Artificial neural networks for prediction of recurrent venous thromboembolism.

BACKGROUND: Recurrent venous thromboembolism (RVTE) is a multifactorial disease with occurrence rates which vary from 13 % to 25 % in 5 years after the initial event. Once a patient the first thrombotic event, the probability of recurrence should be determined, as well as the most adequate anticoagulant therapy. To our knowledge based on the published literature, three statistical models have been proposed to calculate RVTE probability. However, these models present several limitations, such as: limited input variables, lack of external validation and applicability only for patients with a first unprovoked thrombosis. Additionally, some of the models have been recognized to fail in determining RVTE when patients have a low risk of recurrence. OBJECTIVE: An alternative procedure in which three Artificial Neural Network (ANN) models were developed to classify which patients will present RVTE based solely on clinical data. METHODS: Data of 39 clinical factors from 235 patients were used to train several ANN structures. The difference among the three models was its inputs. In ANN 1, the inputs were all 39 factors. In ANN 2, 18 factors determined previously as the main predictors of RTVE using Principal Component Analysis (PCA). Finally, in ANN 3, 15 factors combining PCA results with practical aspects. Different number of hidden layers and neurons, and three optimization algorithms were considered. 5-fold cross validation was also performed. RESULTS: The results showed that all models were capable of performing this task. Different optimization algorithms lead to different results. The best models presented high accuracy. The best structures were 39-10-10-1, 18-10-5-1, and 15-15-10-1 for ANN 1, ANN 2, and ANN 3 models, respectively. The cross-validation showed that the results are consistent. CONCLUSIONS: This work showed that the association of multivariate techniques and ANNs is a powerful tool that can be used to model a complex phenomenon such as RVTE without the restrictions of existing approaches. APPLICATION: After proper validation, these ANN models can be used to help clinicians with decisions regarding VTE treatment.

Evaluation of the immature platelet fraction contribute to the differential diagnosis of hereditary, immune and other acquired thrombocytopenias.

The differential diagnosis of immune (ITP) and hereditary macrothrombocytopenia (HM) is key to patient management. The immature platelet fraction (IPF) represents the subset of circulating platelets with higher RNA content, and has been shown to distinguish hypo- from hyperproliferative thrombocytopenias. Here we evaluated the diagnostic accuracy of IPF in the differential diagnosis between HM and other thrombocytopenias in a population of patients with post-chemotherapy thrombocytopenia (n = 56), bone marrow failure (n = 22), ITP (n = 105) and HM (n = 27). TPO levels were also measured in HM and ITP matched for platelet counts. Platelet counts were similar in all patient groups. Higher IPF values were observed in both ITP (12.3%; 2.4-65.6%) and HM (29.8%; 4.6-65.9%) compared to hypoproliferative thrombocytopenias. IPF values were also higher in HM compared to ITP, yielding a diagnostic accuracy of 0.80 (95%CI 0.70-0.90; P < 0.0001) to distinguish these two conditions. Intra- and inter-assays reproducibility of IPF in HM patients revealed that this is a stable parameter. In conclusion, IPF is increased in HM compared to both ITP and other thrombocytopenias and contributes to the differentiation between ITP and HM. Further studies are warranted to understand the biological rationale of these findings and to its incorporation in diagnostic algorithms of HM.

Performance of a point-of-care device in determining prothrombin time in supra-therapeutic INRs.

INTRODUCTION: Point-of-care (POC) devices have been widely adopted for monitoring prothrombin time (PT) (INR) following the demonstration of their accuracy compared to standard INR determination. However, guidelines suggest confirmation of POC results when INRs increase above therapeutic range, due to concerns regarding possible inferior performance of POC devices in high INR levels. Unfortunately, patients with supra-therapeutic INRs are underrepresented in studies that validated these devices. METHODS: We performed a prospective evaluation of the performance of a POC device in monitoring oral anticoagulation in patients with INR values above 3.5 in a University outpatient anticoagulation clinic. During a 6-month period, 2322 INR determinations were performed with a POC device, and results above 3.5 were immediately repeated on an automated coagulometer. RESULTS: Dual INR determinations by two methods were obtained in 160 visits, with a mean INR from the POC device of 4.52 ± 0.96. Both classical statistics and clinical concordance analysis yielded satisfactory results when the two methods were compared. CONCLUSION: Our results demonstrate that POC devices present good correlation with standard laboratory methods for PT determination in supra-therapeutic INRs and that differences in clinical management do not support the need for systematic confirmation of these results in nonbleeding patients.

