Development of Optimal Techniques for Cryopreservation of Human Platelets: I. Platelet Activation during Cold Storage (at 22 and 8°C) and Cryopreservation

• Published in: Cryobiology 1999-05, Vol.38 (3), p.225-235
• Main Authors: Gao, D.Y., Neff, K., Xiao, H.Y., Matsubayashi, H., Cui, X.D., Bonderman, P., Bonderman, D., Harvey, K., McIntyre, J.A., Critser, J., Miraglia, C.C., Reid, T.
• Format: Article
• Language: English
• Subjects: activation; Anesthesia. Intensive care medicine. Transfusions. Cell therapy and gene therapy; annexin V; Biological and medical sciences; Blood Platelets - cytology; Blood Platelets - physiology; Blood Preservation - methods; Blood. Blood and plasma substitutes. Blood products. Blood cells. Blood typing. Plasmapheresis. Apheresis; Cell Membrane - metabolism; Cell Survival; Cold Temperature; cryopreservation; Cryopreservation - methods; Cryoprotective Agents; Dimethyl Sulfoxide; Ethylene Glycol; human platelets; Humans; In Vitro Techniques; Medical sciences; phosphatidylserine; Phosphatidylserines - blood; Platelet Activation; Platelet Transfusion; Propylene Glycol; Time Factors; Transfusions. Complications. Transfusion reactions. Cell and gene therapy
• ISSN: 0011-2240; 1090-2392
• DOI: 10.1006/cryo.1999.2162

Using the current blood bank storage conditions at 22°C, the viability and function of human platelets can be maintained for only 5 days. This does not allow for the necessary and extensive banking of platelets needed to treat patients afflicted with thrombocytopenia, a side effect of many invasive surgeries such as cardiopulmonary bypass or bone marrow transplantation. The development of optimal techniques for long-term cryopreservation and banking of human platelets would provide the ability to greatly extend the viable life of the platelet and would fulfill an increasing and urgent need in many clinical applications. To determine the optimal techniques for platelet preservation, the expression of an activation marker, phosphatidylserine, on the platelet membrane during storage at 22 and 8°C as well as during the different freezing preservation processes was examined using flow cytometry and annexin V binding assay. Human platelets were identified by both CD41 and light scatter in flow cytometry. In cryopreservation experiments, effects of the following factors on platelet activation were evaluated: (a) cryoprotective agents (CPAs) type: dimethyl sulfoxide (Me2SO), ethylene glycol (EG), and propylene glycol (PG), (b) CPA concentration ranging from 0 to 3 M, and (c) ending temperatures of a slow cooling process at −1°C/min. Our results demonstrated that (a) approximately 50% of platelets were activated on days 7 and 16 at 22 and 8°C, respectively; (b) platelets were not significantly activated after 30-min exposure to 1 M Me2SO, EG, and PG at 22°C, respectively, and (c) there was a significant difference in cryoprotective efficacy among these three CPAs in preventing platelets from cryoinjury. After being cooled to −10°C, 74% of the cryopreserved platelets survived (nonactivated) in 1 M Me2SO solution, while in 1 M EG and 1 M PG solutions, 62 and 42% of the platelets survived, respectively. Using the information that Me2SO consistently yields higher percentages of nonactivated platelets and does not seem to be cytotoxic to platelets for 30-min exposure time, this was found to be the optimal cryoprotective agent for platelets. In addition, significant Me2SO toxicity to platelets was not noted until Me2SO concentrations exceeded 2 M. Finally, a concentration of 1 M Me2SO proved to be the most effective at all cryopreservation ending temperatures tested (−10, −30, −60, and −196°C). In conclusion, under the present experimental conditions, a storage temperatu