Patent Publication Number: US-2022211030-A1

Title: Irradiation of Red Blood Cells and Anaerobic Storage

Description:
CROSS-REFERENCE TO RELATED APPLICATIONS 
     The present application claims priority based on U.S. Provisional Application No. 61/410,684, filed Nov. 5, 2010, which is incorporated herein by reference in its entirety. 
    
    
     BACKGROUND 
     1. Field 
     The present disclosure relates to a storage blood system having an oxygen/carbon dioxide depletion device and a blood storage bag for the long-term storage of red blood cells (RBCs). More particularly, the present disclosure relates to a blood storage system that is capable of removing oxygen and carbon dioxide from the red blood cells prior to storage and gamma and/or X-ray irradiating red blood cells either pre- or post-anaerobic treatment, as well as maintaining oxygen or oxygen and carbon dioxide depleted states during storage, thereby prolonging the storage life and minimizing deterioration of the deoxygenated red blood cells. 
     2. Background of the Art 
     Adequate blood supply and the storage thereof is a problem facing every major hospital and health organization around the world. Often, the amount of blood supply in storage is considerably smaller than the need therefore. This is especially true during crisis periods such as natural catastrophes, war and the like, when the blood supply is often perilously close to running out. It is at critical times such as these that the cry for more donations of fresh blood is often heard. However, unfortunately, even when there is no crisis period, the blood supply and that kept in storage must be constantly monitored and replenished, because stored blood does not maintain its viability for long. 
     Stored blood undergoes steady deterioration which is, in part, caused by hemoglobin oxidation and degradation and adenosine triphosphate (ATP) and 2-3,biphosphoglycerate (DPG) depletion. Oxygen causes hemoglobin (Hb) carried by the red blood cells (RBCs) to convert to met-Hb, the breakdown of which produces toxic products such as hemichrome, hemin and free Fe 3+ . Together with the oxygen, these products catalyze the formation of hydroxyl radicals (OH.cndot.), and both the OH.cndot. and the met-Hb breakdown products damage the red blood cell lipid membrane, the membrane skeleton, and the cell contents. As such, stored blood is considered unusable after 6 weeks, as determined by the relative inability of the red blood cells to survive in the circulation of the transfusion recipient. The depletion of DPG prevents adequate transport of oxygen to tissue thereby lowering the efficacy of transfusion immediately after administration (levels of DPG recover once in recipient after 8-48 hrs). In addition, these deleterious effects also result in reduced overall efficacy and increased side effects of transfusion therapy with stored blood before expiration date, when blood older than two weeks is used. Reduction in carbon dioxide content in stored blood has the beneficial effect of elevating DPG levels in red blood cells. 
     There is, therefore, a need to be able to deplete oxygen and carbon dioxide levels in red blood cells prior to storage on a long-term basis without the stored blood undergoing the harmful effects caused by the oxygen and hemoglobin interaction. Furthermore, there is a need to store oxygen and carbon dioxide depleted red blood cells in bags containing or in a bag surrounded by a barrier film with oxygen and carbon dioxide depletion materials. Furthermore, there is a need to optimize ATP and DPG levels in stored red blood cells by varying the depletion or scavenging constituents prior to and/or during storage depending upon the needs of the recipient upon transfusion. Furthermore, the blood storage devices and methods must be simple, inexpensive and capable of long-term storage of the blood supply. 
     Another issue relates to transfusion-associated graft-versus-host disease (TA-GVHD) which is a rare but nearly fatal complication associated with transfusion therapy in severely immuno-compromised blood recipients (for example, bone marrow transplant recipient, patients receiving aggressive chemotherapy, premature neonates). Prevention of TA-GVHD requires complete removal of, or arrest of the proliferative potential of T-lymphocytes from donor blood. Although leuko reduction filters are widely in use, they are not adequate in prevention of TA-GVHD because it cannot completely eliminate lymphocytes. Thus, lymphocyte inactivation by gamma-irradiation is currently the only recommended method for TA-GVHD prevention. Since it is a nearly fatal side effect of transfusion, some hospitals and countries irradiate every unit of RBC for TA-GVHD prevention. More commonly, RBC units ordered for specific recipients are irradiated before dispensed to the bedside. 
     Accordingly, anaerobically stored RBC must be compatible with gamma- or X-ray irradiation treatment so that anaerobically stored blood can be transfused to patients requiring irradiated RBC. 
     Gamma-irradiation abrogates proliferation of T-lymphocytes by damaging the DNA directly and via reactive oxygen species (ROS), namely hydroxyl radicals produced during gamma-radiolysis of water. Although red blood cells (RBC) do not contain DNA, ROS generated by gamma-irradiation have been shown to cause significant damage to the RBC. The major damage observed includes: i) increased hemolysis; ii) increased K+ leak; iii) reduction in post-transfusion survival; and iv) reduced deformability. Such damage is similar to, but an exaggerated form of storage-induced damage of RBC. The compromised status of RBC is well known to the physicians who administer such compromised RBC. The FDA mandates restricted use of such RBC in terms of shortened shelf life after gamma-irradiation (14 days) and/or 28 days total shelf life for irradiated units. 
     The irradiation of blood components has received increased attention due to increasing categories of patients eligible to receive such blood to prevent transfusion-associated graft versus host disease. However, irradiation leads to enhancement of storage lesions, which could have deleterious effects when such blood is transfused. It is well known in the field that the main deleterious side-effect of radiation on RBC is oxidative damage caused by ROS. 
     Radiation damage to RBC in the presence of oxygen can occur in two ways;
         i) By ROS generated during and immediately after irradiation. ROS can reside in RBC lipid, then attack proteins and lipids in vicinity later during storage, as well as to initiate peroxidation cycle of lipid and protein using oxygen to fuel.   ii) Met-Hb and its denaturation products generated in i) above act as catalysts to further cause ROS-mediated oxidative damage during subsequent extended refrigerated storage of RBC. This is an enhanced version of storage lesion development using O2.       

