Improved blood storage unit and method of storing blood
United States Patent [191 Deindoerfer et al.  
 [ Apr. 1, 1975 IMPROVED BLOOD STORAGE UNIT AND METHOD OF STORING BLOOD [75] Inventors: Fred H. Deindoerfer, Northridge;  
 Jon M. Brake, Burbank, both of Calif.  
 [73] Assignee: American Hospital Supply Corporation, Evanston, Ill.  
 [22] Filed: Mar. 29, 1973 [21] Appl. No.: 345,961  
 Related U.S. Application Data [63] Continuation-impart of Ser. Nos. 194,652, Nov. 1, l97l, Pat. NO. 3,847,378, and Ser. No. 194,689, Nov. 1, 1971, Pat. N0. 3,795,581.  
 [52 U.S. Cl 128/272, l95/1.8, 424/101 [51] Int. C13... A61J l/00, A61K 17/00, C12K 9/00 [58] Field of Search 195/18; 424/101 [56] References Cited UNITED STATES PATENTS 3,703,438 11/1972 Dovgalev 195/].8  
 Primary Examiner-Sam Rosen [57 7 ABSTRACT Blood storage units comprising containers with aqueous preservative solutions therein are improved by incorporating dihydroxyacetone (DHA) together with L-ascorbate (vitamin C) in the preservative solutions. The invention also relates to a method of storing human blood wherein viable red cells are stored in contact with both DHA and L-ascorbate. The preservative solution of the blood storage unit may also contain an anticoagulant such as citrate, a sugar energy source such as dextrose, and. an ATP maintaining agent such as adenine. Where the blood storage unit is to be heat sterilized, as preferred, a blood bag providing a separate compartment for part of the preservative agents prevents deterioration of the preservative agents, the DHA and sugar energy source being heat sterilized separately for later admixture with the ascorbate, citrate anticoagulant and adenine. Separate pH control can also -thereby be provided for the heat sterilization.  
 15 Claims, 7 Drawing Figures SOLUTION B y 2 IMPROVED BLOOD STORAGE UNIT AND METHOD OF STORING BLOOD CROSS-REFERENCES This application is a continuation-inpart of our copending application Ser. Nos. 194.652 and 194.689. both filed Nov. 1. 1971. now US Pat. Nos. 3.847.378 and 3,795,581. respectively.  
 DRAWINGS The accompanying drawing. comprising FIGS. 1 to 7, illustrates one form of a blood collection and storage unit for use in practicing the present invention where the preservative solutions are heat sterilized. The construction and method of use of such blood storage units will be further described in Example IV.  
 BACKGROUND AND SUMMARY The state of the art with respect to biochemical knowledge ofthe chemical makeup and functioning of red cells (erthyrocytes) is summarized in two recent publications: Red Cell Metabolism and Function. edited by George J. Brewer. Plenum Press. 1970; and Red Cell Metabolism. Ernest Beutler. Grune and Stratton. 1971.  
 The principal energy source for red cells is glucose (or equivalent sugar) which is metabolized by the cells through complex biochemical pathways involving enzymaticreactions. The principal pathway. often referred to as the Embden-Meyerhoff pathway, involves the anaerobic breakdown of glucose to pyruvic or lactic acid. An additional pathway is referred to as the direct oxidative shunt or hexose monophosphate shunt.  
  In the Embden-Meyerhoff pathway, the compound l.3-diphosphoglycerate is produced from D-glyceraldehyde-3-phosphate. The 1,3-diphosphoglycerate is converted by interaction with ADP (adenosine diphosphate) to ATP (adenosine triphosphate) and 3. phosphoglycerate, the reaction being catalyzed by phosphoglyceratc kinasc. An alternate by-path also leads to 3-phosphoglycerate. by way of 2.3-diphosphoglycerate (hereinafter referred to as 2,3-DPG or more concisely as DPG) an important regulator of the oxygen affinity of hemoglobin. The complexing of 2.3- DPG with hemoglobin decreases the affinity of oxygen to hemoglobin in a manner essential to the release of oxygen to the body tissues.  
