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Objectives To assess the possible association between inherited thrombophilic alterations and the severity of peripheral arterial disease (PAD). Design A case-control study. Methods We evaluated the presence of G20210A prothrombin (FII) and R506Q FV Leiden mutations, antithrombin, protein C and S deficiencies in 176 patients with PAD at Fontaine's stage II and in 106 patients with critical limb ischaemia (Fontaine's stage III/IV) consecutively referred to our unit. As control group, we studied 209 apparently healthy subjects. Results The prevalence of G20210A prothrombin mutation was similar in PAD patients and controls (odds ratio (OR): 1.361; 95% confidence interval (CI): 0.552–3.355; p = 0.503 after adjustment for age, sex, smoking and presence of diabetes), but was significantly higher in patients with Fontaine's stage III/IV vs. those with stage II and controls (10.4% vs. 3.4% vs. 4.3%; p = 0.02, respectively). According to a logistic multivariate model that included all patients with PAD, the presence of the FII G20210A mutation (OR: 4.621; 95% CI: 1.548–13.789; p = 0.006) was associated with critical limb ischaemia after adjustment for age, sex, smoking, presence of diabetes and the use of platelet aggregation inhibitors. The prevalence of the other thrombophilic alterations was not different in patients with Fontaine's stage III/IV, in patients with stage II and in controls. Conclusion These hypothesis-generating data suggest that the FII 20210A allele may be considered as a genetic marker predisposing critical ischaemia in patients with PAD, justifying larger longitudinal studies.

Introduction Buflomedil hydrochloride is a vasoactive agent widely used, since 1976, for the treatment of peripheral circulatory insufficiency, with good results in cases of chronic occlusive peripheral arterial diseases (Desandre & Coudrec 1980, Galley & De Boysson 1981, Cloarec 1976, Balas & Pagratis 1981, Trubestein & Duong 1981) and of microcirculatory disorders (Fagrell & Hermansson 1980, Courlier, Bergeron & Fouque 1981). Its mechanism of action has not yet been completely elucidated; recently, however, Dormandy and Ernst (1981) have reported that individual venous infusion with Buflomedil caused a marked improvement in red blood cell deform ability, studied with a filtration method, in ten patients with lower limb ischaemia. This suggests that Buflomedil also has an haemorheologic effect. It is a known fact that patients with diabetes are often affected by vascular diseases, both macroand microcirculatory, and it is also known that one of the pathogenetic processes of vascular complications in diabetes consists in complex alterations of the blood flow properties (McMillan 1975, Calwell et al 1979), comprising mainly an increase in blood and plasma viscosity and alterations in the deformability and aggregability of the red cells (McMillan 1976, McMillan, Utterback & La Puma 1978, Juhan et al 1978, SchmidSchonbein & Volger 1976). The purpose of this study was to investigate the effect of the administration of Buflomedil on the rheologic properties of blood in a group of diabetic patients.

SummaryThe aims of the present study were: l) interlaboratory normalization of prothrombin time (PT) testing for anticoagulant therapy control through calibration of customary thromboplastins against international reference materials, and 2) “on field” validation of the advantages offered by expression of results as International Normalized Ratio (INR) as opposedto percentage activity. PT tests were carried out over 8 days on the same normal subjects (16) and patients on oral an ticoagulants (48) in the 9 laboratories of the Bologna area. The use of customary thromboplastins and coagulometers was maintained in all labs throughout the study. The main results were: 1) the interlaboratory CV of the prothrombin ratios obtained for each sample with all customary thromboplastins (5 different brands) was 15%, but was reduced to levels of 5.8 to 8.9 when using constant thromboplastin brands and batches; 2) the International Sensitivity Index (ISI) values obtained in the different labs were only slightly influenced by the use of different coagulometers; 3) comparable ISI values were obtained through direct calibration with the international reference material and through intermediate calibration with a locally selected standard; 4) use of INR values instead of percentage activity greatly reduced interlaboratory variability and significantly improved uniformity of anticoagulation level measurements. thus reducing the possibility of erroneous prescriptions. The Bologna exercise is therefore of educational value for laboratory and community doctors of the area in understanding and accepting the INR system.

