Rous and Turner introduced first anticoagulant preservative in 1916. It consisted of a citrate-glucose solution in which blood from rabbits was stored for two weeks, which prevented anaemia when transfused in another rabbit who had suffered from blood loss. Rous Turner's solution as used for storage of human blood during the First World War (Mollison 1987). The next important development occurred in 1943 during the Second World War when acidifieditrate dextrose (ACD) solution was introduced for clinical use by Loutit and Mollison.In 1957 Gibson et al developed an improved preservative of citrate-phosphate-dextrose (CPD), which was less acidic than ACD and maintained 2,3-diphosphoglycerate (2,3-DPG) level better than in ACD solution. CPD eventually replaced ACD and became commonly used preservative for storage of blood/red cells in liquid form. Shelf-life of blood stored in CPD at 2-4 °C was 21 day.

In 1978 citrate-phosphate-dextrose with adenine (CPDA-1) preservative was developed. The addition of adenine improved the synthesis of adenosine triphosphate (ATP) in the stored blood,which prolonged the storage of blood/red cells at 2-4 °C to 35 days.
APPROVED PRESERVATIVE SOLUTIONS
Table. : Compositions of Preservatives/Anticoagulants

Action of ingredients of anticoagulant solution 

Glucose _ supports ATP generation by glycolytic pathways.

Adenine _ synthesizes ATP, increases level of ATP, extends the shelf-life of red cells to 42 days.

Citrate _ prevents coagulation by chelating calcium.

Sodium di-phosphate preventsfall in pH

RED CELLS PRESERVATION 
The goal of blood preservation is to provide viable and functional blood components for patients requiring blood transfusion. More than 70% of red cells should remain viable in circulation 24hours after transfusion of stored blood in CPDA-1 for 35 days. The blood is stored at 2-6 °C to maintain the optimal viability.
The loss of red cells viability is correlated with the "lesion of storage" due to various biochemical changes:

Decrease in pH

Build up of lactic acid

Decrease in glucose consumption

Decrease in ATP level

Low 2,3-DPG levels

Decrease in pH
When blood is stored at 2-6 °C, glycosis is reduced but does not stop. Giycosis results in the production of lactate, with subsequent decrease in pH. Whole blood collected in CPD has a pH7.20 on day 0 and 6.84 on day 21. Preservative solutions provide buffering capability to minimize pH changes and optimize the storage period.
Loss of Adenosine Triphosphate (ATP)
ATP is associated with the red cells viability. Loss of ATP causes increase in cellular rigidity and decrease in red cell membrane integrity and deformability. A decrease in ATP allows the leak of Na+ and K+ through red cell membrane at levels exceeding those normally seen in vivo. The ATP level in CPDA-1 red cells at day 35 is 45 % (±12) of the initial level.
Decline of 2, 3-Dipnosphoglycerate (2,3-DPG)
A fall in pH in the stored blood results in a decrease in red cell 2,3-DPG level, which results in increase in hemoglobin-oxygen affinity. DPG-depleted red cells have impaired capacity to deliver oxygen to the tissues. The degree of reduction of 2,3-DPG levels depends upon the preservativesolution used. ACD solution has lower pH than that of CPD solution. Thus 2,3-DPG falls within the first few days in ACD where as blood stored in CPD/CPDA-1 maintains adequate levels of2,3-DPG for 10-14 days.
The pathological effects of the transfusion of red cells with low 2,3-DPG levels and increased affinity with oxygen include increase in cardiac output and a decrease in mixed venous PO2 tension. 2,3-DPG levels in transfused blood are important in certain clinical conditions. Myocardial functions improve following transfusion of blood with high 2,3-DPG levels during cardiovascularsurgery. In patients with shock the transfusion of DPG-depleted red cells makes a significant difference in recovery.
After transfusion, the red cells continue to synthesize 2,3-DPG and levels return to expected normal values within 24 hours. The acid-base status of the recipient, phosphorous metabolism anddegree of metabolism influence the rate of restoration of 2,3-DPG.
Adenine
Simon (1962) showed that in CPD solution supplemented with 17 mg (0.25 mm) adenine per 63   of anticoagulant and 25% more dextrose, the survival of red cells 24 hours after transfusionof blood stored for 35 days was 80 ± 6.5 %. Adenine synthesizes ATP and its level is 56.4 ± 15.9%of the initial level in the stored blood for five weeks. Adenine nephrotoxicity due to its unmetabolized product, 2,8-dioxyadenine, is negligible at a level of 15mg/kg body weight. This amount is present in 30 units of fresh CPD adenine (0.5mM/unit) blood or in 60 units each having 0.25 mM adenine The quantity of adenine is less if red cells are used.
Changes inNa+and K+ levels
During refrigerated storage, Na+ and K+ leak through the red cell membrane rapidly. The cells lose and gain Na+, however, the K+ loss is greater than the Na+ gain during storage.
Temperature
The lower temperature keeps the rate of glycolysis at lower limit and minimizes the proliferationof bacteria that might have entered the blood unit during venipuncture or from atmosphere. The rate of diffusion of electrolytes (Na* and K+) across the cell membrane is also less at lower temperature.

