Turbidimetric Determination of Fecal Calprotectin Using Two Table Top Chemistry Analyzers: Mindray BS-200E and Cobas® c111.

BACKGROUND: Fecal calprotectin assays are widely used in diagnosis and monitoring of inflammatory bowel disease (IBD) in patients with suspected IBD. The most frequently used technique is ELISA and microtiter plates. Turbidimetric assays for analysis of fecal calprotectin can significantly reduce turnaround time. Many laboratories may be reluctant to run fecal samples on their large chemistry analyzers. The aim of this study was to evaluate fecal calprotectin particle enhanced turbidimetric immunoassay (PETIA) on smaller chemistry analyzers that could be dedicated for fecal samples. METHODS: The BÜHLMANN fCAL® turbo assay was validated on two table top chemistry analyzers, Mindray BS-200E and cobas® c111. RESULTS: The assay was linear in the range between 20 and 1,900 µg/g with a limit of quantification around 20 µg/g on both instruments. The total coefficient of variation was < 7% in the range between 50 and 1,300 µg/g on both instruments. No antigen excess hook effect was observed up to 18,000 µg/g on the Mindray BS-200E and up to 20,000 µg/g on cobas® c111. The BÜHLMANN fCAL® turbo assay showed a high correlation with the BÜHLMANN fCAL® ELISA. CONCLUSIONS: Running the BÜHLMANN fCAL® turbo on Mindray BS-200E or cobas® c111 chemistry analyzers can provide rapid test results without exposing large routine chemistry analyzers to stool samples.

A novel turbidimetric immunoassay for fecal calprotectin optimized for routine chemistry analyzers.

BACKGROUND: Fecal calprotectin assays are widely used to exclude inflammatory bowel disease (IBD) in patients with suspected IBD. A problem with the fecal calprotectin assays is the rather long test-turnaround times. A particle enhanced turbidimetric immunoassays (PETIA) for fecal calprotectin would reduce test-turnaround times and would permit more laboratories to perform the measurements. The aim of this study was to evaluate a new feces calprotectin PETIA. METHOD: Using routine fecal samples the feces calprotectin PETIA was validated on two chemistry analyzers, Mindray BS-380 and Cobas 501. RESULTS: The assay is linear in the range 11-2000 µg/g, with a limit of quantitation of approximately 10 µg/g. No antigen excess hook effect was observed up to 10 000-15 000 µg/g depending on the instrument used. The turbidimetric method showed a good agreement with the Bühlmann ELISA. The total coefficient of variation was 3%-8% in the 50-100 µg/g range. CONCLUSION: The fecal calprotectin PETIA, fCal Turbo, is well suited for rapid analysis of fecal calprotectin on Mindray BS-380 or Cobas 501 clinical chemistry analyzers. The test results are commutable with Bühlmann fecal MRP8/14 ELISA.

A new turbidimetric immunoassay for serum calprotectin for fully automatized clinical analysers.

Serum and plasma calprotectin concentration is shown to be elevated when neutrophils are activated, and may therefore be used as a marker for inflammatory diseases. A serum calprotectin immunoassay was developed based on calprotectin values observed in samples from the intensive care unit. The polyclonal avian antibodies were raised and affinity purified with calprotectin antigens. The performance was tested and it was observed that the assay was linear in the range 0.3-24.7 mg/L, the limit of quantitation was observed to be lower than 0.3 mg/L, no antigen excess was observed up to 54 mg/L, all CVs were lower than 1.8 % in the precision study, the calibration curve stability was longer than 6 weeks, and there was no significant interference detected for haemoglobin, intralipid or bilirubin. The serum calprotectin immunoassay presented in this paper performs well within the criteria carefully set from the limited clinical experience obtained in both serum and plasma. In addition it is commutable with Bühlmann MRP8/14 ELISA.

Generation of a new cystatin C-based estimating equation for glomerular filtration rate by use of 7 assays standardized to the international calibrator.

BACKGROUND: Many different cystatin C-based equations exist for estimating glomerular filtration rate. Major reasons for this are the previous lack of an international cystatin C calibrator and the nonequivalence of results from different cystatin C assays. METHODS: Use of the recently introduced certified reference material, ERM-DA471/IFCC, and further work to achieve high agreement and equivalence of 7 commercially available cystatin C assays allowed a substantial decrease of the CV of the assays, as defined by their performance in an external quality assessment for clinical laboratory investigations. By use of 2 of these assays and a population of 4690 subjects, with large subpopulations of children and Asian and Caucasian adults, with their GFR determined by either renal or plasma inulin clearance or plasma iohexol clearance, we attempted to produce a virtually assay-independent simple cystatin C-based equation for estimation of GFR. RESULTS: We developed a simple cystatin C-based equation for estimation of GFR comprising only 2 variables, cystatin C concentration and age. No terms for race and sex are required for optimal diagnostic performance. The equation, [Formula: see text] is also biologically oriented, with 1 term for the theoretical renal clearance of small molecules and 1 constant for extrarenal clearance of cystatin C. CONCLUSIONS: A virtually assay-independent simple cystatin C-based and biologically oriented equation for estimation of GFR, without terms for sex and race, was produced.

Comparison between chicken and rabbit antibody based particle enhanced cystatin C reagents for immunoturbidimetry.

We have compared three commercial particle enhanced cystatin C reagents. One of the reagents utilizes chicken antibodies and the other two reagents are rabbit antibody based. We show that the chicken antibody based reagent yields a higher delta absorbance when reacting with the antigen. IgY coupled to latex particles show a strong scatter response even at high antigen concentrations in contrast to the steep decline in scatter previously reported for IgY antibodies in solution. The reagent also showed a low CV for duplicate samples. Laying hens thus seems as an interesting source of antibodies for particle-enhanced immunoassays.

Performance characteristics of a cystatin C immunoassay with avian antibodies.

BACKGROUND: A new particle-enhanced turbidimetric immunoassay (PETIA) with avian antibodies for measuring serum/plasma cystatin C has been developed. The performance characteristics of the assay are described. METHODS: Measurements were performed on a Roche Modular P and on an Abbott Architect ci8200 using Gentian cystatin C immunoassay. RESULTS: Measuring range was 0.3-8.0 mg/L. Reference range was 0.57-1.09 mg/L. Total analysis time was 10 minutes. Linearity was absolute over the whole assay range. Recovery of samples and controls was within 98.6-109.4%. Total imprecision CV, measured over 20 days with two lots, was < or = 4.2%. Comparison with a particle enhanced nephelometric cystatin C immunoassay (PENIA) by linear regression resulted in a slope within 0.97-1.02 and intercept within +/-0.05 mg/L. Interference studies with drugs, anticoagulants, intralipid (< or = 11 g/L), triglycerides (< or = 14 g/L) and bilirubin (< or = 420 mg/L) showed no significant interference. Due to the use of avian antibodies, no interference with rheumatoid factor was observed. No carry-over was detected. Lower detection limit and lower quantification limit (CV < or = 6%) were both below 0.33 mg/L, which is less than the lowest standard. Sample stability was up to one month at 2-8 degrees C. Stability of the reagents at 2-8 degrees C was estimated to be 24 months. Stability of the reagents in use was minimum 9 weeks. CONCLUSIONS: Gentian cystatin C PETIA is shown to have excellent performance between methods . Interference results are improved due to avian antibodies and a broader span of the calibration curve. Avian antibodies are also known to have better immune response than mammalian antibodies towards mammalian antigens.