Cobas Bio Centrifugal Analyzer Research Articles

Formation of high density lipoproteins containing both apolipoprotein A-I and A-II in the rabbit

Human plasma HDLs are classified on the basis of apolipoprotein composition into those that contain apolipoprotein A-I (apoA-I) without apoA-II [(A-I)HDL] and those containing apoA-I and apoA-II [(A-I/A-II)HDL]. ApoA-I enters the plasma as a component of discoidal particles, which are remodeled into spherical (A-I)HDL by LCAT. ApoA-II is secreted into the plasma either in the lipid-free form or as a component of discoidal high density lipoproteins containing apoA-II without apoA-I [(A-II)HDL]. As discoidal (A-II)HDL are poor substrates for LCAT, they are not converted into spherical (A-II)HDL. This study investigates the fate of apoA-II when it enters the plasma. Lipid-free apoA-II and apoA-II-containing discoidal reconstituted HDL [(A-II)rHDL] were injected intravenously into New Zealand White rabbits, a species that is deficient in apoA-II. In both cases, the apoA-II was rapidly and quantitatively incorporated into spherical (A-I)HDL to form spherical (A-I/A-II)HDL. These particles were comparable in size and composition to the (A-I/A-II)HDL in human plasma. Injection of lipid-free apoA-II and discoidal (A-II)rHDL was also accompanied by triglyceride enrichment of the endogenous (A-I)HDL and VLDL as well as the newly formed (A-I/A-II)HDL. We conclude that, irrespective of the form in which apoA-II enters the plasma, it is rapidly incorporated into spherical HDLs that also contain apoA-I to form (A-I/A-II)HDL.

Evidence that endothelial lipase remodels high density lipoproteins without mediating the dissociation of apolipoprotein A-I

Endothelial lipase (EL) is a triglyceride lipase gene family member that has high phospholipase and low triglyceride lipase activity. The aim of this study was to determine whether the phospholipase activity of EL is sufficient to remodel HDLs into small particles and mediate the dissociation of apolipoprotein A-I (apoA-I). Spherical, reconstituted HDLs (rHDLs) containing apoA-I only [(A-I)rHDLs], apoA-II only [(A-II)rHDLs], or both apoA-I and apoA-II [(A-I/A-II) rHDLs] were prepared. The rHDLs, which contained only cholesteryl esters in their core and POPC on the surface, were incubated with EL. As the rHDLs did not contain triacylglycerol, only the POPC was hydrolyzed. Hydrolysis was greater in the (A-I/A-II)rHDLs than in the (A-I)rHDLs. The (A-II)rHDL phospholipids were not hydrolyzed by EL. EL remodeled the (A-I)rHDLs and (A-I/A-II)rHDLs, but not the (A-II)rHDLs, into smaller particles. The reduction in particle size was related to the amount of phospholipid hydrolysis, with the diameter of the (A-I/A-II)rHDLs decreasing more than that of the (A-I)rHDLs. These changes did not affect the conformation of apoA-I, and neither apoA-I nor apoA-II dissociated from the rHDLs. Comparable results were obtained when human plasma HDLs were incubated with EL. These results establish that the phospholipase activity of EL remodels plasma HDLs and rHDLs into smaller particles without mediating the dissociation of apolipoproteins.

A simple inflammation model that distinguishes between the actions of anti-inflammatory and anti-rheumatic drugs

