WO2004112631A1

WO2004112631A1 - A FORMULATION 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

Info

Publication number: WO2004112631A1
Application number: PCT/IN2003/000226
Authority: WO
Inventors: Achutha Kurup Parameswara; Arun Peethambaran; Balagopal Chandrasekhar
Original assignee: TERUMO PENPOL Ltd
Current assignee: TERUMO PENPOL Ltd
Priority date: 2003-06-20
Filing date: 2003-06-20
Publication date: 2004-12-29
Anticipated expiration: 2005-12-20
Also published as: AU2003250529A1

Abstract

SAGM (saline- adenine-dextrose-mannitol) solution is the most widely used preservative solution for storage of RMCs after separation farm whole blood. Even through 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 cells, which increases with storage time. Some of the most harmful changes are a drastic decrease in the level of 2,3 diphosphoglycerate (which is essential for delivery or oxygen to the tissues), increased hemolysis (which releases Hb and K+ from the RBCs into the SAGM, both of which are harmful) and progressive solubilization or di- (2-ethylhexyl) phthalate (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.

Description

TITLE :- A FORMULATION 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

This Invention relates to a formulation and a process for reducing the deterioration of RBCs during storage, particularly the decrease Jn the level of 2, 3DPG.

Blood is usually collected in a triple Bag and stored in certain anticoagulant preservative solutions like ACD (acid-citrate-dextrose), CPD (citrate-phosphate- dextrose) and CPDA (dtrate-phosphate-dextrose-adenine).

An ACD solution may contain

Trisodium citrate (dihydrate) 22.0g Citric acid (anhydrous) 8.0g Dextrose (monohydrate) 24.6g Distilled Water to make up to 1 litre

A CPD solution may contain

Trisodium citrate (dihydrate) 26.3g

Citric acid (anhydrous) 3.27g

Sodium dihydrogen phosphate

(monohydrate) 2.22g

Dextrose (monohydrate) 25.2g

Distilled Water to make up to 1 litre

A CPDA solution may contain -

Trisodium citrate (dihydrate) - 26.3g

Citric add (anhydrous) - 2.99g Sodium dihydrogen phosphate

(monohydrate) - 2.22g

Dextrose (monohydrate) - 31.9g

Atenine - 0.275g

Distilled Water - to make up to 1 litre

A triple blood Bag system with a Primary Bag and two Satellite Bags is used for the collection of whole blood and separation of RBCs. The Primary Bag (Bag A) contains the anticoagulant solution, which is usually CPD. One of the Satellite Bags (Bag B) contains the preservative solution for RBCs, which is usually SAGM having the following composition.

Sodium chloride - 8.77gm

Adenine - 0.30gm

Dextrose (monohydrate) - 9.0gm

Mannitol - 5.25gm

Water - to make up to 1 litre

The other Satellite Bag(Bag C) is empty, i.e. it does not contain any solution.

Blood for the donor is collected in the Primary Bag (Bag A) containing CPD solution and transferred immediately to 4 ± 1° C RBCs are separated by centrlfuging at 270Qrpm for 12 minutes at 4 ± 1° C. The RBCs sediment to the bottom and by using a plasma expresser, the plasma is transferred to the second empty Satellite Bag (Bag C). Then the SAGM solution from the first Satellite Bag (Bag B) is transferred to the Primary Bag (Bag A) containing RBCs. The RBCs are gently mixed with the SAGM solution, kept in a Wood bank refrigerator and maintained at 4 ± lo c. QWe T OF IHVEMTIQN

The plastic Bags normally used for the collection and storage may be PVC Bags which use di-(2-ethyi hexyl) phthalate (D&IP), tri-(2-ethyl hexyl) trimβllltate (TEHTM) or butyl-n-tri-hexyl citrate (BTHC) as the plasticfeer or non-PVC Bags like polyolefin.

