IMPROVED METHOD FOR CRYOPRESERVATION OF HUMAN RED-CELLS IN LIQUID-NITROGEN WITH HYDROXYETHYL STARCH

Refinement of a method described previously made possible routine freezing of full units of packed erythrocytes after separation of platelet rich plasma and buffy coats. The volume frozen was 405 ml which included packed red cells (190-220 ml), plasma (43-73 ml) and cryo-HES [hydroxyethyl starch] (142 ml, final concentration 14% w/v). The units could be frozen with or without shaking by direct immersion in liquid N. Thawing was accomplished by transferring units quickly from liquid N storage to a shaking water bath at 54.degree. C. The average yield from units of red cells was 98.4%. The stability to a 50-fold dilution in 0.15 M NaCl was 87.8%. Thawing rate was the critical variable in producing the most stable thawed cells. Plasma expander HES was usable but the thawed units were more viscous and about 7% less stable. Red cells prewashed with 0.15 M NaCl and frozen without plasma showed no significant changes in cellular yield or stability. The optimum resuspension medium was 3% glucose. A morphologic study of cells fixed in 1% glutaraldehyde revealed that before freezing red cells were partially dehydrated in 14% HES. These were smooth, flat discs. Cells fixed on thawing were extensively dehydrated and seen as large, thin, smooth, flat discs with approximately 10% echinocytes. On dilution with 6% glucose (1:1) these swelled and reverted to biconcave discocytes except for approximately 5% echinocytes. Storage in liquid N measured in groups of 3 units of 15 units for 0, 3, 6, 9 and 12 wk revealed normal postthawed O2 delivery. The greatest measurable effect of freezing red cells in HES was a loss of cellular K+ compensated by a corresponding increase in Na+.