Changes in fluorescence intensity of selected leukocyte surface markers following fixation.
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.


CONCLUSIONS
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.
Stewart
Judith C
JC
Department of Medicine, University of Rochester Medical Center, Rochester, New York 14642, USA.
Villasmil
Michelle L
ML
Frampton
Mark W
MW
D000328:Adult; D015703:Antigens, CD / Q000032:analysis*; D005404:Fixatives / Q000737:chemistry*; D005434:Flow Cytometry; D016650:Fluorescein-5-isothiocyanate / Q000737:chemistry; D005453:Fluorescence; D005456:Fluorescent Dyes / Q000737:chemistry; D005557:Formaldehyde / Q000737:chemistry*; D006801:Humans; D016130:Immunophenotyping / Q000379:methods*; D007962:Leukocytes / Q000276:immunology*; D008875:Middle Aged; D011108:Polymers / Q000737:chemistry*; D013997:Time Factors; D016707:Tissue Fixation / Q000379:methods*
D015703:Antigens, CD; D005404:Fixatives; D005456:Fluorescent Dyes; D011108:Polymers; D005557:Formaldehyde; D016650:Fluorescein-5-isothiocyanate; C003043:paraform