It is known that when RBCs are stored in such Bags containing SAGM, there is deterioration in the quality of red cells, the extent of which increases with the period of storage. A few major deleterious changes observed are : decrease in pH, a very drastic decrease in 2,3 diphosphoglycerate, increased hemolysis with consequent increase In hemoglobin & potassium in the medium, a decrease in the level of adenosine triphosphate, an increase in the level of DB-iP solubilized in to the SAGM containing RBCs and an increase in lipid peroxidation.

The increase in hemolysis is believed to be due to the damage to the red ceil membrane caused by lipid peroxidation.

Any increase in hemoglobin in the storage medium may cause renal failure and other complications when solution containing high levels of hemoglobin Is transfused. Increased levels of potassium in the storage medium may cause hyperkaiemia which may result in cardiac complications.

Decrease in pH is also deleterious since it may adversely affect glycolysis, a major metabolic activity of RBCs.

Another deleterious effect of storage when D&IP plasticized PVC Bags are used, is an increase in the amount of D&IP solubilized in the SAGM containing RBCs, which may cause toxic problems In recipients of RBC transfusion. D&IP has been reported to be toxic particularly to the liver and reproductive organs and is also carcinogenic. Further we have found that D&IP at low levels causes decrease in insulin and cortisol, depletion of fat soluble vitamins (^ D & E), Increase In T₃ & T₄ and more important inhibition of membrane bound Na⁺ - * 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 low level during storage. The invivo restoration of 2,3 DPG depends on the level of 2,3 DPG in the RBCs 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 euro 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 haemoglobin and on oxygen transport invlvo. Hemoglobin binds to oxygen to form oxyhemogiobin which does not release oxygen to the tissues. 2,3 DPG binds strongly to oxyhemogiobin 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 conseq uent i nadeq uate oxygen delivery to the tissues.

During our study of large number of RBCs stored in SAGM solution in blood Bags at 4 ± 1⁰ C, the following pattern of decrease in 2,3 DPG has been observed. DAY Level of 2,3 DPG (μmol/g Hb}

12 - 15

14 3.9 - 9.9

28 0.46 - 1.3

42 0.37 - 0.83

The loss of 2,3 DPG is reversible however, post transfusion restoration of 2,3 DPG does take place, but the time for this depends on the level of 2,3 DPG in the RBCs transfused. If the level is low as is the case after storage after 14 days, it may take more than 24 hours for this recovery. This is the reason why for cardiac or rieuro 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 fell 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 fail to a level below 50% of the 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 glycolysis, which also produces 2,3 DPG from 1,3-diphosphoglycerate by the diphospho glycerate shunt 1,3-diphospho gly erate produced during glycolysis has two metabolic fates, one, It is converted directly to 3-phospho glycerate and continues in the glycolytic pathway to lact^βte. The second one is the shunt pathway where it is converted to 2,3-diphospbo glycerate by the action of the enzyme 2^3-diphospho glycerate mutase. 2,3- diphospho glycerate is then acted on by 2,3-dfphospbo glycerate phosphatase to form 3-phospho glycerate which continues in the glycolytic 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-diphσspho glycerate mutase and its rate of degradation by 2,3-diphospho glycerate phosphatase.

(2) A pH above 7.0 favours 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.

(5) 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 2,3 DPG, decreases haemolysis and helps to maintain pH higher.

(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 hemolysis.

(3) Addition of nicotinic acid also decreases the leaching out of DB P (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 cortisol, depletion of fat soluble vitamins (A, D & E), increase in T₃ &-T* 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 disease, diabetes, 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.

DESCRIPTION OF IilVEMTION

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 has all the three factors i.e. contains nicotinic acid, ascorbic acid and has pH above 7.0, and as stated herelnabove 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 provkied a formulation and a process for reducing the deterioration of RBCs during storage, characterized in that the solution used for their 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 anticoaguter* 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 hβmotytic 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 referenoe 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 Primary Bag A contains CPD solution (49ml for 350ml blood Bag system and 63ml for 450ml blood Bag system).

