A Flow Cytometric Analysis of Damage and Effect of Additives on The Membrane Structure of Frozen Red Blood Cells.
Abstract
Cryopreservation of erythrocytes (RBC) became possible when it was discovered that when mixed with glycerol, RBC could be successfully frozen and thawed. The disadvantages of this method were that glycerol had to be removed from the RBC because it is harmful to the patient and the expense involved. A simple, inexpensive wash procedure has not been developed. The ideal cryoprotectant should be safe for infusion. A one-step technique that eliminates the wash process would have significant impact on transfusion medicine. Protection of RBC from freeze injury and acceptable in vivo survival must also be achieved. The present study was designed to develop a one- step technique using phospholipid-like additives to achieve less than 2% supernatant hemolysis and to develop a flow cytometric assay for quantitation of membrane damage. Cryopreservatives used were hydroxyethyl starch (HES), Viastarch, and Cell Sep. L- carnitine and urea were used as phospholipid-like additives. Supernatant hemolysis was calculated from total and supernatant hemoglobin concentrations. RBC &count and mean corpuscular volume (MCV) were determined on all samples before and after cryopreservation to further evaluate hemolysis. Cryopreservation with Cell Sep resulted in the least amount of hemolysis, but the goal of less than 2% hemolysis, with and without additives, was not achieved. The RBC count did not change significantly, but the MCV was slightly increased in all samples post-thaw. The use of flow cytometric analysis was investigated to quantitate RBC membrane damage. Fluorescent labeled antibodies to intracellular antigens were used to label cells with sufficiently large holes to allow entry and attachment of the labeled antibody. Cells without holes should not allow the labeled antibody to enter and attach. There was no difference in fluorescense of cells that had not been frozen and those that had, disallowing the use of these assays to quantitate membrane damage.
Document Details
- Document Type
- Technical Report
- Publication Date
- Oct 01, 1998
- Accession Number
- ADA354220
Entities
People
- Margaret C. Hawkins
Organizations
- Bowling Green State University