Roche Diagnostics GmbH Research Articles

Effects of microdialysis catheter insertion into the subcutaneous adipose tissue assessed by the SCGM1 system.

To monitor glucose in patients with diabetes continuously a microdialysis-based glucose sensor system (SCGM1 System, Roche Diagnostics GmbH, Mannheim, Germany) is under clinical development. This system allows monitoring of glucose levels in the subcutaneous interstitial fluid of patients with diabetes for a maximum duration of up to 120 h. The aim of the study was to determine the effect of microdialysis catheter insertion on the stability of the SCGM1 System glucose sensor signal. At four study sites, 47 experiments with the prototype of the novel SCGM1 System were performed in 42 patients with type 1 diabetes; two additional experiments were performed in two healthy volunteers. The microdialysis catheter was inserted in the subcutaneous adipose tissue of the patients in order to measure the glucose concentration in the interstitial fluid continuously. The catheter was perfused with a pump rate of 0.3 microL/min. For method comparison capillary blood glucose measurements were performed as reference values. In addition, the skinfold thickness was measured. Out of the total of 49 experiments 34 were usable. The average monitoring time in these experiments was 106.0 +/- 14.3 h (mean +/- SD). However, for this study the data from the first study day were evaluated in more detail. The analysis showed that during the first 12 h after catheter insertion the sensor signal increased 20% in comparison with the capillary blood glucose values (normalized calibration factor). This leads to a lower normalized calibration factor compared with the following study days. It remains stable in the time thereafter. The skinfold thickness showed no significant effect on the sensor signal. The observed increase in sensor signal in the first hours after insertion of the microdialysis catheter was probably due to a local trauma, which can induce an inflammation reaction. Thereafter, the signal registered by the SCGM1 System was stable and free of drift to the end of the experiment.

Continuous glucose monitoring: reliable measurements for up to 4 days with the SCGM1 system.

Continuous glucose monitoring allows patients with diabetes to check their metabolic status throughout the day, including rarely monitored time periods, such as postprandial and nocturnal periods. The performance of a prototype of the novel SCGM1 System (Roche Diagnostics GmbH, Mannheim, Germany) employing the microdialysis technique was evaluated. Forty-two patients with type 1 diabetes participated in the study [29 males/13 females, age 34 +/- 9 years, duration of diabetes 16 +/- 11 years, glycated hemoglobin 7.7 +/- 1.2% (mean +/- SD)]. Forty-seven experiments were performed at four different investigational sites. A microdialysis catheter was inserted into the subcutaneous tissue of the patient's abdominal wall. Glucose was extracted from the interstitial fluid, and glucose levels were determined and stored by the SCGM1 System continuously for the duration of the experiment. Capillary blood glucose was measured frequently (at least 10 times per day) and used for linear retrospective calibration. The patients were instructed to maintain their normal diet and insulin therapy. Thirty experiments (mean duration 103 +/- 18 h) were analyzed in detail. The mean deviation of the calibrated glucose sensor values from the capillary blood glucose values (expressed as percent predicted error sum of squares) was <12.5% in 25 and <20% for all of the 30 experiments analyzed. The percent median absolute difference between the calibrated values and the reference values was <10% in 28 experiments, with a median of 5.8% for all 30 experiments. The error grid analysis of all 30 experiments showed that 99.5% of all 1,195 pairs' values were in zones A and B with only 0.2% in zone C and 0.3% in zone D. Thus the microdialysis technique employed by the SCGM1 System allows precise and accurate continuous glucose monitoring over prolonged periods of time. It appears also that effective monitoring of acute metabolic deteriorations is possible.

Variation of the Hypervariable Region-1 of Mitochondrial DNA in Central-Eastern Italy

Abstract DNA samples were extracted from fresh blood (5 mL in EDTA tube) by alkali bursting of blood cells followed by protein digestion with proteinase K. After a phenol-chloroform extraction, DNA was precipitated with absolute ethanol and dried at room temperature. The DNA pellet was diluted in 100 μL of TBE buffer (Tris HCl, 10mM, pH 7.5; EDTA 1mM). PCR amplification was carried out by using a Perkin Elmer 2400 thermal cycler in 25 μL reaction volume. The product of amplification was purified with a High Pure Product Purification Kit (Roche Diagnostics GmbH, Mannheim, Germany). The sequencing reactions were performed separately on each strand with the primers L15990 and H16401. The hypervariable region-1 (HVR1) sequence analysis was carried out using the A.L.F. semi-automated sequencer (Pharmacia Biotech, Uppsala, Sweden). We observed a total of 44 different haplotypes out of 50 individuals examined, 41 of which were singletons. Only 8 haplotypes (no. 09, 13, 29, 39, 40, 42, 44, and 50) have been already observed among the Italian populations database available at www.hvrbase.de. The parameters of intrapopulation diversity (haplotype diversity 0.990 ± 0.008; mean number of pairwise differences 4.979 ± 2.463) fall into the range of values reported for Italian populations (1). The values of Tajima (1,944, p = 0.015) and Fu's tests (-25.552, p = 0.00) are those expected for populations who have had a large and sudden expansion (2). Applying an exact test for population comparisons implemented in the Arlequin software (3), we obtained statistically significant p-values for the comparison between our sample and those from Tuscany (0.004) Sardinia (0.039). Complete data are available at the e-mail address of the corresponding author upon request.