Isotonic buffered saline used has the following composition :

(a) 70.2mg NaH₂ PO in 1.5ml IP grade water.

(b) 1.45gm Na₂HP0₄ in 34ml IP grade water.

(c) 0.445gm NaCl 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 NaHCOa .

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 autø aved at 15Jbs 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 centrlfuging 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 -fc IP c These parameters are again measured after 28 days in another aliquot

EXAMPLE 1

Experiment 1

Experiment 2

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. Malondialdehyde 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. D&IP is harmful and a lower 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.

Result obtained after 28 days

Parameters (Range obtained) SAGM Modified formulation

23 DPG (μM g Hb) 0.10 - 0.2 11.0 - 14.0 pH 6.86 - 6.94 7.02 - 7.06

Supernatant Hb (mg dl) 150.0 - 310.0 1350 - 250.0 Supernatant tC (mM lltre) 55.0 - 70.0 40.0 - 48.0

ATP (μM/g Hb) 27 - 3.2 26 - 3.1

MDA (nM mlllion cells) 3.40 - 4.1 28 - 3.3

Reduced glutathione (μg/ml) 220 - 228 247 - 250

D&IP solubilized (mg/dl) 6.9 - 8.0 5.5 - 6.73

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.

EXAMPLE 3

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 all 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/dl RBC suspension. Similariy 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 fell in 23 DPG that take 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 D&iP 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.

Claims

CLAIM

1. An apparatus and a process for preparing a formulation 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 Satellite 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 rprri for 12 minutes for separation of plasma and RBCs; e. transfer of said solution C from said Satellite Bag 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 B 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 (n 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 mean® of gentle manual agitation; j. sealing of said Primary Bag A using a tube sealer; k. storage of said Primary Bag A with collected RBC at 4 ± 1 °C

2 A process for preparing a formulation 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 B in the said Satellite Bag B has to following composition :

350ML BLOOD UNIT (350 BLOOD BAG SYSTEM)

Solution in Satellite Bag(B)

Adenine USP - 0.00676 gm

Sodium citrate (dihydrate) IP - 1.117gm

Mannitol IP - 0.42 gm

LAscorbic acid IP - 0.0136gm

Nicotinic acid IP - 0.024 gm

ι6

450ML BLOOD UNIT (450 BLOOD BAG SYSTEM)

Solution in Satellite Bag(B)

Adenine USP - 0.00845 gm

Sodium citrate (dihydrate) IP - 1.396 gm

Mannitol IP - 0.525 gm

L Ascorbic acid IP - 0.017 gm

Nicotinic acid IP - 0.03 gm

Isotonic buffered saline (pH 7.6) - 50ml

3. A process for preparing a formulation for reducing the deterioration of RBCs of blood during storage with particular reference to decrease in the level of 2 diphosphoglycerate as daimed in dai 1, wherein the said solution C in the said Satellite Bag C has the following composition :

350ML BLOOD UNIT (350 BLOOD BAG SYSTEM)

Solution in Satellite Bag(C)

Dextrose (monohydrate) - 0.6544 gm

Water for injection IP - 40ml

OR

450ML BLOOD UNIT (450 BLOOD BAG SYSTEM)

Solution in Satellite Bag(C)

Dextrose (monohydrate) - 0.818 gm

Water for Injection IP - 50ml

4. A process for preparing a formulation 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 -

Trisodium citrate (dihydrate) 26.3g

Citric acid (anhydrous) 299g

Sodium dihydrogen phosphate

(monohydrate) 222g

Dextrose (monohydrate) 25.2g

Water to make up to 1 litre

5. A process for preparing a formulation for reducing the deterioration of RBCs of blood during storage with particular reference to decrease in the level of 23 diphosphoglycerate as claimed in claim 1, wherein autoclaving of step a is done at 15ibs pressure for 15 minutes.

