Title of Invention	AN APPARATUS AND A PROCESS FOR REDUCING THE DETORIORATION OF RBCS OF BLOOD DURING STORAGE WITH PARTICULAR REFERENCE TO DECREASE IN THE LEVEL OF 2,3 DIPHOSPHOGLYCERATE AND FORMULATION EMPLOYED FOR THE SAME
Abstract	SAGM (saline-adenine-dextrose-monitor) solution is the most widely used preservative solution for storage of RBCs after separation form whole blood. Even though the permitted period of storage of RBCs in SAGM solution is 35 days, there is a steady and progressive deterioration in the quality of the quality of the cells, which increases with storage time. Some of most harmful changes are a drastic decrease in the level of 2,3 diphosphogiycerate (which is essential for delivery of oxygen to the tissues), increased haemolysis (which releases Hob and K+ from the RBCIs Into the SAGM; both of which are harmful) and progressive volatilization of di-(2-ethyl hexyl) phthalates (DEHP) [the plasticizer commonly used in the bag] in the medium containing RBCs, which is also harmful. The present formulation prevents the extent of these changes in the RBCs during storage, particularly the drastic decrease in 2,3 DPG.

Title of Invention

AN APPARATUS AND A PROCESS FOR REDUCING THE DETORIORATION OF RBCS OF BLOOD DURING STORAGE WITH PARTICULAR REFERENCE TO DECREASE IN THE LEVEL OF 2,3 DIPHOSPHOGLYCERATE AND FORMULATION EMPLOYED FOR THE SAME

Abstract

SAGM (saline-adenine-dextrose-monitor) solution is the most widely used preservative solution for storage of RBCs after separation form whole blood. Even though the permitted period of storage of RBCs in SAGM solution is 35 days, there is a steady and progressive deterioration in the quality of the quality of the cells, which increases with storage time. Some of most harmful changes are a drastic decrease in the level of 2,3 diphosphogiycerate (which is essential for delivery of oxygen to the tissues), increased haemolysis (which releases Hob and K+ from the RBCIs Into the SAGM; both of which are harmful) and progressive volatilization of di-(2-ethyl hexyl) phthalates (DEHP) [the plasticizer commonly used in the bag] in the medium containing RBCs, which is also harmful. The present formulation prevents the extent of these changes in the RBCs during storage, particularly the drastic decrease in 2,3 DPG.

