Evaluation and application of a high performance liquid chromatographic method for prilocaine analysis in human plasma.

BACKGROUND: Prilocaine, a local anesthetic of the amide type, is frequently applied in substantial doses during tumescent liposuction. Although it cannot be excluded that the subcutaneously infiltrated narcotic may enter the circulation and trigger adverse systemic reactions, prilocaine plasma levels have rarely been measured during routine tumescent surgery. We established and evaluated a high performance liquid chromatography (HPLC) method for analysis of this narcotic and used it to measure the drug in plasma samples drawn in the course of tumescent liposuction with prilocaine local anesthesia. METHODS: After approval by the local ethics committee and written informed consent, 283 heparin plasma samples were collected from 132 patients during and about 6, 12, and 24 hours after tumescent liposuction with prilocaine infused at doses of 19 +/- 5 mg/kg body weight. Calibrators and controls were prepared by spiking blank plasma with prilocaine. Following addition of internal standard and sodium hydroxide, plasma was extracted with diisopropyl ether. For HPLC analysis, dried extracts were dissolved in methanol - 4.35 mmol/L ammonium phosphate, pH7.0, (60:40 v/v) and applied to a Synergy 4 microm Fusion-RP column (250 x 4.6 mm) rinsed with the same buffer. Analytes were detected by absorption at 237 nm. For liquid chromatography mass spectrometry (LC-MS), extracts were dissolved in acetonitrile - 2 mmol/L ammonium acetate - formic acid (5:95:0.2 v/v/v), applied to a Synergy 4 microm Polar-RP column (75 x 2 mm), and eluted with a gradient of acetonitrile in 2 mmol/L ammonium acetate - formic acid. Analytes were detected by an ion trap mass spectrometer with electrospray ionization run in a MS/MS mode. RESULTS: In the HPLC assay established, prilocaine and the internal standard lidocaine eluted at about 14 and 25 minutes, respectively. The limit of detection of prilocaine was 0.002 mg/L, the measurable range extended to 30 mg/L. At prilocaine concentrations between 0.08 and 10.0 mg/L, inter-assay coefficients of variation of 6.2 to 9.9% were obtained. Analyses of plasma pools spiked with variable amounts of prilocaine showed recoveries of 91-101%. Results measured in 20 plasma samples by both HPLC and an independent LC-MS assay agreed acceptably (Y(HPLC) = 0.07 + 1.19x(LC-MS), R 0.98). Prilocaine plasma concentrations measured by HPLC in 132 plasma samples drawn in the late phase of liposuction ranged between 0.01 and 32.0 mg/L, roughly one third of all samples exhibiting levels above 5 mg/L. About 6 hours later, prilocaine levels measured in 46 plasma samples were lower (0.13 - 1.56 mg/L) and decreased further in the evening of the operative day (n = 49, 0.10 - 0.62 mg/L) and on the morning of the first postoperative day (n = 55, 0.03 - 0.25 mg/L). CONCLUSIONS: An HPLC method for determination of prilocaine was established and successfully applied to analysis of this drug in human plasma. Our results clearly indicate that during tumescent liposuction a significant portion of the subcutaneously infiltrated prilocaine enters the circulation, resulting in potentially harmful blood levels in about one third of the patients studied. 6 hours after liposuction, however, all samples exhibited prilocaine plasma levels far below a critical concentration and these levels further decreased in the evening of the day of treatment and on the next morning.

Standardization of autoimmune diagnostics in Germany: activities of the German group in the European Autoimmune Standardization Initiative.

The German Regional Group of EASI was established during the annual Meeting of the German Society of Immunology in Kiel in September 2005. Since this initial informative meeting, an active core group of about a dozen rheumatologists, immunologists, and laboratory specialists has been generating starter projects. In general, these projects do focus on clinically associated diagnostic questions, and do integrate a variety of specialists with profound knowledge in several related subjects. The aims of the German EASI group are to contribute to the definition of standards and to improve patient care. Therefore, the group is establishing guidelines for the diagnosis of autoimmune diseases, to standardize and improve their quality, combining the experience of clinical and laboratory specialists. The diagnostic activities focus currently on systemic lupus erythematosus (SLE) and on rheumatoid arthritis. These activities include laboratory investigations and diagnosis through clinical manifestations. Standardized diagnostics cannot be based solely on vague symptoms and positive laboratory tests. In laboratory diagnostics, standardization and implementation of objective methods for the detection of autoantibodies has been identified as a central challenge. Here, immune fluorescence techniques and the evaluation of RibP are used as first parameters that could improve SLE diagnostics. Furthermore, guidelines and proposals from scientific medical organizations, and in particular from other national EASI groups will be adapted to the German health system. A cornerstone of implementation is the identification and logistic preparation of existing serum banks, the definition of gaps that should be bridged, and, particularly, the definition and collection of adequate control groups. Through these measures, the German EASI group will provide a standardized diagnostic model of autoimmune disorders throughout Europe starting in the field of rheumatology. Diagnostics may become more rational, efficient, faster, and cost-efficient. Patients with autoimmune rheumatic disorders will profit from receiving an earlier and more accurate diagnosis, which again will allow earlier therapeutic intervention and lead to a better long-term clinical outcome.

The Abbott Architect c8000: analytical performance and productivity characteristics of a new analyzer applied to general chemistry testing.

Applying basic potentiometric and photometric assays, we evaluated the fully automated random access chemistry analyzer Architect c8000, a new member of the Abbott Architect system family, with respect to both its analytical and operational performance and compared it to an established high-throughput chemistry platform, the Abbott Aeroset. Our results demonstrate that intra- and inter-assay imprecision, inaccuracy, lower limit of detection and linear range of the c8000 generally meet actual requirements of laboratory diagnosis; there were only rare exceptions, e.g. assays for plasma lipase or urine uric acid which apparently need to be improved by additional rinsing of reagent pipettors. Even with plasma exhibiting CK activities as high as 40.000 U/l, sample carryover by the c8000 could not be detected. Comparison of methods run on the c8000 and the Aeroset revealed correlation coefficients of 0.98-1.00; if identical chemistries were applied on both analyzers, slopes of regression lines approached unity. With typical laboratory workloads including 10-20% STAT samples and up to 10% samples with high analyte concentrations demanding dilutional reruns, steady-state throughput numbers of 700 to 800 tests per hour were obtained with the c8000. The system generally responded to STAT orders within 2 minutes yielding analytical STAT order completion times of 5 to 15 minutes depending on the type and number of assays requested per sample. Due to its extended test and sample processing capabilities and highly comfortable software, the c8000 may meet the varying needs of clinical laboratories rather well.

Lamina propria plasma cells in inflammatory bowel disease: intracellular detection of immunoglobulins using flow cytometry.

This is the first application of flow cytometry for the detection of lamina propria plasma cells and their intracellular immunoglobulins in patients with inflammatory bowel disease compared to healthy controls. The study has been focused on the distribution of IgA, IgG, IgM and the four IgG subclasses. Plasma cells were detected as high CD38 positive cells. For fixation and permeabilisation a single step reagent, Ortho Permeafix, was used. By flow cytometry, in patients with inflammatory bowel disease compared to healthy controls, a higher percentage of IgG+ cells can be observed, in Crohn's disease also a higher percentage of IgM+ cells. Regarding the IgG subclass distribution, patients with Crohn's disease show an increase in IgG2+ cells, patients with ulcerative colitis an increase in IgG1+ and IgG3+ cells. These results do agree with and expand the results of earlier immunohistochemical and functional studies, which are favoured today. For the determination of lymphocyte subset proportions and the detection of intracellular antigens, flow cytometry provides a useful alternative to well-established immunohistochemical methods. By analysing a larger number of cells, this method is more reproducible and less prone to interobserver variations than immunohistochemistry, which needs the pre-selection of a mucosal area, the microscopic scoring of a limited number of cells and the circumvention of high background staining. The optimized flow cytometric protocol used in this study might be a promising tool for further investigations of various purposes.

Evaluation of new oxidation methods for the measurement of bilirubin on the aeroset clinical chemistry analyzer and comparison with methods on the Hitachi 717.

