Journal article
Development and optimization of a diluted whole blood ELISpot assay to test immune function
Journal of immunological methods, Vol.533, 113743
10/2024
DOI: 10.1016/j.jim.2024.113743
PMCID: PMC11398710
PMID: 39147231
Abstract
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.
Details
- Title: Subtitle
- Development and optimization of a diluted whole blood ELISpot assay to test immune function
- Creators
- Ricardo F. Ungaro - University of FloridaJulie Xu - University of MinnesotaTamara A. Kucaba - University of MinnesotaMahil Rao - University of IowaChristian B. Bergmann - University Hospital UlmScott C. Brakenridge - University of WashingtonPhilip A. Efron - University of FloridaMichael D. Goodman - University of CincinnatiRobert W. Gould - University of MinnesotaRichard S. Hotchkiss - Washington University in St. LouisMuxuan Liang - University of FloridaMonty B. Mazer - Rainbow Babies & Children's HospitalPatrick W. McGonagill - University of IowaLyle L. Moldawer - University of FloridaKenneth E. Remy - Rainbow Babies & Children's HospitalIsaiah R. Turnbull - Washington University in St. LouisCharles C. Caldwell - University of CincinnatiVladimir P. Badovinac - University of IowaThomas S. Griffith - University of Minnesota
- Resource Type
- Journal article
- Publication Details
- Journal of immunological methods, Vol.533, 113743
- DOI
- 10.1016/j.jim.2024.113743
- PMID
- 39147231
- PMCID
- PMC11398710
- NLM abbreviation
- J Immunol Methods
- ISSN
- 0022-1759
- eISSN
- 1872-7905
- Publisher
- Elsevier B.V
- Grant note
- National Institutes of Health: GM-132364, GM-142481, GM-140806, GM-126928, GM-133756, GM-134880, GM-1480881 Department of Veterans Affairs: IK6BX006192
This work was supported in part by the National Institutes of Health grants: GM-132364 (L.L.M) , GM-142481 (S.C.B.) , GM-140806 (P.A.E.) , GM-126928 (R.S.H.) , GM-133756 (I.R.T.) , GM-134880 (V.P.B.) , and GM-1480881 (T.S.G.) . V.P.B. is a University of Iowa Distinguished Scholar. T.S.G. is the recipient of a Research Career Scientist award (IK6BX006192) from the Department of Veterans Affairs.
- Language
- English
- Electronic publication date
- 08/13/2024
- Date published
- 10/2024
- Academic Unit
- Critical Care; Stead Family Department of Pediatrics; Pathology; Surgery
- Record Identifier
- 9984696787302771
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