EVALUATION OF WHOLE-BLOOD FLOW CYTOMETRIC DETECTION OF PLATELET BOUNDFIBRINOGEN ON NORMAL SUBJECTS AND PATIENTS WITH ACTIVATED PLATELETS

Activated platelets can be detected by measuring platelet-bound fibrin ogen in a whole blood, flow cytometric assay, using a fluorescently-co njugated polyclonal antibody. Fibrinogen binding to unstimulated plate lets from normal subjects was low in this assay, as was expression of the CD63 antigen. Single cell counting of samples prepared for flow cy tometric analysis showed platelet aggregates do not form during the as say procedure. Immune complexes were not seen, and fibrinogen binding to the platelets was unaffected by the CD32 MAb, IV.3. Artefactual act ivation of the unfixed samples could be minimised by control of phlebo tomy, time and temperature of incubation. Variations in platelet count in the range 140-430 x 10(9) l-1 and in plasma fibrinogen in the rang e 2-6 g l-1 did not affect the assay results. Comparison of fibrinogen binding with expression of CD63 antigen on normal platelets, stimulat ed with agonists in vitro, demonstrated that fibrinogen binding detect s an earlier stage of platelet activation. Platelet bound fibrinogen w as shown to be sensitive in detecting small numbers of activated plate lets in clinical samples in twelve patients on intensive care, four un dergoing haemofiltration. The patients had a significantly higher medi an percentage of circulating platelets with bound fibrinogen (p <0.005 ), but fibrinogen binding was significantly lower (p <0.02) in respons e to 10(-5) M ADP, compared to twelve age-matched normal controls.