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P253 The impact of storage time and freeze–thaw cycles on faecal calprotectin concentration in inflammatory bowel disease patients and controls

C. Caenepeel*1, K. Machiels1, S. Vieira-Silva2, N. Ardeshir Davani1, M. Ferrante1,3, S. Vermeire1,3

1KU Leuven, TARGID, Leuven, Belgium, 2Rega Institute for Medical Research, Microbiology and Immunology, Leuven, Belgium, 3University hospitals Leuven, Gastroenterology and Hepatology, Leuven, Belgium

Background

Faecal calprotectin (FCal) is considered the best surrogate marker of mucosal inflammation and therefore routinely used for diagnosis and follow-up of inflammatory bowel disease (IBD). For practical reasons, freezing the faecal sample prior to FCal extraction would be beneficial. However, freeze–thawing might degrade neutrophils, potentially leading to false-positive FCal measurement. We investigated the effect of multiple freeze–thaw cycles as well as long-term storage on FCal stability in frozen faecal samples and FCal extracts.

Methods

Fresh faecal samples from 10 healthy controls (HC) and 10 active IBD patients were collected in March 2017 and immediately split into five tubes which were processed differently (conditions I–VI, Table 1). During a freeze–thaw cycle, the tubes of all samples were thawed for 1 h, a FCal extract was prepared from one tube. The remaining tubes were stored at −80°C. Bühlmann® Smart Prep Faecal Sample Preparation Kit and Bühlmann® FCALTM ELISA kit were used for FCal extraction and measurement, respectively. From condition I-IV, an additional aliquot of FCal was stored at −20°C for 1.5 years (VI). Statistical analyses were performed in JMP. Linear regression analysis was performed to compare FCal concentrations. Root mean square errors (RMSE) demonstrate the average difference between FCal measurements.

Results

The median FCal concentration in, respectively the HC and IBD group were 30 and 852 µg/g faeces. The RMSE’s comparing the fresh FCal concentrations (I) with the FCal concentrations II-VI are presented in Table 1.

Abstract P253 – Table 1. Characteristics of the different storage conditions (I–VI) and RMSEs of the fresh FCal concentrations (I) compared with FCal concentrations after freeze–thawing (II–IV) and long-term storage (V and VI).

Freeze–thawing resulted in both ascending and descending deviations from the fresh FCal concentration. In HC, FCal concentrations did not exceed 100 µg/g faeces, neither after different freeze–thaw cycles nor after long-term storage, except for one sample that went up to 123 µg/g faeces after three freeze–thaw cycles. One IBD patient switched from a commonly regarded positive calprotectin to a negative calprotectin (254 vs. 154 µg/g faeces) after 1.5 years storage.

Conclusion

Multiple freeze–thaw cycles and long-term storage of faecal samples and FCal extracts influence FCal concentrations only moderately, and without influence on clinical decision-making. The non-consistent variation between different conditions is more likely caused by existing within-stool variability and variation in technical execution, rather than by freeze–thawing or storage duration. For further clinical use and research, freezing and long-term storage are acceptable to perform reliable FCal measurements.