A family with a new congenital dysprothrombinemia is described. The propositus was a 36-yr-old female from the Molise region of Italy who presented with epistaxis, easy bruising, and menometrorrhagia. The main laboratory features of the defect included slight prolongation of prothrombin time, Thrombotest, Normotest, PP test, and partial thromboplastin time. Prothrombin activity was approximately 10% in several one- and two-stage systems employing tissue thromboplastins as activating agents. Using several viper venoms as activating agents low prothrombin levels were also obtained. However, the staphylocoagulase-complexed prothrombin level and immunologic methods yielded levels of about 50%. A line of identity between normal and abnormal prothrombin was seen on immunodiffusion. The migration of the abnormal prothrombin was normal in single and bidimensional immunoelectrophoresis systems. A single abnormal serum fragment was seen on bidimensional immunoelectrophoresis. The brother of the propositus was also affected and presented a similar pattern. Family studies revealed that the mother had approximately 50% prothrombin activity and antigen, whereas the father had 65% prothrombin activity and 100% prothrombin antigen. The propositus's first son showed approximately 50% prothrombin activity and antigen. A second child showed approximately 50% prothrombin activity but normal antigen. We suggest that the propositi are heterozygous for an abnormal prothrombin and heterozygous for “true” prothrombin deficiency. The father and the second child are heterozygous for abnormal prothrombin. The mother and the first child are heterozygous for “true” prothrombin deficiency. The term prothrombin Molise is proposed for this peculiar prothrombin abnormality.

The effects of ticlopidine treatment (250 mg b.i.d. for 21 months) on fibrinogen and other rheological variables, as compared to placebo, were studied in 44 patients with intermittent claudication due to peripheral arterial occlusive disease. Blood samples were collected every 3 months during this double-blind, randomised placebo-controlled trial which lasted 21 months. Consistently lower values of fibrinogen, haematocrit and whole blood viscosity at high and low shear rate levels were found in the ticlopidine group; the intergroup differences were statistically significant at most but not all follow-up examinations. A significant time-related variance was observed in the ticlopidine group for the measured variables, also after correction for the variability found in the placebo group. Thus, the observed changes in the ticlopidine group are mainly treatment related. These effects on fibrinogen and haemorheology may contribute, besides the known antiplatelet activity of the drug, to the clinical improvement reported in a larger group of claudicants to which the present subset of patients belong.

A congenital abnormal fibrinogen was detected in a 30 year old woman presenting several episodes of peripheral arterial thrombosis.The abnormality in fibrin formation is located in the fibrin monomers aggregation, since the fibrinopeptides release and the fibrin stabilization were normal.The SDS polyacrylamide gel electrophoresis of plasmic degradation products, obtained either in the presence or in the absence of calcium, revealed an absence of protective effect of calcium for plasmin degradation of Fibrinogen Haifa. Since, Ca++ protects against further plasmin degradation of the gamma chain, we suggest that the anomaly is located in this part of the fibrinogen molecule.Because the anomaly was discovered at Haifa, Israel, we suggest to call this abnormal fibrinogen : “Fibrinogen Haifa”.

A series of 40 patients with TIA (25 males and 15 females) was thoroughly investigated by means of angiography and computerized tomography, and divided into a group (A) of 15 “sine materia”, and a group (B) of 25 with direct or indirect evidence of vascular occlusive or stenotic changes. Blood viscosity at 230 sec-1 37° was cp 4.2 ± 0.3 in the controls, cp 4.7 ± 0.7 in all patients (p < 0.05) cp 4.98 ± 0.7 in all male patients (p < 0.01 versus male controls), and cp 4.75 ± 0.8 in group B (p < 0.02). Haematocrit and Fibrinogen were also significantly increased in all male patients and in group B. Circulating platelet aggregates (CPA) were increased in 40% of the patients. Almost all patients with elevated CPA were males, with a slight prevalence in group B. Changes in blood viscosity parameters and in platelet aggregation in TIA patients were therefore related both to evidence of vascular lesions, and to sex, since they were found to prevail in male patients of both groups.

Twenty-one thyroprival patients, previously submitted to total thyroidectomy for tumours, were investigated during stabilized L-thyroxine supplementation and at the end of a 20-25 day "no-therapy" period, necessary for a 131I total body scintigraphy. During supplementation therapy a lower than normal mean beta-thromboglobulin (beta-TG) release level was found, the other blood clotting and platelet function tests being normal. After substitution therapy withdrawal, platelet function tests showed reduced adrenalin aggregation, increased collagen threshold aggregating concentrations, decreased beta-TG release values and reduced aggregation to ristocetin, whereas blood clotting tests showed prolonged aPTT values and reduced levels of factor VIII:C and vWf:Ag. We conclude that in acquired hypothyroidism the lowering of factor VIII:C and vWf:Ag (acquired von Willebrand disease) is associated with impaired platelet reactivity not only to ristocetin but also to collagen and especially adrenalin. In the patients investigated these changes were almost completely corrected by substitutive therapy with L-thyroxine at clinically effective doses.

