The coagulation factor V Leiden, MTHFRC677T variant and eNOS 4ab polymorphism in young Chinese population with ischemic stroke.

BACKGROUND: The mechanisms of ischemic stroke in young adults are poorly understood. The main goal of the current investigation was to determine the possible contribution of genes affecting hemostasis, homocysteine metabolism and endothelial function on the occurrence of ischemic stroke in a cohort of young cases and corresponding controls. METHODS: We evaluated in 97 consecutive patients referred to our center between April 2006 and July 2007 for a history of young adult ischemic stroke (age at first event, <45 y) the prevalence of factor V Leiden, 5,10-methylenetetrahydrofolate reductase (MTHFR) C677T and endothelial nitric oxide synthase (eNOS) 4ab gene variants. Ninety-nine subjects age and gender matched (18 to 45 y) from other departments of the Tiantan Hospital served as controls. RESULTS: Homozygosity for the TT genotype of the MTHFR gene was 29 of 97 (29.9%) in patients and 34 of 99 (34.3%) in controls; this difference was not statistically significant (p>0.05, chi2). Variation of factor V Leiden was not detected both in patients and in controls of this cohort. Genotype eNOS 4bb was 90 of 97 (92.8%) in patients and 81 of 99 (81.8%) in controls; this difference was statistically significant (p<0.05, chi2 test). CONCLUSIONS: The eNOS 4ab variant appears to be an important risk factor for ischemic stroke in young Chinese population.

Improvement for identification of heterophile antibody interference and AFP hook effect in immunoassays with multiplex suspension bead array system.

BACKGROUND: A variety of immunoassays including multiplex suspension bead array have been developed for tumor marker detections; however, these assays could be compromised in their sensitivity and specificity by well-known heterophile antibody interference and hook effect. METHODS: Using Luminex® multiplex suspension bead arrays, we modified protocols with two newly-developed solutions that can identify heterophile antibody interference and AFP hook effect. Effectiveness of the two solutions was assessed in serum samples from patients. RESULTS: Concentrations of 9 tumor markers in heterophile antibody positive samples assayed with Solution A, containing murine monoclonal antibodies and mouse serum, were significantly reduced when compared with those false high signals assayed without Solution A (all p<0.01). With incorporation of Solution H (fluorescent beads linked with AFP antigen), a new strategy for identification of AFP hook effect was established, and with this strategy AFP hook effect was identified effectively in serum samples with very high levels of AFP. CONCLUSIONS: Two proprietary solutions improve the identification of heterophile antibody interference and AFP hook effect. With these solutions, multiplex suspension bead arrays provide more reliable testing results in tumor marker detection where complex clinical serum samples are used.

Evaluation of a method for the simultaneous detection of multiple tumor markers using a multiplex suspension bead array.

OBJECTIVE: To achieve higher tumor detection efficiency, we evaluated a multiplex assay for TM analysis based on the Luminex-100 multiplex suspension bead array. DESIGN: The assay simultaneously determined the concentrations of nine TMs in 1114 human serum specimens (546 patients with tumors, 158 patients with non-tumor inflammatory diseases, and 410 normal controls). The nine TMs were AFP, CEA, CA125, CYFRA 21-1, CA242, f-PSA, t-PSA, NSE and free ß-hCG. The multiplex suspension bead assays were compared with conventional methods used in clinical laboratories. RESULTS: The Luminex assay has the same levels of sensitivity, specificity and accuracy in the prediction of positive tumor specimens as conventional methods. CONCLUSION: Multiplex suspension bead arrays have promising applications in clinical laboratories.

Purification and characterization of the human erythrocyte band 3 protein C-terminal domain.

To clarify the function of the hydrophilic carboxyl-terminal tail of human erythrocyte membrane band 3 protein (HEM-B3), we purified two peptides, C1 (Ala893-Val911) and KS4 (Gly647-Arg656), from human erythrocyte band 3 protein preparations. Purified C1 peptides at concentrations from 5 to 80 microM were incubated with fresh human erythrocyte white ghosts. The C1 peptide demonstrated a novel protease activity, which cleaved glycophorin A (GPA) at Leu118-Ser119 in a dose-dependent manner. This activity was eliminated by trypsin. In a control experiment, the KS4 peptide did not cleave GPA under the same conditions. To help substantiate that the band 3 C-terminal tail peptide (C1) alone possesses the protease activity, two experiments were performed. First, the plasmids pGBKT(7)-GPA-Ct and pGADT(7)-AE1-Ct were cotransformed into the yeast strain AH109. The pGBKT(7)-GPA-Ct plasmid contains the cDNA of the 33 amino acid residue section of GPA (Tyr93-Asn125) fused with the pGBKT(7) vector. The plasmid pGADT(7)-AE1-Ct contains the cDNA of the C-terminal 33 amino acid residues of HEM-B3 fused with the GAL4 DNA-binding domain in the pGADT(7) vector. The results of the cotransformation experiment indicated that the C-terminal 33 amino acid residues of HEM-B3 interacted directly with the GPA C-terminal segment defined above. Second, we used a mammalian two-hybrid analysis to confirm the interaction relationship between the band 3 C-terminal segment and the GPA C-terminus. The C-terminus of GPA and the C-terminal 33 amino acid residues of HEM-B3 were subcloned into the DNA-binding domain and transcription activation domain vectors of the two-hybrid system, respectively. They were then cotransfected along with a chloramphenicol acetyltransferse (CAT) reporter vector into HeLa cells. The CAT activity measured in this experiment also indicated that there was interaction between the C-terminal 33 amino acid residues of HEM-B3 and the C-terminus of GPA.