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Changes in fluorescence intensity of selected leukocyte surface markers following fixation
Judith C Stewart1, Michelle L Villasmil, Mark W Frampton
1Department of Medicine, University of Rochester Medical Center, Rochester, New York 14642, USA.
Insights
Fixing leukocytes after staining impacts fluorescence intensity, with changes varying by cell type and marker. Researchers recommend assessing fluorescence stability for each specific cell and marker combination.
Area of Science:
- Immunology
- Cell Biology
- Biotechnology
Background:
- Immunophenotyping of blood leukocytes commonly uses paraformaldehyde fixation before flow cytometry.
- The impact of cell type and marker specificity on fluorescence intensity stability post-fixation is not well understood.
Purpose of the Study:
- To investigate the stability of fluorescence intensity in human leukocytes after staining and paraformaldehyde fixation.
- To determine how cell type and surface marker specificity influence fluorescence changes over time post-fixation.
Main Methods:
- Human whole blood was stained with fluorescein isothiocyanate-labeled antibodies.
- Unfixed and fixed samples were analyzed by flow cytometry at multiple time points (0-96 hours).
- Fluorescence intensity was measured and converted to molecules of equivalent soluble fluorochrome.
Main Results:
- Fixation led to decreased forward and side scatter, necessitating gating adjustments.
- Autofluorescence increased significantly in fixed samples, particularly in monocytes.
- Marker-associated fluorescence decreased variably (5-39%) depending on cell type and marker after autofluorescence correction.
Conclusions:
- Fluorescence intensity changes post-staining and fixation are cell type and marker-dependent.
- Assessing fluorescence stability for each cell type and marker is crucial.
- The influence of fixation on cell autofluorescence must be considered in flow cytometry analysis.
Background:
Immunophenotyping of blood leukocytes often involves fixation with paraformaldehyde prior to cytometry analysis. However, the influence of cell type and marker specificity on the stability of fluorescence intensity after fixation has not been well studied.
Methods:
Human whole blood was stained using a panel of fluorescein isothiocyanate-labeled antibodies to surface markers. Unfixed and fixed samples were analyzed by flow cytometry at 0, 2, 4, 6, 24, 48, and 96 h after staining. Fluorescence measurements were converted to molecules of equivalent soluble fluorochrome for comparison.
Results:
Fixation caused a significant decrease in both forward and side scatter at 48 h which required gating adjustments to achieve resolution of cell populations. The autofluorescence increased progressively in fixed samples (ninefold at 96 h for monocytes). Variable decreases in marker-associated fluorescence became apparent after correction for autofluorescence. The magnitude of the decrease at 96 h varied with cell type and marker, from 5% for CD32 on monocytes to 39% for CD16 on neutrophils.
Conclusion:
The change in fluorescence intensity following staining and fixation of leukocytes varies with cell type and surface marker. Fluorescence stability should be determined for each cell type and marker used, and the confounding effects of fixation on cell autofluorescence should be considered.
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