Full Text	This invention relates to an apparatus and a process for reducing the deterioration of RBCs during storage, particularly the decrease in the level of 2, 3DPG and formulation employed for the same. Blood is usually collected in a triple Bag and stored in certain anticoagulant preservative solutions like AGO (acid-citrate-dextrose), CPD (citrate-phosphate-dextrose) and CPDA (citrate-phosphate-dextrose-adenine). Decrease in pH is also deleterious since it may adversely affect giycoiysis, a major metabolic activity of RBCs. Another deleterious effect of storage when DEHP plasticized PVC Bags are used, is an increase in the amount of DEHP solubilize in the 5AGM containing RBCs, which may cause toxic problems in recipients of RBC transfusion. DEHP has been reported to be toxic particularly to the liver and reproductive organs and is also carcinogenic. Further we have found that DEHP at low levels causes decrease in insulin and Cortical, depletion of fat soiubie vitamins (A, 0 & E), increase in T3 & T4 and more important inhibition of membrane bound Na+ - K+ ATPase. The last observation is significant since it is now known that there is inhibition of this enzyme activity in a variety of pathologic conditions like neurodegenerative disorders, cardiovascular disease, diabetes, cancer etc. The decrease in the level of ATP is not very drastic, but the level of 2,3 DPG falls to a very tow level during storage. The invivo restoration of 2,3 DPG depends on the level of 2,3 DPG in the RBO; transfused. If the level is very low, then it may take a long time, some time over 20hrs to restore the original level, A major purpose of transfusion of RBCs is to restore oxygen supply to the tissues. This is particularly so in transfusions with compromised circulation in the heart or brain, or in massive loss of blood. This is also more particularly important In infants, since inadequate delivery of oxygen can result in development of hypoxy/ischemic encephalopathy, the major cause of long term neuro developmental problem in children. A major factor involved in ensuring adequate delivery of oxygen to the tissues is 2,3 DPG concentration in the RBCs. 2,3 DPG has a regulatory effect on the oxygen affinity of hemoglobin and on oxygen transport In vivo. Hemoglobin binds to oxygen to form ox hemoglobin which does not release oxygen to the tissues. 2,3 OFG binds strongly to ox hemoglobin and this binding decreases the affinity of hemoglobin to oxygen with consequent release of oxygen to the tissues. Therefore an adequate level of 2,3 DPG in the RBCs is essential particularly in critical situations like cardiac surgery, brain surgery or in case of massive hemorrhage. During storage of RBCs in SAGM, the red cells usually become depleted of 2,3 DPG after 1-2 weeks which causes increase in the Hb oxygen affinity with consequent inadequate oxygen delivery to the tissues. During our study of large number of RBCs stored in SAGM solution in blood Bags at 4 ± 1° C, the following pattern of decrease in 2,3 DPG has been observed. Th« loss of 2,3 DPG Is n»v«rsibl«» however, post transfusion restoration of 2,3 DPG does taice place, but tine time for this depends on the level of 2,3 DPG in the RBCs transfused If the level is low as is the raze after storage after 14 days, it may take more than 24 hours for this recovery. This is the reason why for cardiac or neuron surgery, fresh blood is always preferred. From a practical point of view, a shelf life of 14 days and not 35 days is preferred in critical situations, because of the drop in the 2,3 DPG levels. Compared to the fall In 2,3 DPG, the fall in ATP level is not so dramatic. The pattern of change in concentration of ATP in the RBCs in our studies is given below :- ATP level is a measure of the metabolic activity of the RBCs, Maintaining of red cell viability is relatively closely associated to the cellular concentration of ATP. Since ATP level does not fait to a level below 50% of th« original level, post transfusion restoration of ATP does not take much time, as Is the case with the situation in 2,3 DPG. ATP is produced by RBCs during glycol sis, which also produces 2,3 DPG from 1,3-diphosphogiycerate by the diphosphonic glycerate shunt. 1,3-diphospho glycerate produced during glycol sis has two metabolic fates, one, it is converted directly to 3-phospho glycerate and continues In the glycol tic pathway to lactate. The second one is the shunt pathway where it is converted to 2,3-diphospho glycerate by the action of the enzyme 2,3-diphospho glycerate mutate. 2,3-diphospho glycerate is then acted on by 2,3-diphospho glycerate phosphates to from 3-phospho glycerate which continues in the glycol tic pathway. The factors which control the concentration of 2,3 DPG in the RBCs are :- (1) The balance between its rate of synthesis by 2,3-diphospho glycerate mutate and its rate of degradation by 2,3-diphospho glycerate phosphates. (2) A pH above 7.0 favors the synthesis of 2,3 DPG while a pH below 7.0 favours its decomposition. (3) When RBCs are stored In SAGM solution, the pH steadily decreases. (4) Adenine, even though it favors increase in concentration of ATP, has a deleterious effect on the concentration of 2,3 DPG. C5) Citrate in the medium for RBC storage helps to keep intracellular pH higher since the citrate ion is not permeable. These observations indicate that keeping the intracellular pH higher can increase the concentration of 2,3 DPG, We had earlier made the following observations. (1) Addition of nicotinic acid in the preservative medium for RBCs, Increases the concentration of 2 (2) Addition of an optimum level of ascorbic acid significantly increases the concentration of 2,3 DPG. The concentration has to be optimum, because above a particular concentration it decreases ATP and Increases haemolysis, (3) Addition of nicotinic acid also decreases the leaching out of OEHP (the plasticizer used in PVC Bags) into medium for RBCs storage. This is important since DEHP at low levels has now been found by us to cause decrease in insulin and Cortical, depletion of fat soluble vitamins (A, 0 &. E), increase in T3 &.T4 and more important inhibition of membrane bound Na* - K^ ATPase (under publication). The last observation is significant since it is now known that there is inhibition of this enzyme activity in a variety of pathologic conditions like neurodegenerative disorders, cardiovascular diseasing, deadheads, cancer etc. (4) Incorporation both nicotinic acid and ascorbic acid has an additive effect, in that the beneficial effects are more than the effect of each added separately. (5) Based on these observation we have developed modified formulation for the preservation of RBCs. This modification has been achieved by (1) Maintaining the pH of the preservative solution for RBCs at 7.6. This is made possible by autoclaving the dextrose solution separately in the second Satellite Bag C since dextrose solution at alkaline pH develops a yellow to red color on autoclaving, which is not acceptable. The triple Bag with CPD solution in the Primary Bag A, preservative solution containing the various ingredients to be mentioned below except dextrose in Satellite Bag B and the dextrose solution in the Satellite Bag C is now autoclaved as per the standard procedure. The dextrose solution after autoclaving is transferred from the Satellite Bag C to the rest of the solution at pH 7.6 In the first Satellite Bag B. Blood is collected from the donor in the Primary Bag A. (2) Incorporating ascorbic acid and nicotinic acid at certain optimum concentration in each case to the medium. This formulation is effective in minimizing the deleterious effects, which occur during storage of RBCs in blood Bags. Thus, according to one aspect of the present invention, there is provided a formulation for better storage and preservation of RBCs characterized in that it has a pH above 7.0, It contain nicotinic acid and ascorbic acid at optimum levels. None of the preservative