Cobas Bio Centrifugal Analyzer Research Articles

Adaptation of coenzyme stimulation assays for the nutritional assessment of vitamins B1, B2 and B6 using the Cobas Bio centrifugal analyser.

Adaptation of coenzyme stimulation assays for the nutritional assessment of thiamine, riboflavin and pyridoxine on the Cobas Bio centrifugal analyser are described. Whole blood was collected into acid-citrate dextrose, which preserves the erythrocytes, prior to assay for several days. Washed erythrocytes stored at -70 degrees C and subsequently thawed, showed altered enzyme activities. The methods offer improved precision over existing procedures and take advantage of the high throughput capabilities of the instrumentation.

Kinetic enzymatic method for automated determination of HDL cholesterol in plasma.

A sensitive fixed-time kinetic enzymatic method for the measurement of high density lipoprotein cholesterol is described, as adapted for the Cobas Bio centrifugal analyser (Hoffmann LaRoche). The Boehringer Mannheim cholesterol esterase/cholesterol oxidase/peroxidase/3,4-dichlorophenol kinetic reagent was modified by the inclusion of 2,4,6-tribromo-3-hydroxybenzoic acid which reacts with hydrogen peroxide and 4-aminophenazone to produce a quinone-imine dye with a greater molar absorptivity than that produced with phenol. The method has been developed for the determination of HDL fractions isolated with polyethylene glycol 6000, for which a reagent of high sensitivity is required. The method is linear to at least 3.88 mmol/l of HDL cholesterol and the coefficients of variation for within-run and day-to-day precision were less than 3.0%. Correlation of the kinetic HDL cholesterol method with an equilibrium method was good (R = 0.9980). The assay is rapid, inexpensive and large numbers of specimens can be processed conveniently.

Screening of factor VIII:C levels in blood donors.

A new chromogenic peptide substrate method, modified for assay with semi-micro tubes, Cobas Bio centrifugal analyser and microplates, was used for screening of F VIII:C in blood donors. The precision of the assays is high and the costs are reasonable. The assay with microplates is especially suitable for selection of donors for a plasma programme and for quality control in blood banks.

Quality assessment of determinations of serum fructosamine in 33 clinical chemistry laboratories.

We investigated the analytical performance of fructosamine measurement in 33 routine clinical chemistry laboratories. We prepared 12 human control sera spanning a range of nondiabetic and diabetic fructosamine concentrations and assigned target values by using commercial reagents and a Cobas Bio centrifugal analyzer. Each control serum was analyzed by participating laboratories on two separate occasions. Averaged results were compared with target values, and systematic error was determined from slope and intercept regression coefficients. Overall, we found no significant (p greater than 0.05) slope or intercept bias, although slight nonlinearity was found with bichromatic instruments. Random error was determined from the standard error of estimate and from duplicate measurements. We found a median interlaboratory CV of 5.36% and similar analytical performance with all automated instruments used. The median random error with manual techniques was 18.1%. We conclude that fructosamine measurement as currently performed in routine laboratories is sufficiently accurate and reliable to function as an index of blood glucose control in diabetic patients.

Immunonephelometric measurement of urinary albumin by a Cobas Bio centrifugal analyzer with fluorescence attachment.

Immunonephelometric measurement of urinary albumin by a Cobas Bio centrifugal analyzer with fluorescence attachment.

Immunoturbidimetric techniques for quantifying apolipoproteins CII and CIII.

Immunoturbidimetric assays for determination of serum apolipoproteins CII and CIII (apo CII and CIII) were developed for use on the Cobas-Bio centrifugal analyzer with commercially available antisera and reference sera. Within-run and between-run imprecision (CVs) for apo CII was respectively 3.3 and 6.2%, and for apo CIII 2.75 and 5.2%. Addition to sera of as much as 50 g of hemoglobin or 0.15 g of bilirubin per liter did not interfere with determinations of apolipoproteins. Concentrations of apo CII and CIII were measured in sera from 40 normolipidemic and hyperlipidemic subjects by these immunoturbidimetric methods (x) and by radial immunodiffusion assays (y). Apo CII and CIII gave slopes of 0.93 and 0.86, y-intercepts of 7.4 and 9.5 mg/L, and correlation coefficients of 0.96 and 0.94, respectively. Reference intervals for the two apolipoproteins were established with sera from 100 normolipidemic subjects. The distribution of apo CII and CIII among the various lipoprotein fractions for normal subjects differed significantly (p less than 0.005) from that for hypertriglyceridemic subjects.

