ThromboFix platelet stabilizer: advances in clinical platelet analyses by flow cytometry?

Platelet flow cytometry is restricted by spontaneous platelet activation in unfixed samples or by significant alterations of platelet performance caused by the use of fixatives. Aim of this study was to evaluate the new platelet stabilizer ThromboFix for clinical diagnostics.

Whole blood samples with or without addition of a weak (ADP) or strong (TRAP-6) agonist were fixed with either ThromboFix or paraformaldehyde (PFA) and stored for different periods of time for up to 7 days. Samples were then incubated with either CD41 and CD62P or CD42b and CD45 monoclonal antibodies and analyzed by flow cytometry.

The numbers of platelets, microparticles and aggregates remained stable for 7 days after treatment with ThromboFix but not with PFA due to an increasing aggregate formation after 3 days. Platelet activation was restricted to less than 1% of CD62P positive events in resting samples without a significant difference compared to an unfixed reference sample. Fixation, however, significantly reduced CD62P expression after stimulation (P < 0.05). Stabilized by ThromboFix, the level of platelet activation remained unchanged in resting and ADP stimulated samples for 7 days but decreased moderately with time after a strong stimulation with TRAP-6 (P < 0.01). After PFA fixation, intact CD62P antigen disappeared from the platelet surface within hours (P < 0.01). ThromboFix reduced the formation of platelet-leukocyte conjugates significantly (P < 0.05) and, in contrast to PFA, failed to stabilize the already formed conjugates.

In clinical situations without immediate access to a flow cytometer, ThromboFix is helpful in the flow cytometric analysis of the platelet activation marker CD62P. It should not be used for the investigation of platelet-leukocyte conjugate formation.