Rapid and simultaneous typing of hemoglobin S, hemoglobin C, and seven Mediterranean beta-thalassemia mutations by covalent reverse dot-blot analysis: application to prenatal diagnosis in Sicily

The molecular lesions causing beta-thalassemia in Sicily can be subdivided into two groups. One that occurs at a 71% frequency and consists of the beta 39, IVS 1,110 and IVS 1,6 mutations and the other group at a 20% frequency comprising the -87, beta s, IVS 1,1 and IVS 2,745 mutations. The identification of all these mutations by polymerase chain reaction (PCR) and conventional dot-blot hybridization has been time consuming and expensive. In this article, we describe the implementation of the reverse dot-blot (RDB) hybridization as a rapid nonradioactive method for the identification of the nine most frequent molecular lesions in the beta-globin gene (-87, beta s, beta c, IVS 1,1, IVS 1,6, IVS 1,110, beta 39, IVS 2,1, IVS 2,745) in Sicily. Sixty prenatal diagnoses were performed by this RDB assay, each of which was confirmed by dot-blot/ASO hybridization; thus demonstrating the accuracy of the RDB. The main advantage of this assay is the rapid typing of an individual's DNA for many mutations in a single working day. Because the mutations in this assay are representative for the Mediterranean region, this mutational panel can also be extended to the screening of beta-thalassemia from other Mediterranean regions.     

Inhibition of tissue factor/factor VIIa activity in plasma requires factor X and an additional plasma component

A study was carried out to explore requirements for the inhibition of tissue factor-factor VIIa enzymatic activity in plasma. Reaction mixtures contained plasma, 3H-factor IX or 3H-factor X, tissue factor (vol/vol 2.4% to 24%), and calcium. Tissue factor-factor VIIa activity was evaluated from progress curves of activation of factor IX or factor X, plotted from tritiated activation peptide release data. With normal plasma, progress curves exhibited initial limited activation followed by a plateau indicative of loss of tissue factor-factor VIIa activity. With hereditary factor X-deficient plasma treated with factor X antibodies, progress curves revealed full factor IX activation. Adding only 0.4 micrograms/mL factor X (final concentration) could restore inhibition. Inhibition was not observed in purified systems containing 6% to 24% tissue factor, factor VII, 0.5 micrograms/mL, factor IX, 13 micrograms/mL, and factor X up to 0.8 micrograms/mL, but could be induced by adding barium-absorbed plasma to the reaction mixture. Thus, both factor X and an additional material in plasma were required for inhibition. The amount of factor X needed appeared related to the concentration of tissue factor; adding more tissue factor at the plateau of a progress curve induced further activation. These results also indicate that inhibited reaction mixtures contained active free factor VII(a). Preliminary data suggest that inhibition may stem from loss of activity of the tissue factor component of the tissue factor- factor VII(a) complex.     

The influence of previous low birth weight on birth weight, gestational age, and anthropometric measurements in the current pregnancy.

The effect of a previous low birth weight birth (less than 2750 g) was examined using a series of regression analyses. Effects on birth weight were partitioned into those associated with preterm delivery (128 g) and term delivery (178 g). Among term births, a mean difference of 107 g was associated with a previous birth of less than 2750 g, even after controlling for other risk factors including smoking, drug and alcohol use, maternal race, size, and hypertension. The pattern of measurements seen after a previous birth of less than 2750 g included significantly smaller head, chest, abdomen, arm, and thigh circumferences, but an insignificant impact on skinfold thicknesses and no significant effect on length measurements.     

Coxsackievirus-cell interactions that initiate infection in porcine ileal explants

Summary Coxsackievirus B 5 (CB 5) labeled with tritiated uridine was used to trace the interaction of the virus with explant cultures of porcine ileum. Similarly labeled human poliovirus 1 (PO 1), which is not specifically retained by porcine tissue, was used as a control. The explant procedure employed could maintain ileal tissue in a differentiated state for up to 48 hours. Porcine ileum was acquired from both young (4–6 week-old) and adult (9–11 month-old) animals. Inoculated explants of either absorptive or lymphoid tissue were incubated at temperatures selected to permit either viral adsorption or penetration and elution to occur. Retention of radioactive virus was quantitated by liquid scintillation counting and localized by autoradiography. Only in absorptive tissue explants from young animals did adsorption of CB 5 at 6°C exceed penetration at 37°C. This suggested that incubation at 6°C may not be an appropriate condition for studying enterovirus adsorption in explants. CB 5 penetrated most efficiently into lymphoid tissue explants from young animals, indicating that these tissues could discriminate between CB 5 and PO 1. In explants from adults, CB 5 penetrated equally well into lymphoid and absorptive tissues. Virus penetrated into the absorptive epithelial cells and, possibly, the lamina propria near the villous tips. Low efficiency of penetration, and the non-critical function of these target cells, may help account for the characteristic lack of gastrointestinal symptoms in enterovirus infections.     

Limitations of the polymer two phase system for detection of viruses

Summary The aqueous polymer two phase technique has been evaluated in the detection of seven types of enteroviruses. Using primary monkey kidney cells as the host system, the two phase technique was found to be a significant aid to detection of each of the poliovirus types, Coxsackie virus type B-3, and, in the presence of fluid maintenance medium only, Coxsackie virus type A-9. The method was found to be worse than no treatment of samples containing Coxsackie virus type B-2 and ECHO virus type 6. The lower phase, into which the viruses concentrate, is strongly inhibitory to these two agents and to influenza A (PR 8) virus, suggesting that the effect might extend to other viruses as well. Users of this procedure in environmental virology studies should be aware that their results may be biased significantly by the selective action of the dextran sulfate employed in this technique.     

Use of vaginal polymorphonuclear to epithelial cell ratios for the prediction of preterm birth

OBJECTIVE: To compare the use of midtrimester vaginal polymorphonuclear cell (PMN) counts on Gram-stain preparations with PMN to epithelial cell (EPI) ratios for the prediction of subsequent spontaneous preterm birth. METHODS: From a prospective cohort study, 83 women with a spontaneous preterm birth at less than 35 weeks of gestation (cases) were compared with a control group of 108 women who delivered at term. Vaginal Gram-stain preparations were obtained at the initial study visit (20-25 weeks). Vaginal PMN and epithelial EPI cell counts were assessed on 5 nonadjacent oil immersion fields under x 1,000 magnification. Vaginal PMN/EPI ratios for each evaluated slide field were calculated to control for intraslide variation in cellular density. RESULTS: Mean delivery gestational age was 31.5 +/- 3.5 weeks for the cases and 39.8 +/- 1.2 weeks for the controls. Mean PMN counts were similar in the cases (13 +/- 20 cells per oil immersion field) and the controls (10 +/- 14 cells per oil immersion field) (P = .17). The mean PMN/EPI ratio, however, was significantly higher among the cases (3.4 +/- 6.0) than among the controls (1.8 +/- 2.4, P = .01). When PMN counts and PMN/EPI ratios were dichotomized by the 95th percentile cutoff (based on control values), the PMN/EPI ratio was significantly associated with subsequent development of spontaneous preterm birth at less than 35 weeks of gestation (odds ratio 3.8, 95% confidence interval 1.3-11.2), whereas PMN counts were not (odds ratio 2.1, 95% confidence interval 0.7-6.1). CONCLUSION: Midtrimester vaginal PMN counts on Gram-stain preparations were not significantly associated with subsequent spontaneous preterm birth in this population. However, the ratio of PMN to EPI counts, which provides internal standardization of variation in slide cellular density, was significantly higher in women with subsequent spontaneous preterm birth at less than 35 weeks.