The activated seven lupus anticoagulant assay detects clinically significant antibodies.

Lupus anticoagulants are a heterogeneous group of autoantibodies detected by their effects on phospholipid-dependent coagulation assays. Persistent lupus anticoagulants are associated with thrombotic disease, but not all are clinically significant. Antibody heterogeneity and reagent and test variability dictate that at least 2 tests, of different types, should be used to screen lupus anticoagulants. The objective of this study was to investigate whether the activated seven lupus anticoagulant assay detects clinically significant antibodies. Eighty-two patients with antiphospholipid syndrome (APS) and 32 with systemic lupus erythematosus + positive for activated seven lupus anticoagulant and who were without thrombosis, who were positive by activated seven lupus anticoagulant assay, were investigated for lupus anticoagulants by dilute Russell's viper venom time, dilute activated partial thromboplastin time, and Taipan snake venom time, and for anticardiolipin antibodies. Fifty-seven of the APS patients were positive for lupus anticoagulants in multiple assays, 25 in activated seven lupus anticoagulant alone. Fourteen of the latter group were previously positive in other antiphospholipid antibodies assays, and 11 had only been positive for lupus anticoagulants by activated seven lupus anticoagulant. Twenty-eight had elevated anticardiolipin antibodies, 6 of whom were from the group that was positive in activated seven lupus anticoagulant only. Eight of the systemic lupus erythematosus + lupus anticoagulants (without thrombosis) patients were positive for lupus anticoagulant by activated seven lupus anticoagulant alone and had only been positive in activated seven lupus anticoagulant previously, and none had elevated anticardiolipin antibodies. The remaining 24 patients were lupus-anticoagulant positive in multiple assays, and 9 had elevated anticardiolipin antibodies. Dilute Russell's viper venom time and Dilute activated partial thromboplastin time are widely used to detect lupus anticoagulants and are considered to detect clinically significant antibodies. Activated seven lupus anticoagulant detected antibodies in APS patients who were positive by these assays and also lupus anticoagulants undetectable by the dilute Russell's viper venom time/dilute activated partial thromboplastin time reagents used, demonstrating its utility as a first-line or second-line assay.

Characterisation of five factor XI mutations.

A large scale factor XI (FXI) mutation screening program identified a number of novel candidate mutations and previously reported mutations and polymorphisms. Five potential missense mutations were selected for further study; these included two novel missense mutations - Met-18Ile (p.Met1Ile) and Met102Thr (p.Met120Thr), two previously reported missense mutations - Tyr133Ser (Tyr151Ser) and Thr575Met (Thr593Met), and one amino acid substitution previously reported as a polymorphism - Arg378Cys (Arg396Cys). The substitutions were recreated by the site-directed mutagenesis of a FXI cDNA and stably expressed in a BHK-570 cell line. Subsequent analysis of both the conditioned media and cell lysates showed that three of the substitutions, Met-18Ile, Met102Thr andTyr133Ser, prevented secretion of the mutated protein from the transfected cell line, resulting in a cross-reactive material negative (CRM-) phenotype. The remaining two mutants, Thr575Met and Arg378Cys, secreted significant levels of FXI into the conditioned media; however, these mutant FXIs were shown to have negligible factor IX activation activity in an APTTbased assay. These results confirmed all five of the missense mutations as being causative of factor XI deficiency, despite one having been previously reported as a polymorphism (Arg378Cys) and one (Tyr133Ser) as a mild mutation - FXI:C 38 U/dl in a homozygous patient.

Spectrum of factor XI (F11) mutations in the UK population--116 index cases and 140 mutations.

Factor XI deficiency is an autosomal bleeding disorder of variable severity. It is particularly common in the Ashkenazi Jewish population, the result of two founder mutations - E117X and F283L. Recent studies have shown the causative mutations of Factor XI deficiency, outside the Ashkenazi Jewish population, to be highly heterogeneous. We have studied 116 index cases, mostly from an ethnically diverse UK population, in order to better understand the spectrum of mutations responsible for factor XI deficiency. A total of 140 causative mutations of the F11 gene were identified in 109 patients. Fifty-five (39.3%) of the mutations were one of three common mutations--E117X (Type II), F283L (Type III), or C128X. The remaining 85 (60.7%) mutations comprised at least 57 variants including 31 novel mutations and whole gene deletions. This large study reconfirms that, despite the presence of founder mutations in discrete populations, factor XI deficiency remains a highly heterogeneous disease at the molecular level. .

