Platelet Activation Test in Unprocessed Blood (Pac-t-UB) to Monitor Platelet Concentrates and Whole Blood of Thrombocytopenic Patients

Abstract

Background: Platelet concentrate transfusion is the standard treatment for hemato-oncology patients to compensate for thrombocytopenia. We have developed a novel platelet activation test in anticoagulated unprocessed blood (pac-t-UB) to determine platelet function in platelet concentrates and in blood of thrombocytopenic patients.

Methods: We have measured platelet activity in a platelet concentrate and in anticoagulated unprocessed blood of a post-transfusion thrombocytopenic patient.

Results: Our data show time-dependent platelet activation by GPVI agonist (collagen related peptide; CRP), PAR-1 agonist (SFLLRN), P2Y12 agonist (ADP), and thromboxane receptor agonist (U46619) in a platelet concentrate. Furthermore, pac-t-UB showed time-dependent platelet activation in unprocessed blood of a post-transfusion patient with thrombocytopenia. Testing platelet function by different agonists in relation to storage show that 3-day-old platelet concentrates are still reactive to the studied agonists. This reactivity rapidly drops for each agonists during longer storage.

Discussion: Pac-t-UB is a novel tool to estimate platelet function by different agonists in platelet concentrates and in unprocessed blood of thrombocytopenic patients. In the near future, we will validate whether pac-t-UB is an adequate test to monitor the quality of platelet concentrates and whether pac-t-UB predicts the bleeding risk of transfused thrombocytopenic patients.

Keywords: Flow cytometry; Platelet concentrates; Platelet function; Platelet storage; Platelet transfusion; Platelet, Platelet activation; Platelets; Thrombocytopenia.

Figures

Fig. 1

Flow cytometry technique to measure platelet activation in whole blood. a Platelets are separated from red blood cells and white blood cells by forward-side scatter characteristics. Platelet activation can be quantified by fluorescence intensity in the platelet gate of RPE-conjugated antibody binding to the platelet activation marker P-selectin or FITC-conjugated antibody against fibrinogen. b Resting platelets show no signal of platelet activation markers, while c activated platelets show high intensity of platelet activation markers. The concentration-dependent platelet reactivity experiments to different agonists (ADP (d, e), SFLLRN (f, g), XL-CRP (h, i) and U46619 (j, k)) show dose-dependent reaction curves both for P-selectin expression (d, f, h, j) and for fibrinogen binding (e, g, I, k). The experiments in figure 1 are done in six healthy donors.

Fig. 2

Determination of the concentration agonist (SFLLRN, ADP, XL-CRP, and U46619) to study time-dependent platelet activation of each agonist was done in 6 healthy donors. Serial dilutions of SFLLRN, CRP, ADP, and U46619 were used to measure platelet activation by P-selectin expression on the platelet surface. From the dose-response curves we titrated 4.5 µmol/l ADP, 6 µmol/l SFLLRN, 200 ng/ml CRP and 18 µmol/l U46619 as optimal concentrations for further experiments (a–d). e shows the reactivity of platelets of three healthy donors to a serial dilution of SFLLRN using a freshly prepared reaction mix that was stored in complete reaction mix for one week, while f shows the identical experimental setting using reaction mix that was stored in complete reaction mix for 1 week.

Fig. 3

Forward sideward scatter plot (a-c) of flow cytometry measurements of platelet activation by SFLLRN (PAR1 agonist) in platelet concentrates. Time response curves of platelet activation by 4.5 µmol/l ADP (d, e), 6 µmol/l SFLLRN (f, g), 200 ng/ml CRP (h, i), and 18 µmol/l U46619 (j, k) were used to measure platelet activation by RPE-conjugated anti-P-selectin antibody binding to the platelet surface (d, f, h, j), and FITC-conjugated anti-fibrinogen antibody binding to platelets (e, g, I, k).

Fig. 4

Platelet activation of platelet concentrates by 4.5 µmol/l ADP (a, b), 6 µmol/l SFLLRN (c, d), 200 ng/ml XL-CRP (e, f), and 18 µmol/l U46619 (g, h) after 20 min reaction time. Platelet function in platelet concentrates was measured at day 1, 2, 3, 6, 7 and 10.

Fig. 5

Forward sideward scatter plots (a–c) of flow cytometry measurements of platelet activation by SFLLRN (PAR1 agonist) in blood of a platelet concentrate transplanted patient with cell count of 20 × 109/l. Time response curves of platelet activation by 4.5 µmol/l ADP (d, e), 6 µmol/l SFLLRN (f, g), 200 ng/ml CRP (h, i) and 18 µmol/l U46619 (j, k) were used to measure platelet activation by RPE-conjugated anti-P-selectin antibody binding to the platelet surface (d, f, h, j), and FITC-conjugated anti-fibrinogen antibody binding to platelets (e, g, I, k).