To determine the effects of anti-inflammatory and anti-rheumatic drugs on paw swelling and changes in plasma levels of acute phase proteins (APPs) during acute inflammation in the rat. Inflammation was induced in rats by the injection of adjuvant and the animals were bled five days later and plasma levels of seromucoid, haptoglobin, caeruloplasmin and albumin were determined spectrophotometrically using a Cobas-bio centrifugal analyzer. The effects of daily administration of a variety of drugs used to treat arthritis were determined on paw swelling and APP levels. Injection of the adjuvant induced a pronounced change in APP levels which correlated with the increase in paw swelling. In general, the NSAIDs tested significantly reduced paw swelling and significantly increased levels of haptoglobin and caeruloplasmin in a dose-related manner. Two dose-levels of steroids were administered, the higher dose reduced swelling, and reduced levels of seromucoid, haptoglobin and caeruloplasmin, but raised albumin levels; the lower dose also reduced paw swelling, but the only change in APPs was increased albumin levels. Anti-rheumatic drugs such as gold salts reduced levels of some APPs (seromucoid, haptoglobin and caeruloplasmin) without reducing paw swelling. Immunomodulators had a variety of effects on inflammation and APPs depending on mechanism of action. The different classes of anti-inflammatory/anti-rheumatic drug tested show distinct profiles of activity against APPs and paw swelling. These differential effects may result from modulation of cytokine activity.

Immunoturbidimetry of serum apolipoproteins A-I and B on the Cobas Bio centrifugal analyzer: Method validation and reference values

We describe an immunoturbidimetric assay for estimating the concentration of apolipoproteins (apo) A-I and B in serum with a Cobas Bio centrifugal analyzer. Several analytical variables were studied: pH, temperature, wavelength, reaction time, sample pretreatment, antisera dilution, concentration of NaCl, phosphate buffer, and PEG 6000; a good optical response was obtained with small volumes of sample and antisera. Imprecision was low, with intrarun coefficient of variance (CV) from 1.1 to 2.8% and interrun CV from 1.8 to 3.2%. We demonstrated a good correlation between our method and radial immunodiffusion for apo A-l ( r = 0.975) and apo B ( r = 0.980). Reference values were calculated from a sample of 494 healthy subjects, and separate intervals for the ratio of apo A-l to apo B in gender and age subgroups were established.

Enzymatic method for assaying calcium in serum and urine with porcine pancreatic alpha-amylase

We developed a kinetic assay for calcium in serum and urine, based on the activation of porcine pancreatic alpha-amylase (EC 3.2.1.1) with 2-chloro-4-nitrophenyl-alpha-maltotrioside as substrate. The kinetic generation of 2-chloro-4-nitrophenol, monitored at 405 nm, was proportional to the concentration of calcium in serum and urine. The assay was developed and evaluated with the Cobas Bio centrifugal analyzer. The average within-run and between-day imprecision (CV) was 0.96/1.26% and 1.07/1.63%, respectively, for serum calcium and 1.50/2.54% and 1.70/2.64%, respectively, for urine calcium. Results of the proposed method (y) correlated well with those determined by atomic absorption spectrophotometry (x): y(serum) = 1.005x + 0.028 mmol/L (Sy/x = 0.058, r = 0.974, n = 50), and y(urine) = 1.017x-0.115 mmol/L Sy/x = 0.30, r = 0.981, n = 25). We also present data showing that the method is highly sensitive, rapid, relatively free of interference, and amenable to automation.

A sensitive determination of uric acid in serum using uricase/catalase/formaldehyde dehydrogenase coupled with formate dehydrogenase

We developed and evaluated an assay for serum uric acid based on the uricase (EC 1.7.3.3)-catalase (EC 1.11.1.6)-formaldehyde dehydrogenase (FADH, EC 1.2.1.46) method coupled with formate dehydrogenase (formate: NAD oxidoreductase, FDH, EC 1.2.1.2). Formate dehydrogenase from Pseudomonas oxalaticus catalyzes the formation of NADH from formate produced by FADH. Owing to the NADH and formate oxidase activity of the FDH itself, the full reaction curve is not linear, but gradually decreases. The formation of NADH is not stoichiometric with formate removal, but is strictly proportional to it. To overcome this decrease of extinction, we added hydroxylamine hydrochloride to the FDH. The sensitivity of the full reaction in the presence of FDH was about 1.8 times that without FDH. Analysis with a Cobas Bio centrifugal analyzer revealed a linearity of up to 3.56 mmol/L. The uricase-catalase-alcohol dehydrogenase method correlated well with the uricase-peroxidase-chromagen method. Our method is more sensitive than other methods.

