What happens before the sample hits the cytometer

In most cytometry labs, it's routine to process samples hours, or even days, after collection. But the clock starts ticking the moment blood is drawn. Cells degrade, populations shift, and data quality quietly erodes.
Sneak peek, within 72 hours of the blood draw, fresh blood samples see a:
48%
Drop in cell count
88%
Decrease in monocyte frequency
48%
Drop in neutrophil frequency
See the Study

Experimental Design

Blood sample degradation analysis over 72 hours.

Sample Collection

Whole blood from three healthy donors was collected in sodium heparin vacutainers. Each specimen was split into eight aliquots: four for live processing and staining, and four for fixation.

Timeline

We analyzed samples at time 0 (immediately after draw), 24, 48 and 72 hours after draw.

Live Samples

Samples designated for live processing were diluted in ACK lysis buffer to lyse red blood cells (RBC). Samples were subsequently counted, and stained with a 25-marker antibody cocktail (spectral flow cytometry). After staining, samples were fixed with 1.5% PFA and analyzed.

Analysis Equipment

Analysis was performed on a 5 laser Cytek® Aurora spectral flow cytometer.

Panel Design

Spectral flow cytometry panel design was adapted from Cytek® 25-color Immunoprofiling Assay. The final panel consists of 25 markers with unique spectral signatures and does not contain a viability stain. The panel included markers for:

T cells
B cells
NK cells
Monocytes
Dendritic cells (DCs)
Neutrophils

By 24 hours, you're already losing nearly 20% of your sample.

Look below to see how cell counts change in the first 24, 48, and 72 hours after the blood draw.
Fresh blood sample cell counts drop overtime. Cell loss starts early and doesn't slow down:
  • 15% of cells are lost in 24 hours.
  • 33% of cells are lost in 48 hours.
  • 48% of cells are lost in 72 hours.

During this period, not all cell populations are lost equally.

Fresh blood samples see variable loss across immune cell populations, making it impossible to predict the patient's true biology at T = 0.
Not all cell populations are lost at the same rate. Notably, there's:
  • An 88% drop in monocytes: from ~20% to just 2.7% of nongranulocytes by 72 hours.
  • A 48% drop in neutrophils: from ~80% to 40% of total leukocytes over the same period.

CD66b is hit hard in fresh samples, making it difficult to identify neutrophils.

Missed neutrophils lead to distorted immune profiles that inflate T, B, NK, and Dendritic Cell frequencies.

Why does fresh blood sample degradation matter for your team?

Degradation distorts patient immune profiles
When cells die or surface markers decay, immune profiles shift, creating results that reflect technical variation 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 or validate biomarkers.

Want to dig deeper into fresh blood sample degradation?

See this data, compared to the results of samples fixed with TokuKit, in our recently published whitepaper.

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