Sodium citrate

Blood samples from eight healthy, fasting Japanese volunteers (6 men and 2 women with a mean age of 41.50 ± 13.53 years) were collected, as previously described [9 (link),10 (link)]. The body mass indexes of the participants were unknown, but their general health was good. None of the volunteers had taken drugs, including antithrombotic drugs, within two weeks of the study. Blood samples were collected from 9 a.m. to 11 a.m. in plastic tubes containing 3.2% sodium citrate (Terumo Co., Tokyo, Japan).

In stable CAD patients and in acute phase MI patients, blood samples were collected before the administration of unfractionated heparin. The administration timing of antiplatelet agents was left to the discretion of attending physician. In MI patients, blood samples were collected again at discharge as the chronic phase. The blood sample was collected into plastic tubes containing 3.2% sodium citrate (Terumo, Tokyo, Japan). It was allowed to stand for 1 to 3 hours, of which 480 µL was mixed with 20 µL of 0.3 mol/L CaCl
₂containing 1.25 mg/mL of corn trypsin inhibitor immediately before measurement.

The primary outcome variable was platelet aggregation evaluated one hour after aspirin intake. Platelet aggregometry was performed using two different whole blood tests; multiple electrode aggregometry (Multiplate Analyzer; Roche Diagnostics International LDT, Rotkreuz, Switzerland) and VerifyNow Aspirin (Accumetrics Inc., San Diego, CA, USA). Blood was collected in 3.6 mL (Multiplate Analyzer) and 2.7 mL (VerifyNow Aspirin) tubes containing 3.2% sodium citrate (Terumo, Leuven, Belgium), and all analyses were completed within two hours of sampling.
Multiplate Analyzer is an impedance aggregometer used for multiple electrode aggregometry [17] (link). Agonist solutions were delivered using an automatic pipette and contained arachidonic acid or collagen at final agonist concentrations of 1.0 mmol/L and 3.2 µg/mL (Roche Diagnostics International LDT, Rotkreuz, Switzerland). Aggregation was reported as arbitrary aggregation units plotted against time and the area under the aggregation curve was measured (aggregation units × min). The VerifyNow instrument is based on turbidimetric optical detection of platelet aggregation, and the VerifyNow Aspirin assay employs arachidonic acid as the agonist. Results are reported as arbitrary Aspirin Reaction Units.

This single-center observational study was approved by the Ethics Committee of Kagoshima University Hospital (approval no. 170178–2). The study was conducted in compliance with the Declaration of Helsinki, and written informed consent to participate was obtained from patients or their close relatives. We included 10 adult patients admitted to the ICU of Kagoshima University Hospital between November 2017 and October 2019 who required a platelet transfusion. We excluded patients administered drugs that affect the hemostatic system, such as antiplatelet agents or anticoagulants. The use of anticoagulants for the maintenance of arterial lines or extracorporeal devices was permitted. The need for platelet transfusion was determined in accordance with our standard clinical practice, independent of the present study.
Blood was drawn from the radial arterial line before and after platelet transfusion. The samples were anticoagulated with EDTA (Becton Dickinson Co., Fukushima, Japan), 3.2% sodium citrate (Terumo, Tokyo, Japan), or hirudin (Roche Diagnostics GmbH, Mannheim, Germany) and were used for blood cell counts, thromboelastometry, T-TAS, and multiplate aggregometry. After platelet transfusion, platelet concentrates remaining in the transfusion tube were collected and analyzed for their thrombogenic potential.