Underestimation of plasma level of factor V coagulant activity and fibrinogen concentration together with prolonged prothrombin time, activated partial thromboplastin time and thrombin time can result from pre-analytical very low calcium level in citrated sample tube.

INTRODUCTION: Pre-analytical phase is a critical step in the haemostasis laboratory cycle. Numerous variations affect tests results, and it is crucial to detect them in order to reject improper specimens before reporting test results. Comparing to prior results or requesting, a repeat sample can help in pre-analytical irregularity assessment. METHODS: Each time a sample addressed to our laboratory displayed aberrant results or discordant with a prior report, another specimen was asked and both were analysed through calcium (Ca) level, prothrombin time (PT), activated partial thromboplastin time (APTT), thrombin time (TT), fibrinogen concentration, factor II, factor VII+X and factor V coagulant activity measurements. Among these, all the primary citrated samples from inpatients without anticoagulant treatment, displaying very low calcium level ('Ca 0' samples), were selected for this 2 years study. RESULTS: A total of 17 samples could be identified. Ca level in their paired repeat samples was always >1.00 mmol/L. Coagulation testing for 'Ca 0' samples showed a significant prolongation of PT, APTT, TT and a significant decrease for fibrinogen concentration and factor V coagulant activity. CONCLUSION: We identified factor V coagulant activity, as the parameter with the most important variation in case of very low calcium level in presumed citrated sample tubes probably contaminated with EDTA.

Increased risk for heart valve disease associated with antiphospholipid antibodies in patients with systemic lupus erythematosus: meta-analysis of echocardiographic studies.

BACKGROUND: Heart valve disease (HVD) is frequent in patients with systemic lupus erythematosus (SLE), and the role of antiphospholipid antibodies (aPL) is controversial. Thus, our objective was to estimate the risk of HVD, including Libman-Sacks endocarditis, associated with aPL in patients with SLE. METHODS AND RESULTS: Studies were selected if they investigated the association between aPL and HVD in SLE patients and if aPL-negative patients were included for comparison. Data sources were MEDLINE, Embase, Cochrane Library, hand search, contact with investigators, and reference lists of studies, without language restrictions. Data on study and patient characteristics, risk estimates, and study quality were independently extracted by 2 investigators. Pooled effect estimates were obtained by using the DerSimonian-Laird method. Of 234 identified abstracts, 23 primary studies (15 cross-sectional, 7 cohort, 1 case-control) met inclusion criteria, including 1656 SLE patients and 508 cases of HVD. Compared with SLE patients without aPL (n=988), the overall pooled odds ratios for HVD and Libman-Sacks endocarditis in aPL-positive patients (n=668) were 3.13 (95% confidence interval, 2.31 to 4.24) and 3.51 (95% confidence interval, 1.93 to 6.38), respectively. The risk of HVD depending on aPL subtypes was the highest for lupus anticoagulant at 5.88 (95% confidence interval, 2.92 to 11.84) and IgG anticardiolipin antibodies at 5.63 (95% confidence interval, 3.53 to 8.97). CONCLUSIONS: Overall, the presence of aPL in SLE patients is significantly associated with an increased risk for HVD including Libman-Sacks endocarditis. The risk conferred by IgG anticardiolipin antibodies is as strong as by lupus anticoagulant. Systematic echocardiographic examinations in SLE patients with aPL should be performed.

Human immunodeficiency virus-associated thrombotic microangiopathies: clinical characteristics and outcome according to ADAMTS13 activity.