In order to evaluate slimming effects on certain metabolic (cholesterol, triglycerides, basal insulinaemia) and haemorheologic (haematocrit, fibrinogen, whole blood viscosity and plasma viscosity) rates, we studied 24 obese subjects (15 female and nine male) aged 25-58, with BMI ranging from 35.5 to 67, before and after a hypocaloric diet period involving a 20-kg weight loss. All subjects underwent blood sampling to assess: OGTT, plasma proteins, serum total cholesterol, HDL-cholesterol, serum triglycerides, haematocrit, whole blood viscosity at high and low shear rate with and without correction of 45 per cent haematocrit, plasmatic capillary viscosity and fibrinogen level. Our study showed no significant changes in plasma proteins, serum HDL-cholesterol, haematocrit, fibrinogen and whole blood viscosity at high and low shear rate, while basal insulinaemia, total cholesterol, triglycerides, low shear rate corrected at 45 per cent haematocrit blood viscosity and capillary plasmatic viscosity are significantly reduced. However the analysis of possible relation between the differences of assessed rates before and after slimming has shown no significant correlations. In conclusion, we can say that the slimming of very obese subjects improves blood and plasma viscosity, but the mechanism by which this improvement occurs is not the one which usually affects the determination of these rates.

A pilot open controlled study of FOY was performed in 20 intensive care patients (pts, age 18-63) with DIC diagnosed with standard laboratory criteria (at least 3 of the following: Normotest 70%, fibrinogen 150 mg%, AT III 80%, FDP 20 ug/ml, platelets 150000). Besides the usual treatments, FOY was given to 10 pts (FOY G.) by continuous i.v. infusion (1mg/kg/h) for up to 7 days, while in 10 control pts (Hep.G.) the treatment included low dose s.c. heparin. Blood clotting tests were performed at admission to the study and daily for 7 days; we consider here results obtained at baseline and at the 4th (7 survivors in FOY G. and 10 in Hep.G.) and the 7th day (6 surv. in FOY G. and 9 in Hep. G.). Statistical evaluation was made by means of the twotailed Wilcoxon test for non parametric paired data. In the FOY G. depressed baseline AT III and plasminogen (Plgn) activities (61.8+/-5.3% and 57+/-5.5% respectively) significantly increased at 4th day (92+/-11.2% and 83+/-3.1% p

In order to establish whether injection sclerotherapy of oesophageal varices could bring about a worsening of the coagulation abnormalities of patients with cirrhosis, the platelet count and the coagulation profile were monitored prior to, then 30 min and 18 h after, the injection in 8 patients undergoing 18 sclerotherapy sessions. Under basal conditions the platelet count, prothrombin activity, normotest and antithrombin III were all reduced; fibrinogen was in the low, and partial thromboplastin time in the high, normal range. A significant shortening of PTT and a further reduction in the platelet count, in the normotest and in fibrinogen occurred after 30 min. On one occasion laboratory evidence of disseminated intravascular coagulation was observed. After 18 h most parameters approached basal values, but the normotest remained persistently reduced. Even though a transient activation of the coagulation process, with consumption of platelets and the liver-dependent clotting factors took place after sclerotherapy in most cases, leading in one to self-limiting disseminated intravascular coagulation, haemorrhagic complications did not occur in our patients. These results suggest that injection sclerotherapy did not lead to clinically significant deterioration of coagulation even in patients with abnormal coagulative function. The observed changes appeared to be self-limiting and confined to the laboratory level in all cases.

The rationale of the hemorheologic approach to the pathophysiological knowledge and to the treatment of obstructive arterial diseases of the limbs is discussed in this paper. Indeed, the hemodynamic concepts are the natural frame in which the hemorheologic phenomena have to be considered. Proceeding from this background, it seems evident that the viscosity of blood and its major determinants have a limited role in the regulation of blood flow and pressure gradient through a stenotic artery. On the contrary, viscosity factors are of outstanding significance in the regulation of blood flow in the microcirculation, especially in presence of a reduced perfusion pressure and of exhausted arteriolar vasodilation capacity. Consequently, therapeutic measures capable of modifying one or more viscosity determinants may improve blood flow in the microcirculation of territories related to a stenotic artery. Baseline alterations of viscosity or viscosity determinants may be found in patients with obstructive arterial disease, being classified as primary, associated or secondary to arterial obstruction and ischemic damage. However, the presence of such baseline changes is not relevant to the indication of hemorheologic treatments.

The pharmacodynamic properties of a new LMWH (alfa-LMWH) were investigated in 8 healthy volunteers after single subcutaneous administrations of 7,500, 15,000 and 30,000 anti-XaU doses at weekly intervals. Anti-Xa and anti-IIa heparin activities were monitored together with aPTT, thrombin time, bleeding time and euglobulin lysis time. No relevant changes in bleeding time or major side-effects were ever recorded. A group of 26 patients submitted to gynaecological surgery were then investigated to determine the dosage schedule for prophylaxis of post-operative deep vein thrombosis. Two subgroups received daily subcutaneous doses of 7,500 and 15,000 anti-XaU alfa-LMWH respectively, beginning 2 h before surgery; the third subgroup received 5,000 IU calcium heparin three times daily over the seven postoperative days. The following tests were peri-operatively monitored: anti-Xa heparin activity, aPTT, PT, fibrinogen, Antithrombin III. No differences in intra-operative bleeding or side-effects were recorded. On the basis of the levels of anti-Xa heparin activity and the negligible effects on aPTT, the dose of 7500 anti-XaU was selected at single daily administration for thromboprophylaxis in gynecological surgery.

