Albumin activation of urinary amylase as determined with the Du Pont aca.

Protein activation of urinary alpha-amylase (EC 3.2.1.1) activity was observed during an evaluation of the Du Pont aca procedure for the determination of urinary alpha-amylase. This activation effect became constant for urinary albumin concentrations exceeding 1.50 g/liter. It is recommended that urinary alpha-amylase be analyzed with sufficient albumin added to maximize this effect. The aca alpha-amylase procedure is compared to an amyloclastic method for both serum and urine analysis. Expected ranges are presented for the aca method for serum and urinary amylase, amylase clearance, and the amylase clearance/creatinine clearance ratio.     

Abbott TDx, Dade Stratus, and Du Pont aca automated digoxin immunoassays compared with a reference radioimmunoassay method

Automated digoxin immunoassays with the Abbott TDx, Dade Stratus, and Du Pont aca were evaluated against an RIA similar to the Centers for Disease Control Candidate Reference Method. All three automated methods correlated poorly with the reference method. Particularly disconcerting were the falsely increased values (up to 0.6 micrograms/L) obtained by the Stratus for blank specimens. We discuss the inaccuracies of the results in terms of sample matrix effects, calibrator problems, lot-to-lot reagent variation, and our laboratory requirements. We strongly support the acceptance of the CDC Candidate Reference Method for digoxin and the establishment of a standardized protocol for determining the accuracy of digoxin measurements.     

Development and analytical performance of a functional assay for fibrinogen on the Du Pont aca analyzer

A method for determination of functional fibrinogen has been developed for the Du Pont aca discrete clinical analyzer. This fully automated assay is based on the rate of fibrin turbidity formation when thrombin is added to the test sample. The rate of this reaction is enhanced by added calcium chloride and dextran. The assay range is from 0.50 to 8.00 g/L. Within-run reproducibility studies at the medical decision level of 1.0 g/L showed a CV of 2.5%. There was no interference by heparin up to 10 USP units per milliliter of plasma.     

Abbott radiative energy attenuation method for quantifying ethanol evaluated and compared with gas-liquid chromatography and the Du Pont aca

Quantification of ethanol by a radiative energy attenuation (REA) technique was evaluated and compared with results by gas-liquid chromatography (GLC) and by the Du Pont aca. Within-assay CVs were less than 5.5%. Between-assay CVs ranged from 1.9% to 6.0% for serum and blood controls at concentrations of 0.5, 1.0, and 2.5 g/L. We observed no cross reactivity with methanol, isopropanol, or acetone, and analytical recovery of ethanol from serum averaged 101%. For the three-method comparison we performed parallel determinations of 156 blood, 92 serum, and 54 urine samples containing a wide range of ethanol concentrations. Linear regression analysis of the REA results vs those of GLC or aca yielded the following: for serum, REA = 1.03 GLC -0.03 (r = 0.998), REA = 1.13 aca -0.04 (r = 0.999); for blood, REA = 0.97 GLC + 0.05 (r = 0.994), REA = 0.99 aca + 0.06 (r = 0.996); and for urine, REA = 1.01 GLC -0.03 (r = 0.998). We also discuss the clinical and forensic use of the REA method for ethanol.     

Particle-enhanced turbidimetric inhibition immunoassay for theophylline evaluated with the Du Pont aca

We evaluated the Du Pont Particle-Enhanced Turbidimetric Inhibition Immunoassay (PETINIA) for theophylline. The imprecision (CV) of the assay was less than 4.7% between-run and less than 3.6% within-run for theophylline concentrations between 5 and 30 mg/L. Standard curves for the assay were linear for theophylline concentrations from 0 to 46 mg/L and were stable throughout the study (i.e., for at least three months). The monoclonal antibody against theophylline used in this assay increases specificity; of the possibly interfering drugs, metabolites, and anticoagulants tested, only 1,3-dimethyluric acid and EDTA showed measurable effects. Bilirubin (less than 300 mg/L), hemoglobin (less than 6 g/L), or lipemia (triglycerides less than 6 g/L) does affect the quality of the assay. Analytical recovery of theophylline added to serum (5 to 40 mg/L) averaged 98% (range 93% to 112%). Comparison of results for patients' sera by the PETINIA method with those by enzyme immunoassay (EMIT) and by "high-performance" liquid chromatography yielded slopes and intercepts not significantly different from 1.0 and 0.0, respectively, and correlation coefficients ranging from 0.986 to 0.995.     