     On the other hand, there is ample literature suggesting ROS as a major culprit in causing deterioration of red blood cell (RBC) during refrigerated storage at blood banks, and that storing RBC under anaerobic condition significantly reduce such damages. Studies have shown that irradiated red blood cells that are oxygen and oxygen and carbon dioxide depleted are equivalent or healthier (in terms of K+ leakage, hemolysis and oxidized proteins/lipids) in comparison to non-irradiated and non-oxygen and carbon dioxide depleted blood and non-oxygen and carbon dioxide depleted irradiated blood. In the context of the present application, the higher concentration of potassium in RBC storage media was at levels that indicated red blood cell damage. The present disclosure applies the finding of compatibility of gamma-irradiation with anaerobically stored blood, as well as the protective effects of anaerobic conditions in enhancing ATP, DPG and in reducing oxidative damage during refrigerated storage, to substantially reduce the negative or deleterious effect of gamma- and X-ray irradiation of RBCs in the presence of oxygen. 
     U.S. Pat. No. 5,362,442 to Kent describes adding a scavenger to bind free radicals such as ethanol. U.S. Patent No. 61875572 to Platz et al. describes adding chemical sensitizers; U.S. Pat. No. 6,482,585 to Dottori and U.S. Pat. No. 6,403,124, also to Dottori, describe adding L-carnitine or an alkanoul derivative to reduce RBC cell membrane damage induced by irradiation. These additives are not required to prevent the deleterious effects of irradiation on RBCs when treated anaeorobically. 
     SUMMARY 
     A method and system for gamma or X-ray irradiation of RBC under anaerobic or anaerobic and CO 2  depleted conditions, and extended refrigerated storage of such RBC under anaerobic or anaerobic and/or CO 2  depleted conditions using an oxygen and/or CO 2  depletion device. 
     A method and system for removing plasma with or without platelets, adding an additive solution (e.g., nutrient and/or metabolic supplements) to the concentrated RBC, filtering out leukocytes and/or platelets via a leuko reduction filter, removing oxygen and/or CO 2  from the filtered RBC, and gamma irradiating or X-ray irradiating the oxygen and/or CO 2  filtered RBC either prior to or during storage thereof. The preferred range of gamma irradiation is a minimum of between about 25 Gy to 50 Gy. 
     Gamma or X-ray irradiating RBC under anaerobic or anaerobic and CO 2  conditions (ambient to 1° C.) defined as less than 20% SO 2  (oxygen-saturation of hemoglobin), more preferably less than 5%, and most preferably less than 3%. 
     Storing gamma or X-ray irradiated (either under anaerobic or anaerobic and CO 2  conditions) RBC for extended time at 1-6° C. under anaerobic condition defined as less than 20% SO 2  (oxygen-saturation of hemoglobin), more preferably less than 5%, and most preferably less than 3%. 
     Gamma or x-ray irradiating RBC under anaerobic or anaerobic and CO 2  depleted conditions (ambient to 1° C.) defined as less than 20% SO 2  (oxygen-saturation of hemoglobin), more preferably SO 2 &lt;5%, and most preferably SO 2 &lt;3% and pCO 2 &lt;10 mmHg; pCO 2 &lt;5 mmHg; pCO 2 &lt;1 mmHg. 
     Gamma or x-ray irradiating RBC under aerobic conditions (ambient to 1° C.) and then removing oxygen or oxygen and carbon dioxide from the irradiated RBC to levels defined as less than 20% SO 2  (oxygen-saturation of hemoglobin), more preferably SO 2 &lt;5%, and most preferably SO 2 &lt;3% and pCO 2 &lt;10 mmHg; pCO 2 &lt;5 mmHg; pCO 2 &lt;1 mmHg. The gamma or x-ray irradiation under aerobic conditions and removal of oxygen or oxygen and carbon dioxide can be performed before placing blood for extended storage, or within 24 hr of blood collection, between 1 through 7 days after blood collection or beyond 7 days 
     Using older blood, defined as blood stored for more than one week, and exposing such blood to gamma or x-ray irradiating RBC under aerobic conditions (ambient to 1° C.) and then removing oxygen or oxygen and carbon dioxide from the irradiated RBC to levels defined as less than 20% SO 2  (oxygen-saturation of hemoglobin), more preferably SO 2 &lt;5%, and most preferably SO 2 &lt;3% and pCO 2 &lt;10 mmHg; pCO 2 &lt;5 mmHg; pCO 2 &lt;1 mmHg. 
     Using older blood, defined as blood stored for more than one week, and removing oxygen or oxygen and carbon dioxide from such older blood and exposing such blood to Gamma or x-ray irradiation at wherein the levels of oxygen and carbon dioxide are levels defined as less than 20% SO 2  (oxygen-saturation of hemoglobin), more preferably SO 2 &lt;5%, and most preferably SO 2 &lt;3% and pCO 2 &lt;10 mmHg; pCO 2 &lt;5 mmHg; pCO 2 &lt;1 mmHg. 
     Storing gamma or X-ray irradiated or pre-irradiated RBC (either under anaerobic conditions with or without CO 2  depletion) RBC for extended time at 1-6° C. under anaerobic or anaerobic and CO 2  depleted condition defined as less than 20% SO 2  (oxygen-saturation of hemoglobin), more preferably less than 5%, and most preferably 3% and less than pCO 2 &lt;10 mmHg; pCO 2 &lt;5 mmHg; pCO 2 &lt;1 mmHg. 
     A preferred embodiment includes a blood storage system comprising: a collection vessel for red blood cells; an oxygen or oxygen/carbon dioxide depletion device; tubing connecting the collection vessel to the oxygen or oxygen/carbon dioxide depletion device and the storage vessel for red blood cells that can be gamma or X-ray irradiated and stored under anaerobic or anaerobic and CO 2  depleted condition for extended time. 
     Preferably, the anaerobic or anaerobic and CO 2  condition is measured as an oxygen-saturation of hemoglobin of less than 20% SO 2 , preferably about 5% or less, and most preferably about 3% or less. 
     The oxygen or oxygen/carbon dioxide depletion device comprises: a cartridge; a plurality of gas permeable hollow fibers or sheets extending within the cartridge from an entrance to an exit thereof, wherein the hollow fibers or gas-permeable films are adapted to receiving and conveying red blood cells; and an amount of an oxygen scavenger or both oxygen scavenger and a carbon dioxide scavenger packed within the cartridge and contiguous to and in between the plurality of hollow fibers. 
     Preferably, the oxygen or oxygen/carbon dioxide depletion device comprises: a cartridge; a plurality of hollow fibers or gas-permeable films extending within the cartridge from an entrance to an exit thereof, wherein the hollow fibers or gas-permeable films are adapted to receiving and conveying red blood cells; and a low oxygen or a low oxygen and carbon dioxide environment is created outside the hollow fibers by flowing an inert gas in-between the hollow fibers. 
     The blood storage system further comprising a leuko reduction filter disposed between the collection vessel and the oxygen/carbon dioxide depletion device. The blood storage system further comprising an additive solution vessel in communication with the collection vessel. The blood storage system further comprising a plasma vessel in communication with the collection vessel. 
     A method for storing red blood cells, the method comprising: removing oxygen or oxygen and carbon dioxide from red blood cells to produce anaerobic red blood cells; and storing irradiated RBC with either gamma- or X-ray, thereby producing irradiated anaerobic red blood cells; and storing the irradiated anaerobic or anaerobic and CO 2  depleted red blood cells. 
     The irradiated anaerobic or irradiated anaerobic and CO 2  depleted red blood cells are preferably stored at a temperature from between about 1° C. to about 6° C. under anaerobic conditions. 
     The present disclosure also provides for a device and method of removing carbon dioxide (CO 2 ) in addition to oxygen (O 2 ) prior to or at the onset of anaerobic or anaerobic and CO 2  depleted storage and/or gamma or X-ray irradiation. 
     The present disclosure provides for a blood collection system that incorporates an oxygen or oxygen/carbon dioxide depletion device having an oxygen or oxygen and carbon dioxide sorbent in combination with a filter or membrane to strip oxygen or oxygen and carbon dioxide from the blood during transport to the storage bag, wherein the oxygen/carbon dioxide depleted blood is gamma or X-ray irradiated either prior to or during storage. 
     The present disclosure further provides for a system to deplete the oxygen or oxygen and carbon dioxide from collected red blood cells that includes an (optional additive solution), an oxygen or oxygen and carbon dioxide depletion device, and a blood storage bag that maintains the red blood cells in an oxygen or oxygen and carbon dioxide depleted state after gamma- or X-ray irradiation. 
     The present disclosure and its features and advantages will become more apparent from the following detailed description with reference to the accompanying drawings. 
    
