Tom Nilsen

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 imparticle 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 ( 11g/L), triglycerides ( 14 g/L) and bilirubin (420mg/L) antibodies, no interference with rheumatoid factor was observed. No carry-over was 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°C. Stability of the reagents at 2–8°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.

Comparison between Chicken and Rabbit Antibody Based Particle Enhanced Cystatin C Reagents for Immunoturbidimetry

Abstract 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.

Serum calprotectin levels in elderly males and females without bacterial or viral infections

OBJECTIVES Calprotectin is released from activated leukocytes and calprotectin can thus be used as a marker for leukocyte activation. Faeces calprotectin is not only used as a marker for inflammatory bowel disease but can also be used to detect leukocyte activation in other body fluids. The aim of the present study was to study serum calprotectin levels in non-infected elderly individuals to establish reference intervals for the marker. METHODS Serum calprotectin was analyzed by immunoturbidimetry in 75 year old females and males without known infections. Individuals with CRP>20mg/L were excluded as this could indicate a subclinical infection. The calprotectin levels in the remaining 713 individuals were used to calculate reference values for this population. The Spearman rank correlations between calprotectin and 27 other laboratory biomarkers were also investigated. RESULTS There was a strong positive Spearman rank correlation between calprotectin and CRP (p<0.000001) and alkaline phosphatase (p<0.000001). There were also significant negative correlations between calprotectin and ApoA1 and direct HDL-cholesterol. CONCLUSIONS The reference interval for serum-calprotectin for all study subjects was 0.3-2.6 mg/L. Leukocyte alkaline phosphatase contributes to serum alkaline phosphatase levels.

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

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.

Determination of cerebrospinal fluid cystatin C on Architect ci8200

BACKGROUND Human cystatin C is a cysteine protease inhibitor produced by all nucleated cells in the body and the protein is present in all body fluids. The concentration in cerebrospinal fluid (CSF) is considerably higher than in plasma. Cystatin C levels seem to influence the development of Alzheimer disease (AD) and low levels in the brain are associated with an increased risk for AD. The aim of this study was to develop a high throughput assay for the quantification of cystatin C in CSF. METHODS Antigen excess, imprecision, interference, linearity, recovery, sample stability and reference values were evaluated on Architect ci8200 (Abbott Laboratories, Abbott Park, IL, USA). RESULTS The assay had an antigen-excess limit at 23 mg/L and was linear over the range of 0.84 to 8.33 mg/L. Results > 8.33 mg/L were automatically rerun in a higher dilution. Within-run coefficient of variation (CV) was 1.71, 1.10 and 0.79%, between day CV was 1.71, 0.39 and 1.45%, between-run CV was 0.58, 0.66 and 0.48%, and total CV was 2.49, 1.34 and 1.72% at cystatin C concentrations of 1.39, 3.17 and 6.28 mg/L, respectively. The recovery was 97-102%. No interference at a 7.5% deviation level was observed for 8.5 g/L of hemoglobin or 800 mg/L (1368 micromol/L) of bilirubin. Reference values for cystatin C in cerebrospinal fluid obtained with this method were 2.42-14.33 mg/L.

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.

Performance of plasma calprotectin as a biomarker of early sepsis: a pilot study

AIM To determine the performance of plasma calprotectin as a marker of sepsis on intensive care unit (ICU) admission and as a marker of mortality day 30 post-ICU admission. MATERIALS & METHODS Consecutive ICU patients were allocated to: sepsis (n = 15), postoperative inflammation (n = 23) and intoxication without inflammation (n = 7) groups. RESULTS Calprotectin was 4.3 (2.6-8.2; mg/l; median [interquartile range]) in the sepsis, 2.8 (1.6-4.4) in the postoperative and 0.7 (0.4-1.6) in the intoxication groups. Area under the receiver operating characteristic curve for sepsis versus intoxication group was: 0.95, for sepsis versus postoperative groups: 0.65 and for survivors versus nonsurvivors: 0.70. CONCLUSION Calprotectin was a sensitive marker of systemic inflammation, is a potential sepsis marker and performed well as mortality predictor in this pilot study.

Extraction, isolation, and concentration of calprotectin antigen (S100A8/S100A9) from granulocytes

Calprotectin is a promising biomarker for granulocyte activation. It is mainly measured in faeces as a marker for inflammatory bowel disease. A limitation is that there is no widely accepted calibrator.