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 permeates red cells fairly rapidly during freezing is most effective in protecting the human red cells.
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
Preservation and Storage of Blood
concentration glycerol [20% weight in volume (w/v)] in the final concentration of cryopreservative. Most blood banks use the high glycerol technique.
Table 3.5 ADVANTAGES OF HIGH CONCENTRATION GLYCEROL TECNIQUE OVER LOW CONCENTRATION GLYCEROL TEHNIQUE
Advantages High Glycerol Low Glycerol
Initial freezing temperature -80 °C -196 °C
Need to control freezing No Yes
Type of freezer Mechanical Liquid nitrogen
Maximum storage temperature -65 °C -120 °C
Shipping requirement Dry ice Liquid nitrogen
Frozen cells are deglycerolized before transfusion. Removal of glycerol is achieved by systematically replacing the cryoprotectant with decreasing concentrations of saline. The cells are washed with 12% saline, followed by 1.6% saline, with a final wash with 0.2% dextrose in normal 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 CPDA-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:
1 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.
2 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 total of 25 mEq/1 of monobasic and disodium phosphates to produce a pH of approximately 6.8 (Meryman et al. 1972)
Prior of 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 satellite bag 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 partially glycerolized 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 -65(‘C.
TRANSFUSION MEDICINE Technical Manual
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 250 ml 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 bath of 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 deglycerolization 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 solution containing glucose. Glycerol contents must be reduced to 1-2% otherwise they will haemolyse on 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 disodium 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 chloride solution 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-life of 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 centrifugation in the IBM blood processor. Protocols for each instrument should be followed as advised by the manufacturers.
Clinical consideration
1. Deglycerolized red cells are comparable in volume and Hct to standard unit of red cells. 2. 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.
3. 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.
Preservation and Storage of Blood
Indications for the use of frozen red cells
1 Freezing of rare blood groups enables long-term storage and supply on a regional and national basis.
2 Storage of blood for patients with antibodies against high frequency antigens. 3 Storage of blood for autotransfusion, specially in patients with rare blood group.
4 Prevention of non-haemolytic febrile transfusion reaction in patients sensitized to leucocytes, platelets or plasma protein.
5 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
1. 5% trisodium citrate (Na3C6H507)
I. weigh 100 g of trisodium citrate II. place in 2 Litre volumetric flask
III. half fill with distilled water and mix until crystals are dissolved. IV. make up to 2 litre with distilled water and mix
2. 12% glycerol solution
Dissolve 120 ml analytical grade glycerol in 880 ml of 5% trisodium citrate solution 3. 60% glycerol solution
Dissolve 600 ml analytical grade glycerol in 400 ml of 5% trisodium citrate solution Freezing Procedure :
1 Blood is collected in CPDA-1 double pack. The pack is centrifuged at 3000 X g for 7 min and the plasma is removed.
2 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.
3 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.
4 Dispense into labeled 10 ml sterile plastic/glass tubes (15X100 mm)
5 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. 6 When frozen, tubes may be stored at -20° C.
Procedure for thawing and deglycerolization: 1 Thaw red cells in water bath at 37° C.
2 Prepare 25 cm strips of dialysing tubing by soaking in 0.9% saline. Seal one end by folding. 3 Transfer thawed red cells to dialysing tubing. Seal both ends by folding and clamp with paper
clip.
4 Suspend tubing by means of paper clip supported by an applicator stick in a large beaker having 0.99c saline (approximately 2 L).
Note: Cells in 4 dialysing tubings can be immersed in the same beaker for dialysing.
TRANSFUSION MEDICINE Technical Manual
5 Dialyze for at least 1 -2 h at RT or overnight at 4° C.
6 Transfer cells from tubing to labelled tubes, spin and wash cells with 0.9% saline until supernatant is clear.
7. Add an equal volume of AB serum to packed cells. 8. 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.