The aim of this study was to investigate the relationship between recurrent miscarriages and factor V Leiden, prothrombin G20210A and C677T methylenetetrahydrofolate reductase (MTHFR) mutations. In this case-control study the prevalence of factor V Leiden, prothrombin G20210A and C677T methylenetetrahydrofolate reductase mutations was determined in a consecutive series of 80 recurrent miscarriage patients and 100 controls. Fifteen of 80 recurrent miscarriage patients and four out of 100 controls carried the factor V Leiden mutation (19 versus 4%, P = 0.003, odds ratio 5.5, 95% confidence interval (CI): 1.7-17). Seven of 80 recurrent miscarriage patients and two of 100 controls were carriers of the prothrombin G20210A mutation (9 versus 2%, P = 0.038, odds ratio 4.6, 95% CI: 0.9-23.2). Six of 80 recurrent miscarriage women and 15 of 100 controls were homozygotes for the C677T MTHFR mutation (8 versus 15%, P = 0.134, odds ratio: 0.4, 95% CI: 0.1-1.2). Our results suggest that the presence of factor V Leiden and prothrombin G20210A polymorphism, but not MTHFR C677T homozygosity, could be additional risk factors for recurrent miscarriages. Furthermore, it was suggested that the prevalence of factor V Leiden and prothrombin G20210A mutations is more prominent in second trimester, primary fetal losses and it is independent of the existence of additional pathology predisposing to recurrent fetal losses.

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Dear Sir, Protein S (PS) deficiency is a risk factor for venous thromboembolism. However, accurate measurement and interpretation of PS levels is problematic [1]. Functional assays for PS, which measure the APC cofactor activity of PS in a clot-based system, offer a rapid and readily automated method for detecting reduction in biological activity. Several kits for functional PS assay are now commercially available, and UK NEQAS thrombophilia exercises have identified significant differences in results obtained by different methods [2]. It is possible these differences may affect assay sensitivity and influence the diagnosis of PS deficiency. We report here a study of three widely used functional PS kits, including sensitivity to defects in the PROS1 gene, and to the FV Leiden (FVL) defect.Plasma was collected from 23 subjects with genetically confirmed defects in the PROS1 gene. Prothrombin time (PT), activated partial thromboplastin time (APTT), lupus screens and FVL status in these subjects were normal. Plasma was also obtained from 13 subjects with the FV Leiden defect (nine heterozygous, four homozygous) and 20 normal donors. Blood was collected into 1/10th volume 0.105 M buffered trisodium citrate, centrifuged twice (10 min, 2000 Â g), and the resulting platelet-poor plasma was stored between À55 and À70 8C. Nine subjects with familial thrombophilia were recruited as donors for the UK NEQAS proficiency testing program, and plasma was collected by plasmapheresis into CPD anticoagulant as previously described [3], and also into 0.105 mM trisodium citrate.Total and free PS assays were performed as previously described [4]. Functional PS was measured using three commercial kits according to the manufacturers' instructions. IL test Protein S assay kit (Instrumentation Laboratories, Warrington, UK) was used on an ACL 300R, Stago Staclot (Diagnostica Stago, Asnières, France) and Biopool Bioclot (Biopool AB, Umeå, Sweden) Protein S assay kits were tested on a Sysmex CA6000 instrument. All tests were performed in duplicate. Plasma dilutions were employed to give results within the linear portion of each reference curve (between 25% and 100%). A common reference plasma (SSC secondary plasma standard lot no. 1) was used for each assay. This plasma had previously been distributed in a UK NEQAS exercise (median PS activity level 81 U dL À1 , n ¼ 114). This median value was used in the study, since the standard had not been assigned a potency for PS activity. Plasmas from each manufacturer (Instrumentation Laboratories Calibrant plasma, Biopool normal control plasma, Stago Unicalibrator) were also tested using each kit. Assay precision was determined using a normal lyophilized plasma (PS activity approximately 75 U dL À1 ). Between run coefficients of variation obtained with this sample were: IL kit 6.0%, Stago kit 10.2%, Biopool kit 13.9%. Reference curves were prepared for each assay between 100% and 25%; in all cases, linearity was acceptable over this range of activity (r > 0.98). The following reference ranges were ...

Thrombocytopenia is a serious situation that causes anxiety in doctors and patients due to its relationship with serious hemorrhagic manifestations. Pseudothrombocytopenia, however, is a situation without clinical interest because it is an in vitro decrease in platelet count. Pseudothrombocytopenia is related to the ethylenediaminetetraacetate (EDTA)-dependent exposure of antigenic determinants of platelet membrane glycoproteins gpIIb-IIIa and the subsequent reaction of common antibodies with these receptors. To our knowledge, a similar situation in Greece has not been reported. The main characteristic of pseudothrombocytopenia due to EDTA is that the fall in platelet count is time-dependent and it disappears after the use of anticoagulant other than EDTA (e.g., citrate sodium or heparin). We present seven cases of pseudothrombocytopenia revealed when we used automatic hematologic analyzers and EDTA as an anticoagulant. After the use of another anticoagulant (e.g., citrate sodium) the platelet count in the same hematologic analyzer was normal.