MLS 306

BLOOD BANK
ANTICOAGULANTS AND
PRESERVATIVES

AKINBO D.B. Lecture Series

 History of blood banking
• The primary hindrance to successful transfusions in the early days
of transfusion was clot formation in the course of donation.

• Numerous methods were employed in the early practice to

prevent clotting but with little success and they include:

- Side by side transfusion,

- Rapid transfer by funnel or syringes,

- Collection either by scarification or venesection & defibrinating by

rapid stirring,

- Delaying coagulation by using paraffin flask.

 History of blood banking
• The wars resulted in the introduction of indirect transfusion.

• There are three physiological components of RBCs crucial for

normal erythrocyte survival and function and defects in any
of these components will result in reduction in RBC Survival
below the normal 120 days. They are:

- Normal chemical composition and structure of RBC

membrane;
- Hemoglobin structure and function;
- RBC metabolism

• Albert Hustin and Luis Agote discovered in 1914 that the

addition of sodium citrate to blood prevented it from clotting
(the concept of anti-coagulation).
 History of blood banking
• Francis Peyton Rous and J.R. Turner in 1916 added glucose to
sodium citrate to allow for storage of blood for sometime before
transfusing and this heralded storage of human blood during the
First World War using the Rous-Turner's solution.

• During the Second World War, Loutit and Mollison introduced

Acidified Citrate Dextrose (ACD) solution in 1943.

• Gibson and colleagues developed citrate-phosphate-dextrose

(CPD) in 1957 and this eventually replaced ACD, to become
commonly used preservative for blood/red cells storage in liquid
form with a shelf-life of 21days at 2-4°C.

• Citrate-phosphate-dextrose with adenine (CPDA-1) was introduced

in 1968 by Shields and the addition of adenine improved ATP
synthesis in the stored blood, thereby prolonging the blood/red
cells storage to 35 days at 2-4°C.
 Anticoagulants and preservatives
• Anticoagulants: substances that prevents blood clot
formation

• Preservatives: substances added to a specimen to prevent

changes in the constituents of a specimen

• RBCs in-vivo are carried and protected by the plasma at an

optimum temperature, pH, glucose and metabolic waste
disposal with a life span of 120 days.

• Storage lesions: A set of biochemical & biomechanical

changes that occur during blood storage leading to
decreased viability of the cells & its physiological functions.
 Storage lesions
• RBCs experience decreased ATP and 2,3 DPG levels, a drop in pH to
acidic level, poor functioning of Na-K+ Pump as K+ accumulates in
stored blood, oxidative damage with change in the structure of
Band3 and Lipid peroxidation.

• RBCs lose membrane lipids, affecting deformability and osmotic

fragility, there are morphological changes as the disc changes to
echinocytes & spherocytes subsequently.

• Platelets lose their discoid shape, microscopic platelet aggregate

formation occurs, fragmentation, appearance of disintegrated,
‘balloon’ forms subsequently.

• In order for anticoagulants and blood preservatives to be

considered effective, they must therefore ensure the viability and
stability of the products, inhibit growth of microorganisms and
prevent clotting of the product.
Anticoagulants and preservatives solution
• Citrate: The first anticoagulant preservative introduced in 1916 (World War
1) by Rous and Turner and initially used for rabbit blood storage.

• Blood stored up to 12 days in citrate-sucrose solution prevented anemia

post-transfusion.

• It is a calcium-chelating agent and prevents coagulation by interfering with

calcium-dependent steps in the coagulation cascade. Citrate retards
glycolysis.

• Disadvantages:
• - Citrate has affinity for magnesium ions- hypomagnesaemia in the setting
of massive transfusion,
• - Citrate toxicity: in new born without adequate calcium store and with
premature liver
• - Hyperglycemia is seen during massive transfusion in orthotopic liver
transplant in massive transfusion and is considered to be a cause of cardiac
arrhythmia
Anticoagulants and preservatives solution
• Citrate dextrose: The dextrose provides nutrient for stored
red cells metabolism via the Anaerobic glycolysis pathway
and was found to be marginally better in diminishing lysis in
human blood cells.

• Advantages of Citrate dextrose:

- Most efficient and available anticoagulant for greater part of 20
years.

• Disadvantages of Citrate dextrose:

- Required dilution of blood to about three times its original
volume (1:3).
- Could not be heat sterilized because sugar caramelized.
- Risk of bacterial contamination from open mixing of ingredients
and addition of solution to the bottle.
Anticoagulants and preservatives solution
• Acid-Citrate-Dextrose (ACD): Loutit and Mollison in
1943 (world war II) showed simple acidification of
citrate dextrose prevented caramelization of sugar
during autoclaving.