solutions for RBCs hitherto known contains nicotinic acid and ascorbic acid or has pH above 7.0, and as stated hereinabove it was an important finding for us to see that the incorporation of nicotinic acid and ascorbic acid and maintaining pH above 7.0 of the preservative solution considerably reduces the deterioration of RBCs during storage. Thus, according to another aspect of the present Invention, there is provided an apparatus and a process for reducing the deterioration of RBCs during storage, characterized in that the solution used for the preservation of blood is modified by the incorporation of nicotinic acid, ascorbic acid and keeping pH above 7.0. The concentration of nicotinic acid used in the anticoagulant solution may vary, and it may preferably be in the range of 5 to 8 mg/dl of RBC suspension. However the optimum concentration was found to be 6.8 mg/dl of RBC suspension. The concentration of ascorbic acid used in the preservative solution also may vary, and it may preferably be in the range of 3 to 5 mg/dl RBC suspension. However the optimum concentration with minimum hemolytic effect and maximum beneficial effects was found to be 4.0 mg/dl RBC suspension. The pH of the preservative solution can vary form 7.0 to 8.0, but the optimum pH was found to be at 7.6. The invention will now be illustrated with reference to the following non-limitative examples. The following procedure gives details of the formulation and how it is carried out. A triple blood Bag system is used which had a Primary Bag A and two Satellite Bags B & C, The PrimatY Bag A contains CPD solution (49ml for 350mi blood Bag system and 63ml for 450ml blood Bag system). Isotonic buffered saline used has the following composition : (a) 70.2mg NaHz PO4 In 1.5ml IP grade water. (b) 1.45gm NazHPO4 in 34ml IP grade water. (c) 0.445gm NaCI in 25ml IP grade water. Mix (a) and (b); Adjust pH to 7.6., mix 25ml of this with 25ml of (c) and adjust pH to 7,6 with NaHCOs. In the above table, nicotinic acid can be replaced by nicotinamide. The Bag system containing the respective solution in the Primary Bag A and the Satellite Bags B & C is autoclaved at 151bs pressure for 15 minutes. Results obtained with the modified formulation and SAGM solution in a few typical cases are given below. Blood is collected from the donor by usual procedure in to the Primary Bag A. Then the solution from Satellite Bag C Is transferred to the solution in the Satellite Bag B, RBCs are separated by centrifuging at 2700rpm for 12 minutes and the plasma separated is transferred to the empty Satellite Bag C using a plasma expressor. Then the mixed solution in the Satellite Bag B is transferred to the Primary Bag containing RBCs and mixed. Then the two Satellite Bags are removed and the Primary Bag A sealed using a tube sealer. Various biochemical parameters are studied in an aliquot immediately after mixing (0 day) and the Bags are kept at 4 ± 1° C. These parameters are again measured after 28 days in another aliquot. Note ; 1. 2,3 DPG Is essential for maintaining oxygen delivery to the tissues. The higher its concentration, the better. 2. pH should be above 7.0 for optimum glycolysis and higher concentration of 2,3 DPG. 3. Plasma Hb and plasma K+ are measures of hemolysis. The extent of hemolysis should be low. 4. ATP is a measure of metabolic activity of red cells. 5. Malondiaidehyde is a measure of lipid peroxidation and its higher concentration indicates higher lipid peroxidation, which is harmful. 6. Reduced glutathione Is an antioxidant, which protects against the harmful effects of lipid peroxidation. The higher its concentration, the higher is the protective effect. 7. DEHP is harmful and a tower level in the solution is desirable. The results given for the 2 experiments are typical of all experiments carried out. A total number of 10 experiments Is carried out for the modified formulation and CPDA solution and the results obtained in every case are similar. The range of variation in the different parameters in different experiments are given below. EXAMPLE 2 The details of the experiments are the same except that nicotinic acid was incorporated in the preservative solution at a concentration of 5 mg/dl RBC suspension and ascorbic acid was incorporated at a concentration of 3 mg/dl RBC suspension. Similar changes as in example 1 are observed In all the parameters studied. However the extent of beneficial effects observed are lower compared to example 1. The details of the experiments were the same except that nicotinic acid was incorporated in the preservative solution at a concentration of 8 mg/dl RBC suspension and ascorbic acid was incorporated at a concentration of 6 mg/dl RBC suspension. Similar changes as in example 1 were observed in ait the parameters studied. However the extent of beneficial effects observed are lower compared to example 1 and hemolysis was more. EXAMPLE 4 Keeping the concentration of nicotinic acid constant, the concentration of ascorbic acid is varied for 3 to 6 mg/di RBC suspension. Similarly the concentration of ascorbic acid Is kept constant while that of nicotinic acid was varied from 5-8 mg/dl RBC suspension. Optimum beneficial effects are obtained when the concentration of nicotinic acid is 6.8 mg/dl RBC suspension and that of ascorbic acid 4.0 mg/dl RBC suspension. The modified solution is thus effective in preventing the drastic fail in 2,3 DPG that taka place during storage of RBCs. The level of 2,3 DPG Is maintained almost at the same level as on the day 0. There is also significant decrease in the leaching of DEHP from the Bag surface to the RBC suspension. PH Is well maintained over 7.0. Hemolysis and extent lipid peroxidation are lower. ATP level is not significantly altered. 1. An apparatus and a process For reducing the deterioration of RBCs of blood during storage with particular reference to decrease in the level of 2,3 diphosphoglycerate comprising of: i. an apparatus of Triple Blood Bag system which has a Primary Bag A, first Satellite Bag B and second Satellite Bag C, the said Primary Bag A containing a solution A which is an anti¬coagulant solution, the said first Satiated Bag B containing a solution B which is a preservative formulation, and the said second Satellite Bag C containing a solution C which is a dextrose solution ; and ii. a process including the following steps : a. autoclaving of solutions In said Primary Bag A, said Satellite Bag B and said Satellite Bag C; b. collection of donor blood into said Primary Bag A; c. said Primary Bag A with collected blood maintained at a temperature 4 ± 1 °C; d. centrifugation of collected blood in said Primary Bag A at 2700 rpm for 12 minutes for separation of plasma and RBCs; e. transfer of said solution C from said Satellite Bay C to said Satellite Bag B such that the Satellite Bag B contains a mixture of said solution B and said solution C and said Satellite Bag C being empty at the end of this step; f. mixing of said solution C with said solution B in said Satellite Bag 6 by means of agitation to form a mixed preservative solution at the end of this step; g. transfer of said plasma so separated in the said Primary Bag A in step d as above using plasma expresser into empty Satellite Bag C such that said Primary Bag A contains only RBCs at the end of this step; h. transfer of mixed solution contained in said Satellite Bag B from step f into said Primary Bag A containing only RBCs of step g; I. mixing of RBCs contained in said Primary Bag A with the solution transferred from said Satellite Bag B by means of gentle manual agitation; j. sealing of said Primary Bag A using a tub© sealer; k. storage of said Primary Bag A with collected RBC at 4 ± 1°C; 2. An apparatus and a process for reducing the deterioration of RBCs of blood during storage with particular reference to decrease in the level of 2,3 diphosphogiycerate as claimed in claim 1, wherein the said solution B in the said Satellite Bag B has to following composition : 3. An apparatus and a/process for reducing the deterioration of RBCs of blood during storage with particular reference to decrease in the level of 2,3 diphosphoglycerate as claimed in claim 1, wherein the said solution C in the said Satellite Bag C has the following composition : 4. An apparatus and a process for reducing the deterioration of RBCs of blood during storage with particular reference to decrease in the level of 2,3 diphosphoglycerate as claimed in claim 1, wherein said solution A i.e. the CPD solution comprises of the following composition : A CPD solution may contain - Tribolium citrate (dehydrate) - 26.3g 5. An apparatus and a process for reducing the deterioration of RBCs of blood during storage with particular reference to decrease in the level of 2,3 diphosphoglycerate as claimed in claim 1, wherein autoclaving of step a is done at ISIbs pressure for 15 minutes. 