Influence of cryopreservation and mechanical stimulation on equine Autologous Conditioned Plasma (ACP^®)

Summary

Objective: To determine the influence of cryopreservation at two different temperatures on platelet concentration, growth factor (GF) levels and platelet activation parameters in equine ACP^®; moreover, to determine if adding mechanical ACP^® stimulation to freeze-thaw activation amplifies GF release from platelets. Material and methods: Firstly, blood from five horses was used to prepare ACP^®. Platelet, platelet derived growth factor BB (PDGF-BB) and transforming growth factor β₁ (TGF-β₁) concentrations as well as mean platelet volume (MPV) and mean platelet component (MPC) were determined in fresh and corresponding ACP^® samples after 2 months cryopreservation at –20 °C and –80 °C, respectively. Secondly, ACP^® was prepared from blood of nine horses. Half of ACP^® was activated using one freeze-thaw-cycle at –20 °C, whereas the rest was first vortexed. Their PDGF-BB and TGF-β₁ concentrations were subsequently determined. Results: Platelet concentration significantly decreased after –80 °C cryopreservation. PDGF-BB level augmented significantly after both storage methods, whereas TGF-β₁ concentration was not significantly altered. MPV significantly increased after –20 °C cryopreservation. Both storage regimens induced a significant MPC decrease. No significant differences in GF concentrations between the vortexed and nonvortexed samples were detected. Discussion: Both cryopreservation methods induced platelet activation, but storage at –80 °C apparently harmed the platelets without generating higher GF release than –20 °C. The mechanical stimulation process could not enhance GF release in subsequently frozen-thawed ACP^®. Conclusion and clinical relevance: Storage of ACP^® at –20 °C could be useful in equine practice, but, before this procedure can be recommended, further qualitative tests are needed. The mechanical stimulation technique should be adjusted in order to increase platelet activation.

Zusammenfassung

Gegenstand und Ziel: Untersucht wurde der Einfluss der Kryokonservierung bei verschiedenen Temperaturen auf die Konzentration von Thrombozyten und Wachstumsfaktoren (WF) sowie die Thrombozyten-Aktivierungsparameter in equinem ACP^® (Autologous Conditioned Plasma). Zudem wurde geprüft, ob eine mechanische ACP^®-Stimula tion vor einer Einfrier-Auftau-Aktivierung die Freisetzung von WF aus den Blutplättchen verstärkt. Material und Methoden: Zunächst erfolgte die Herstellung von ACP^® aus Blut von fünf Pferden. Die Kon zentrationen von Thrombozyten, Platelet Derived Growth Factor BB (PDGF-BB) und Transforming Growth Factor β₁ (TGF-β₁) sowie das mittlere Thrombozytenvolumen (Mean Platelet Volume, MPV) und die mittlere Thrombozytendichte (Mean Platelet Component, MPC) wur den in frischen und jeweils in für 2 Monate bei –20 °C und –80 °C kryokonservierten ACP^®-Proben gemessen. Außerdem wurde ACP^® aus dem Blut von neun Pferden vorbereitet. Eine Hälfte des ACP^® wurde mit einem Einfrier-Auftau-Zyklus bei –20 °C aktiviert, der Rest wurde vor diesem Vorgang mit einem Rüttler geschüttelt. Dem schloss sich die Bestimmung der PDGF-BB- und TGF-β₁-Konzentration in den Proben an. Ergebnisse: Nach Kryokonservierung bei –80 °C verringerte sich die Thrombozytenkonzentration signifikant. Beide Gefriertemperatu ren induzierten einen signifikanten Anstieg der PDGF-BB-Konzentra tion, ohne den TGF-β₁-Gehalt signifikant zu beeinflussen. Das MPV stieg nach der Kryokonservierung bei –20 °C signifikant an. Die MPC nahm bei beiden Gefriertemperaturen signifikant ab. Die geschüttelten und nicht geschüttelten Proben differierten bezüglich der WF-Konzentration nicht signifikant. Diskussion: Beide Gefriertemperaturen induzierten eine Thrombozytenaktivierung, aber die Lagerung bei –80 °C schädigte die Zellen, ohne höhere WF-Konzentrationen als bei –20 °C zu erzeugen. Die mechanische Stimulation konnte die WF-Ausschüttung in eingefrorenem-aufgetautem ACP^® nicht verstärken. Schlussfolgerung und klinische Relevanz: Die Kryokonservierung von ACP^® bei –20 °C könnte ein praxistaugliches Verfahren darstellen. Vor einer Empfehlung bedarf es weiterer Qualitäts tests. Die mechanische Stimulation muss angepasst werden, um die Thrombozyten stärker aktivieren zu können.

Keywords

Platelet concentrate - cryopreservation - activation - horse

Schlüsselwörter

Thrombozytenkonzentrat - Kryokonservierung - Aktivierung - Pferd

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