B-002 Method Validation of Cystatin-C in the Serum of Dogs

Abstract

Abstract Objective The objective of this study is to validate the new Siemens Cystatin-C reagent for analysis of dog serum on the ADVIA 1800 analyzers. Methodology The ADVIA 1800 analyzer was used in the validation of Cystatin-C in Dog serum. Whole blood samples were collected in tubes free of anti-coagulant and centrifuged for 10 min at 2400 rpm. Serum was separated after centrifugation using appropriate separation techniques. Testing disciplines for the validation included intra-assay precision, inter-assay precision, accuracy, linearity, limit of quantitation (LOQ), limit of detection (LOD), reference interval, carryover, correlation, and stability. Intra-assay was determined by analyzing two biological samples and the three quality control levels of BioRad Liquichek Immunology Control 10 consecutive times (in duplicate) within a single run. Analysis of two biological samples and three levels of BioRad Liquichek Immunology Control six times for six days over a 10-day period was conducted to determine inter-assay precision. Accuracy was calculated using the data from the inter-assay precision testing. Specimen samples and Siemens Cystatin C-2 Calibrator were diluted and analyzed in duplicate for linearity. To determine LOQ calibrator was diluted to produce a value at the low end of the reportable range and tested six times within the same run. LOD was determined by assaying the appropriate blank 10 times within the same run. The reference interval was determined by analyzing 21 serum samples. Through analyzing 10 serum samples on both ADVIA 1800 analyzers correlation between analyzers could be determined. The carry-over of the assay was determined by analyzing the high-level quality control material followed by the low-level quality control material three consecutive times. Stability of serum samples was tested at room temperature, refrigerated, and frozen (at −20°C) for various durations of storage. Results The measurement of Cystatin-C in Dog serum met the acceptance criteria for intra-assay precision, inter-assay precision, accuracy, linearity of dilution, LOD, LOQ, carry-over, and stability as articulated in the results. The intra-assay precision and inter-assay precision in specimen and control material %CV was within ±20%. For accuracy the total error observed (TEobs) was within ±20% and the mean was within the acceptable range specified by the manufacturer. For linearity of dilution, the coefficient of determination was ≥0.9000 and TEobs for each dilution was lower than ±20%. The LOD was calculated by adding the mean concentration and 3 times the standard deviation. LOQ was set at the lowest concentration measured at which the %CV, %bias, and TEobs was within ±20%. The reference interval was set to the 2.5th to 97.5th percentile interval. The carry-over was accepted as it was ≤2%. Correlation between the two ADVIA 1800 analyzers was determined to describe any bias in result interpretation. Stability was found to be acceptable at all storage conditions with a mean difference within ±20%, except freeze/thaw #2. Conclusion Cystatin-C in Dog serum met the acceptance criteria for all required validation parameters. The ADVIA 1800 analyzer can be used for preclinical studies to test Cystatin-C in dog serum.