Heterophilic Antibody Interference with CARDIAC T Quantitative Rapid Assay

Point-of-care troponin assays are promoted as providing rapid, reliable results to support best practice management of acute chest pain (1). We report a case of false-positive cardiac troponin T (cTnT) results that caused inappropriate clinical management. A 46-year-old man consulted his general practitioner after 10 h of central chest pain and tingling in the left arm after a heavy fall. An electrocardiogram showed possible ST elevation. A repeat electrocardiogram in the local hospital showed sinus rhythm and an early repolarization pattern. Whole-blood cTnT (lithium heparinate) was increased both on admission and the next day in a CARDIAC T Quantitative Rapid Assay (third generation; Roche Diagnostics). The patient was a moderately heavy smoker, was mildly hypercholesterolemic, and had a family history of ischemic heart disease. The patient was transferred to the tertiary hospital where a coronary angiogram and plasma (lithium heparinate) cTnT (T STAT, third generation; Roche Diagnostics) were normal (Table 1⇓ ). Creatine kinase and lactate dehydrogenase concentrations in the two samples taken at the local hospital and two samples taken at the tertiary hospital were within the appropriate reference intervals (data not shown). The patient was diagnosed with presumed musculoskeletal chest pain. View this table: Table 1. cTnT results obtained by the CARDIAC T Quantitative Rapid and Troponin T STAT methods. The CARDIAC T sandwich assay uses a test strip and two murine monoclonal antibodies to human cTnT. A reader records the intensity of the reflectance … [↵][1]bAddress correspondence to this author at: Roche Diagnostics GmbH, Evaluation, Primary and Cardiac Care (Department DD-EN), Sandhofer Strasse 116, D-68163 Mannheim, Germany. [1]: #xref-corresp-2-1

Patent Law. Claim Construction. U. S. District Court for the District of Massachusetts Finds Conflict on Federal Circuit and Holds That Product-by-Process Claims Are Not Limited by Their Process Steps. Trustees of Columbia University v. Roche Diagnostics GmbH, 126 F. Supp. 2d 16 (D. Mass. 2000)

Patent Law. Claim Construction. U. S. District Court for the District of Massachusetts Finds Conflict on Federal Circuit and Holds That Product-by-Process Claims Are Not Limited by Their Process Steps. Trustees of Columbia University v. Roche Diagnostics GmbH, 126 F. Supp. 2d 16 (D. Mass. 2000)

Evaluation of the Analytical and Clinical Performance of the Elecsys® CA 15-3 Immunoassay

Although the sensitivity and specificity of CA 15-3 in primary diagnosis of breast cancer is limited, CA 15-3 measurement has found widespread use in monitoring disease progression and response to therapy [reviewed in Ref. (1)]. A new assay for determination of CA 15-3 on Elecsys® immunoanalyzers has been introduced by Roche Diagnostics GmbH. The analytical performance of this assay was tested in an international multicenter evaluation in 11 evaluation sites, and the clinical performance was judged in 4 of these laboratories. The results are described in this report. Elecsys CA 15-3 is a sandwich immunoassay that uses biotinylated 115D8 and ruthenylated DF3 as capture and detection antibodies, respectively, and electrochemiluminescence as the detection technology. Details of the test principle have been described previously (2). The test is standardized against the Enzymun-Test CA 15-3 (Roche Diagnostics GmbH) in the laboratories of the manufacturer. The analytical evaluation was carried out according to a standardized protocol of the European Committee for Clinical Laboratory Standards (3). The results of within-run imprecision (21 replicates per analysis) and between-day imprecision (1 replicate analyzed on 21 different days) studies are available as a data supplement at Clinical Chemistry Online (http://www.clinchem.org/content/vol47/issue12). The medians of the within-run and between-day CVs were 2.5–3.8% and 5.0–5.9%, respectively, at all concentrations tested (8.4–251.7 kilounits/L) for human sera and control sera. Confirmation of target values assigned to control sera was tested in an interlaboratory survey and was 97% for PreciControl Tumor Marker Level 1 (18.4 kilounits/L) and 96% for Level 2 (58.6 kilounits/L). The medians of all participants of an interlaboratory survey of the German Society for Clinical Chemistry were confirmed for each of the two control samples used (11.8 and 16.8 kilounits/L) by 99% (on the basis of the medians of all participants in the Elecsys study); thereby demonstrating the excellent agreement …