    
     
       BRIEF DESCRIPTION OF THE DRAWINGS 
         FIG. 1 a    illustrates the components of a gamma irradiated, disposable blood anaerobic storage system of the present disclosure. 
         FIG. 1 b    illustrates the components of a second embodiment of a gamma irradiated, disposable blood anaerobic storage system of the present disclosure. 
         FIG. 2 a    illustrates the components of an embodiment of a disposable blood anaerobic storage system that are used in conjunction with RBC irradiation in which red blood cells are irradiated during anaerobic storage. 
         FIG. 2 b    illustrates the components of a second embodiment of a disposable blood anaerobic storage system that are used in conjunction with RBC irradiation. 
         FIG. 3  illustrates a pre-storage oxygen/carbon dioxide depletion device of the present disclosure. 
         FIG. 4  illustrates a first embodiment of a blood storage bag having a storage bag with a secondary outer oxygen film containing an oxygen sorbent in a pocket. 
         FIG. 5 a    illustrates a pre-storage oxygen/carbon dioxide depletion bag having a blood storage bag with a large sorbent sachet enclosed in gas-permeable, red blood cell compatible polymers in contact with the RBCs. 
         FIG. 5 b    illustrates a third embodiment of a blood storage bag having a storage bag a laminated oxygen film barrier with a large sorbent in contact with the RBCs. 
         FIG. 6 a    illustrates a fourth embodiment of a blood storage bag having a secondary configured secondary outer barrier bag surrounding an inner blood storage bag having an oxygen sorbent. 
         FIG. 6 b    illustrates a fifth embodiment of a blood storage bag having a secondary outer barrier bag surrounding an inner blood storage bag having a large oxygen sorbent sachet enclosed in a gas permeable, red blood cell compatible polymers in contact with RBCs. 
         FIGS. 7 a  through 7 c    illustrate an embodiment of a depletion device that depletes oxygen and carbon dioxide from red blood cells prior to storage by a flushing inert gas or inert gas/CO 2  mixture of defined composition around a hollow fiber inside the assembly. 
         FIGS. 8 a  through 8 c    illustrate another embodiment of a depletion device that depletes oxygen and carbon dioxide from red blood cell prior to storage. 
         FIGS. 9 a  through 9 c    illustrate another embodiment of a depletion device that depletes oxygen and carbon dioxide from red blood cells prior to storage wherein oxygen and CO 2  is scavenged by scavenger materials in the core of the cylinder, surrounded by hollow fibers. 
         FIGS. 10 a  through 10 c    illustrate another embodiment of a depletion device that depletes oxygen and carbon dioxide from red blood cells prior to storage wherein oxygen and CO 2  is scavenged by scavenger materials surrounding cylinders of hollow fibers enveloped in gas permeable, low water vapor transmission material. 
         FIG. 11  illustrates a plot of flow rate of RBC suspension per minute versus oxygen partial pressure for the depletion devices of  FIGS. 7 a  through 7 c   ,  FIGS. 8 a  through 8 c   ,  FIGS. 9 a  through 9 c    and  FIGS. 10 a    through  10   c.    
         FIGS. 12 a  through 12 h    illustrate plots of the effect of oxygen and oxygen and carbon dioxide depletion on metabolic status of red blood cells during refrigerated storage. 
         FIG. 13  illustrates an effect of gamma-irradiation on K+ leak rates from RBC (as measured by free K+ concentrations in RBC suspending media after storage). 
     
    
    