6. An apparatus and a process for reducing the deterioration of RBCs of blood during storage with particular reference to decrease in the level of 2,3 diphosphoglycerate as claimed in claim 1, wherein the said Primary Bag A containing collected blood In the said solution A from step b is immediately transferred in step C to be stored at temperature 4 ± 1 °C after collection from donor and the process of RBCs and plasma separation is done at 4 ± 1 °C immediately thereafter by centrifugation as in step d. 7. An apparatus and a process for reducing the deterioration of RBCs of blood during storage with particular reference to decrease in the level of 2,3 diphosphoglycerate as claimed in claim 1, wherein in step e and In step f, the said solution C in the said Satellite Bag C is transferred into the said Satellite Bag B containing the said solution B until the said Satellite Bag C is empty and thereafter mixed gently by agitating manually the said Satellite Bag B continuously during the mixing. 8. An apparatus and a process for reducing the deterioration of RBCs of blood during storage which particular reference to decrease in the level of 2,3 diphosphoglycerate as claimed In claim 1, wherein the said mixed preservative solution of step h for RBC storage formulation in the said Satellite Bag B to be transferred in step h to said Primary Bag A has pH level in the range 7-8. 9. An apparatus and a process for reducing the deterioration of RBCs of blood during storage with particular reference to decrease In the level of 2,3 diphosphogiycerate as claimed in claim' 1, wherein in the said mixed preservative solution of step h, formulation for RBC storage has optimum PH level at 7.6. 10. An apparatus and a process for reducing the deterioration of RBCs of blood during storage with particular reference to decrease in the level of 2,3 diphosphogiycerate as claimed in claim 1, wherein the said mixed preservative formulation of step h for RBC storage, the concentration of the nicotinic add is in the range of 5 - 8 mg/dl blood collected. 11. An apparatus and a process for reducing the deterioration of RBCs of blood during storage with particular reference to decrease in the level of 2,3 diphosphoglycerate as claimed in claim 1, wherein in the said mixed preservative formulation of step h for RBC storage, the optimum concentration of nicotinic acid is 6.8 mg/dl of blood collected. 12. An apparatus and ^process for reducing the deterioration of RBCs of blood during storage with particular reference to decrease In the level of 2,3 diphosphoglycerate as claimed in claim 1, wherein in the said mixed preservative formulation of step h for RBC storage, the concentration of ascorbic acid is in the range of 3 - 5 mg/dl blood to be collected. 13. An apparatus and a process for reducing the deterioration of RBCs of blood during storage with particular reference to decrease in the level of 2,3 diphosphoglycerate as claimed in claim 1, wherein in the said mixed preservative formulation of step h for RBC storage, the optimum concentration of ascorbic acid is 4.0 mg/dl blood collected. 14. An apparatus and a process for reducing the deterioration of RBCs of blood during storage with particular reference to decrease In the level of 2,3 diphosphoglycerate as claimed in claim 1, wherein the said mixed preservative formulation of step h for RBC storage, is characterized in that the optimum pH is 7.6, optimum nicotinic acid concentration is 6.8 mg/dl blood collected and optimum ascorbic acid concentration is 4.0 mg/di blood collected. 15. An apparatus and, a process for reducing the deterioration of RBCs of blood during storage with particular reference to decrease in the level of 2,3 diphosphoglycerate as claimed in claim 1, wherein the said mixed preservative formulation of step h for RBC storage, Is characterized in that the pH is the range of 7 - 7.8, contains nicotinic acid in the range of 5 - 8 mg/dl blood collected and contains ascorbic acid in the range 3-6 mg/dl blood collected. 16. An apparatus and a process for reducing the deterioration of RBCs of blood during storage with particular reference to decrease in the level of 2,3 diphosphoglycerate as claimed in claim 2, wherein the said nicotinic acid in said solution B in Said Satellite Bag B, can be replaced by nicotinamide. 17. An apparatus and a process for reducing the deterioration of RBCs of blood during storage wlth particular reference to decrease in the level-of 2,3 diphosphoglycerate as claimed in claim 1, wherein apparatus includes a Triple Blood Bag comprising of a Primary Bag A containing anti-coagulant solution A, a first Sateiiite Bag B containing preservative solution B, and a second Sateiiite Bag C containing dextrose solution C. 18. An apparatus and a proces for reducing the deterioration of RBCs of blood during storage with particular reference to decrease in the level of 2,3 diphosphoglycerate as claimed in claim 1, wherein the process includes independent autoclaving of solutions A, B and C in the said Bags A, B and C respectively, collection of donor blood in the said Primary Bag A at desired temperature, separation of plasma from RBC by centrifugation, transferring of said solution C from the said Sateiiite Bag C into the said Sateiiite Bag B to form a mixed preservative solution in the said Sateiiite Bag B, the rejected plasma from the said Primary Bag A is collected In empty Satellite Bag C, thereafter transferring of mixed preservative solution from the said Satellite Bag B Into the said Primary Bag A containing the RBCs in the said Primary Bag A Is sealed and through stored at desired temperature. 19. An apparatus and process reducing the deterioration of RBCs of blood during storage with particular reference to decrease in the level of 2,3 diphosphoglycerate as claimed in claim 1 & claim 2, the isotonic buffered saline of said solution B has the following ingredients mixed in a sequence such that the Ingredients Include (a) 70.2mg NaH2 PO4 In 1.5ml IP grade water. (b) 1.45gm Na2HPOA in 34ml IP grade water. (c) 0.445gm NaCI in 25ml IP grade water. and sequence of mixing includes the steps : Mix (a) and (b); Adjust pH to 7.6., Mix 25ml of this with 25ml of (c). & adjust pH to 7.6 with NaHCOs. 20, An apparatus and a process for reducing the deterioration of RBCs of blood during storage with particular reference to decrease in the level of 2,3 diphosphogiycerate as claimed in claim 1, wherein the biochemical parameters in the stored blood of step k studied in an aliquot after 28 days of storage of RBCs are ; for RBC storage) 21. An apparatus and a process for reducing the deterioration of RBCs of blood during storage with particular reference to decrease in the level of 2,3 diphosphogtycerate as claimed in claim 1, and as described in the complete specification herein. 22. An apparatus and a (process for reducing the deterioration of RBCs of blood during storage with particular reference to decrease in the level of 2,3 diphosphogtycerate as claimed in claim 1, and as iiiustrated in the accompanying drawings.