  In human blood. the normal level of 2,3-DPG is within the range from 12 to 18 micromoles 2.3-DPG per gram of hemoglobin. Beutler gives a more precise figure: 15.36 i 1.98 micromoles 2.3-DPG/g. hemoglobin (Red Cell Metabolism supra, p. 99). In the body, under usual conditions, sufficient 2,3-DPG is produced by the red cells in the metabolism of glucose by the EmbdenMeyerhoff pathway to provide the required amount for proper oxygen-hemoglobin-tissue transfer. For reasons that are not understood, however. the 2,3- DPG content of red cells in stored blood decreases to subnormal levels interfering with oxygen released by the cells, even though blood is stored under refrigerator conditions (l-6 C.) in admixture with an anticoagulant solution containing dextrose (or equivalent sugar) as the principal energy source for the red cells. Therefore. although the red cells remain viable. contain sufficient ATP, and provide a satisfactory survival rate (70 percent or more after 24 hours), the subnormal 2,3-DPG content of the red cells may actually cause a decrease in the oxygen delivered to the tissues for several hours after the transfusion, and as long as 24 hours may be required for the transfused red cells to be restored to normal 2.3-DPG levels for effieent delivery of oxygen to the tissues. (Dawson. The Hemoglobin Function of Blood Stored at 4 C..&#34; pp. 3()53l7, in Red Cell Metabolism and Function. supra).  
  The problem of administering stored blood deficient in 2.3-DPG is rendered more acute under many clinical conditions. such as patients in septic shock. patients receiving large volumes of stored blood. and infants. particularly premature infants. with infection or the respiratory disease syndrome. since the 2.3-DPG levels of the patients blood may already be depressed. and further depression may occur on administration of the low 2.3-DPG level blood.  
  Since the recognition of the function of 2.3-DPG as an oxygen release regulator for hemoglobin. and the recognition that depressed levels of 2.3DPG can occur in the body and under in vitro storage of blood. there has been a widespread search for chemical additives or other means of maintaining. or even increasing. the 2.3-DPG content of red cells. It has been found that frozen blood stored under very cold conditions (viz. C.) can be stored for many months without significant change in2.3-DPG levels. However. because of the added expense in freezing blood and storing it in the frozen conditions. the use of frozen blood has not become a commercial blood storage practice. The almost universal procedure in the United States at the present time is to combine the freshly collected blood with an anticoagulant solution containing dextrose, such as a citrate-dextrose solution or a citratephosphate-dextrose solution, and then to store the blood under refrigeration at a substantially constant temperature within the range from 1 to 6 C. Following this procedure, blood bank storage is approved up to 21 days. and if the blood is not administered by that time. it usually&#39;must be discarded.  
  In our copending applications, Ser. Nos. 194,652 and 194.689. cited-above. we have disclosed a blood storage unit and method of blood storage wherein dihydroxyacetone (DI-IA) is incorporated in the preservative solution and maintained in contact with the red cells of the blood during storage for the purpose of maintaining and/or increasing the 2.3-DPG content of the red cells. Subsequent to our discovery of the effect of DHA on DPG levels of red cells, DR. Ernest Beutler. City of Hope Medical Center, Duarte, Calif. has found that the mechanism of action of the DHA involves triokinase enzyme activity. a type of enzyme activity which had not previously been known to exist in red cells. By the postulated mechanism, dihydroxyacetone is converted to dihydroxyacetone phosphate by the mediation of triokinase enzyme activity. and the dihydroxyacetone phosphate enters the main metabolic pathway of the red cells.  
  Dr. Ernest Beutler has also reported that L-ascorbic acid (vitamin C) has a positive effect on the maintenance of DPG levels in stored blood, but that the mechanism of action of -L-ascorbate is unknown: Transfusion Congress, American Association of Blood Banks, XXV Annual Meeting, Aug. 27-Sept. 2, 1972; and Western Society of Clinical Research, Mleeting Feb. 3-5, 1972, Carmel. Calif. As reported by Dr. Beutler, red cells stored with L-ascorbate use significantly less dextrose than controls, and the intracellular pH is significantly higher. Further, during storage of the red cells with ascorbate, less lactate and more pyruvate is formed from the sugar energy source. It therefore appears that the mechanism iof action of ascorbate in maintaining DPG levels, although not fully understood. is quite different from the mechanism of action of DHA. Our discovery, which forms an important part of the present invention, was therefore unexpected: namely, that DHA and L-ascorbic acid (vitamin C) can function synergistically in maintaining and/or increasing DPG levels of red cells in stored blood, especially where the blood is stored for 2 or 3 weeks or longer, such as storage periods of from 3 to 6 weeks.  