Fibrinogen (Fgn) and haemorheologic parameters were serially measured in 44 pts (38 males, age 59+/−7) with claudicatio intermittens due to peripheral arterial obliterative disease (PAOD) treated for 24 months with Ticlopidine (T group, 250 mg b.i.d. p.os), or placebo (P group), in a double blind randomized controlled study, part of a larger clinical trial. Fgn (immunodiffusion), haematocrit (Ht, micromethod), whole blood viscosity (BV, Contraves LS 30, at 37 C) at high (94.5) and low (0.2 sec-1) shear rates (s.r.) and plasma viscosity (PV, Contraves LS 30) were measured three-monthly. The data were evaluated by means of ANOVA and Student's t test. The values in groups T and P did not differ at baseline except for greater BV at high s.r. in group P (p< 0.05). During the observation period circannual variations appeared especially in group T, where summer-time values for most parameters (Fgn, Ht, PV, low s.r. BV) were lower (significance from p

A series of 102 cerebrovascular patients (CVP) investigated with aortic arch angiography, carotidography and CAT scan, were classified clinically in STROKE (55) and TIA (47) and pathologically as having (IAL, 51) or having no (NIAL, 51) identifiable arterial lesions. Coagulation and hemorheologic tests were performed at least 3 months after STROKE or 1 month after a TIA episode. All CVP as compared to controls (84) had significantly higher fibrinogen (Fg), F VIII AHF and RAg, blood (BV) and plasma (PV) viscosity, and poorer erythrocyte deformability (ED, as filtered erythrocyte volume, FEV), but no differences in euglobulin lysis time (ELT) even after venostasis (ELTV) and in circulating platelet aggregates (CPA, as 1/PAR). IAL vs NIAL CVP had higher Fg (mg% 285±74 vs 241±64; p

The aims of the present study were: 1) interlaboratory normalization of prothrombin time (PT) testing for anticoagulant therapy control through calibration of customary thromboplastins against international reference materials, and 2) "on field" validation of the advantages offered by expression of results as International Normalized Ratio (INR) as opposed to percentage activity. PT tests were carried out over 8 days on the same normal subjects (16) and patients on oral anticoagulants (48) in the 9 laboratories of the Bologna area. The use of customary thromboplastins and coagulometers was maintained in all labs throughout the study. The main results were: 1) the interlaboratory CV of the prothrombin ratios obtained for each sample with all customary thromboplastins (5 different brands) was 15%, but was reduced to levels of 5.8 to 8.9 when using constant thromboplastin brands and batches; 2) the International Sensitivity Index (ISI) values obtained in the different labs were only slightly influenced by the use of different coagulometers; 3) comparable ISI values were obtained through direct calibration with the international reference material and through intermediate calibration with a locally selected standard; 4) use of INR values instead of percentage activity greatly reduced interlaboratory variability and significantly improved uniformity of anticoagulation level measurements, thus reducing the possibility of erroneous prescriptions. The Bologna exercise is therefore of educational value for laboratory and community doctors of the area in understanding and accepting the INR system.

In 20 patients with liver cirrhosis and prolongation of thrombin (Th) and/or thrombin coagulase (ThC) times, fibrin polymerisation was followed at 350 nm after adjustment of fibrinogen concentration. The optical-density values at 10’ (OD 10) were grossly abnormal both with Th and with ThC, but no correlation was found between OD 10 and Th or ThC times. Polymerisation was re-tested after fibrinogen purification with 6 alanine and glycine. ThC time was generally shortened (p < 0.005) although in no case restored to normal. The OD 10 values improved (p < 0.001), but became normal in only a half of the cases, while remaining totally abnormal in 2 cases: PAA gel electrophoresis showed an abnormal band of very low m.w. fibrinogen in 2 out of 10 cases investigated. These results suggest that defective fibrin polymerisation in liver cirrhosis cannot be attributed only to acquired dysfibrinogenaemia. Other features like partially degraded fibrinogen and, especially, still unknown antipolymerizing factor(s)in plasma contribute to the disorder of iibrin formation.