A chromogenic assay for coagulation factor VII: analytical performance characteristics and application in several diseases.

A chromogenic assay for coagulation factor VII has been adapted for use on a Cobas Bio centrifugal analyzer. The interassay coefficients of variation ranged from 4.1% to 14%. There was no effect on the factor VII amidolytic activity after freezing and thawing of the samples. The test was linear up to 200% factor VII. The correlation with a chronometric method for prothrombin time was acceptable (r = 0.97, y = 1.05 x + 5.5). The reference ranges for males (54%-138%) and females (56%-200%) were significantly different. The factor VII amidolytic activities were significantly elevated in males with non-insulin-dependent diabetes mellitus and in male hypertensives. No statistically significant increase was observed for female patients rather a decrease in females with insulin-dependent diabetes mellitus. Only in the male hypertensive patients and in the male patients with non-insulin-dependent diabetes mellitus were noticeable proportions of increased values seen (39.4% and 12.0%). Nevertheless, the availability of a precise, rapid and easy to perform test for the determination of factor VII might be useful in the study of the role of this factor in the development of cardiovascular disease.

A rapid procedure for the measurement of elastase inhibitory function of plasma and bronchoalveolar lavage fluid

We describe a method to measure human leukocyte elastase (HLE) inhibitory capacity and compared it with porcine pancreatic elastase (PPE) inhibitory capacity and with a turbidimetric method using a specific antibody to alpha-1 antitrypsin (AAT), all performed on a Cobas Bio centrifugal analyser. This assay used methoxysuccinyl-dialanine-proline-valine-p-nitroanilide as substrate in the presence of 0.01% Brij 35, an HLE enzyme activator. Samples containing commonly used anti-coagulants and serum could be used in the assay, except for those containing heparin which strongly inhibited HLE. This assay was used to determine the functional AAT concentrations in plasma from a number of normal volunteers and patient groups, and was compared to the immuno-turbidimetric AAT assay. No difference in the proportion of functional to immuno-turbidimetric AAT was noted between any of the groups studied except for the adult respiratory distress syndrome (ARDS), where this percentage was reduced (p less than 0.05). An increase in both immuno-turbidimetric and functional AAT was seen for children (both p less than 0.01), in emphysema (p less than 0.05 and p less than 0.01 respectively) and ARDS (both p less than 0.05) when compared to adult non-smokers. This assay was also used to determine the HLE inhibitory capacity of serum and bronchoalveolar lavage (BAL) fluid from normal volunteer smokers (n = 4) and non-smokers (n = 4), and in the serum and BAL fluid from patients with ARDS (n = 5). Serum AAT was 94% functional in non-smokers (91% with PPE functional assay) and 96% in smokers (97% with PPE assay).(ABSTRACT TRUNCATED AT 250 WORDS)

Enzymatic method for phenylketonuria screening using phenylalanine dehydrogenase

An enzymatic method using phenylalanine dehydrogenase (PheDH;EC 1.4.1.-) for the determination of phenylalanine in blood-spot specimens has been developed for the Cobas Bio centrifugal analyser. Method accuracy was established by correlation of results from blood spots with phenylalanine values obtained by quantitative aminoacid analysis on simultaneously collected plasma specimens. The phenylalanine dehydrogenase method demonstrated linearity to 2500 mumol/L and had recoveries of 89.4 +/- 4.6% (mean +/- SEM). The between-run precision using blood-spot specimens was 9.8% at 285 mumol/L. In the context of a routine newborn screening program, the enzymatic method was found to have a lower false-positive rate (1% vs 3%) when evaluated against the classical fluorimetric Autoanalyser method.

Assay of urease-inhibiting activity in serum from children infected with Helicobacter pylori.

