Whole blood fixation and cryopreservation procedures allow cellular analysis by flow cytometry for up to 30 days after cell isolation (TECH2P.763)

Abstract

Abstract Background: Pre-clinical and clinical immunotyping of whole blood (WB) by flow cytometry (FC) is commonly used to assess immune system changes. Current recommendations require FC analysis of pre-stained samples to be completed within 24 h, imposing logistical constraints. Development of sample preservation methods for FC is crucial for extending this technology to locations not equipped with FC and high-throughput studies. Here, we tested a sample preservation method to extend the time window for FC analysis. Methods: Paired fresh WB samples from 4 Rhesus macaques were untreated or stimulated (100 µg/ml LPS, 37oC, 22 h), surface stained, fixed, permeabilized, and intracellular (IC) stained immediately or after preservation by freezing (-20oC; 5, 7, 15, 30 days). Mean fluorescence intensity (MFI) was quantified (BD FACSAria II). Statistical analysis between frozen and fresh samples was performed by the Friedman test with significance at p≤0.05. Results: There were no significant changes over 30 d in MFI from surface markers (CD3, p=0.69; CD4, p=0.32; CD8, p=0.11) or IC cytokines in either lymphoid (IL-8, p=0.98; IL-10, p=0.33; TNFα, p=0.32) or myeloid populations (IL-8, p=0.76; IL-10, p=0.23; TNFα, p=0.14). Conclusion: In frozen WB cells, FC revealed surface marker fluorescence can be maintained for up to 30 days with stable staining of IC markers. These findings have implications for the use of FC in locations with resource constraints, as well as in high-throughput analysis.