No abstract

The present study, using robotized DNA isolation and quantitative PCR based on the Neisseria meningitidisspecific capsular transport A gene, demonstrates the ease, rapidity, specificity, and sensitivity of quantifying neisserial DNA in plasma (n ‫؍‬ 65) and cerebrospinal fluid (CSF) (n ‫؍‬ 12) from patients with systemic meningococcal disease. We found a close correlation between the levels of neisserial DNA and lipopolysaccharides in plasma (r ‫؍‬ 0.905) and in CSF (r ‫؍‬ 0.964). The median concentration of neisserial DNA in plasma in 23 patients with persistent shock was 2 ؋ 10 7 copies/ml, versus <10 3 copies/ml in 42 nonshock patients. Furthermore, quantitative PCR made possible estimates of the total number of meningococci in plasma, as opposed to conventional blood cultures, suggesting about 1,000 dead meningococci for every viable bacterium. Finally, with logistic regression analyses, neisserial DNA may predict a patient's disease severity and outcome at hospital admission. The number of meningococci in plasma and CSF appears to be the main determinant of the lipopolysaccharide levels, clinical presentation, and outcome.

Background: Quantitative reverse transcription-PCR (RT-PCR) used to detect small changes in specific mRNA concentrations is often associated with poor reproducibility. Thus, there is a need for stringent quality control in each step of the protocol. Methods: Real-time PCR-based calibration curves for a target gene, tissue factor (TF), and a reference gene, ␤-actin, generated from PCR amplicons were evaluated by running cDNA controls. In addition, the reverse transcription step was evaluated by running mRNA controls. Amplification efficiencies of calibrators and targets were determined. Variances within and between runs were estimated, and power statistics were applied to determine the concentration differences that could reliably be detected. Results: Within-and between-run variations (CVs) of cDNA controls (TF and ␤-actin), extrapolated from reproducible calibration curves (CVs of slopes, 4.3% and 2.7%, respectively) were 4 -10% (within) and 15-38% (between) using both daily and "grand mean" calibration curves. CVs for the ␤-actin mRNA controls were 12% (within) and 19 -28% (between). Estimates of each step's contribution to the total variation were as follows: CV RT-PCR , 28%; CV PCR , 15%; CV RT , 23% (difference between CV RT-PCR and CV PCR ). PCR efficiencies were as follows: ␤-actin calibrator/target, 1.96/1.95; TF calibrator/ target, 1.95/1.93. Duplicate measurements could detect a twofold concentration difference (power, 0.8). Conclusions: Daily PCR calibration curves generated from PCR amplicons were reproducible, allowing the use of a grand mean calibration curve. The reverse transcription step contributes the most to the total variation. By determining a system's total variance,

No abstract

SummaryAt the end of a diagnostic right heart catheterization ten patients received an intravenous infusion of l-adrenaline which gradually increased the arterial plasma adrenaline concentration from resting physiological values to high values as seen during myocardial infarction, pheochromocytoma and hypoglycemia. Blood was sampled from the brachial artery, femoral vein and hepatic vein. During the adrenaline infusion venous beta-thromboglobulin concentrations increased 23% from 61 ± 5 to 80 ± 7 μg/l (mean ± SE), arterial platelet counts 20% from 212 ± 17 to 253 ± 25 × 109/l and arterial platelet volume 4% from 7.25 ± 0.20 to 7.56 ± 0.21 femtoliter. All changes were significant at the 5% level. Thus, acute increments of arterial plasma adrenaline significantly stimulated the blood platelet parameters studied.