Quantitative method for determining serum alkaline phosphatase isoenzyme activity I. Guanidine hydrochloride: new reagent for selectively inhibiting major serum isoenzymes of alkaline phosphatase.

The potential use of the protein denaturant guanidine hydrochloride to inhibit selectively the enzyme activity of serum alkaline phosphatase isoenzymes from liver, bone, intestine, and placenta was investigated. Inhibition of each isoenzyme was shown to be dependent on time and concentration of inhibitor. In the presence of 0.3 mol/l (28.7 g/l) guanidine hydrochloride for 170 seconds 14%, 47%, and 90% of the total alkaline phosphatase activity remained in samples of bone, liver, and intestinal origins, respectively. In contrast, the activity of the placental isoenzyme increased by 24%. The degree of inhibition was shown to be independent of total alkaline phosphatase activity. Investigations were performed at 37 degrees C using the Cobas Bio centrifugal analyser. We conclude that this reagent has several practical advantages over urea as a selective inhibitor of alkaline phosphatase isoenzymes, including a faster and more reproducible inhibition at a much lower reagent concentration.

Comparison of the Amicon Centrifree Micropartition System with the Sartorius SM 13249E Centrisart I Device to Determine Protein Free Phenytoin Concentrations

The Amicon Centrifree micropartition system was compared with the Sartorius SM 13249E Centrisart I ultrafiltration device to determine non-protein-bound phenytoin levels. Phenytoin was measured by an enzyme multiplied immunoassay technique (EMIT), adapted to a Cobas Bio centrifugal analyzer, and by a radiometric assay. From pooled human plasma with total phenytoin concentrations of 19.1, 44.0, and 95.1 microM, the following percentages of free phenytoin levels in the ultrafiltrate were obtained with Amicon: 10.6 +/- 1.3, 10.5 +/- 0.4, and 8.8 +/- 0.2. The corresponding figures with Sartorius were 5.4 +/- 0.8, 5.4 +/- 0.5, and 4.9 +/- 0.4, respectively (n = 5). Similar results were obtained with 3H-labeled phenytoin, ruling out the possibility that the discrepancies were due to interference with the EMIT assay. Phenytoin binding to the Amicon membrane was less than or equal to 5% of the free phenytoin concentration, whereas the Sartorius membranes absorbed 80%. With the Amicon device, the free phenytoin concentration remained constant as serial ultrafiltrate volumes from the same sample were collected, whereas with Sartorius, free phenytoin increased from 2.5 +/- 1.8% of the total concentration in the first fraction to 13.4 +/- 1.6% in the last collection (n = 6). The results indicate that the Amicon Centrifree filter is an excellent tool to obtain protein-free phenytoin ultrafiltrates, whereas the Sartorius Centrisart I device is not suited for this purpose because of excessive adsorption of phenytoin.

Immunoturbidimetric assays of apolipoproteins A, AI, AII, and B in serum.

In these immunoturbidimetric assays developed for apolipoproteins A, AI, AII, and B we used the Cobas-Bio centrifugal analyzer and commercially available antisera and calibrators. Within-day and within-run precision ranged from 1.0 to 5.8% for the four assays. The addition of bilirubin, up to 150 mg/L, or hemoglobin, 50 g/L, to sera did not interfere with the measurement of these apolipoproteins. Comparison of results of the immunoturbidimetric assays with those of radial immunodiffusion assays yielded slopes of 1.002, 0.822, and 0.957 and intercepts of -0.02, 0.47, and 0.03 g/L for apolipoproteins B, A, and AI, respectively. Reference intervals for the four apolipoproteins were determined by using sera from 193 apparently healthy individuals.

Spectrophotometry of carnitine in biological fluids and tissue with a Cobas Bio centrifugal analyzer.

A spectrophotometric method for carnitine has been adapted to the Cobas Bio centrifugal analyzer. The addition of carnitine to a system containing carnitine acetyltransferase (EC 2.3.1.7) and acetyl-CoA gives rise to the formation of CoA. The system is coupled to 5,5'-dithiobis(2-nitrobenzoate) (DTNB). Assay response varied linearly with concentration of carnitine over a wide concentration range. The total CV was 5.5% for a carnitine concentration in serum of 58.0 mumol/L. Analytical recovery of carnitine added to a serum sample was 93%. No interference was found in icteric, not grossly hemolyzed, lipemic, or uremic sera. Comparison with a radioenzymatic method showed that results correlated well (r greater than 0.965) but the present method gave values proportionally greater by 10 to 25% for samples of plasma, dialysis fluid, urine, and muscle tissue. Advantages over the original spectrophotometric assays involving DTNB include low reagent costs, rapidity, simplicity, and reproducibility. However, this modification is not as sensitive and probably not as specific as the radioenzymatic methods.

