Anticoagulants

I am Dr.Gayathri.A.M. MD Student in SCTIMST, Trivandrum.

1. ANTICOAGULANT-PRESERVATIVE SOLUTIONS PRESENTATION NO.5 Dr.Gayathri.A.M .

2. ANTICOAGULANT : Chemical that prevent clotting of blood PRESERVATIVE : a chemical added to any specimen to prevent changes in the constituents of it In the body, blood is in fluid state due to optimal temperature, pH, proper nourishment to cells and detoxification. INTRODUCTION

3. Before the discovery of anticoagulants, defibrinated blood was used(whipping & twirling of collected blood) Direct transfusions happened which needed great surgical expertise Birchoff (1835) did transfusion of defibrinated blood in animals In JOHN HOPKINS HOSPITAL : collected blood was agitated in Erlenmeyer flask with glass beads till clot is fully formed & clot was removed; remaining liquid portion with RBCs poured into another container through a sterile gauze & and used for transfusion Till 1920’s defibrinated blood was used for transfusion HISTORY

4. Transfusion reactions occurred because of damage of RBCs due to trauma to collected blood during defibrination CURTIS & DAVIS (1911): used Y-shaped paraffin coated cannula tied into veins of both donor & recipient & a syringe was attached to neck of cannula KIMPTON BROWN (1913): used paraffin coated glass cylinders with horizontal side tube for suction BERNHIEM : Syringe , U Shaped Tubes for needle attachment & 2 way Stopcock Apparatus UNGER : Syringe, 4 way outlet Stopcock(blood aspiration, injection to recipient & for flush out) Curtis & Davis Bernheim

5. JOHN BRAXTON HICKS(LONDON,1868): First to experiment with chemical methods to prevent coagulation of blood ; phosphate of sodas(blood kept fluid but patient died of shock) Swiss Physiologists ARTHUR & PAGES(1890): Connect calcium with blood clotting on addition of any small amount of organic salts LANDOIS(1892) : Suggested Hirudin from leeches A.E.WRIGHT(1894) : Suggested non-toxic citrates; that bind enough ca without causing convulsions [21 years back before it was used] SATTERLEE & HOOKER(1914): Used Hirudin but found to have a narrow therapeutic window& difficulty in obtaining ERA OF ANTICOAGULANTS

6. WEIL(NY,1914) : Citrated blood can be preserved in icebox for long term basis HUSTIN(BELGIUM,1914) : Reported use of sodium citrate & glucose AGOTE(ARGENTINA,1915) : Citrate added blood can be kept for longer time LEWISOHN(NY,1915 ): Minimum amount of 0.2% citrate can prevent clotting & relatively safer in massive transfusions with 2.5litre of citrated blood ALBERT HUSTIN LUIS AGOTE RICHARD WEIL RICHARD LEWISOHN LEWISOHN & ROSENTHAL(1933): Explained that hyperpyrexial reactions occuring is because of bacterial growth in distilled water which was being used as a solvent for anticoagulant

7. YEAR PERSON ANTICOAGULANT REMARKS 1869 Braxton Hicks Sodium Phosphate 1 st example of bl ood preservation research 1914 Hustin Sodium Citrate Citrate used 1 st time 1915 Lewisohn Sodium Citrate Determined minimum amount needed for anticoagulation and demonstrated non toxicity in small amounts 1916 Rous and Turner Citrate -dextrose 1 st anticoagulant 1917 Robertson Citrate-Dextrose Used RT solution to build blood bank in Harvard medical unit YEAR PERSON ANTICOAGULANT REMARKS 1943 Loutit and Mollison (ACD)Acid citrate dextrose Lowered the pH to 5 to make ACD 1950 Carl Walter & W P Murphy Plastic blood bags Easy transportation & component preparation possible 1950 Audrey Smith Cryo preservation of red cells using glycerol Long term storage 1957 Gibson Citrate phosphate dextrose Cells passively lose phosphate , so by adding phosphate this loss can be prevented 1957 Gabrio and collegues Inclusion of nucleotides Important for red cell metabolism YEAR PERSON ANTICOAGULANT REMARKS 1968 Shields CPDA-1 Showed that Adenine (preservative) markedly improved WB storage 1970 Beutler Concept of additive system 1979 Hogman (SAG)Saline , adenine, glucose 5 weeks storage without adverse effects of high ph 1980 Lovric CP2D in primary bag with additive solution composed of SAG , sodium citrate , citric acid and sodium phosphate 1985 ---- SAGM Shelf life reduced to42 days CHRONOLOGY OF DISCOVERIES

