Design and application of a 23-gene panel by next-generation sequencing for inherited coagulation bleeding disorders.

INTRODUCTION: Molecular testing of Inherited bleeding coagulation disorders (IBCDs) not only offers confirmation of diagnosis but also aids in genetic counselling, prenatal diagnosis and in certain cases genotype-phenotype correlations are important for predicting the clinical course of the disease and to allow tailor-made follow-up of individuals. Until recently, genotyping has been mainly performed by Sanger sequencing, a technique known to be time consuming and expensive. Currently, next-generation sequencing (NGS) offers a new potential approach that enables the simultaneous investigation of multiple genes at manageable cost. AIM: The aim of this study was to design and to analyse the applicability of a 23-gene NGS panel in the molecular diagnosis of patients with IBCDs. METHODS: A custom target enrichment library was designed to capture 31 genes known to be associated with IBCDs. Probes were generated for 296 targets to cover 86.3 kb regions (all exons and flanking regions) of these genes. Twenty patients with an IBCDs phenotype were studied using NGS technology. RESULTS: In all patients, our NGS approach detected causative mutations. Twenty-one pathogenic variants were found; while most of them were missense (18), three deletions were also identified. Six novel mutations affecting F8, FGA, F11, F10 and VWF genes, and 15 previously reported variants were detected. NGS and Sanger sequencing were 100% concordant. CONCLUSION: Our results demonstrate that this approach could be an accurate, reproducible and reliable tool in the rapid genetic diagnosis of IBCDs.

Oral anticoagulant treatment: risk factors involved in 500 intracranial hemorrhages.

Intracranial bleeding is the most severe complication caused by anticoagulant or antiplatelet treatment. The increasing use of this therapy, especially in older people, makes the balance between clinical benefit and bleeding risk an important consideration. A retrospective study of all consecutive 500 intracranial hemorrhages in the West Valladolid area, approximately 220,000 people, during the period 1998 to 2004, was performed. In relation to mortality, predisposing conditions were included, such as age, antithrombotic treatment, arterial hypertension, cancer, blood diseases, vascular malformations, and traumatisms. The incidence of intracranial hemorrhage was 310 per 100,000 per year with a mortality of 30%. Higher mortality was found in antiplatelet-treated patients (44.9%) than in anticoagulated patients (31.1%). This may be related to a different mean age of 78 vs. 71 years. Arterial hypertension was the most frequent risk factor (45.1% in nontreated patients, 60% anticoagulated, and 75.5% antiplatelet). The relative risk of intracranial bleeding in anticoagulated patients was 11.2 (p < 0.001) with an incidence of 0.03% and a median of 14 months since treatment began. The median INR was 3.3. In 40% of the patients the previous five controls were in range. Strict consideration of indications criteria joined to a better control of risk factors may avoid intracranial bleeding episodes.

Tratamiento de la coagulopatía en la sepsis severa / Treatment of coagulopathy in severe sepsis

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Disnea, tos y masa pulmonar / Eighty-five year old male patient with a subcutaneous mass in the right costal area

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Degree of hypercoagulability and hyperfibrinolysis is related to organ failure and prognosis after burn trauma.

Severely burned patients often present a hypercoagulability situation. However, its magnitude, time course, and relationship with organ failure and outcome remains to be established. Forty-three patients were studied on the first and seventh day after burn for hypercoagulability and fibrinolysis parameters. A hypercoagulability and hyperfibrinolysis state was found the first day after burn demonstrated by high levels of activated factor VII (VIIa, p<0.01), thrombin-antithrombin III complex (TAT, p<0.01), tissue plasminogen activator (t-PA, p<0.001) and D dimer (DD, p<0.01) and low levels of antithrombin III (ATIII, p<0.01), protein C (PC, p<0.01), plasminogen (PG, p<0.001) and alpha2 antiplasmin (AP, p<0.001). A paradoxical coexisting hypofibrinolysis was found as suggested by a low global fibrinolytic activity in the euglobulin plasma fraction fibrin plate assay (FA, p<0.01) and high levels of tissue plasminogen activator inhibitor type 1 (PAI-1, p<0.01). On day 7, a less marked hypercoagulability situation was found, with low ATIII (p<0.01) and PC (p<0.01), persisting the hypofibrinolytic situation observed on the first day. Non-survivors (NS) showed higher levels of VIIa (p<0.01), TAT (p<0.05) and t-PA (p<0.05), and lower levels of ATIII (p<0.05), PC (p<0.05) and AP (p<0.001) than survivors (S) on the first day. Also, there was a positive correlation of Marshall organ failure score with ATIII, (r2=0.49, p<0.001), PC, (r2=0.14, p<0.045) and PG levels, (r2=0.41, p<0.0003). Severely burned patients show a state of transient disseminated intravascular coagulation, related to the development of organ failure and outcome.

Efficacy of D-dimer and total fibrin degradation products evaluation in suspected pulmonary embolism.

