Absolute quantitative real-time polymerase chain reaction for the measurement of human papillomavirus E7 mRNA in cervical cytobrush specimens.

BACKGROUND: Few reports of the utilization of an accurate, cost-effective means for measuring HPV oncogene transcripts have been published. Several papers have reported the use of relative quantitation or more expensive Taqman methods. Here, we report a method of absolute quantitative real-time PCR utilizing SYBR-green fluorescence for the measurement of HPV E7 expression in cervical cytobrush specimens. RESULTS: The construction of a standard curve based on the serial dilution of an E7-containing plasmid was the key for being able to accurately compare measurements between cervical samples. The assay was highly reproducible with an overall coefficient of variation of 10.4%. CONCLUSION: The use of highly reproducible and accurate SYBR-based real-time polymerase chain reaction (PCR) assays instead of performing Taqman-type assays allows low-cost, high-throughput analysis of viral mRNA expression. The development of such assays will help in refining the current screening programs for HPV-related carcinomas.

Measurement of the vascular input function in mice for DCE-MRI

DCE-MRI is an important technique in the study of small animal cancer models because its sensitivity to vascular changes opens the possibility of quantitative assessment of early therapeutic response. However, extraction of physiologically descriptive parameters from DCE-MRI data relies upon measurement of the vascular input function (VIF), which represents the contrast agent concentration time course in the blood plasma. This is difficult in small animal models due to artifacts associated with partial volume, inflow enhancement, and the limited temporal resolution achievable with MR imaging. In this work, the development of a suite of techniques for high temporal resolution, artifact resistant measurement of the VIF in mice is described. One obstacle in VIF measurement is inflow enhancement, which decreases the sensitivity of the MR signal to the presence of contrast agent. Because the traditional techniques used to suppress inflow enhancement degrade the achievable spatiotemporal resolution of the pulse sequence, improvements can be achieved by reducing the time required for the suppression. Thus, a novel RF pulse which provides spatial presaturation contemporaneously with the RF excitation was implemented and evaluated. This maximizes the achievable temporal resolution by removing the additional RF and gradient pulses typically required for suppression of inflow enhancement. A second challenge is achieving the temporal resolution required for accurate characterization of the VIF, which exceeds what can be achieved with conventional imaging techniques while maintaining adequate spatial resolution and tumor coverage. Thus, an anatomically constrained reconstruction strategy was developed that allows for sampling of the VIF at extremely high acceleration factors, permitting capture of the initial pass of the contrast agent in mice. Simulation, phantom, and in vivo validation of all components were performed. Finally, the two components were used to perform VIF measurement in the murine heart. An in vivo study of the VIF reproducibility was performed, and an improvement in the measured injection-to-injection variation was observed. This will lead to improvements in the reliability of quantitative DCE-MRI measurements and increase their sensitivity.

Effect of continuous gastric enteral feeding on tonometric measurement of gastric intramucosal carbon dioxide levels in healthy volunteers

Purpose. The aim of this research was to evaluate the effect of enteral feeding on tonometric measurement of gastric regional carbon dioxide levels (PrCO2) in normal healthy volunteers. Design and methods. The sample included 12 healthy volunteers recruited by the University Clinical Research Center (UCRC). An air tonometry system monitored PrCO2 levels using a tonometer placed in the lumen of the stomach via orogastric intubation. PrCO2 was automatically measured and recorded every 10 minutes throughout the five hour study period. An oral dose of famotidine 40 mg was self-administered the evening prior to and the morning of the study. Instillation of Isocal® High Nitrogen (HN) was used for enteral feeding in hourly escalating doses of 0, 40, 60, and 80 ml/hr with no feeding during the fifth hour. Results . PrCO2 measurements at time 0 and 10 minutes (41.4 ± 6.5 and 41.8 ± 5.7, respectively) demonstrated biologic precision (Levene's Test statistic = 0.085, p-value 0.774). Biologic precision was lost between T130 and T140 40 when compared to baseline TO (Levene's Test statistic = 1.70, p-value 0.205; and 3.205, p-value 0.042, respectively) and returned to non-significant levels between T270 and T280 (Levene's Test statistic = 3.083, p-value 0.043; and 2.307, p-value 0.143, respectively). Isocal® HN significantly affected the biologic accuracy of PrCO2 measurements (repeated measures ANOVA F 4.91, p-value <0.001). After 20 minutes of enteral feeding at 40 ml/hr, PrCO2 significantly increased (41.4 ± 6.5 to 46.6 ± 4.25, F = 5.4, p-value 0.029). Maximum variance from baseline (41.4 ± 6.5 to 61.3 ± 15.2, F = 17.22, p-value <0.001) was noted after 30 minutes of Isocal® HN at 80 ml/hr or 210 minutes from baseline. The significant elevations in PrCO2 continued throughout the study. Sixty minutes after discontinuation of enteral feeding, PrCO2 remained significantly elevated from baseline (41.4 ± 6.5 to 51.8 ± 9.2, F = 10.15, p-value 0.004). Conclusion. Enteral feeding with Isocal® HN significantly affects the precision and accuracy of PrCO2 measurements in healthy volunteers. ^