Table : Biochemical Changes in Stored Blood

Additive Solutions

Traditional preservatives were put into use when whole blood was the major product. With the advent of component therapy use of red cells increased. This resulted in several problems in preservation. In preparing red cell concentrates 40% adenine and glucose present in CPDA-1 solution is removed with plasma and there is decrease in viability of the red cells, particularly in the last two weeks of storage. Red cell concentrates relatively void of plasma are more viscous and difficult to infuse in emergency situations. To overcome this problem red cell concentratesare prepared with hematocrits of less than 80%. This allows adequate plasma to remain for red cells nourishment and to improve flow properties. This results in lower plasma yields, affecting fresh frozen plasma and cryoprecipitate production.
The use of additive solutions allows recovery of maximum amount of plasma and preparation of red cells unit with a final hematocrit of about 60%. This new blood collection system has a primary bag containing a standard anticoagulant (CPD) and a satellite bag containing an additive solution. Blood is collected in the primary bag containing anticoagulant solution. After the plasmais removed from the whole blood into another empty satellite bag, the additive solution is added to the red cells, thus providing nutrients to red cells for improved viability. The red cells can be stored for six weeks at 2-6°C. The additive solution should be added to red cells within 72 hours since phlebotomy.
Three additive solutions are available (1) Adsol ( AS-1 ) [Baxter Laboratories], (2) Nutricel (AS-3) [Medsep Corporation, formerly Cutter Biologicals] and Optisol (AS-5) [TerumoCorporation |.
Table : Composition of Additive Solutions

One major benefit of the additive system is increase in the level of ATP, and red cells viability is enhanced, extending the shelf-life of the red cells to 42 days. This facilitates better inventory control of blood as well as wider use in pre-deposit autologous donations. More than 80% red cells survive in circulation 24 hours after transfusion of blood stored for 42 days. The additive solutions do not increase 2,3-DPG levels. Additive solution having mannitol are not routinely used for exchange or neonatal transfusion. There is no restriction on the use of additive solutions in any other type of transfusion recipients. In addition, the use of additive solutions allows extraction of more plasma/platelet rich plasma for optimal production of platelets, factor VIII yields and fresh frozen plasma.
Table : Biochemical Changes in Stored Additive Red Cells