Full Text

This invention relates to an apparatus and a process for reducing the deterioration of RBCs during storage, particularly the decrease in the level of 2, 3DPG and formulation employed for the same.
Blood is usually collected in a triple Bag and stored in certain anticoagulant preservative solutions like AGO (acid-citrate-dextrose), CPD (citrate-phosphate-dextrose) and CPDA (citrate-phosphate-dextrose-adenine).

Decrease in pH is also deleterious since it may adversely affect giycoiysis, a major metabolic activity of RBCs.
Another deleterious effect of storage when DEHP plasticized PVC Bags are used, is an increase in the amount of DEHP solubilize in the 5AGM containing RBCs, which may cause toxic problems in recipients of RBC transfusion. DEHP has been reported to be toxic particularly to the liver and reproductive organs and is also carcinogenic. Further we have found that DEHP at low levels causes decrease in insulin and Cortical, depletion of fat soiubie vitamins (A, 0 & E), increase in T3 & T4 and more important inhibition of membrane bound Na+ - K+ ATPase. The last observation is significant since it is now known that there is inhibition of this enzyme activity in a variety of pathologic conditions like neurodegenerative disorders, cardiovascular disease, diabetes, cancer etc.
The decrease in the level of ATP is not very drastic, but the level of 2,3 DPG falls to a very tow level during storage. The invivo restoration of 2,3 DPG depends on the level of 2,3 DPG in the RBO; transfused. If the level is very low, then it may take a long time, some time over 20hrs to restore the original level,
A major purpose of transfusion of RBCs is to restore oxygen supply to the tissues. This is particularly so in transfusions with compromised

circulation in the heart or brain, or in massive loss of blood. This is also more particularly important In infants, since inadequate delivery of oxygen can result in development of hypoxy/ischemic encephalopathy, the major cause of long term neuro developmental problem in children.
A major factor involved in ensuring adequate delivery of oxygen to the tissues is 2,3 DPG concentration in the RBCs. 2,3 DPG has a regulatory effect on the oxygen affinity of hemoglobin and on oxygen transport In vivo. Hemoglobin binds to oxygen to form ox hemoglobin which does not release oxygen to the tissues. 2,3 OFG binds strongly to ox hemoglobin and this binding decreases the affinity of hemoglobin to oxygen with consequent release of oxygen to the tissues. Therefore an adequate level of 2,3 DPG in the RBCs is essential particularly in critical situations like cardiac surgery, brain surgery or in case of massive hemorrhage.
During storage of RBCs in SAGM, the red cells usually become depleted of 2,3 DPG after 1-2 weeks which causes increase in the Hb oxygen affinity with consequent inadequate oxygen delivery to the tissues.
During our study of large number of RBCs stored in SAGM solution in blood Bags at 4 ± 1° C, the following pattern of decrease in 2,3 DPG has been observed.

Th« loss of 2,3 DPG Is n»v«rsibl«» however, post transfusion restoration of 2,3 DPG does taice place, but tine time for this depends on the level of 2,3 DPG in the RBCs transfused If the level is low as is the raze after storage after 14 days, it may take more than 24 hours for this recovery. This is the reason why for cardiac or neuron surgery, fresh blood is always preferred. From a practical point of view, a shelf life of 14 days and not 35 days is preferred in critical situations, because of the drop in the 2,3 DPG levels.
Compared to the fall In 2,3 DPG, the fall in ATP level is not so dramatic. The pattern of change in concentration of ATP in the RBCs in our studies is given below :-

ATP level is a measure of the metabolic activity of the RBCs, Maintaining of red cell viability is relatively closely associated to the cellular concentration of ATP. Since ATP level does not fait to a level below 50% of th« original level, post transfusion restoration of ATP does not take much time, as Is the case with the situation in 2,3 DPG.
ATP is produced by RBCs during glycol sis, which also produces 2,3 DPG from 1,3-diphosphogiycerate by the diphosphonic glycerate shunt. 1,3-diphospho glycerate produced during glycol sis has two metabolic fates, one, it is converted directly to 3-phospho glycerate and continues In the glycol tic pathway to lactate. The second one is the shunt pathway where it is converted to 2,3-diphospho glycerate by the action of the enzyme 2,3-diphospho glycerate mutate. 2,3-diphospho glycerate is then acted on by 2,3-diphospho glycerate phosphates to from 3-phospho glycerate which continues in the glycol tic pathway.
The factors which control the concentration of 2,3 DPG in the RBCs are :-

(1) The balance between its rate of synthesis by 2,3-diphospho glycerate mutate and its rate of degradation by 2,3-diphospho glycerate phosphates.
(2) A pH above 7.0 favors the synthesis of 2,3 DPG while a pH below 7.0 favours its decomposition.
(3) When RBCs are stored In SAGM solution, the pH steadily decreases.

(4) Adenine, even though it favors increase in concentration of ATP, has a
deleterious effect on the concentration of 2,3 DPG.
C5) Citrate in the medium for RBC storage helps to keep intracellular pH higher since the citrate ion is not permeable.

These observations indicate that keeping the intracellular pH higher can increase the concentration of 2,3 DPG,
We had earlier made the following observations.
(1) Addition of nicotinic acid in the preservative medium for RBCs, Increases the concentration of 2 (2) Addition of an optimum level of ascorbic acid significantly increases the concentration of 2,3 DPG. The concentration has to be optimum, because above a particular concentration it decreases ATP and Increases haemolysis,
(3) Addition of nicotinic acid also decreases the leaching out of OEHP (the plasticizer used in PVC Bags) into medium for RBCs storage. This is important since DEHP at low levels has now been found by us to cause decrease in insulin and Cortical, depletion of fat soluble vitamins (A, 0 &. E), increase in T3 &.T4 and more important inhibition of membrane bound Na* - K^ ATPase (under publication). The last observation is significant since it is now known that there is inhibition of this enzyme activity in a variety of pathologic conditions like neurodegenerative disorders, cardiovascular diseasing, deadheads, cancer etc.

