Inclusion Of Internal Controls Research Articles

Hepatic Glucose Production, Ureagenesis, and Lipolysis Quantified using the Perfused Mouse Liver Model.

The liver has numerous functions, including nutrient metabolism. In contrast to other in vitro and in vivo models of liver research, the isolated perfused liver allows the study of liver biology and metabolism in the whole liver with an intact hepatic architecture, separated from the influence of extra-hepatic factors. Liver perfusions were originally developed for rats, but the method has been adapted to mice as well. Here we describe a protocol for in situ perfusion of the mouse liver. The liver is perfused antegradely through the portal vein with oxygenated Krebs-Henseleit bicarbonate buffer, and the output is collected from the suprahepatic inferior vena cava with clamping of the infrahepatic inferior vena cava to close the circuit. Using this method, the direct hepatic effects of a test compound can be evaluated with a detailed time resolution. Liver function and viability are stable for at least 3 h, allowing the inclusion of internal controls in the same experiment. The experimental possibilities using this model are numerous and may infer insight into liver physiology and liver diseases.

Inhibition monitoring in veterinary molecular testing.

Many of the sample matrices typically used for veterinary molecular testing contain inhibitory factors that can potentially reduce analytic sensitivity or produce false-negative results by masking the signal produced by the nucleic acid target. Inclusion of internal controls in PCR-based assays is a valuable strategy not only for monitoring for PCR inhibitors, but also for monitoring nucleic acid extraction efficiency, and for identifying technology errors that may interfere with the ability of an assay to detect the intended target. The Laboratory Technology Committee of the American Association of Veterinary Laboratory Diagnosticians reviewed the different types of internal controls related to monitoring inhibition of PCR-based assays, and provides information here to encourage veterinary diagnostic laboratories to incorporate PCR internal control strategies as a routine quality management component of their molecular testing.

Simultaneous Multiplex Detection and Confirmation of the Proteinaceous Toxins Abrin, Ricin, Botulinum Toxins, and Staphylococcus Enterotoxins A, B, and C in Food

Detection of proteinaceous toxins in complex heterogeneous mixtures requires highly specific and sensitive methods. Multiplex technology employing multiple antibodies that recognize different epitopes on a toxin provides built-in confirmatory analysis as part of the initial screen and thereby increases the reliability associated with both presumptive positive and negative results. Polyclonal and monoclonal antibodies were obtained for abrin, botulinum toxins, ricin, and Staphylococcus enterotoxins A, B, and C (SEA, SEB, and SEC). Food samples were spiked with the toxins either individually or mixed and analyzed following 40-fold dilution. Abrin, botulinum toxin A complex, ricin, and SEB displayed limits of detection in the original food samples ranging from 0.03 to 1.3 microg/mL, from 0.03 to 0.07 microg/mL, from 0.01 to 0.1 microg/mL, and from <0.01 to 0.03 microg/mL, respectively. Redundancy, that is, multiple antibodies for each toxin, some recognizing different epitopes or displaying different binding affinities, provided a "fingerprint" for the presence of the toxins and built-in confirmation, thus reducing the likelihood of false-positive and false-negative results. Inclusion of internal controls, including a unique protein, helped control for variations in dilution. Paramagnetic microspheres facilitated the detection of analyte in foods containing particulate matter incompatible with the use of filter plates normally used in the wash steps of assays employing standard polystyrene microspheres.

Quantitative FISH analysis and in vitro suspension cultures of erythroid cells from maternal peripheral blood for the isolation of fetal cells.

Several techniques for the enrichment of nucleated fetal red blood cells present in maternal blood have been reported. Here we describe the use of a quantitative fluorescence in situ hybridization (FISH) method and in vitro suspension cultures of erythroid cells from newborn cord blood and maternal peripheral blood. Together with a rapid high performance liquid chromatography (HPLC) method, that allows us to determine as few as 100 cells containing haemoglobin F (HbF), we have scrutinized the reported enrichment methods for fetal nucleated cells in peripheral maternal blood. One hundred FISH analyses on maternal peripheral blood were performed. The method comprises a cell lysis method for depletion of red cells with minimal losses of nucleated cells, uniform numbers of cells (750 000 cells each) on microscopic slides, and inclusion of internal controls to monitor the efficacy of hybridization. Twenty-six cultures of pure erythroid progenitor cells from maternal peripheral blood were analysed for the expansion of fetal cells. To generate these in vitro cultures, nucleated cells from 10-20 ml of peripheral blood from 26 pregnant women were grown in media containing growth factors and hormones to yield over 10(7) of immature erythroid cells within two weeks. Of those, 13 cultures were from pregnancies with confirmed male fetuses. A total of approximately 8x10(8) maternal cells were added into tissue culture medium for these 13 cultures, resulting in about 2x10(8) nearly pure erythroid cells after two weeks. Whereas fetal cells, alone or added into cultures of peripheral blood, grow rapidly and can be detected quantitatively, we could not find any fetal cells in cultures from maternal blood. Likewise, in 7.5x10(7) peripheral blood cells probed by FISH analysis (half of which were from pregnancies with male fetuses) no single Y chromosome was detected. In summary, suspension cultures of erythroid cells can be established routinely and easily. With the quantitative FISH technique used, 750 000 cells per slide can be screened reliably for cells with Y chromosomes. However, the stringent quality-criteria and most elaborate methods indicate that fetal cells in maternal peripheral blood can not be found using the current technology.