     DETAILED DESCRIPTION OF THE PREFERRED EMBODIMENT 
     RBCs do not require oxygen for their own survival. It was shown previously that when RBCs were stored in blood bank refrigerator (1-6° C.) under anaerobic or anaerobic and CO 2  depleted conditions, they demonstrated significantly improved post-transfusion recovery after 6-week storage compared to the conventionally stored controls. The mechanisms of reduction in storage lesions under anaerobic or anaerobic/CO 2  depleted conditions have been described and direct evidences demonstrated. It is, at least in part, due to reduction in oxidative damages in the presence of O 2  caused by ROS during refrigerated storage. 
     Because gamma- or X-ray irradiation exacerbate oxidative damage on treated RBC, storing irradiated RBC under anaerobic and, optionally, CO 2  depleted condition is not expected to intensify the damage; it is also expected to prevent damage resulting from ROS generated during irradiation by depriving O 2  that fuels those reactions. 
     Effectiveness of gamma- or X-ray irradiation is not dependent on the presence of oxygen. In contrast, anaerobic condition is shown to be more effective in causing damage to DNA (and thus inhibiting proliferation of lymphocytes). Furthermore, absence of O 2  during and/or immediately after gamma- or X-ray irradiation will reduce O 2 -fueled oxidative damages to RBC induced by hydroxyl radicals and ROS produced by radiolysis of water with gamma- or X-rays. 
     Referring to the drawings and in particular to  FIG. 1 a   , a disposable blood anaerobic storage system is shown and referenced using reference numeral  10 . The blood storage system includes an oxygen/carbon dioxide depletion device  100  (OCDD  100 ), an anaerobic blood storage bag  200  and an additive solution bag  300 . OCDD  100  removes oxygen and/or carbon dioxide from red blood cells traveling through it. The system also contains a leuko reduction filter  400 . Components conventionally associated with the process of blood collection are a phlebotomy needle  410 , a blood collection bag  420  containing an anti-coagulant and a bag  430  containing plasma. Tubing can connect the various components of the blood storage system  10  in various configurations (one embodiment shown). Tube  440  connects collection bag  420  with leuko reduction filter  400 . Tube  441  connects additive solution bag  300  with collection bag  420 . Tube  442  connects plasma bag  430  with collection bag  420 . Tube  443  connects leukoreduction filter  400  with OCDD  100 . Tube  414  connects OCDD  100  with blood storage bag  200 . Blood storage system  10  is preferably a single-use, disposable, low cost system. As filtered and oxygen or oxygen and carbon dioxide depleted blood passes from OCDD  100  to blood storage bag  200 . Blood stored in bag  200  will be gamma and/or X-ray irradiated during storage via device  453 . Bag  200  containing oxygen depleted or oxygen and carbon dioxide depleted RBC is placed into device  453  and exposed to gamma and/or X-ray radiation. Alternatively, pre-anaerobic blood stored in collection bag  421  can be gamma and/or X-ray irradiated via device  445  before passing through OCDD  100  and stored in bag  200 , as shown in  FIG. 1 b   . In  FIG. 1 b   , bag  420  could also be gamma and/or X-ray irradiated in an irradiating device  445  prior to passing through leukoreduction filter  400 . 
     Oxygen or oxygen/carbon dioxide depletion device  100  removes the oxygen from collected RBCs prior to the RBCs being stored in blood storage bag  200 . The oxygen content in RBCs must be depleted from oxy-hemoglobin because more than 99% of such oxygen is hemoglobin-bound in venous blood. Preferably, the degree of oxygen saturation is to be reduced to less than 4% within 48 hours of blood collection. The oxygen depletion is preferably accomplished at room temperature. The affinity of oxygen to hemoglobin is highly dependent on the temperature, with a p50 of 26 mmHg at 37° C. dropping to ˜4 mmHg at 4° C. Furthermore, this increase in O 2  affinity (Ka) is mainly due to reduction in O 2  release rate (k-off), resulting in an impractically low rate of oxygen removal once RBC is cooled to 4° C. Thus, it places a constraint on oxygen stripping such that it may be preferable to accomplish it before RBC are cooled to storage temperatures of 1° C. to 6° C. 
     In addition to oxygen depletion, carbon dioxide depletion has the beneficial effect of elevating DPG levels in red blood cells. Carbon dioxide exists inside RBCs and in plasma in equilibrium with HCO 3   −  ion (carbonic acid). Carbon dioxide is mainly dissolved in RBC/plasma mixture as carbonic acid and rapid equilibrium between CO 2  and carbonic acid is maintained by carbonic anhydrase inside RBC. Carbon dioxide is freely permeable through RBC membrane, while HCO 3   −  inside RBC and plasma is rapidly equilibrated by anion exchanger (band 3) protein. When CO 2  is removed from RBC suspension, it results in the known alkalization of RBC interior and suspending medium. This results from removal of HCO 3   −  inside and outside RBC; cytosolic HCO 3   −  is converted to CO 2  by carbonic anhydrase and removed, while plasma HCO 3   −  is removed via anion exchange inside RBC. Higher pH inside RBC is known to enhance the rate of glycolysis and thereby increasing ATP and DPG levels. ATP levels are higher in Ar/CO 2  (p&lt;0.0001). DPG was maintained beyond 2 weeks in the Argon purged arm only (p&lt;0.0001). Enhanced glycolysis rate is also predicted by dis-inhibition of key glycolytic enzymes via metabolic modulation and sequesterization of cytosolic-free DPG upon deoxygenation of hemoglobin as a result of anaerobic condition. DPG was lost at the same rate in both control and Ar/CO 2  arms (p=0.6) despite thorough deoxygenation of hemoglobin, while very high levels of ATP were achieved with OFAS3 additive ( FIGS. 12 a -12 d   ). 
     Referring to the drawings, and in particular to  FIG. 2 a   , another embodiment of a disposable blood anaerobic storage system is shown and referenced using reference numeral  500 . The anaerobic conversion system includes an oxygen or oxygen/carbon dioxide depletion device  515  (OCDD) and an anaerobic blood storage bag  528 . OCDD  515  removes oxygen or oxygen and carbon dioxide from red blood cells traveling through it. Tubing connects the various components of the blood storage system  500 . Tube  512  connects to RBC concentrate prepared by using an additive solution (e.g., AS1, AS3, AS5, SAGM, MAPS, etc.) and storing in bag  528  by passing aforementioned RBC concentrate from collection bag  510  through OCDD  515 . Tubes  518  and  520  connect OCDD  515  with blood storage bag  528 . Blood storage system  500  is preferably a single-use, disposable, low cost system. Oxygen and/or carbon dioxide depleted blood is gamma and/or X-ray in blood storage bag  528  via device  553  and subsequently stored for later transfusion. 
     Alternatively, blood in collection bag  510  may be gamma- or X-ray irradiated via device  551  prior to oxygen or oxygen and carbon dioxide depletion and low temperature storage, as shown in  FIG. 2 b   .  FIG. 2 b    applies to the scenario in which blood bag  510  contains older, for example 2 day old blood, that is then irradiated and depleted of oxygen or oxygen and or carbon dioxide, and stored. 
     Referring to  FIG. 3 , an oxygen or oxygen/carbon dioxide depletion device (OCDD)  101  contains an oxygen sorbent  110 . OCDD  101  is a disposable cartridge  105  containing oxygen sorbent  110  and a series of hollow fibers  115 . Oxygen sorbent  110  is a mixture of non-toxic inorganic and/or organic salts and ferrous iron or other materials with high reactivity toward oxygen. Oxygen sorbent  110  is made from particles that have significant absorbing capacity for O 2  (more than 5 ml O 2 /g) and can maintain the inside of cartridge  105  to less than 0.01% which corresponds to PO 2  less than 0.08 mmHg. Oxygen sorbent  110  is either free or contained in an oxygen permeable envelope. OCDD  101  of the present disclosure must deplete approximately 100 mL of oxygen from a unit of blood. 
     After oxygen and, optionally, carbon dioxide have been stripped from RBCs in the OCDD of  FIG. 3 , RBCs are stored in a blood storage bag  200 . The oxygen content of RBC suspended in additive solution  300  must be reduced to equal to or less than 4% SO 2  before placing them in refrigerated storage. Further, oxygen depleted RBC must be kept in an anaerobic state and low carbon dioxide state throughout entire storage duration. 
     RBCs pass through an oxygen permeable film or membrane, that may be formed as hollow fibers  115  of  FIG. 3 . The membrane or films may be constructed in a flat sheet or hollow fiber form. The oxygen permeable films can be non porous materials that are capable of high oxygen permeability rates (polyolefins, silicones, epoxies, polyesters, etc.) and oxygen permeable membranes are hydrophobic porous structures. These may be constructed of polymers (e.g., polyolefins, Teflon, PVDF, or polysulfone) or inorganic materials (e.g., ceramics). Oxygen depletion takes place as RBC pass through hollow fibers  115 . Oxygen permeable films or oxygen permeable membranes may be extruded into sheets or hollow fibers  15 . Accordingly, hollow fibers  115  and sheets may be used interchangeably. OCDD provides a simple structure having a large surface area to remove oxygen and maintain constant flow of blood therethrough. The oxygen depletion or removal is accomplished by irreversible reaction of ferrous ion in oxygen sorbent  110  with ambient oxygen to form ferric oxide. OCDD  101  does not need agitation for oxygen removal and can be manufactured easily to withstand centrifugation as part of a blood collection system as necessary. 
     Referring to  FIGS. 7 a  through 7 c    and  FIGS. 8 a  through 8 c   , examples of flushing depletion devices are disclosed. The depletion devices function to deplete, O 2  and CO 2 , or O 2  alone, or O 2  with specific levels of CO 2  by supplying appropriate composition of flushing gas. Gases appropriate for depletion devices are, for example, Ar, He, N 2 , Ar/CO 2 , or N 2 /CO 2 . 
       FIGS. 9 a  through 9 c  and 10 a  through 910 c   , also disclose scavenging depletion devices. Depletion takes place with the use of scavengers or sorbents and without the use of external gases. In both types of depletion devices however, carbon dioxide depletion in conjunction with oxygen depletion is effective to enhance DPG and ATP, respectively, prior to storage in blood storage bags. 
     Referring to  FIGS. 7 a  through 7 c   , a depletion device  20  is shown. Depletion device  20  includes a plurality of fibers  25 , approximately 5000 in number, through which red blood cells flow. Plurality of fibers  25  are surrounded by a plastic cylinder  30 . Plastic cylinder or cartridge  30  contains a gas inlet  35  and a gas outlet  40  through which a flushing gas or a combination of flushing gases, such as those mentioned above, are supplied to remove carbon and/or oxygen from blood. Specifications for depletion device  20  are shown in Table 1 below at second column. 
     