Human immunodeficiency virus (HIV) infection is a risk factor for thrombotic microangiopathy (TMA). We sought whether a severe deficiency in ADAMTS13, the enzyme specifically involved in the cleavage of von Willebrand factor, was associated with specific presenting features and outcome in HIV-associated TMA. In this prospective, multicentre, case-control study, 29 patients of 236 in the French Network on TMA had an HIV-associated TMA. Seventeen patients with severe ADAMTS13 deficiency (ADAMTS13 <5% HIV(+) group) were compared to 12 patients with a detectable ADAMTS13 activity (ADAMTS13 >or=5% HIV(+) group). HIV(+) patients were also compared to 62 patients with idiopathic TMA, either with (45 patients, ADAMTS13 <5% idiopathic group) or without (17 patients, ADAMTS13 >or=5% idiopathic group) severe ADAMTS13 deficiency. ADAMTS13 <5% HIV(+) patients had less AIDS-related complications than ADAMTS13 >or=5% HIV(+) patients (23.5% versus 91.6%, respectively, P = 0.0005) and their median CD4(+) T cell count was higher (P = 0.05). TMA-associated death rate was higher in ADAMTS13 >or=5% HIV(+) patients than in ADAMTS13 <5% HIV(+) patients (50% versus 11.7%, respectively, P = 0.04). In ADAMTS13 <5% patients, TMA-associated death rate was comparable between HIV(+) and idiopathic patients (15.5% in idiopathic patients, P-value was non-significant). By contrast, TMA-associated death rate in ADAMTS13 >or=5% HIV(+) patients was higher than in idiopathic patients (11.7% in idiopathic patients, P = 0.04). In conclusion, HIV-associated TMA with severe ADAMTS13 deficiency have less AIDS-related complications and a higher CD4(+) T cell count. TMA prognosis is better and comparable to this of idiopathic forms.

[Evaluation of the automated coagulation analyser ACL TOP (instrumentation laboratory)]. / Evaluation de l'automate d'hémostase ACL TOP (Instrumentation Laboratory).

The ACL TOP is a fully automated random access analyser for coagulometric, chromogenic ou immunologic measurements. The aim of the present study was to evaluate its performances. The tests performed were: prothrombin time (PT), activated partial thromboplastin time (aPTT), prothrombin time derived fibrinogen (FibD), factor VIII (FVIII), antithrombin (AT) and free protein S antigen (PS). The comparison study was performed by analyzing patients samples in duplicate with ACL TOP and the analyser MDA II (BioMérieux) or CA 6000 (Dade-Behring). Imprecision (CV%) was < 4% for routine tests and < 5% for FVIII, AT and PS, except a between-run imprecision < 7,5% for low AT levels. The measured lower limits of linearity for FVIIII, AT and PS were satisfactory. The correlation observed between the ACL TOP and the other analysers was strict for PT and AT and good for TCA and FibD. Triglyceride levels > or = 6 mmol/L interfered with PT and FibD measurements. No sample or reagent carryover was observed in the conditions of the study. Overall, the ACL TO is an analyser with very satisfactory analytical and technical performances and is well suited for routine and specialized coagulation laboratories.

Circulating procoagulant microparticles in women with unexplained pregnancy loss: a new insight.

One of the frequently proposed mechanisms for pregnancy losses refers to uteroplacental thrombosis. However the contribution of classical thrombotic risk factors remains questionable and, if real, does not account for a large number of pregnancy losses. The aim of this study was to investigate the presence of circulating procoagulant microparticles, a new marker of cell activation already associated with various prothrombotic clinical settings. Microparticles were assessed by an original prothrombinase assay on platelet depleted plasma obtained from 74 women with a history of pregnancy loss without apparent cause and 50 controls. Patients were studied at least 2 months after the last obstetrical event and were classified into 2 groups: 49 women with at least 3 consecutive spontaneous abortions at or before the 10th postmenstrual week and 25 with at least one fetal death beyond the 10th postmenstrual week. Among the 74 patients, 41 had increased levels of circulating microparticles, 29 belonging to the group of early pregnancy loss (59%) and 12 to the group of late pregnancy loss (48%). The high prevalence of increased levels of procoagulant microparticles in both groups makes this new marker very promising for the understanding, follow up and therapeutical handling of pregnancy loss.

Thrombotic risk factors in pulmonary hypertension.