The dilution effect of equal volume mixing studies compromises confirmation of inhibition by lupus anticoagulants even when mixture specific reference ranges are applied.

INTRODUCTION: One of the recommended criteria for the laboratory diagnosis of lupus anticoagulants (LA) is demonstration of inhibitory activity. This is confirmed by performing mixing tests with normal plasma, usually in a 1:1 ratio, and demonstrating persistence of an abnormal clotting time in the screening test with significant confirmatory test reduction. However, the mixing with normal plasma can dilute the antibodies to undetectable levels and generate apparent negative results. No guidelines or consensus exist in how to interpret mixing study results. PATIENTS AND METHODS: The present study assessed the 1:1 mixing study results from 600 patients with a thrombotic history positive for LA demonstrated in neat plasma by individual assays, or combinations, of dilute Russell's viper venom time, dilute activated partial thromboplastin time, activated seven lupus anticoagulant assay and Taipan snake venom time, plus confirmatory tests. Mixing tests were assessed initially using locally derived neat plasma reference ranges and subsequently with mixture specific ranges. RESULTS: The mixture specific ranges had lower upper limits. Of the total LA positive results, 32.5% were positive in the mixing studies when neat plasma reference ranges were applied, and a further 11.2% demonstrated LA activity when using the mixture specific ranges. The remaining 56.3% had been diluted such that they did not elevate the screening test above the upper limit of normal or generated minimal prolongation with an insignificant difference between the screen and confirmatory test result sufficient to confirm LA activity. CONCLUSIONS: The significant impact of the dilution effect in 1:1 mixing studies emphasises the limitations of mixing studies as a vehicle for confirmation of inhibition by LA antibodies.

The significance of published polymorphisms in 14 cases of mild factor VII deficiency.

Factor VII (FVII) plays a critical role in the initiation of blood coagulation, and patients with dysfunctional or reduced levels of this protein are susceptible to mucosal bleeding. There is poor correlation between the clinical presentation and the phenotypic data; and in cases of a mild bleeding tendency, mild to moderate reductions in both FVII antigen and activity may be overlooked. The prevalence of FVII deficiency may therefore be underestimated. Polymorphic differences throughout the FVII gene are associated with variations in plasma FVII antigen and activity levels. This study highlights the significance of mild FVII deficiency, and examines the importance of seven previously published polymorphisms in such patients.

Determination of the urinary aglycone metabolites of vitamin K by HPLC with redox-mode electrochemical detection.

We describe a method for the determination of the two major urinary metabolites of vitamin K as the methyl esters of their aglycone structures, 2-methyl-3-(3'-3'-carboxymethylpropyl)-1,4-naphthoquinone (5C-aglycone) and 2-methyl-3-(5'-carboxy-3'-methyl-2'-pentenyl)-1,4-naphthoquinone (7C-aglycone), by HPLC with electrochemical detection (ECD) in the redox mode. Urinary salts were removed by reversed-phase (C18) solid-phase extraction (SPE), and the predominantly conjugated vitamin K metabolites were hydrolyzed with methanolic HCl. The resulting carboxylic acid aglycones were quantitatively methylated with diazomethane and fractionated by normal-phase (silica) SPE. Final analysis was by reversed-phase (C18) HPLC with a methanol-aqueous mobile phase. Metabolites were detected by amperometric, oxidative ECD of their quinol forms, which were generated by postcolumn coulometric reduction at an upstream electrode. The assay gave excellent linearity (typically, r2 > or = 0.999) and high sensitivity with an on-column detection limit of < 3.5 fmol (< 1 pg). The interassay precision was typically 10%. Metabolite recovery was compared with that of an internal standard [2-methyl-3-(7'-carboxy-heptyl)-1,4-naphthoquinone] added to urine samples just before analysis. Using this methodology, we confirmed that the 5C- and 7C-aglycones were major catabolites of both phylloquinone (vitamin K1) and menaquinones (vitamin K2) in humans. We propose that the measurement of urinary vitamin K metabolite excretion is a candidate noninvasive marker of total vitamin K status.

