1. 1. A Post-graduate Credit Seminar On ARTIFICIAL BLOOD Speaker: Patel Mehul D. Reg. No. 04VETMA011942013 Course No. : VPY-691 Major Advisor Dr. A. Lateef Minor Advisor Dr. B.S. Chandel DEPARTMENT OF PHYSIOLOGY AND BIOCHEMISTRY
2. 2. CONTENTS Introduction History Types : i. Perfluorocarbons ii. Hemoglobin-based Products Advantage of Artificial Blood Other Promising Technique Artificial Blood Controversy Future Scope Conclusion
3. 3. 3 INTRODUCTION
4. 4. Blood is circulated in the body through blood vessels by the pumping action of the heart. With lungs, arterial blood carries oxygen from inhaled air to the tissues of the body, and venous blood carries carbon dioxide, a waste product of metabolism produced by cells, from the tissues to the lungs to be exhaled. WHOLE BLOOD RED BLOOD CELLS or Erythrocytes (93%) WHITE BLOOD CELLS or Leucocytes (0.16%) AGRANULOCYTS Lymphocytes- 20-40% of white cells MonocyteS 1-10% white cells GRANULOCYTES Neutrophils 50-70% of white cells Eosinophils ~ 3% of white cells Basophils ~ 1% of white cells SOLID FRACTION (Cells-45%) PLATELETS or Thrombocytes (6-7%) FLUID FRACTION (Plasma-55%) 5 What is blood?
5. 5. Blood has many functions : Transportation Regulation of Body temperature and pH Protection against blood loss through clotting Protection against diseases through phagocytic white blood cells and antibodies 6
6. 6. Blood is the life (Riess, 2001) When a patient looses too much blood due to trauma or surgery, the blood volume and RBCs will be reduced In such an emergency situation, doctors will often give patients volume expanders to make up for lost blood volume. This helps to restore normal blood pressure and lets the remaining red blood cells continue to carry oxygen. If it is not enough, doctors can give patients blood transfusions to replace some of the lost blood. 7 Clinical perspective of blood transfusion?
7. 7. Do animals need blood transfusion? Animals need blood transfusions for the same reasons that humans do, including surgery, trauma and diseases. (http://k9bloodbank.com) 8 Usually, the need for blood transfusions is acute, as in acute hemorrhage Transfusions are also appropriate in treatment of acute or chronic anemias. Animals with hemostatic disorders often require repeated transfusions (Cotter, 2013)
8. 8. What are blood types? Another protein, an antigen, may be found on some red blood cells - called Rh factor. Blood cells that have Rh factor are RhD positive, those that don't are RhD negative. 9
9. 9. Animals Blood types Dogs DEA 1.1, 1.2, 3, 4, 7, and Dal. (DEA system=Dog Erythrocyte antigen system) DEA 1.1 negative= Universal Donor DEA 1.1 positive=Universal Recipient (Wardrop, 2007) Cats The main blood group system in cats is the A-B grouping. Cats can be either type A, type B or type AB. (http://www.catblooddonors.com/for-cat-owners/about-cat-blood- transfusions.php) Cattle The polymorphic systems in cattle include the A, B, C, F, J, L, M, S, and Z polymorphisms. (http://en.wikipedia.org/wiki/Blood_type_non-human) Horses There are eight major recognized blood groups in horses Viz., A, C, D, K, P, Q, U and T. ( http://en.wikipedia.org/wiki/Blood_type_non-human) Blood types in animals
10. 10. When transfusion is required, cross matching should be performed or a universal donor may be used. Cross-match should be performed in the following situations: 1) Naturally occurring antibodies to foreign blood group antigens. This occurs in the cat. In this species, a cross-match should be performed on the first and every transfusion Type A cats should receive only type A blood and type B cats should receive only type B blood. However type AB cats can receive either Type A or Type B blood with minimal or no clinical reactions (Henset, 2011) 11
