Compositions and methods for the storage of red blood cells

Abstract

The present invention provides an aqueous composition for storage of red blood cells consisting essentially of: adenine; dextrose; at least one nonmetabolizable membrane-protectant sugar; and a specifically defined pH buffering system. Also provided are improved methods for preserving red blood cells and methods for increasing the viability, membrane retention, and recoverability while suppressing apoptosis, hemolysis, and post-reinfusion clearance of stored red blood cells which utilize the novel compositions.

1 . A method of preserving red blood cells (RBCs) for a storage period comprising:
 (a) mixing a sample of collected whole blood containing the RBCs to be stored and plasma with an anticoagulant solution, thereby forming a suspension of collected whole blood;   (b) treating the suspension of collected whole blood to deplete the plasma and concentrate the RBCs, thereby forming packed RBCs;   (c) mixing the packed RBCs with an amount of an aqueous composition sufficient to form a suspension of RBCs having about 35% to about 70 RBCs by volume;   (d) cooling the suspension of RBCs to about 1 to about 6° C.; and   (e) storing the cooled suspension of RBCs according to standard bank procedures,   
       wherein the aqueous composition consists essentially of:
 adenine; 
 dextrose; 
 at least one nonmetabolizable membrane-protectant sugar; and a pH buffering system, 
 wherein the pH buffering system comprises a combination of physiologically acceptable buffering agents including at least one agent providing bicarbonate anions, at least one agent providing phosphate anions, and at least one agent providing sodium cations, wherein the pH buffering system is present in an amount sufficient for the composition to be operable to maintain a pH of a red blood cell (RBC) suspension to which the composition is added at a value sufficient to establish and maintain during a storage period a reaction equilibrium in the red blood cell that favors glycolysis over synthesis of 2,3-diphosphoglycerate (DPG) from 1,3-DPG, thereby generating a net gain in adenosine tri phosphate (ATP) synthesis with respect to the reaction equilibrium during the storage period. 
 
     
     
         2 . The method of preserving RBCs as recited in  claim 1  wherein the suspension of RBCs is suitable for direct infusion into a patient in need of such an infusion. 
     
     
         3 . The method of preserving RBCs as recited in  claim 1  wherein the at least one nonmetabolizable membrane-protectant sugar is mannitol. 
     
     
         4 . The method of preserving RBCs as recited in  claim 1  wherein the at least one agent providing sodium cations is selected from the group consisting of sodium bicarbonate, disodium phosphate, and combinations thereof. 
     
     
         5 . The method of preserving RBCs as recited in  claim 1  wherein the at least one agent providing bicarbonate anions is sodium bicarbonate. 
     
     
         6 . The method of preserving RBCs as recited in  claim 1  wherein the at least one agent providing phosphate anions is selected from the group consisting of sodium phosphate, disodium phosphate, trisodium phosphate, and combinations thereof. 
     
     
         7 . The method of preserving RBCs as recited in  claim 1  wherein the at least one agent providing phosphate ions to the aqueous composition is disodium phosphate. 
     
     
         8 . The method of preserving RBCs as recited in  claim 1  wherein the combination of physiologically acceptable buffering agents additionally comprises at least one agent providing a physiologically acceptable cation selected from the group consisting of hydrogen, potassium, ammonium, magnesium, and combinations thereof. 
     
     
         9 . The method of preserving RBCs as recited in  claim 1  wherein the composition has an osmolarity of from about 200 to about 310 mOsm. 
     
     
         10 . The method of preserving RBCs as recited in  claim 9  wherein the osmolarity is from about 221 to about 280 mOsm. 
     
     
         11 . The method of preserving RBCs as recited in  claim 10  wherein the osmolarity is about 270 mOsm. 
     
     
         12 . The method of preserving RBCs as recited in  claim 1  wherein the composition has a pH of from about 8 to about 9. 
     
     
         13 . The method of preserving RBCs as recited in  claim 12  wherein the pH is from about 8.2 to about 8.8. 
     
     
         14 . The method of preserving RBCs as recited in  claim 13  wherein the pH of the composition is from about 8.4 to about 8.6. 
     