 DETAILED DESCRIPTION In practicing the present invention, approved types of blood collection and preservation containers are preferred. Either glass or plastic containers can be utilized, providing they meet the USP. requirements. (See US. Pharmacopeia XVIII, pages 887 and 923.) The containers will be sized for receiving and storing a predetermined volume of blood, such as 1000 ml., 500 ml., etc. Typically, the containers will have an internal volume adapted for receiving 500 ml. /2 I.) of blood together with 70 to 125 ml. of anticoagulant solution. In other words, the containers can have an internal volume of around 570 to 625 ml. The containers will also be equipped with means for introducing the fresh blood as it is collected, and for delivery of the blood in transfusion. Such transfusion and infusion assemblies used with the blood collection and storage units should meet U.S.P. requirements. (See US. Pharmacopeia XVIII, p. 887).  
  As in the established practice, the anticoagulant solution for admixture with blood collected in the containers should contain an anticoagulant substance to prevent coagulation of the blood, preferably, also. a sugar energy source for the red cells in addition to the DHA. The preferred anticoagulant is citrate ions&#34; which may be supplied by sodium citrate, or mixtures of citric acid and sodium citrate. The quantities to be employed can be the same as in present practice (see U.S. pharmacopeia XVIII, pages 47-49).  
  The sugar energy source for the red cells is preferably dextrose. However, it is known that other sugars are equivalent to dextrose for this purpose, including fructose, mannose, and galactose. The amount of dextrose or equivalent sugar employed can be the same as in present practice (see US. Pharmacopeia XVIII, pages 47-48). More specifically, from about 1.7 to 1.9 grams dextrose based on dextrose monohydrate can be uti- 1 lized per 500 mililiters of blood.  
  In practicing the present invention, the blood collection and preservation unit should contain at least and preferably at least 10, millimoles (mM) DHA per liter of blood. Consequently, when the unit is designed to collect 500 ml. of blood, at least 2.5 and preferably 5 mM of DHA will be incorporated in the aqueous anticoagulant solution. While there does not appear to be any critical upper limit on the content of DHA, there appears to be no reason to exceed I00 mM DHA per liter of blood. When the container is designed for 500 ml. of blood, therefore, it will not be necessary to incorporate more than 50 mM of DHA in the anti-coagulant solution. Where the DHA is being utilized for 2,3-DPG maintenance, and a sugar energy source is provided, as in present practice, it will usually not be necessary to employ more than 30 mM of DHA per liter of blood, or 15 mM per 500 ml. of blood.  
  Since red cells occupy approximately one-third the volume of whole blood, it will be appreciated that the red cells in storage will be in contact with an aqueous solution containing from 7.5 to mM of DHA per liter of solution, or preferably l5 to 45 mM DHA per liter of solution. Preferably. the DHA is incorporated in the blood immediately after its collection.  
  For cooperation with the DHA in maintaining and/or increasing the DPG content of the red cells of the blood, the present invention utilizes L-ascorbic acid (vitamin C) as a cooperating additive. The L-ascorbic acid may be incorporated in the preservative solution in its free acid form, or as a water-soluble, bloodcompatible, non-toxic ascorbate salt. For example, the sodium salt of L-ascorbic acid can advantageously be used to obtain the same effect as adding ascorbate as free L-ascorbic acid. As used subsequently herein, therefore, the term L-ascorbate or, ascorbate is intended to refer to and include the L-ascorbate moiety both as free acid and in salt form, either form being biologically equivalent for the purposes of the present invention.  
  On the basis of L-ascorbate content, the preservative solution for admixture with the stored blood should provide 0.5 to 20 mM of L-ascorbate per liter of blood. For example, where the preservative solution is for admixture with substantially 0.5 liters of whole blood, from 0.25 to 10 mM L-ascorbate should be used in combination with 2.5 to 50 mM Dl-IA. Preferably, from 1 to 10 mM of L-ascorbate per liter of blood is employed. For example, when the preservative solution is to be added to substantially 0.5 liters of blood, it can advantageously contain from 0.25 to 10 mM L- ascorbate together with 2.5 to 50 mM DHA. The red cells will therefore be stored in contact with an aqueous solution containing from 0.75 to 30 mM L-ascorbate per liter of solution, or preferably from 1.5 to 15 mM L-ascorbate per liter of solution.  
  The DHA-ascorbate preservative solution also preferably contains adenine. For example, from 0.1 to 1.0 mM adenine can be incorporated in the preservative solution per liter of predetermined blood volume. In other words, where the preservative solution is for admixture with substantially O.5 liters of blood, the amount of adenine can range from 0.05 to 0.5 mM.  