In order to provide a basis for obtaining further information concerning the host response to Helicobacter pylori urease, four assay methods for detecting urease-inhibiting activity in serum were examined. A quantitative assay, established in a COBAS BIO centrifugal fast analyzer and based on detection of the consumption of NADH by glutamate dehydrogenase stimulated by ammonia production, was considered most suitable for large-scale serological work. Serum samples from 63 children (aged 5 to 16 years), 28 of whom had seropositive H. pylori gastritis, were assayed. One of the serum samples in this latter group showed significant inhibitory activity. This serum sample was one of 13 in the seropositive group known to bind to urease antigen. It showed no inhibitory activity against Bacillus pasteurii or jack bean urease. Protein A binding and heat treatment indicated that the inhibitory activity was immunoglobulin G mediated. The patient from whom this sample was collected showed no distinctive features in his illness. The COBAS BIO analyzer-based urease inhibition assay provides a new tool for studying one aspect of the host response to H. pylori infection.

Enzymatic Assay of Inorganic Phosphate with Use of Sucrose Phosphorylase and Phosphoglucomutase

We developed a new enzymatic method for the assay of inorganic phosphate (Pi) by using sucrose phosphorylase (SP; EC 2.4.1.7) and phosphoglucomutase (PGM; EC 5.4.2.2). Pi is transferred to sucrose by SP, producing alpha-D-glucose 1-phosphate (G1P) and alpha-D-fructose. G1P is transphosphorylated by PGM in the presence of alpha-D-glucose 1,6-bisphosphate to form alpha-D-glucose 6-phosphate, which is oxidized by NAD+ and glucose-6-phosphate dehydrogenase (EC 1.1.1.49) to form 6-phosphogluconate (6PG) and NADH. Finally, the oxidation of 6PG by NAD+, catalyzed by 6-phosphogluconic dehydrogenase (EC 1.1.1.44), yields D-ribulose 5-phosphate and NADH. Thus two molecules of NADH are formed for each molecule of Pi, and the reaction is monitored at 340 nm. The Km values of SP for Pi and sucrose were 4.44 and 5.31 mmol/L, respectively. The best buffer was 1,4-piperazinediethanesulfonic acid (PIPES) at 50 mmol/L and pH 6-7. Implementing this method with a Cobas-Bio centrifugal analyzer allowed us to measure Pi accurately and precisely.

Quantifying intermediary metabolites in whole blood after a simple deproteinization step with sulfosalicylic acid

We assessed the reproducibility, recovery, and stability of several circulating metabolites--glucose, pyruvate, lactate, alanine, glutamate, glutamine, 3-hydroxybutyrate, acetoacetate, and glycerol--in the presence of sulfosalicylic acid (SSA), which was used to deproteinize blood. The assays, which involved reactions linked to NADH/NAD+, were carried out at 37 degrees C and measured at 355 nm with a Cobas-Bio centrifugal analyzer. The intra- and interbatch CVs were less than 2.1%, except for the interbatch CV for 3-hydroxybutyrate at low concentration (15-30 mumol/L), which was 5.4%. Analytical recovery of metabolites added to blood ranged from 96.4% to 103.0%. Of the metabolites studied, all were stable at -20 degrees C for 90 days in the SSA-blood extract, except for glutamine and acetoacetate, which progressively decreased with time. We conclude that these nine circulating metabolites can be satisfactorily measured after a single deproteinizing step with SSA. This single-step procedure has several advantages over many of the currently used methods.

Semiquantitation of cannabinoid immunoassays? A reexamination of the EMIT 20-ng/mL assay.

Concerns regarding the ability to semiquantitate drugs-of-abuse urine immunoassay results, particularly THC-COOH, prompted us to reexamine EMIT results. The Syva EMIT d.a.u. cannabinoid 20-ng/mL assay was performed on the Cobas Bio centrifugal analyzer, and positive samples were confirmed by Toxi-Lab or GC/MS. Of 39 specimens tested, 17 were confirmed positive. However, four specimens were not accurately semiquantitated by EMIT. These four had very high levels (greater than 100 ng/mL) of the primary metabolite but yielded EMIT results between the low and medium calibrator (20-75 ng/mL). Linearly studies confirmed that the absorbance changes with the EMIT immunoassay plateau near the medium calibrator. In addition, we obtained false positive EMIT results due to sample carryover when samples containing 500 ng/mL (or greater) of THC-COOH preceded a negative specimen. Significant carryover was not observed when concentrations up to 1,300 ng/mL were used with the 100-ng/mL immunoassay, and this effect may be explained by the higher cutoff used. Because EMIT cannabinoid results on the Cobas Bio analyzer can be affected by carryover and a hook effect, they should not be reported even semiquantitatively.

