Laboratory-based quantitative calprotectin measurement Ascitic calprotectin in ascites was assayed using a commercially-available selleck inhibitor ELISA (B��hlmann Laboratories AG, Sch?nenbuch, Switzerland) and following the manufacturer��s instructions. Briefly, 10 ��L aliquots of the supernatant samples were diluted 1:50 in incubation buffer and 100 ��L was applied to a microtiter plate coated with a monoclonal capture antibody highly specific for the calprotectin heterodimeric and polymeric complexes. After incubation, washing and further incubation with a detection antibody conjugated to horseradish peroxidase, the tetramethylbenzidine chromogenic substrate was added. The reaction was terminated by a stop solution and the absorbance (optical density at 450 nm) was measured by spectrophotometry.
The measuring range of the test was 0.2-12 ��g calprotectin/mL ascites with an intra- and interassay coefficient of 4.7% and 11.3%, respectively. Point-of-care quantitative calprotectin measurement The Quantum Blue? quantitative calprotectin lateral flow assay (B��hlmann Laboratories AG) was used for the point-of-care (POC) measurement of ascitic calprotectin. The Quantum Blue? reader is currently marketed for 2500 USD ($), and the test cartridges cost 20 USD per sample and analysis. Aliquots of 60 ��L 1:10 diluted ascites samples (20 ��L ascites in 180 ��L extraction buffer) were pipetted respectively onto the sample loading port of the test cartridge. After a 12 min incubation, the test cartridge was quantitatively read by the Quantum Blue? Reader. The measurement range of the lateral flow test was 0.
38-3.8 ��g calprotectin/mL ascites, with an interassay coefficient of 15.6%. Specimens with concentrations above this measurement range were further diluted with extraction buffer. In addition, a random subgroup of samples (n = 17) was immediately measured by POC, without first performing the centrifugation step of processing. These results were compared to the results from the POC measurements obtained in the laboratory setting after processing and storage. Statistical analysis All statistical analyses were performed using the SPSS software package, version 19.0 (SPSS Inc., Chicago, IL, United States). A P-value of less than 0.05 indicated statistical significance. Intergroup comparisons were made using the Mann-Whitney U test and the ��2 test where appropriate. Correlations between numerical data were determined with the Spearman��s rank correlation coefficient. All hypothesis testing was two-tailed. The Bland-Altman plot was used to assess agreement between ELISA test results and POC test results, in which the differences between the results of the two tests Anacetrapib for each individual patient were plotted against the corresponding mean of the two readings.