57 It’s about time: rapid on-site fixation of whole blood samples prior to high-dimensional flow cytometric analysis
Bibliographic record
Abstract
Background Currently, about 70-90% of flow cytometry is performed on fresh blood samples, posing a challenge for clinical trials: trial sites must be near a flow cytometry laboratory for fresh blood to be processed, usually within 24-48 hours of sample collection. This limited time window is prohibitive, especially for sites that require shipping of samples to a distant laboratory1 resulting in the loss of >10% of specimens. Here, we use a commercially available fixation method to fix whole blood samples after collection. We then profile the samples using a 20+-marker flow cytometry panel to investigate the effects of sample fixation and storage at -80°C on immune cell composition and activation status. Data was compared to control samples collected within the same blood draw, and processed freshly according to current convention. Methods Whole blood samples from healthy donors (n=3) were collected into sodium heparin vacutainers and split into two – one control and one test sample. Control samples underwent red blood cell lysis and were stained before fixation with 1.6% paraformaldehyde. For test samples, whole blood specimens were fixed in Stable-Lyse Stable-Store reagent following manufacturer’s protocol and stored in -80°C for up to one month. Test samples were thawed, washed, permeabilized, and stained with a 20+ marker cytometry panel. Data acquisition and unmixing were performed on the Cytek Aurora spectral cytometer. Gating was performed on CellEngine. The frequencies and median fluorescence intensities (MdFI) of markers in over 30 immune populations were compared between control (live cell staining) and test samples (fixed). Results Compared to control samples, preservation of whole blood samples in Stable-Lyse Stable-Store and storage at -80C resulted in comparable frequencies for the majority of immune cell populations. While MdFI varied, correlation analysis of gated immune populations resulted in a Pearson coefficient of 0.975 between test and control samples, highlighting preservation of immune cell composition in fixed test samples. Consistency was also observed for assessment of activation status, as illustrated by an average of <10% change in the frequency of PD-1+ cells within T-cell subsets (CD4+ or CD8+ T-cells) when comparing test to control samples. Conclusions Preservation helps to reduce loss of specimens by increasing the shipping window and minimizes batch variability by allowing for central processing. We hereby report on the performance of our high-dimensional spectral flow cytometric assay to robustly detect 30+ immune cell populations in live and fixed samples. Reference Verschoor CP, Kohli V. Cryopreserved whole blood for the quantification of monocyte, T-cell and NK-cell subsets, and monocyte receptor expression by multi-color flow cytometry: a methodological study based on participants from the canadian longitudinal study on aging. Cytometry A 2018 May;93(5):548-555.
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How this classification was reachedexpand
Full frame machine prediction
Teacher imitationNot calibrated prevalence, not ground truth. Human validation pending. The Gemma side is a direct model label for every work in the frame, read from the title-only record. The Codex side is a classifier learned from the 10,348 direct Codex labels and calibrated to design-weighted sample rates; fields without enough sample support carry no Codex call. Candidate is the union of the two sides; consensus is their intersection. These outputs are machine_predicted_unvalidated and are not human labels.
Distilled classifier scores by category (both heads)
| Category | Codex | Gemma |
|---|---|---|
| Metaresearch | 0.001 | 0.001 |
| Meta-epidemiology (narrow) | 0.001 | 0.001 |
| Meta-epidemiology (broad) | 0.001 | 0.001 |
| Bibliometrics | 0.001 | 0.001 |
| Science and technology studies | 0.001 | 0.001 |
| Scholarly communication | 0.002 | 0.001 |
| Open science | 0.001 | 0.001 |
| Research integrity | 0.001 | 0.002 |
| Insufficient payload (model declined to judge) | 0.023 | 0.012 |
Machine scores (provisional)
The two teacher heads of the student model, read on this work. A score orders the frame for review; it never asserts a category, and the validation status ships verbatim with every row.
Baseline scores from an immature model (maturity gate not passed, 7 training rounds). Scores rank; they never assert a category.
score_only:v0-immature-baseline · verbatim from the scoring run: score_only means the number may rank works, and no category label ships from itClassification
machine, unvalidatedMachine predicted; a candidate call from one source (direct Gemma or distilled Codex), not a consensus.
How this classification was reached, model by model and score by score, is at the end of the page under "How this classification was reached".