Elisabetta Sacchi

High Risk of Cerebral-Vein Thrombosis in Carriers of a Prothrombin-Gene Mutation and in Users of Oral Contraceptives

BACKGROUND Idiopathic cerebral-vein thrombosis can cause serious neurologic disability. We evaluated risk factors for this disorder, including genetic risk factors (mutations in the genes encoding factor V and prothrombin) and nongenetic risk factors (such as the use of oral contraceptive agents). We compared the prevalence of these risk factors in 40 patients with cerebral-vein thrombosis, 80 patients with deep-vein thrombosis of the lower extremities, and 120 healthy controls. The G1691A mutation in the factor V gene and the G20210A prothrombin-gene mutation, which are established genetic risk factors for venous thrombosis, were studied. We also assessed the use of oral contraceptives and other risk factors for thrombosis. RESULTS The prevalence of the prothrombin-gene mutation was higher in patients with cerebral-vein thrombosis (20 percent) than in healthy controls (3 percent; odds ratio, 10.2; 95 percent confidence interval, 2.3 to 31.0) and was similar to that in patients with deep-vein thrombosis (18 percent). Similar results were obtained for the mutation in the factor V gene. The use of oral contraceptives was more frequent among women with cerebral-vein thrombosis (96 percent) than among controls (32 percent; odds ratio, 22.1; 95 percent confidence interval, 5.9 to 84.2) and among those with deep-vein thrombosis (61 percent; odds ratio, 4.4; 95 percent confidence interval, 1.1 to 17.8). For women who were taking oral contraceptives and who also had the prothrombin-gene mutation (seven patients with cerebral-vein thrombosis but only one control), the odds ratio for cerebral-vein thrombosis rose to 149.3 (95 percent confidence interval, 31.0 to 711.0). CONCLUSIONS Mutations in the prothrombin gene and the factor V gene are associated with cerebral-vein thrombosis. The use of oral contraceptives is also strongly and independently associated with the disorder. The presence of both the prothrombin-gene mutation and oral-contraceptive use raises the risk of cerebral-vein thrombosis further.

Myocardial blood flow and infarct size after CD133+ cell injection in large myocardial infarction with good recanalization and poor reperfusion: Results from a randomized controlled trial

Objective Large acute ST-elevation myocardial infarction (STEMI) sometimes leaves extensive ischemic damage despite timely and successful primary angioplasty. This clinical picture of good recanalization with incomplete reperfusion represents a good model to assess the reparative potential of locally administered cell therapy. Thus, we conducted a randomized controlled trial aimed at evaluating the effect of intracoronary administration of CD133+ stem cells on myocardial blood flow and function in this setting. Methods Fifteen patients with large anterior STEMI, myocardial blush grade 0–1 and more than 50% ST-elevation recovery after optimal coronary recanalization (TIMI 3 flow) with stenting were randomly assigned to receive CD133+ cells from either bone marrow (group A) or peripheral blood (group B), or to stay on drug therapy alone (group C). The cells were intracoronary injected within 10–14 days of STEMI. Infarct-related myocardial blood flow (MBF) was evaluated by NH3 positron emission tomography 2–5 days before cell administration and after 1 year. Results MBF increased in the infarct area from 0.419 (0.390–0.623) to 0.544 (0.371–0.729) ml/min per g in group A, decreased from 0.547 (0.505–0.683) to 0.295 (0.237–0.472) ml/min per g in group B and only slightly changed from 0.554 (0.413–0.662) to 0.491 (0.453–0.717) ml/min per g in group C (A vs. C: P = 0.023; B vs. C: P = 0.066). Left ventricular volume tended to increase more in groups B and C than in group A, ejection fraction and wall motion score index remained stable in the three groups. Conclusion These findings support the hypothesis that intracoronary administration of bone marrow-derived, but not peripheral blood-derived CD133+ cells 10–14 days after STEMI may improve long-term perfusion.

A rapid assay for ristocetin cofactor activity using an automated coagulometer (ACL 9000).