  For refrigeration storage, as described above, the conjoint action of the DHA-ascorbate combination of the present invention in maintaining DPG levels is accentuated as the length of the storage period increases. After storage of about 2 to 3 weeks, the synergistic cooperation of the L-ascorbate and the DHA becomes the predominant effect. With the DHA-ascorbate combination of the present invention, blood may be stored while maintaining acceptable DPG levels for periods of time over 3 weeks and up to 5 to 6 weeks. Data demonstrating the remarkable synergism of DHA and L- ascorbate during the extended storage of blood is presented below in Example I. Where adenine is incoporated in the preservative solution, as preferred, the ATP (adenosine triphosphate) content of the red cells can also be maintained at a satisfactory level during such extended storage periods.  
  The DHA-ascorbate combination of this invention can be utilized at preservative pI-ls from neutrality (approximately pI-I 7.0) down to acid pI-Is as low as 5.0.  
 PHs on the acid side may be advantageous. For example. an admixture of the preservative solution with the blood. a pH in the range of 5.3 to 5.9, such as a pH of substantially 5.6, is particularly advantageous.  
  Where the preservative solutions are sterilized by heat (autoclaving), as preferred. it has been discovered that the decomposition of the DHA and the ascorbate can be minimized by dividing the preservative solution into two separate solutions for purposes of sterilization, the solutions being recombinable for admixture with the blood within the blood collection container. Specifically it has been discovered that ascorbate when heat sterilized tends to be decomposed by DHA and also by dextrose. Consequently, it is preferred to provide the blood storage unit with a separate compartment containing an aqueous solution of DHA and dextrose, the blood bag, or other compartment, containing an aqueous solution of the ascorbate. The DHAdextrose aqueous solution component has been found to be most sta ble when heat sterilized at a pH within the range from 3.8 to 4.2, such as a pH of substantially 4.0. This pH is therefore preferred. The ascorbate containing solution component can advantageously have a pH of 5.3 to 5 .9, such as a pH of substantially 5.6. This component can also contain the citrate anticoagulant and the adenine, all of these ingredients being substantially stable under heat sterilization in admixture with each other under the stated pH. Alternatively, however, all ingredients of the preservative solution can be combined, and the aqueous solution can be sterilized by passing it through a sterilization filter before being filled into the blood storage container. This procedure, however, is more difficult and expensive than heat sterilization.  
  Various aspects of the present invention are further illustrated by the specific examples set out below:  
 EXAMPLE I This example describes actual laboratory experi ments and reports the data obtained, which demonstrate the synergistic effect of dihydroxyacetone (DHA) and L-ascorbic acid (vitamin C) on 2,3-diphosphoglycerate (DPG) in stored blood. In three separate experiments, blood from a single donor was divided into four portions. One was stored with CPD-adenine,  
 one with CPD-adenine-ascorbate, one with CPD- adenine-DHA, and one with CPD-adenine-ascorbate- DHA. The concentrations of the components were as follows: CPD-adenine, CPD (citrate-phosphatedextrose) per U.S.P. XVlll, pg. 48-49, and adenine, 0.5 mM per liter of blood; L-ascorbic acid (L- ascorbate), 100 mg. per each 100 ml. blood; and dihydroxyacetone (DHA), 20 mM per liter blood. The pH of the preservative solution was 5.6.  
  Samples were stored in 100 ml. plastic blood bags at 4 C. and sampled at intervals. DPG was determined by the enzymatic method of Prins and Loos, as described in Red Cell Metabolism and Function, ed. G. J. Brewer, pp. 227-288 (Plenum Press, 1970).  
  In Experiment N0. 1, blood was drawn into heparin (2115 U.S.P. units/500 ml. blood). Forty ml. of blood were transferred to the sterile 100 ml. plastic bags containing 6 ml. of CPD-adenine. 1n Experiments 2 and 3, blood was drawn in CPD-adenine m]. anticoagulant/500 ml. of blood). Aliquots of the blood were then transferred aseptically to sterile ml. plastic bags.  
  A 10% solution of DHA was prepared and sterilized by autoclaving. It was added to selected bags in a ratio of 0.5 ml. per 500 ml of blood. A solution of L- ascorbate was prepared by dissolving 5 grams of L- ascorbic acid in 100 ml. of water and adjusting to pH 5.5 with l N sodium hydroxide. It was sterilized by filtering through a 0.22 micron sterilizing filter, and was added to selected bags in a ratio of l ml. per 50 ml. of blood.  