8. AUDREY SMITH PEYTON ROUS PATRICK MOLLISON JOHN G GIBSON

9. IN-VITRO STUDIES Stored RBC studied using biochemical and morphologic measures Safer studies Lower expense Standard used: Hemolysis less than 1% in US and 0.8% in Europe RBC STORAGE STUDIES IN VIVO STUDIES Measuring RBC recovery and survival in volunteers (HUMAN TRIALS) Are of greater than minimal risk Require scientific, medical and radiation physics oversight Clear standards:- 75% survival after24 hours and hemolysis less than 1 % Disadvantage :- need for stable population of volunteers

10. STORAGE LESIONS ATP 2,3 BPG Constellation of changes associated with irreversible erythrocyte damage and reduced post transfusion survival following storage at 2 -6 O c They have effect on : Post transfusion viability reflected in ATP levels Hemoglobin function reflected by 2,3 DPG

11. PHYSICAL BIOCHEMICAL Morphological changes-Disc changes to echinocytes and then to spherocytes Decrease in 2,3 DPG-Decrease in pH-Shift of O2 dissociation curve to left-Decrease release of O2 from Hb to tissues Change in deformability due to loss in membrane lipids Electrolytes-poor functioning of Na-K+ Pump-accumulation of K+ in stored blood Increase in osmotic fragility Oxidative damage with change to the structure of Band 3 and Lipid peroxidation STORAGE LESIONS PHYSICAL Loss of heat labile coagulation factors ie factor V and VIII(30% and 20% respectively) Apoptic changes with racemization of membrane phospholipids

12. CONSEQUENCES OF STORAGE LESIONS These events risk compromising the safety and efficacy of long stored RBC Reduces their capacity to carry and release oxygen Promotes release of potentially toxic intermediates Negatively influences on physiological rheology

13. COMMON ANTICOAGULANTS USED IN HEMATOLOGY EDTA SODIUM CITRATE HEPARIN OXALATES SODIUM FLUORIDE IODOACETATE

14. EDTA Its used in a concentration of 1 to 2 mg/ml of blood It may be used for both hematology and chemistry tests, and is the preferred anticoagulant for blood cell counts (CBC), ABO Blood grouping. Cytotoxic and weakly genotoxic

15. SODIUM CITRATE The ratio is one part of 3.8% aqueous solution to nine parts of whole blood. Its used for blood coagulation and platelet function studies (PT &PTT) and can be used for Erythrocyte sedimentation rate (ESR).

16. OXALATES Inhibit blood coagulation by forming insoluble complex with calcium K oxalate: concentration of 1-2 mg/ml of blood NH 4 + & K+ oxalate mixture in the ratio of 3:2, and 2 mg / ml of blood is the required amount Ammonium and/or potassium oxalate does not cause shrinkage of erythrocytes, consists of three parts by weight of NH 4 + oxalate, which causes swelling of the erythrocytes, balanced by two parts of K oxalate which causes shrinkage

17. SODIUM FLUORIDE weak anticoagulant but is often added as preservative for blood glucose together with potassium oxalate as anticoagulant It is effective at a concentration of 2mg/ml blood It inhibits the enzyme enolase (sodium fluoride) thus inhibiting glycolysis . Na fluoride/K oxalates are mixed in the ratio 1:3 IODOACETATE Sodium iodoacetate at concentration of 2mg/ml is an effective antiglycolytic agent and a substitute for sodium fluoride.  It is a potent (suicide)  inhibitor  of G3P dehydrogenase Formalin, toluene, 6N HCl , Boric acid, thymol , chloroform are used as urine preservatives for 24 hour urine

18. Stopper color Additive Notes Red No additive Used for blood bank, some chemistries. Collection of serum 10-15 min is required to allow blood to clot before centrifugation Lavender (purple) EDTA Collection of whole blood ( binds calcium) COLOUR CODE FOR TUBES WITH DIFFERENT ANTICOAGULANTS

19. Stopper color Additive Notes Green Sodium or lithium heparin Inhibits thrombin activation. chemistry studies Light blue Sodium citrate Coagulation studies (bind calcium) (PT &PTT) (ESR).