We have set up a rapid assay for measuring the activity of the von Willebrand factor ristocetin cofactor (VWF : RCo) using an automated coagulometer (ACL 9000; Instrumentation Laboratory, Lexington, Massachusetts, USA) and commercially available lyophilized platelet reagents (Dade-Behring, Marburg, Germany). Since VWF : RCo tested with the coagulometer (ACL-VWF : RCo) did not require loading, calculation of von Willebrand factor (VWF) activity or pretreatment of samples (calibration standard and tested plasmas), we thought it might be useful for rapid, automatic screening of VWF activity. To assess the precision and the potency of this ACL-VWF : RCo, we tested the assay in this laboratorys internal normal and abnormal controls, in a group of 67 healthy individuals and in 28 patients with different types of von Willebrand disease (VWD). We compared the ACL-VWF : RCo findings with those of the standard agglutination test (Agg-VWF : RCo), normalizing both assays against VWF antigen (VWF : Ag) measured by ‘automatic’ and standard enzyme-linked immunosorbent assay ‘in-house’ methods, to calculate the VWF : RCo/VWF Ag ratios. The within-assay and between-assay repeatability of the automatic ACL-VWF : RCo gave coefficients of variation < 5%, and reproducibility in normal and abnormal laboratory controls gave coefficients of variation of 7.9 and 9.3%, respectively. In VWD patients the results were equivalent to those of the standard Agg-VWF : RCo assay but, because of the good sensitivity, the ACL-VWF : RCo was more accurate in evaluating the VWF : RCo and establishing the VWF : RCo/VWF : Ag ratios in VWD patients with low VWF levels. Moreover, this ACL-VWF : RCo is faster than Agg-VWF : RCo, providing results of calibration curves and of 10 patient samples within 15 min. We can conclude that this automatic ACL-VWF : RCo gives a reliable and useful measure of VWF activity and can be proposed as a screening test for rapid diagnosis of VWD even in patients with low VWF levels in plasma.

Plasma factor VII levels are influenced by a polymorphism in the promoter region of the FVII gene.

Genetic factors play a role in determining the variability of plasma factor VII (FVII) levels in healthy individuals. There is also evidence that high serum lipids are associated with high FVII levels in plasma. In the promoter region of the human FVII a DNA polymorphism has been described, originating from a decanucleotide insert present in the less frequent allele. This biallelic system, reflecting the absence (AA) or presence (Aa) of the decanucleotide, can be detected by a DNA enzyme immunoassay of PCR products. We evaluated the association between the polymorphic alleles and the levels of FVIL:Ag and FVIL:C in 100 healthy individuals and in 19 hypertriglyceridemic individuals. Among healthy individuals, mean FVII:Ag and FVIL:C levels of those with the homozygous genotype (A/A; mean FVIL:Ag 112%, mean FVIL:C 109%) were significantly higher (P < 0.001) than the mean levels of those with the heterozygous genotype (A/a, mean FVIL:Ag 80%, mean FVIL:C 90%; P < 0.001). Similar genotype-associated differences for FVIL:Ag and FVIL:C were found in individuals with triglycerides above 250 mg/dl (P < 0.05). FVIL:C and FVIL:Ag levels were positively related to triglycerides only in individuals without the insert (P < 0.01); there was no significant relationship in those carrying the allele with the insert (A/a; P = 0.43 and 0.08). Our findings of genotype-associated differences in FVII levels and interactions with triglycerides are similar to those obtained with the amino acid dimorphism at position 353 of the factor VII protein.

HELLP syndrome and factor V Leiden

The association of thrombophilia and obstetrical complications is documented and well consistent with the hypothesis of an insufficient placental perfusion due to fibrin deposition as a major underlying pathophysiological mechanism. Factor V Leiden is one of the most frequent thrombophilic mutations. A high prevalence of this mutation has recently been reported in a group of 21 German women with haemolysis, elevated liver enzymes, low platelets (HELLP) syndrome. In this respect, we studied the prevalence of factor V Leiden in 18 women who were consecutively diagnosed at our Department of Obstetrics and Gynaecology as having HELLP syndrome, between 1995 and 1999. Women were tested either at the time of diagnosis or months or years after delivery for coagulation parameters, protein C (PC), protein S (PS), antithrombin III, lupus-like anticoagulant, anticardiolipin antibodies (ACA), activated protein C (APC) resistance and detection of the G1691A mutation (factor V Leiden). In all women, the parameters studied were normal and in none of the investigated cases was the G1691A mutation found. HELLP being a severe form of preeclampsia, we think that the reported association between factor V Leiden and HELLP may reflect the well-known association with preeclampsia.