11. 11. 2) Sensitization of an animal to foreign red cell antigens, in a species without naturally occurring antibodies. This is the situation in the dog and horse, resulting in the production of acquired antibodies. In these species, a cross-match does not need to be performed on the first transfusion the animal receives but should be performed at subsequent transfusions. Also in cow, buffalo, sheep, goat and pig, a single, unmatched whole blood transfusions are generally safe (Weiss and Wardrop, 2010) 12
12. 12. 13 Blood collection : The donor cow restrained in a head bail and given a small injection of sedation if needed. Blood collection takes around 20-30 minutes. Blood transfusion : The recipient cow restrained by halter (and in a head bail if possible). Blood transfusion takes around 20 minutes. http://www.franklinvets.co.nz/Dairy++BeefSheep/Farm+Services/Animal+Health/theileria.html
13. 13. Blood transfusions can be critical, life-saving procedures. Blood needs to be readily availableand that requires a blood bank. Do animas have blood bank ? 14
14. 14. India's 1st government blood bank for dogs started in Chennai by the Tamil Nadu Veterinary and Animal Sciences University (TANUVAS) in 2010. (http://timesofindia.indiatimes.com/india/Indias-1st-blood-bank-for-dogs- opens-in-TN/articleshow/5861905.cms) 15 Even in veterinary field, there are several blood banks which store animal blood for future transfusion. Animal Blood Bank
15. 15. 16 Volunteer donor dog during a donation: The dog lies comfortably on his side and a needle is placed in his jugular vein to extract the blood. The procedure takes about 15 to 20 minutes in most dogs.
16. 16. Drawbacks: According to Riess (2001) blood transfusion carries and will always carry a certain level of risk. Blood has to be kept cool (4 C ), and it has a shelf life of 42 days (Lockwood et al.,2003) Prolonged RBC storage before transfusion increases multiorgan failure and mortality in patients (Eldad et al., 2010) The effects of prolonged storage on red cells include decreased deformability; depletion of 2,3-diphosphoglycerate (2,3-DPG); increased adhesiveness and aggregability; reduction in the concentrations of nitric oxide and adenosine triphosphate etc (Koch et al., 2008) 17
17. 17. Blood supply shortages : A country needs a minimum stock of blood equal to 1 per cent of its population. The total recorded blood collection in India is 4 million units, which meet only 40% of need against a least requirement of 10 million units. (World Health Organization, 2008) Safety of blood supply and risk of infections Risk of transmission of HIV, Hep B, Hep C, and other blood borne diseases Mistransfusions due to error Blood Group matching required. (Scott et al., 1997) Cost Donations, screening, storage, and administration ~ 800-1200 Rs. (Maiti, 2012) 18
18. 18. The term artificial blood is really a misnomer. The complexity of blood is far too great to allow for absolute duplication in a laboratory. (Kresie, 2001) Artificial blood is a product made to act as a substitute for blood for the transportation of oxygen throughout the body. (Shalini, 2012) It is also called as Blood Substitute or Arificial Oxygen Carrier. 19 This is where artificial blood comes in
19. 19. The most promising artificial blood products are 1. Perflourocarbons (PFCs) and 2. Haemoglobin based oxygen carriers (HBOCs). (Mohankrishna et al., 2011) The delivery of oxygen by oxygen carriers of the these two classes of have both benefits and risks which are unique to their class. (Tremper et al., 1980) 20
20. 20. 21 HISTORY OF ARTIFICIAL BLOOD
21. 21. In 1616, when William Harvey first described the circulation of blood In 1665, the first recorded successful blood transfusion on dog by Richard Lower 22 (http:// www.redcrossblood.org) History
22. 22. Many materials used for transfusion that include beer, urine, milk, plant resins, and sheep blood. In 1854, patients were injected with milk to treat Asiatic cholera. 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) 23 Contd...
23. 23. In 1909, Karl Landsteiner classified human blood into four different groups: A, B, AB, and O. A fourth group AB was discovered the following year. (Squires, 2002) Karl Landsteiner 24 Contd...
