C. De Cunto2, A.I. Leuci1, G. Grammatico1, C. Roveta1, G. Inzaina1, F.E.O. Ferrara1, M. Pirovano2, F. Magro2
1 CDI Centro Diagnostico Italiano SpA, Via Saint Bon, 20, 20147 Milano, Italy
2 Sentinel CH. SpA, Via Robert Koch, 2, 20152 Milano, Italy
Calprotectin is the main protein expressed by neutrophil cells and it’s considered one of the most interesting biomarkers of inflammation. Due to its district specificity, the measurement of calprotectin in stools is widely used to detect inflammatory process in the intestinal tract and it’s mainly used to differentiate inflammatory bowel diseases (IBD) from non-inflammatory disorders like irritable bowel syndrome (IBS). The aim of the study was to evaluate the analytical performances of Sentinel new generation CALiaGold® and to complete the Design Validation of the product in an external clinical lab and under routine conditions on fully automated SENTiFIT® 270 Analyzer.
CALiaGold® is a complete and integrated solution dedicated to the Clinical Lab for the quantitative measurement of Calprotectin in human feces.
It comprises of a barcoded Sampling Tube with universal shape and dimensions, Latex enhanced Immunoturbidimetric reagent, single high-level Calibrator with instrumental automatic dilution and Quality Controls. All reagents are liquid and ready to use.
Evaluation of the analytical performances of the second generation CALiaGold® has been based on respective CLSI evaluation guidelines by using 3 different production lots. Design Validation was performed at CDI Centro Diagnostico Italiano under preapproved and formally released protocol. Statistical analysis was performed with Analyse-it.
Analytical sensitivity
Lowest limit of the Analytical Measuring Range (AMR) was based on Limit of Quantitation.
Acceptance Criteria were defined as the analyte concentration with a ≤20% total imprecision CV%. LOQ
study format was: 8 dilutions x 10 replicates x 3 days. Worst cases are reported in the plot.
Lowest Limit of the AMR was 18.5 μg/g.
Prozone
Prozone of the second generation CALiaGold® was evaluated with 3 reagent lots. Acceptance criteria were defined as no false reportable values from 2200 μg/g up to 6000 μg/g.
Acceptance Criteria were met with all three lots.
Imprecision
Imprecision of the assay was evaluated in terms of Intra-imprecision as well as Total Imprecision on 3 reagent lots. Total imprecision study format was: 5 samples x 2 replicates x 20 run across 30 days.
Acceptance criteria were: ≤8% as total Imprecision CV%.
Method comparison
Accuracy of the second generation CALiaGold® was evaluated as per CLSI guidelines by Method Comparison against the first generation CALiaGold®.
The study has been done on SENTiFIT® 270 Analyzer with 3 reagent lots. Study format was based on at least 100 fecal samples and independent sampling between the two methods.
External design validation
Second generation CALiaGold® was compared to the previous CALiaGold® version in an external clinical laboratory on 306 patients’ samples from the routine.
BiasPlot of the samples around the clinically relevant range under 650 μg/g delivered a not significant mean Bias of -5,64 μg/g.
Linearity
Linearity of the second generation CALiaGold® was evaluated with 3 reagent lots.
Acceptance criteria was defined as ±10% in terms of relative bias and ±5 μg/g as absolute Bias. The assay was proved to be linear up to 2200 μg/g.
The study proved that the second generation CALiaGold® met specifications in terms of accuracy ±10% relative Bias or ±5.0 μg/g as absolute bias and total imprecision < 8.0% CV%.
External Design Validation proved substantial equivalence in comparison to the first CALiaGold® generation under routine conditions. Automatic dilution of the Calibrator on SENTiFIT® 270 and SENTiFOB® analyzers completes Sentinel offer of a fully integrated solution to the clinical labs for the measurement of fecal Calprotectin.