Thrombotic lesions are consistently observed in chronic thromboembolic pulmonary hypertension (CTEPH) and frequently found in primary pulmonary hypertension (PPH). It remains unknown, however, whether thrombosis is related to defects of the antithrombotic pathway or to previous vascular injury. This study therefore analysed the frequency of both hereditary and acquired thrombotic risk factors in CTEPH and PPH. One hundred and forty-seven consecutive patients with CTEPH investigated in the author's institution were compared to 99 consecutive patients with PPH. In 116 CTEPH patients and 83 PPH patients, phospholipid-dependent antibodies (antiphospholipid antibodies and lupus anticoagulant) were analysed by both immunological and clotting assays. In patients enrolled since 1994 (46 CTEPH and 64 PPH), hereditary thrombotic risk factors were also determined. Antithrombin, protein C and protein S activities were measured by functional assays. Mutations of factor V and factor II were identified by polymerase chain reaction. The prevalence of hereditary thrombotic risk factors was not increased in patients with either PPH or CTEPH. In contrast, a high frequency of phospholipid-dependent antibodies was observed in PPH (10%) and more notably in CTEPH (20%). Moreover, in PPH, antibodies were present only in low titre whereas in CTEPH, half of the patients with antiphospholipid antibodies had high titres. In addition, in CTEPH all but one of the patients with lupus anticoagulant also had antiphospholipid antibodies. The most striking finding of this study was the high prevalence of phospholipid-dependent antibodies but their clinical relevance appears to be different in primary pulmonary hypertension and chronic thromboembolic pulmonary hypertension. In primary pulmonary hypertension, these antibodies in low titre probably reflect endothelial dysfunction. In contrast, in chronic thromboembolic pulmonary hypertension the presence of antibodies in high titre associated with lupus anticoagulant, underlines the role of thrombosis in the pathogenesis of this condition.

Lymphocyte P-glycoprotein expression and activity before and after rifampicin in man.

It has recently been shown that P-glycoprotein (P-gp) is inducible by rifampicin in the human gut as shown in intestinal biopsies. The present study was performed in order to test the hypothesis that human peripheral lymphocytes can be used to assess such an inducibility. We also assessed inter- and intra-individual variability of P-gp expression and activity in peripheral lymphocytes. Blood samples from 13 healthy volunteers were collected 1.7, 14 and 19 days after inclusion. Rifampicin treatment (600 mg/day) was administered from day 15 to day 18. Lymphocyte P-gp expression was measured at the messenger RNA level by semi-quantitative RT-PCR and at the protein level by immunostaining flow cytometry. P-gp activity was determined by flow cytometry with rhodamine 123 efflux. Cytochrome P4503A4 (CYP3A4) inducibility was measured by comparing the urinary metabolic ratio of 6beta-hydroxycortisol/cortisol on day 14 and 19, Lymphocyte P-gp expression and activity was not induced by rifampicin, while it increased CYP3A4 activity from 5.0 +/- 4.0 to 22.9 +/- 16.6 (P < 0.001). There was a 3 - 4-fold inter-individual variability and a 3 - 44 % intra-individual variability of lymphocyte P-gp expression and activity. Peripheral lymphocytes are not an appropriate material to assess P-gp inducibility in humans. P-gp shows significant inter- and intra-individual variability in human lymphocytes.

Screening for identified and unidentified protein C pathway defects by the Agkistrodon contortrix venom test in consecutive patients.

To determine the real performance of the Agkistrodon contortrix venom (ACV) screening test for protein C (PC) pathway defects, we studied 400 consecutive patients referred for the study of personal venous thrombosis or for family study. All 82 patients with factor V Arg 506 Gln (FV R506Q) (n = 82), 6 patients with activated PC resistance without FV R506Q, 17 patients with PC deficiencies, and 9 patients with combined defects were identified by an abnormal ACV result. Three of 6 protein S deficiencies overlapped the normal range. Among the 280 patients without a PC pathway defect, 63 of 193 with thrombosis and 18 of 87 asymptomatic subjects (relatives of patients with thrombosis) had an abnormal ACV result. A significant linear inverse relationship was observed between the ACV results and factor VIII. However, 31 of 63 patients (49%) with thrombosis and 15 of 18 (83%) asymptomatic subjects with an abnormal ACV had a normal factor VIII level. This test, with a 100% negative predictive value, is reliable for screening for PC deficiencies and for FV R506Q and can be used safely as an exclusion test. Moreover, the test may be useful to indicate, in the PC pathway, unidentified risk factors for venous thrombosis.