Rapid and specific cyclosporine assay for the Du Pont aca discrete clinical analyzer performed directly on whole blood

A rapid, sensitive, and specific enzyme immunometric assay for cyclosporine in whole blood has been developed for the aca analyzer, using a monoclonal antibody from Sandoz, Ltd. Between-run CVs ranged from 6.5 to 7.6% for samples containing between 60 and 400 ng/mL cyclosporine. Sensitivity was better than 25 ng/mL in the assay, which has an effective upper range of greater than 600 ng/mL. Two correlation studies compared cyclosporine values from the Du Pont method to those determined by HPLC procedures in three hospital laboratories. The results from a total of 120 whole blood samples with CsA between 20 and 800 ng/mL showed excellent correlation between the methodologies. HPLC and Du Pont CsA values from 10 day serial studies also correlated well for samples from a kidney, kidney-pancreas, heart, and two liver transplant patients. We conclude that the Du Pont CSA assay provides accurate and reproducible results in a convenient format in less than 30 minutes.     

Determination of amylase activity in serum by using a wheat germ inhibitor with the Du Pont aca

A rapid procedure for determining salivary- and pancreatic-type amylase (EC 3.2.1.1) in serum by incorporating a wheat germ inhibitor (from Triticum aestivum) was developed for the Du Pont aca IV analyzer. Under optimal assay conditions, activities of salivary and pancreatic amylase were inhibited by 93% and 19%, respectively. The 95% central reference interval for the percentage of inhibition of serum amylase was 38-84%. Patients with acute pancreatitis showed less than 26% inhibition of amylase after addition of the wheat germ extract, reflecting the prevalence of pancreatic-type amylase in this disorder.     

Determination of serum aspartate aminotransferase with pyridoxal 5'-phosphate in the Technicon SMAC and Du Pont aca compared and correlated with the IFCC recommended method

Inexpensive improvements in continuous-flow analytical apparatus can eliminate some perplexing inconsistencies and limitations in the use of the Technicon SMAC instrument for measuring the activity in serum of aspartate aminotransferase (EC 2.6.1.1) in the presence of pyridoxal 5'-phosphate. In addition, a calibration procedure based on values for this enzyme obtained by a modified IFCC method can be used to calibrate both the Technicon SMAC and the Du Pont aca instruments to produce excellent correlation between the two.     

Colorimetric, enzymatic, and liquid-chromatographic methods for serum uric acid compared.

We describe high-performance liquid chromatography in conjunction with electrochemical detection as a possible reference method for serum uric acid. Separation was effected on a column packed with "Vydac" strong anion-exchange resin, with use of a detection potential of +0.80 V vs. an Ag/AgCl reference electrode. Results were linearly related to concentration up to 1.0 g/liter, and no interferences were seen. Assay of human sera gave within-run and day-to-day coefficients of variation of 0.83% and 1.1%, respectively; analytical recoveries averaged 100%. Comparison of the new procedure (x) with the phosphotungstate and uricase methods (y) showed the following linear regression and correlation coefficients for results: y equal 0.963x + 0.219 (r = 0.995), and y = 0.991x + 0.165 (r = 0.999), respectively. As compared to these methods, the procedure we describe is more accurate, because of the selective detection system based on retention time and redox potential. Samples can be analyzed at the rate of 20/h. No deproteinization is required.     

