How does extended fixation in TokuKit's Buffer One impact cell population frequencies?

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.

What did we find?

Neutrophil, Monocyte, T, B, NK, and Dendritic cell population frequencies showed a <25% change from baseline when fixed for up to 1 hour.

Some immune cell subsets showed a >25% change from baseline when fixed for greater than 15 minutes.

What do we recommend?

We recommend that blood is transferred from Buffer One to Buffer Two after 15 minutes, particularly when conducting biomarker studies with deeper immune profiling. For TBNKM profiling, teams have more flexibility, with results remaining within a 25% change from baseline for up to 1 hour of fixation.

Download the results here

Experimental Design

Evaluating the impact of extended time left in Buffer One on cell population frequencies.

Sample Collection + Storage Conditions

Whole blood was collected from one healthy donor was aliquoted and incubated in Buffer One for 15 mins, 30 mins, 1 hr or 2 hrs before transfer to Buffer Two.
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After the transfer, the samples were stored at –80 °C, thawed, and analyzed with our 41-marker mass cytometry panel within the same week.

Panel Design

A 41-marker mass cytometry panel was applied to assess the immune cell populations visualized in the gating strategy below.

Instrument

Cytometry analysis was performed on a CyTOF® Helios.

Major immune cell population frequencies maintained after 1 hour of fixation.

After 1 hour of fixation, Neutrophil, Monocyte, T, B, NK, and Dendritic cell frequencies remained within 25% of those measured at the 15-minute baseline.

Immune cell subsets are best maintained with a 15 minute fixation period.

Fixation periods longer than 15 minutes resulted in >25% changes in some immune cell subset frequencies, relative to the 15-minute baseline.

Why does pressure testing TokuKit matter for your team?

Establish clear standards for trial sites

By defining the steps that impact immune cell frequencies the most, you can guide trial sites to focus on what’s critical and maintain reliable sample collection between operators.

Define your tolerance window

Knowing the conditions where immune cell frequencies stay stable gives you a clear standard for deciding whether to include or exclude a sample from analysis.

Interpret findings with clarity

Understand which shifts in immune cell frequencies are due to differences in sample handling between operators and which are associated with a patient's response to therapy.

Want to learn more?

Ask us how teams are using TokuKit for reliable sample collection, and what’s coming next in our series of TokuKit pressure-testing studies.

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References

^{1 Miltenyi Biotec.}^{MACS® handbook: Human cells and organs — Human cell sources: Blood (human)}^{[Internet]. [cited 2025 Mar 3]. Available from:}^{https://www.miltenyibiotec.com/US-en/support/macs-handbook/human-cells-and-organs/human-cell-sources/blood-human.html}²^{González-Mancera MS, Bolaños NI, Salamanca M, Orjuela GA, Rodríguez AN, González JM. Percentages of CD4⁺CD8⁺ double-positive T lymphocytes in the peripheral blood of adults from a blood bank in Bogotá, Colombia.}^{Turk J Haematol}^{. 2020;37(1):36-41. doi:10.4274/tjh.galenos.2019.2019.0256}³^{Huang C, Bi J. Expression regulation and function of T-Bet in NK cells.}^{Front Immunol}^{. 2021;12:761920. doi:10.3389/fimmu.2021.761920}