Influences of Fixatives on Flow Cytometric Measurements of Platelet P-Selectin Expression andFibrinogen Binding

Abstract

Sample fixation is an important issue in flow cytometric platelet assays. However, previous reports were less than consistent regarding the influence of sample fixation on the assays. We evaluated the effects of formaldehyde and paraformaldehyde fixation on platelet P-selectin expression and fibrinogen binding using whole-blood flow cytometry and a Coulter EPICS XL-MCL cytometer. Fluorescent-labeled whole-blood samples were diluted with HEPES-buffered saline or fixed with formaldehyde (0.2, 0.5, and 1.0%) or paraformaldehyde (0.5, 1.0, and 2.0%). Platelet P-selectin expression was 1.1±0.3% and 39.6±13.7% in unfixed resting and 10 −5 M ADP stimulated samples, respectively. Resting P-selectin expression was not significantly altered by 0.2 or 0.5% formaldehyde fixation, but was slightly decreased by 1.0% formaldehyde fixation or PFA fixation. Formaldehyde fixation caused small increases of P-selectin expression in ADP-stimulated samples. Compared to platelet fibrinogen binding of unfixed resting (4.5±2.1%) and ADP-stimulated (56.7±22.6%) samples, formaldehyde or paraformaldehyde fixation had no significant influence on resting samples, but mildly increased fibrinogen binding in stimulated samples. Unfixed samples were stable for 2 h. Fixed samples were generally stable for at least 6 h, but not thereafter. Thus, formaldehyde and paraformaldehyde have mild but complex influences on platelet P-selectin expression and fibrinogen binding measurements. To evaluate the stabilities of unfixed and fixed samples, samples were analyzed after different durations (0, 1, 2, 4, 6, 12, and 24 h) of storage at 4°C in the dark. The results suggest that sample manipulation without fixation may be used when the samples are analyzed within 2 h, and that fixation with 0.5–1.0% formaldehyde or paraformaldehyde seems to be preferable when sample analysis is delayed. Effects of fixation should be carefully evaluated when establishing flow cytometric platelet assays in every laboratory.