Heparin
Heparin prevents coagulation by inactivating the prophylactic activity of thrombin after complexing with AT 111 and thrombin. 1000 IU of heparin is equal to 10 mg heparin but IU and mg are not strictly interchangeable because commercial preparations of heparin vary in composition.
Dose of heparin for anticoagulation is 0.5-2.0 IU/ml. of blood e.g. approximately 500 IU of heparin for 500 ml of blood. Heparinized blood should be used within 24 hours. Earlier heparinized blood was used in pen heart surgery but now usually it is not used as extracorporeal pumps are now usually primedwith crystalloids and not with blood. The effect of heparin can be neutralized with protamine sulphate. 1 mg of protamine sulphate neutralizes 1 mg of heparin e.g. to neutralize 5000 units of heparin (50 mg), 5 ml of 1 % solution of protamine sulphate will be needed.
Rejuvenate Solutions 
Rejuvenate solutions having phosphate, inosine, glucose, pyruvate and adenine increase the levels of 2,3-DPG and ATP in stored red cells. These solutions can be added at any time between 3 days post collection and 3 days after expiration of red cells. The solution is added directly to the red cells, mixed and incubated at 37 °C for one hour. The rejuvenated red cells are either washed with saline (2 Litres of unbuffered 0.9% NaCl) and can be kept at 2-6°C, however, they should be transfused with in 24 hours after washing or they are glycerolized for keeping red cells in frozen state to improve the quality of red cells.
Red blood cells rejuvenation solution, 50 ml sterile vial (Rejuvesol, Cytosol Laboratories,Braintree, MA) is commercially available. The rejuvenation process is expensive and time consuming and is rarely used.
Plastic Bags 
Many other factors may limit the viability of transfused red cells. One of the factors is also the plastic material used for the bags. The plastic material should be sufficiently permeable to CO2 in order to maintain higher pH during storage. Currently the blood is stored in plastic bags made of polyvinyl chloride (PVC) with plasticizer, di-(2-ethylhexyl) phthalate (DEHP). It is known that DEHP leaches from plastic into plasma and cell membrane during storage and may be harmful to the patient. The accumulation of excessive amount of acid due to glycosis even at low storage temperature is also a major problem in liquid preservation of red cells. So there is need to develop improved plastic blood bags as well as preservative solutions.
RED CELL FREEZING 
Smith in 1950 reported that glycerol could prevent freezing injury in human red cells and that red cells, mixed with glycerol could be frozen without damage.
Effect of Freezing 
It is believed that freezing damages red cells due to the intracellular ice formation and probably to some extent due to hypertonicity. If glycerol (cryoprotective agent) is added to the cells they can be frozen and thawed without damage (Polge et al. 1949). The effect of the glycerol is probably due to the fact that it limits ice formation and provides liquid phase in which salts are distributed as cooling proceeds excessive hypertonicity is also avoided (Lovelock, 1953). Glycerol which termeates red cells fairly rapidly during freezing is most effective in protecting the human redcells.
Frozen red cells are primarily used for autologous transfusion and the storage of rare group blood. For freezing red cells a cryoprotective agent is added to red cells that are less than 6 days old. Glycerol is used most commonly and is added to the red cells slowly with vigorous shaking so that glycerol permeates into the red cells. The cells are rapidly frozen and stored in a freezer.The freezing and storage temperature depends on the concentration of glycerol. Two concentrations are used to freeze red cells, a high concentration glycerol |40% weight in volume (w/v) and a low concentration glycerol [20% weight in volume (w/v)] in the final concentration of cryopreservative. Most blood banks use the high glycerol technique.
Table :  ADVANTAGES OF HIGH CONCENTRATION GLYCEROL TECNIQUE OVER LOW
CONCENTRATION GLYCEROL TEHNIQUE

Frozen cells are deglycerolized before transfusion. Removal of glycerol is achieved by systematically replacing the cryoprotectant with decreasing concentrations of saline. The cellsare washed with 12% saline, followed by 1.6% saline, with a final wash with 0.2% dextrose innormal saline. The shelf life of thawed red cells stored at 2-6 °C is 24 hours. Commercially available Cell Washing System manufactured by several companies can be used.
Generally cells are glycerolized and frozen with in 6 days of collection of blood in CPD or PDA-1. Red cells stored in additive solutions can be frozen up to 42 days.
The frozen red cells can be stored for 10 years. The outdating period of the thawed red cells stored at 2-6°C is 24 hours.
Method of freezing and preservation of red cells in frozen state:

Glycerolized red cells having final concentration of 40% W/V of glycerol can be frozen at -80°C over a period of 30 min using mechanical refrigeration, then can be preserved at -60 to-65°C for 10 years.