20. Stopper color Additive Notes Gray Sodium fluoride & potassium oxalate : inhibits enolase ( phosphopyrovate dehydrogenase ) Sodium iodoacetate : inhibits glocose-3-phosphate dehydrogenase For glucose determination in chemistry (stabilize glucose in plasma) Yellow Acid citrate dextrose (ACD) For use in blood bank studies, HLA phenotyping , DNA and paternity testing (preserves red cells)

21. HEPARIN Prevents coagulation by inactivating the prophylactic activity of thrombin after combining with AT 111 and thrombin. 1000 IU of heparin is equal to 10 mg (commercially available as IU) Dose of heparin for anticoagulation is 0.5-2.0 IU/ml of blood (approx.500 IU of heparin for 500 ml of blood) Heparinized blood should be used within 24 hours. Earlier heparinized blood was used in pen heart surgery but now usually it is not used as extracorporeal pumps are now usually primed with crystalloids and not with blood. The effect of heparin can be neutralized with Protamine sulphate 1 mg of protamine sulphate neutralizes 1 mg of heparin (to neutralize 5000 units of heparin [50 mg], 5 ml of 1 % solution of protamine sulphate will be needed) Heparin is used in cord blood collection apart from CPD

22. CITRATE DEXTROSE First anticoagulant preservative Introduced in 1916 (World War 1) by Rous and Turner Initially used to store rabbit blood Blood stored up to 12 days in citrate-sucrose solution prevented anemia post transfusion. Dextrose was found to be marginally better in diminishing lysis in human blood ADVANTAGES : Most efficient and available anticoagulant for greater part of 20 years DISADVANTAGES : Required dilution of blood to about three times its original volume (1:3) Could not be heat sterilized because sugar got caramelized Risk of bacterial contamination from open mixing of ingredients and addition of solution to the bottle

23. ACD (ACID-CITRATE-DEXTROSE) 1943(world war II) Loutit and Mollison showed that simple acidification of citrate dextrose prevented caramelization of sugar pH of citrate dextrose was lowered to make it 5 ACD first used in 1:4 ratio but later concentrated to use in 1:7 ratio ACD blood was the basis of building national blood systems in British commonwealth and USA during World War II ADVANTAGES : Simplified sterilization procedure Reduced volume of preserving solution Enhanced preservative properties Increased shelf to 21 days Minimal effects on acid base balance DISADVANTAGES : Levels of 2,3 DPG lost early within 1st week

24.  ACD is used during procedures such as  plasmapheresis  instead of  heparin Two different solutions (Solution A and B) are defined by the  United States Pharmacopeia CONTENTS (g) ACD-A ACD-B Total Citrate 20.59 to 22.75 12.37 to 13.67 Dextrose 23.28 to 25.73 13.96 to 15.44 Sodium 4.90 to 5.42 2.94 to 3.25

25. CITRATE PHOSPHATE DEXTROSE John .G . Gibson developed a method for estimating in vivo red cell survival using double isotope procedure with 51 Cr and 32 P to study the effect of blood preservative solution & noted existence of high energy phosphate compound in blood collected in CPD (later found to be 2,3 DPG) Because of the lower pH in the ACD preservative, most of the 2,3-DPG is lost early in the first week of storage & thus CPD came into widespread use in the US in 1970s because it was superior for preserving 2,3 BPG since it has a higher pH Even in CPD, RBCs become low in 2,3-DPG by the second week. Shelf-life of blood stored in CPD at 2-4 °C was 28 days with <30% RBC loss

26. ADVANTAGES : Better maintenance of 2,3DPG Higher pH (5.6) Improved ATP synthesis CPD with 16 mMol /L phosphate increased the fraction of RBC recovered after 3 weeks of storage from75% with ACD to more than 79%,and after 4 weeks only slightly lower than storage in ACD

27. CITRATE PHOSPHATE DEXTROSE ADENINE (CPDA- 1) CPDA - 1 was developed in 1968 and shown to permit whole blood storage for 5 weeks However regulatory questions regarding safety of adenine (especially development of uric acid stones) delayed licensure of CPDA 1 in USA for 11 years In those 11 years blood banking shifted from whole blood storage to manufacture of components

28. CITRATE PHOSPHATE DEXTROSE ADENINE 2 (CPDA 2) Beutler and West showed that packed RBC stored in CPDA - 1 ran out of glucose sooner than whole blood stored in CPDA 1. They suggested addition of more glucose to original anticoagulant solution making CPDA -2 This meant that plasma and platelets contained large amounts of unnecessary sugar 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