Evaluation of the PG-Numeric assay for semi-automated analysis for phosphatidylglycerol in amniotic fluid

We evaluated the performance of the PG-Numeric (PGN; Isolab, Inc.) enzymatic assay for phosphatidylglycerol (PG) adapted for semi-automated analysis on the Cobas-Bio (Roche) centrifugal analyzer. We evaluated precision, linearity, and potential interference from endogenous glycerol. In addition, we compared the results of the PGN assay with those for two other PG assays. Amniostat-FLM (AFLM; Hana Biologics) and phosphatidylglycerol/sphingomyelin ratio (PG/S), on amniotic fluid samples from 51 patients. Within-run and total CVs for the PGN test (n = 80) were respectively 8.6% and 11.4% for PG at 2.6 mumol/L, and 4.5% and 6.2% at 7.3 mumol/L. The PGN test exhibited no significant deviation from linearity between PG concentrations of 0.4 and 15 mumol/L. Endogenous glycerol up to 500 mumol/L did not affect PGN results significantly. Results of the PGN assay compared favorably with those of the other PG assays for tests of fetal lung maturation. The Cobas-Bio adaptation of the PGN test performed well analytically and clinically, and allowed the determination of PG in less than 1 h.

A study of the CEDIA digoxin immunoassay

We evaluated the CEDIA digoxin immunoassay (Microgenics, Inc., Concord, CA), as performed with the Cobas-Bio centrifugal analyzer. In assays of sera with known concentrations of digoxin, the enzyme activity, measured when two beta-galactosidase (EC 3.2.1.23) fragments were combined according to the assay format, was proportional to the digoxin concentration. Results of assays of sera containing 0 to 3 micrograms of digoxin per liter correlated well when compared with an RIA method: CEDIA, microgram/L = 1.00 x RIA - 0.06 microgram/L (n = 90, r = 0.95, Sxy = 0.05). Duplicate assays of three control sera containing 0.8, 2.2, or 3.3 micrograms/L, each analyzed 20 times a day with each group of patients' samples, gave within-run CVs of 1-3% and day-to-day CVs of 3-12%. The reconstituted CEDIA reagents were stable for at least a month at 5 degrees C. As many as 25 samples and controls can be assayed in half the time needed to complete a similar number of RIA measurements with comparable results.

Optimized methods to measure acetoacetate, 3‐hydroxybutyrate, glycerol, alanine, pyruvate, lactate and glucose in human blood using a centrifugal analyser with a fluorimetric attachment

Optimized methods are described for the analysis of glucose, lactate, pyruvate, alanine, glycerol, D-3-hydroxybutyrate and acetoacetate in perchloric acid extracts of human blood using the Cobas Bio centrifugal analyser. Glucose and lactate are measured using the photometric mode and other metabolites using the fluorimetric mode. The intra-assay coefficients of variation ranged from 0.7 to 4.1%, except with very low levels of pyruvate and acetoacetate where the coefficients of variation were 7.1 and 12% respectively. All seven metabolites can be measured in a perchloric acid extract of 20 μl of blood. The methods have been optimized with regard to variation in the perchloric acid content of the samples. These variations arise from the method of sample preparation used to minimize changes occurring in metabolite concentration after venepuncture.