Novel insights into the development of atherosclerosis in hemophilia A mouse models.

BACKGROUND: Cardiovascular diseases in aging people with hemophilia (PWH) represent a growing concern. The underlying hypocoagulability probably provides a protective effect against acute thrombus formation, but the limited data available show no preventive effect against the development of atherogenesis in PWH. Atherosclerosis-prone mice are attractive tools for the study of atherosclerosis development, and may provide insights into disease progression in PWH. METHODS: Severe hemophilia A (factor VIII-deficient [FVIII(o)]) mice were crossed with mice lacking apolipoprotein E (ApoE(-/-)) or mice lacking the LDL receptor (LDLR(-/-)), and then compared to hemostatically normal littermate controls. After mice had received atherogenic diets for 8, 22 or 37 weeks, atherosclerotic lesion size and phenotypic characterization were analyzed in the aortic sinus and whole aortas. RESULTS: ApoE(-/-)/FVIII(o) mice showed a time-dependent protective effect against the development of atherosclerosis, beginning after 22 diet-weeks and persisting to 37 diet-weeks in both the aorta sinus and whole aorta as compared with ApoE(-/-) mice. Notably, the FVIII deficiency did not influence the progression of atherosclerosis in the FVIII(o)/LDLR(-/-) model as compared with controls at early or late time points. CONCLUSIONS: Hypocoagulability ameliorates vascular disease in the ApoE-deficient model in a lipid-independent manner. Interestingly, FVIII deficiency did not affect the development of atherosclerosis in LDLR(-/-) mice. In contrast to the ApoE model, the LDLR model resembles the lipid profile that is commonly observed in humans with atherosclerosis. These findings, to a certain extent, support the notion of atherosclerosis development in the complete absence of FVIII.

Evaluation of the host response to endotoxemia of FVIII and FIX deficient mice.

For several years, coagulation has been implicated in the pathogenesis of sepsis. However, results from clinical trials with natural anticoagulants, as well as studies with knock-out mice for specific coagulation factors yielded conflicting results on the role of coagulation in the pathogenesis of sepsis. The aim of this study was to evaluate the impact of severe The factor VIII:C (FVIII:C) and factor IX:C (FIX:C) deficiency on a lipopolysaccharide (LPS)-induced murine model of sepsis. FVIII:C and FIX:C deficient mice, and their haemostatic normal littermate controls were challenged with LPS, and several parameters of the host response were evaluated: seven-day survival experiments were performed using two dose levels of LPS; biochemical and histological markers of tissue damage, coagulation parameters, and pro-inflammatory cytokines were evaluated at baseline and after 3 h and 6 h after an injection of LPS. Severe FVIII and FIX deficiency were compatible with normal survival in experimental sepsis. In addition, LPS-induced tissue damage and coagulation activation were similar in FVIII or FIX deficient mice compared to their respective controls. A lower release of pro-inflammatory cytokines was observed in FIX but not in FVIII deficient mice. Severe FIX or FVIII deficiency does not protect mice from mortality or from tissue damage in the endotoxemia model, supporting the hypothesis that FVIII and FIX are not critical to the pathogenesis of experimental sepsis.

High frequency of vitamin B12 deficiency in a Brazilian population.

OBJECTIVE: There are few studies regarding vitamin B12 deficiency in developing countries. In Brazil, a late diagnosis of vitamin B12 deficiency progressing to severe neurological damage is common. Thus, the aim of the present study was to verify the frequency of vitamin B12 deficiency in two Brazilian populations (elderly and adult participants) and to compare different methods of vitamin B12 deficiency detection. DESIGN: Five hundred participants were recruited from health centres from south-east Brazil and were separated into two groups: 60 years old or more and 30-59 years old. Vitamin B12 and folate concentrations were measured using electrochemiluminescence immunoassay (ECI) and RIA. Methylmalonic acid (MMA) was measured by LC coupled to tandem MS. Full blood counts were acquired using standard methods. RESULTS: All participants had normal blood count results and mean cell volume less than 99 fl; none of them presented folate deficiency according to the results, which were all greater than 3 ng/ml. Cobalamin levels less than 200 pmol/l were identified by one of the two or by both methods in 7.2 % of the participants aged 60 years or more and 6.4 % of the participants aged 30-59 years. MMA levels were higher in older subjects (P = 0.007) compared with younger subjects. A greater correlation of MMA v. RIA was observed than of MMA v. ECI (P = 0.0017 v. P = 0.014). MMA quantification estimated that cobalamin deficiency was present in more than 11 % of the subjects for both studied groups. CONCLUSIONS: The study shows that vitamin B12 deficiency is frequent in Brazilian adults and suggests that RIA is more sensitive than ECl for measuring cobalamin levels.

