Anticoagulants and blood preservatives

Dr R Amita
Assistant Professor
Dept of Transfusion Medicine
Anti coagulants and Blood
Preservatives

 From ancient times, blood was called “living force” of the
body.
 Greeks and romans committed suicide by letting out blood
 Early times, man recognised that loss
of blood is associated with weakness
and disease
 Lost blood replaced by direct drinking
blood from young & healthy animal
or man

Albert Hustin and Luis Agote
discover that
adding sodium citrate to blood
will prevent it from Clotting
ANTI COAGULATION!!!

Francis Peyton Rous and J R Turner
added glucose to sodium citrate;
allowed blood to be STORED for
sometime before transfusing
BLOOD PRESERVATION!!!

Anticoagulants and blood
preservatives

 Anticoagulant- any substance that prevents blood clotting
 Preservative- any substance added to a specimen to
prevent changes in the constituents of a specimen
 In vivo, RBCs are carried and protected by the plasma,
– Optimum temperature, pH, glucose, disposal of metabolic
waste
– life span is 120 days

Storage lesions
 A set of biochemical &biomechanical changes which occur during
storage leading to decreased viability of the cell & its
physiological functions .
 RBCs:
– decreased ATP and 2,3DPG levels, pH- acidic
– poor functioning of Na-K+ Pump-accumulation of K+ in stored blood
– Oxidative damage with change in the structure of Band3 and Lipid
peroxidation
– Loss in membrane lipids affects deformability and osmotic fragility
– Morphological changes-Disc changes to echinocytes & to spherocytes
 Platelets:
– Loss of discoid shape, microscopic platelet aggregate formation,
fragmentation, appearance of disintegrated, ‘balloon’ forms

To ensure that blood retains its in vivo
environment
 Anticoagulants
 Preservative
 Characteristics of blood bag
 Storage temperature
 Shipping/transport conditions
Anticoagulants and blood preservatives
• To ensure the viability and stability of the products
• To inhibit growth of microorganisms
• To prevent clotting of the product

Chronology of Anticoagulants
year person anticoagulant remarks
1916 Rous and Turner Citrate dextrose RT soln 12 days
1943 Loutit and Mollison Acid citrate dextrose pH 5
1957 Gibson Citrate phosphate dextrose Prevents PO4 loss
1957 Gabrio Added nucleotides RBC metabolism
1968 Sheilds CPDA-1
1970 Beutler Additive solutions
1979 Hogman SAG 5 weeks storage
1980 Lovric CP2D (1⁰ bag) + SAG (additive)

Citrate
 Calcium-chelating agent
 Prevents coagulation by
interfering with calcium-
dependent steps in the
coagulation cascade
Citrate dextrose
 The dextrose provide
nutrient for red cells
Disadv:
 dilution 1:3
 Cannot be heat sterilized
 Risk of infection

Acid-citrate-dextrose
 Citric acid, sodium citrate, and dextrose
(pH 5)
 Shelf life of 21 days
 Acid pH does not help in maintaining
2,3-DPG levels
 Prevents caramelization of sugar
 First used in 1:4 ratio but later
concentrated to use in 1:7 ratio
 Used in apheresis procedure
Citrate-phosphate-
dextrose
 Alkaline pH and PO4
help in maintaining 2,3-
DPG
 Shelf life of 28 days
 CPD is commonly used

Added Nucleotides
 ADENOSINE : Restores ATP, but
marked hypotensive effect.
 INOSINE : generates ATP, but
hypoxanthine formed degrades
to uric acid
 GUANOSINE: Used in PAGGS-M
which provides 7 weeks of RBC
storage with recovery of 74 %
Citrate-phosphate-dextrose-
adenine (CPDA-1)
 Improved ATP synthesis
 Longer shelf life (35
days)
 Disadv: uric acid stones

Citrate Phosphate Dextrose
Adenine 2 (CPDA 2)
 PRC stored in CPDA - 1
ran out of glucose very
soon compared to CPD
 CPDA -2 contains more
glucose than CPDA-1
 Disadv: plasma and
platelets loaded with
sugar
CP2D
 100% more glucose than
CPD and 60% more than
CPDA-1
 used with an additive
solution (AS3): doesnt
contain glucose

Blood : Anticoagulant Ratio
 Volume of anticoagulant –preservative solution is 1/7 the
volume of collected blood
 14 ml of CPD/CPDA is used in preserving 100 ml blood
 63 ml for a 450 ml collection
 49 ml for 350 ml collection
 At the end of the collection , venous blood (pH 7.35)
mixed with anticoagulant-preservative solution (pH 5.0 to
5.6) with resulting pH of 7.05 in the mixture.

