Blood substitues

artificial blood

1. N.VEERARAGAVAN II YEAR PG BLOOD SUBSTITUES

2. INTRODUCTION The transfusion of blood and blood products has become commonplace since the first successful transfusion in 1818. Although the incidence of severe transfusion reactions and infections is now very low, in recent years it has become apparent that there is an immunological price to be paid from the transfusion of heterologous blood , leading to increased morbidity and decreased survival in certain population groups (trauma, malignancy). Supplies are also limited, and therefore the use of blood and blood products must always be judicious and justifiable for clinical need

3. HISTORY

4. 1961- plaletet concentrates are reconized to reduce mortality from hemorrhaging in cancer patients 1972- process of apheresis. 1983- stanford blood centre done screening centre for AIDS 2002- west nile virus identified as transfusion – transmissible

5. INDICATIONS acute blood loss , to replace circulating volume and maintain oxygen delivery perioperative anaemia , to ensure adequate oxygen delivery during the perioperative phase symptomatic chronic anaemia , without haemorrhage or impending surgery.

6. METHODS BLOOD PRODUCTS ARTIFICIAL BLOOD OR BLOOD SURRGOATE

7. BLOOD PRODUCTS Whole blood blood components :red cell concentrates, platelet concentrates, fresh frozen plasma and cryoprecipitate plasma derivatives :albumin, coagulation factors and immunoglobulins.

8. Key Elements Donors are chosen to exclude anyone whose blood may harm the recipient Each donation is tested to establish the ABO and RhD group of the donor’s red cells.

9. Screening tests H epatitis B H epatitis C HIV-1 HIV-2 HTLV Syphilis . Donations are leukodepleted as a precaution against Creutzfeldt – Jakob disease (this may also reduce the immunogenicity of the transfusion)

10. Whole blood consists of red blood cells, white blood cells and platelets Whole blood has a shelf life of 35 days. Citrate phosphate dextrose adenine (CPDA-1) is an anticoagulant preservative in which blood is stored at 1°C to 6°C. The storage at 1°C to 6°C assists preservation by slowing the rate of glycolysis approximately 40 times the rate at body temperature.

11. Citrate is an anticoagulant (prevents clotting by binding calcium). Phosphate serves as a buffer. Dextrose is a red cell energy source Adenine allows RBCs to resynthesize adenosine triphosphate (ATP).

12. Advantages Rich in coagulation factor Metabolically active Disadvantage Limited resource Poor source of platelets

13. Packed RBC Packed red cells are produced by removing between 150-200ml of citrated plasma from a unit of whole blood. Haematocrit = 60-70%. Storing red cells just above freezing allows survival for up to 42 days. – but unfortunately decreases 2,3-DPG – ruins the platelets and neutrophils.

14. The administration of packed RBCs is facilitated by reconstituting them with a crystalloid or colloid ; however, not all crystalloids are suitable .(5% dextrose in water ) If the solution contains calcium, clotting occurs . Solutions recommended for reconstituted packed erythrocytes are 5% dextrose in 0.4% saline, 5% dextrose in 0.9% saline, 0.9% saline, and Normosol -R with a pH of 7.4.

15. A unit of whole packed red cells will raise the hematocrit by 3% and the hemoglobin by 1-1.5 gm/ dL OTHER PRBC PRODUCTS Irradiated packed red cells Washed packed red cells Cryopreserved packed red cells

16. Platelet Concentrates Component : platelets, 50 ml plasma cellular components that help in the clotting process. Platelets are stored for up to five days at room temperature . Indication – used if there is a platelet disorder – when massive blood loss has occurred

17. Platelets last for 3-5 days if stored on an agitator at 22°C and at a pH of between 6.2 and 7.8. Each bag has a volume of 250-350ml .(apheresis-PC) Platelets should be inspected prior to infusion and packs should be rejected, or referred for further opinion, if there is any unexpected appearance such as discolouration . Platelets are not usually cross-matched with the recipient, but where possible ABO specific platelets should be used.

18. ASA GUIDELINESS Patients with severe thrombocytopenia (<20,000 cells/mm3) and clinical signs of bleeding usually require platelet transfusion. However , patients may have very low platelet counts (much less than 20,000 cells/mm3) and not have any clinical bleeding. Patients such as these probably do not need platelet transfusions .