Prevalence of antiphospholipid-related antibodies in unselected patients with history of venous thrombosis.

Antiphospholipid antibodies (aPL) are heterogeneous and are now accepted to be mainly phospholipid-protein-dependent antibodies. Although these antibodies are classically associated with thrombosis, their clinical relevance remains to be established. The subgroups of antibodies characterized by their proteic targets were reported to be more appropriate thrombotic markers. We analysed the prevalence of a large panel of antiphospholipid-related antibodies (aPLR), comprising antibodies directed to phospholipid-protein complexes and to different protein cofactors (beta2GPI, prothrombin, annexin V and protein S), in 122 consecutive unselected patients who had experienced at least one venous thrombotic event. The presence of lupus anticoagulants was assessed with an integrated assay using hexagonal phase phospholipids. Two types of aPL (APA and anti-beta2GPI-PL) were measured using a mixture of phospholipids containing cardiolipin and goat serum or human beta2GPI, respectively, as a source of protein cofactor. Our results show a similar prevalence, close to 15%, of lupus anticoagulants, APA and anti-beta2GPI-PL. In contrast, antibodies to beta2GPI were detected in only 8% of the patients, and very few patients had antibodies directed to other proteins. Of the 35 patients having at least one positive aPLR, 17 were classified as severe, because they had recurrent or early onset of thrombosis (< 35 years). The distribution of aPLR between severe and mild cases was not significantly different except for lupus anticoagulants. Our results clearly indicate that lupus anticoagulant is the only aPLR test to be strongly associated with the severity of thrombosis.

Cell damage at the origin of antiphospholipid antibodies and their pathogenic potential in recurrent pregnancy loss.

Antiphospholipid antibodies (APA) are associated with thrombosis, thrombocytopenia and fetal loss but they occur in a variety of diseases. Despite many efforts, a correlation between the specificity of particular subgroups of APA and particular clinical situations remains to be established. The antigens at the origin of APA remain to be identified. We discuss here the possible links between cell apoptosis or necrosis, leading to plasma membrane alterations, and the occurrence of APA in response to sustained stimulation. The pathogenic potential of APA is also considered with respect to recurrent pregnancy loss.

The tissue thromboplastin inhibition test in the detection of lupus anticoagulants: importance of a correction factor eliminating the influence of fibrinogen level.

Tissue thromboplastin inhibition test (TTI) is a highly sensitive but poorly specific test used in the diagnosis of lupus anticoagulant (LA) which is usually requested for patient with inflammatory status. Therefore, we investigated the influence of fibrinogen level on TTI using Recombiplastin (Ortho), Neoplastine (Diagnostica Stago) and Thromborel S (Behring), in a group of 84 patients with fibrinogen levels ranging from 1.6 to 11.8 g/l. The highly significant (p < 0.0001) positive correlation observed between fibrinogen level and TTI with the 3 thromboplastins allowed the calculation of a new TTI value corrected for fibrinogen level named TTIfg. TTIfg was defined as TTI - ("S" x fibrinogen level) where "S" was the slope of the regression line defining TTI as a function of fibrinogen values. In the group studied, the low TTI specificity, respectively 64%, 67% and 75% with Recombiplastin, Neoplastine and Thromborel S, was greatly improved to respectively 95%, 93% and 95% when results were expressed as TTIfg. TTIfg remained as sensitive as TTI when evaluated in a group of 38 patients with previously diagnosed LA.

Anticoagulant response to Agkistrodon contortrix venom (ACV test): a new global test to screen for defects in the anticoagulant protein C pathway.