• pH of citrate dextrose was lowered to 5.

• ACD was first used in 1:4 ratio but later concentrated

to use in 1:7 ratio.

• It is used in apheresis procedure and has a shelf-life of

21 days.
Anticoagulants and preservatives solution
• Advantages of ACD:
- Simplified sterilization procedure
- Reduced volume of preserving solution
- Enhanced preservative properties
- Increased shelf to 21 days
- Minimal effects on acid base balance

• Disadvantages of ACD:
- Levels of 2,3 DPG lost early within 1st week because of the
acidic Ph.
Anticoagulants and preservatives solution
• Citrate-Phosphate-Dextrose (CPD): Was developed by John .G .
Gibson for estimating in-vivo red cell survival using double isotope
procedure with 51Cr and 32P to study the effect of blood
preservative solution.

• Gibson reported existence of high energy phosphate compound in

blood collected in CPD (later found to be 2,3 DPG) as cells
passively lose phosphate.

• Adding sodium phosphate to ACD, reduced phosphate loss by

decreased gradient in phosphate concentration between the
inside and outside of stored RBC.

• Advantages of CPD: - Better maintenance of 2,3DPG via a more

alkaline pH (5.6) and improved ATP synthesis with a shelf life of 28
days.
 Nucleotides of Anticoagulants and
preservatives solution
1. Adenosine

•Advantage: It restores ATP, and this is likely due to its phosphorylation

to AMP and subsequent conversion to ATP.

•Disadvantage: Adenosine was found to have marked hypotensive

effect and was never used due to its toxicity.

2. Inosine: Adenosine deaminase rapidly converted adenosine to

inosine.

•Advantages: Excellent effect on cell viability via its ATP generation.

•Disadvantages: Hypoxanthine formed from inosine degraded to uric

acid.
 Nucleotides of Anticoagulants and
preservatives solution
3. Guanosine: Has also been used as an additive to ACD based
blood preservative but usefulness as practical additive has not yet
been established.

4. Adenine: Addition of adenine to cells resulted in restoration of

red cell ATP, shape and post-infusion viability with a possible
exception in the case of G6PD deficient donors.

Adenine seems to increase ADP levels upon incorporation to the

solution, thereby driving glycolysis towards synthesis of ATP.

ATP-dependent cytoskeleton control red cell membrane shape and

rigidity.
 Nucleotides Anticoagulants and
preservatives solution
• Citrate Phosphate Dextrose Adenine (CPDA- 1): Developed in
1968 and permit whole blood storage for 5 weeks with
improved ATP synthesis.

- Disadvantage of CPDA- 1: development of uric acid stones.

• Citrate Phosphate Dextrose Adenine 2 (CPDA 2): Packed RBC

stored in CPDA-1 ran out of glucose sooner than whole blood
stored in CPDA 1 and the glucose concentration of the CPDA-1
solution was therefore increased to form the CPDA-2.

- Disadvantage of CPDA- 2: Makes plasma and platelets loaded

with large amounts of unnecessary sugar.
Anticoagulants and preservatives solution
• CP2D: It has 100% more glucose than the CPD
and 60% more than CPDA-1 and its high
glucose content is necessary because it is used
with an additive solution that does not
contain sufficient glucose –AS3.
Anticoagulants and preservatives solution
• Blood : Anticoagulant Ratio

- Routinely, the volume of anticoagulant–nutrient solution is 1/7,

- 14 ml of CPD/CPDA is used to preserve 100 ml of blood,

- 63 ml for a 450 ml blood collection,

- 70 ml for 500 ml blood collection,

- Venous blood with pH of 7.35 mixes with anticoagulant-nutrient

solution with pH 5.0-5.6 to yield a mixture of pH 7.05 after the
entire collection process.
Anticoagulants/Preservatives Constituents
 Recent Advances in Anticoagulants
and preservatives solution
• Additive solutions: Preserving solutions added to RBCs after
removal of the plasma with/without platelets. The advent of
component therapy resulted in an increase in PRC usage in
the 1970s.

• The removal of the plasma component during the

preparation of RBC concentrates removed much of the
nutrients, about 40% Adenine and glucose present in
anticoagulants needed to maintain RBCs during storage.

• The new product packed RBCs became more viscous and

difficult to infuse in emergency situation, thus decreased
viability particularly in the last 2 weeks of storage.
 Additive solutions
• Using additive solutions allowed maximum recovery of
plasma and preparation of RBC units with final haematocrit
of approximately 60%.

• The use of additives also overcame the problem of high

viscosity of RBC concentrates.