Carrier detection and feasibility of prenatal diagnosis of hemophilia B by multiplex polymerase chain reaction

SummarySince direct diagnosis based on detection of factor IX mutations by direct sequencing is currently performed in a few laboratories only, restriction fragment length polymorphisms are still useful for carrier detection and prenatal diagnosis of hemophilia B. We analyzed 29 women from 20 families at risk of hemophilia B with three intragenic (MnlI,DdeI,TaqI) and two extragenic (HhaI,BamHI) restriction fragment length polymorphisms, all by the polymerase chain reaction. This analysis confirmed 10 possible carriers and excluded 16 as carriers. The diagnosis was made in 17 of the 20 families (85%). The combination of three polymorphisms indicated thatMnlI/DdeI/HhaI andTaqI/DdeI/HhaI were the most informative (15/20 families, 75% and 14/20 families, 70%). For carrier detection with the most useful polymorphism combination (TaqI/DdeI/HhaI) we devised a multiplex polymerase chain reaction that uses two or three sets of primers, allowing carrier diagnosis in one step with 99% reliability. Our results indicate that the combination of five restriction fragment length polymorphisms makes carrier detection and prenatal diagnosis possible for 85% of families at risk of hemophilia B, with 99% reliability. With multiplex polymerase chain reaction such diagnoses can be obtained faster.

Supportive transfusion therapy in cancer patients with acquired defects of hemostasis

Bleeding occurs in approximately 10% of patients with cancer: supportive transfusion therapy with Platelets Concentrates (PC), Fresh Frozen Plasma (FFP) and plasma-derived or recombinant concentrates is often required for the cessation and prevention of the bleeding episodes. The most frequent causes of bleeding in cancer is thrombocytopenia followed by liver insufficiency with or without vitamin K deficiency, disseminated intravascular coagulation (DIC) and the inappropriate or excessive use of anticoagulants. Other acquired hemostatic defects such as acquired hemophilia (AHA) and acquired von Willebrand syndrome (AVWS) are rare but they can be life-threatening. Thrombocytopenia in cancer patients may be the consequence of marrow invasion, chemotherapy or platelet auto-antibodies; patients with severe hypoproliferative thrombocytopenia, must be treated with PC and carefully followed to assess refractoriness to PC. The management of the other acquired defects of hemostasis usually requires the use of FFP and specific plasma-derived or recombinant concentrates. PC, FFP and plasma-derived concentrates can induce complications and/or adverse events in cancer patients: these include mainly allergic (ALR) or anaphylactic reactions (ANR), Transfusion-Associated Graft-Versus-Host Disease (TA-GVHD), Trasfusion-transmitted bacteriemia (TTB), Transfusion-Related Acute Lung Injury (TRALI), Acute Hemolytic Transfusion Reactions (AHTR), Febrile Non Hemolytic Transfusion Reactions (FNHTR). Therefore, modifications such as leukocyte-reduction and irradiation of the blood components to be transfused in cancer patients are recommended to reduce the risk of these complications.

Interleukin-6 is a determinant of PAI-1 levels in diabetic subjects with the 4G allele at position -675 of the PAI-1 gene

Interleukin-6 is a determinant of PAI-1 levels in diabetic subjects with the 4G allele at position –675 of the PAI-1 gene -

Diagnosis of hemophilia a in a female subject by using restriction fragment length polymorphisms linked to the factor VIII gene

SummaryA 6-year-old girl, daughter of a male patient with moderate hemophilia A (factor VIII 3%), was referred to our Center because she also had very low levels of factor VIII (4%). The proband’s brother has mild hemophilia A (7%); the mother (29%) is a possible carrier, no other case of hemophilia A being reported in her family. Hence, the factor VIII deficiency found in the girl is consistent either with a carrier state with extreme lyonization in favour of the hemophilic gene or with homozygosity for the hemophilia gene. To distinguish these possibilities, we studied the segregation of three restriction fragment length polymorphisms (RFLPs) linked to the factor VIII gene in the 4 members of the family. Employing one intragenic (FVIII-BclI) and two extragenic (St14-TaqI and Dx13-Bg1II) RFLPs, we showed that the proband has inherited from both parents the defective gene, being therefore homozygous for the hemophilia gene.