Microparticles in deep venous thrombosis, antiphospholipid syndrome and Factor V Leiden.

Microparticles (MPs) are blebs released from cellular surfaces during activation/apoptosis. They are procoagulant, pro-inflammatory and could contribute to pathogenesis of deep venous thrombosis (DVT). This study compared the number, cellular origin and procoagulant activity of MPs on DVT patients in different clinical situations: at diagnosis (n = 9, 5F/4M; mean age = 41.11), 1-3 years after warfarin withdrawal (n = 10, 7F/3M; mean age = 32.90), associated to antiphospholipid syndrome (APS; n = 11, 9F/2M; mean age = 33.82), or asymptomatic carriers of Factor V Leiden (FVL; n = 7, 7F/0M; mean age = 34.00) vs healthy controls (CTR). The quantification and characterization were performed by flow cytometry using CD235, CD61, CD45, CD31, CD14, CD45, anti-TF and Annexin V. The plasmatic procoagulant activity was investigated by prothrombin fragment 1 + 2 (F1 + 2) determination. The MPs procoagulant activity was analyzed by D-dimer (DD2) and Thrombin Generation Test (TGT) on a healthy pool of plasmas adjusted or not by their number (10,000 MPs). The MPs percentages were not different between the groups, but absolute number was increased in patients 1-3 years after warfarin withdrawal vs CTR (P = 0.02). There was no difference of the MPs cellular origin comparing patients to controls. TGT using 10,000 MPs was lower on these patients (P = 0.01). APS patients showed a reduction of plasmatic procoagulant activity (P = 0.004), but they were under warfarin therapy. DD2 in the presence of MPs, independently of its number, was higher in patients with DVT at diagnosis (P < 0.0001). MPs of patients with spontaneous DVT at diagnosis can promote coagulation activation demonstrated by increased DD2. Even the increased MPs from patients 1-3 years after thrombotic episode generated lower amount of thrombin, they can have a protective effect by activation of Protein C anticoagulant pathway.

Three novel mutations in the activin receptor-like kinase 1 (ALK-1) gene in hereditary hemorrhagic telangiectasia type 2 in Brazilian patients.

Hereditary hemorrhagic telangiectasia (HHT) or Osler-Rendu-Weber disease is a systemic fibrovascular dysplasia with an autosomal dominant inheritance pattern. Mutations in two genes, endoglin and ALK-1, are known to cause HHT, both of which mediate signaling by transforming growth factor beta ligands in vascular endothelial cells. Ten patients were analyzed. Diagnosis of HHT was carried out by means of family history, recurrent bleeding, and the presence of multiple telangiectases lesions. Conformation-sensitive gel electrophoresis analyses with consistent abnormal migration patterns were cloned and sequenced using the MegaBace 1000 DNA automated analyzer. Three novel mutations were identified in the coding sequence of the ALK-1 gene in five patients and their families, which demonstrated clinical manifestations of HHT type 2. These mutations included a G insertion and a T deletion of single base pairs in exons 3 and 7, as well as missense mutations in exons 7 and 8 of the ALK-1 gene. These data indicate that loss-of-function mutations in a single allele of the ALK1 locus are sufficient to contribute to defects in maintaining endothelial integrity. We suggest the high rate of mutation detection and the small size of the ALK-1 gene make genomic sequencing a viable diagnostic test for HHT2.

Polymorphism of the human platelet antigen-5 system is a risk factor for occlusive vascular complications in patients with sickle cell anemia.

