Method validation guidelines, which provide an organizational structure for the design and evaluation of a validation procedure, are presented for a wide range of pharmaceutical applications. The validation guidelines are based on the analyte concentration/sample matrix combination to which the method will be applied. These guidelines include the selection of appropriate validation parameters, design considerations for evaluation, and a discussion of acceptance guidelines for the determination of acceptable method performance. A set of tables is included which illustrates the selection and testing procedure and tailors the entire validation process to the specific characteristics of the determination to be made.
Global temperature increases are predicted to have pronounced negative effects on the metabolic performance of both terrestrial and aquatic organisms. These metabolic effects may be even more pronounced in intertidal organisms that are subject to multiple, abruptly changing abiotic stressors in the land-sea transition zone. Of the available studies targeting the intertidal environment, emphasis has largely been on water-breathing model organisms and this selective focus resulted in limited reliable forecasts on the impact of global warming on primarily air-breathing intertidal species. We investigated the thermal sensitivity of six phylogenetically related fiddler crab species that occupy different microhabitats on intertidal shores from south America and east Asia to test how bimodal-breathing intertidal ectotherms cope with thermal stress. We examined the metabolic physiology and thermal limits of the crabs by measuring their cardiac function and oxygen consumption along a thermal gradient. Their specific thermal microhabitat was also appraised. We found that subtropical fiddler crab species inhabiting vegetated microhabitats have lower upper lethal temperatures and therefore greater thermal sensitivity in comparison to their tropical counterparts. Additionally, females exhibited higher oxygen consumption and lower lethal temperatures in comparison to males. Our results contradict previous predictions that species from higher latitudes that experience greater temperature variability have broader latitudinal distributions, greater phenotypic plasticity and lower thermal sensitivity. Furthermore, the higher thermal sensitivity demonstrated by female fiddler crabs with respect to males strongly suggests a role of both gametogenesis and physiological dimorphism on the thermal performance of tropical and subtropical intertidal organisms. These observations ultimately, advocates for further studies on sex-biased and development-biased thermal sensitivity before drawing any generalizations based on a single sex or life stage.
Recently, a new set of parameters produced by the Beckman-Coulter’s GEN’S, LH500, LH750 and the latest LH780 automated blood counters, derived from leukocyte and red cell’s morphological data by means of the VCS technology are available and can be printed as research data together with the leukocyte differential and reticulocyte analysis. With the use of these Research Population Data (RPD) parameters several recent publications have shown their usefulness for the diagnosis of malaria (Fourcade et al), sepsis (Chaves ), apoptosis detection (Lab hem 2005) and the diagnosis and differentiation of lymphoptoliferative disorders (Silva M et al) In order to evaluate the analytical behaviour of these newly described red blood cell and leukocyte morphological parameters in routine bood samples and in the Beckman-Coulter’s calibration and control 5C, we conducted a study to evaluate the paramters’ precision and performance during short and long term sample’s storage: Precision: 10 samples from normal individuals were analyzed 10 times and the CV for each parameter was calculated Short term stability stability at 4C and 22C: 5 normal samples were evaluated from the time of blood drawing until 5 hours later, both at room temperature (22C) and at 4 C Long term stability at 4C: 15 normal samples were evaluated at various times after blood drawing, from 4 hours (which was found to be the best time for sample analysis in the short term stability study, above) up to 77 hours after blood withdrawal. Control and Calibration with 5C: We evaluated the performance of the research parameters in the 5C by running the same batch of 5C control in 5 different counters from 4 different centers, comparing the results with those from 5 normal individuals Stability in a period of time: We have evaluated every week during two months 15 normal samples, recording the temperature and the avergae of all RPD values. RESULTS Precission: The CV% for the Mean Volume, Conductivity and Scatter of the different leukocyte types was always less than 1%, with an average of 0,6%. The CV% for the Standard Deviation of Volume, Conductivity and Scatter varied between 2.45% and 7.7%, wit an average of 4.9%. The CV% of any SD always being higher than the primary parameter. Short term stability: We observed no significant differences between samples kept at 4C and 22C during the first 5 hours after blood drawing. During the first two hours, however, minor fluctuations in results occurred, always <5%, being preferable to test the samples after blood stabilization, between 2 and 5 hours after withdrawal. Long term stability: The stability of the research parameters after 9 hours is best when the samples are kept at 4C, but the differences are not clinically significant up to 17 hours and, for some parameters, up to 24 hours after blood drawing. The comparison of the RPD research parameters from normal samples and from the Beckman’s 5C control showed good correlation in the different counters and the different centers. When the results from the 5C were higher or lower, the results from the normal samples were concordantly higher or lower respectively, The correlation coefficient was 0.98 for the Mean Volume and 0.92 for the Mean Conductivity The Stability of RPD values was acceptable during the period evaluated (2 m.)with changes lower than 3%. CONCLUSIONS: The results of the present study show that from the analytical stand point, precission, stability and possibility of calibration, these Research Population data are equivalent to those of other laboratory tests performed by modern instruments.
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