57 It’s about time: rapid on-site fixation of whole blood samples prior to high-dimensional flow cytometric analysis
Notice bibliographique
Résumé
<h3>Background</h3> 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 laboratory<sup>1</sup> 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. <h3>Methods</h3> 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). <h3>Results</h3> 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. <h3>Conclusions</h3> 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. <h3>Reference</h3> 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. <i>Cytometry A</i> 2018 May;<b>93</b>(5):548-555.
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Comment cette classification a été obtenuedéplier
Prédiction distillée sur la base complète
Imitation des enseignantsNi prévalence calibrée, ni vérité terrain. Validation humaine à venir. Apprise à partir de 10 348 étiquettes directes de Codex et de 10 348 étiquettes directes de Gemma. Le mode candidate est l'union des têtes enseignantes seuillées; le consensus est leur intersection. Ces sorties portent le statut machine_predicted_unvalidated et ne sont ni des étiquettes humaines ni des étiquettes directes de modèles de pointe.
Scores Codex et Gemma par catégorie
| Catégorie | Codex | Gemma |
|---|---|---|
| Métarecherche | 0,000 | 0,000 |
| Méta-épidémiologie (sens strict) | 0,000 | 0,000 |
| Méta-épidémiologie (sens large) | 0,000 | 0,000 |
| Bibliométrie | 0,000 | 0,001 |
| Études des sciences et des technologies | 0,000 | 0,000 |
| Communication savante | 0,000 | 0,000 |
| Science ouverte | 0,000 | 0,000 |
| Intégrité de la recherche | 0,000 | 0,000 |
| Charge utile insuffisante (le modèle a refusé de juger) | 0,000 | 0,000 |
Scores machine (provisoires)
Les deux têtes enseignantes du modèle étudiant, lues sur ce travail. Un score ordonne la base pour la relecture; il n'affirme jamais une catégorie, et le statut de validation accompagne chaque rangée tel quel.
Scores de référence d'un modèle non mature (critères de maturité non atteints, 7 itérations). Un score ordonne; il n'affirme jamais une catégorie.
score_only:v0-immature-baseline · tel quel depuis la passe de notation : score_only signifie que le nombre peut ordonner les travaux, et qu'aucune étiquette de catégorie n'en découleClassification
machine, non validéePrédiction automatique; un appel candidat d’une seule tête enseignante, pas un consensus.
Le détail, modèle par modèle et score par score, se trouve en fin de page sous « Comment cette classification a été obtenue ».