24. 24. During World War I, galactoso- gluconic acid was used to extend plasma. World War II, human plasma was used to replace blood and to save soldiers from hemorrhagic shock. Eventually, this led to the establishment of blood banks by the American Red Cross in 1947. (Kirschman and Ruth , 2005) 25 Contd...
25. 25. 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) However, the established blood bank system in developed countries worked so well that research on blood substitutes waned in those countries. It received renewed interest when the shortcomings of the blood bank system were discovered during the Vietnam conflict. This prompted some researchers to begin looking for hemoglobin solutions (Jani et al., 2012) 26 Contd...
26. 26. 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 Minimal side effects Survivability over a wider range of storage temperatures Long shelf life Cost efficient (Squires, 2002) 27
27. 27. 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 28(Jani et al.,2012)
28. 28. Perfluorocarbons 29
29. 29. Perfluorocarbons (PFCs) Peruorocarbons are low-molecular-weight linear or cyclic hydrocarbons in which hydrogen atoms of the carbon chain have been substituted by uorine atom, leading to total chemical inertness and a complete lack of metabolism in vivo. (Jahr et al., 2007) 1st Generation Fluosol-DATM 2nd Generation OxyfluorTM OxygentTM 30 3rd Generation PerftoranTM PHER-O2 TM
30. 30. 31 Fluosol-DATM OxygentTM PerftoranTM
31. 31. The PFC particles are about 0.2 microns in diameter (1/40th of RBC size), with a perfluorocarbon core and a thin lecithin phospholipids as a coating (Mohankrishna et al., 2011) The perfluorocarbons are not hydrosoluble and administered as emulsions called perfluorocarbon emulsions (PFCEs) (Squires, 2002) 32
32. 32. Perfluorocarbons do not have the oxygen-bonding properties but act as simple solvents. The transport and liberation of gases based on their physical solubility, and the quantity of gas dissolved linearly related to its partial pressure. (Jahr et al., 2007) They Can dissolve more oxygen than the biological blood. (Cabrales and Intaglietta, 2013) They carry much less oxygen than hemoglobin-based products (Jani et al., 2012) 33 Physiology of carrying oxygen
33. 33. Structure of PFC 34(Tao and Ghoroghchian, 2014)
34. 34. Ingredients Quantity (%) Perfluoro-octyl bromide 28 FO-9982 12 Yolk lecithin 2.4 DSPE-50 H 0.12 Distilled water 57.48 Table 1. Composition of PFC based blood substitute (Mitsuhiro et al., 2005) 35 Where, DSPE= Distearoyl phosphatidyl ethanolamine (ammonium salt) FO =Perfluoroalcohol esters with oleic acid
35. 35. The first generation of PFCEs developed was Fluosol-DATM in 1989. Fluosol-DATM was approved by US FDA in human, but withdrawn later because of marginal benefits and development of flu-like symptoms (Castro and Briceno, 2010) The second generation of PFCEs developed were OxyfluorTM and OxygentTM, with improved lipophilicity (Modery et al., 2013) 36 Developments in PFCEs
36. 36. However, OxyfluorTM was terminated after early clinical trials due to its severe side effects OxygentTM was also terminated, because of an increased incidence of stroke in coronary bypass patients The third generation PFCEs are PerftoranTM and PHER-O2 TM Pulmonary complications has been reported with the use of PerftoranTM and, PHER-O2 TM is in reasearch (Modery et al., 2013) 37 Contd
37. 37. Table 2. Current status of PFC based products Name Sponsor Status OxygentTM Alliance pharmaceuticals (USA) Discontinued OxycyteTM Oxygen biotherapeutics (USA) Discontinued PHER-O 2TM Sanguine Corp (USA) In research PerftoranTM PERFTORAN (Russia) Approved in Russian clinical application. 38
38. 38. Cheap and easy to manufacture in large quantities Can be stored at room temperature for more than 1 year Can be mixed safely with any blood group without the need to check first The molecules are smaller than red blood cells, allowing them to bypass arterial blockages and penetrate small capillaries with ease, delivering oxygen to areas which need it most Effect of chemotherapy or radiation in tumour treatment can be enhance when patient pretreated with PFCs (Jani et al., 2012; Singh et al.,2012) Benefits of Perfluorocarbon based products 39