       
         
           
               
               
               
               
             
               
                   
                 TABLE 1 
               
               
                   
                   
               
               
                   
                 Prototype 
                 External Gas 
                 Externa Gas 
               
               
                   
                 Specification 
                 Pathways 
                 Pathways 
               
               
                   
                   
               
             
            
               
                   
                 Prototype Serial #: 
                 Device 20 
                 Device 45 
               
               
                   
                 Fiber Type: 
                 Celgard 
                 Celgard 
               
               
                   
                   
                 200/150-66FPI 
                 200/150-66FPI 
               
               
                   
                 Number of Fibers: 
                 5000 
                 5000 
               
               
                   
                 Active Length of 
                 13 
                 28 
               
               
                   
                 Fibers (cm): 
                   
                   
               
               
                   
                 Fiber OD 
                 200 
                 200 
               
               
                   
                 (microns): 
                   
                   
               
               
                   
                 Fiber ID 
                 150 
                 150 
               
               
                   
                 (microns): 
                   
                   
               
               
                   
                 Total Length of 
                 15 
                 30 
               
               
                   
                 Fibers 
                   
                   
               
               
                   
                 Active Fiber 
                 0.4084 
                 0.8796 
               
               
                   
                 Surface Area 
                   
                   
               
               
                   
                 (m2): 
               
               
                   
                   
               
            
           
         
       
     
     Referring to  FIGS. 8 a  through 8 c   , a depletion device  45  is shown. Depletion device  45 , like device  20  of  FIGS. 7 a  to 7 c   , includes a plurality of fibers  50 , approximately 5000 in number, through which red blood cells flow. Plurality of fibers  50  are surrounded by a plastic cylinder  55 . Plastic cylinder  55  contains a gas inlet  60  and a gas outlet  65  through which a gas or a combination of gases, such as those mentioned above are supplied to remove oxygen or oxygen and carbon dioxide from blood. Specifications for depletion device  45  are shown in Table 1 above in the third column. The active surface area of depletion of device  45  is twice that of device  20  because device  45  is twice as long as device  20 . 
       FIGS. 9 a  through 9 c    disclose a depletion device  70  having a core  75  containing scavenging materials for either O 2 , or both O 2  and CO 2 . Core  75  is packed by a gas permeable film with very low liquid permeability. Hollow fibers  80  are wound around core  75 , and a plastic cylinder  82  contains and envelopes hollow fibers  80 . In this particular embodiment, the active surface area for depletion is approximately 0.8796 m 2  as shown in Table 2 below at the second column. 
     
       
         
           
               
               
               
               
             
               
                   
                 TABLE 2 
               
               
                   
                   
               
               
                   
                   
                 Center Core 
                 10 individual 
               
               
                   
                 Prototype 
                 125 grams 
                 Bundles 
               
               
                   
                 Specification 
                 Sorbent 
                 200 grams Sorbent 
               
               
                   
                   
               
             
            
               
                   
                 Prototype Serial 
                 Device 70 
                 Device 85 
               
               
                   
                 #: 
                   
                   
               
               
                   
                 Fiber Type: 
                 Celgard 
                 Celgard 
               
               
                   
                   
                 200/150-66FPI 
                 200/150-66FPI 
               
               
                   
                 Number of 
                 5000 
                 5000 
               
               
                   
                 Fibers: 
                   
                   
               
               
                   
                 Active Length 
                 13 
                 28 
               
               
                   
                 of Fibers (cm): 
                   
                   
               
               
                   
                 Fiber OD 
                 200 
                 200 
               
               
                   
                 (microns): 
                   
                   
               
               
                   
                 Fiber ID 
                 150 
                 150 
               
               
                   
                 (microns): 
                   
                   
               
               
                   
                 Total Length of 
                 15 
                 30 
               
               
                   
                 Fibers 
                   
                   
               
               
                   
                 Active Fiber 
                 0.8796 
                 0.8796 
               
               
                   
                 Surface Area 
                   
                   
               
               
                   
                 (m2): 
               
               
                   
                   
               
            
           
         
       