Three different assays for fibrin/fibrinogen degradation products (FDP) were evaluated in patients with suspected pulmonary embolism (PE) as rapid screening tests with the aim of evaluating whether they could be used either as a substitute of ventilation/perfusion lung scanning or to supplement scintigraphy in patients in whom the scan was inconclusive (low or intermediate probability). D-Dimer by enzyme-linked immunosorbent assay (ELISA) and latex and total FDP by ELISA were measured prospectively in 85 consecutive patients with suspected PE. With a cutoff of 500 ng/ml, D-dimer by ELISA presented a 96% sensitivity and a 42% specificity, with positive and negative predictive values of 49 and 96%, respectively. D-Dimer by latex, also with a cutoff of 500 ng/ml showed a 93% sensitivity and 29% specificity, with positive and negative predictive values of 43 and 89%. For total FDP, with a cutoff of 900 ng/ml, the sensitivity and specificity were 96 and 26% respectively, with positive and negative predictive values of 42 and 93%. A normal assay may have reduced the necessity of a ventilation/perfusion only in 28% patients with D-dimer ELISA, 21% with D-dimer latex and 17% with total FDP ELISA and with a possible error of 4, 11 and 7%, respectively.(ABSTRACT TRUNCATED AT 250 WORDS)

High plasma levels of plasminogen activator inhibitor 1 (PAI-1) in polycythemia vera and essential thrombocythemia are associated with thrombosis.

We attempted to determine if a hypercoagulability state exists in patients with polycythemia vera (PV) and essential thrombocythemia (ET). We studied the hematocrit level, platelet count, use of any antiaggregant drugs, thrombotic or bleeding accidents and plasma levels of antithrombin III, protein C, total protein S, free protein S, vWF:Ag (Von Willebrand's factor related antigen), thrombin-antithrombin complexes, D-dimer, fibrinolytic activity, tissue plasminogen activator, plasminogen and PAI-1 in 33 patients (19 with ET and 14 with PV). PAI-1 plasma concentration was significantly higher in, both ET and PV patients than in the control group, and were higher in those patients with previous thrombotic episodes than in asymptomatic patients or with previous bleeding episodes. Increasing age was associated to more thrombotic episodes while younger patients presented with more hemorrhagic complications. A linear correlation between platelet count and PAI-1 levels in PV patients (r = 0.44, p < 0.05) and ET patients (r = 0.30, p < 0.05) was found. Fibrinolytic activity in patients with ET was significantly decreased when compared to the control group. A hypofibrinolytic state could be an additional factor which could be used as a predictive index of the thrombotic or bleeding tendency in each patient.

[Checking oral anticoagulation in capillary blood]. / Control de la anticoagulación oral en sangre capilar.

PURPOSE: To check out the reproducibility and costs of prothrombin time (PT) determination as a control of oral anticoagulant therapy (OAT) in plasma and capillary blood. PATIENTS AND METHODS: The study was carried out in two phases: along two years, 1,700 patients with OAT were controlled, 700 of them in the hospital outpatient clinic. In 149 patients INR was simultaneously determined in both capillary and venous blood. The 700 patients receiving acenocoumarin who had been controlled in 1991 according to the conventional plasma-sample fashion, were controlled in the second year (i.e., 1992) by means of capillary blood testing, a comparison of the costs of each method and the need for anticoagulant drugs being undertaken. Venous blood PT was assessed with reagent thromboplastin (Tromborel S) in an Electra-1000 (MLA) system. An automated Trombotrack system was used for the capillary blood tests using Thrombotest as current procedure. The results were expressed as INR in both methods. The statistical evaluation of the results was carried out by means of Student's t, variance analysis, and correlation study. RESULTS: No significant differences were found in the anticoagulation intervals attained from venous or capillary blood samples. No significant differences were seen in 87 patients on whom the test was repeated in two samples drawn from a single capillary puncture. The weekly OAT doses of 30 patients along six months were analysed. The need for anticoagulant drugs was similar (17.4 vs 17.2 mg/patient/week). The mean INR in 1991 was 2.82 and the mean drug-need was 15.24 mg/week, whereas in 1992 the mean INR was 2.86 and the need for anticoagulant was 15.49 mg/week. The costs of the conventional method were 103.6 Pta, this being 70 Pta for capillary blood, which means a 32% savings. CONCLUSIONS: OAT control by means of PT performed on capillary blood must be considered a substitutive method for the venous blood assay due to its efficacy, simplicity and lower costs.

[Familial thrombophilia due to resistance to activated protein C]. / Trombofilia familar debida a resistencia a la proteína C activada.

Resistance to activated protein C (RAPC) has been described recently as a cause of trombophilia; this may justify up to 50% of thromboembolic disease without predisposing cause in patients under 45 years. A 29 years-old male with a previous deep venous thrombosis (DVT) in the lower left limb three years earlier, developed a DVT in the right lower limb after a trauma of the knee that required immobilization, was associated to pulmonary thromboembolism diagnosed by gammagraphic methods. The phlebographic study showed femoro-iliaco-caval venous thrombosis. The proband's father and a younger brother had a previous history of thrombotic episodes. The following tests, were performed in the proband and relatives: prothrombin time, aPTT, thrombin time, fibrinogen, (Von Clauss), antithrombin III (chromogenic), protein C and protein S (coagulometry and ELISA), plasminogen (chromogenic) and lupus anticoagulant (ITT, dRVVT, aCL). RAPC was evaluated in two different samples. The proband study was performed under oral anticoagulation treatment (OAT). Control groups were: 21 blood donors and 12 OAT patients. The results showed a decreased response to APC in the proband (ratio 1.5) and relatives: father (1.4), brothers (1.5 and 1.5), while the mother was within the normal range (> or = 2). In normal controls and OAT patients the ratio was over 2. No other abnormalities were detected in the assays performed. It is concluded that RAPC is the cause of this familial trombophilia. RAPC should be included in the evaluation study of trombophilia.