In this collaborative study we have investigated the coexistence of two different markers of fibrinogen to fibrin conversion in 19 patients with liver cirrhosis, confirmed with liver biopsy, and classified according to standardized criteria into 11 moderate and 8 severe cases. Sepharose gel filtration of plasma allowed separation of fibrinogen-like material (FLM) into high m.w. (HMWF), peak, and low m.w. (LMWF) fractions, quantitated as percent (w/w) of the total FLM eluted. FPA was assayed radioimmunologically.HMWF was markedly increased in the whole group of cirrhotics (av ± SD 10.4% ± 2.77) , and in both moderate and severe patients, vs controls (6.9% ± 1.39; n=16; p < 0.01 for each comparison). LMWF was moderately increased in patients (6.8% ± l.84) vs controls (5.3% ± 1.35; p < 0.05).FPA levels (controls: median (m) ng/ml 2.2; m confidence limits (mcl) 1.4-2.5; range (r) < 0.6 to 4.1) showed an increase in cirrhotics (m 2.8; mcl 2.64-3.35, rThe lack of correlation between FPA and HMWF although possibly related to half-life differences, suggests that, besides thrombin effect, other mechanisms for excess formation and/or accumulation of HMWF, as dysproteinaemia, dys- fibrinogenaemia, or impaired clearance, should be considered.

Positivity of paracoagulation tests in a previously studied group of 80 patients with chronic active liver disease did not exceed 5-10% of the cases. In the present study, plasma samples from 20 cases of decompensated liver cirrhosis, assessed by liver biopsy, were investigated by means of agarose cromatography. Fibrinogen related materials were measured immunologically and by Staphylococcal Clumping Test.First appearance of fibrinogen-like materials occurred at earlier fractions in cirrhotic patients in comparison with normal controls. The relative amount of soluble fibrin monomer complexes (SFMC) as referred to total fibrinogen was significantly increased. No correlation was found between the amount of SFMC and the severity of fibrinogen polymerisation defect.Circulating SFMC are therefore present in severe liver cirrhosis. However, DIC may not be the only proposed explanation for this finding.

An increased content in sialic acid in the carbohydrate moiety of fibrinogen (fibrinogen sialic acid, FSA) as described in a number of different liver patients has been accounted for the polymerization defect often found in conditions of liver damage.In 22 cases of liver cirrhosis divided into “moderate” (n = 11) and “severe” (n = 11) according to standardized criteria, FSA was measured with the method of Warren after purification of fibrinogen with β-alanine and glycine, and expressed in mg per 100 mg of fibrinogen.In the whole group of cirrhotics FSA was 0.79 mg ± 0.17 (controls n = 10, FSA 0.70 mg ± 0.13, p > 0.05). However, FSA was markedly increased in “severe” cases (0.90 mg ± 0.16) both vs controls (p < 0.001) and vs “moderate” cases (0.69 mg ± 0.10, p < 0.005). Thrombin time (TT) although prolonged in cirrhotics (p < 0.001) was not significantly different between the subgroups. FSA correlated inversely with fibrinogen level (p < 0.01) but did not correlate with TT.These results show that the increase in FSA is a feature of severe liver cirrhosis especially in presence of a low fibrinogen level. Although probably involved in the polymerization defect, this increase alone cannot properly explain the prolongation of TT which is known to occur also in absence of serum FDP.

A comparison of different methods for AT III was performed in 65 patients. Of these, 45 affected by thrombotic diseases (TD) or under oral contraceptives (0C) were selected on the basis of the finding of low values of “serum” AT III (von Kaulla, AT III S), while 20, under oral anticoagulants, (AC) had abnormally high values of AT III S. The methods used were: AT III S, Xa I, reptilase defibrination (RD), immunologic (Imm), Coatest S 2238 (Chr).In the 45 cases (TD+OC) with low AT III S (≤ 70%), a low AT III level was confirmed with Imm in 20% of the cases, RD also in 20%, XaI in 15,5%, and Chr in 4,4%. Imm values were correlated (p < 0.05) with RD values. The variability of AT III S was completely different from that of all other methods. In AC patients with increased values of AT III S, there was no significant change in AT III with any other method.In conclusion, our results show a marked discrepancy between AT III S and the other methods used, suggesting heterogeneity of AT III S method. AT III S values are in fact related not only to AT III “in vivo”, but also to its “in vitro” consumption rate and possibly to other clotting inhibitors. However, the clinical usefulness of AT III S as an index of “hypercoagulability” remains to be investigated.

The haemorheologic condition was evaluated in 43 obese children and 35 controls. In 18 of the obese children and in 21 controls the euglobulin lysis time (ELT) was also studied. Blood viscosity at 94.5 and at 0.204 s-1 shear rates, plasma viscosity, fibrinogen and erythrocyte filtration time were significantly higher in obese than in control children. No significant differences were observed in haematocrit levels. Triglycerides, non-esterified fatty acids (NEFA), pre-beta-lipoprotein and insulin rates were all significantly higher in obese than in control children. There were no significant differences in glycaemia and in haemoglobin A1 values. ELT, both basal and after stimulation with 1-deamino-8-D-arginine-vasopressin (DDAVP), was significantly higher in the obese than in control children. The haemorheologic disturbances together with alterations of the haemostatic balance and fibrinolysis may be an important risk factor for the development of vascular changes at paediatric age.