Optimized calcium/phosphorus solubility in a parenteral nutrition solution containing dicarboxylic amino acids and cysteine.

The solubility of calcium and phosphorus was studied in neonatal parenteral nutrition solutions containing dicarboxylic amino acids and cysteine. Experimental amino acid solutions containing aspartic acid, glutamic acid, and cysteine in concentrations from 0.5% to 2.0% were studied with dextrose concentrations of 5-20% plus standard electrolyte, vitamin, and trace element additives. Solutions were held at room temperature for 24 hr prior to incubation in a 37 degrees C water bath for 30 min. The pH of each solution was determined. Precipitation was detected by light scattering on a Cobas Bio centrifugal analyzer. An absorbance greater than 0.015 at 600 nm was considered evidence of precipitation. The pH of test solutions ranged between 5.7 and 6.4. Increasing amino acid concentration produced an improvement in calcium/phosphate solubility. Change in dextrose concentration had a lesser effect. Solutions of 10% dextrose with 2% amino acids contained 40 mEq/liter calcium and 17 mmol/liter phosphorus without precipitation. Administered at 150 ml/kg/day, such a solution would provide 120 mg/kg body weight/day calcium and 80 mg/kg/day phosphorus, approximately the daily in utero accretion rates during the last trimester. This is not readily achieved in comparable solutions of previous amino acid formulations.

Screening of Factor VIII :C Levels in Blood Donors

A new chromogenic peptide substrate method, modified for assay with semi-micro tubes, Cobas Bio centrifugal analyser and microplates, was used for screening of F VIII : C in blood donors. The precision of the assays is high and the costs are reasonable. The assay with microplates is especially suitable for selection of donors for a plasma programme and for quality control in blood banks.

Turbidimetry of rheumatoid factor in serum with a centrifugal analyzer.

We describe the simple, rapid turbidimetry of IgM rheumatoid factor in human serum by use of the Cobas-Bio centrifugal analyzer. Heat-aggregated human IgG is used as the antigen. The immunoturbidimetric reaction is monitored at 340 nm for 300 s, and the changes in absorbance after the antigen is added are used to prepare the standard curve. Test results are calculated from the stored curve and reported in int. units/mL, based on comparison with the WHO reference serum for rheumatoid factor. There is no interference from bilirubin (up to 340 mumol/L) or hemoglobin (up to 5600 mg/L). Serum samples with a triglyceride concentration greater than 2.20 mmol/L must be cleared of lipids before analysis. The standard curve is linear from 30 to 500 int. units/mL. Precision, accuracy, linearity, and sensitivity are quite acceptable. The CV was generally less than 5% for different concentrations of rheumatoid factor. Results agree well with those by a rate-nephelometric procedure on the Beckman ICS system (rs = 0.932). However, both correlate poorly with a modified classical Waaler-Rose test. Of 47 patients with rheumatoid arthritis, 34 had IgM rheumatoid factor in their serum, but the measured value did not reflect the activity of the disease.

A semiautomated procedure for urinary D-glucaric acid using a centrifugal analyzer.

The most commonly used method for quantifying D-glucaric acid, the enzyme inhibition method, is a two-step procedure. In the first step of the present study, urine was boiled at acidic pH to convert glucaric acid to 1,4-glucarolactone, which inhibits beta-glucuronidase activity. In the second step, inhibition of beta-glucuronidase by the prepared urine was measured. This is an indirect measure of the D-glucaric acid concentration. The second step was adapted to a Cobas-Bio centrifugal analyzer. Values on a reference population obtained by the manual method were highly correlated with values obtained by the semiautomated method described here (r = 0.976, p = 0.25), and precision was comparable by the two methods. The throughput increased from 8 to 20 samples per day when the automated method was used.

