Stability of Hemostasis Parameters in Whole Blood, Plasma, and Frozen Plasma: Literature Review and Recommendations of the SFTH (French Society of Thrombosis and Haemostasis).

Preanalytical sample management is critical for a proper assessment of hemostasis parameters, and may differ depending on prescribed tests or additional tests considered to be necessary after initial results. Although there is quite vast literature on this issue, the Working Group of the French Society of Thrombosis and Haemostasis (SFTH) deemed it necessary to make an in-depth literature review and propose recommendations for the proper handling of samples prior to hemostasis assays. This extensive assessment is accessible on-line in French at the SFTH website. Here, a more synthetic view of these recommendations is proposed, supported by easy-to-use tables. The latter respectively deal with the stability of whole blood or fresh plasma, frozen samples, and proper handling of samples forwarded on dry ice. Procedures are classified as recommended, acceptable, not conformed and lacking data. This work involved the retrieval of 125 references, first screened by a working group of 6 experts, then reviewed by 20 other experts in the field. The highly detailed conditions summarized in these tables will hopefully help hemostasis laboratories to secure the conditions recommended for sample collection and transportation. Moreover, as some conditions clearly lacked recommendations, this review can open new fields of investigation for hemostasis preanalytics.

Comparative study of coagulation and thrombin generation in the portal and jugular plasma of patients with cirrhosis.

Portal vein thromboses are frequent in cirrhotic patients and may be favoured by hypercoagulability in the splanchnic venous system. The coagulation balance and thrombin generation (TG) were evaluated in platelet-free plasma obtained from portal and systemic blood samples in 28 cirrhotic patients while undergoing transjugular intrahepatic porto-systemic shunt. TG assay (TGA) was performed with all samples from cirrhotic patients and with plasma samples from 14 healthy controls, with varying concentrations of tissue factor and phospholipids, with or without thrombomodulin. Screening tests and specific assays were also performed and activated partial thromboplastin time was shorter in portal plasma samples with higher FVIII and lower protein C levels, well correlated with Child-Pugh scores, and higher D-dimers and F1+2 levels However, all TGA parameters were similar in portal and jugular samples, possibly due in part to similar concentrations of factor II and antithrombin in these two sites of plasma sampling. TGA showed lower thrombin peaks and endogenous thrombin potential values in cirrhotic plasma compared to those of healthy controls. Importantly, a resistance to thrombomodulin that well correlated with factor VIII and PC levels, was evidenced in all samples from patients with cirrhosis, and was more significant in those from severely affected cases. This study therefore supports the existence of a relative hypercoagulability in the portal vein of cirrhotic patients that is likely due to protein C/S deficiency and to high FVIII levels.

Factor v Leiden homozygous genotype and pregnancy outcomes.

OBJECTIVE: To assess the rate of early (first trimester) and late (second and third trimester) fetal loss in women who are factor V Leiden homozygous. METHODS: Between December 1995 and February 2007, consecutive, unrelated white women who were factor V Leiden homozygous and who had been pregnant at least once were recruited from 10 French hemostasis units. For reasons of comparison, we included women who were factor V Leiden heterozygous and a group of noncarriers. The frequency of early and late fetal loss was assessed retrospectively and compared among the three groups. The effect of concomitant thrombophilic abnormalities was evaluated. The overall pregnancy outcome was reported. RESULTS: We analyzed 240 thromboprophylaxis-free pregnancies in 95 women who were factor V Leiden homozygous, 425 in 195 women who were factor V Leiden heterozygous, and 182 in 73 women who were noncarriers. The risk of late fetal loss was higher in women who were homozygous (13/95, 13.7%) compared with those who were noncarriers (1/73, 1.4%, odds ratio 11.41, 95% confidence interval 1.46-89.46, P=.002), whereas it was similar in women who were heterozygous and in noncarriers (6/195, 3.1% compared with 1/73, 1.4%, P=.68). The percentage of women with early fetal loss was similar in the three groups (P=.81). The live-birth rate was 80%, 84%, and 85%, respectively, for women who where homozygous, heterozygous, and noncarriers (P=.88). CONCLUSION: The factor V Leiden homozygous genotype increases the risk of late fetal loss. However, the overall likelihood of a positive outcome is high in our series of women who were homozygous. LEVEL OF EVIDENCE: III.

Thrombin generation in first-degree relatives of patients with venous thromboembolism who have factor V Leiden. A pilot study.

The thrombin generation test appears to be a highly sensitive and specific test in the detection of thrombophilia in patients with venous thromboembolism. We aimed to determine the accuracy of the thrombin generation test to detect factor V Leiden and/or other prothrombotic states in first-degree relatives of patients with venous thromboembolism and factor V Leiden. Sixty-two first-degree relatives of 21 index cases were tested for factor V Leiden, the G20210A prothrombin gene mutation and thrombin generation. Information about oestrogen therapy and previous VTE was also collected. The normalized Thrombomodulin sensitivity ratio (n-TMsr) was defined as the ratio of endogenous thrombin potential determined in the presence and absence of thrombomodulin which was normalized against the same ratio determined in normal control plasma. The mean n-TMsr was 1.37 (+/- 0.33) in the 45 relatives with one or more prothrombotic state (factor V Leiden, G20210A prothrombin mutation, oestrogen therapy or hormonal therapy) and 1.02 (+/- 0.34) in the 17 relatives without prothrombotic state (p = 0.001). The positive predictive value was 90.3 (95%CI, 73.1-97.4). In relatives with an abnormal n-TMsr, the adjusted odds ratio for having a prothrombotic state was 8.3 (95%CI, 1.9-36.9) and the adjusted odds ratio for having the factor V Leiden was 14.3 (95%CI, 2.9-71.2). An abnormal thrombin generation test appears highly predictive for having factor V Leiden and/or other prothrombotic states in first-degree relatives of patients with venous thromboembolism and factor V Leiden.