Glycerolized red cells having final concentration of 20% W/V of glycerol can be frozen at - 196°C using liquid nitrogen for 2-3 minutes and can be preserved in the gas phase of liquid nitrogen at -120°C for 3 years.

High glycerol solution (40% W/V concentration). The glycerolizing solution consists of 6.2 M glycerol solution that contains 57 gm% glycerol, 1.6 gm% Na lactate, 0.03 gm% KCl and a totalof 25 mEq/1 of monobasic and disodium phosphates to produce a pH of approximately 6.8 (Meryman et al. 1972)
Prior to glycerolization, whole blood in CPD solution, fresh or stored at 4°C for no more than 3-4 days, is centrifuged at 3000x g for 7 min; supernatant plasma is expressed into satellitebag and used for preparation of components or freezed. Appropriate volume of 6.2 M glycerol solution is added in two stages e.g. 300 ml when the weight of the packed red cells is 150-230 g.\First 100 ml of glycerolizing solution is added to the cells in the collecting bag with vigorous shaking. After at least two minutes of equilibration, the remainder glycerol solution and the partiallyglycerolized cells are transferred to a 850 ml polyolefin bag (Hebia blood bag). The bag is centrifuged, the supernatant is expressed and the red cells are frozen at -80°C using a deep freezer.
They can then be stored at -60 to -65C. 
Low Glycerol Solution (20% W/V cencentration)The glycerolizing solution consists of 35.0 gm% glycerol, 2.88% mannitol, and 0.65g sodium chloride.
Whole blood in CPD is centrifuged at 3000x g for seven minutes. Plasma is taken off and freezed. Low glycerol solution equal in volume to the red cells (e.g. 250 ml of solution for 250ml of red cells) is added with vigorous shaking. The glycerolized red blood cells are transferred to a polyolefin plastic bag and kept in aluminium container which is then placed upright in a bathof liquid nitrogen at -196°C and then stored at -120°C in liquid nitrogen vapour.
Thawing and Deglycerolizing
Frozen red cells are thawed in a water bath at 37°C for about 10 min. Glycerol must be properly removed from the thawed cell to avoid haemolysis in vivo and/or in vitro. The intracellular environment of glycerolized cells is hypertonic relative to plasma and the first solution used for reglycerolization must be also somewhat hypertonic. This allows the glycerol to begin diffusing out of the red cells while the intracellular environment remains hypertonic. Subsequently followed by washing with solution progressively less hypertonic and finally with isotonic electrolyte solutioncontaining glucose. Glycerol contents must be reduced to 1-2% otherwise they will haemolyse in contact with plasma.
The 40% W/V glycerolized red cells are diluted with 150 ml of 12g% sodium chloride buffered to about pH 7.2 with 0.15% disodium phosphate and allowed to equilibrate for 5 minutes. Then wash with one to two litres of 1.6g% sodium chloride solution buffered to pH 7.2 with 0.03g% of isodium phosphate and finally with one litre of 0.9g% sodium chloride solution containing 0.2g%glucose buffered with 0.065g% disodium phosphate to a pH of about 6.8 (isotonic glucose solution). The washed cells are finally suspended in isotonic glucose solution and ready for transfusion. The shelf-life of the processed unit is 24h.
The 20% W/V glycerolized red cells are diluted with 500 ml of 3.2g% sodium chloride solution buffered to about pH 7.2 and then washed with 1-2 litres of 0.9g% sodium chloridesolution containing 0.2g% glucose buffered with 0.065g% disodium phosphate to pH 6.8. The washed cells are finally suspended in isotonic glucose saline and ready for transfusion. Shelf-lifeof the processed unit is 24 h.
The washing can be carried out either by continuous flow washing in the Haemonetic Blood processor or by continuous flow washing in Fenwal Elutramatic system or serial centrifugationin the IBM blood processor. Protocols for each instrument should be followed as advised by the manufacturers.
Clinical consideration

Deglycerolized red cells are comparable in volume and Hct to standard unit of red cells.

Deglycerolized red cells consist of red cells in electrolyte solution. Virtually all plasma, anti-coagulants and most of the leucocytes and platelets have been removed.