SummaryIn 107 asymptomatic and untreated patients with inherited syndromes associated with thrombophilia (antithrombin III, protein C and protein S deficiencies), we compared in parallel two plasma peptides which reflect activation of the common coagulation pathway: the prothrombin fragment 1 + 2 (F1+2) and fibrinopeptide A (FPA). Both F1+2 and FPA were measured with simple, commercially available ELISA methods. High levels of F1+2 or FPA were found in about one fourth of the patients as a whole. When patients were divided according to the type of inherited thrombophilie syndrome, it appeared that F1+2 was more frequently elevated in protein C and protein S deficiencies than in antithrombin deficiency; and that, in general, it was no more frequently elevated than FPA. Although our data confirm the existence of a procoagulant imbalance in inherited thrombophilie syndromes due to defects of natural anticoagulant proteins, they do not confirm that such imbalance can be more frequently diagnosed by measuring F1+2 levels, particularly in patients with antithrombin deficiency.

SummaryIntroduction D-dimer assay, generally evaluated according to cutoff points calibrated for VTE exclusion, is used to estimate the individual risk of recurrence after a first idiopathic event of venous thromboembolism (VTE). Methods Commercial D-dimer assays, evaluated according to predetermined cutoff levels for each assay, specific for age (lower in subjects

SummaryHeparin-induced thrombocytopenia (HIT) is a life-threatening complication of heparin treatment; the prognosis depends on early and accurate diagnosis, and prompt start of alternative anticoagulants. Because of high sensitivity, the commercially available immunologic assays are widely used, though not suited to be run on single samples and with a turnaround time of 2–3 hours. We evaluated two new, rapid, automated, semi-quantitative chemiluminescent immunoassays in HIT suspected patients: HemosIL® AcuStar HIT-IgG(PF4-H) (specific for IgG anti- PF4/heparin antibodies) and HemosIL® AcuStar HIT-Ab(PF4-H) (detecting IgG, IgM and IgA anti-PF4/heparin antibodies) (both from Instrumentation Laboratory). A total of 102 patients with suspected HIT were included; HIT was diagnosed in 17 (16.7%). No false negative cases were observed using either the HemosIL AcuStar HIT-IgG(PF4-H) or the HITAb(PF4-H) assay (sensitivity and negative predictive values = 100%; negative likelihood ratios

SummaryFew data are available on thrombophilic risk factors and progression of atherosclerotic peripheral arterial disease (PAD). Thrombophilic alterations can be an aggravating factor when arterial stenoses are present. In a cross-sectional study, we evaluated the presence of the thrombophilic factors fibrinogen, homocysteine, factor (F)VIII, lupus anticoagulant (LAC), FII G20210A, and FV R506Q mutations in 181 patients with PAD at Fontaine’s stage II (claudication), in 110 patients with critical limb ischaemia (CLI), and in 210 controls. Fibrinogen was higher in patients with CLI vs. those with claudication and controls (427.9 ± 10.5 vs. 373.1 ± 5.2 vs. 348.9 ± 7.0 p=0.001, respectively). Homocysteine and FVIII were higher in patients with PAD than in controls, but were similar in patients with CLI and claudication. The prevalence of LAC increased in patients with CLI vs. those with claudication and controls (21.4% vs. 7.8% vs. 5.2% p

Background The optimal dose of low-molecular-weight-heparin (LMWH) to prevent venous thromboembolism (VTE) after bariatric surgery remains controversial. Aim The aim of this study was to evaluate the pharmacodynamic parameters of two doses of the LMWH parnaparin administered to patients undergoing bariatric surgery. Methods Patients were enrolled in a multicentre, open label, pilot study and were randomised to receive 4250 IU/day [n = 36; 30 females; median age: 38 years (23-56); median BMI: 46.7 Kg/m2 (36.5-58.8)] or 6400 IU/day [n = 30; 24 females; median age: 42 years (22-63); median BMI: 43.7 Kg/m2 (36.1-64.1)] of parnaparin s.c. for 7-11 days. The pharmacodynamic effects of parnaparin were analysed by measuring the anti Factor Xa activity on day 0 (12 hours after the first parnaparin injection), day 4 and day 6 after surgery (before and 4 hours after parnaparin administration). Results In 98.3% of patients receiving 4250 IU/day the peak anti-Xa levels were in the range of 0.1-0.4 IU/ml. Higher anti-Xa levels were observed in patients receiving 6400 IU/day: in 62.3% of these patients the peak anti-Xa levels were greater than 0.4 IU/ml. The anti-Xa levels measured 4 hours after injection on days 4 and 6 were not statistically correlated with BMI for either dose of-parnaparin (p = 0.077 and p = 0.401 for 4250 or 6400 IU/day, respectively). Conclusion The dose of 4250 IU/day seems adequate to achieve prophylactic anti-Xa levels in morbid obese patients undergoing bariatric surgery. Conversely, most of the patients receiving 6.400 IU/day show anti-Xa levels higher than the recommended prophylactic values.