We evaluated analytical and performance quality of the new oxidation methods for direct and total bilirubin on the Abbott Aeroset clinical chemistry analyzer. Within-day imprecisions for Abbott Aeroset assays ranged from 0.7 to 2.9% and between-day imprecisions from 2.1 to 7.3%. Inaccuracies as compared with the control "target values" for the Jendrassik-Gróf method showed deviations of -18.2 to +4.2%. Limits of detection were determined and showed very low values of < or = 0.25 micromol/l and dilution linearities were confirmed up to > 300 micromol/l. A method comparison for 100 patient samples with established Jendrassik-Gróf and DPD methods on the Roche Hitachi 717 showed good linearities between the investigated methods (r > or = 0.995). Due to slopes that ranged from 0.829 to 0.950, reference ranges for the oxidation methods differ slightly from those of established Roche Jendrassik-Gróf methods, but results can be adapted by the introduction of converting factors. In conclusion, the oxidation bilirubin assays revealed convincing analytical and performance qualities for medical needs that were similar or even better than for established methods. Application of the oxidation methods on the Aeroset clinical chemistry analyzer also improves laboratory efficiency by increasing throughput, speed of obtaining results and lowered sample and reagent volumes compared to established methods.

Evaluation of two Mg2+-selective electrodes by means of a flow-through device.

Monitoring the ionized magnesium (Mg2+) concentration in critically ill patients can prevent development of serious and potentially fatal complications. The analyzers KONE Microlyte 6 (KONE Instruments, Espoo, Finland) and NOVA CRT (NOVA Biomedical, Waltham, MA, USA) provide the discontinuous measurement of Mg2+ and were evaluated in several studies. It was our objective to integrate the Mg2+-selective electrodes into a device for continuous on-line measurements. This device is suitable not only for research but also for a specific evaluation of electrode characteristics. It allowed us to compare the genuine electrodes and reference systems independently of their specific analyzers. Precision, accuracy, response time, limit of detection, drift and interferences were assessed by continuous flow-through measurements and discussed in comparison to the results of previous studies. The NOVA electrode proved to be superior regarding accuracy, sensitivity and selectivity, especially with respect to calcium. It was demonstrated that current commercial serum-like control materials were not appropriate for quality control of the assessed Mg2+-electrodes. However, despite the fact that the electrodes are commercially used for discontinuous measurements, both sensor types can be used for continuous on-line measurements in an extracorporeal circulation in a rat model. The NOVA electrode showed superior characteristics with this application as well. This study should also be understood as a contribution to the development of devices for online analyzers used in point-of-care-testing.

Immunity against diphtheria and tetanus in German blood donors.

After the recent diphtheria epidemics in Eastern Europe in the early 1990s, we re-evaluated the diphtheria and tetanus immunity of 321 German blood donors (192 men and 129 women). The mean antitoxin levels of all blood donors in this study, measured by commercial ELISA, revealed a questionable protection (0.1-1.0 IU/ml) against diphtheria. In 1994, 66.4% were without immunity against diphtheria (55.0% in 1997/98), 32.1% (41.5% in 1997/98) showed questionable protection and only 1.5% (3.5% in 1997/98) had protective antitoxin levels. The evaluation of tetanus immunity revealed only 0.5% (1.1% in 1997/98) of the subjects with no protection and 9.1% (8.5% in 1997/98) with questionable protection. For this reason, we conclude that the diphtheria epidemics only lead to an insufficient improvement of the immunization status in a healthy German population.

Determinación de la concentración de lisoenzima en saliva: variaciones con la edad / Determination of lysozyme concentration in saliva variation with age

Se obtuvieron muestras de saliva de 59 personas sanas en edades comprendidas entre 8 y 24 años. La determinación de la concentración de lisozima se efectuó por los métodos microbiológicos e inmunológico, de forma absoluta en el primero y de forma relativa en el segundo. El coeficiente de corelación entre ambos métodos fue de r=0,752. No hubo diferencia significativa en la concentración de lisozima entre los diferentes grupos de edades en ambos métodos. Las variaciones intraclase fueron marcadas, pero se observó una tendencia a la disminución de las variaciones con el aumento de la edad. Los resultados obtenidos muestran que es dificil, si se parte sólo de la valoración de la concentración de lisozima, llegar a conclusiones sobre el estado de inmunidad local en la cavidad bucal, sobre todo en la infancia, donde ocurren grandes variaciones individuales