(4) Incorporation both nicotinic acid and ascorbic acid has an additive effect, in that the beneficial effects are more than the effect of each added separately.
(5) Based on these observation we have developed modified formulation for the preservation of RBCs.
This modification has been achieved by
(1) Maintaining the pH of the preservative solution for RBCs at 7.6. This is made possible by autoclaving the dextrose solution separately in the second Satellite Bag C since dextrose solution at alkaline pH develops a yellow to red color on autoclaving, which is not acceptable. The triple Bag with CPD solution in the Primary Bag A, preservative solution containing the various ingredients to be mentioned below except dextrose in Satellite Bag B and the dextrose solution in the Satellite Bag C is now autoclaved as per the standard procedure. The dextrose solution after autoclaving is transferred from the Satellite Bag C to the rest of the solution at pH 7.6 In the first Satellite Bag B. Blood is collected from the donor in the Primary Bag A.

(2) Incorporating ascorbic acid and nicotinic acid at certain optimum concentration in each case to the medium.
This formulation is effective in minimizing the deleterious effects, which occur during storage of RBCs in blood Bags.
Thus, according to one aspect of the present invention, there is provided a formulation for better storage and preservation of RBCs characterized in that it has a pH above 7.0, It contain nicotinic acid and ascorbic acid at optimum levels. None of the preservative solutions for RBCs hitherto known contains nicotinic acid and ascorbic acid or has pH above 7.0, and as stated hereinabove it was an important finding for us to see that the incorporation of nicotinic acid and ascorbic acid and maintaining pH above 7.0 of the preservative solution considerably reduces the deterioration of RBCs during storage.
Thus, according to another aspect of the present Invention, there is provided an apparatus and a process for reducing the deterioration of RBCs during storage, characterized in that the solution used for the preservation of blood is modified by the incorporation of nicotinic acid, ascorbic acid and keeping pH above 7.0.

The concentration of nicotinic acid used in the anticoagulant solution may vary, and it may preferably be in the range of 5 to 8 mg/dl of RBC suspension. However the optimum concentration was found to be 6.8 mg/dl
of RBC suspension.
The concentration of ascorbic acid used in the preservative solution also may vary, and it may preferably be in the range of 3 to 5 mg/dl RBC suspension. However the optimum concentration with minimum hemolytic effect and maximum beneficial effects was found to be 4.0 mg/dl RBC suspension.
The pH of the preservative solution can vary form 7.0 to 8.0, but the optimum pH was found to be at 7.6.
The invention will now be illustrated with reference to the following non-limitative examples.
The following procedure gives details of the formulation and how it is carried out.

A triple blood Bag system is used which had a Primary Bag A and two Satellite Bags B & C, The PrimatY Bag A contains CPD solution (49ml for 350mi blood Bag system and 63ml for 450ml blood Bag system).

Isotonic buffered saline used has the following composition :
(a) 70.2mg NaHz PO4 In 1.5ml IP grade water.
(b) 1.45gm NazHPO4 in 34ml IP grade water.
(c) 0.445gm NaCI in 25ml IP grade water.
Mix (a) and (b); Adjust pH to 7.6., mix 25ml of this with 25ml of (c) and adjust pH to 7,6 with NaHCOs.
In the above table, nicotinic acid can be replaced by nicotinamide.
The Bag system containing the respective solution in the Primary Bag A and the Satellite Bags B & C is autoclaved at 151bs pressure for 15 minutes.

Results obtained with the modified formulation and SAGM solution in a few typical cases are given below.
Blood is collected from the donor by usual procedure in to the Primary Bag A. Then the solution from Satellite Bag C Is transferred to the solution in the Satellite Bag B, RBCs are separated by centrifuging at 2700rpm for 12 minutes and the plasma separated is transferred to the empty Satellite Bag C using a plasma expressor. Then the mixed solution in the Satellite Bag B is transferred to the Primary Bag containing RBCs and mixed. Then the two Satellite Bags are removed and the Primary Bag A sealed using a tube sealer. Various biochemical parameters are studied in an aliquot immediately after mixing (0 day) and the Bags are kept at 4 ± 1° C. These parameters are again measured after 28 days in another aliquot.

Note ;
1. 2,3 DPG Is essential for maintaining oxygen delivery to the tissues. The higher its concentration, the better.
2. pH should be above 7.0 for optimum glycolysis and higher concentration of 2,3 DPG.
3. Plasma Hb and plasma K+ are measures of hemolysis. The extent of hemolysis should be low.
4. ATP is a measure of metabolic activity of red cells.
5. Malondiaidehyde is a measure of lipid peroxidation and its higher concentration indicates higher lipid peroxidation, which is harmful.
6. Reduced glutathione Is an antioxidant, which protects against the harmful effects of lipid peroxidation. The higher its concentration, the higher is the protective effect.
7. DEHP is harmful and a tower level in the solution is desirable.
The results given for the 2 experiments are typical of all experiments carried out. A total number of 10 experiments Is carried out for the modified formulation and CPDA solution and the results obtained in every case are similar. The range of variation in the different parameters in different experiments are given below.

EXAMPLE 2
The details of the experiments are the same except that nicotinic acid was incorporated in the preservative solution at a concentration of 5 mg/dl RBC suspension and ascorbic acid was incorporated at a concentration of 3 mg/dl RBC suspension. Similar changes as in example 1 are observed In all the parameters studied. However the extent of beneficial effects observed are lower compared to example 1.

