25-Marker Spectral Flow Panel for Immune Profiling in Fixed Whole Blood
Magee & Lancaster et al. CYTO (2025)
Key Findings
Fresh samples processed by cytometry within 72 hours of the blood draw see:
A 48% drop in cell count.
A -88% change in the population frequency of Monocytes.
A -48% change in the population frequency of Neutrophils.
TokuKit-fixed samples showed strong correlations with fresh samples processed at T = 0: R = 0.97 for immune cell frequencies and R = 0.91 for functional subset frequencies.
TokuKit-fixed samples processed on our 25-marker, 146 cell population spectral flow panel see single digit inter- and intra-run coeffficients of variation, compared to the 25% standard.
Overview
This poster describes the validation of a 25-color spectral flow cytometry panel designed for the identification of 146 human immune phenotypic and functional marker subpopulations. Whole blood specimens from three healthy donors were collected in sodium heparin vactuainers, fixed with TokuKits, and then processed with the panel. We assessed the cell population and functional subset frequencies in TokuKit-fixed specimens at 24, 48, and 72 hours after preservation and freezing.
Methods
Sample Collection: We collected whole blood from three healthy donors using 10 mL sodium heparin vacutainers and split each specimen into two aliquots for live or fixed processing.
Live Sample Processing: Live aliquots were RBC-lysed using ACK buffer, counted, stained with a 25-marker spectral flow cytometry panel, fixed with 1.5% PFA, and analyzed on a 5-laser Cytek® Aurora.
Fixed Sample Processing: Fixed aliquots were stabilized using Teiko’s TokuKit, which includes SmartTube® Stable Lyse and Stable Store buffers. Samples were incubated, frozen at -80°C, thawed, permeabilized, stained with the same 25-marker panel, and analyzed on the same instrument.
Panel Design: The spectral flow panel was adapted from Cytek’s 25-color Immunoprofiling Assay, with substitutions made for antibody clones and fluorophores incompatible with fixation. See the list of antibodies and their respective clones below.
TokuKit Preserves Cell Count and Population Frequencies Over Time
Cell counts in live whole blood specimens decreased steadily over the first 72 hours after the blood draw, with approximately 50% of cells lost by the end of that period. In contrast, fixing whole blood specimens with TokuKit preserved cell counts throughout the 72-hour window.
Line graph of cell counts (10e6) per milliliter (mL) of blood for both live and fixed samples over time.
Over the same 72-hour period, neutrophil and monocyte populations in live whole blood specimens lost 48% and 88% of their cell counts, respectively. In contrast, TokuKit-fixed samples maintained consistent cell population frequencies throughout the time period.
Immune cell frequencies calculated as percent of non-granulocytes (B cell, T cell, NK cell, Monocyte, and Dendritic cell populations) or leukocytes (Neutrophil) for three whole blood donors over time.
Phenotypic Immune Cell Populations are Comparable between Live and TokuKit-fixed Whole Blood Specimens
We compared immune cell population and functional marker subset frequencies between live and TokuKit-fixed specimens, observing a correlation of R = 0.97 for population frequencies and R = 0.91 for functional subset frequencies.
Correlation of population frequencies between TokuKit-fixed and live samples.
Correlation of functional marker subset frequencies between TokuKit-fixed and live samples.
Panel optimization is required for fixed whole blood samples
We optimized the panel to improve staining of TokuKit-fixed specimens. For example, we started by comparing CD127 density plots between live and TokuKit-fixed samples. After adding a permeabilization step and switching the antibody clone, we observed comparable staining in the fixed samples.
We applied a similar approach to γδTCR, comparing fixed sample staining with and without the added permeabilization step.
A) Density plots comparing CD127 staining in live samples versus TokuKit samples with and without permeabilization and change in antibody clone. (B) Density plots comparing γδTCR staining in live samples versus TokuKit samples with and without permeabilization.
Phenotypic immune cell populations are comparable between TokuKit processed and live whole blood samples
We optimized the panel for TokuKit-fixed samples and then compared the density plots of immune cell populations across major lineages. As shown below, the staining patterns appeared similar.
Density plots of immune cell populations and subsets (B Cells, T Cells, NK Cells, Dendritic Cells, and Monocytes) compared between live and TokuKit-fixed samples.
TokuKit samples maintain single-digit inter- and intra-run precision
We tested the panel’s inter- and intra-run precision across three healthy donors and achieved single-digit coefficients of variation for phenotypic immune cell frequencies, functional marker frequencies, and functional marker expression.
Table of inter-run and intra-run precision measurements for phenotypic immune cell frequencies, and functional marker frequencies reported as median CV (%).
.png)