High F1.2 fragment of prothrombin, thrombin-antithrombin III complex (TAT) and soluble fibrin plasma levels demonstrate hypercoagulability induced during loco-regional thrombolytic therapy with rt-PA.

In order to investigate the coagulation and fibrinolysis state in arterial peripheral thrombosis and thrombolysis, we studied 33 consecutive patients (mean age = 65, range: 28-88), 25 males and 8 females diagnosed of acute or subacute lower limb arterial thrombosis, treated with an intrathrombus infusion of rt-PA (0.1 mg/Kg/h) for three hours. Plasma levels of antithrombin III (AT-III), protein C (PC), plasminogen (Pg) and alpha 2-antiplasmin (AP), total and free protein S (PS), thrombin-antithrombin III complex (TAT), F1.2 fragment of prothrombin (F1.2), fibrinogen (Fg), soluble fibrin monomers (FM), tissue-plasminogen activator (t-PA), plasminogen activator inhibitor 1 (PAI-1), total fibrinogen/fibrin degradation products (TDP) and D dimer (DD) were determined prior to the therapeutic regime, at the end of the treatment, and 24 hours later. Levels of AT-III and protein C were somewhat low during the complete study. There was an increase in t-PA, TDP and D Dimer and a decrease of fibrinogen, alpha 2-antiplasmin and plasminogen at 3 hours. An elevation of TAT, fibrin monomers and F1.2 levels was found at three hours. A positive correlation between TAT and F1.2 was observed (r = 0.57, p < 0.05). There was also a positive correlation between soluble fibrin and TAT (r = 0.59, p < 0.05) and with F1.2 (r = 0.56. p < 0.05). These latter facts reflect an hypercoagulable situation induced during loco-regional thrombolytic therapy.

[Current status of thrombolysis. The need for coadjuvant antithrombotic therapy]. / Estado actual de la trombolisis. Necesidad de la terapéutica antitrombótica coadyuvante.

Thrombolytic therapy yields a 80% repermeabilitation in the acute myocardium infarct if applied soon. In other thrombotic sites such as deep venous thrombosis, pulmonary thromboembolism and peripheral arterial thrombosis, with no large cooperative randomized clinical trials, its usefulness is clear, tough selectively. The high rethrombosis frequency is the major drawback of this therapy. The concomitant use of an anti-thrombotic and/or anti-aggregant agent, even tough the possible higher risks of hemorrhages, seems a key element to improve the results obtained with thrombolysis.

Time course of hemostatic abnormalities in sepsis and its relation to outcome.

OBJECTIVES: To investigate the time course and the relation to prognosis of coagulation and fibrinolytic abnormalities in patients with septic shock. PATIENTS AND METHODS: Forty-eight consecutive patients admitted to the medical ICU with the diagnosis of septic shock (diagnosed by defined criteria) were studied. Mortality was 25 of 48. Mean age was 57 +/- 7.3 years. Blood samples were obtained on days 1, 4, and 7 after hospital admission to measure tissue-type plasminogen activator antigen (t-PA), urokinase-type plasminogen activator (u-PA), plasminogen activator inhibitor antigen (PAI-1), plasminogen, alpha 2-antiplasmin, fibrinogen, antithrombin III, protein C, protein S, thrombin-antithrombin complexes (TAT), D-dimer, and von Willebrand factor-related antigen (vWF:Ag). RESULTS: All patients showed marked abnormalities in both the coagulation and fibrinolytic systems. There were signs of coagulation activation and elevation of both activators and inhibitors of fibrinolysis. Nonsurvivors showed lower levels of protein C and antithrombin III and higher concentration of TAT than survivors. While both t-PA and PAI-1 concentrations were high in survivors and nonsurvivors, only survivors showed a progressive normalization of both parameters during the study period. Low plasminogen levels and plasminogen/alpha 2-antiplasmin ratio were found in both groups, presenting a trend toward normalization only in survivors. The differences reported were not apparent at the time of hospital admission. CONCLUSIONS: Septic shock is characterized by coagulation activation and fibrinolysis activation and inhibition. Nonsurvivors present a particular hemostatic profile characterized by a more marked activation of coagulation and a more intense inhibition of fibrinolysis. None of the abnormalities studied was significantly different between survivors and nonsurvivors at the time of hospital admission. In the presence of fibrin formation, nonsurvivors present a maintained imbalance in the fibrinolytic response determined by higher PAI-1 plasma concentration, probably contributing to their poor outcome.