29. CP2D 100% more glucose than CPD and 60% more than CPDA-1 The high glucose content of CP2D is necessary because it is used with an additive solution (AS-3)that does not contain sufficient glucose

30. CONTENTS ( mMol /L) ACD-A ACD-B CPD CPDA-1 CP2D CITRIC ACID 35 21 14 14 14 SODIUM CITRATE 97 58 116 117 117 DEXTROSE 136 81 141 142 284 MONO SODIUM PHOSPATE - - 15.8 16 16 ADENINE - - - 2 - pH 5 5.6 5.6 5.6 VOLUME RATIO USED 1:7 1:4 1:7 1:7 1:7 A/c to ROSSI’s

31. ADDED NUCLEOTIDE ADENOSINE : ADVANTAGE : Restoration of ATP. This was assumed to be due to its phosphorylation to AMP and subsequent conversion to ATP DISADVANTAGE :Adenosine was found to have marked hypotensive effect. It was never used due to its toxicity GUANOSINE : Used in PAGGS-M which provides 7 weeks of RBC storage with recovery of 74 % Guanosine has also been used as an additive to ACD based blood preservative but usefulness as practical additive has not yet been established

32. INOSINE : Adenosine deaminase rapidly converted adenosine to Inosine Inosine + P1  Ribose 1 phosphate (R-1-P) + Hypoxanthine R-1-P is readily converted to F-6- P by pentose shunt which feeds into glycolytic pathway leading to generation of ATP ADVANTAGES : Excellent effect on viability DISADVANTAGES : Hypoxanthine formed from inosine degraded to uric acid INOSINE can serve as indispensible part of rejuvenation solution that are used to revitalize red cells after they have lost their ATP and 2,3 DPG

33. DIHYDROXYACETONE Introduced by Brake and Deindoefer in 1972 Increased maintenance of 2,3 DPG levels MOA : RBC contain enzyme triokinase with capability of phosphorylating DHA to DHAP.

34. OXALATE AND ASCORBATE Effect of Ascorbate on red cell 2,3 DPG discovered in 1972- found to increase 2,3 DPG levels Studies by Kandler et al show that pure ascorbate has no effect at all. The oxalate that contaminates it is responsible for increase in 2,3 DPG Oxalate precipitates renal calculi even in small quantities , so its use not practical

35. XANTHONE DERIVATIVES Hyde et al,(1984) studied effect of tricyclic acid 2 ethoxy-6-5( tetrazolyl ) xanthone (BWA825C) on O 2 dissociation curve of Hb They found that it not only exerted an effect on purified Hb solution but also increased 2,3 DPG levels during storage Related compounds BWA440C and BWA827C were also found to be effective in maintaining 2,3 DPG levels Little is known about the toxicity of these compounds to permit their use clinically

36. INORGANIC PHOSPHATES Phosphate exerts multiple metabolic effects on erythrocytes and when high concentrations are added to blood preservatives ,levels of both 2,3 DPG and ATP are enhanced Larger quantities have profound effect on ATP preservation but do not have corresponding advantageous effect on post infusion viability

37. PYRUVATE Pyruvate makes NAD available from NADH through Lactate dehydrogenase reaction, the addition of pyruvate to red cells may facilitate metabolism through the glyceraldehyde dehydrogenese step of glycolysis Pyruvate exerts a modest favorable effect on 2,3 DPG Pyruvate undergoes various polymerization reactions and therefore unstable .Hence, little practical use as component of blood preservative Used in Rejuvenating solutions

38. CITRATE Prevents coagulation by chelating calcium Retards glycolysis Side effects: Has affinity for magnesium ions- hypomagnesaemia in the setting of massive transfusion has been reported 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, citrate considered to be a cause of cardiac arrhythmia MECHANISM OF ACTION OF VARIOUS COMPONENTS DEXTROSE Improves red cell viability Provides energy for ATP synthesis Decreases rate of hydrolysis of phosphorus

39. SODIUM BIPHOSPHATE It acts as a buffer to control decrease in pH from the generation of lactic acid Cells passively lose phosphate, so by adding phosphate this loss can be reduced The incorporation of adenine seems to increase ADP levels thereby driving glycolysis towards synthesis of ATP As ATP-dependent cytoskeleton control red cell membrane shape and rigidity , addition of adenine to cells restores shape and post transfusion viability .Adenine provides a substrate for ATP synthesis in RBC resulting in improved viability Adenine approved by FDA in August 1978 ADENINE