19. Individuals who have undergone trauma or require surgery need higher platelet counts, probably 100,000 cells/mm3, to maintain adequate hemostasis. Laboratory determinations and clinical evaluations must be taken into account before a decision to transfuse platelets is made.

20. Fresh Frozen Plasma FFP is collected as the supernatant after centrifuging a donation of whole blood. It contains all the plasma proteins, Particularly factors V and VIII, which gradually decline during the storage of blood. shelf life – 2 years It is the first-line therapy in the treatment of coagulopathic haemorrhage and used in reversal of warfain therapy

21. Collection and storage of FFP It is frozen within 8 hours at -40 ° C t0 -50 °C Under these conditions, the loss of Factors V and VIII is kept to a minimum. Frozen packs are brittle and should be handled with care. The frozen plasma can be thawed using a dry oven (10 mins ), microwave (2-3 mins ) or a water bath (20mins). Thawed FFP is best used immediately but may be stored at 4°C and infused within 24 hours.

22. Cryoprecipitate Cryoprecipitate is a supernatant precipitate of FFP rich in factor VIII and fibrinogen. It is stored at −30°C with a two year shelf life. It is given in low fibrinogen states or factor VIII deficiency. Uses Factor VIII deficiency or hemophilia A Treatment of fibrinogen deficiencies

23. Transfusion reactions Hemolytic Reactions Allergic Reactions Febrile Reactions Bacterial Contamination Circulatory Overload Hypothermia Graft Versus Host Disease (GVHD) Transfusion related acute lung injury (TRALI)

24. ARTIFICAL BLOOD

25. Artificial Blood Artificial blood or blood surrogate is a substance used to mimic and fulfil some functions of biological blood,usually in the oxygen carrying sense. Main aim is to provide an alternative to blood transfusion,which is transferring blood based products from one person to another . It does not contain plasma,RBCs or WBCs.

26. VOLUME EXPANDERS Crystalloids Colloids OXYGEN CARRYING BLOOD BASED PRODUCTS Haemoglobin based oxygen carriers Perflurocarbon oxygen carriers

27. Colloids Solutions that contain large molecules that don't pass the cell membranes. 5% albumin 25% albumin 10% dextran 6% dextran

28. Crystalloids Solutions that contain small molecules that flow easily across the cell membranes, allowing for transfer from the bloodstream into the cells and body tissues It is subdivided into: Isotonic Hypotonic Hypertonic

29. Isotonic solutions Types of isotonic solutions include: 0.9 % sodium chloride (0.9% NaCl) lactated Ringer's solution 5 % dextrose in water Ringer's solution

30. Hypotonic solutions • 0.45% sodium chloride (0.45% NaCl ), 0.33% sodium chloride , 0.2 % sodium chloride, and 2.5% dextrose in water Hypotonic fluids are used to treat patients with conditions causing intracellular dehydration, when fluid needs to be shifted into the cell , such as: 1. Hypernatremia 2. Diabetic ketoacidosis 3. Hyperosmolar hyperglycemic state.

31. HYPERTONIC SOLUTIONS 3 % sodium chloride ( 3% NaCl ): May be prescribed for patients in critical situations of severe hyponatremia. Patients with cerebral edema may benefit from an infusion of hypertonic sodium chloride 5% Dextrose with normal saline (D5NS): Which replaces sodium, chloride and some calories

32. Why artificial blood ? Increasing demand Decreasing supply Safety Infectious disease transmission Transfusion reactions Immunosuppression Cost

33. History In 1616, when William Harvey first described the circulation of blood In 1665, the first recorded successful blood transfusion on dog by Richard Lower Many materials used for transfusion that include milk, plant resins, and sheep blood. In 1854, patients were injected with milk to treat Asiatic cholera.