Additive Solutions:
 Need- With advent of component therapy , PRC usage increased (in 1970s).
 40%Adenine and glucose present in anticoagulants was removed in
preparation of RBC.
 Remaining RBC became more viscous and difficult to infuse in emergency
situation and decreased viability particularly in last 2 weeks of storage.
 Use of additive solutions allowed maximum recovery of plasma and
preparation of RBC units with final haematocrit of about 60%
 1stGeneration: SAG and BAGP-M 2ndGeneration: SAG-M and PAGGS-M
 3rd Gen: AS-1 and PAGGG-M 4thGen: AS-2 and Erythrosol-1
 5th Gen: AS-3 and Erythrosol-2 6th Gen: Erythrosol-81 7th Gen: EAS-64

 SALINE-ADENINE GLUCOSE (SAG ): maintains 83% viability after 35 days storage
 MANNITOL : acts as a membrane stabilizer and reduces hemolysis to acceptable levels
 BICARBONATE ADENINE GLUCOSE PHOSPHATE MANNITOL (BAGP-M)
 ERYTHROSOL-1: Uses half strength citrate (0.5 CPD)
 ERYTHROSOL-2: Uses full strength CPD and alkaline solution with disodium phosphate Solution. 7
week storage
 EAS -81 EXPERIMENTAL ADDITIVE SOLUTION
 PHOSPHATE ,ADENINE, GLUCOSE, GUANOSINE, SODIUM CHLORIDE, MANNITOL (PAGGS-M)
 Advantages
– Increased level of ATP and red cell viability
– Increase in shelf life of red cells to 42 days
– Extraction of more plasma/platelet rich plasma for optimal production of platelets, factor VIII yields
and FFP
 Disadvantage
– do not maintain 2,3DPG throughout the storage time Therefore , blood stored in additive solutions
is not given routinely to newborn infants

Rejuvenation Solutions
 Stored RBCs, even at the end of their allowable shelf-life can be
rejuvenated with FDA – approved rejuvenation solution.
 PIPA (Sodium Pyruvate, Inosine, Adenine, Dibasic Sodium Phosphate
and Monobasic Sodium Phosphate)
 Increases 2,3 DPG & ATP levels in stored RBCs.
 Added at any time between 3 days post collection and 3 days after
expiry
 Added directly to PRC, mixed and incubated at 37⁰C for 1 hour and
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
 Used in autologous donations/rare blood groups

Cryopreservation
 Process of preserving biologic structure and/ function of
living systems by freezing and storage at ultra low
temperatures
 Cryoprotectants
 Nonpenetrating : viscous solution,reduce optimum cooling
velocity.
– Eg: Lactose ,Sucrose, Albumin, Hydroxyl Ethyl Starch(HES),
Polyvinyl Pyrrolidone (PVP)
 Penetrating :Prevent damage during slow freezing and
thawing. Eg: Glycerol , DMSO

Cryoinjury:
 Fatal effects of freezing
– Intracellular ice crystal formation
– Denaturation of proteins by high solute concentrations
– Osmotic stress during the freezing and thawing process
 AUDREY SMITH (1950) Mother of cryobiology
 Glycerol prevents freezing injury human RBC
– Relatively nontoxic
– Readily permeates cell at 37⁰C
 Add slowly with vigorous shaking.
 Frozen red cells stored for 10 years
 Frozen RBC has to be deglycerolized before transfusion
 Shelf life of thawed RBC is 24 hours

