title: A 10-minute prototype assay for tissue degradation monitoring in clinical specimens.
abstract: We recently identified alpha II spectrin as a Tissue Degradation Indicator (TDI) and demonstrated that intrinsic spectrin-breakdown levels reliably reveal tissue degradation status in biospecimens. With the present study, we introduce an in vitro biological assay to mimic the endogenous spectrin-breakdown process and serve as degradation monitor (DM). By initiating the DM at the time of specimen collection and by attaching the DM to respective specimens, specimen degradation can be assessed by DM readout without specimen consumption. Using a protease inhibitory assay and protease-targeted immunoassays, we identified calpain as the protease responsible for degradation-induced spectrin breakdown. To recapitulate spectrin degradation in vitro, we developed several enzymatic assays in test tubes by incubating recombinant spectrins and synthetic Fluorescence Resonance Energy Transfer (FRET)-based spectrin peptides with purified human and porcine calpains. The in vitro assays reliably performed in different environments for a limited time due to loss of calpain activity. To maintain longer calpain activity, we introduced cultured cells as calpain providers into the in vitro assays. Under a variety of degradative conditions, including 4°C, 13°C, 23°C, 29°C, 37°C, freezing, and freeze-thaw steps, we compared the use of this prototype DM to the intrinsic spectrin cleavage assay (ISCA) in specimen degradation assessment using animal models. A strong correlation (r=0.9895) was detected between the DM-revealed degradation and the ISCA-revealed degradation. Notably, the DM-based degradation assessment takes only 10min and does not jeopardize the tissue itself, whereas the ISCA-based degradation assessment needs to sacrifice tissues and takes several hours to accomplish. Our data suggests the application of an in vitro degradation monitor for fast, real time, and non-invasive assessment of specimen degradation. This observation could lead to a transformative product dedicated to biospecimen quality control. This study also addresses critical, yet unmet needs for developing a universal standard for specimen degradation measurement.
authors.affiliation: Department of Immunology, Tianjin Key Laboratory of Cellular and Molecular Immunology, Key Laboratory of Educational Ministry of China, School of Basic Medical Sciences, Tianjin Medical University, Tianjin 300052, PR China; Department of Pathology, Yale University School of Medicine, New Haven, CT 06520, United States.
authors.forename: Jia
authors.initials: J
authors.lastname: Sun
authors.affiliation: Department of Pathology, Yale University School of Medicine, New Haven, CT 06520, United States.
authors.forename: Catherine
authors.initials: C
authors.lastname: Kil
authors.affiliation: Department of Pathology, Yale University School of Medicine, New Haven, CT 06520, United States.
authors.forename: Michael C
authors.initials: MC
authors.lastname: Stankewich
authors.affiliation: Department of Immunology, Tianjin Key Laboratory of Cellular and Molecular Immunology, Key Laboratory of Educational Ministry of China, School of Basic Medical Sciences, Tianjin Medical University, Tianjin 300052, PR China.
authors.forename: Zhi
authors.initials: Z
authors.lastname: Yao
authors.affiliation: Department of Pathology, Yale University School of Medicine, New Haven, CT 06520, United States. Electronic address: li.jie@yale.edu.
authors.forename: Jie
authors.initials: J
authors.lastname: Li
authors.affiliation: Department of Pathology, Yale University School of Medicine, New Haven, CT 06520, United States. Electronic address: alexander.vortmeyer@yale.edu.
authors.forename: Alexander O
authors.initials: AO
authors.lastname: Vortmeyer
mesh_terms: D000818:Animals; D002154:Calpain / Q000378:metabolism*; D002478:Cells, Cultured; D059472:Exome; D031541:Fluorescence Resonance Energy Transfer / Q000379:methods*; D057809:HEK293 Cells; D006801:Humans; D051379:Mice; D008810:Mice, Inbred C57BL; D046228:Microarray Analysis / Q000379:methods*; D011994:Recombinant Proteins / Q000235:genetics / Q000378:metabolism; D015203:Reproducibility of Results; D017422:Sequence Analysis, DNA; D013048:Specimen Handling; D013049:Spectrin / Q000378:metabolism*; D013552:Swine
chemical_list: D011994:Recombinant Proteins; D013049:Spectrin; D002154:Calpain
keywords: Alpha II spectrin; Calpain; Degradation; Fluorescence Resonance Energy Transfer (FRET); Specimen quality