34. Other materials that were tried during the 1800s include hemoglobin and animal plasma .( Chang , 2004 ) In 1883,there was a creation of Ringer's solution. In research using part of a frog's heart, Sydney Ringer, found that the heart could be kept beating by applying the solution . (Hoffman et al ., 1990 )

35. In 1966, experiments with mice suggested a new type of blood substitute , perfluorocarbons (PFCs). In 1968, the rat's blood replaced with a PFC emulsion and it lived for a few hours and recovered fully after blood was replaced. ( Sarkar, 2008 )

36. Ideal Artificial Blood Increased availability that would rival that of donated blood, even surpass it Oxygen carrying capacity, equaling or surpassing that of biological blood Volume expansion Universal compatibility: elimination of cross matching Pathogen free: elimination of blood contained infections

37. Minimal side effects Survivability over a wider range of storage temperatures Long shelf life Cost efficient (Squires, 2002)

38. Types of Blood Substitutes 1 ) Perfluorocarbons (PFCs), chemical compounds which can carry and release oxygen 2) Haemoglobin -based oxygen carriers (HBOCs) derived from humans , animals, or artificially via recombinant technology

39. Haemoglobin -based oxygen carriers Hboc are prepared from devasted RBC, bacteria,bovine . High-level production of recombinant Hb using simple  Escherichia coli  expression system has been reported by Hoffman et al  in 1990. Human hemoglobin is obtained from donated blood that has reached its expiration date One unit of hemoglobin solution can be produced for every 2 units of discarded blood

40. Hemoglobin ( Hb ) The structure of Hb was determined in 1959 by Max Perutz . Molecular weight 64.5 kDa Tetrameric protein comprised of two α and two ß globin subunits that fold into compact quaternary structure (α 2 ß 2). Each α and ß subunit contain an iron- heme group that binds to oxygen molecule allowing for transport.

41. Research on hemoglobin-based fluids dates back to the 1920s when the stroma of the cells was lysed to obtain hemoglobin Problems with free hemoglobin include osmotic diuretic effects renal toxicity coagulation abnormalities short half-life vasoactive effect

42. Two types Acellular Hboc Cellular Hboc

43. Acellular Types

44. Cross-linked HBOC - intramolecular covalent bonds were formed between globin chains in order to prevent their detachment . Polymerised HBOC- Hb molecules are cross-linked intermolecularly to increase the molecular size.  Conjugated HBOC- Inert polymers are attached to the surface of Hb molecules.

45. Hemopure One of those products is HBOC-201 ( Hemopure ; Biopure Corporation ), made from bovine blood. It is universally compatible and is stable at room temperature for up to 3 years. P atients who received Hemopure had an increased number of serious adverse events. The vasoconstrictive properties of Hemopure may have caused myocardial infarction in susceptible patients Studies have been proposed to coinfuse a nitric oxide donor such as nitroglycerin in a fixed ratio, in a single bag compound or as separate infusions( OPK BIOTECH)

46. Oxyglobin A pp ro v e d f o r v e t e r in a r y u s e . It c o nsis t s o f c h em i ca l l y s t a b i lize d b o v i n h a e m oglo b i n i n a b alanc e d s alt s o luti on and c o n t a i n s n o r e d b lo od ce l l s . T h e cr os s - l i n k e d h ae m o g l o b i n , s e v era l t e t r ame r s b o u n d t o g e th e r , w o r k s b y circ u l a t i on i n t h e p l a s m a a n d s u p p l y i n g o x y g e n t o t i ss u e s . Drawback :   risk of transmission of diseases 

47. Polyheme Outdated human donated blood ( pyridoxylated human Hb ) shelf life of about a year at room temperature They reported that patients can be resuscitated with PolyHeme , without using stored blood, for up to 6 units in 12 hours after injury Drawback: increased number of myocardial infarctions

48. Conjugated HBOCs I nert polymers are attached to the surface of Hb molecules . Due to unique characteristics, low toxicity, and lack of immunogenicity or antigenicity in body, PEG can be the best polymer for conjugation. Hemospan is a PEG-conjugated Hb , which is under clinical trial as an oxygen carrier. This modification has been shown to increase the circulation half-life of the product.