39. 39. Adverse Effects Of PFC Allergy Especially 1st Gen Repeated doses may cause hepatic engorgement Retained in RE system Decrease platelet count Impaired neutrophil function Early: Headache Late: Flu like symptoms (Dietz et al., 1996; Jahr et al., 2007; ) 40
40. 40. Hemoglobin-based oxygen carriers 41
41. 41. Understanding Hemoglobin (Hb) : The structure of Hb was determined in 1959 by Max Perutz. Molecular weight 64.5 kDa Tetrameric protein comprised of two and two - globin subunits that fold into compact quaternary structure ( 2 2). Each and subunit contain an iron-heme group that binds to oxygen molecule allowing for transport. 42
42. 42. 43
43. 43. A Hb molecule carries a maximum of four oxygen molecules. Various factors such as low pH and high CO2 and high 2,3-diphosphoglycerate (DPG) level in the tissues, cause a lower oxygen affinity state facilitating oxygen offloading. As oxygen is being unloaded, CO2 binds to Hb, resulting in carbamino-Hb. 44 Contd
44. 44. Local conditions in the lungs including higher O2, higher pH, and lower 2,3 DPG level, cause Hb to shift back to the higher oxygen affinity state. Such a transition favors CO2 release, which is then exhaled. 45 Contd
45. 45. HBOCs In search for an alternative to PFCs, considerable efforts have been made in the development of acellular Hb based oxygen carriers 46
46. 46. To prepare acellular Hb, Hb is derived from human or bovine blood by chemical modifications or from bacteria host systems by genetic recombinantion (Alayash, 2014) 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 (Lesley, 2001) 47 HBOCs (Hemoglobin-based oxygen carriers) Once obtained from any of these sources, the hemoglobin must be purified and modified to decrease its toxicity and increase its effectiveness
47. 47. The first clinical study with free hemoglobin resulted in nephrotoxicites. The hemoglobin used was found to have erythrocyte membrane stromal lipids as well as bacterial endotoxins. (Shalini, 2012) To side-step these problems, stroma-free hemoglobin was developed, but new problems arose, o 1) too short intravascular half life o 2) too high affinity for oxygen (Jean, 2001) 48
48. 48. 1) Stroma free Hb had too short of an intravascular half life because tetrameric Hb ( 2 2) dissociated into dimmers that were filtered by the kidneys and excreted in the urine. 2) Stroma free Hb had too high of an oxygen affinity because 2,3-DPG was lost during the purification process. (Shalini, 2012) To increase the intravenous half life of hemoglobin solutions, manufacturers had to develop methods to stabilize the hemoglobin tetrameric structure and increase its size. Additional modifications in the hemoglobin, such as pyridoxylation, will create a product with near-normal oxygen-binding affinity. (Lesley, 2001) 49
49. 49. (Mohankrishna et al., 2011)50 Stabilization of stroma free Hb
50. 50. (A,B) Tetrameric stabilization by intramolecular crosslinking between the two or (C) The effective molecular weight of Hb can be increased by conjugating it to polyethylene glycol. (D) Polymerized Hb may be produced through polyfunctional crosslinking agents. E) Hb can also be encapsulated into liposomes 51
51. 51. Intramolecular cross-linking Because the alpha/beta (-) dimers are relatively stable, the goal of intramolecular modification is to cross-link the two alpha (-) or beta (-) subunits and stabilize the association of the two alpha/beta (-) dimers. The popular cross-linkers used are 3,5-dibromosalicyl fumarate (DBBF) and nor-2-formylpyridoxal 5-phosphate (NFPLP). (Mohankrishna et al., 2011) The cross-linking not only prevents tetramer dissociation, but also reduces the affinity of Hb for O2. 52