  The results of these experiments are shown in Table A. After 3 weeks of storage, the synergism of DHA and ascorbate on DPG levels is revealed. For this purpose, synergism can be measured when the DPG level of the DHA plus ascorbate sample exceeds the sum of the DPG level of ascorbate alone plus DHA alone. At 3 weeks, such synergism was measured in one of three experiments. At 4 weeks and 5 weeks, the synergism was measured in 2 out of 3 experiments. At 6 weeks, synergism was measured in all 3 experiments.  
  In Table B, the same data are recalculated as difference values, sample minus control. This isolates the effect on DPG due to the additive from the effect due to TABLE A Effect of Ascorbate. DHA and the Combination of Ascorbate/DHA on DPG Levels of Blood Stored in CPD-Adenine Storage Time DPG (9? of initial) Additive (weeks) Exp. No. 1 Exp. No. 2 Exp. No. 3 Average None 3 12 12 22 15.3 Ascorbate 3 18 66 71 51 .7 DHA 3 67 71 95 77.7 DHA Ascorbate 3 137* 109 150 132* None 4 10 15 14 13 Ascorbate 4 14 71 60 48.3 DHA 4 41 26 25 30.7 DHA Ascorbate 4 1 19* 86 125* None 5 l3 8 15 12 Ascorbate 5 25 39 56 40 DHA 5 28 1O 20 19.3 DHA Ascorbate 5 125* 48 96* None 6 13 1 1 19 14.3 Ascorbate 6 32 13 50 31.7 DHA 6 5 8 20 1 1 DHA Ascorbate 6 88* 36* 84* 693* *Sum of DPG value for ascorbate and DHA alone is less than DPG value: for ascorbate and DHA together.  
 TABLE B Effect of Ascorbatc. DHA and the Combination of Ascorhatc /DHA on Differential DPG Levels of Blood Stored in CPD-Adenmc um ol&#39; DPG \alucs of ascorhate and DHA separately is less than DPG \aluc of the combination of the two. Calculated as the difference in DPG between the sample and the control CPD-adenine.  
 1e CPD-adenine preservative. The synergism is even lore clearly evidenced in these results; namely syner- .stic action is disclosed in 2 out of 3 experiments at 3 &#39;eeks, and in 3 out of 3 experiments at 4, 5, and 6 eeks. It is therefore apparent that synergistic cooperaon of DHA and ascorbate in maintaining DPG levels I stored blood provides a means for greatly improving 1e quality of the blood.  
 EXAMPLE 11 In one embodiment, the invention may be practiced follows:  
 To prepare a CPD-adenine-ascorbate- .hydroxyacetone system, dissolve the following chemals in 800 ml of water for injection U.S.P. and add ater to make one liter of solution: sodium citrate dihyate 30.8 grams (g), dextrose (anhydrous) 22.2 g. dildroxyacetone 14.7 g, adenine 0.55 g, L-ascorbic acid 1 g, and sodium biphosphate monohydrate 2.22 g. ierilize by filtration through a 0.22 micron sterilizing ter. Using aseptic technique fill 70 milliliters (ml) to sterile blood bags of volume capacity for collection 500 ml of blood. Pack the prepared units in, metal ms under nitrogen until needed for blood collection 1d storage use. Where the blood in admixture with the DHA and vcorbate is stored for periods beyond 1 week. as prerred, it is desirable to invert the storage containers at ast at the end of each week of storage. In one prerred procedure, the storage containers are inverted lily, or at least 5 days per week. Such inversion serves provide a mild agitation of the contents of the blood 1g. thereby maintaining the red cells in more uniform mtact with the solution of DHA and ascorbate. This ill help to assure that the combined effects of the HA and the ascorbate are maximized.  
 EXAMPLE [11 will be subsequently described in detail in Example IV. In general, the unit consists ofa 500 ml. blood bag with a 15 ml. pilot tube attached. The solution for the blood bag (Solution A) is prepared by dissolving the following in 800 ml. of water and adding water to make one liter final volume: sodium citrate dihydrate 38.3 g., adenine 0.68 g., ascorbic acid 11.1 g., and sodium biphosphate monohydrate 2.76 g.  
  With the pilot tube clamped off, 56.4 ml. of this solution is filled through a donor tube into the blood bag. Then cyclohexane is applied to the end of the donor tube, and it is inserted into a needle adaptor with attached needle. This seals the needle to the tubing.  