Germline mosaicism resulting in the transmission of severe hemophilia B from a grandfather with a mild deficiency.

We report a family in which the normal pattern of X-linked inheritance of hemophilia B (Factor IX deficiency) is complicated by mosaicism in the proband's maternal grandfather. The proband, an infant with severe Factor IX deficiency, was initially thought to be a sporadic case. Testing of other family members identified his mother as a carrier of the disorder, and his asymptomatic maternal grandfather as having very mild FIX deficiency. The causative familial mutation was identified as a two base pair deletion (AG within codons 134-135) in the Factor IX gene. The grandfather was shown to be "heterozygous" for the deletion. Karyotype analysis confirmed him to be 46XY thereby ruling out Klinefelter syndrome. The proband's aunt, who as the daughter of a man with hemophilia is theoretically an obligate carrier, was found not to carry this familial mutation, and thus not to be a carrier of hemophilia B. The grandfather must therefore be an X chromosome somatic and germline mosaic, with consequent segregation of the affected and non-affected Factor IX genes. This observation underlines the importance of confirming carrier status even in those individuals assumed to be obligate carriers, and has implications for genetic counseling.

An Alu-mediated 31.5-kb deletion as the cause of factor XI deficiency in 2 unrelated patients.

Factor XI deficiency (MIM 264900) is an autosomal bleeding disorder of variable severity. Inheritance is not completely recessive as heterozygotes may display a distinct, if mild, bleeding tendency. Recent studies have shown the causative mutations of factor XI deficiency, outside the Ashkenazi Jewish population, to be highly heterogeneous. We studied 39 consecutively referred patients with factor XI deficiency to identify the molecular defect. Conventional mutation screening failed to identify a causative mutation in 4 of the 39 patients. Epstein-Barr virus (EBV)-transformed cells from these 4 patients were converted from a diploid to haploid chromosome complement. Subsequent analysis showed that 2 of the patients had a large deletion, which was masked in the heterozygous state by the presence of a normal allele. We report here the first confirmed whole gene deletion as the causative mutation of factor XI deficiency, the result of unequal homologous recombination between flanking Alu repeat sequences.

Heterogeneity of Russell's viper venom affects the sensitivity of the dilute Russell's viper venom time to lupus anticoagulants.

A number of studies have shown that commercial dilute Russell's viper venom time (DRVVT) reagents vary in their sensitivity for lupus anticoagulant (LA) detection. The differences in performance are considered to be predominantly due to antibody heterogeneity and a wide variation in phospholipid content, and also the techniques and clot detection methods employed. The present study compared the LA detection rates of five different Russell's viper venom (RVV) preparations using identical phospholipid reagents to assess the contribution of venom heterogeneity to LA detection by DRVVT. Initial analysis of 300 samples sent for thrombophilia screening identified 48 (16.0%) LAs by DRVVT and confirmatory tests with a single RVV reagent. Subsequent DRVVT analysis of all 300 samples using the other four venom reagents, with confirmatory tests on samples with elevated screen ratios, revealed a further 38 LAs in a variety of combinations of reagents. Only 15 of 86 (17.4%) of LAs were detected with all five RVV reagents. Significant biological variation of RVV exists due to differences between Russell's viper subspecies and a variety of environmental and physiological factors. The clear variations in diagnostic performance between alternative RVV preparations are most probably due to biological venom heterogeneity, and also differences in the manufacturing processes.

Tinzaparin sodium for thrombosis treatment and prevention during pregnancy.