BACKGROUND: Polymorphisms of platelet membrane glycoproteins such as human platelet antigen (HPA)-1b, HPA-2b, the -5T/C Kozak sequence and C807T have been described as risk factors for vascular disease. Vaso-occlusion episodes are a common feature of sickle cell anaemia (SCA), leading to complications such as stroke, acute chest syndrome, avascular head femur necrosis and priapism. Complex interactions are involved in vaso-occlusion, and activated platelets may play an important role. These data raised the question of whether platelet polymorphisms could be implicated in occlusive vascular complications (OVC) of SCA. MATERIALS AND METHODS: In this study, 97 patients with SCA were analysed in two groups: 34 patients presenting with OVC (SCA-VC) and 63 without these complications (SCA-N). The distribution of the HPA-1, -2 and -5 systems, as well as C807T dimorphism and -5T/C Kozak sequence alleles, was evaluated using DNA-based methods. RESULTS: Patients of the SCA-VC group showed a higher frequency of the HPA-5b allele (0.324) compared with those of the SCA-N group (0.111) (chi2 = 13.19, P = 0.0002). None of the other polymorphisms, isolated or associated as haplotypes, demonstrated any correlation with the development of OVC in these patients. CONCLUSIONS: The findings of this study suggest that the HPA-5b allele is a genetic risk factor for the development of OVC in patients with SCA. This allele could be explored as a target for the development of new therapeutic approaches.

Antithrombin deficiency in Brazilian patients with venous thrombosis: molecular characterization of a single splice site mutation, an insertion and a de novo point mutation.

The prevalence of antithrombin (AT) deficiency in 342 unselected Brazilian patients with venous thrombosis was 1.16%, which increased to 3% when only patients under the age of 50 or with a familial history of thrombosis were considered. In two patients, a clinical (contraceptive use) or genetic risk factor (factor V Leiden and C677T in the methylene tetrahydrofolate reductase gene [MTHFR]) was identified and corroborated the hypothesis that an interaction of factors accounted for the appearance of thrombosis. However, no risk factor other than AT deficiency was identified in one patient with an important clinical and family history of spontaneous thrombosis. Three mutations were identified in these patients: a G-->A transition in intron 5 at position +1 (5'-->3'), three base insertions corresponding to arginine at position 5383 in exon 3A, and a G-->A transition at 13328, corresponding to an Ala404Thr de novo mutation. The polymorphisms in the genes coding for coagulation factors XII and XIII and fibrinogen normally associated with an increased risk for venous thrombosis were not related to thrombosis in these patients. This is the first study in South America to assess the prevalence of AT deficiency and to report the molecular characterization of the mutations involved.

The impact of the search for thrombophilia risk factors among antiphospholipid syndrome patients with thrombosis.

Thrombosis is a major clinical feature of the antiphospholipid syndrome. Interactions between genetic and acquired factors could contribute to thrombosis development. In this study, we evaluated 40 patients with antiphospholipid syndrome and thrombosis, 31 primary and nine secondary to systemic lupus erythemathosus, to estimate the carrier rates of factor V Leiden, 20210A --> G prothrombin variant and 677C --> T in the MTHFR gene. Protein C, protein S and antithrombin were measured in 30 patients, with a median of 100.66 +/- 23.86, 93.57 +/- 36.44 and 98.8 +/- 5.67%, respectively. None of the patients were deficient on these natural anticoagulants. No significant variation was found between the patient group and the controls, regarding the prevalence of homozygotes for the mutated 677T allele (2.5 versus 5.4%), or heterozygotes for factor V Leiden (0 versus 0.7%). Despite the fact that these mutations are relatively common in Brazilian thrombophilic patients, its low prevalence in this cohort of patients suggest that these genetic alterations are not risk factors for thrombosis in antiphospholipid syndrome. The prevalence of the mutated allele 20210A of the prothrombin gene was higher in patients when compared with controls (5 versus 0.7%; P = 0.01), suggesting that prothrombin variant could increase the risk of thrombosis in patients with antiphospholipid syndrome.

C282Y mutation in the HLA-H gene is not a risk factor for patients with myocardial infarction.

BACKGROUND: Iron is suspected to play a role in the development of atherosclerosis and in the progression of the disease, and consequently in myocardial infarction. Authors of a recent study identified a mutation in HLA-H gene, C282Y, that is an excellent marker for hemochromatosis, which is the most common cause of iron overload. There is a high prevalence of carriers of heterozygous hemochromatosis, most of whom are asymptomatic even with abnormalities of iron metabolism. OBJECTIVE: To study C282Y mutation in the HLA-H gene of 173 survivors of myocardial infarction matched with 172 controls by age, race, and sex, and 119 patients upon diagnosis of acute myocardial infarction. METHODS: Identification of the mutation was performed by PCR amplification of the DNA fragment followed by Rsal digestion. RESULTS: The prevalence of heterozygotes for the mutated allele both among patients and among controls was 1.74%. None of the 119 patients studied upon diagnosis was a carrier of the mutation. CONCLUSION: Our data suggested that the most common cause of iron overload is not associated with myocardial infarction.

