To account for cases where samples might be left on the bench at a clinical trial site and fixed for longer than 15 minutes, we ran a study evaluating the impact of extended fixation—15 minutes, 30 minutes, 1 hour, and 2 hours—on both population frequency and functional subset readouts.
When processing blood samples with TokuKit, the recommended time in Buffer One is 15 minutes. Clinical teams recognize that, in practice, samples may remain in Buffer One longer than this recommendation and want to understand how extended time affects immune cell population frequencies and functional marker readouts. Teams ask whether results remain consistent at 20 minutes, 30 minutes, or longer, and at what point extended time begins to impact cytometry data.
To evaluate the impact of extended time in Buffer One, we assessed immune cell population frequencies and functional marker subset frequencies across multiple fixation durations.
Whole blood from a healthy donor was incubated in Buffer 1 for 15 minutes, 30 minutes, 1 hour, or 2 hours prior to transfer into Buffer 2. After transfer, samples were stored at −80°C, thawed, and analyzed within the same week using a 41-marker mass cytometry panel. Cytometry analysis was performed on a CyTOF® Helios cytometer.
Results showed that neutrophil, monocyte, T cell, B cell, NK cell, and dendritic cell population frequencies remained within 25% of the 15-minute baseline when fixed for up to 1 hour. Some immune cell subsets showed >25% deviation from baseline when fixation exceeded 15 minutes.
This data supports transferring blood from Buffer 1 to Buffer 2 after a 15-minute incubation for deep immune profiling, while TBNKM-focused profiling remains stable for up to 1 hour, providing clear guidance for site handling.
Read more about the experimental design and data: https://teiko-labs.com/time-in-buffer-one
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