Development of optimal techniques for cryopreservation of human platelets - I. Platelet activation during cold storage (at 22 and 8 degrees C) and cryopreservation

Using the current blood bank storage conditions at 22 degrees C, the viabil ity 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 i nvasive surgeries such as cardiopulmonary bypass or bone marrow transplanta tion. 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, phosphatidy lserine, on the platelet membrane during storage at 22 and 8 degrees C as w ell as during the different freezing preservation processes was examined us ing flow cytometry and annexin V binding assay. Human platelets were identi fied by both CD41 and light scatter in flow cytometry. In cryopreservation experiments, effects of the following factors on platelet activation were e valuated: (a) cryoprotective agents (CPAs) type: dimethyl sulfoxide (Me2SO) , ethylene glycol (EG), and propylene glycol (PG), (b) CPA concentration ra nging 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, respectiv ely; (b) platelets were not significantly activated after 30-min exposure t o 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 so lution, while in 1 M EG and 1 M PG solutions, 62 and 42% of the platelets s urvived, respectively. Using the information that Me2SO consistently yields higher percentages of nonactivated platelets and does not seem to be cytot oxic to platelets for 30-min exposure time, this was found to be the optima l cryoprotective agent for platelets. In addition, significant Me2SO toxici ty to platelets was not noted until Me,SO concentrations exceeded 2 M. Fina lly, a concentration of 1 M Me,SO proved to be the most effective at all cr yopreservation ending temperatures tested (-10, -30, -60, and -196 degrees C). In conclusion, under the present experimental conditions, a storage tem perature of 8 degrees C appeared to be much better than 22 degrees C. Altho ugh 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 survi val, (C) 1999 Academic Press.