[Whole blood flow cytometry for detection of activated platelets. I. Platelet identification, blood collection and storage].
In order to assess platelet activation by flow cytometry and activation-dependent monoclonal antibodies, a minute volume(2.5 microliters) of citrated whole blood was mixed for 15 minutes with a cocktail of monoclonal antibodies(MoAb), including PAC1, (a MoAb specific for fibrinogen receptors), and a MoAb against CD62P, (an alpha-granule membrane protein which associates with platelet surface membranes when platelets are activated). After fixation with 1% formaldehyde, the percentage of platelets positive for PAC1 and/or MoAb-CD62P was measured by flow cytometry. Even in resting platelets without stimulation, about 15% of platelets were found to be PAC1-positive and 0.6% of platelets were CD62P-positive. If there was a one-minute delay after needle puncture and before blood collection, the number of PAC1-positive platelets was increased compared to that of blood obtained immediately after puncture. If blood was allowed to stand at room temperature, there was a gradual increase in the number of PAC1-positive platelets, and after 60 minutes, this increase became statistically significant. The addition of Iloprost, (a prostacyclin analogue), immediately after venipuncture did not completely prevent the increase in PAC1-positive platelets after 60 minutes. If the inhibitor was added after first incubating for 60 minutes, however, the percentage of PAC1-positive platelets was reduced to preincubation value. Although flow cytometry is a simple and powerful tool to assess platelet activation, there remain several methodological problems to be resolved before this method may be employed in routine clinical use.
Shimizu
M
M
Department of Laboratory Medicine, School of Medicine, Tokai University, Isehara 259-1193.
Ando
Y
Y
Takei
M
M
Miyachi
H
H
Tanaka
Y
Y
Kawada
T
T
D000328:Adult; D001792:Blood Platelets / Q000187:drug effects / Q000502:physiology*; D001800:Blood Specimen Collection; D005260:Female; D005434:Flow Cytometry / Q000379:methods*; D006801:Humans; D016285:Iloprost / Q000494:pharmacology*; D008297:Male; D015539:Platelet Activation / Q000502:physiology*; D010975:Platelet Aggregation Inhibitors / Q000494:pharmacology*; D010976:Platelet Count; D013997:Time Factors
D010975:Platelet Aggregation Inhibitors; D016285:Iloprost