     
       FIGS. 10 a  through 10 c    disclose a depletion device  85  containing fiber bundles  87  enclosed in gas permeable film with very low liquid permeability. Fiber bundles  87  are surrounded by scavenger materials  89  for either O 2  or both O 2  and CO 2 . Fiber bundles  87  and scavenger materials  89  are contained within a plastic cylinder  90 . The active surface area for depletion is approximately 0.8796 m 2  as shown in Table 2 above at the third column. 
       FIG. 11  is a plot of the performance of flushing depletion devices  20  and  45  and scavenging depletion devices  70  and  85 . The data of  FIG. 11  was plotted using the following conditions: Hematocrit, 62% (pooled 3 units of pRBC), and 21° C. at various head heights to produce different flow rates. Oxygen/carbon dioxide scavenger (Multisorb Technologies, Buffalo, N.Y.) was activated with adding 5% and 12% w/w water vapor for device  79  and device  85 , respectively. Data are plotted with flow rate (g RBC suspension per min) vs. pO 2  (mmHg). 
     In the oxygen/carbon dioxide depletion devices disclosed herein, a plurality of gas permeable films/membranes may be substituted for the plurality of hollow fibers. The films and fibers may be packed in any suitable configuration within the cartridge, such as linear or longitudinal, spiral, or coil, so long as they can receive and convey red blood cells. 
       FIG. 11  shows that lowest oxygen saturation is achieved using devices  45  and  85 . Device  45  exhibits a larger active surface area exposed to gases along length of fibers  50 . Device  85  also has a long surface area of exposure to scavenging materials. Device  85  has bundles  87  surrounded by scavenging materials  89 . The space occupied by scavenging materials  89  between bundles  87  promotes dispersion of oxygen and carbon dioxide from red blood cells contained in fiber bundles  87 , thus aiding scavenging of oxygen and carbon dioxide from red blood cells. 
     A further use of the depletion devices is to add back oxygen and or carbon dioxide prior to transfusion by flushing with pure oxygen or air. This use is for special cases, such as massive transfusions, where the capacity of the lung to re-oxygenate transfused blood is not adequate, or sickle cell anemia. 
     Similarly, depletion devices can be used to obtain intermediate levels or states of depletion of oxygen and carbon dioxide depending needs of the patient to obtain optimal levels in the transfused blood depending upon the patients needs. 
     Referring to  FIG. 4 , a blood storage bag  200  according to a preferred embodiment of the present disclosure is provided. Blood bag  200  has an inner blood-compatible bag  250  (preferably polyvinyl chloride (PVC)), and an outer barrier film bag  255 . The material of bag  250  is compatible with RBCs. Disposed between inner bag  250  and outer oxygen barrier film bag  255  is a pocket that contains an oxygen/carbon dioxide sorbent  110 . Barrier film bag  255  is laminated to the entire surface of inner bag  250 . Sorbent  110  is contained in a sachet  260 , which is alternately referred to as a pouch or pocket. Sorbent  110  is optimally located between tubing  440  that leads into and from bag  200 , specifically between inner bag and outer oxygen barrier film bag  255 . This location will ensure that oxygen disposed between these two bags will be scavenged or absorbed. Oxygen sorbent is ideally located in a pouch or pocket  260  and not in contact with RBCs. Oxygen sorbent may also be combined with CO 2  scavengers or sorbents, enabling sorbent  110  to deplete both oxygen and carbon dioxide at the same time. 
     Referring to  FIGS. 5 a  and 5 b   , blood storage bags  201  and  202  are configured to store RBCs for extended storage periods of time. Inner blood storage bags  205  are preferably made from DEHP-plasticized PVC and are in contact with RBCs. DEHP-plasticized PVC is approximately  200  fold less permeable to oxygen compared to silicone. However, PVC is insufficient as an oxygen barrier to maintain the anaerobic state of RBCs throughout the storage duration. Therefore, blood storage bags  201  and  202  are fabricated with outer transparent oxygen barrier film  206  (e.g., nylon polymer) laminated to the outer surface inner blood bag  205 . This approach, as well as one shown in  FIG. 3 , uses accepted PVC for blood contact surface (supplying DEHP for cell stabilization) at the same time prevents oxygen entry into the bag during extended storage. 
     In  FIG. 5 a   , a small sachet  210  containing oxygen/carbon dioxide sorbent  110  enveloped in oxygen-permeable, RBC compatible membrane is enclosed inside of laminated PVC bag  205  and in contact with RBCs. Small sachet envelope  210  is preferably made from a silicone or siloxane material with high oxygen permeability of biocompatible material. Sachet envelope  210  has a wall thickness of less than 0.13 mm thickness ensures that O 2  permeability ceases to become the rate-limiting step. PVC bag  205  may also contain carbon dioxide scavengers. 
     Referring to  FIG. 5 b   , bag  202  has a similar configuration to bag  201  of  FIG. 4 a   . However, bag  202  has a large sorbent  215  enclosed inside of PVC bag  205 . Large sorbent  215  preferably has a comb-like configuration to rapidly absorb oxygen during extended storage. The benefit of laminated bags of  FIGS. 4 a  and 4 b    is that once RBCs are anaerobically stored in bags, no further special handling is required. Similarly, bag  202  may contain carbon dioxide scavenger to provide carbon dioxide-scavenging in addition to oxygen-scavenging capability. 
     Referring to the embodiments of  FIGS. 6 a  and 6 b   , RBCs are stored in secondary bags  301  and  302 , respectively, in order to maintain an anaerobic storage environment for RBC storage. Secondary bags  301  and  302  are transparent oxygen barrier films (e.g., nylon polymer) that compensate for the inability of PVC blood bags  305  and  320 , respectively, to operate as a sufficient oxygen barrier to maintain RBCs in an anaerobic state. Secondary bags  301  and  302  are made with an oxygen barrier film, preferably a nylon polymer or other transparent, flexible film with low oxygen permeability. 