OBJECTIVE: This study was undertaken to assess the pharmacodynamic profile, safety, and efficacy of tinzaparin during pregnancy. STUDY DESIGN: Fifty-four pregnant women, 12 for treatment of thrombosis and 42 for thromboprophylaxis, received tinzaparin by once daily injection. Four-hour postdose anti-Xa results were analyzed by use of repeated measures statistical methods. RESULTS: One woman (3.4%) on the 175 anti-Xa U/kg dose and three women (20%) on the 50 anti-Xa U/kg dose required a dose increase during the initial dose titration phase to achieve target anti-Xa activity. No thrombotic events occurred. CONCLUSION: The 175 anti-Xa U/kg dose is appropriate for treatment and for high-risk thromboprophylaxis throughout pregnancy. In pregnant women at moderate risk of thrombosis, a higher tinzaparin dose is required than in the nonpregnant state and 75 anti-Xa U/kg appears to be appropriate. The majority of women do not need a dose increase with advancing gestation.

Fibronectin promotes VEGF-induced CD34 cell differentiation into endothelial cells.

BACKGROUND: Adult endothelial progenitor cells (EPC) may be a useful source for engineering the endothelialization of vascular grafts. However, the optimal factors that promote differentiation of EPCs into endothelium remain to be elucidated. The goal of this current report was to determine which extracellular matrix (ECM) protein might modulate or enhance the effects of EPCs on differentiation into mature endothelium. METHODS: Human EPCs (CD34(+) cells) were cultured in ECM-coated six-well plates in MCDB-131 medium containing vascular endothelial growth factor (VEGF), insulin-like growth factor-1, and basic fibroblast growth factor. After 21 days, differentiated endothelial colonies were confirmed by immunofluorescence for von Willebrand factor (vWF) and vascular-endothelial (VE)-cadherin and mRNA expression of the endothelial markers Flk-1, vWF, and VE-cadherin. Cell migration toward the VEGF-matrix protein combinations was also measured. RESULTS: As judged by positive staining for endothelial markers vWF and VE-cadherin, the combination of VEGF with fibronectin (FN) produced significantly more endothelial colonies (P <.05) than did collagens I or IV or vitronectin. Defined fragments of FN did not enhance VEGF-mediated effects. Fibrinogen produced intermediate stimulation of differentiation. FN also enhanced VEGF-mediated CD34(+) cell migration. Blockade of alpha5beta1, but not alphavbeta3 or alphavbeta5, inhibited both VEGF-mediated CD34(+) cell differentiation and migration. CONCLUSIONS: VEGF and FN together significantly promote the migration and differentiation of CD34(+) cells. This synergism is specific to FN and the alpha5beta1 integrin. Combinations of VEGF and FN may be useful in promoting differentiation of circulating endothelial progenitors into endothelial cells for tissue engineering. Clinical relevance Treatment of injured or diseased tissues with adult stem cells is a promising approach. In particular, bone marrow derived circulating endothelial progenitors (CEP's) have been shown to differentiate into endothelial cells in vitro and promote tissue revascularization of ischemic limbs and myocardium in vivo. Because of the relative ease of obtaining CEP's and as well as its high proliferative rate, CEP's may have clinical potential for endothelialization of prosthetic vascular grafts and revascularization of injured myocardium. However, there is a need to better understand the molecular pathways involved in the proliferation and differentiation of CEP's to take full advantage of its clinical potential.

Prevalence of hyperhomocysteinemia and the MTHFR C677T polymorphism in patients with arterial and venous thrombosis from North Western Russia.

Conflicting data from Western European and USA population studies led us to investigate hyperhomocysteinemia (HHcy), the methylenetetrahydrofolate reductase (MTHFR) C677T polymorphisms and thrombotic disease in North Western Russia. Plasma total homocysteine (tHcy) levels, MTHFR C677T genotype, selected life style determinants and haemostatic factor activity were determined in patients with arterial (n = 33), venous (n = 40), arterial + venous (n = 11) thrombosis and healthy controls (n = 30). We found raised median tHcy levels in all patient groups vs. controls (p < 0.05), with odds ratios (95% CI) for vascular disease among patients with HHcy (defined as > 15 micromol/l) of 3.9 (0.6 - 14.3), 4.8 (1.2 - 18.8) and 15.8 (2.8 - 87.3) respectively. tHcy levels were a function of MTHFR C677T genotype, and all patients with tHcy levels > 30 micromol/l had the MTHFR C677T homozygous substitution. Elevated tHcy levels (p < 0.05) were identified in smokers and coffee drinkers, with the degree of elevation dependent on MTHFR C677T genotype. Of the studied haemostatic parameters increased factor VIII activity and vWF antigen and activity was observed in HHcy subjects. We conclude that HHcy and MTHFR C677T genotype are positively associated with arterial and venous thrombotic disease in the population of North Western Russia.

