Incorporation of fluorescein conjugated function-spacer-lipid constructs into the red blood cell membrane facilitates detection of labeled cells for the duration of ex-vivo storage

Abstract

Please cite this article as: Ki, Katrina K., Flower, Robert L., Faddy, Helen M., Dean, Melinda M., Incorporation of fluorescein conjugated function-spacer-lipid constructs into the red blood cell membrane facilitates detection of labelled cells for the duration of ex-vivo storage, Journal of Immunological Methods (2016Methods ( ), doi: 10.1016Methods ( /j.jim.2016 This is a PDF file of an unedited manuscript that has been accepted for publication. As a service to our customers we are providing this early version of the manuscript. The manuscript will undergo copyediting, typesetting, and review of the resulting proof before it is published in its final form. Please note that during the production process errors may be discovered which could affect the content, and all legal disclaimers that apply to the journal pertain. A C C E P T E D M A N U S C R I P T ACCEPTED MANUSCRIPT A C C E P T E D M A N U S C R I P T ACCEPTED MANUSCRIPT 2 ABSTRACTThe contribution of ex-vivo storage duration of packed red blood cells (PRBC) to patient outcomes and transfusion-related immunomodulation (TRIM) remains a broadly debated area in transfusion medicine. Kode TM Technology with fluorescein conjugated function-spacerlipid (FSL-FLRO4) constructs is a tool that can aid in-vitro visualisation and tracking of red blood cells (RBC) during routine storage. FSL-FLRO4 is incorporated into the RBC membrane without altering cell function. In this study, we explore the suitability of this technology to label clinical grade PRBC, and to determine if the label would be retained during ex-vivo storage. Firstly, to confirm feasibility and assess the limit of detection of FSL-FLRO4 on PRBC at date of expiry (42 days post-collection), we tracked the binding of FSL-FLRO4 on PRBC at weekly intervals during routine storage. Over the time course, all cells remained labelled with FSL-FLRO4, although a decrease in the intensity of labelling was observed (P<0.0001). We then further investigated differences in FSL-FLRO4 labelling during RBC storage by labelling separated light-young-and dense-old-RBC from the same PRBC unit. There were no differences in the capacity of FSL-FLRO4 to label these different RBC subsets. Together these data demonstrate FSL-FLRO4 is a suitable reagent for labelling PRBC at any point during routine storage. This technology will facilitate the development of immunoassays and transfusion models focussed on addressing the mechanisms involved in TRIM.

Applications for kodecytes in immunohaematology

“…The ability to observe cells (and viruses) in vivo , their biodistribution and fate and then recover them back for analysis after being in the circulation for 24 h is also achievable using kodecytes . Kodecytes labelled with FSL‐FLRO4 (a fluorescent construct) have also been used to enable observations of young and old red cells during storage . There are also many other in vitro R&D uses for kodecytes including immobilization of cells onto surfaces and dissecting antibody responses via agglutination and solid‐phase antibody assays .…”