Preservation of Blood Principles and its Applications in Blood Banking

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History
Before the era of preservative solutions, direct artery to vein
blood transfusion was practiced.
In the year 1910, Fleig described the procedure of storage of
red cells.
In 1914, Hustin transfused first citrated blood to humans
and the safety of citrate was described.
In 1915, Weil found out a procedure and citrated blood was
stored in a ice box.
In 1916, Rous and Turner used citrate sucrose solution, and
later found that dextrose was better than sucrose. Rous and
Turner’s solution was used for storage of human blood during
the First World War.
In 1937, the first modern blood bank was established in Cook
County Hospital, Chicago by Benard Fantus as director and
Oswald Robertson as an advisor.
In 1943, Loutit and Mollison introduced acidified citrate
dextrose (ACD) solution. This solution was used during the
Second World War for clinical use.
In 1957, Gibson et al developed citrate phosphate-dextrose
(CPD) which is:
• Less acidic than ACD
• 2,3-diphosphoglycerate (2,3-DPG) levels are better in CPD
than in ACD solution.
• CPD has eventually replaced ACD
• Shelf-life of blood stored in CPD at 2–4°C is 21 days.
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Preservation of Blood,
Principles and its Applications
in Blood Banking
104

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Preservation of Blood, Principles and its Applications
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In 1978, Nakao et al and Simon used citrate-phosphate-
dextrose with adenine (CPDA-1). 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.
PRESERVATIVES SOLUTIONS
Acid Citrate Dextrose (ACD)
Trisodium citrate
1.485 g
Citric acid
0.540 g
Dextrose
1.687 g
In a volume of 67.5 mL for 450 mL of blood.
pH after collection is 7.0, falls to 6.7 with storage. Shelf life
is 21 days.
Citrate Phosphate Dextrose (CPD)
Trisodium citrate
1.66 g
Citric acid (monohydrate)
0.206 g
Sod. dihydrogen monophosphate
0.140 g
Dextrose
1.61 g
In a volume of 63 mL for 450 mL of blood.
Higher pH and phosphate enhances 2,3-DPG content.
pH 7.2 on day 0, falls to 6.8 during storage day 21. Shelf life
is 21 days.
Citrate Phosphate Dextrose A-1 (CPDA-1)
Trisodium citrate
1.66 g
Citric acid (monohydrate)
0.206 g
Sod. dihydrogen monophosphate
0.140 g
Dextrose
2.01 g
Adenine (0.25 mM)
0 .017 g
In a volume of 63 mL for 450 mL of blood, shelf life is
35 days.
Action of Ingredients of Anticoagulant Solutions
Dextrose: Supports ATP generation by glycolytic pathways and
prevents lysis of cells.

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Essentials of Blood Banking and Transfusion Medicine
Adenine: Synthesizes ATP, extends shelf life of red cells to 35 days.
Citrate: Prevents coagulation by chelating calcium.
Sodium diphosphate: Prevents fall in pH.
Changes occurring with stored blood are the following:
• Loss of red cell viability is due to:
– Decrease in pH
– Built up of lactic acid
– Decrease in glucose
– Decrease in ATP level
– Low 2,3 DPG levels
• Red cell 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 circula-
tion 24 hours after transfusion of stored blood in CPDA-1
for 35 days.
The blood is stored at 2–6°C to maintain the optimal viability.
• pH changes: When blood is stored at 2–6°C, glycolysis is
reduced but does not stop.
Glycolysis results in the production of lactate with subse-
quent decrease in pH.
Whole blood collected in CPD has a pH of 7.2 on day 0 and
6.84 on day 21.
Preservative solutions provide buffering capability to
minimize pH changes and optimize the storage period.
• 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.
Decrease in ATP allows the leak of Na+ and K+ through the 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.
• 2,3-diphosphoglycerate (2,3-DPG): With fall in pH, red cell
2,3-DPG level decreases which increases haemoglobin-oxygen
affinity.

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Preservation of Blood, Principles and its Applications
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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 preservative solution used.
ACD solution has lower pH than that of CPD solution.
Thus 2,3-DPG falls within the first few days in ACD, whereas
blood stored in CPD/CPDA-1 maintains adequate levels of
2,3-DPG for 10–14 days.
• After transfusion, the red cells continue to synthesize ATP.
2,3-DPG levels return to expected normal values within
24 hours.
2,3-DPG levels in transfused blood are important in certain
clinical conditions.
The pathological effects of low 2,3-DPG levels with increased
affinity with oxygen increases cardiac output.
Myocardial function improves with transfusion of blood
high in 2,3-DPG levels during cardiovascular surgery.
In patients with shock, the transfusion of 2,3-DPG-depleted
red cells makes a significant difference in recovery.
• Adenine: CPD solution supplemented with 17 mg (0.25 mm)
adenine per 63 mL of anticoagulant and 25% more dextrose,
the survival of red cells 24 hours after transfusion of blood
stored for 35 days may be around 80%.
Adenine synthesizes ATP and its level is 56.4 ± 15.9% of the
initial level in the stored blood for five weeks.
Adenine causes nephrotoxicity due to its unmetabolized
product, 2,8-dioxyadenine, however, toxicity is negligible
with 15 mg/kg body weight.
• Changes in Na+ and K+ levels: During storage, Na+ and K+
leak through the red cell membrane rapidly. The cells loose
K+ 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 proliferation of
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 a lower temperature.

