Development of a charged-coupled device-based light-scattering instrument for the detection of C-reactive protein using particle-enhanced immunoassay.

A novel light-scattering instrument has been developed for rapid detection of immunoreactions in test latex particle-enhanced immunoassays. The detector consists of a flat-field grating and a charge-coupled device mounted on a rotating platform, and the detector measures a continuous spectrum from 350 nm to 735 nm at 440 polar angles with a resolution of 0.5 degrees. Optimal detection for rates of immunoreaction were determined by intensity of scattered light at different angles. Instrumental precisions were all shown to fall within 5% of the target relative standard deviation limits. The accuracy of the instrument was confirmed using monodispersed latex particles of known size and shape. The initial results showed the possibility of a sensitive and accurate detection of C-reactive protein throughout the range of clinical interest, thus demonstrating a significant potential for biomedical applications.

Plasma preparation from whole blood using ultrasound.

A technique to efficiently separate plasma from human whole blood is described. Essentially, 3-mL samples are held on the axis of a tubular transducer and exposed for 5.7 min to an ultrasonic standing wave. The cells concentrate into clumps at radial separations of half wavelength. The clumps grow in size and sediment under gravity. A distinct plasma/cell interface forms as the cells sediment. The volume of clarified plasma increases with time. The separation efficiencies of transducers of 29-mm and 23-mm internal diameters driven, by test equipment, at radial resonances of 3.4 and 1.5 MHz, respectively, were compared. The average efficiency of separation was 99.6% at 1.5 MHz and 99.4% with the 3.4-MHz system. The cleared plasma constituted 30% of the sample volume at 1.5 MHz and 25% at 3. 4 MHz. There was no measurable release of haemoglobin or potassium into the suspending phase, indicating that there was no mechanical damage to cells at either frequency. A total of 114 samples from volunteers and patients were subsequently clarified in a 1.5-MHz system driven by an integrated generator. The average efficiency of clarification of blood was 99.76% for the latter samples. The clarification achieved is a significant improvement on that previously reported (98.5%) for whole blood exposed to a planar ultrasonic standing wave field (Peterson et al. 1986). We have, therefore, now achieved a six-fold reduction of cells in plasma compared to previous results.

Clarification of plasma from whole human blood using ultrasound.

There has been interest for a number of years in the possibility of separating blood into cells and plasma by methods other than centrifugation, so that the plasma can be analysed on-line. Cells in whole blood normally occupy about 45% of the suspension volume. It has been shown with a number of different cell types, such as yeast and bacteria, that for concentrations of this order the cells are not as efficiently harvested by ultrasound as those for lower concentrations. In this study, removal of cells from 3-4 ml whole blood volumes has been examined in ultrasonic standing wave fields from tubular transducers driven at a frequency of 1.6 MHz. Samples of whole human blood (n = 11) from two volunteers have been processed by three tubular transducers where high levels of cell removal, 99.7% on average, have been demonstrated with high reproducibility between samples as well as for different transducers.

Development of an automated on-line analysis system using flow injection, ultrasound filtration and CCD detection.

The design and construction of an automated on-line analysis system is described with reference to applications in bioprocess control, clinical, and environmental analysis. The new system is built around three main elements: ultrasound filtration, a micro flow injection analysis (FIA) system, and direct readout spectrophotometry. The advantages of three on-line ultrasound filtration devices, developed for clarification of water, human blood and mammalian cell culture samples, are described. The filters avoid off-line centrifugation and do not suffer from the blockage problems associated with conventional filters. The separation efficiency of the ultrasound filters is also discussed. The delivery system is based on a gas driven FIA technique, using helium to avoid bubble formation, with the carrier and reagent flow being controlled by computer switched solenoid valves. Direct readout spectrometers are used, based on charge coupled devices (CCDs) covering the wavelength range 200-900 nm. These detectors provide near instantaneous capture of full spectra, allowing several analytes to be monitored simultaneously, and are much smaller than conventional spectrophotometers. Optical fibres are used to link the light source to the detector via a flow cell in the FIA system. Software to run the entire system was developed using the LabVIEWtrade mark graphical programming environment, enabling rapid development of the control system and user interface. The integration of these components has shown significant improvement in the application of FIA techniques to on-line analysis.

A visual programming environment for bioprocess control.

The paper introduces the use of a visual programming environment (LabVIEW) to program custom control functions for bioprocess research. The time taken for a bioprocess scientist to program new functions compared well with typical times expected for experienced programmers using conventional languages. Experienced LabVIEW programmers will develop applications significantly faster. The package described was flexible, easy to use and was ideally suited to developing new applications for control of bioprocesses. It was demonstrated with the development of a system to control specific growth rate in a fed-batch culture.

Orthodontic treatment need and the dental aesthetic index.

The Dental Aesthetic (DAI) was devised as a measure of dental appearance and based on lay opinions as opposed to professional assessments of need. The DAI is calculated from the weighted scores of ten occlusal variables. These same variables have been used in other malocclusion indices intended to measure morphological deviations from normality. It is, therefore, important to ask if the DAI might be used to screen both deviations of dental appearance and deviations of occlusion from accepted norms. The original DAI design was based on analysis data from permanent dentitions. However, a large percentage of orthodontic assessments are carried out in the mixed dentition. Therefore, the present study includes a sample of mixed dentitions to test the effectiveness of the DAI in this group. The DAI was tested against the subjective assessments of severity made by orthodontists. Sensitivity and negative predictive power were high. Performance in the mixed dentition group was slightly lower than the permanent dentition group. It is concluded that while the DAI is not sufficiently reliable for assessment of treatment need in the individual, its sensitivity and negative predictive power might provide a useful screening mechanism which reflects both the concerns of patients regarding appearance and the general treatment recommendations of orthodontists. Testing on much larger samples is now required.