Method of isolating and cryopreserving human white cells from whole blood
AssigneeThe United States of America as represented by the Secretary of the Navy
InventorsFabian J. Lionetti, Stephen M. Hunt
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Abstract
A method of salvaging granulocytes, or white cells, from buffy coat of ceifuged whole blood is provided. The sequence of operations leading to the separation red cells, white cells, platelets and plasma is presented in a flow diagram of a bag system for the cryopreservation of leukocytes, with freezing of the white cells accomplished by the introduction of a combination of hydroxyethyl starch (HES), which functions as both a sedimenting agent and a cryoprotective agent, and dimethylsulfoxide (DMSO), a cryoprotective agent. A preferred combination is 4% HES with 5% DMSO. White cell separation is interfaced with collection methods for plasma and platelets so as to conserve all major cell types.
Claims10
A method of salvaging human white blood cells from the buffy coat of centrifuged human whole blood comprising:
- isolating and removing by centrifuging the platelet rich plasma and a selected volume of the buffy coat of the topmost red cells from a blood collection bag containing a unit of whole blood;
- preparing a 40% weight/volume solution of hydroxyethyl starch in water and further diluting this solution in an isotonic solution of salts containing 148 mequiv per liter each of cations Na.sup.+ 140, K.sup.+ 5 and Mg.sup.2.sup.+ 3, and anions including chloride 98, acetate 27 and gluconate 23 to dilutions on the order of from 10 to 16% of the 40% weight/volume solution;
- obtaining a concentration of 4% hydroxyethyl starch at a hematocrit of 0.30 from said diluted solution by admixing selected volumes of concentrated hydroxyethyl starch and sedimenting out the red cells in the cell mixture;
- siphoning off the red cells from said mixture of separated and concentrated cells into a sterile bag for storage;
- admixing with the remaining white cells a sterile solution of 10% dimethylsulfoxide in said isotonic solution with said sedimented white cells;
- freezing said white cells and storing the frozen white cells in an environment maintained at substantially -80.degree. C;
- thawing said concentration of white cells,
- admixing with said thawed cells a sterile wash solution containing an isotonic solution as defined above but adjusted to a pH of 7.4 containing citrate-phosphate-dextrose in a 10 ml volume;
- removing the supernatant layer of substantially 8.0 ml;
- and
- resuspending the residue of white cells in said adjusted isotonic solution by gentle agitation.
A system for salvaging human white cells from human whole blood comprising:
- centrifuging whole blood and obtaining a selected volume of the buffy coat of the topmost red cells thereof;
- sedimenting said buffy coat red cells with hydroxyethyl starch and dimethylsulfoxide in an isotonic solution of salts;
- separating said red cells from said solution and admixing with the remaining white cells a sterile solution of dimethylsulfoxide in said isotonic solution;
- freezing said white cells and storing said frozen white cells in an environment maintained at substantially -80.degree. C;
- thawing said frozen white cells and washing said thawed white cells in a solution containing an isotonic solution of salts;
- removing the supernatant layer of said isotonic solution of salts and white cells;
- and
- resuspending the residue of white cells in said adjusted isotonic solution by gentle agitation.
Dependencies are inferred from the claim text.