Cryopreservation of feline red blood cells in liquid nitrogen using glycerol and hydroxyethyl starch.

Abstract

The objective of this study was to evaluate the techniques and short-term effects of cryopreservation of feline red blood cells (RBCs) in liquid nitrogen using glycerol or hydroxyethyl starch (HES) as a cryoprotectant. Feline RBCs were manually mixed with either 20% glycerol or 12.5% HES and frozen for 24 h in liquid nitrogen. The samples were thawed and glycerolized samples were manually washed. Success of the freeze/thaw process was determined by recovery rate of RBCs and evaluation of morphological changes using scanning electron microscopy (SEM). A unit of canine packed RBCs was also subjected to the same methodology to evaluate the cryopreservation handling technique. Feline RBCs preserved with 20% glycerol had a high recovery rate (94.23 ± 1.25%) immediately after thawing. However, the majority of the cells were lost during the washing process, with a final packed cell volume of <1%. A recovery rate was unable to be assessed for samples preserved with HES owing to the high viscosity of the mixture. SEM revealed significant morphological changes after glycerol was added to the feline RBCs. Although these morphological changes were partially reversed after thawing, the majority of the RBCs were lost during the washing process. Minimal morphological changes were noted in the HES sample. Similar results were noted with the canine RBCs. The described manual technique for cryopreservation using glycerol was not able to successfully preserve feline or canine RBCs. In the present study, it was difficult to make conclusions about the efficacy of HES. Further studies evaluating HES as a cryoprotectant are warranted.