6. A process for preparing a formulation 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. A process for preparing a formulation 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. A process for preparing a formulation for reducing the deterioration of RBCs of blood during storage with particular reference to decrease in the level of 23 diphosphoglycerate as daimed 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. A process for preparing a formulation 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 solution of step h, formulation for RBC storage has optimum PH level at 7.6.

10. A process for preparing a formulation 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, the concentration of the nicotinic add is in the range of 5 - 8 mg/dl blood collected.

11. A process for preparing a formulation 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 A process for preparing a formulation for redudng the deterioration of RBCs of blood during storage with particular reference to decrease in the level of 2,3 diphosphoglycerate as daimed 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. A process for preparing a formulation for reducing the deterioration of RBCs of blood during storage with particular reference to decrease In the level of 2,3 diphosphoglycerate as daimed in daim 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. A process for preparing a formulation for reducing the deterioration of RBCs of blood during storage with particular reference to decrease in the level of 2 diphosphoglycerate as daimed in daim 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 add concentration is 6.8 mg/dl blood collected and optimum ascorbic adά concentration is 4.0 mg/dl blood collected.

15. A process for preparing a formulation 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 add in the range of 5 - 8 mg/dl blood collected and contains ascorbic add in the range 3 -6 mg/dl blood collected.

16. A process for preparing a formulation for reducing the deterioration of RBCs of blood during storage with particular reference to decrease in the level of 23 diphosphoglycerate as claimed in claim 2 wherein the said nicotinic add in said solution B in Said Satellite Bag B, can be replaced by nicotinamide.

2D

17. An apparatus 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 apparatus includes a Triple Blood Bag comprising of a Primary Bag A containing anti-coagulant solution A a first Satellite Bag B containing preservative solution B, and a second Satellite Bag C containing dextrose solution C.

18. A process for preparing a formulation for reducing the deterioration of RBCs of blood during storage with particular reference to decrease in the level of diphosphoglycerate as claimed in claim 1, wherein the process includes independent autoclaving of solutions A ^B and C in the said Bags J 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 Satellite Bag C into the said Satellite Bag B to form a mixed preservative solution in the said Satellite 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 stored at desired temperature.

19. A process for preparing a formulation for reducing the deterioration of RBCs of blood during storage with particular reference to decrease in the level of diphosphoglycerate as daimed in daim 1 & daim 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 P0 in 1.5ml IP grade water.

(b) 1.45gm Na₂HP0₄ in 34ml IP grade water.

(c) 0.445gm NaCI in 25ml IP grade water.

and sequence of mixing indudes 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 .

2D. A process for preparing a formulation 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 biochemical parameters In the stored blood of step k studied in an aliquot after 28 days of storage of RBCs are :

Parameters (Range obtained) SAGM^* Modified formulation

23 DPG (μM g Hb) 0.10 - 0.2 11.0 - 14.0

pH 6.86 - 6.94 7.02 - 7.06

Supernatant Hb (mg dl) 150.0 - 310.0 13S0 - 250.0

Supernatant * (mM/litre) 55.0 - 7CX0 40.0 - 48.0

ATP (μM g Hb) 27 - 3.2 26 - 3.1

MDA (nM/million cells) 3.40 - 4.1 28 - 3.3

Reduced glutathione (μg/ml) 220 - 228 247 - 250

DEHP solubilized (mg/dl) 6.9 - aθ 5.5 - 6.73

"(formulation now in use for RBC storage)

21. An anticoagulant preservative formulation 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 daim 1, where in the formulation has a pH. above 7.0 and it contains nicotinic acid is in the range of 5 - 8 mg/dl blood to be collected and ascorbic add is in the range of 3 - 5 mg/dl blood to be collected .

22. A process for preparing a formulation for redudng the deterioration of RBCs of blood during storage with particular reference to decrease in the level of 2,3 diphosphoglycerate as daimed in daim 1, and as described in the complete specification herein.

23. A process for preparing a formulation 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, and as illustrated in the accompanying drawings.