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Essentials of Blood Banking and Transfusion Medicine
• Heparin: Heparin prevents coagulation by inactivating the
prophylactic activity of thrombin after complexing with
antithrombin III.
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.
Additive Solutions
Additive solutions were discovered in 1983. This system has a
primary bag containing CPD and a satellite bag containing
100 ml of additive solution. The additive solution is added to
only red cells, as only they are benefited with this procedure.
Plasma and platelets are separated first. The additive solution
is added to the remaining red cells within 72 hours of phlebo-
tomy. With this procedure:
• Shelf life of RBCs is extended to 42 days
• ATP levels are maintained
Following are the additive solutions which are in use:
• SAG, SAGM/SAGMAN (saline, adenine, glucose, mannitol)
• Adsol (AS-1)
• Nutricel (AS-3)
• Optisol (AS-5).
Composition of some of the additive solutions is given below.
Additive solution AS-1
Dextrose
2.20 g
Adenine (0.25 mM)
27 mg
Mannitol
750 mg
Sodium chloride
900 mg
In a volume of 100 mL
Additive solution AS-3
Trisodium citrate
588 mg
Sod. dihydrogen monophosphate
276 mg
Citric acid (monohydrate)
42 mg
Dextrose
1.10 g
Adenine (0.25 mM)
30 mg
Mannitol
—
Sodium chloride
410 mg
In a volume of 100 mL

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Preservation of Blood, Principles and its Applications
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Additive solution AS-5
Trisodium citrate
—
Sod. dihydrogen monophosphate
—
Citric acid (monohydrate)
—
Dextrose
900 mg
Adenine (0.25 mM)
30 mg
Mannitol
525 mg
Sod. chloride
877 mg
In a volume of 100 mL
SAGM is the currently used additive solution in Europe.
AS-1, AS-5 and AS-3 are now licensed in USA.9
Rejuvenate Solutions
To increase the levels of 2,3-DPG and ATP in stored blood,
rejuvenation solution can be added. These solutions:
• Have phosphate, inosine, glucose, pyruvate and adenine.
• Increase the levels of 2,3-DPG and ATP.
• Solution added at any time after 3 days collection up to
3 days after expiration of shelf life.
• Rejuvenation solution is added to red blood cells, incubated
for 1 hr and washed.
• 50 mL sterile solution (rejuvenol, cytosol laboratories,
braintree, MA) is commercially available.
• The rejuvenation process is expensive and time consuming
and is rarely used.
Note: For composition of rejuvenol, refer to topic on blood
and blood components.
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. If not
removed at a controlled rate, the pH value of the blood is
reduced which shortens the life of blood components.
• Blood is stored in plastic bags made of polyvinyl chloride
(PVC) with plasticizer, di-(2-ethylhexyl) phthalate (DEHP).

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Essentials of Blood Banking and Transfusion Medicine
• New plastic bags made of polyolefin with new plasticizer
(Baxter’s PL 732) and thin walled PVC with tri-(2-ethylhexyl)
trimellate plasticizer (TOTM) [Baxter’s PL 1240 and Cutter
CLX] are available with higher permeability of gases (O2 and
CO2) and platelets can be stored for 5 days.
Red Cell Freezing
Freezing damages red cells due to the intracellular ice formation
and to some extent due to hypertonicity.
Red cells mixed with glycerol could be frozen without damage.
If glycerol (cryoprotective agent) is added to the cells, they
can be frozen and thawed without damage.
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.
The cells are rapidly frozen and stored in a freezer. The freezing
and storage temperature depends on the concentration of glycerol.
Frozen red cells are used in autologous transfusion and the
storage of blood with rare blood groups.
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).
Most blood banks use the high glycerol technique.
Platelet Preservation
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.

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• New plastic bags maintain pH and function up to about
7 days. However, it is recommended to store platelets for
5 days only from the date of collection.
Granulocyte Preservation
• 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 site is better if transfused within
8 hours of storage than that of granulocytes stored for 24 hours.
Fresh Frozen Plasma (FFP)
Shelf life of FFP is 12 months at –18°C or lower. After thawing,
FFP can be stored at 2–6°C for 6 hours before transfusion. FFP
contains plasma proteins and all coagulation factors including
factor VIII.
Recovered Plasma
Recovered plasma is plasma that can be separated from donors
red cells at any time up to 5 days after the expiration of blood.
This contains all stable coagulation factors including fibrinogen
and factor IX. For details refer to topic on “Blood and Blood
Components”.
Cryoprecipitate
Cryoprecipitate 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 6 hours. For details refer to topic on “Blood and Blood
Components”.
Transporting Blood and Blood Components within 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 the blood bag in the
blood bank refrigerator if there is possibility that blood/red
cells will not be transfused immediately.

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Essentials of Blood Banking and Transfusion Medicine
Transporting platelets: Transporting of stored platelets to the
transfusion facility should use a well-insulated container, with
no ice, to maintain the temperature between 20° and 24°C.
Transporting of frozen components
• Fresh frozen plasma and cryoprecipitate must be transported
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 of the well-insulated container.
• Check breakage during transportation.
Storage of Blood
• Temperature range: 2°–6°C except for platelets with a
temperature range of 22°–24°C.
• Refrigerator for storing blood should have a fan for circulat-
ing air to ensure uniform temperature.
• System should be there to monitor and record the tempera-
ture continuously.
• Temperature recording charts should be changed regularly.
• There should be alarms.
• In case where blood has to be transported (camps), blood
should be packed in cold boxes surrounded by ice packs.
Advantages of lower temperature
• Keeps the rate of glycolysis at a lower limit.
• Minimizes the proliferation of bacteria.
• Reduces the rate of diffusion of electrolytes (Na and K) across
the cell membrane.
• Blood should be administered within half an hour of
receiving from the blood bank.
General Requirements for Storage of Blood and Blood Products
• The anticoagulant/preservative solutions with proper
temperature are essential for maintaining the viability and
stability of the blood and its products.
• Separate areas should be reserved for storing untested and
tested products.
• No food should be stored in the refrigerators and freezers.