New concepts in systemic autoimmunity testing

Diagnosis of systemic autoimmune diseases is highly complex, and it is becoming increasingly difficult to make assumptions about the functional roles and diagnostic significance of autoantibodies. The latter is mainly due to the fact that results from different assay systems are not interchangeable. A laboratory "gold standard" which helps the clinician to differentiate irrelevant autoimmune phenomena from significant autoimmune diseases at an early stage, is clearly missed. To meet this challenge, a rheuma entrance screening (RES) assay toolbox is proposed based on fully-automated enzyme immunoassay (EIA) technology on one system for the clinical and routine laboratory. The RES concept is intended to cover the most important syndromes of systemic rheumatic diseases, i.e. collagenosis, early rheumatoid arthritis, early osteoarthritis, anti-phospholipid syndrome and inflammation. The serological part of diagnosis of these diseases comprises testing for anti-nuclear antibodies (ANA), rheumatoid factor (RF), low levels of C-reactive protein (CRP), and disease-specific anti-phospholipid antibodies, e.g. anti-beta-2 glycoprotein I (anti- g 2 GPI). To eliminate the known problems of varying assay systems in this field, a novel, objective, rapid and reproducible approach to screen for such analytes in patient serum or plasma more efficiently is the application of EIAs on the fully-automated immunoassay analyser COBAS ® CORE (Roche Diagnostics GmbH, Mannheim, Germany). The combined use of the RES (COBAS ® CORE HEp2 ANA EIA, COBAS ® CORE RF EIA Quant, COBAS ® CORE CRP EIA Quant and COBAS ® CORE Anti- g 2 GPI EIA) is intended for patients sent to the laboratory with the primary suspicion of harbouring a systemic rheumatic disease.

TIF-IA, the factor mediating growth-dependent control of ribosomal RNA synthesis, is the mammalian homolog of yeast Rrn3p.

Cells carefully modulate the rate of rRNA transcription in order to prevent an overinvestment in ribosome synthesis under less favorable nutritional conditions. In mammals, growth-dependent regulation of RNA polymerase I (Pol I) transcription is mediated by TIF-IA, an essential initiation factor that is active in extracts from growing but not starved or cycloheximide-treated mammalian cells. Here we report the molecular cloning and functional characterization of recombinant TIF-IA, which turns out to be the mammalian homolog of the yeast factor Rrn3p. We demonstrate that TIF-IA interacts with Pol I in the absence of template DNA, augments Pol I transcription in vivo and rescues transcription in extracts from growth-arrested cells in vitro.

Observer Dependency of the SimpliRed D-dimer Assay in 81 Consecutive Patients with Suspected Pulmonary Embolism

The SimpliRed D-dimer assay is a whole blood agglutination test for the exclusion of pulmonary embolism (PE) and has been advocated as a reliable rapid bedside alternative to more time-consuming quantitative assays. However, widely differing negative predictive values (77–100%) are reported. This study assessed the intra- and interobserver variability as a possible cause for this wide accuracy range and evaluated the accuracy of the SimpliRed test in 81 consecutive patients with clinically suspected PE. Absolute D-dimer concentration was measured using the Tinaquant (Roche Diagnostics GmbH, Mannheim, Germany) assay. Every assay was immediately scored by one observer and then photographed. Photos were randomised and read twice by two other independent observers. The intraobserver reproducibility was good: kappa=0.81 and 0.73. However, we found poor interobserver reproducibility with kappa= 0.47. This seems due to the fact that the SimpliRed test proved difficult to interpret in the 0.3–1.3 μg/mL plasma concentration D-dimer range, which contained 32/81 (40%) patients, 8 of whom had PE. Using pulmonary angiograms and ventilation perfusion lung scans with a concurrent spiral computed tomography scan as a gold standard (25 patients had PE), SimpliRed's accuracy to exclude PE was moderate, with negative predictive values of 0.78 and 0.84. In particular, the two observers scored the SimpliRed test as normal in 12 of 25 and 5 of 25 patients with proven PE. We conclude that SimpliRed is strongly observer-dependent. This explains the varying sensitivities for the detection of venous thromboembolism as reported in previous studies.