     
         15 . The method of preserving RBCs as recited in  claim 14  wherein the pH of the composition is about 8.5. 
     
     
         16 . The method of preserving RBCs as recited in  claim 1  wherein the buffering system has a buffering capacity in the red blood cell (RBC) suspension to which the composition is added which increases by 2 mEq between a pH of 6.5 and 7.2 over a storage period of 6 weeks. 
     
     
         17 . The method of preserving RBCs as recited in  claim 1  wherein the composition is operable to maintain the pH of the red blood cell (RBC) suspension to which the composition has been added at between about 6.4 and about 7.4. 
     
     
         18 . The method of preserving RBCs as recited in  claim 1  wherein the composition is operable to maintain the pH of the red blood cell (RBC) suspension to which the composition has been added at between 7.0 and less than about 7.2. 
     
     
         19 . The method of preserving RBCs as recited in  claim 1  wherein the composition is operable to maintain the pH of the red blood cell (RBC) suspension to which the composition has been added at a value greater than about 7.1 and less than 7.2. 
     
     
         20 . The method of preserving RBCs as recited in  claim 1  wherein the composition comprises adenine in an amount of about 1-3 mM, dextrose in an amount of from about 20 to about 115 mM, nonmetabolizable membrane-protectant sugar in an amount of about 15 to about 60 mM, sodium bicarbonate in an amount from about 20 to about 130 mM, and disodium phosphate in an amount of from about 4 to about 20 mM. 
     
     
         21 . The method of preserving RBCs as recited in  claim 20  wherein the composition comprises adenine in an amount of about 2 mM, dextrose in an amount of from about 60 to about 100 mM, nonmetabolizable membrane-protectant sugar in an amount of about 40 to about 60 mM, sodium bicarbonate in an amount of from about 22 to about 40 mM, and disodium phosphate in an amount of from about 7 to about 15 mM. 
     
     
         22 . The method of preserving RBCs as recited in  claim 21  wherein the composition comprises adenine in an amount of about 2 mM, dextrose in an amount of about 80 mM, nonmetabolizable membrane-protectant sugar in an amount of about 55 mM, sodium bicarbonate in an amount of about 26 mM, and disodium phosphate in an amount of about 12 mM, and further wherein the composition has a pH of about 8.5. 
     
     
         23 . The method of preserving RBCs as recited in  claim 1  wherein the volume ratio of the composition to the collected whole blood is about 1:4.5. 
     
     
         24 . The method of preserving RBCs as recited in  claim 1  wherein the volume of the composition is about 110 mL and the volume of the collected whole blood is about 500 mL. 
     
     
         25 . A method of improving red blood cell (RBC) membrane maintenance and suppressing RBC apoptosis during a storage period, the method comprising: storing the RBCs during the storage period in a suspension to which the composition as recited in  claim 1  has been added. 
     
     
         26 . A method of improving RBC membrane maintenance and suppressing RBC apoptosis during a storage period as recited in  claim 25 , wherein an indicator of membrane maintenance is a measure of microvesicle concentration on the RBC at the end of a storage period, and wherein the microvesicle concentration is reduced by about 50 to 75 percent. 
     
     
         27 . A method of decreasing red blood cell (RBC) fragility and suppressing RBC hemolysis during a storage period, the method comprising: storing the RBCs during the storage period in a suspension to which the composition as recited in  claim 1  has been added. 
     
     
         28 . A method of increasing viability of red blood cells (RBCs) subsequent to a storage period and after infusion into a patient in need of such an infusion, and decreasing a rate of post-infusion clearance of the RBCs by the patient, the method comprising: storing the RBCs during the storage period in a suspension to which the composition as recited in  claim 1  has been added. 
     
     
         29 . The method of preserving RBCs as recited in  claim 1 , wherein the volume ratio of the composition to the collected whole blood is in accordance with standard blood bank procedures. 
     
     
         30 . A method of preserving red blood cells (RBCs) for a storage period comprising:
 (a) mixing a sample of collected whole blood containing the RBCs to be stored and plasma with an anticoagulant solution, thereby forming a suspension of collected whole blood;   (b) treating the suspension of collected whole blood to deplete the plasma and concentrate the RBCs, thereby forming packed RBCs;   (c) mixing the packed RBCs with an amount of an aqueous composition sufficient to form a suspension of RBCs having about 27% to about 70% RBCs by volume;   (d) cooling the suspension of RBCs to about 1 to about 6° C.; and   (e) storing the cooled suspension of RBCs according to standard bank procedures,   wherein the aqueous composition consists essentially of:
 adenine; 
 dextrose; 
 at least one nonmetabolizable membrane-protectant sugar; and a pH buffering system, 
   wherein the pH buffering system comprises a combination of physiologically acceptable buffering agents including at least one agent providing bicarbonate anions, at least one agent providing phosphate anions, and at least one agent providing sodium Cations, wherein the pH buffering system is present in an amount sufficient for the composition to be operable to maintain a pH of red blood cell (RBC) suspension to which the composition is added at a value sufficient to establish and maintain during a storage period a reaction equilibrium in the red blood cell that favors glycolysis over synthesis of 2,3-diphosphoglycerate (DPG) from 1,3-DPG, thereby generating a net gain in adenosine tri phosphate (ATP) synthesis with respect to the reaction equilibrium during the storage period.   
     
     
         31 . A method of preserving red blood cells (RBCs) for a storage period comprising:
 (a) mixing a sample of collected whole blood containing the RBCs to be stored and plasma with an anticoagulant solution, thereby forming a suspension of collected whole blood;   (b) treating the suspension of collected whole blood to deplete the plasma and concentrate the RBCs, thereby forming packed RBCs;   (c) mixing the packed RBCs with an amount of an aqueous composition sufficient to form a suspension of RBCs which has a percentage of RBCs by volume within the range produced by standard blood bank procedures;   (d) cooling the suspension of RBCs to about 1 to about 6° C.; and   (e) storing the cooled suspension of RBCs according to standard bank procedures,   
       wherein the aqueous composition consists essentially of:
 adenine; 
 dextrose; 
 at least one nonmetabolizable membrane-protectant sugar; and a pH buffering system, 
 wherein the pH buffering system comprises a combination of physiologically acceptable buffering agents including at least one agent providing bicarbonate anions, at least one agent providing phosphate anions, and at least one agent providing sodium cations, wherein the pH buffering system is present in an amount sufficient for the composition to be operable to maintain a pH of a red blood cell (RBC) suspension to which the composition is added at a value sufficient to establish and maintain during a storage period a reaction equilibrium in the red blood cell that favors glycolysis over synthesis of 2,3-diphosphoglycerate (DPG) from 1,3-DPG, thereby generating a net gain in adenosine tri phosphate (ATP) synthesis with respect to the reaction equilibrium during the storage period. 
 
     
     
         32 . A method of preserving red blood cells (RBCs) for a storage period comprising:
 (a) mixing a sample of collected whole blood containing the RBCs to be stored and plasma with an anticoagulant solution, thereby forming a suspension of collected whole blood;   (b) treating the suspension of collected whole blood to deplete the plasma and concentrate the RBCs, thereby forming packed RBCs;   (c) mixing the packed RBCs with a volume of aqueous composition ranging from about 60 mL to about 400 mL;   (d) cooling the suspension of RBCs to about 1 to about 6° C.: and   (e) storing the cooled suspension of RBCs according to standard bank procedures,   
       wherein the aqueous composition consists essentially of:
 adenine; 
 dextrose; 
 at least one nonmetabolizable membrane-protectant sugar; and a pH buffering system, 
 wherein the pH buffering system comprises a combination of physiologically acceptable buffering agents including at least one agent providing bicarbonate anions, at least one agent providing phosphate anions, and at least one agent providing sodium cations, wherein the pH buffering system is present in an amount sufficient for the composition to be operable to maintain a pH of a red blood cell (RBC) suspension to which the composition is added at a value sufficient to establish and maintain during a storage period a reaction equilibrium in the red blood cell that favors glycolysis over synthesis of 2,3-diphosphoglycerate (DPG) from 1,3-DPG, thereby generating a net gain in adenosine tri phosphate (ATP) synthesis with respect to the reaction equilibrium during the storage period.