40. CITRIC ACID Prevents glucose caramelization during autoclaving Provides optimal pH with citrate for red cells

41. BLOOD : ANTICOAGULANT RATIO Volume of anticoagulant –nutrient solution is normally 1/7 volume of collected blood 14 ml of CPD/CPDA is used in preserving 100 ml blood 63 ml for a 450 ml collection 70 ml for 500 ml collection At the end of the collection , venous blood with pH of 7.35 mixes with anticoagulant-nutrient solution with pH 5.0 to 5.6 with resulting pH of 7.05 in the mixture

42. ADDITIVE SOLUTIONS REJUVENATION SOLUTIONS RED CELL FREEZING CURRENT TRENDS IN RBC PRESERVATION

43. The additive system concept was developed by Beutler in the 1970s, and implementation of specific solutions was initiated in the early 1980s by Lovric in Australia and Högman in Sweden Additive solutions are preserving solutions that are added to the RBCs after removal of the plasma with/without platelets Removal of the plasma component during the preparation of RBC concentrates (high hemocrit preparations esp.) remove much of the nutrients needed to maintain RBCs during storage Removal of substantial amounts of adenine and glucose present in anticoagulant-preservative solution lead to a decrease in viability, particularly in the last 2 weeks of storage ADDITIVE SOLUTIONS Void of plasma with high hematocrits makes units to be more viscous and difficult to infuse, especially in emergency situations Additive solutions reduce hematocrits from around 70 to 85% to around 50 to 60% Additive solutions employed in the systems were composed of standard ingredients used intravenously: saline, dextrose, and adenine Additive solutions are of: First generation & second generation First Generation consists of: AS-1, AS-3 ,AS-5 (USA) & MAP(Japan ) Second Generation consists of: BAGPM, only approved are PAGGS-M, AS-7; PAGGGSM, ErythroSol-1,2 & 4

44. Högman’s System : Standard CPD in primary bag with the additive solution containing saline, adenine, and glucose (SAG), further modified with the addition of mannitol (SAGM), which protected against storage-related hemolysis SAGM~ Adsol AS 1 Lovric : doubled dextrose concentration in the CPD (CP2D)and used it in connection with an additive solution composed of saline, adenine, glucose, trisodium citrate, citric acid & Na 3 PO 4 Formulations by Lovric and Högman provided the basis for the three additive solutions (USA): AS-1 ( Adsol ), AS-3 ( Nutricel )& AS-5 ( Optisol )

45. AS-1 : SAGM (retard hemolysis) coupled with CPD (in primary bag) AS-3 : SAG but at different concen . & in addition to sodium phosphate, sodium citrate & citric acid ; AS-3 is coupled with CP2D (in 1 bag) AS-5 : SAGM at different concen . and uses CPD in the primary bag All of these additive solutions are approved for 42 days of storage of RBCs None of the additive solutions maintain 2,3-DPG throughout the storage time As with RBCs stored only with primary anticoagulant-preservatives, 2,3-DPG is depleted by 2 weeks of storage Post storage survival rates of greater than 80 percent & with less than 1 percent hemolysis.

46. First Generation : to replace the volume and sugar lost with plasma removal and add the adenine necessary for storage beyond 3 weeks Mannitol reduced hemolysis and increased the osmolarity of the solution further Second Generation : attempts to rebalance the final suspending solution and a search for additional nutrients for the packed RBC concentrates BAGP was the result of the original attempt by Beutler to preserve both ATP and 2,3-DPG by raising the pH(resulted in high concen of 2,3 BPG at the expense of ATP with no improvement in storage conditions)

47. pH >7.2 <6.4 Bifunctional enzyme diphosphoglycerate mutase – phosphatase converts almost all 1,3-DPG into 2,3-DPG,depriving the cell of new ATP Activities of hexose kinase and Phosphofructo kinase , are too low to support ATP production 6.4-7.2 Hb , the mineral salts in the suspension & bicarbonate all serve to buffer the protons produced by glycolysis

48. FIRST GENERATION ADDITIVE SOLUTION Phosphate (2mmol /100ml) in additive solution buffers about 1mmol of additional protons Bicarbonate (2mmol /100ml) : protonated to make carbonic acid, converted to CO2 and water by red cell carbonic anhydrase , and buffer 2mmol of protons as the CO2 diffuses out of the plastic bag

