Diagnostic Performance of a New Rapid Lateral Flow Immunoassay in Patients Suspected of Heparin-Induced Thrombocytopenia and Its Clinical Consequences.

We prospectively evaluated the diagnostic accuracy of a new rapid assay (STic Expert HIT) alone or in combination with a clinical score in 90 HIT-suspected patients. The 4Ts score was calculated, and ELISA and serotonin-release assay (SRA) were performed; the average time taken for test results were 2 and 5 days for ELISA and SRA, respectively. The STic test was performed in our laboratory as an evaluation exercise and the result was available in 1 hour, but results were not communicated to the clinicians so as to not influence management. Diagnostic performance of STic test was assessed, alone and in combination with 4Ts score. HIT was diagnosed in 19 patients. The sensitivity, specificity, and positive and negative predictive values for the STic test alone were 95%, 92%, 75%, and 98%, respectively, with an accuracy of 92%. The likelihood ratio for positive and negative results with the STic test was 11.2 and 0.06. The combination of the 4Ts score and the STic test results had a negative predictive value of 100% and a negative likelihood ratio of 0. The favorable performance of the STic test may allow for the rapid exclusion of HIT in combination with a low to intermediate pretest clinical probability. During the subsequent year, using the STic test in real time to rapidly exclude the diagnosis, we observed a 50% reduction in danaparoid administration in HIT-suspected patients.

Transfusions, major bleeding, and prevention of venous thromboembolism with enoxaparin or fondaparinux in thoracic surgery.

Enoxaparin 40 mg/day (E) or fondaparinux 2.5 mg/day (F) are recommended to prevent venous thromboembolism (VTE) in medical and surgical patients at risk. Over the two years after switching from E to F in our 35-bed department of pulmonology and thoracic surgery, an increase in the number of transfusions was observed. A retrospective explanatory investigation was undertaken. Hospitalised patients in the two years before and after switching from E to F were compared. The files of all transfused patients were reviewed. A blinded independent committee adjudicated major bleeding events. In the investigated time period, the overall transfusion rate increased from 1.8% of 2,989 patients to 3.1% of 3,085 patients (p=0.002). Mean ages (58.4 vs. 59.1 years), proportions of surgical patients (63.6% vs. 58.4%), cancer patients (72.1% vs. 69.5%), and treated patients (≥ 1 dose of E or F: 51.8% vs. 52.5%) were similar. The number of medical patients transfused while receiving E or F did not increase significantly (0.9% vs. 1.3%, RR=1.45 [0.66-3.17], p=0.35). The number of surgical patients transfused postoperatively while receiving E or F increased significantly (0.7% vs. 1.9% of all surgical patients, relative risk [RR]=2.75 [1.45-5.23], p=0.001), including a significant increase in transfusions for major bleeding (0.2% vs. 0.9%, RR=5.97 [1.74-20.4], p<0.001). A multivariate analysis did not find confounding factors. The incidence of symptomatic postoperative pulmonary embolism remained very low (0.05% vs. 0.17%). In conclusion, in thoracic surgery patients, switching from enoxaparin to fondaparinux to prevent VTE was associated with a significant increase in the risk of postoperative major bleeding. A causal relationship appears plausible.

Fetal intracerebral hemorrhage in familial thrombophilia.

We describe a fetal intracerebral hemorrhage associated with familial thrombophilia. Intraventricular and intraparenchymal hemorrhage of the left cerebral hemisphere was diagnosed at 22 weeks of gestation. Neuropathologic examination demonstrated a large germinal zone hemorrhage and ischemic changes secondary to bleeding. The fetus manifested a heterozygous Leiden mutation of the factor V gene, and a heterozygous F76L mutation of the protein C gene. The literature indicates that the coexistence of genetic risk factors of thrombophilia and fetal intracerebral hemorrhage is rare, except in the context of pregnancy-related complications. The utility of laboratory screening is discussed.

Activation of PAK1/2 during the shedding of platelet microvesicles.

Simultaneously to phospholipid flip-flop that supports the procoagulant activity of activated platelets, blebs, supported by actin reorganization, are formed at the plasma membrane and generate microvesicles. The molecular mechanism of microvesicle shedding from activated platelets implicates Ca influx and Ca-dependent protease, calpain. We previously demonstrated that the formation of lamellipodias and filopodias associated with platelet shape change involved the reorganization of actin filaments through a Cdc42/Rac1/p21-activated kinase (PAK)-dependent pathway. Here, we investigated whether platelet blebbing also depends on the Cdc42/Rac1/PAK pathway. Exposure of platelets in vitro to either a mixture of thrombin receptor-activating peptide (TRAP) and collagen or the Ca ionophore A23187 in the presence of Ca generates microvesicles that can be identified by flow cytometry. The calpain inhibitor, calpeptin, diminished microvesicle formation induced by the Ca ionophore A23187, confirming the role of calpain in this process. PAK1/2 is cleaved in a calpain-dependent manner, and calpeptin prevents this cleavage and allows a transient activation of the kinase. Inhibition of Cdc42 and Rac1 by toxin B from Clostridium difficile, that suppresses PAK1/2 activation induced by TRAP and collagen or by A23187 in the presence of calpeptin, decreases polymerization of actin, lamellipodia and filopodia formation and interferes with the shedding of microvesicles. We conclude that the Rac1/Cdc42/PAK pathway controls actin reorganization that is necessary for microvesicle shedding.