  The solution for the pilot tube (Solution B) is prepared by dissolving the following in 800 ml. of water and bringing to a final volume of one liter: dextrose (anhydrous) 105.6 g. and dihydroxyacetone (66.9 g.). The pH is adjusted to 4 by adding 1 N sodium hydroxide. The separate compartment provided by the pilot tube is connected at its inner end to the blood bag, by a releasably clamped tubing. Then 15.4 ml. of this solution is added to the pilot tube, through the short filling tubing connected to the outer end of the pilot tube. This filling is then heat sealed.  
  The bag unit can be used as follows: After opening the can, the bag is removed and the clamp between the pilot tube and the bag is opened. The pilot tube is squeezed, forcing Solution B into the main bag. The clamp on the pilot line is closed, and the bag is agitated to mix Solutions A and B thoroughly. The needle protector is removed, and a venipuncture was made by the usual technique in a human volunterr. After 500 ml. (530 g.) of blood is collected, the clamp on the donor line is closed. The bag is stored on its side in a 4 C. refrigerator, and agitated to resuspend the red cells in the plasma, as described in Example 11.  
 EXAMPLE IV In the accompanying drawings, there is shown a blood storage unit which is adapted for the practice of the present invention. The figures of this drawing are related as follows:  
  FIG. I is an elevational view of a complete blood storage unit ready for the collection of blood;  
  FIG. 2 is a perspective view of one of the two clamps of the unit of FIG. 1;  
  FIG. 3 is an exploded elevational view of the needle adaptor and needle cover of the unit of FIG. 1;  
  FIG. 4 is a detailed view showing the clamped portion of one of the tubes of FIG. 1;  
  FIG. 5 illustrates the appearance of the clamped portion of the tube of FIG. 4 immediately after the removal of the clamp;  
  FIG. 6 illustrates the appearance of the clamped portion of the tube of FIG. 4 after the clamp has been removed and the tube opened for the flow of liquid; and  
  FIG. 7 is a sectional view taken on line 77 of FIG. 5 showing the tube in collapsed condition as it would appear when clamped or before opening the tube for liquid flow.  
  As referred to in Example III, the blood storage unit includes a standard flexible plastic blood bag 10 having a blood storage compartment 11 therein, and a pilot tube 12 providing a separate smaller liquid storage compartment 13 therein. As indicated on FIG. 1, compartment 11 contains Solution A while compartment 13 contains Solution B. It will be understood that these solutions may be prepared and incorporated in these compartments as described in Example Ill.  
  Although the constructional details of the blood bag unit of FIG. I are conventional and well known in the blood collection and storage art, they will be briefly described in order that the use of the blood storage unit for the purpose of the present invention may be clearly understood. Blood bag 10 which may be formed by a heat sealing procedure from a suitable plastic sheet material such as polyvinylchloride is provided with an inlet 14 connected to an inlet tube 15. As illustrated, tube 15, which may be longer than illustrated ifdesired, connects to a Yconnector 16. From the Y-connector there extends a blood collection tube 17 having a needle assembly 18 at the outer end thereof and a line clamp 19 thereon adjacent a slidable sleeve 20. As shown more clearly in FIG. 3, the needle assembly 18 includes a hub 19, a needle and a needle cover or protector 21. From connector 16 there also extends a tube 22 which connects to the inner end of the enlarged pilot tube 12 and with the compartment 13 therein. On tube 22, there is also provided a line clamp 19 and an adjacent sleeve 20. It will be understood that the tube 17 and 22 may be longer than shown if desired. At the other end of the pilot tube 20, compartment 13 connects to a short filling tube 23.  
  As indicated in Example III, Solution A will be filled into compartment 11 through tube 17 before the needle assembly 18 is attached to the outer end thereof, the clamp 19 on line 17 being open during this filling operation, while the clamp 19 on line 22 is closed. Following the filling of Solution A through tube 17, clamp 19 can be moved to closed position and needle assembly 18 attached. As shown more clearly in FIG. 2, clamp 19 provides an enlarged opening 19a through which the tubing can extend without being clamped, and this opening communicates with the restricted slot 19b within which the tubing is clamped to a temporarily sealed condition.  