49. MP4 is another PEG– Hb conjugate designed as an oxygen carrier. This product did not cause vasoconstriction in animal models, and its efficacy to deliver oxygen to hypoxic tissues was demonstrated; MP4 is now under human clinical trials. In addition to PEG, other polymers have been used to conjugate Hb , including benzene tetracarboxylate dextran,hydroxyethyl starch (called HRC 101), and albumin

50. CELLULAR HBOC’s Third-generation hemoglobin substitutes have begun to address the deficiencies of earlier formulations .   Hb is encapsulated in a cell-like structure The mixing of phospholipids and cholesterol in the presence of free hemoglobin forms a sphere with hemoglobin in the center . Encapsulation of Hb by a phospholipid layer (liposome-encapsulated Hb [LEH]) prolonged its half-life and shelf-life comparing to acellular products

51. These liposomes have oxygen dissociation curves similar to red cells, and administration can transiently achieve high circulating levels of hemoglobin and oxygen-carrying capacity. Research is still in the preclinical testing stage; progress in prolonging the half-life and elucidating the effects on the immune system, particularly reticuloendothelial sequestration, is crucial before clinical testing can begin . Hb vesicle is a PEGylated product with increased serum half-life and decreased recognition by the immune system

52. Clinical applications One of the main problems limiting the application of these products is their inability to convert Fe 3+  to Fe 2+ , which is an important function of RBCs Met- Hb with low oxygen-carrying capacity was produced, showing that such complications can be avoided by attaching reducing agents to Hb surface in this product series

53. Current status of Hb0c

54. Perfluorocarbons Perfluorochemicals (PFCs) are colorless, inert, and apparently nontoxic liquids with low boiling point temperatures and are insoluble in water and alcohol First demonstration on O2 capacity by Clark in 1966

55. PFCs have two challenges to overcome for use as blood substitutes . The first is that the liquid form is immiscible in water; thus, PFCs must be suspended as microdroplets with the use of emulsifying agents. The second is that unlike hemoglobin, the oxygen that is dissolved in PFCs has a linear relationship to the partial pressure of oxygen, whereas hemoglobin has a sigmoidal disassociation curve favoring full loading at normal atmospheric oxygen levels. Thus, the FIO2 that has to be applied is too high..

56. Fluosol -DA Fluosol -DA was the first accepted PFC-based RBC substitute, which is an emulsion of perfluorodecaline and perfluorotripropylamine .  Oxygen-carrying capacity of Fluosol -DA is only 7.2% at 37°C, which is lower than RBCs The use of this product entails complications such as pulmonary reactions supposedly due to complement activation by the emulsifying agent in Fluosol -DA and can be prevented by steroid injection

57. Second generation PFC’s Formulated to allow more oxygen-carrying capacities, with alterations in the emulsion properties. Such new compounds can also be stored at 4° C, whereas previous solutions had to be frozen. OxyFlour ™ and Oxygent ™ are among the second-generation PFC-based blood substitutes which are rejected by clinical trials due to some side effects such as complications in determining the effective dose for OxyFlour ™ administration and also increased risk of stroke following administration of Oxygent ™ . 

58. However, except some changes in clotting factors, no specific interaction between blood components and administered PFCs has been reported.  Administration of PFC-based products can result in mild thrombocytopenia (10%–15% reduction in platelet count) as well as flu-like syndrome

59. Third generation PFCEs Perftoran and PHER-O2 Pulmonary complications has been reported with the use of Perftoran and , PHER-O2 is in reasearch ( Modery et al ., 2013 )

60. Current status PFC

61. Other Promising Technique There is a possibility of using stem cells as a means of producing an alternate source of transfusable blood. A study performed by Giarratana et al. (2013) describes a large scale ex-vivo production of mature human blood cells using hematopoietic stem cells. A team of IIT-Madras scientists from the department of engineering design has been successful in creating enough red blood cells from stem cells . ( Narayan, 2013) To date, the use of red blood cells (RBCs) produced from stem cells in vitro has not proved for routine transfusion .( Kim, 2014 )

62. CONCLUSION Blood supply demand are increasing as compared to blood donations in the world. Artificial blood is especially useful in circumstances when donor RBC units are unavailable or when transfusion of real blood is not an acceptable option . Two distinctly different classes of oxygen carriers are being developed, each capable of transporting and delivering oxygen to peripheral tissues.

63. Most of the initial attempts at synthesizing blood substitutes were not favorable because of significant adverse effects. However , Considering the need, there are several companies still working on the production of a safe and effective artificial blood substitute. Though , there are many challenges in this aspect, advancing science and technology may result in development of better blood substitutes in future for overcoming the need for biological blood transfusions in the operative and trauma settings.

64. references Sabiston textbook of surgery Bailey & love short practice of surgery Journals