As specific assays used to identify defects in the protein C (PC) anticoagulant pathway are laborious and expensive, we describe here a global test to screen for these defects. This assay is expressed as the ratio of two activated partial thromboplastin times, one in the absence and one in the presence of 0,125 U/ml of the PC activator of Agkistrodon contortrix venom (ACV). Eight of the 168 healthy volunteers of the control group exhibited an ACV ratio below the lower normal limit of 3.37 [6 subjects with the mutation Arg 506 to Gln in their factor V gene (FV R506Q) and one with PS deficiency]. 128 patients who have had at least one episode of deep-vein thrombosis were retrospectively studied. All patients carrying FV Q506R (n = 48), PC deficiency (n = 14) or combined defects, i.e. FV Q506R and PC deficiency (n = 4) or FV Q506R and PS deficiency (n = 3), had ACV ratios < 3.37. PS deficient plasmas (n = 20) exhibited ACV ratios which overlapped normal range. ACV ratios of one out of seven patients with antithrombin deficiency, and 10% of patients without identified defect in the PC anticoagulant pathway (n = 30) were < 3.37. An ACV ratio raised to 3.70 could lead to a test identifying all patients with a defect in the PC anticoagulant pathway.

Detection of bilayer phospholipid-binding antibodies using flow cytometry.

Antiphospholipid antibodies (APL) are usually detected using solid-phase immunoassays, where cardiolipin is the most common capture antigen. Phosholipids are believed to adopt a monolayer organization when coated onto polystyrene after evaporation of the solvent. However, bilayer phospholipids are probably those evidenced as microparticles or cell fragments circulating in vivo under various pathological circumstances. The surface density of monolayer phospholipids on polystyrene is six times lower than that of bilayer phospholipids. In order to assess the influence of phospholipid organization on the detection of APL, we prepared glass microspheres coated with bilayer phospholipids (cardiolipin, phosphatidylcholine, cholesterol). Such lipospheres enabled us to study the binding of antibodies in 1:100 diluted plasma samples from patients with anticardiolipin antibodies of IgG isotype previously diagnosed by ELISA. Among the 39 plasma samples analysed by flow cytometry, 17 showed positive IgG binding to lipospheres. Only four additional samples became positive when adding 20 micrograms/ml apolipoprotein H. The specificity of the binding was demonstrated by complete reversibility with 1.4 microM annexin V and with a large excess of liposomes of the same composition. The absence of correlation between liposphere and ELISA results suggests that different subgroups of antibodies are detected depending on the method. The detection of APL using bilayer phospholipids is an original assay and may represent a more physiopathological approach to the specificity of APL.

Monocyte vesiculation is a possible mechanism for dissemination of membrane-associated procoagulant activities and adhesion molecules after stimulation by lipopolysaccharide.

Endotoxin-stimulated monocytes can elicit a dual procoagulant response. They express tissue factor and expose phosphatidylserine in the outer leaflet of the plasma membrane. Tissue factor, a membrane glycoprotein, is the cellular trigger of blood coagulation reactions. Phosphatidylserine is an essential anionic phospholipid for surface amplification of thrombin generation. In this study the distribution of these two procoagulant entities between activated monocytes and derived microparticles was assessed after stimulation by LPS. The presence of CD14, CD11a, and CD18, and possible associated adhesion potential were examined, particularly on microparticles. Tissue factor was evidenced by using a specific functional assay and flow cytometry. Phosphatidylserine exposure was monitored through its catalytic activity in a thrombin generation assay and by flow cytometry with the use of FITC-conjugated annexin V, a protein probe of anionic phospholipids. CD14, CD11a, and CD18 were detected by flow cytometry. The interaction of microparticle CD11a/CD18 with intracellular adhesion molecule-1 was demonstrated by using immobilized recombinant intracellular adhesion molecule-1 fusion protein. The major part of tissue factor and phosphatidylserine-dependent procoagulant activity was associated with microparticles after LPS stimulation. This was confirmed by flow cytometry. The presence of functional CD11a/CD18, and CD14 on microparticles testifies to an associated adhesion potential. These results show that membrane vesiculation could be responsible for dissemination of inducible monocyte procoagulant activities and suggest that derived microparticles could also participate in endothelium stimulation. This emphasizes the role of monocyte as a central element in the coupling between inflammation/infection and thrombosis.