PROT1 whole-blood fixation

The purpose of the study was to evaluate the impact of fixation of blood samples on detection of immune cell subsets by flow cytometry, with additional analysis of the robustness of the fixation during different conditions that could occur if samples are obtained in resource poor-settings.
Conclusion:
In conclusion, we show that utilizing fixed buffy coat (FixedBC) with the fixation agent PROT1 is a viable alternative to the PBMC isolation, since it improves the storage conditions, facilitates sampling protocols, leads to miniaturization of blood-volume required, and reduces the need for specialized personnel, equipment and technical sources, also reducing user variability. It ensures reproducibility and reliability on future blood studies when performing further upstream analysis, such as of mass cytometry or flow cytometry. However, we also show that blood cell fixation can affect marker recognition in addition to cell permeabilization. It is therefore recommended that working antibody panels are evaluated prior to fixation.
Notes:
First, antibody binding to cell surface markers on cryopreserved PBMC (cPBMC) and Fixed buffy coat (FixedBC) was evaluated. Additionally, from the markers studied, the least affected were chosen to assemble two different panels: - monocytes, dendritic cells (DCs) and B cells - natural killer (NK) cells and T cells We compared the mean fluorescence intensity (MFI) of the positive population from cPBMC and FixedBC. In addition, we tested a range of different conditions regarding blood cell fixation: time of fixation, temperature of fixation and temperature of storage after fixation. In summary, 24 conditions were studied and further compared with the manufacturer?s optimal condition. CST beads (BD Biosciences) were run weekly to monitor the performance of the flow cytometer, according to the manufacturer instructions. CST beads were always run at least within 24h prior to the experiment.