Relationships between optical aggregometry (type born) and flow cytometry in evaluating ADP-induced platelet activation.
BACKGROUND
Platelet response to activating agents is used to monitor the efficacy of anti-aggregation therapies. The aim of our study has been to demonstrate the existence of relationships between early events of ADP-induced platelet activation, measured by flow cytometry and platelet-rich plasma aggregation, quantified by optical aggregometry.


METHODS
We evaluated peripheral blood of 12 donors. The following parameters were quantified by cytometry after stimulation with adenosine diphosphate (ADP) (0.5, 1, 2, 5, 10, 20 muM): CD62P (P-selectin) and PAC-1 expression, and cytosolic Ca(2+) mobilization. Aggregation was measured by optical aggregometry. We also studied 13 patients, undergoing coronary stenting, treated with aspirin (before procedure) or with aspirin plus clopidogrel (after procedure). We evaluated CD62P and PAC-1 expression, aggregation, and vasodilator-stimulated phopshoprotein phosphorylation (platelet reactivity index, PRI).


RESULTS
Flow procedures were more sensitive than aggregometry, with a lowest interindividual variability. Linear relationships existed in donors between CD62P expression and Ca(2+) mobilization (P < 0.0001), and between aggregation and Ca(2+) mobilization (P < 0.0001). Linear relationships existed between aggregation and CD62P expression, as percentage (P < 0.0001), or relative fluorescence intensity (RFI) (P < 0.0001). Exponential equations related aggregation and PAC-1 expression, as percentage (P < 0.0001), or RFI (P < 0.0001). Linear relationships between aggregation and CD62P expression (as percentage) existed in the patients before (P = 0.0022) and after procedure (P = 0.0020). Exponential relationships between aggregation and PAC-1 expression (as percentage) existed before (P = 0.0012) and after procedure (P = 0.0024). Linear correlations related aggregation response predicted on CD62P expression, and measured aggregation inhibition after clopidogrel (P = 0.0013) as well as predicted aggregation and PRI inhibition (P = 0.0031).


CONCLUSIONS
Tight relationships between aggregation and cytometric quantification of platelet markers in whole blood, in particular CD62P, allow to predict aggregation response to ADP from flow data in patients treated with aspirin alone or with aspirin plus clopidogrel.
Sbrana
Silverio
S
Laboratory of Hematology and Flow Cytometry, CNR Institute of Clinical Physiology, Massa, Italy. sbrana@ifc.cnr.it
Della Pina
Francesca
F
Rizza
Antonio
A
Buffa
Manuela
M
De Filippis
Rossella
R
Gianetti
Jacopo
J
Clerico
Aldo
A
D000244:Adenosine Diphosphate / Q000494:pharmacology*; D000328:Adult; D000368:Aged; D000369:Aged, 80 and over; D001241:Aspirin / Q000627:therapeutic use; D001792:Blood Platelets / Q000187:drug effects* / Q000378:metabolism; D015815:Cell Adhesion Molecules / Q000378:metabolism; D000077144:Clopidogrel; D004305:Dose-Response Relationship, Drug; D016903:Drug Monitoring / Q000379:methods; D004359:Drug Therapy, Combination; D054641:Dual Specificity Phosphatase 2 / Q000378:metabolism; D005260:Female; D005434:Flow Cytometry / Q000379:methods*; D006801:Humans; D019044:Image Cytometry / Q000379:methods*; D008297:Male; D008840:Microfilament Proteins / Q000378:metabolism; D008875:Middle Aged; D019007:P-Selectin / Q000378:metabolism; D010750:Phosphoproteins / Q000378:metabolism; D010766:Phosphorylation; D015539:Platelet Activation / Q000187:drug effects*; D010974:Platelet Aggregation / Q000187:drug effects*; D010975:Platelet Aggregation Inhibitors / Q000627:therapeutic use; D015203:Reproducibility of Results; D012680:Sensitivity and Specificity; D013988:Ticlopidine / Q000031:analogs & derivatives / Q000627:therapeutic use; D000098662:Vasodilator-Stimulated Phosphoprotein
D015815:Cell Adhesion Molecules; D008840:Microfilament Proteins; D019007:P-Selectin; D010750:Phosphoproteins; D010975:Platelet Aggregation Inhibitors; D000098662:Vasodilator-Stimulated Phosphoprotein; D000244:Adenosine Diphosphate; D000077144:Clopidogrel; C517032:DUSP2 protein, human; D054641:Dual Specificity Phosphatase 2; D013988:Ticlopidine; D001241:Aspirin