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.

High prevalence of reduced thrombin generation and/or decreased platelet response in women with unexplained heavy menstrual bleeding

“…We used an agonist‐induced platelet activation test, which quantifies platelet activation with P‐selectin expression, which is a marker of platelet alpha‐granule release . Using this test, we found impaired platelet function in 14 (24%) women with HMB without other gynaecologic aetiology or pathophysiology.…”

“…Agonist‐induced platelet P‐selectin expression and αIIbβ3 activations were measured using phycoerythrin (PE)‐conjugated monoclonal mouse antihuman‐P‐selectin antibody (BD Bioscience) and fluorescein phycoerythrin (FITC)‐conjugated polyclonal sheep antihuman fibrinogen (Dako) . Whole blood was 10‐fold diluted (5 μL: 45 μL) in HEPES‐buffered saline (HBS; 10 mmol/L HEPES, 150 mmol/L NaCl, 1 mmol/L MgSO4, 5 mmol/L KCl, pH 7.4), containing 20 μg/mL FITC‐labelled antifibrinogen, 20 μg/mL PE‐labelled anti‐P‐selectin, with either 30 μmol/L ADP, 625 ng/mL XL‐CRP and 10 μmol/L PAR‐1 agonist TRAP (SFLLRN) to study agonist specific responses . The assay kits were prepared in large badges and stored at −20°C for a maximum of 2 months in nonsterile tubes (Brand GMBH, Wertheim, Germany).…”

“…nist TRAP (SFLLRN) to study agonist specific responses. 19 The assay kits were prepared in large badges and stored at −20°C for a maximum of 2 months in nonsterile tubes (Brand GMBH, Wertheim, Germany).…”

“…In addition, it requires expert laboratory skills, it needs dedicated equipment, and it has a high intra‐ and interbatch and technician variability within and between tests . In this study, we have used agonist‐induced platelet activation measured in whole blood . Similar to LTA, this test triggers platelet activation with different agonists, but it quantifies platelet activation with surface markers (P‐selectin expression and α IIb β 3 activation) instead of aggregation.…”

“…17,18 In this study, we have used agonist-induced platelet activation measured in whole blood. 19 Similar to LTA, this test triggers platelet activation with different agonists, but it quantifies platelet activation with surface markers (P-selectin expression and α IIb β 3 activation) instead of aggregation. In our hands, platelet function testing with flow cytometry is less laborious than LTA, because the reaction mix is prepared in advance and stored stable as ready to use reaction mix for at least 6 months at −20°C.…”