Fibrinogen Aalpha-Thr312Ala and factor XIII-A Val34Leu polymorphisms in idiopathic venous thromboembolism.

INTRODUCTION: Fibrinogen Aalpha-Thr312Ala and Factor XIII Val34Leu polymorphisms have been shown to modify fibrin clot structure and function. However, clinical studies have yielded conflicting results on their possible association with venous thromboembolism (VTE). METHODS: We studied the association between these two polymorphisms and VTE in a hospital-based case-control study. We also assessed whether an independent or interactive association exists between Aalpha-fibrinogen Thr312Ala and FXIII Val34Leu polymorphisms and VTE. Fibrinogen Aalpha-Thr312Ala and FXIII Val34Leu polymorphisms were determined after PCR and restriction endonuclease digestion in 286 patients with idiopathic VTE and 286 age- and gender-matched controls. Results were analysed using a conditional logistic regression model for matched series. RESULTS: The Fg-Aalpha 312Ala allele was associated with higher risk of VTE (OR 1.5; 95% CI: 1.1 to 2.2, p=0.01) while the FXIII 34Leu allele appeared protective (OR 0.7; 95% CI: 0.6 to 0.9, p=0.02). Both alleles demonstrated an independent association with idiopathic VTE after adjustment for Factor V Leiden and G20210A prothrombin polymorphisms. There was no interaction between the fibrinogen Aalpha-Thr312Ala and FXIII Val34Leu polymorphisms for the risk of VTE. CONCLUSION: In this case-control study, the fibrinogen Fg-Aalpha 312Ala allele was associated with an increased risk of VTE. The FXIII 34Leu allele was also significantly associated with a lower risk of VTE without any interaction between the two polymorphisms studied.

Increased prevalence of factor V Leiden in patients with retinal vein occlusion and under 60 years of age.

Retinal vein occlusion (RVO) is a multifactorial disease involving vessel damage, stasis, viscosity and thrombosis. Conflicting findings on hereditary thrombophilic risk factors have been reported and their impact on RVO features remains to be defined. The aim of the present study was to evaluate the prevalence of hereditary thrombophilic risk factors (HTRF) and characteristics of RVO in patients with or without HTRF. The design of the study was a prospective, observational case series. Two hundred and thirty-four patients with RVO were included consecutively. A French healthy population of the same region was studied as control group. The HTRF studied were protein C (PC), protein S (PS) and antithrombin (AT) deficiencies, factor V Leiden (FVL) and factor II 20210A polymorphisms. Chi-Square was used for comparison with the healthy subjects and between RVO patient with and without HTRF according to localisation (branch vs. central), type of RVO (ischemic or non-ischemic), recurrence, age at first event and classical vascular risk factors. Twenty-two patients had HTRF (12 FV Leiden heterozygotes, 9 FII 20210A heterozygotes and 1 PS deficiency). No AT or PC deficiency was detected. Frequencies of PS deficiency, FVL and FII 20210A allele were similar to the reference population as well as to published data in the general caucasian population. Eighty-six patients experienced their first episode before the age of 60 years. Systemic hypertension, glaucoma and angina were significantly less frequent in patients with RVO before 60 years. Fourteen of the 22 patients with one HTRF (64%) experienced their first episode of RVO before the age of 60 years compared to 72 of 212 without HTRF (34%) (p = 0.006). Heterozygote status for FV Leiden was significantly more frequent in patients who had experienced their first episode of RVO before 60 years (p = 0.027). In conclusion, this study suggests a role of FV Leiden in the occurrence of RVO in patients younger than 60 years who exhibit fewer acquired vascular risk factors than in older patients.

Ischaemic strokes and homozygosity for the alpha2 807T allele of the platelet collagen receptor in young monozygotic twins.

A nucleotide 807T variant of the glycoprotein Ia gene that correlates with increased platelet surface levels of the platelet collagen receptor alpha2beta1 was recently found to be associated with an increased risk of ischaemic stroke in younger patients. We report the history of twins who developed ischaemic strokes and were shown to be homozygous for the alpha2 807T allele. The twins developed ischaemic strokes at the ages of 23 and 33 years, one of them with recurrent events. They had no conventional risk factors. Cardiac and vascular investigations were normal and no aetiology could be found. There was a family history of cerebrovascular disease. Genotyping of glycoprotein alpha2 C807 T was performed and both twins were found to be homozygous for the 807T allele. This allele probably contributed to the occurrence of strokes in these young men. Further prospective studies are needed to evaluate whether screening for this polymorphism should be considered in young patients with unexplained stroke, particularly when a positive family history was found.