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Using the current blood bank storage conditions at 22 degrees 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 degrees 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 degrees C/min. Our results demonstrated that (a) approximately 50% of platelets were activated on days 7 and 16 at 22 and 8 degrees C, respectively; (b) platelets were not significantly activated after 30-min exposure to 1 M Me2SO, EG, and PG at 22 degrees 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 degrees 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 degrees C). In conclusion, under the present experimental conditions, a storage temperature of 8 degrees C appeared to be much better than 22 degrees C. Although the potential chemical toxicity of 1 M Me2SO, EG, or PG is negligible, 1 M Me2SO was found to be optimum for cryopreservation of human platelets. PG has the least cryoprotective function for low-temperature platelet survival.

1. (1) Determination of the decrease in light absorbance of platelet suspensions appeared to be a useful screening test in the search for optimal conditions for the cryopreservation of platelets. This decrease was due to morphological damage of the platelets. 2. (2) Cryopreservation studies on human platelets included: variation of type and concentration of cryoprotectant, variation of cooling rate, and investigation of a “two-step” cooling method (rapid cooling to a specific subzero temperature, followed by rapid cooling to −196 °C). 3. (3) Best results were obtained with dimethyl sulfoxide (DMSO) and N,N-dimethylacetamide (DMAC) at external cooling rates from 1 to 50 °C/min.

Summary During the past decade efforts to preserve platelets have indicated that storage at 4°C will not maintain functional integrity. The current anticoagulants are acceptable with the proposed modification of ACD solutions. Plastic equipment has been of value to prepare platelet concentrates rapidly. We need to have better plastic materials for the projected temperatures used for storage. While the Cr51 platelet label has been of great value to evaluate methodology, further data are required to determine if isotope labeling of frozen platelets can be used to assess hemostasis. The two preservatives currently used for freezing need comparative studies to define acceptance in a national program. At the present time preservation studies continue to be done in an empiric fashion. Only when we understand platelet membrane functions more completely can we define the cryophysiology of this blood component.