Background: Red blood cell (RBC) preservation is essential to transfusion medicine. Many blood group reference laboratories need a method to preserve rare blood samples for serologic testing at a later date. This study offers a comparison of three common cryoprotective agents and protocols used today: bulk preservation with glycerol and droplet freezing with sucrose-dextrose (S+D) or polyvinylpyrrolidone (PVP).Study Design and Methods: Human blood from 14 volunteers was collected and frozen at set intervals over 2 weeks with PVP, S+D, or glycerol. The frozen RBCs were later thawed and the percentage of surviving RBCs was determined. Detailed protocols and an instructional video are supplied.Results: Over a 2-week period, RBCs preserved with glycerol and thawed with a widely used protocol showed a recovery of 41 ± 16% (mean ± standard deviation) while those thawed with a modified glycerol protocol showed a recovery of 76 ± 8%. RBCs preserved by droplet freezing with S+D showed a recovery of 56 ± 11% while those preserved by droplet freezing with PVP showed a recovery of 85 ± 6%. Recovery values were similar with ethylenediaminetetraacetic acid or heparin anticoagulants, differing freezing rates, and varying droplet volumes.Conclusion: Droplet freezing with PVP offered the greatest recovery. While bulk freezing with glycerol can also be effective, droplet freezing may be a more convenient method overall. It requires less effort to thaw, needs much less storage room, and allows blood group laboratories to be frugal with thawing rare samples.