• The additive solution used is SAGM (saline, adenine,

glucose, mannitol) when CPD anticoagulant is in the
primary bag
 Additive solutions
• Saline-Adenine Glucose (SAG ): Formulated in 1978
and one of the first generation additive solution.

• SAG represented an attempt to replace the volume

and sugar lost with plasma removal and add the
adenine necessary for storage beyond three weeks.

• It is a simple medium that allows storage of RBC after

removal of most of the plasma and maintains viability
of 83% after 35 days of storage.
 Additive solutions
• Addition of Mannitol: Storage of RBCs in electrolyte
solutions caused haemolysis of the cells.

• The Na and K ions gradually equilibrated across the

red cell membrane and with loss of osmotic gradient
and osmotic pressure of intracellular Hb, unbalanced
by that of plasma proteins, resulted in swelling and
haemolysis.

• Mannitol was used to substitute osmotic activity of

albumin and it acts as a membrane stabilizer, reducing
haemolysis to acceptable levels.
 Additive solutions
• Bicarbonate Adenine Glucose Phosphate Mannitol (BAGP-M):
Stabilizes 2,3-PDG by the loss of CO2.

• Erythrosol-1: Uses half strength citrate (0.5 CPD), increases

intracellular pH and utilises more alkaline to maintain ATP.

• Erythrosol-2: Uses full strength CPD and alkaline solution with

disodium phosphate Solution to achieve successful 7 weeks storage.

• Experimental Additive Solution (EAS-81): Maintains high

concentration of red cell ATP, limited haemolysis with mannitol and
preserves 2,3 DPG concentration.

• Phosphate, Adenine, Glucose, Guanosine, Sodium Chloride,

Mannitol (PAGGS-M): Preserved RBC for 7 weeks with 74%
recovery.
 Additive solutions
• Advantages:
- Enhances red cell viability via increase ATP level,
- Increased red cell shelf life of 42 days,
- More plasma/platelet-rich plasma extraction for optimal
platelets, factor VIII yields and FFP,
- Allows for room temperature storage of whole blood for
close to 8 hours,
- Increases 2,3 DPG level in transfused blood

• Disadvantage:
- Unable to maintain 2,3DPG throughout the storage time and
therefore not routinely transfused to newborn infants
 Recent Advances in Anticoagulants
and preservatives solution
• Rejuvenation Solutions: They are used for rejuvenating stored
RBCs, even at the end of their allowable shelf-life e.g. PIPA.

• Increases 2,3 DPG and ATP levels in stored RBCs and can be added
at any time between 3 days post-collection and 3 days after expiry.

• It is directly added to packed RRC, mixed and incubated at 37⁰C for

1 hour, washed with saline (2 litres of unbuffered 0.9% Nacl) and
kept at 2-6⁰C.

• Rejuvenated RBCs should be transfused within 24 hours after

washing. It is used in autologous donations/rare blood groups.

• The process is expensive, cumbersome and rarely used.

 Recent Advances in Anticoagulants
and preservatives solution
• Cryopreservation: the process of preserving biological structure
and/or function of living systems by freezing and storage at ultra
low temperatures and there are 2 types of Cryoprotectants.

- Non-penetrating: increases solution viscosity and reduce

optimum cooling velocity e.g. Dextrose , lactose ,sucrose,
albumin, hydroxylethyl starch (HES), polyvinyl pyrrolidone (PVP).

- Penetrating: they prevent damage during slow-freezing and

thawing e.g. Glycerol , DMSO

• Both non-penetrating and penetrating CPA can be used for RBC

freezing and the two common agents in clinical use are Glycerol
and Dimethylsulphoxide (DMSO).
 Cryopreservation
• Mechanism of action: The penetrating cryoprotectant enters the
cells and prevents damage by cellular dehydration due to its
osmotic force thereby preventing intracellular ice formation and
subsequently mechanical injury to RBC.

• Fatal effects of freezing:

– Intracellular ice crystal formation.
– Denaturation of proteins by high solute concentrations.
– Osmotic stress during the freezing and thawing process.

• Glycerol prevents freezing injury of human RBCs due to its

relatively nontoxic nature and ability to readily permeate cell at
37⁰C.
 Platelets Anticoagulants and
preservatives solution
• The viability of platelets is best preserved with fresh
heparinized blood but causes extreme adhesion of platelets.

• EDTA also preserves platelet integrity. However it is

cardiotoxic and EDTA anticoagulated platelets are rapidly
removed from circulation.

• Citrate-phosphate Dextrose (CPD or CP2D) are standard anti-

coagulant-preservative solutions for blood collection and the
recovery time are similar after storage for 72 hours in either

• Apharesis platelets can be collected into citric acid ,

trisodium citrate and dextrose solutions with pH 5, and
prevents clumping of platelets.