     Referring to  FIG. 6 a   , a small oxygen/carbon dioxide sorbent  310  is disposed between a PVC barrier bag  305  and secondary bag  306  to remove slowly diffusing oxygen.  FIG. 6 a    is similar to the preferred embodiment of the blood bag of  FIG. 4  except that secondary bag  306  is separate from and not bonded to bag  305  in this embodiment. PVC bag  305  including ports are enclosed in secondary barrier bag  305 . Oxygen sorbent  310  may optionally contain carbon dioxide scavengers to provide both oxygen and carbon dioxide scavenging capability. 
     Referring to  FIG. 6 b   , a secondary bag  302  contains a large sachet  325  inside of PVC bag  320 . Sachet  325  is filled with either oxygen or oxygen/carbon dioxide sorbent  110 . Sachet  325  is a molded element with surface texture to increase the surface area. Sachet  325  has a comb-like geometry for rapid oxygen or oxygen/carbon dioxide depletion. Sachet  325  acts rapidly to strip oxygen or oxygen/carbon dioxide from RBCs prior to refrigeration and storage of RBCs in place of OCDD of  FIG. 3 . However, with this configuration, agitation is necessary, therefore sachet  325  must possess a large surface area, high oxygen or oxygen/carbon dioxide permeability and mechanical strength to withstand centrifugation step during component preparation and the prolonged storage. Sachet  325  is preferably made from materials such as 0.15 mm thick silicone membrane with surface texture to increase the surface area. Sachet  325  may be made from materials such as PTFE or other fluoropolymer. Sachet  325  may have a rectangular shape such, such as, for example, a 4″×6″ rectangle, although other sizes are possible, for the anaerobic maintenance. Sachet  325  may contain carbon dioxide scavengers in addition to oxygen scavengers to provide oxygen and carbon dioxide scavenging capability. 
     The embodiments of  FIGS. 6 a  and 6 b    are easily made from off-shelf components except for sachet  325  of  FIG. 6 b   . In order to access RBCs for any testing, secondary bags  301  and  302  must be opened. Unless the unit is transfused within short time, RBC must be re-sealed with fresh sorbent for further storage. (1 day air exposure of storage bag would not oxygenate blood to appreciable degree, since PVC plasticized with DEHP has relatively low permeability to oxygen). 
     In  FIGS. 5 a , 5 b , 6 a  and 6 b   , the PVC bag is preferably formed with the oxygen barrier film, such as a SiO 2  layer formed with the sol-gel method. A portion of the sheet material will be sealed on standard heat sealing equipment, such as radiofrequency sealers. Materials options may be obtained in extruded sheets and each tested for oxygen barrier, lamination integrity, and seal strength/integrity. 
     For each of the several embodiments addressed above, an additive solution from bag  300  is provided prior to stripping oxygen and carbon dioxide from the RBCs is used. The additive solution  300  preferably contains the following composition adenine 2 mmol/L; glucose 110 mmol/L; mannitol 55 mmol/L; NaCl 26 mmol/L; Na 2 HPO 4  12 mmol/L citric acid and a pH of 6.5. Additive solution  300  is preferably an acidic additive solution OFAS3, although other similar additive solutions could also be used that are shown to enhance oxygen/carbon dioxide-depleted storage. OFAS3 has shown enhanced ATP levels and good in vivo recovery as disclosed herein. While OFAS3 is a preferred additive solution, other solutions that offer similar functionality could also be used. Alternatively, additive solutions used currently in the field, such as AS1, AS3, AS5, SAGM, and MAPS can also be used. Additive solutions help to prevent rapid deterioration of RBCs during storage and are typically added prior to RBCs being made anaerobic. 
     Additionally, we envision that the OCDD and storage bags  100  and  200  can be manufactured independent of other components of the disposable, anaerobic blood storage system (i.e., every item upstream of and including leuko reduction filter  400  in  FIG. 1 a   ). 
     It is within the scope of the present disclosure to remove oxygen from the RBCs or to strip oxygen and carbon dioxide from the blood prior to storage in the storage bags. An oxygen scavenger can be used to remove the oxygen from the RBCs prior to storage in the blood bags. As used herein, “oxygen scavenger” is a material that irreversibly binds to or combines with oxygen under the conditions of use. For example, the oxygen can chemically react with some component of the material and be converted into another compound. Any material where the off-rate of bound oxygen is zero can serve as an oxygen scavenger. Examples of oxygen scavengers include iron powders and organic compounds. The term “oxygen sorbent” may be used interchangeably herein with oxygen scavenger. As used herein, “carbon dioxide scavenger” is a material that irreversibly binds to or combines with carbon dioxide under the conditions of use. For example, the carbon dioxide can chemically react with some component of the material and be converted into another compound. Any material where the off-rate of bound carbon dioxide is zero can serve as a carbon dioxide scavenger. The term “carbon dioxide sorbent” may be used interchangeably herein with carbon dioxide scavenger. For example, oxygen scavengers and carbon dioxide scavengers are provided by Multisorb Technologies (Buffalo, N.Y.) or Mitsubishi Gas Chemical Co (Tokyo, Japan). Oxygen scavengers may exhibit a secondary functionality of carbon dioxide scavenging. Such materials can be blended to a desired ratio to achieve desired results. 
     Carbon dioxide scavengers include metal oxides and metal hydroxides. Metal oxides react with water to produce metal hydroxides. The metal hydroxide reacts with carbon dioxide to form water and a metal carbonate. For example, if calcium oxide is used, the calcium oxide will react with water that is added to the sorbent to produce calcium hydroxide 
       CaO+H 2 O→Ca(OH) 2  
 
