Protein biomarkers in exfoliated cells collected from the human rectal mucosa: implications for colorectal disease detection and monitoring

Abstract

Colorectal disease biomarkers in stool are actively explored, but instability of biomolecules in faeces constitutes a problem. Collection of exfoliated cells from the surface of the rectal mucosa provides an alternative to stool-based methods. We aimed to develop an original approach allowing preservation and quantification of protein biomarkers in exfoliated material and tested it in a pilot clinical study. A novel method of cell and protein preservation in ammonium sulphate-rich buffers was developed using cultured human cells and applied to exfoliated cell samples collected from 139 faecal occult blood test (FOBT)-positive patients prior to colonoscopies. Protein biomarkers comprising calprotectin, eosinophil-derived neurotoxin (EDN), dimeric pyruvate kinase type M2 (M2PK), soluble cytokeratin-18, d-dimer and glyceraldehyde 3-phosphate dehydrogenase were quantified using enzyme-linked immunosorbent assays with parallel cytological and immunocytochemical analysis. Long-term preservation of cells and their protein constituents at ambient temperature was achieved using buffers containing saturated ammonium sulphate. Application of this approach to exfoliated cell samples allowed consistent protein quantification. Calprotectin, EDN, M2PK, soluble cytokeratin 18 and d-dimer showed dramatic increase in a few cases of inflammatory bowel disease (IBD) detected among trial participants. Cytological signs of inflammation were also present in these samples. Application of exfoliated cells collected from the surface of the rectal mucosa provides a reliable method for quantifying protein biomarkers of gastrointestinal diseases. Our preliminary results obtained in a limited number of cases indicate that the approach might be especially useful for IBD diagnosis and monitoring, but further studies are needed to assess its diagnostic value.