49. Second Generation AS : Its formulation and pH balance almost double the amount of ATP energy available to stored red cells by depressing diphosphoglycerate mutase activity while sustaining glycolysis PAGGS-M : mannitol , the initials stand for phosphate, adenine, glucose, guanosine & saline Guanosine : GTP was detected in red cells and known to decrease during storage (may inhibit primitive coagulation enzyme transglutaminase )

50. FIRST GENERATION SECOND GENERATION 1. SAG 1. BAGP-M 2. SAG-M 2. PAGGS-M 3. AS-1 3.PAGGG-M 4. AS-2 4.Erythrosol-1 5. AS-3 5.Erythrosol-2 6.Erythrosol-81 7.EAS-64

51. Composition of Additive Solutions CONTENTS (per 100ml ) AS-1 AS-3 AS-5 Dextrose 2200 1100 900 Adenine 27 30 30 Monobasic sodium phosphate 276 Mannitol 750 525 NaCl 900 410 877 Sodium citrate 588 Citric acid 42 Primary bag anticoagulant CPD CP2D CPD

52. BIOCHEMICAL CHARACTERISTICS CONTENTS AS-1 AS-3 AS-5 Storage period (days) 42 42 42 pH (at 37 C) 6.6 6.5 6.5 24hr Survival (%) 83 85.1 80 2,3BPG (% initial) 68 67 68.5 Hemolysis (%) 0.5 0.7 0.6

53. PLATELET ADDITIVE SOLUTION PAS : Crystalloid nutrient media used in place of plasma for platelet storage. They replace 60-70% of plasma in platelet components, so the amount of storage plasma can be decreased have a lower risk for allergic transfusion reactions & TRALI PAS III received FDA approval Recovery range = 35% - 71% Survival range = 4.5 – 7.0 days

54. The changes that lead to loss of viability by cumulative oxidative damages occurring during storage are largely reversible by a process called rejuvenation (metabolic recharging of red cells at the end of their storage period) Hogman showed that rejuvenating red cells at the end of six weeks storage in SAG-M increased their 24-hour in-vivo recovery from 77 to 89% Initial rejuvenate solutions had phosphate, inosine , glucose, pyruvate and adenine (PIGPA) This rejuvenation is a strictly metabolic recharging of red cells at the end of their storage period They can be rejuvenated by incubation in a high-pH solution of phosphate, inosine , pyruvate & adenine ( PIPA,Rejuvesol,etc ) for 2 hours REJUVENATE SOLUTIONS

55. It increase the levels of 2,3-DPG and ATP in stored red cells & can be added at any time between 3 days post collection and 3 days after expiration of red cells and increases in-vivo recovery of stored RBCs, probably by allowing them to internalize negatively charged membrane phospholipids that would otherwise signal clearance by macrophages Return of the normal distribution of phospholipids also prevents red cells from participating in plasma coagulation reactions Rejuvenation does not reverse the oxidative damage to band3 of the cell membrane, desialation of glycoproteins , or loss of membrane

56. 50ml solution is added directly to a unit of red cells, mixed and incubated at 37 °C for one hour The rejuvenated red cells are either washed with saline (2 Litres of unbuffered 0.9% NaCl ) and can be kept at 2-6°C, however, they should be transfused with in 24 hours after washing or they are glycerolized for keeping red cells in frozen state to improve the quality of red cells Red blood cells rejuvenation solution, 50 ml sterile vial ( Rejuvesol ) is commercially available; used primarily to salvage liquid-stored RBCs (also autologous RBCs) that have reached outdate Rejuvenated RBCs can be frozen with glycerol as the cryo -protecting solution; rare units and O-type units are primarily treated for subsequent cryopreservation Because the processing including the washing procedure is currently accomplished with open systems that are not specifically designed to prevent the entrance of bacteria, federal regulations require that rejuvenated/ frozen RBCs are used within 24 hours of thawing Rejuvenation process is expensive and time consuming and is rarely used

57. RED CELL FREEZING 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 Glycerol, Dimethyl sulfoxide (DMSO) & Hydroxyethyl Starch(HES) (cryoprotective agent) is added to red cells they can be frozen and thawed without damage by intracellular ice formation and hypertonicity Glycerol limits ice formation and provides liquid phase in which salts are distributed as cooling proceeds thereby avoiding excessive hypertonicity Frozen red cells are primarily used for autologous transfusion and the storage of rare group blood Cryoprotective agent is added to red cells that are less than 6 days old Glycerol (used commonly) 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. High concentration glycerol [40% weight in volume] and a low concentration glycerol [20% weight in volume] in the final concentration of cryopreservative Most blood banks use the high glycerol technique.