Clinical effectiveness of the Du Pont aca measurement of creatine kinase MB in serum from patients in a coronary-care unit

We evaluated the clinical effectiveness of measuring creatine kinase (CK; EC 2.7.3.2) isoenzyme MB and lactate dehydrogenase (LD; EC 1.1.1.27) isoenzymes in diagnosis of acute myocardial infarction. We used an agarose electrophoresis method to measure CK and LD isoenzymes and the Du Pont aca column method to measure CK-MB. Serial blood specimens were drawn from 100 patients consecutively admitted to our Coronary Care Unit. Because of the low diagnostic specificity for CK-MB measurements by both agarose electrophoresis and the discrete-analysis method, as compared with reported values, we re-evaluated our isoenzyme data by using Receiver Operating Characteristic curves. Such analysis of the data established optimal decision levels of greater than or equal to 25 U/L and greater than or equal to 18 U/L plus greater than or equal to 6% of total CK for serum CK-MB measured by the agarose electrophoresis and the aca methods, respectively, and an optimal decision level of greater than or equal to 0.92 for the ratio of LD 1/2 measured after agarose electrophoresis. At these decision levels we obtained a sensitivity of 100%, 100%, and 95% and a specificity of 94%, 92%, and 90% for CK-MB (agarose electrophoresis), CK-MB (aca), and the LD 1/2 ratio, respectively.     

Fluoroimmunoassays for procainamide and N-acetylprocainamide compared with a liquid-chromatographic method

We measured procainamide and its active metabolite, N-acetylprocainamide (NAPA), in 80 sera from 37 patients by a new fluorimmunoassay procedure and an established "high-performance" liquid-chromatographic method. Additive and proportional differences between the methods were 0.07 mg/L and 9%, respectively, for procainamide and 0.62 mg/L and 16% for NAPA. Between-day CVs by the chromatographic and immunoassay methods, respectively, were 3.9% and 2.2% for procainamide at a concentration of 6 mg/L, and 5.1% and 1.2% for NAPA (14 mg/L). We applied a modification of the fluoroimmunoassay for determination of procainamide concentrations, using sera obtained during a pharmacokinetic study, and demonstrated excellent agreement with the chromatographic method.     

High-performance liquid-chromatographic method compared with a modified radioimmunoassay of cotinine in plasma

Cotinine is a sensitive and specific biochemical marker of exposure to cigarette smoke. We describe a simple solid-phase extraction of cotinine from plasma before quantification by HPLC. Extraction recovery was 97.9% +/- 11.0% for plasma concentrations of 5-400 micrograms/L. Baseline separation of cotinine and caffeine was achieved within 11 min of injection onto a C18 reversed-phase column. The mobile phase was citric acid/dibasic potassium phosphate (30 mmol/L each, pH 6.0) containing 100 mL of acetonitrile per liter. Within-day and day-to-day precision (CV) were 4.7% and 8.4%, respectively. We also describe a modification of the Nicotine Metabolite RIA kit (Diagnostic Products Corp.) for quantifying cotinine in plasma. Recovery of cotinine from supplemented plasma was within 10% of the expected value with this RIA kit. Interassay precision averaged 8.1% for samples in the range 50-400 micrograms/L; intra-assay precision averaged 3.6% at 230 micrograms/L and 8.7% at 53 micrograms/L. Correlation between the two methods was RIA = 1.13 HPLC + 14.8 (n = 128, r = 0.957, P less than 0.001). Both methods are technically simple to perform.     

Development and clinical evaluation of a microcentrifugal analyzer method for determining creatine kinase MB isoenzyme

We have adapted to a microcentrifugal analyzer an immunoinhibition assay for measuring the activity of creatine kinase MB by using an inhibitory antibody for the M monomer. The method actually measures half the MB activity, but results are not multiplied by two because atypical isoenzymes of creatine kinase, including BB, IgG-BB, and the isoenzyme derived from mitochondria, are also detected, if they are present. Results correlated well with an electrophoresis method for 36 serum samples. Myocardial infarction was assessed in 175 patients admitted to our coronary-care unit, with respect to sensitivity (100%) and specificity (98%) when a decision point of 100 U/L (30 degrees C) was chosen for total creatine kinase activity (dithiothreitol-activated) and 6 U/L (30 degrees C) for the isoenzyme (by immunoinhibition). Atypical isoenzymes are easily recognized and confirmed by electrophoresis when the MB activity (by immunoinhibition) exceeds 6 U/L and 20% of the total creatine kinase activity.