Cryopreservation of human whole blood allows immunophenotyping by flow cytometry up to 30Â days after cell isolation

Immunophenotyping of whole blood (WB) by flow cytometry (FC) is used clinically to assess a patient's immune status and also in biomedical research. Current protocols recommend storage of immunolabeled samples at 4 °C with FC analysis to be completed within seven days. This data acquisition window can be extended to up to one year post-labeling, but this requires cryopreservation of the samples at ultra-low temperatures (≤− 80 °C or in liquid nitrogen). In this study we optimized a standardized cryopreservation protocol to enable preservation of immunolabeled, human WB samples at − 20 °C for FC and tested its effectiveness after 0, 5, 15 or 30 days. Analysis of stored samples shows that this protocol effectively preserves immunolabeled WB samples and that the duration of storage has no effect on morphology, viability or frequency of WB cell subpopulations, and that the intensity of fluorescent signal from labeled extracellular markers is fully preserved for at least 15 days, and up to 30 days for some markers. We demonstrate that using this protocol, we are able to differentiate resting versus activated WB cells as demonstrated by detection of significantly increased expression of CD11b by myeloid cells in WB samples pretreated with LPS (100 μg/mL for 12 h). Finally, we show that this method allows for labeling and detection of the intracellular cytokine (IL-8) up to 30 days following cryopreservation from myeloid cells, in previously labeled and cryopreserved WB samples.