Dr R AmitaAssistant Professor

Dept of Transfusion Medicine

Anti coagulants and Blood Preservatives

Unknown Roman poet 43 BC

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

Give me blood.. Ha ha ha

19th Century

20th Century

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 conditionsAnticoagulants 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 Anticoagulantsyear person anticoagulant remarks

1916 Rous and Turner Citrate dextrose RT soln 12 days1943 Loutit and Mollison Acid citrate dextrose pH 51957 Gibson Citrate phosphate dextrose Prevents PO4 loss1957 Gabrio Added nucleotides RBC metabolism1968 Sheilds CPDA-11970 Beutler Additive solutions1979 Hogman SAG 5 weeks storage1980 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 cellsDisadv: 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 StateBest 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.