Development of a photometric assay for activated partial thromboplastin time and its application to the cobas (R) biocentrifugal analyzer

We describe a two-step procedure for APTT that can be performed on photometric devices. It includes preincubation of diluted plasma with ellagic acid and phospholipids and a starting reagent that contains calcium and a chromogenic peptide substrate for thrombin, Tos-Gly-Pro-Arg-pNA. Reaction time is recorded from addition of the starting reagent until thrombin formation occurs, and a prefixed amount of substrate is cleaved. The pattern of sensitivity to clotting factors and heparin was similar to clotting assays and the substrate used did not interfere with the activity of factor Xa. An application of the method was made for the Cobas (R) Bio centrifugal analyzer. Absorbance readings were sent to an external computer and were transformed into reaction times by a computer program. Although the results are independent on fibrinogen concentrations, from kinetic data of the reaction curve fibrinogen concentrations can be estimated. Correlation studies showed good correspondence to clotting methods (r = 0.92, n = 53) as well as an excellent precision (CV 3% for inter-assays, n = 15) and high throughput of samples (>100/h) in the automated assay.

Effect of heat treatment on results for biochemical analysis of plasma and serum.

Incubation of serum and plasma samples at 56 degrees C for 30 min inactivates the HTLV-III virus. We assessed the effect of this procedure on results of routine biochemical tests by dividing samples of serum and plasma into two, heat-treating one while the other remained at room temperature. Samples were then analyzed in an SMA 16/60, an Astra-8, an Analox glucose analyzer, a Cobas Bio centrifugal analyzer, and manually for salicylate and acetaminophen (paracetamol). Most of the differences produced by heat treatment were not clinically significant, although heated samples proved unsuitable for use in assay of some commonly measured enzymes. Serum evidently is preferable to plasma for this procedure, and heat-treated serum samples can validly be used for most routine analyses. Thus this procedure makes safer the analysis of samples from patients with suspected or proven acquired immune deficiency syndrome (AIDS).

A rapid, simple method for the micro-estimation of purine nucleoside phosphorylase activity in peripheral blood lymphocytes

Giblett and coworkers showed that a congenital deficiency of the enzyme purine nucleoside phosphorylase (PNP), which catalyses the reversible conversion of inosine to hypoxanthine, resulted in depressed T-cell but normal B-cell immunity [l]. This enzyme has since been shown to play a key role in the maturation of lymphocytes [2] and is used as a non-membrane biochemical marker in lymphoid malignancies [3]. Most PNP assays are compfex and involve the use of radioactive isotopes and chromatographic separation of product(s) and substrate. In this manuscript a rapid, simple spectrophotometric method for PNP estimation in lymphocytes using a Cobas-Bio centrifugal analyzer is described and compared with a radioisotopic assay. The spectrophotometric assay measures the PNP catalyzed conversion of inosine to hypoxanthine by estimating the rapid conversion of hypoxanthine to uric acid with xanthine oxidase.

A cost-effective system for performing therapeutic drug assays. I. Optimization of the theophylline assay.

We have developed a cost-effective system for performing drug assays by using the COBAS BIO centrifugal analyzer and EMIT reagents. With the theophylline assay as a model, we were able to optimize both instrument and chemical parameters to allow assay of 2470 samples with a 100-test EMIT kit. This decreased current reagent costs from +2.00 per test for the Syva manual method to 9 cents per test for our proposed method. Within-run and between-run CVs are typically less than 3% and 6%, respectively.

Benzethonium chloride method for proteins adapted to centrifugal analysis.

A benzethonium chloride method for measuring protein concentrations in several types of body fluids has been adapted to the Cobas Bio centrifugal analyzer. The ability of this analyzer to add two reagents stepwise, measuring absorbances after each addition, is essential for proper blanking of urine specimens. The method has excellent curve stability and precision and yields results that correlate well with those of four other protein methods.

Enzymatic measurement of serum glycerol using a COBAS-BIO centrifugal analyzer

An enzymatic method for the measurement of glycerol using glycerol kinase coupled to pyruvate kinase and lactate dehydrogenase was adapted to a COBAS-BIO centrifugal analyzer. Routine standardization is not required under the optimized conditions since the calculation factor derived from the measurement of pure glycerol standard was found to be identical to the theoretical value. Excellent correlation between the method and the DuPont ACA triglyceride method was obtained. The coefficients of variation for within-run and day-to-day precision were less than 4%. Only 2 microL of serum is required, making this an excellent means for monitoring therapeutic glycerol administration in premature infants with hydrocephalus. The method has the sensitivity to measure glycerol concentrations as low as 0.1 mmol/L. This, together with the rapid analysis time (28 specimens in 10 min), makes it suitable for the correction of glycerol interference in the measurement of serum triglyceride.