Influences of fixatives on flow cytometric measurements of platelet P-selectin expression and fibrinogen binding

Sample fixation is an important issue in flow cytometric platelet assays. H owever, 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 fibrino gen binding using whole-blood flow cytometry and a Coulter EPICS XL-MCL cyt ometer. Fluorescent-labeled whole-blood samples were diluted with HEPES-buf fered saline or fixed with formaldehyde (0.2, 0.5, and 1.0%) or paraformald ehyde (0.5, 1.0, and 2.0%). Platelet P-selectin expression was 1.1+/-0.3% a nd 39.6+/-13.7% in unfixed resting and 10(-5) M ADP stimulated samples, res pectively. Resting P-selectin expression was not significantly altered by 0 .2 or 0.5% formaldehyde fixation, but was slightly decreased by 1.0% formal dehyde fixation or PFA fixation. Formaldehyde fixation caused small increas es 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 signif icant 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, formaldeh yde and paraformaldehyde have mild but complex influences on platelet P-sel ectin expression and fibrinogen binding measurements. To evaluate the stabi lities of unfixed and fixed samples, samples were analyzed after different durations (0, 1, 2, 4, 6, 12, and 24 h) of storage at 4 degreesC in the dar k. The results suggest that sample manipulation without fixation may be use d 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 analys is is delayed. Effects of fixation should be carefully evaluated when estab lishing flow cytometric platelet assays in every laboratory. (C) 2000 Elsev ier Science Ltd. All rights reserved.