58. CRYO INJURY

59. Characteristics 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

60. Frozen cells are deglycerolized before transfusion Removal of glycerol is achieved by systematically replacing the cryo-protectant 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. 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. Red cells stored in additive solutions can be frozen up to 42 days

61. DMSO DMSO protects the cells by: 1) partially solublizing the membrane so that it is less prone to puncture, 2) interrupting the lattice of the ice, so that fewer crystals form.  10% concentration and is usually combined with saline or serum albumin  also used in the banking of cord blood cells

62. DMSO TOXICITY DMSO's systemic toxicity is considered low It has effects on coagulation; anticholinesterase activity; DMSO- induced histamine release by mast cells DMSO can decrease membrane thickening and induces temporary water pores when used at low concentrations. Side effects of infusion of DMSO- cryopreserved cells include nausea, emesis, chills, rigors, and cardiovascular events  DMSO also shows neurotoxic effects including encephalopathy, when stem cells were infused into cancer patients, as well as gastrointestinal effects  DMSO is also directly cell toxic, affecting cell viability, inducing apoptosis and differentiation & decrease membrane thickening and induces temporary water pores when used at low concentrations. At higher concentration it induces disintegration of the bilayer structure of the lipid membrane

63. HYDROXY ETHYL STARCH A synthetic modified polymer based on purified starch Large molecules of HES serve as a non-penetrating cryoprotectant ; this effect of depends on its ability to absorb water molecules and keep these thermally inert in glassy state without experiencing any phase transition during cooling HES influences the viscosity of solutions and decreases the cooling rate required for optimal survival during vitrification , increases propensity for supercooling and kinetically inhibits ice formation (HES can absorb up to 0.5 g water per 1 g of HES) Accumulation of HES in the extracellular space, initially increases extracellular viscosity reduces the rate at which water can be withdrawn from the cells preventing osmotic stress and damage HES has been used in the cord blood banking industry for the separation of blood into its individual components

64. HES HES is widely used as a plasma volume substitute due to its colloidal osmotic pressure which increases viscosity of plasma and whole blood and facilitates delivering of oxygen by red blood cells, in hemodilution treatment to enhance the microcirculation and for peripheral arterial stenosis treatment (w/w) HES 6%,11.5% 12%,14% : used for storage of RBC according to different studies

65. HES TOXICITY HES administration leads to increased serum amylase concentration of up to 5 times the initial value. However, this increase does not affect the pancrease or lipase activity and therefore seems to have no pathological relevance Osmotic nephrosis -like lesions in renal transplant patients Pruritus on chronic administration bleeding complications due to decreased factor VIII/von Willebrandt factor, platelet function defects and incorporation into fibrin clots, probably due to dilution effects Large HES molecules can cause detrimental effects on rheological parameters of blood

66. MECHNISM OF CRYOPRESERVATION Upon cryopreservation extracellular (1) and intracellular (2) water crystallises to ice which can lead to damage including to membranes (3) DMSO opens cell walls and leads to water being removed from the cell (4) while DMSO enters HES in turn binds extracellular water (5) and establishes a concentration gradient which removes water from the cell (6), and can thereby confine ice formation away from the cell (7) In addition, it is considered to stabilize the cell membrane (8) but normally without entering the cell

67. INDICATIONS FOR THE USE OF FROZEN RED CELLS Freezing of rare blood groups enables long-term storage and supply on a regional and national basis. Storage of blood for patients with antibodies against high frequency antigens. Storage of blood for autotransfusion , specially in patients with rare blood group. Prevention of non-haemolytic febrile transfusion reaction in patients sensitized to leucocytes, platelets or plasma protein. Prevention of sensitization against HLA antigens in potential recipients of tissue transplants.

68. REFERENCES AABB TECHNICAL MANUAL 18 TH EDITION ROSSI’s PRINCIPLES OF TRANSFUSION MEDICINE 5 TH EDITION www.mahasbtc.com/preservation-and-storage-blood MODERN BLOOD BANKING & TRANSFUSION PRACTICES (DENISE M HARMENING) Transfusion and Apheresis Science 46 (2012) 137–147

69. THANK YOU