clarified physiologic role]

Heparin Cofactor II (HCII) is a glycoprotein in human plasma which inactivates thrombin rapidly in the presence of dermatan sulfate. Inhibition occurs by formation of a stable equimolar complex between HCII and thrombin. HCII association with thrombotic events has not always been observed, thus decreased HCII does not appear to be a strong risk factor for thromboembolic events. Reduced HCII levels have been detected in different clinical conditions, such as hepatic failure, disseminated intravascular coagulation, thalasemina, sickle cell anemia. Increased physiological levels have been found in pregnant women and oral contraception. In our laboratory, we measured HCII plasmatic levels in the normal Buenos Aires city population and in patients under different clinical conditions, such as sepsis, diabetis, burns, oral anticoagulation and in patients treated with heparin, hyperhomcysteinemia in whom septic and diabetic patients showed decreased values. HCII thrombin inhibition possibly takes place in extravascular sites where dermatan sulfate is present. HCII activity would be important in the regulation of wound healing, inflammation, or neuronal development.

Impaired clot lysis by rt-PA catalyzed mini-plasminogen activation.

The fibrinolytic system contains a proenzyme plasminogen (Plg) which is converted to plasmin (Plm) by the action of Plg activators. Physiological Plg activators are: tissue-type plasminogen activator (t-PA) and urokinase-type plasminogen activator. Plg was shown to be further cleaved by leukocyte elastase producing several fragments, one of which is called mini-plasminogen (mini-Plg) or neo-plasminogen Val442. In this paper we studied whether mini-Plg is able to produce clot lysis when it is activated by rt-PA in purified systems and in Plg depleted normal plasma. We found that mini-Plg clot lysis time was longer than that of Plg. Clot lysis times were 2.3 minutes +/- 0.06 for Plg and 9.8 minutes +/- 0.1 for mini-Plg. Mini-Plg is less efficient than Plg in producing clot lysis at all studied concentrations (0.1-1.2 microM). In Plg depleted normal human plasma mini-Plg is unable to produce complete clot lysis in presence of rt-PA. Although mini-Plg can be activated to mini-Plm by rt-PA, these results show that the activation process is insufficient to produce an efficient clot lysis.

Plasminogen: an important hemostatic parameter in septic patients.

BACKGROUND: Previously we observed in some but not all septic patients a low plasma concentration of plasminogen. OBJECTIVES: To investigate prospectively whether plasma levels of plasminogen or the ratio of plasminogen to alpha-2-antiplasmin have a prognostic value for survival from sepsis and to study the variation of other hemostatic parameters during septicemia. PATIENTS: The study population consisted of 45 consecutive patients with septicemia, 15 non-septic patients from the same intensive care unit and 30 healthy volunteers. MEASUREMENTS AND MAIN RESULTS: Plasminogen concentrations were significantly lower (p < 0.001) in plasma of septic patients (median 0,62 IU/ml range: 0.15-1,06) than in plasma of healthy controls (median 1.00 IU/ml, range: 0.75-1.10) or of non-septic intensive care patients (median 1.00 IU/ml, range: 0.82-1.08). Among the other parameters tested, plasminogen activator inhibitor (PAI-1) antigen concentration and PAI activity were similar in septic and non-septic intensive care patients, but higher than in healthy controls. Concentrations of elastase-alpha-1-protease inhibitor or of thrombin-antithrombin complexes were higher in septic patients than in non-septic intensive care patients or healthy controls. A degraded form of plasminogen of 38 kDa was detected by Western blot analysis in the plasma of septic patients, but not in plasma of non-septic intensive care patients or controls. Plasminogen alone or the ratio of plasminogen to antiplasmin were good markers for survival from septicemia. E.g. for plasminogen at a cut off of 0.65 IU/ml, sensitivity was 90.5% and specificity 66.7%, whereas for the ratio of plasminogen over antiplasmin at a cut off ratio of 0,65 IU/ml, sensitivity was 95.2% and specificity 70.8%. CONCLUSION: Plasminogen or the ratio of plasminogen to antiplasmin are sensitive markers for survival in patients with septicemia.

Heparin cofactor II in diabetic patients.

Levels of heparin cofactor II (HCII) activity and antigen and electrophoretic pattern were studied both in normal subjects and in type I diabetic patients with high and normal levels of glycosylated haemoglobin. There was a significant reduction in HCII activity (83 +/- 7%) in patients with high levels of glycosylated haemoglobin compared with controls (95 +/- 17%; P < 0.001). However, plasma HCII antigen levels were not decreased in these patients.