  Also, as indicated in Example III. Solution B is introduced into the separate compartment 13 through the filler tube 23 with the clamp 19 on line 22 in closed position. After the filling operation. the filler tube 23 may be heat sealed as indicated at 24. During heat sterilization which may be carried out as described in Example III, the clamps 19 on lines 17 and 22 may remain closed. For collection of blood, the protector 21 will be removed from the needle 20, clamp 19 opened and the tube held in oepn condition by means of sleeve 20. The blood from the donor will then be transferred through lines 17. and 15 to the compartment 11. After the blood has been collected, clamp 19 on line 17 may again be moved to closed position. Either prior to the collection of the blood or subsequent thereto, Solution B may be mixed with Solution A and with the blood in compartment 11 by opening clamp 19 and moving sleeve 20 to hold tube 22 in open condition. Since the pilot tube 20 is formed of a flexible plastic material. it can be squeezed to provide a pump action forcing Solution B through tubes 22 and 15 into compartment 1]. Tube 12 may also be elevated to assist this transfer by gravity flow. After the transfer of Solution B to compartment 11, the clamp 19 on line 22 may again be moved to closed position. Where it is desired to make the unit more compact for storage of the collected blood, and after the blood and Solution B are both in compartment 11, the tubeils&#39;may be heat sealed, as indicated at 25 and then clipped off, as indicated at 26.  
  The procedure for manipulating the clamp 19 and the sleeve 20 in relation to a tube T, such as the tubes 17 or 22 of FIG. 1, is illustrated in FIGS. 4 to 7. In FIG. 4, clamp 19 is shown in its raised or clamping position, the tube T being squeezed to a temporarily sealed condition by its engagement in the slot 19b. to open the tube, clamp 19 is moved in relation to tube T so that the tube extends through the larger opening 19a, and is then moved away from the previously clamped portion by sliding it down the tube. As shown in FIGS. 5 and 7, the clamped portion 27 of the tube T tends to remain sealed after removal of the clamp 19. It can be opened by squeezing it between a thumb and forefinger. After opening, the sleeve 20 is pushed over the previously clamped portion of the tube to hold the tube in open condition. This position is illustrated by FIG. 6. Since such use and manipulation of such clamps and sleeves are well known in the blood collection and administration art, it is not believed to be necessary to further describe them herein.  
  Conveniently, all of the components of the blood collection and storage units of FIG. 1 can be formed of suitable plastic materials. For example, bag 10, pilot tube 12, tubes 15, 17, 22 and 23 and Y connector 16 may be formed of polyvinyl chloride, slide clamps 19 of nylon or other relatively rigid thermoplastic, and hub 19 and protector 21 of polyvinyl chloride or other suitable thermoplastic. Needle 21 is preferably formed of stainless steel of a standard needle size, such as a l6 gauge needle.  
  It will be understood, as shown, that bag 10 is provided with the standard hanging loops and perforations, for example, as indicated at 28 and 29. The top of the bag is also provided with a pair of tubular connector outlets 30 having their outer ends closed by tear-off caps 31. For administration of the blood to a patient, one of the caps 31 can be removed, and a blood administration set connected to one of the tubes 30.  
  It will be apparent to those skilled in the art that the blood collection unit of FIG. 1 can be modified in various ways while still being usable for the practice of the present invention. For example, the pilot tube 12 may be replaced by a small separate bag, or bag can be manufactured with two compartments, and means provided for opening a seal between the two compartments to mix Solutions A and B after completion of the heat sterilization.  
 EXAMPLE V This example describes laboratory experiments demonstrating that DHA and ascorbate can be added to blood after one week of storage, resulting in the maintenance of high DPG levels for 6 weeks. Five hundred ml. of human blood were collected in a blood bag containing 70 ml. of CPD-adenine (composition given in Example 1). Four 35-ml. aliquots of the blood were transferred to sterile 100 ml. blood bags, one bag serving as a control and the others being used in other experiments. The bags were stored at 4 C. for 1 week, and then DHA mM/l.) and L-ascorbate (5.7 mM/l.) were added to one bag as follows: a sterile injection site (a spike with a rubber septum attached) was placed in one of the ports of the blood bag. Then using a sterile syringe, the following solutions were injected into the large blood bag: 3.8 ml. of a 2 molar solution of DHA, sterilized by autoclaving at 250 F. for 10 minutes, and 7.6 ml. of a 5 percent solution of L- ascorbic acid adjusted to pH 5.6 with sodium hydroxide and sterilized by filtration through a 0.22 micron sterile filter. All bags were mixed daily except weekends.  
  The results of DPG assays of the blood are shown in Table C. The control showed a rapid fall in DPG levels after the first week, while the blood supplemented with DHA and ascorbate at 1 week of storage maintained normal or higher than normal DPG levels for 6 weeks.  
 TABLE C DPG Levels in Blood Collected in CPD-Adenine With and Without Addition of DHA/Ascorbate After One Week of Storage at 4 C.  