Summary Introduction The use of adapted cut-off values in the elderly, combined with clinical probability (PTP), increases the proportion of patients in whom venous thromboembolism (VTE) can be safely excluded, compared with the conventional cut-off value of 500 μg/L fibrinogen equivalent units (FEU). We evaluated the clinical performance of three different approaches to establish cut-off values for a D-dimer assay whose results are expressed in D-dimer units (D-DU). Methods HemosIL D-dimer HS assay (Instrumentation Laboratory) was performed in 279 consecutive outpatients with suspected deep venous thrombosis (DVT) and nonhigh PTP. Results Considering patients >60 years, the number of negative D-dimer results increased using the modified (376 ng/mL if ≥60 years) and the age-adjusted cut-off (age years × 5 ng/mL if >50 years) compared to the conventional one (230 ng/mL for all patients; 54.6%, 58.2%, and 25.0%, respectively), with no false-negative results. The higher increase was observed in patients >80 years (43.9%, 56.1%, and 8.8%, respectively). Conclusion For the HemosIL D-dimer HS, the use of specific cut-off values in older subjects with suspected DVT and nonhigh PTP increases the number of patients in whom DVT can be safely excluded.

Background Post-thrombotic syndrome (PTS) is the most common complication of deep vein thrombosis (DVT), but few data are available on the risk factors for PTS. Aims To assess whether the time-course of D-dimer, FVIII, and thrombotic burden are related to PTS development. Methods Patients (n = 59) with proximal DVT of the lower limbs (age 64; range:20-88 years; male 56%) were enrolled on the day of diagnosis (D0) and all received heparin for 5-7 days, overlapped and followed by vitamin K antagonists (VKA) for 3 months. Whole-leg compression ultrasound examination was conducted on D0 and 7 (D7), 30 (D30), and 90 (D90) days afterwards, when blood samples were also taken for D-dimer (STA Liatest) and FVIII (chromogenic assay) testing. Thrombotic burden was defined at each time point according to a score, which considered thrombosis extent and occlusion degree. Villalta score was evaluated at D30, D90, and D180. Results At D90, 12 patients developed PTS (Villalta score ≥ 5) and the median Villalta score was 1 (IQR 0.3-3.0) and was not correlated with either D-dimer or FVIIII time course. At D180, 13 patients had PTS and they had similar thrombotic score at D0, D30 to those without PTS, but higher at D90 (7.6 ± 5.1 vs. 3.2 ± 3.6; p = 0.011). Thrombotic score at D90 was correlated with Villalta score at D90 (rho = 0.374, p = 0.009) and at D180 (rho = 0.436, p = 0.006). Conclusions Thrombotic burden after 90 days of VKA is correlated with PTS.

Inherited and acquired thrombophilic alterations in patients with superficial vein thrombosis of lower limbs

It was our aim to assess whether factor V Leiden (FVL) and G20210A prothrombin (FII) mutation are associated with the presence of residual vein obstruction (RVO) after a standard course of anticoagulation for a first episode of idiopathic proximal deep-vein thrombosis (DVT) of the lower limbs, with or without symptomatic pulmonary embolism (PE). Patients were enrolled in two prospective multicentre studies: PROLONG and PROLONG II. RVO was detected by compression ultrasonography according to the method of Prandoni on the day of anticoagulation withdrawal. Patients were also screened for FVL and FII mutation. The presence of FVL and/or FII mutation was determined in 872/963 (90.5%) patients, in 753 of whom RVO was assessed. FVL was significantly less frequent among subjects with isolated PE (7/176:4%) than among patients with either DVT and PE (15/133:11.3%; p=0.0018) or isolated DVT (89/563:15.8%; p

Dear Sirs, Skov et al. object that the effect of the variant allele rs7294 which is associated with higher warfarin requirements is not relevant in our dosing algorithm, its effect being significant at the univariate but not at the multivariate analysis. Skov et al. also show that there is not a complete linkage disequilibrium but a significant correlation between rs 7294 and the variant allele rs 9934438 which is associated with warfarin sensitivity, based on the data of their study [1]. The frequency of the variant allele rs 7294 was 9.6% in their study vs 7.3% in our derivation group and 10% in the validation group. The partial linkage disequilibrium between the rs 7294 and the rs 9934438 is also consistent with data previously published by Wadelius et al [2]. In the algorithm recently elaborated by Pavani et al. [3], various parameters were included in the multiple linear regression model, and they concluded that the incorporation of VKORC1*3 (rs 7294) and VKORC1*4 (6009 C/T) could help in a precise prediction of therapeutic warfarin dose. Moreover, in a recent meta-analysis [4], Jorgensen et al. showed that for the Caucasian ethnic group, the pooled effect estimate of the VKORC SNP rs 7294 on warfarin dosing was nonsignificant for heterozygotes versus wild-types, but was statistically significant for mutant-types versus wild-types. In our study [5], we chose to employ a simultaneous multivariate regression as our analysis was exploratory and hypothesis generating. The loss of significance of the effect of rs 7294 at the multivariate analysis could be due to our limited sample size, as acknowledged in our discussion, and larger studies are required to confirm our algorithm.