Hyperhomocysteinemia and B-vitamin status after discontinuation of oral anticoagulation therapy in patients with a history of venous thromboembolism.

Although hyperhomocysteinemia is an established risk factor for venous thromboembolism there is no consensus for routine determination of circulating homocysteine in the UK, either at the beginning or end of oral anticoagulation therapy. The purpose of this study was to evaluate the prevalence of hyperhomocysteinemia and its relationship to folate and vitamin B12 status in subjects with venous thromboembolism 4 weeks after discontinuation of warfarin therapy. In 78 consecutively recruited patients, plasma homocysteine was significantly higher (p < 0.001) and red cell folate significantly lower (p = 0.03) than in controls. Plasma vitamin B12 was similar in both groups. Strikingly, 38.5% of patients had hyperhomocysteinemia (> 15 micromol/l). Retrospective analysis revealed a significant positive association between plasma total homocysteine and duration of warfarin therapy (p < 0.001) but a negative, though non-significant (p = 0.06), trend with warfarin dose. The results do not suggest any direct interaction between warfarin and plasma homocysteine but raise the possibility of reduced intake of a common food source of folate and vitamin K. One possibility is the shortage of green-leafy vegetables since patients are often advised to limit their intake of this major source of vitamin K. On the basis of this study we suggest that homocysteine screening should be carried out at the time that patients begin warfarin therapy.

Glanzmann's thrombasthenia proposed optimal management during surgery and delivery.

Glanzmann's thrombasthenia (GT) is an autosomal recessive disorder of platelet function. Conventional management is by platelet transfusion, given before invasive interventions. Alloimmunization resulting in platelet refractoriness and an unpredictable response to platelet infusion have provided particular management difficulties in the past. More recently recombinant (r)VIIa (Novoseven) has a valuable role in the treatment of platelet function disorders. Treatment of a patient with GT during two pregnancies and spinal surgery is reported. An algorithm is presented to provide a structured and consistent approach to treatment.

Genetic variations observed in arterial and venous thromboembolism--relevance for therapy, risk prevention and prognosis.

We undertook genetic and biochemical assays in patients with arterial (n = 146) and venous (n = 199) thromboembolism and survivors of pulmonary embolism (n = 58) to study causation and gene-life style interactions. In the clinical material from North Western Russia, factor V Leiden was found to be a risk factor in venous thrombosis (OR = 3.6), while the methylenetetrahydrofolate reductase (MTHFR) C677T mutation was a significant variable in both venous (p = 0.03) and arterial thrombosis (p = 0.004). Homocysteine levels were determined (n = 84) and hyperhomocysteinemia correlated with the T allele of the MTHFR gene, and with smoking and coffee consumption. Vitamin supplementation reduced homocysteine levels dependent on MTHFR genotype (36% TT, 25% CT, 22% CC). In pulmonary embolism patients, frequency of the -455G/A beta-fibrinogen dimorphism was studied. Carriers of this allele were significantly underrepresented (p < 0.02) among pulmonary embolism survivors (34.5%) compared to controls (56.7%). Additionally, -455AA homozygotes were found in 11.7% controls but only 1.7% of pulmonary embolism patients (p = 0.006). In venous and arterial thrombosis cases, MTHFR and homocysteine data led to effective dietary supplementation with a reduced risk of disease progression. Results from the pulmonary embolism study may indicate that screening tests for the -455G/A beta-fibrinogen genetic variation could be of prognostic value, and may point the way for novel anticoagulation strategies.

