Development and optimization of a diluted whole blood ELISpot assay to test immune function.

Autor: Ungaro RF; Sepsis and Critical Illness Research Center and Department of Surgery, University of Florida College of Medicine, Gainesville, FL, United States of America., Xu J; Department of Urology, University of Minnesota, Minneapolis, MN, United States of America., Kucaba TA; Department of Urology, University of Minnesota, Minneapolis, MN, United States of America., Rao M; Department of Pediatrics, University of Iowa, Iowa City, IA, United States of America., Bergmann CB; University Hospital Ulm, Clinic for Trauma Surgery, Hand, Plastic, and Reconstructive Surgery Albert-Einstein-Allee 23, Ulm, Germany., Brakenridge SC; Department of Surgery, University of Washington, Seattle, WA, United States of America., Efron PA; Sepsis and Critical Illness Research Center and Department of Surgery, University of Florida College of Medicine, Gainesville, FL, United States of America., Goodman MD; Department of Surgery, University of Cincinnati, Cincinnati, OH, United States of America., Gould RW; Department of Anesthesiology, University of Minnesota, Minneapolis, MN, United States of America., Hotchkiss RS; Department of Anesthesiology, Washington University, St. Louis, MO, United States of America., Liang M; Department of Biostatistics, University of Florida, Gainesville, FL, United States of America., Mazer MB; Department of Pediatrics, UH Rainbow Babies and Children's Hospital, Cleveland, OH, United States of America., McGonagill PW; Department of Surgery, University of Iowa, Iowa City, IA, United States of America., Moldawer LL; Sepsis and Critical Illness Research Center and Department of Surgery, University of Florida College of Medicine, Gainesville, FL, United States of America., Remy KE; Department of Pediatrics, UH Rainbow Babies and Children's Hospital, Cleveland, OH, United States of America., Turnbull IR; Department of Anesthesiology, Washington University, St. Louis, MO, United States of America; Immune Functional Diagnostics, LLC, St. Louis, MO, United States of America., Caldwell CC; Department of Surgery, University of Cincinnati, Cincinnati, OH, United States of America., Badovinac VP; Department of Pathology, University of Iowa, Iowa City, IA, United States of America., Griffith TS; Department of Urology, University of Minnesota, Minneapolis, MN, United States of America; Center for Immunology, University of Minnesota, Minneapolis, MN, United States of America; Minneapolis VA Health Care System, Minneapolis, MN, United States of America. Electronic address: tgriffit@umn.edu.
Jazyk: angličtina
Zdroj: Journal of immunological methods [J Immunol Methods] 2024 Oct; Vol. 533, pp. 113743. Date of Electronic Publication: 2024 Aug 13.
DOI: 10.1016/j.jim.2024.113743
Abstrakt: Sepsis remains a leading cause of death worldwide with no proven immunomodulatory therapies. Stratifying Patient Immune Endotypes in Sepsis ('SPIES') is a prospective, multicenter observational study testing the utility of ELISpot as a functional bioassay specifically measuring cytokine-producing cells after stimulation to identify the immunosuppressed endotype, predict clinical outcomes in septic patients, and test potential immune stimulants for clinical development. Most ELISpot protocols call for the isolation of PBMC prior to their inclusion in the assay. In contrast, we developed a diluted whole blood (DWB) ELISpot protocol that has been validated across multiple laboratories. Heparinized whole blood was collected from healthy donors and septic patients and tested under different stimulation conditions to evaluate the impact of blood dilution, stimulant concentration, blood storage, and length of stimulation on ex vivo IFNγ and TNFα production as measured by ELISpot. We demonstrate a dynamic range of whole blood dilutions that give a robust ex vivo cytokine response to stimuli. Additionally, a wide range of stimulant concentrations can be utilized to induce cytokine production. Further modifications demonstrate anticoagulated whole blood can be stored up to 24 h at room temperature without losing significant functionality. Finally, we show ex vivo stimulation can be as brief as 4 h allowing for a substantial decrease in processing time. The data demonstrate the feasibility of using ELISpot to measure the functional capacity of cells within DWB under a variety of stimulation conditions to inform clinicians on the extent of immune dysregulation in septic patients.
Competing Interests: Declaration of competing interest M.B.M., K.E.R., and I.R.T. are members of Immune Functional Diagnostics, LLC (IFDx LLC) and receive no direct financial compensation. IFDx LLC is developing predictive metrics in critical illness and this technology is evaluated in this research. S.C·B, L.L.M., R.S.H., and the University of Florida may receive royalty income based on a technology developed by S.C.B. and others and licensed by Washington University in St. Louis to IFDx LLC. That technology is evaluated in this research. C.C.C. and the University of Cincinnati may receive royalty income based on a technology developed by C.C.C. and others and licensed by Washington University in St. Louis to IFDx LLC. That technology is evaluated in this research.
(Published by Elsevier B.V.)
Databáze: MEDLINE
Externí odkaz: