Statistical analysis of results obtained by real-time PCR for improvement of absolute quantification of target sequences.

BACKGROUND: Real-time quantitative PCR is increasingly used in clinical laboratories. Genomic DNA or plasmids containing cloned target sequences are necessary to generate data for standard curves. These data must be analysed to obtain the relative or absolute quantity of the target concentration in a sample. The method chosen for data analysis can strongly influence results of the quantification. Absolute quantification is important especially in clinical settings. For different reasons estimating the copy number of the gene of interest based on DNA concentration measurements is vague and tends toward overestimation, especially if cell lines are used. METHODS: Data gained by limiting dilution and multiple-tube approach were analyzed using our new Poisson distribution based software and were compared with results from DNA concentration measurement. Data from different cell sources (peripheral blood mononuclear cells and two cell lines) were compared: RESULTS: Limiting dilution and multiple-tube approach analyzed by a Poisson distribution simplifies and improves the generation of standard curves for real time PCR if cell lines are used. The absolute target copy number in a sample, the standard deviation, and a 95% confidence interval are calculated by the software. CONCLUSIONS: With this easy to use program a target copy number can be reliably quantified. The program is available free of charge from: http://www.medizin.uni-greifswald.de/InnereC/index.php?id=18 (link will be activated after acceptance of the paper).

Incidence of microperforation for surgical gloves depends on duration of wear.

BACKGROUND: The use of sterile gloves is part of general aseptic procedure, which aims to prevent surgical team members from transmitting infectious agents to patients during procedures performed in an operating room. In addition, surgical gloves also protect team members against patient-transmitted infectious agents. Adequate protection, however, requires that the glove material remain intact. The risk of perforations in surgical gloves is thought to correlate with the duration of wear, yet very few prospective studies have addressed this issue. METHODS: We prospectively collected 898 consecutive pairs of used surgical gloves over a 9-month period in a single institution. After surgical team members wore the gloves during surgical procedures, the gloves were examined for microperforations using the watertight test described in European Norm 455, part 1. The gloves were analyzed as a pair; if 1 glove had a perforation, the pair was considered to be perforated. In addition, we evaluated the use of a hand cream that contained a suspension of cornstarch and ethanol to determine its potential influence on the rate of microperforation. RESULTS: Wearing gloves for 90 minutes or less resulted in microperforations in 46 (15.4%) of 299 pairs of gloves, whereas wearing gloves for 91-150 minutes resulted in perforation of 54 (18.1%) of 299 pairs, and 71 of (23.7%) of 300 pairs were perforated when the duration of wear was longer than 150 minutes (P = .05). Subgroup analysis revealed no significant difference in the rates of microperforation for surgeons (56 [23.0%] of 244 pairs of gloves perforated), first assistants (43 [19.0%] of 226 pairs perforated), and surgical nurses (53 [20.5%] of 259 pairs perforated). Of 171 microperforations, 114 (66.7%) were found on the left hand glove (ie, the glove on subjects' nondominant hand), predominantly on the left index finger (55 [32.3%]). The use of the hand cream had no influence on the rate of microperforation. CONCLUSION: Because of the increase in the rate of microperforation over time, it is recommended that surgeons, first assistants, and surgical nurses directly assisting in the operating field change gloves after 90 minutes of surgery.