title: Optimal cellular preservation for high dimensional flow cytometric analysis of multicentre trials.
abstract: High dimensional flow cytometry is best served by centralized facilities. However, the difficulties around sample processing, storage and shipment make large scale international studies impractical. We therefore sought to identify optimized fixation procedures which fully leverage the analytical capability of high dimensional flow cytometry without the need for complex cell processing or a sustained cold chain. Whole blood staining procedure was employed to investigate the applicability of fixatives including Cyto-Chex® Blood Collection tube (Streck), Transfix® (Cytomark), 1% and 4% paraformaldehyde to centralized analysis of field trial samples. Samples were subjected to environmental conditions which mimic field studies, without refrigerated shipment and analyzed across 10 days, based on cell count and marker expression. This study showed that Cyto-Chex® demonstrated the least variability in absolute cell count relative to samples analyzed directly from donors in the absence of fixation. Transfix® was better at preserving the marker expression among all fixatives. However, Transfix® caused marked increased cell membrane permeabilization and was detrimental to intracellular marker identification. Paraformaldehyde fixation, at either 1% or 4% concentrations, was unfavorable for cell preservation under the conditions tested and thus not recommended. Using these data, we have created an online interactive tool which enables researchers to evaluate the impact of different fixatives on their panel of interest. In this study, we have identified Cyto-Chex® as the optimal cellular preservative for high dimensional flow cytometry in large scale studies for shipped whole blood samples, even in the absence of a sustained cold chain.
authors.affiliation: Singapore Immunology Network, Agency for Science, Technology and Research, 8A Biomedical Grove, Immunos Building, Level 3, Biopolis 138648, Singapore. Amanda_Ng@immunol.a-star.edu.sg
authors.forename: Amanda A P
authors.initials: AA
authors.lastname: Ng
authors.forename: Bernett T K
authors.initials: BT
authors.lastname: Lee
authors.forename: Timothy S Y
authors.initials: TS
authors.lastname: Teo
authors.forename: Michael
authors.initials: M
authors.lastname: Poidinger
authors.forename: John E
authors.initials: JE
authors.lastname: Connolly
mesh_terms: D015415:Biomarkers / Q000097:blood*; D001772:Blood Cell Count / Q000379:methods; D001793:Blood Preservation / Q000379:methods*; D003080:Cold Temperature; D005404:Fixatives / Q000592:standards; D005434:Flow Cytometry / Q000379:methods*; D006801:Humans; D015337:Multicenter Studies as Topic / Q000379:methods; D011310:Preservatives, Pharmaceutical / Q000592:standards*; D011933:Reagent Kits, Diagnostic; D015203:Reproducibility of Results
chemical_list: D015415:Biomarkers; D005404:Fixatives; D011310:Preservatives, Pharmaceutical; D011933:Reagent Kits, Diagnostic