In vivo survival and functions of red cells are comparable to fresh drawn liquid stored red cells because 2,3-DPG and oxygen dissociation curves are normal.

Indications for the use of frozen red cells

Freezing of rare blood groups enables long-term storage and supply on a regional and national basis.

Storage of blood for patients with antibodies against high frequency antigens.

Storage of blood for autotransfusion, specially in patients with rare blood group.

Prevention of non-haemolytic febrile transfusion reaction in patients sensitized to leucocytes,platelets or plasma protein.

Prevention of sensitization against HLA antigens in potential recipients of tissue transplants.

FREEZING AND THAWING OF RED CELLS (REAGENT CELLS)
USING HIGH GLYCEROL CONCENTRATION SOLUTIONS

Solutions required

5% trisodium citrate (Na3C6H507)

weigh 100 g of trisodium citrate

place in 2 Litre volumetric flask

half fill with distilled water and mix until crystals are dissolved.

make up to 2 litre with distilled water and mix

12% glycerol solution

Dissolve 120 ml analytical grade glycerol in 880 ml of 5% trisodium citrate solution

60% glycerol solution

Dissolve 600 ml analytical grade glycerol in 400 ml of 5% trisodium citrate solution

Freezing Procedure :

Blood is collected in CPDA-1 double pack. The pack is centrifuged at 3000 X g for 7 min and the plasma is removed.

12% glycerol solution equal to half the volume of the packed cells is added to the packed cells, mixed and allowed to equilibrate at RT for 10 min.

60% glycerol solution equal to half the volume of the packed cells is added to the pack and mixed and allowed to equilibrate for 10 min.

Dispense into labeled 10 ml sterile plastic/glass tubes (15X100 mm)

Freeze at -40° C, keep it in lowest part of freezer and switch freezer to rapid freeze or place in top of liquid nitrogen tank, i.e. vapour phase for 5-10 min.

Note: Tubes should be placed in a metal rack for faster freezing.

When frozen, tubes may be stored at -20° C.

Procedure for thawing and deglycerolization:

Thaw red cells in water bath at 37° C.

Prepare 25 cm strips of dialysing tubing by soaking in 0.9% saline. Seal one end by folding.

Transfer thawed red cells to dialysing tubing. Seal both ends by folding and clamp with paper dlip.

Suspend tubing by means of paper clip supported by an applicator stick in a large beaker saving 0.99c saline (approximately 2 L).

Note: Cells in 4 dialysing tubings can be immersed in the same beaker for dialysing.
Dialyze for at least 1 -2 h at RT or overnight at 4° C.

Transfer cells from tubing to labelled tubes, spin and wash cells with 0.9% saline until supernatant is clear.

Add an equal volume of AB serum to packed cells.

Stopper lubes and store at 4"C.

PLATELETPRESERVATION
The preservation of viable and functional platelets depends on the following factors: 
Temperature
Platelets should be stored at 22-24° C (controlled temperature) with continuous gentle agitation in platelet incubator and agitator.