Laboratory and therapeutic control in the Thrombosis Centre Rotterdam using chromogenic prothrombin time tests

Growing interest is observed in chromogenic substrate assays, because of their better precision, performance and possibilities for automation. Applied to a Cobas Bio centrifugal analyzer, we compared Nycotest-Chrom (N-test) and Thromboquant-PT (Tbq) with Thrombotest (TT). Precision tests were performed with samples from 4 plasma pools: normal (45 sec), low (100 sec), middle (150 sec) and high (200 sec) segments of the therapeutic range of TT. N-test had the best precision profile in both intraassay and interassay determinations compared with Tbq. Both chromogenic substrate assays were better than TT in this respect. By orthogonal regression a provisional therapeutic range was calculated from 312 determinations and later adjusted in a confirmation experiment with natural logarithm regression in 946 determinations. Compared with a TT range of 105-180 sec, the therapeutic ranges were 71-120 sec for N-test and 63-103 sec for Tbq. In a clinical therapeutic control phase, 110 patients were randomized in equal proportions to two groups A and B. Every 2 weeks for a period of 16 weeks, blood samples were tested for N-test, Tbq and TT. In group A, dose adjustment was based on N-test, and in group B on Tbq. The monitoring physician was blinded for Tbq and TT in group A and for N-test and TT in group B. No differences were found between the groups for mean TT, N-test, Tbq or mean dosage, nor differences were found in complication rate. A definite therapeutic range was complied using sensitivity/specificity curves together with their 95% confidence limits. Given TT 105-180 sec, the therapeutic range of N-test is from 80 (95% confidence limits: 77-82) to 110 (95% confidence limits: 108-115) sec, and of Tbq from 68 (95% confidence limits: 66-71) to 95 (95% confidence limits: 91-98) sec. It was concluded that the two chromogenic substrate assays performed equally safe in the monitoring of patients on oral anticoagulant therapy, despite differences in diagnostic correspondence with the reference TT.

Automated analysis of urinary hydroxyproline

4-Hydroxyproline released during collagen breakdown is not reutilised and its excretion in urine therefore reflects collagen breakdown [l]. Excretion of hydroxyproline is increased during accelerated collagen turnover such as growth [2,3], bone diseases [1,4] and tumors [5,6]. Almost all endogenous 4-hydroxyproline in man is confined to collagen, where it constitutes about 14% of the total amino acid content [7]. Quantitation of urinary 4-hydroxyproline is therefore helpful in the diagnosis and management of a variety of diseases [1,7]. Over 90% of the hydroxyproline excreted in urine is peptide bound [l]. Q uantitation of total urinary hydroxyproline therefore involves acid hydrolysis of the hydroxyproline-containing peptides. We have previously described a HPLC method for analysis of hydroxyproline in urine [8]. In this study we have modified the HPLC method to improve the accuracy of hydroxyproline measurement. The conventional spectrophotometric method by Ehrlich’s reaction after chloramine T oxidation to pyrrole [9] was also adapted to a Cobas Bio Centrifugal Analyzer for quick and automated analysis of large number of specimens.

An improved pyrogallol red-molybdate method for determining total urinary protein.

We adapted the pyrogallol red-molybdate method for total urinary protein to the Cobas Bio centrifugal analyzer. The method is simple, rapid, sensitive, and inexpensive. Addition of 25 mg of sodium dodecyl sulfate per liter to the reagent modifies protein reactivities so that the chromogenicity of human gamma globulins is the same as that of albumin. Results by this method and a comparison method that included gel filtration and a modified biuret reaction correlated well (r = 0.951).

Cost-effective assays for use in monitoring carbamazepine, phenobarbital, and phenytoin in serum.

Using Syva "EMIT" reagents and the Cobas Bio centrifugal analyzer, we have developed cost-effective assays for therapeutic drug monitoring of carbamazepine, phenobarbital, and phenytoin. With these optimized methods one can assay up to 2470 samples with a single 100-test EMIT kit with good precision and accuracy. Between-batch CVs, based on results of repeat analysis of routine patients' samples, were 4.6%, 7.0%, and 5.3%, respectively. Results correlated well with those by methods previously used [gas chromatography and fluorescence polarization (Abbott TDx)], but there was a negative bias (-10.9%) of our method for phenobarbital results in patients' samples, as compared with those obtained with the TDx.