BACKGROUND: The optimal duration of anticoagulant treatment after venous thromboembolism (VTE) should be evaluated in relation to bleeding risk. This assessment is particularly difficult with elderly patients, because of their increased risk of both recurrences and hemorrhages. Bleeding risk stratification models have been proposed, but their predictive ability in very elderly patients is unknown. We aimed to assess six bleeding stratification models in this setting, by using information available in our dataset. PATIENTS AND METHODS: Patients aged >/= 80 years receiving vitamin K antagonists (VKAs) for the secondary prevention of VTE were eligible for this prospective cohort study. All patients were followed at Italian anticoagulation clinics for monitoring of VKA treatment. Risk factors for bleeding were collected, and major bleeding events and mortality were documented during follow-up. The association of bleeding events with the available risk factors was tested by means of Cox regression analysis; the c-statistic was used to quantify the predictive validity of the classification schemes. RESULTS: A total of 1078 patients (37.2% males; mean age, 84 years) were enrolled in the study, for a total observation period of 1981 patient-years. The rate of major bleeding was 2.4 per 100 patient-years (47 events; one was fatal). The mortality rate was 5.2 per 100 patient-years. None of the considered risk factors were significantly associated with bleeding events. The predictive validity of the risk stratification models was low, and the most accurate model was not specifically developed for VTE patients (HEMORR2 HAGES, c-statistic 0.60, 95% confidence interval 0.49-0.70). CONCLUSIONS: Bleeding risk stratification models appear to have little accuracy in very elderly VTE patients.

Background Congenital thrombophilia is a risk factor for venous thromboembolism (VTE). Whether it is associated with increased risk of arterial disease is today a matter of debate. We aimed to look for early signs of atherosclerotic alterations in carriers of inherited thrombophilic alterations (ITA). Methods Between January 2006 and September 2008 ultrasonography assessment of the carotid arteries with measurement of intima-media thickness (IMT), and determination of the ankle/brachial pressure index (ABI), was performed in: a) 161 carriers of ITA (deficiency of antithrombin, protein C or S, factor V Leiden or prothrombin G20210A mutations), 84 of whom with previous VTE, and b) 180 subjects without ITA, matched for age, sex and previous VTE. All subjects were Results Carotid plaques were found in 8 subjects [3 (1.9%) with ITA]. Increased IMT values (> 1 mm) were detected in 6 subjects with and 1 without thrombophilia (p = 0.055). The prevalence of IMT values > 90th percentile was not different in subjects with/without thrombophilia (15.2% vs 11.6%, p = 0.416). At multivariate analysis only age was significantly associated with increased odds ratios for IMT values > 90th percentile. No subjects had abnormal ( Conclusions The present study, the first to investigate the presence of atherosclerotic markers in relatively young subjects with inherited thrombophilia, did not find a particular prevalence of signs of early atherosclerotic markers in these subjects.

Introduction D-dimer testing is widely used in conjunction with clinical pretest probability (PTP) for venous thromboembolism (VTE) exclusion. We report on a multicenter evaluation of a new, automated, latex enhanced turbidimetric immunoassay [HemosIL® D-Dimer HS 500, Instrumentation Laboratory (IL)]. Materials and Methods 747 consecutive outpatients with suspected proximal deep vein thrombosis (DVT, n = 401) or pulmonary embolism (PE, n = 346) were evaluated at four university hospitals in a management study with a 3 month follow-up. Samples were tested at each center using the new D-dimer assay on an automated coagulation analyzer [ACL TOP (IL)], with clinical cut-off for VTE at 500 ng/mL (FEU). Results The sensitivity and negative predictive value (NPV) were 100% for all PTP subgroups (no false negative results); for both sensitivity and NPV the lower limit of the 95% CI in patients with moderate/low PTP was higher than 95%. The overall specificity was 45.1% (95%CI: 41.1-49.3%). Higher specificity value was recorded in the low PTP subgroup [49.2% (95%CI: 41.7-56.7)]. No significant differences were found between patients suspected of having DVT or PE; sensitivity and NPV were 100%. The reproducibility of the assay was good, being the total CVs% less than 10% for D-dimer concentration near the clinical cut-off. Conclusions The new, highly sensitive D-dimer assay proved to be accurate when used for VTE diagnostic work-up in outpatients. Based on 100% sensitivity and NPV and lower limit of the 95% CI higher than 95%, the assay can be used as a stand-alone test in patients with non high PTP.