pH
pH should be above 6.0.
Plastic Bag
Maintenance of pH and function of platelets depend on the permeability of the storage bag to oxygen and carbon dioxide. Platelets stored in bags made of polyvinyl chloride (PVC) with plasticizer di-(2-ethylhexyl) phthalate (DEHP) have shelf life of 3 days. New plastic bags made of polyolefin with no plasticizer (Baxter's PL 732) and thin walled PVC with tri-(2-ethylhexyl)trimellate plasticizer (TOTM) [Baxter's PL 1240 and Cutter CLX] maintain pH and functions up to about 7 days. However it is recommended to store platelets in new bags for 5 days only from the date of collection of blood
The pooled platelets can be stored for 4 hours at 22-24° C before they are transfused.
FRESH FROZEN PLASMA (FFP)
Shelf life of FFP is 12 months at - 18°C or lower. After thaw FFP can be stored at 2-6° C for 12 hours before transfusion. If FFP can not be used with in 1 year or thawed plasma is not used within 12 hours it is re-designated as single donor plasma which can be stored farther for 4 years at -18° C or lower.
SINGLE DONOR PLASMA
Shelf-life of Single donor plasma is 5 years at -18° C or lower. Thawed plasma can be stored at 2-6 °C for 5 days before transfusion
CRYOPRECIPITATE (Factor VIII)
Croprecipitate can be stored for 12 months at -18° C or lower. Thawed Cryoprecipitate can be stored for 6 hours at 2-6° C and pooled cryoprecipitate kept at 2-6° C should be used within 4hours
GRANULOCYTES 
The shelf life of granulocytes is 24 hours at 22-24°C. They do not need agitation. Post transfusion recovery of granulocytes in circulation and migration into inflammatory loci is better if transfused with in 8 hours of storage than that if granulocytes stored for 24 hours. 
SHIPPING OF BLOOD PRODUCTS
The temperature of blood and blood products should be monitored during shipment from one place to another. Freshly drawn blood, which will not be used for preparing platelets, should be transported at the temperature between 2-10°C. Units from which platelets are to be made must be transported at temperature 20-24° C.
Transporting Red Cells/Whole Blood
Transporting red cells or whole blood from one facility to another requires that the temperature of the blood during shipment be kept between 2-10°C. Sturdy, well insulated carriers with some type of refrigerant are used. Wet ice is a good refrigerant for red cell shipment. Ice should be putat the top inside the container so that the cool moves down through the shipping box. In climates that are extremely warm or where the shipment journey will be lengthy, ice may be placed at the bottom also of the container. Ice should not come in direct contact with blood at any time because it can cause hemolysis of the red cells or blood.
Transporting red cells/whole blood with in the hospital
The blood and red cells should be transported within the hospital in insulated carrier or in cold insulated boxes if the ambient temperature is more than 25° C. Instructions should be given to keep in refrigerator if there is possibility that blood/red cells will not be transfused immediately.
Shipping of Platelets
Shipping of stored platelets to the transfusion facility should use a well-insulated container, with no ice, to maintain the temperature between 20-24° C.
Shipping of Frozen Components
Fresh frozen plasma and cryoprecipitate must be shipped at -18° C or below. Dry ice is used to maintain the frozen state. Dry ice should be kept at the bottom and at the top inside the well-insulated container. These frozen products are fragile, Insulate the product by dry packing material or plastic air bubbles to check breakage during shipment.
General Requirements for Storage of Blood Products
The anticoagulant/preservative solutions are not the sole variables in maintaining the viability and stability of the blood and its products. Physical storage conditions best suited for products maximum survival are also important.
All storage equipment, whether a refrigerator, a freezer or a platelet environmental chamber should have the following facilities:

Refrigerators for storing blood/red cells should have a fan for circulating air to ensure proper temperature 2-4° C in all compartments.

Refrigerators, freezers and platelets incubators should have a system to monitor and record  the temperature continuously. Temperature recording charts should be changed regularly.They should also have alarm systems with audible signals. These facilities should have battery back up.

Separate areas should be reserved for storing untested, tested and quarantined products.

No food should be stored in the refrigerators and freezers.

Table : Blood Component Storage Summary

INSPECTION OF BLOOD
Blood must be inspected prior to transferring the units from one facility to another or issuing for patient use. The blood should be inspected to check for possible bacterial contamination, whichmay produce abnormal colour in the red cells or plasma. Units are inspected for hemolysis, visible clots, or purple, brown and red plasma. Abnormal units should not be issued and the cause should be investigated. Plasma with a green hue need not to be rejected because this is caused by exposure of bilirubin pigments to the light.
Prior to issue plasma in both the main bag and segments should be visually inspected for hemolysis or discolouration. Yercinia enterocolitica, a bacterium, can grow at 4°C, the PO of the unit decreases and the blood is hemolyzed. This metabolic activity in the unit is not duplicated in the sealed segments. When the segments of the contaminated units are cultured, they are found to be sterile, while the blood from the unit may grow yercinia entrocolitica. This causes change of colour in the unit but not the colour of the segment. In case of hemolysed blood the colour of blood both in bag and tubing is changed to purple.