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Can refrozen TokuKit-fixed samples be used without compromising cytometry readouts?

Yes. Immune cell population and functional subset frequencies are preserved in TokuKit-fixed blood samples up to two refreeze cycles.
Immune Cell Populations Preserved Up To  Two Refreeze Cycles
Immune cell population frequency differences remained within ±3% across all major cell populations
  • <0.5%
    Mean absolute difference in immune cell population frequencies between refrozen and non-refrozen TokuKit-fixed samples.
  • 100%
    All major immune cell populations (T, B, NK, monocytes, DCs) remained within +/- 3% of non-refrozen control samples.
  • ≤2.8%
    Maximum observed difference in immune cell population frequencies across TokuKit-fixed samples subjected to one or two refreeze cycles.
One Refreeze Cycle vs. No Refreeze
Two Refreeze Cycles vs. No Refreeze
Functional Marker Frequencies Are Stable Across Two Refreeze Cycles
  • <3%
    Mean absolute difference in functional subset frequencies between refrozen and non-refrozen TokuKit-fixed samples.
  • ±13%
    95% of functional marker subset frequency differences remained within +/- 13%.  
  • ±25%
    Custom panel functional marker subsets (e.g. IFNg, CD80, CD68, perforin, and others) remained within maximum observed deviation across refreeze cycles.
One Refreeze Cycle vs. No Refreeze
Two Refreeze Cycles vs. No Refreeze

Experimental Design

Evaluation of TokuKit-fixed blood samples processed after 0, 1, or 2 refreeze cycles.
Experimental set up for investigating the impact of refrozen/thawed TokuKit fixed samples

Sample Collection:

Whole blood was collected from 1 healthy donor and processed under 2 biological conditions:
- Unstimulated (to assess immune cell population frequencies)
- Stimulated (to assess functional marker frequencies)

Stimulation Blend:

Stimulated samples were prepared as a blend, consisting of PMA/PTI-activated whole blood spiked with PHA-stimulated PBMCs and Daudi cells, followed by fixation using TokuKit (SLST)

Experimental Conditions:

Refreeze cycles:  
- 0 refreeze cycles: TokuKit-fixed samples (control)
- 1 refreeze cycle: TokuKit-fixed samples refrozen once (experimental)
- 2 refreeze cycles: TokuKit-fixed samples refrozen twice (experimental)

Timeline:

Initial fixation: Whole blood samples collected in sodium heparin, transferred to TokuKit, and frozen at −80°C.

Refreeze simulation*: Samples were thawed, washed, counted, aliquoted, then refrozen in 10%DMSO/CSB at −80°C.

All samples were subsequently thawed and processed on the same day and in the same run

Panel Design

A 43-marker mass cytometry panel was applied to assess the immune cell populations visualized in the gating strategy below

43-marker CyTOF panel used as part of this experiment

Instrument

Cytometry analysis was performed on a CyTOF Helios.

How do I refreeze TokuKit-fixed samples for later use?

Once TokuKit-fixed samples are thawed completely at room temperature, proceed with standard washing, counting, and aliquoting steps.

For storage, adjust cell density to 1 million cells per 100 uL of CSB per cluster tube and mix 1:1 with 20% DMSO/CSB (final concentration of DMSO = 10%). Mix well and store samples at −80 °C for later use.

Click here for the CSB formulation.

Why does sample preservation matter for your team?

Degradation distorts patient immune profiles
When preservation methods degrade or introduce artifacts, immune profiles shift, creating results that reflect preservation instability rather than a patient's true biology.
Failed samples waste time and budget
Degraded samples often fail QC, requiring costly redraws, repeat shipments, and delays in data delivery.
Inconsistencies undermine PD insights
Differences in how long samples sit before processing introduce noise, making it harder to detect real pharmacodynamic signals, mechanism of action, or biomarkers of response.

Want to learn more?

Ask us about the results of our TokuKit freeze/refreeze study—and what studies with TokuKit are coming next.
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