Platelets
 Stored at 22-24⁰C in platelet agitator cum incubator
 Continuous gentle agitation must be maintained on a flat bed agitator: if not
rapid fall in pH due to lactate and decrease in O2 consumption
 pH: above 6.0 Plasma (50-60 ml) is needed for storage
 Shelf life extended from 3 days to 5 days (1981) at room temperature due to
improved platelet storage bags that allowed more efficient gas exchange.
 ANTICOAGULANTS:
 Platelet viability best preserved with fresh heparinized blood
 EDTA : Preserves platelet integrity Disadv: Cardiotoxic , rapidly removed from
circulation
 Citrate-phosphate Dextrose (CPD or CP2D)
 Apharesis platelets : collected into solutions containing citric acid , trisodium
citrate and dextrose. pH 5, prevents clumping of platelets

Platelet Additive Solutions(PAS):
 Synthetic mediums introduced to replace a significant portion of
plasma volume in a platelet component
 PAS being used in Europe since 1991
 Primary Ingredients
– Citrate- anticoagulant
– Acetate- substrate for oxidative metabolism
– Sodium chloride- isotonicity and osmotic strength
– Phosphate-stimulate glycolysis and maintain of pH
– Magnesium/Potassium- decreases platelet activation , improves
morphology score , decreases lactate production
 Tulli’s solution, Tyrode’s medium, Plasma- Lyte A

 Adv: Reduction of allergic reactions and febrile transfusion reactions
– Facilitates ABO incompatible platelet transfusions
– Plasma can be diverted to other uses such as fractionation
– PAS supports 7 days storage of platelets
– Reduced levels of anti-HLA, HNA antibodies and TRALI
Storage Of Platelets In Frozen State
Best results found using DMSO (Dimethyl sulfoxide ) as cryoprotective agent
Stored satisfactorily in liquid nitrogen (-196c) or at -80c deep freeze

Approved Preservative Solutions (FDA)
 ACD - approved for collection of RBC, platelets & FFP
 CPD- approved for 21 days storage
 CP2D- approved for 21 days storage
 CPDA-1- approved for 35 days storage
 Additive Solutions
– Adsol (AS-1)-Fenwal Laboratories
– Nutricel(AS-3)-Medsep Corporation
– Optisol (AS-5) Terumo Corporation
– SAG-M with CPD
 REJUVENATION SOLUTIONS
– Rejuvesol (Cytosol Laboratories)
– PIPA(Phosphate , Inosine, Pyruvate , Adenine )
 PLATELET ADDITIVE SOLUTION Intersol-FDA

Transportation & Storing of Blood &
Blood Components Prior to Transfusion
 Blood Cold Chain
– System for storing & transporting blood & blood
components
– Maintenance of correct temperature at all times from
collection from donor to administration to the patient.
– Blood bank refrigerators ,Plasma freezers, platelet
agitator cum incubators, blood transport boxes
 Any break in the blood cold chain: increased risk to
the recipients

WHOLE BLOOD AND RED CELLS
 Storage at a temperature between +2 degree C to +6
degree C in a blood bank refrigerator.
 Blood bank refrigerators: in built temperature monitoring,
alarm devices and a cooling fan
 Shelf life ranges from 35- 42 days.
 Issue of whole blood & red cells:
– blood transport box or insulator carrier
– temperature <10 degree C
– should be transfused within 30 min of taking out from BbR

Fresh Frozen Plasma and Cryoprecipitate
 Stored at -40⁰ C or colder
 Shelf life one year
 Thawed before transfusion (plasma thawing bath at 30 - 37⁰C )
 Once thawed, FFP and CP should be infused within 30 minutes.
 Transported in a blood transport box, temperature between 2 to
6⁰C.
 If not immediately required, keep at 2 to 6⁰C & transfused within
24hrs (FFP) and 4hrs (CP)
 Once thawed, do not refreeze FFP or CP.

Platelets – Platelet Rich Plasma (Prp)/Platelet
Concentrate (Pc)
 Platelets should be prepared within 8 hrs of phlebotomy
 Whole blood for separation of platelets should be kept at 20-
24⁰C
 Stored at 22 -24⁰C in platelet agitator cum incubator
 Shelf life : 5 days
 Transported in a blood transport box, temperature 22 -24⁰C
 Never refrigerate
 Transfuse as soon as possible.

Anticoagulants and blood preservatives