Eighteen unrelated patients with factor XI deficiency, four novel mutations and a 100% detection rate by denaturing high-performance liquid chromatography.

Factor XI (FXI) deficiency is an autosomal bleeding disorder of variable severity. Inheritance is not completely recessive as heterozygotes may display a distinct, if mild, bleeding tendency. Eighteen unrelated FXI-deficient patients were screened blind by fluorescent single-stranded conformation polymorphism (F-SSCP) analysis and denaturing high-performance liquid chromatography (dHPLC). Mutations were detected in 14 of the 18 patients ( approximately 78%) by F-SSCP and in all 18 patients by dHPLC. Dideoxy sequencing confirmed the mutations in all 18 patients: eight of the mutations being novel (four of which were in previously reported patients). This showed dHPLC to be a highly sensitive, reliable technique for mutation screening in heterogeneous disorders.

The Ecarin time is an improved confirmatory test for the Taipan snake venom time in warfarinized patients with lupus anticoagulants.

The Taipan snake venom time using dilute phospholipid as a screening test with a platelet neutralization procedure as a confirmatory test has been shown to be a sensitive and specific approach to detection of lupus anticoagulants. Taipan venom is largely insensitive to the effects of ongoing warfarin anticoagulation and this can be useful in detection of lupus anticoagulants in patients receiving this treatment. This study compared the use of the platelet neutralization procedure with the Ecarin time as confirmatory tests for the Taipan snake venom time, the Ecarin venom fraction being insensitive to both lupus anticoagulants and the effects of oral anticoagulants. Screening and confirmatory test data were assessed for phospholipid dependence by three different mathematical methods and there was no advantage in using the Ecarin time in detection of 'uncomplicated' lupus anticoagulants. In lupus anticoagulant-positive warfarinized patients, the Ecarin time achieved higher detection rates than the platelet neutralization procedure irrespective of the method used to assess correction. The Ecarin time confirmed lupus anticoagulants in all of those samples that generated elevated Taipan snake venom time ratios whereas the platelet neutralization procedure identified only 33%. Taipan snake venom time plus Ecarin time offers good diagnostic precision for lupus anticoagulant detection in a group of patients where lupus anticoagulant identification is difficult due to ongoing anticoagulation.

A prospective study of recombinant activated factor VII administered by continuous infusion to inhibitor patients undergoing elective major orthopaedic surgery: a pharmacokinetic and efficacy evaluation.

After surgery in haemophilia, haemostasis is difficult to maintain in the presence of an antifactor VIII antibody. This study assessed the pharmacokinetics of recombinant activated factor VII (rFVIIa) and its efficacy in securing post-operative haemostasis in haemophiliacs with inhibitors. Continuous infusion of rFVIIa was evaluated for elective major orthopaedic surgery in nine patients with neutralizing antibodies to FVIII and at high risk of bleeding. After an initial preoperative bolus of 90 micro g/kg, rFVIIa was infused at a fixed rate of 50 micro g/kg/h for a median of 20 d (range 7-20 d). The median plasma FVII coagulant activity (FVII:C) at 24 h, 72 h and 20 d after surgery was 38 IU/ml (range 22-169 IU/ml), 45 IU/ml (range 17-88 IU/ml) and 31 IU/ml (range 27-46 IU/ml) respectively. The median plasma FVIIa:C at the same time points was 51 (range 24-211), 63 (range 22-99) and 44 (range 28-76) IU/ml respectively. Median total rFVIIa clearance remained stable during the rFVIIa continuous infusion period and was 40 (range 9-70), 34 (range 17-86) and 48 (range 32-55)ml/kg/h at the end of 24 h, 72 h and 20 d infusion respectively. Post-operatively, there were bleeds in six patients, which settled readily after a single bolus of rFVIIa (60 micro g/kg). There was a good clinical outcome for all patients. These data indicate that rFVIIa infusion at 50 micro g/kg/h achieves continuous plasma FVII procoagulant activity in excess of 30 IU/ml (12-15 nmol/l) and provides adequate haemostatic control for inhibitor patients during major orthopaedic surgery.