The details of the experiments were the same except that nicotinic acid was incorporated in the preservative solution at a concentration of 8 mg/dl RBC suspension and ascorbic acid was incorporated at a concentration of 6 mg/dl RBC suspension. Similar changes as in example 1 were observed in ait the parameters studied. However the extent of beneficial effects observed are lower compared to example 1 and hemolysis was more.
EXAMPLE 4
Keeping the concentration of nicotinic acid constant, the concentration of ascorbic acid is varied for 3 to 6 mg/di RBC suspension. Similarly the concentration of ascorbic acid Is kept constant while that of nicotinic acid was varied from 5-8 mg/dl RBC suspension. Optimum beneficial effects are obtained when the concentration of nicotinic acid is 6.8 mg/dl RBC suspension and that of ascorbic acid 4.0 mg/dl RBC suspension.
The modified solution is thus effective in preventing the drastic fail in 2,3 DPG that taka place during storage of RBCs. The level of 2,3 DPG Is maintained almost at the same level as on the day 0. There is also significant decrease in the leaching of DEHP from the Bag surface to the RBC suspension. PH Is well maintained over 7.0. Hemolysis and extent lipid peroxidation are lower. ATP level is not significantly altered.

1. An apparatus and a process For reducing the deterioration of RBCs of blood during storage with particular reference to decrease in the level of 2,3 diphosphoglycerate comprising of:
i. an apparatus of Triple Blood Bag system which has a Primary Bag A, first Satellite Bag B and second Satellite Bag C, the said Primary Bag A containing a solution A which is an anti¬coagulant solution,
the said first Satiated Bag B containing a solution B which is a preservative formulation, and
the said second Satellite Bag C containing a solution C which is a dextrose solution ; and
ii. a process including the following steps :
a. autoclaving of solutions In said Primary Bag A, said Satellite
Bag B and said Satellite Bag C;
b. collection of donor blood into said Primary Bag A;
c. said Primary Bag A with collected blood maintained at a
temperature 4 ± 1 °C;

d. centrifugation of collected blood in said Primary Bag A at
2700 rpm for 12 minutes for separation of plasma and RBCs;
e. transfer of said solution C from said Satellite Bay C to said
Satellite Bag B such that the Satellite Bag B contains a
mixture of said solution B and said solution C and said
Satellite Bag C being empty at the end of this step;
f. mixing of said solution C with said solution B in said Satellite
Bag 6 by means of agitation to form a mixed preservative
solution at the end of this step;
g. transfer of said plasma so separated in the said Primary Bag
A in step d as above using plasma expresser into empty
Satellite Bag C such that said Primary Bag A contains only
RBCs at the end of this step;
h. transfer of mixed solution contained in said Satellite Bag B from step f into said Primary Bag A containing only RBCs of step g;
I. mixing of RBCs contained in said Primary Bag A with the solution transferred from said Satellite Bag B by means of gentle manual agitation;
j. sealing of said Primary Bag A using a tub© sealer;
k. storage of said Primary Bag A with collected RBC at 4 ± 1°C;

2. An apparatus and a process for reducing the deterioration of RBCs of blood during storage with particular reference to decrease in the level of 2,3 diphosphogiycerate as claimed in claim 1, wherein the said solution B in the said Satellite Bag B has to following composition :

3. An apparatus and a/process for reducing the deterioration of RBCs of
blood during storage with particular reference to decrease in the level of
2,3 diphosphoglycerate as claimed in claim 1, wherein the said solution C in
the said Satellite Bag C has the following composition :

4. An apparatus and a process for reducing the deterioration of RBCs of
blood during storage with particular reference to decrease in the level of 2,3
diphosphoglycerate as claimed in claim 1, wherein said solution A i.e. the
CPD solution comprises of the following composition :
A CPD solution may contain -
Tribolium citrate (dehydrate) - 26.3g

5. An apparatus and a process for reducing the deterioration of RBCs of blood during storage with particular reference to decrease in the level of 2,3 diphosphoglycerate as claimed in claim 1, wherein autoclaving of step a is done at ISIbs pressure for 15 minutes.
6. An apparatus and a process for reducing the deterioration of RBCs of blood during storage with particular reference to decrease in the level of 2,3 diphosphoglycerate as claimed in claim 1, wherein the said Primary Bag A containing collected blood In the said solution A from step b is immediately transferred in step C to be stored at temperature 4 ± 1 °C after collection from donor and the process of RBCs and plasma separation is done at 4 ± 1 °C immediately thereafter by centrifugation as in step d.
7. An apparatus and a process for reducing the deterioration of RBCs of blood during storage with particular reference to decrease in the level of 2,3 diphosphoglycerate as claimed in claim 1, wherein in step e and In step f, the said solution C in the said Satellite Bag C is transferred into the said Satellite Bag B containing the said solution B until the said Satellite Bag C is

empty and thereafter mixed gently by agitating manually the said Satellite Bag B continuously during the mixing.
8. An apparatus and a process for reducing the deterioration of RBCs of blood during storage which particular reference to decrease in the level of 2,3 diphosphoglycerate as claimed In claim 1, wherein the said mixed preservative solution of step h for RBC storage formulation in the said Satellite Bag B to be transferred in step h to said Primary Bag A has pH level in the range 7-8.
9. An apparatus and a process for reducing the deterioration of RBCs of blood during storage with particular reference to decrease In the level of 2,3 diphosphogiycerate as claimed in claim' 1, wherein in the said mixed preservative solution of step h, formulation for RBC storage has optimum PH level at 7.6.
10. An apparatus and a process for reducing the deterioration of RBCs of
blood during storage with particular reference to decrease in the level of 2,3 diphosphogiycerate as claimed in claim 1, wherein the said mixed preservative formulation of step h for RBC storage, the concentration of the nicotinic add is in the range of 5 - 8 mg/dl blood collected.