 DPG (mM/g Hb) Storage Time C PD-ad C PDad+DHA+ascorbate For practicing the method described in Example V, the blood can be collected in any standard blood storage bag or container, and at the time of collection, mixed with a standard anti-coagulant containing citrate ions and a sugar energy source such as dextrose. For example, the CPD anti-coagulant described in Example I can be employed, and, if desired, adenine may also be included, as described in Example I. The container should be provided with means for subsequently introducing an aqueous solution of dihydroxyacetone and L-ascorbate. For example, a solution for addition to 0.5 liters of blood can be prepared by dissolving 0.90 grams of DHA and 0.44 grams of L-ascorbic acid in ml. of  
 water, and then subjecting the solution to sterile filtration.  
 We claim:  
  1. A blood storage unit comprising a container for receiving and storing a predetermined volume of blood and preservative solution admixable with the blood stored in said container, said preservative solution being sterile and providing a sugar energy source and an anti-coagulant for preserving said blood, said preservative solution also providing for cooperative admixture with said stored blood an amount of dihydroxyacetone (DHA) equal to 5 to millimoles (mM) per liter of said predetermined blood volume together with an amount of L-ascorbate equal to 0.5 to 20 mM per liter of said predetermined blood volume.  
  2. The blood storage unit of claim 1 wherein said DHA is present in an amount of from 10 to 30 mM of DHA per liter of said predetermined blood volume.  
  3. The blood storage unit of claim 1 wherein said preservative solution also provides adenine in an amount equal to from 0.1 to 1.0 mM per liter of said predetermined blood volume.  
  4. The blood storage unit of claim 1 wherein said preservative solution provides from 1 to 10 mM of said L- ascorbate per liter of said predetermined blood volume.  
  5. The method of maintaining the 2,3-diphosphoglycerate (2,3-DPG) content of viable red cells of whole human blood, comprising incorporating in said whole blood from 5 to 100 millimoles (mM) of dihydroxyacetone (DHA) per liter of said blood together with 0.5 to 20 mM of L-ascorbate per liter of said blood, and holding said blood with said red cells in contact with said DHA and L-ascorbate for sufficient time to maintain their 2,3-DPG content at a level resulting from the synergistic action of said DHA and said L-ascorbate.  
  6. The method of claim 5 wherein said DHA and said L-ascorbate are incorporated in said blood in amounts of from 15 to 45 mM DHA and 1.5 to 15 mM ascorbate per liter of blood. I  
  7. The method of maintaining 2,3-diphosphoglycerate (2,3-DPG) content of the red cells of whole human blood under storage conditions, comprising adding to said whole blood from 5 to 100 millimoles (mM) of dihydroxyacetone (DHA) and from 0.5 to 20 mM of L- ascorbate per liter of blood, and storing said DHA and ascorbate containing blood without freezing at a temperature below 10 C.  
  8. The method of claim 7 wherein from 10 to 30 mM of said DHA and from 1 to 10 mM of said L-ascorbate are added to said blood immediately after the collection thereof.  
  9. The method of claim 7 in which said blood is stored for a period of from 3 to 6 weeks.  
  10. A preservative solution for addition to stored blood, comprising a sterile aqueous solution of dihydroxyacetone (DHA) and L-ascorbate, said solution containing from 0.5 to 20 mM of&#39;said L-ascorbate per each 5 to 100 mM of said DHA.  
  11. A preservative solution for addition to substantially 0.5 liters of whole blood, comprising a sterile aqueous solution containing from 2.5 to 50 mM dihydroxyacetone together with 0.25 to 10 mM of L- ascrobate.  
  12. A heat-sterilized blood storage unit, comprising a container for receiving and storing a predetermined volume of blood, a first sterile aqueous preservative solution in said container, means providing a separate compartment, a second sterile aqueous preservative solution in said compartment, means permitting said second solution to be introduced into said container for admixture with said first solution and with said predetermined volume of blood. said first and second solusource and said DHA being contained only in the other of said first and second solutions.  
  13. The blood storage unit of claim 12 wherein said DHA is present in an amount of from 10 to 30 mM of DHA per liter of said predetermined blood volume.  
  14. The blood storage unit of claim 12 wherein one of said preservative solutions also contains adenine in an amount equal to from 0.1 to 1.0 mM per liter of said predetermined blood volume.  
  15. The improved blood storage unit of claim 13 wherein said one preservative solution contains from I to 10 mM of said L-ascorbate per liter of said predetermined blood volume.