Thrombozytenfunktionsmessung im Schafblut: Optimierung der Aggregationsmessung im Multiplate Analyser und Einfluss der Probenlagerung

The examination of haemostasis in sheep is primarily important considering their role in various experimental models including general surgical, trauma surgical, cardiovascular surgical and sepsis or other shock studies. Platelet aggregometry is one of the main in vitro standard techniques for the evaluation of platelet functions. The intention of the first part of the present study was to examine whether the new whole blood impedance aggregometer (Multiplate ™ 5.0 Analyzer, Dynabyte) is a valuable tool to examine platelet aggregation in sheep, to optimise agonist concentrations and to determine reference values for ovine blood. In the first experiment, different concentrations of several agonists (adenoindiphosphat [ADP], collagen, thrombinrezeptor-activating peptid [TRAP-6], ristocetin and arachidonic acid) were added to hirudin-anticoagulated blood of six (TRAP, ristocetin, arachidonic acid) or ten (ADP and collagen) healthy sheep to optimize the agonist concentrations. The following concentrations have been used: - ADP: 1, 1.5, 2, 2.5, 3, 4, 5, 10 and 20 µmol/l; - collagen: 0.5, 1, 1.5, 2, 2.5, 3, 4, 5, 10 and 20 µg/ml; - TRAP-6: 32 and 160 µmol/l; - ristocetin: 0.2 and 1 mg/ml; - arachidonic acid: 0.25, 0.5, 1 and 2 mmol/l. TRAP-6, ristocetin, and arachidonic acid are unsuitable agonists for induction of whole blood impedance aggregation in ovine platelets (median measurement signals < 5 % of the median measurement signals achieved with ADP or collagen). Based on the results of the examinations using ADP and collagen as agonists, further measurements were performed on 30 sheep with selected agonist concentrations (ADP: 3, 4, 5 and 10 µmol/l; collagen: 3, 4 and 5 µg/ml). The lowest agonist concentration leading to the maximum aggregation measurement signal, minimal inter-individual variation and the highest precision based on the internal measurements in duplicate were used as selection criteria. However, 20 % of the measurements with ADP had to be repeated. Reference values based on the total number of 40 healthy sheep revealed a wide variation of measurement values. In ten healthy sheep (n=10), hirudin-anticoagulated blood and in comparison citrate-anticoagulated blood (with and without the addition of calcium chloride) were measured using different concentrations of ADP and collagen. Hirudin-anticoagulated blood generated significantly higher measurement signals. Knowledge of changes of the haemostatic function in stored ovine blood are important with respect to diagnostic and therapeutic aspects as well the use of ovine blood in ex vivo models, e.g. for tests of thrombogenicity. The aim of the second part of this study was to investigate how storage of whole ovine blood for three days influences platelet function and whether the storage temperature affects the results. Whole blood of ten healthy sheep was stored in citrate-phosphate-dextrose-adenosine (CPDA)-1-stabilized transfer bag system on a wobbler, five blood bags at room temperature (20-25°C) and five blood bags at refrigerator temperature (4°C). Immediately after blood withdrawal and after 1, 2, 3, 4, 5, 6, 8, 12, 24, 48 and 72 hours of storage the platelets were counted automatically as well as visually. The number and size of platelet aggregates were counted visually. Additionally, the platelet function was examinated by impedance aggregometry (using 5 and 10 µmol/l ADP as well as 5 µg/ml collagen) as well as by turbidimetric aggregometry (using 10 and 20 µmol/l ADP as well as 5 and 10 µg/ml collagen). Furthermore, a resonance thrombogram (RTG) was prepared and different parameters of the plasmatic coagulation were measured: the prothrombin time, activated partial thromboplastin time, thrombin time, and the fibrinogen concentration using the Jacobssen method. On each day before taking the blood for the storage experiments citrate-anticoagulated blood was taken from the equal sheep and the same measurements were carried out to get comparison values. After storage for 24 hours at 4°C the platelet count decreased significantly, whereas it remained stable at room temperature. A low percentage of platelet aggregates was present immediately after the withdrawl of the blood sample into the transfer bag. After five hours of storage at 4°C approx. 50–60 % of the platelets were within aggregates, whereas only approx. 20–30 % of the platelets formed aggregates in blood which was stored at 20–25°C for up to 72 hours. In addition, platelet aggregates were larger, i.e. consisted of more platelets, when storage was performed at 4 °C. A decrease of aggregability measured with impedance aggregometry was only seen in blood stored at 4 oC (after 4 hours with collagen, after 24 hours with ADP). Maximum turbidimetric aggregation values of the blood stored at 4°C decreased significantly after 5 to 24 hours, dependent on agonist and agonist concentration. In contrast, maximum turbidimetric aggregation did not decrease before a storage time of 24 hours, if blood bags were stored at room temperature. There was no significant change of any of the investigated parameters of the plasmatic coagulation when compared to initial values. A significant shortening of RTG-r at individual times in blood which was stored at room temperature and a prolongation of RTG-p at the end of the observation period in blood stored at 4°C were the only obvious storage-induced changes of the RTG. In conclusion, based on the results of the first part of this study the investigated method of whole blood impedance aggregometry is well appropriate for the examination of ovine platelets in hirudin-anticoagulated blood using the agonists ADP and collagen in the optimal concentrations (ADP: 4–5 µmol/l; collagen : 4–5 µg/ml). In the second part of the study, the most relevant storage-induced change was the functional loss and high percentage of platelets within aggregates in ovine blood stored at refrigerator temperature. This fact has to be taken into account when blood transfusion in sheep is performed, because blood bags are stored at refrigerator temperature to maintain quality of red blood cells. However, when the short-term preservation of haemostasis and platelet function is the primary aim, e.g. for ex vivo experiments, room temperature is the preferred storage temperature providing a relatively stable material for one working day.