Development of a novel immunoassay for the assessment of plasma Gas6 concentrations and their variation with hormonal status.

BACKGROUND: Gas6 is a vitamin K-dependent antiapoptotic protein that has been implicated in cardiovascular pathophysiology. We report the development and validation of an ELISA for Gas6, and the variation of plasma Gas6 with hormonal status in a study designed to evaluate the effect of oral contraception on plasma markers. METHODS: After validation of the main stages of the ELISA assay, we measured plasma Gas6 concentrations in 94 male and 88 female healthy volunteers ages 18 to 38 years. Forty-five of the women then received an oral contraceptive, which contained ethinylestradiol and levonorgestrel, for 3 months before a new measurement was performed at the same time point in their menstrual cycles. RESULTS: Interassay imprecision was 5.8%-11.8%, and the detection limit was 5.9 microg/L. Mean Gas6 plasma concentrations were significantly lower in men (52.0 microg/L) than in women not receiving oral contraceptives (63.8 microg/L, P <0.001). In the women who received oral contraceptives, Gas6 concentrations decreased after 3 months of therapy from 63.6 microg/L to 51.9 microg/L (P <0.001). CONCLUSIONS: We have developed a simple and reproducible ELISA assay for measuring plasma Gas6 concentrations, which vary with sex and are decreased by oral contraceptive use. These results suggest regulation of plasma Gas6 concentrations by sex hormones. Future clinical studies may require participants to be stratified by sex.

Polymorphonuclear leukocyte and monocyte activation induced by plasma from patients with heparin-induced thrombocytopenia in whole blood.

Heparin-induced thrombocytopenia (HIT), a severe complication of heparin therapy, results from platelet activation by heparin-dependent antibodies. Previously, we have shown that plasma from patients with HIT (HIT plasma) induces leukocyteplatelet aggregation in blood. In this report, we examined leukocyte activation by HIT plasma and the contribution of heparin and platelets to this activation, in whole blood. Degranulation of leukocytes from HIT patients was evaluated as a leukocyte activation marker. We showed that polymorphonuclear leukocytes (PMN) and monocytes were the leukocyte subpopulations involved in platelet-leukocyte aggregation induced by HIT plasma in healthy donor blood. PMN and monocyte activation, reflected by increased surface expression of the CD11b adhesion molecule, was induced by HIT plasma in a heparin-dependent manner. The CD11b increase induced by HIT plasma was observed on PMN only when they were associated with platelets. Moreover, the increased CD11b expression on monocytes and PMN correlated strongly with the degree of platelet adhesion to these cells. Degranulation of leukocytes from HIT patients and control subjects (non-HIT heparin-treated patients and healthy subjects) was evaluated in vivo by measuring the plasma myeloperoxidase concentration. HIT plasma contained higher myeloperoxidase concentrations than control plasma, suggesting leukocyte degranulation during HIT. In conclusion, this study provides the first evidence that PMN activation is induced by HIT plasma. HIT plasma induced PMN and monocyte activation in a heparin-dependent manner. In whole blood, platelet association with monocytes and PMN, and the activation of these leukocytes by HIT plasma were interrelated. Finally, leukocyte degranulation could be involved in HIT physiopathology.

Mutations in the SDHB gene are associated with extra-adrenal and/or malignant phaeochromocytomas.

Germ-line mutations in the genes encoding succinate dehydrogenase complex subunits B (SDHB) and D (SDHD) have been reported in familial paragangliomas and apparently sporadic phaeochromocytomas (ASP), but the genotype-phenotype relationships of these mutations are unknown. Eighty-four patients (all but 2 followed up for 8.8 +/- 5.7 years) with ASP (57 with adrenal tumors, 27 with extra-adrenal, multiple, malignant, or recurrent tumors) were screened for the major susceptibility genes for phaeochromocytoma (RET, VHL, SDHD, and SDHB). Thirty-three tumors were available for molecular analysis, enzyme assays, and immunohistochemistry. No (0%) RET and 2 (2.4%) VHL mutations were detected. Only two coding single nucleotide polymorphisms in the SDHD gene (G12S and H50R) were found in 6 patients (7%). Conversely, six deleterious mutations in the SDHB gene were identified in 8 patients (9.5%). Ectopic site and recurrence or malignancy were strongly associated with SDHB mutations (7 of 8, 87%, versus 20 of 76, 26%; P = 0.001). Somatic DNA analysis indicated a loss of heterozygosity at chromosome 1p36 (SDHB locus) in 16 of 33 cases (48%). A loss of heterozygosity at the SDHB locus was found in all tumors with SDHB mutation, and assays of respiratory chain enzymes showed a complete loss of complex II catalytic activity. The vascular architecture of tumors with SDHB mutations displayed features typical of malignancy. These data strongly suggest that SDHB gene is a tumor suppressor gene and that the identification of germ-line mutations in SDHB gene in patients with ASPs should be considered as a high-risk factor for malignancy or recurrence.