     The calcium hydroxide will react with carbon dioxide to form calcium carbonate and water. 
       Ca(OH) 2 +CO 2 →CaCO 3 +H 2 O
 
     It will be appreciated that scavengers can be incorporated into storage receptacles and bags in any known form, such as in sachets, patches, coatings, pockets, and packets. 
     If oxygen removal is completed prior to introduction of the RBCs to the blood storage device, then it can be accomplished by any method known in the art. For example, a suspension of RBCs can be repeatedly flushed with an inert gas (with or without a defined concentration of carbon dioxide), with or without gentle mixing, until the desired oxygen and or carbon dioxide content is reached or until substantially all of the oxygen and carbon dioxide has been removed. The inert gas can be argon, helium, nitrogen, mixtures thereof, or any other gas that does not bind to the hememoiety of hemoglobin. 
     The OCDDs and various storage bags of the present disclosure can be used in varying combinations. For example, OCDD  101  of  FIG. 3  can be used with blood bag of  FIG. 4, 201  of  FIG. 5 a    or  301  of  FIG. 6 a   . When oxygen is depleted by in-bag sachet  215  of  FIG. 6 b   , it can be stored as in  FIG. 6 b    or oxygen/carbon dioxide-depleted content transferred to the final storage bag such as  FIG. 4 ,  FIG. 5 a    or  FIG. 6 a    for extended storage. Other combinations and configurations are fully within the scope of the present disclosure. 
     The present disclosure also provides another embodiment of a blood storage device. The device is a sealed receptacle adapted to retain and store red blood cells. The receptacle has walls formed from a laminate. The laminate has (a) an outer layer of a material substantially impermeable to oxygen or oxygen and carbon dioxide, (b) an inner layer of a material compatible with red blood cells, and (c) an interstitial layer between the outer layer and the inner layer. The interstitial layer is of a material having admixed therein an amount of an oxygen scavenger or an oxygen/carbon dioxide scavenger. The layers preferably take the form of polymers. A preferred polymer for the outer layer is nylon. A preferred polymer for inner layer is PVC. The polymer of the interstitial layer should provide effective adhesion between the inner and outer layers and provide effective admixture of oxygen scavengers or oxygen/carbon dioxide scavengers therein. Useful polymers for the interstitial layer include, for example, olefin polymers, such as ethylene and propylene homopolymers and copolymers, and acrylic polymers. 
     The present disclosure also provides another embodiment of a blood storage system. The system has a collection bag for red blood cells; a unitary device for depleting oxygen or oxygen and carbon dioxide and reducing leukocytes and/or platelets from red blood cells; a storage bag for red blood cells; and tubing connecting the collection bag to the unitary device and the unitary device to the storage bag. A feature of this embodiment is that the functions of depleting oxygen or oxygen and carbon dioxide and reducing leukocytes and/or platelets from red blood cells are combined into a single, unitary device rather than require separate devices. For instance, unitary device can take the form of a single cartridge. Leukocyte and/or platelet reduction is typically carried out by passing red blood cells through a mesh. In this embodiment, a mesh can be incorporated into either the flushing or the scavenging oxygen or oxygen/carbon dioxide depletion device disclosed herein. The mesh is preferably located within the device so that leukocyte and/or platelet reduction takes place prior to the onset of flushing or scavenging. 
     The following are examples of the present disclosure and are not to be construed as limiting. 
     EXAMPLES 
       FIGS. 12 a  through 12 h    show the results of a 3-arm study showing: a control (aerobic OFAS3 with no O 2  or CO 2  depletion), anaerobic OFAS3 (both O 2  and CO 2  depleted with pure Ar), and O 2  only depleted with 95% Ar and 5% CO 2  (CO 2  is not depleted). 
     Whole blood was collected into CP2D (Pall), centrifuged 2K×G for 3 minutes, plasma removed, and additive solution AS-3 (Nutricel, Pall), or experimental OFAS3 added. The unit was evenly divided into 3 600 mL bags. 2 bags were gas exchanged ×7 with Ar or Ar/CO 2 , transferred to 150 mL PVC bags and stored 1° C. to 6° C. in anaerobic cylinders with Ar/H 2  or Ar/H 2 /CO 2 . One control bag was treated in the same manner without a gas exchange and stored 1° C. to 6° C. in ambient air. Bags were sampled weekly for up to 9 weeks. 
     The plots of  FIGS. 12 a , 12 c , 12 e  and 12 g   : use the additive solution OFAS3 (200 mL; experimental, proprietary) and the plots of  FIGS. 12 b , 12 d , 12 f  and 12 h   , use the AS-3 additive solution. Comparing additive solutions, effects of CO 2  depletion on DPG levels were similar. OFAS3 showed higher ATP when oxygen was depleted (±CO 2 ), and O 2  depletion alone showed significant enhancement of ATP compared to aerobic control. AS-3 additive exhibited no significant enhancement of ATP when O 2  alone was depleted. 
       FIGS. 12 a  and 12 b   : DPG levels during storage. DPG levels were maintained for over 2 weeks, when CO 2  was removed in addition to oxygen. 
       FIG. 12 c   : ATP levels during storage with OFAS3. Highest ATP levels were achieved with OFAS3 RBC when O 2  only was depleted. For O 2 /CO 2  depletion, intermediate levels of ATP were observed compared to the control while very high DPG levels were attained during first 2.5 weeks. Very high levels of ATP may suggest higher rate of 24-hour post transfusion recovery. Therefore, extent of carbon dioxide and oxygen depletion levels may be adjusted to meet the specific requirement of the recipient. DPG levels can be maintained very high (at the expense of ATP) for purposes of meeting acute oxygen demand of recipient. Conversely, very high ATP levels may allow higher 24-hour recovery rate (lower fraction of non-viable RBC upon transfusion) thereby reducing the quantity of blood needed to be transfused (up to 25% of RBC are non-viable). More importantly, this would benefit chronically transfused patients who may not demand highest oxygen transport efficiency immediately after transfusion (DPG level recovers in body after 8-48 hours) who suffers from toxic iron overloading caused by non-viable RBCs. 
       FIG. 12 d   : ATP levels during storage with AS3. Highest ATP levels were achieved with AS3 RBC when O 2  only was depleted. No significant differences in ATP levels where observed with control and O 2  depletion alone. 
       FIGS. 12 e  and 12 f   : pH of RBC cytosol (in) and suspending medium (ex). Immediately after gas exchange (day 0), significant rise in pH (in and ex) was observed only when CO 2  was depleted together with O 2 . Rapid rates of pH decline observed with CO 2 /O 2  depleted samples were caused by higher rates of lactate production ( FIGS. 12 g  and 12 h   ). 
       FIGS. 12 g  and 12 h   : Normalized (to hemoglobin) glucose and lactate levels during storage with OFAS3 and AS3. Higher rates of glucose depletion and lactate productions correspond to high DPG levels observed in panels A and B. Legends for symbols/lines are same for both panels. OFAS3 additive contains similar glucose concentration with ×2 volume resulting in higher normalized glucose levels. 
       FIGS. 12 a  and 12 c    taken together, suggest that extent of increases (compared to control) of ATP and DPG levels may be adjusted by controlling level of CO 2  depletion, when O 2  is depleted. Higher glucose utilization and lactate production were observed with enhanced DPG production ( FIG. 12 g   ). This may be also effective with AS3 additive, since similar trend in glucose utilization and lactate production were observed ( FIG. 12 h   ). 
       FIG. 13  shows a graph comparing the effect of gamma irradiation on aerobic and anaerobic RBC.  FIG. 13  shows an control unit, RBC that are aerobic and not gamma-irradiated (Unit A, black filled solid line), aerobic RBC that are gamma-irradiated (Unit B; control plus gamma irradiation indicated by a filled circle with dotted line) and an anaerobically depleted RBC unit that has been gamma-irradiated (Unit C; Anaerobic+γ, open circle and solid line). Unit B and Unit C are irradiated and Unit A is non-irradiated and aerobic RBC. The constituent of the blood that is being measured is potassium. The amount of leakage of potassium (K+) from RBC that is measured in the storage media is an indicator of health of the RBC. Therefore, in the context of the present application, a greater level of concentration of potassium in RBC storage media, is indicative of a greater level of RBC damage relative to a lower level of concentration of potassium in RBC storage media. 
       FIG. 13  indicates that gamma irradiation induced a high rate of K+leakage during the first week for Unit B and Unit C. K+ leakage rates after days eight and fifteen, were similar for all units. Significantly, the difference between K+ leakage between Unit B and Unit C increases beyond the twenty-second day of storage. The results indicate that this trend could exist for several more days. Accordingly, the use of anaerobic depletion and gamma irradiation may permit the extension of current FDA storage limit of twenty-eight days for anaerobically depleted and gamma irradiated blood prepared after component separation. 
     Irradiating RBC for immuno-compromised individuals is a necessity. The present results show that irradiated RBC that were also oxygen depleted did not increase K+ leakage rates, an indicator of RBC damage. The benefits of oxygen depleted RBC including increased levels of ATP and DPG-2,3 are not negatively impacted by the irradiation. 
     In graph above, four ABO Rh identical units (in AS3 additive, leukoreduced; standard RBC concentrate obtained from American Red Cross) are pooled. The three units were used for above-graphed experiment from the pooled unit after it was sub-divided into 4 fractions within 24 hours of blood collection and stored at 1-6° C. 
     Although the present disclosure describes in detail certain embodiments, it is understood that variations and modifications known to those skilled in the art that are within the disclosure. Accordingly, the present disclosure is intended to encompass all such alternatives, modifications and variations that are within the scope of the disclosure as set forth in the disclosure.