THE MEASUREMENT OF HUMANISTIC AND DEHUMANISTIC BEHAVIORS IN HOSPITAL NURSING ENVIRONMENTS

The purpose of this investigation was to develop a reliable scale to measure the social environment of hospital nursing units according to the degree of humanistic and dehumanistic behaviors as perceived by nursing staff in hospitals. The study was based on a conceptual model proposed by Jan Howard, a sociologist. After reviewing the literature relevant to personalization of care, analyzing interviews with patients in various settings, and studying biological, psychological, and sociological frames of reference, Howard proposed the following necessary conditions for humanized health care. They were the dimensions of Irreplaceability, Holistic Selves, Freedom of Action, Status Equality, Shared Decision Making and Responsibility, Empathy, and Positive Affect.^ It was proposed that a scale composed of behaviors which reflected Howard's dimensions be developed within the framework of the social environment of nursing care units in hospitals. Nursing units were chosen because hospitals are traditionally organized around nursing care units and because patients spend the majority of their time in hospitals interacting with various levels of nursing personnel.^ Approximately 180 behaviors describing both patient and nursing staff behaviors which occur on nursing units were developed. Behaviors which were believed to be humanistic as well as dehumanistic were included. The items were classified under the dimensions of Howard's model by a purposively selected sample of 42 nurses representing a broad range of education, experience, and clinical areas. Those items with a high degree of agreement, at least 50%, were placed in the questionnaire. The questionnaire consisted of 169 items including six items from the Marlowe Crowne Social Desirability Scale (Short Form).^ The questionnaire, the Social Environment Scale, was distributed to the entire 7 to 3 shift nursing staff (603) of four hospitals including a public county specialty hospital, a public county general and acute hospital, a large university affiliated hospital with all services, and a small general community hospital. Staff were asked to report on a Likert type scale how often the listed behaviors occurred on their units. Three hundred and sixteen respondents (52% of the population) participated in the study.^ An item analysis was done in which each item was examined in relationship to its correlation to its own dimension total and to the totals of the other dimensions. As a result of this analysis, three dimensions, Positive Affect, Irreplaceability, and Freedom of Action were deleted from the scale. The final scale consisted of 70 items with 26 in Shared Decision Making and Responsibility, 25 in Holistic Selves, 12 in Status Equality, and seven in Empathy. The alpha coefficient was over .800 for all scales except Empathy which was .597.^ An analysis of variance by hospital was performed on the means of each dimension of the scale. There was a statistically significant difference between hospitals with a trend for the public hospitals to score lower on the scale than the university or community hospitals. That the scale scores should be lower in crowded, understaffed public hospitals was not unexpected and reflected that the scale had some discriminating ability. These differences were still observed after adjusting for the effect of Social Desirability.^ In summary, there is preliminary evidence based on this exploratory investigation that a reliable scale based on at least four dimensions from Howard's model could be developed to measure the concept of humanistic health care in hospital settings. ^

The number and timing of time -dependent covariate measurements for the Cox regression model

The problem of analyzing data with updated measurements in the time-dependent proportional hazards model arises frequently in practice. One available option is to reduce the number of intervals (or updated measurements) to be included in the Cox regression model. We empirically investigated the bias of the estimator of the time-dependent covariate while varying the effect of failure rate, sample size, true values of the parameters and the number of intervals. We also evaluated how often a time-dependent covariate needs to be collected and assessed the effect of sample size and failure rate on the power of testing a time-dependent effect.^ A time-dependent proportional hazards model with two binary covariates was considered. The time axis was partitioned into k intervals. The baseline hazard was assumed to be 1 so that the failure times were exponentially distributed in the ith interval. A type II censoring model was adopted to characterize the failure rate. The factors of interest were sample size (500, 1000), type II censoring with failure rates of 0.05, 0.10, and 0.20, and three values for each of the non-time-dependent and time-dependent covariates (1/4,1/2,3/4).^ The mean of the bias of the estimator of the coefficient of the time-dependent covariate decreased as sample size and number of intervals increased whereas the mean of the bias increased as failure rate and true values of the covariates increased. The mean of the bias of the estimator of the coefficient was smallest when all of the updated measurements were used in the model compared with two models that used selected measurements of the time-dependent covariate. For the model that included all the measurements, the coverage rates of the estimator of the coefficient of the time-dependent covariate was in most cases 90% or more except when the failure rate was high (0.20). The power associated with testing a time-dependent effect was highest when all of the measurements of the time-dependent covariate were used. An example from the Systolic Hypertension in the Elderly Program Cooperative Research Group is presented. ^