11. An apparatus and a process for reducing the deterioration of RBCs of blood during storage with particular reference to decrease in the level of 2,3 diphosphoglycerate as claimed in claim 1, wherein in the said mixed preservative formulation of step h for RBC storage, the optimum concentration of nicotinic acid is 6.8 mg/dl of blood collected.
12. An apparatus and ^process for reducing the deterioration of RBCs of
blood during storage with particular reference to decrease In the level of 2,3 diphosphoglycerate as claimed in claim 1, wherein in the said mixed preservative formulation of step h for RBC storage, the concentration of ascorbic acid is in the range of 3 - 5 mg/dl blood to be collected.

13. An apparatus and a process for reducing the deterioration of RBCs of blood during storage with particular reference to decrease in the level of 2,3 diphosphoglycerate as claimed in claim 1, wherein in the said mixed preservative formulation of step h for RBC storage, the optimum concentration of ascorbic acid is 4.0 mg/dl blood collected.
14. An apparatus and a process for reducing the deterioration of RBCs of blood during storage with particular reference to decrease In the level of 2,3 diphosphoglycerate as claimed in claim 1, wherein the said mixed

preservative formulation of step h for RBC storage, is characterized in that the optimum pH is 7.6, optimum nicotinic acid concentration is 6.8 mg/dl blood collected and optimum ascorbic acid concentration is 4.0 mg/di blood collected.
15. An apparatus and, a process for reducing the deterioration of RBCs of blood during storage with particular reference to decrease in the level of 2,3 diphosphoglycerate as claimed in claim 1, wherein the said mixed preservative formulation of step h for RBC storage, Is characterized in that the pH is the range of 7 - 7.8, contains nicotinic acid in the range of 5 - 8 mg/dl blood collected and contains ascorbic acid in the range 3-6 mg/dl blood collected.
16. An apparatus and a process for reducing the deterioration of RBCs of blood during storage with particular reference to decrease in the level of 2,3 diphosphoglycerate as claimed in claim 2, wherein the said nicotinic acid in said solution B in Said Satellite Bag B, can be replaced by nicotinamide.
17. An apparatus and a process for reducing the deterioration of RBCs of blood during storage wlth particular reference to decrease in the level-of 2,3 diphosphoglycerate as claimed in claim 1, wherein apparatus includes a Triple Blood Bag comprising of a Primary Bag A containing anti-coagulant

solution A, a first Sateiiite Bag B containing preservative solution B, and a second Sateiiite Bag C containing dextrose solution C.
18. An apparatus and a proces for reducing the deterioration of RBCs of blood during storage with particular reference to decrease in the level of 2,3 diphosphoglycerate as claimed in claim 1, wherein the process includes independent autoclaving of solutions A, B and C in the said Bags A, B and C respectively, collection of donor blood in the said Primary Bag A at desired temperature, separation of plasma from RBC by centrifugation, transferring of said solution C from the said Sateiiite Bag C into the said Sateiiite Bag B to form a mixed preservative solution in the said Sateiiite Bag B, the rejected plasma from the said Primary Bag A is collected In empty Satellite Bag C, thereafter transferring of mixed preservative solution from the said Satellite Bag B Into the said Primary Bag A containing the RBCs in the said Primary Bag A Is sealed and through stored at desired temperature.
19. An apparatus and process reducing the deterioration of RBCs of blood during storage with particular reference to decrease in the level of 2,3 diphosphoglycerate as claimed in claim 1 & claim 2, the isotonic buffered saline of
said solution B has the following ingredients mixed in a sequence such that the Ingredients Include
(a) 70.2mg NaH2 PO4 In 1.5ml IP grade water.

(b) 1.45gm Na2HPOA in 34ml IP grade water.
(c) 0.445gm NaCI in 25ml IP grade water.
and sequence of mixing includes the steps :
Mix (a) and (b); Adjust pH to 7.6., Mix 25ml of this with 25ml of (c). & adjust pH to 7.6 with NaHCOs.
20, An apparatus and a process for reducing the deterioration of RBCs of blood during storage with particular reference to decrease in the level of 2,3 diphosphogiycerate as claimed in claim 1, wherein the biochemical parameters in the stored blood of step k studied in an aliquot after 28 days of storage of RBCs are ;

for RBC storage)
21. An apparatus and a process for reducing the deterioration of RBCs of blood during storage with particular reference to decrease in the level of 2,3 diphosphogtycerate as claimed in claim 1, and as described in the complete specification herein.
22. An apparatus and a (process for reducing the deterioration of RBCs of blood during storage with particular reference to decrease in the level of 2,3 diphosphogtycerate as claimed in claim 1, and as iiiustrated in the accompanying drawings.

Documents:

994-mas-2001 abstract.pdf

994-mas-2001 claims.pdf

994-mas-2001 correspondence others.pdf

994-mas-2001 correspondence po.pdf

994-mas-2001 description (complete).pdf

994-mas-2001 drawing.pdf

994-mas-2001 form-1.pdf

994-mas-2001 form-26.pdf

994-mas-2001 form-5.pdf

994-mas-2001 form-9.pdf

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Patent Number

193608

Indian Patent Application Number

994/MAS/2001

PG Journal Number

30/2009

Publication Date

24-Jul-2009

Grant Date

18-Apr-2005

Date of Filing

10-Dec-2001

Name of Patentee

TERUMO PENPOL LTD

Applicant Address

IX/132, SASTHAMANGALAM, TRIVANDRUM 695 010,

Inventors:

#	Inventor's Name	Inventor's Address
1	PARAMESWARA ACHUTHA KURUP	R&D DEPARTMENT OF BIOLOGICAL SCIENCES, TERUMO PENPOL LIMITED, TRIVANDRUM
2	PEETHAMBARAN ARUN	R&D DEPARTMENT OF BIOLOGICAL SCIENCES, TERUMO PENPOL LIMITED, TRIVANDRUM
3	CHANDRASEKHAR BALAGOPAL	TERUMO PENPOL LIMITED, IX/1323, SASTHAMANGALAM, TRIVANDRUM

PCT International Classification Number

A01N1/0

PCT International Application Number

N/A

PCT International Filing date

PCT Conventions:

#	PCT Application Number	Date of Convention	Priority Country
1			NA