Inherited thrombophilia as a risk factor for the development of ischemic stroke in young adults.

INTRODUCTION: Several recent studies have analyzed a possible effect of thrombophilia risk factors such as factor V Leiden, the prothrombin variant (allele 20210 A), and homozygosity for thermolabile methylenetetrahydrofolate reductase (MTHFR-T) on the development of ischemic stroke (IS). In the present study, we determined the role of these prothrombotic polymorphisms in the early onset of arterial IS or cerebral venous thrombosis (CVT) in a group of young Brazilian adults of Caucasian and African descent. MATERIALS AND METHODS: We conducted a cross-sectional study of 167 survivors of IS (153 patients with arterial IS and 14 cases of CVT; 66 men: 101 women; 124 of Caucasian and 43 of African origin; median age: 32.6 years; range: 15 to 45 years) and compared the prevalence of inherited thrombophilia risk factors with a control group of 225 sex and age matched individuals of the same ethnic background. To determine the interaction with atherogenic risk factors, the following diagnoses were considered: hypertension, hyperlipoproteinemia, diabetes mellitus, smoking status and use of oral contraceptives. RESULTS: In the arterial IS group, no significant variation was found between patients and controls of Caucasian origin regarding the prevalence of factor V Leiden (P = 0.92), the prothrombin variant (P = 0.13) or homozygosity for MTHFR-T (P = 0.61). Among Brazilians of African descent, 10.3% were homozygous for MTHFR-T, which was significantly elevated, odds ratio of 5.9 (95% CI: 0.88 to 49.15). In the CVT group, two Caucasian patients (20%) were heterozygous for the prothrombin variant, odds ratio of 9.7 (95% CI: 0.95 to 89.71) and one patient was carrier of factor V Leiden (P = 0.49). No prothrombotic polymorphism was identified in patients with CVT of African descent. All women in the CVT group were in use of oral contraceptives or in the post-partum state. DISCUSSION: Inherited thrombophilia risk factors were not found to increase the risk of arterial IS among young patients of Caucasian descent. However, a potential role of homozygosity for MTHFR-T was observed in a small group of patients of African origin. The analysis of patients with CVT revealed an increased risk due to the prothrombin gene variant or oral contraceptive use. Further studies including all incoming patients with IS are necessary to evaluate the impact of inherited thrombophilia risk factors on early mortality.

Allelic frequencies of three VNTRs in intron 40 of the human von Willebrand factor gene in types 1, 2, and 3 von Willebrand disease patients and controls of a Brazilian population.

Intron 40 of the human von Willebrand factor (vWF) gene contains a polymorphic region with three variable-number tandem repeats (VNTRs), type (ATCT)n. In the present report, we evaluated the allelic frequencies of these three VNTRs in a population constituted by 51 Brazilian Caucasian and 25 Types 1, 2, and 3 von Willebrand disease (vWD) patients, and performed segregation analysis in eight families affected by vWD Types 1 and 2. Three pairs of primers were used to amplify independently nucleotides 1640-1794 (VNTR 3), 1890-1991 (VNTR 1), and 2215-2396 (VNTR 2) from intron 40. The observed heterozygosities (0.86, 0.66, and 0.66 for VNTRs 3, 1, and 2, respectively) were in accordance with the expected heterozygosities derived from the allele frequencies (0.81, 0.64, and 0.70, respectively). Although the three VNTRs were highly polymorphic, VNTR 3 showed the highest values of heterozygosity [Haemostasis 25 (1995) 264; Hum. Mol. Genet. 1 (1992) 287.].

Frequencies of platelet-specific alloantigen systems 1-5 in three distinct ethnic groups in Brazil.

The human platelet antigen (HPA) systems are related to immune platelet disorders as well as to the development of occlusive vascular disease. Several distinct biallelic HPA systems are known, and a heterogeneous distribution of HPA alleles has been described among distinct ethnic groups. In this study we genotyped 320 carefully selected individuals from three distinct ethnic groups in Brazil (Caucasians, Blacks and Amazonian Indians) for the HPA-1, -2, -3, -4 and -5 systems. A similar prevalence for all HPA alleles was found in Brazilians of Caucasian and Black descent. These data contrast with those reported for similar ethnic groups in other countries. Among the Amazonian Indians, no b allele of the HPA-1, -4 and -5 systems was identified. The data presented here could be useful in the diagnosis of alloimmune platelet disease, in genetic counselling and in the development of screening programmes for HPA-related diseases.