52. 52. Addition of 2,3 DPG analogs such as pyridoxal-5-phospoate can fix the too high oxygen affinity of stroma-free hemoglobin. The more DPG in the cell the more oxygen delivered to the tissue. The less DPG; the less oxygen delivered to tissues. Pridoxylated stroma-free hemoglobin has nearly normal oxygen affinity (p50 = 22-24 mmHg) (Jean, 2001) E.g., HemopureTM (Hemoglobin Glutamer-250 (bovine) or HBOC 201) 53
53. 53. Polymerized Hemoglobin Polymerization of Hb through intermolecular cross- linking increases the size of molecules through the formation of Hb oligomers. In the process multiple Hb proteins are linked together through the use of dialdehydes, such as glutaraldehyde and glycoaldeyde. (Betts and Whittet, 1962) E.g., PolyHemeTM 54
54. 54. Conjugated Hemoglobin Conjugation of Hb is the binding of Hb to a biocompatible polymer, such as polysaccharide, in order to increase its overall size. In a specific case of pegylation, multiple polyethylene glycol (PEG) chains are added to the Hb protein as a means to increasing the molecule's size. 55(Mohankrishna et al., 2011; Shalini, 2012)
55. 55. 56 Size increases from 3 nm to 15 nm once pegylated Hb conjugation with PEG appears to protect the molecule from renal excretion. Conjugating Hb with a macromolecule extends the intravascular circulation time of a HBOC. E.g. HemospanTM (Shalini, 2012)
56. 56. Hemoglobin Vesicles (Hemoglobin encapsulated vesicles) The encapsulation of Hb is based on the idea of recreating the natural properties of RBC without the presence of blood group antigens Encapsulated Hb is often referred to as hemosome The process of involves the encapsulation of Hb within lipid vesicles using a solution of phospholipids. Lipid membrane allow better diffusion of O2 and Co2 57 (Shalini, 2012)
57. 57. Recombinant Hemoglobin With advances in recombinant DNA technologies, specially modified Hb may be produce from microorganisms, like E. coli Recombinant human hemoglobin was produced in E. coli using an expression vector containing two mutant human globin genes. One was a low oxygen affinity mutant, and the other fused -globins. These recombinant hemoglobin products advanced to clinical trials, but it was stopped due to vasoconstriction and other harmful effects. 58 (Mohankrishna et al., 2012)
58. 58. Table 3. HBOC Products Name Sponsor Description HemopureTM Biopure Corp It is made of chemically stabilized, cross-linked bovine haemoglobin in a salt solution. Hemopure is approved for Phase III trials in the United States and South Africa (Stefan et al., 2007) OxyglobinTM Biopure Corp It consists of chemically stabilized bovine haemoglobin in a balanced salt solution. Oxyglobin is aproved by US FDA and European Commission for veterinary use particularly for routine use in canine anaemia (Jahr et.al.,2007) PolyHemeTM Northfield Laboratories human hemoglobin-based oxygen- carrying blood but discontinued due to adverse effects. HemospanTM Sangart It was produced in powder form, which could then be mixed into liquid form and transfused immediately but discontinued due to adverse effects HemotechTM HemoBiotech Hemotech is currently approved for Phase I trials. 59
59. 59. Hemopure and oxyglobin 60
60. 60. Characteristics Biopures Oxygen Therapeutics Red Blood Cells Storage Room temperature (2o to 30o C) Refrigerated Shelf life 36 months 42 days Preparation Ready to use Testing, typing and crossmatching Compatibility Universal Type specific Effectiveness Immediate oxygen delivery Dependent on length of storage Purity Processed to remove infectious agents Tested and screened for infectious agents Raw material Bovine haemoglobin Human Blood Cost $ 600 - 800 $ 125 - 425 (http://biomed.brown.edu) 61 Table 4. Biopures Oxygen Therapeutics vs. RBCs
61. 61. Table 5. Current developmental status of HBOCs Product type Product name Developer Source and /or technology Status Cross-linked Hb HemAssist TM Baxter (USA) cross-linked human Hb Discontinued Optro (rHb) TM Somatogen (USA) Recombinant Hb Discontinued Polymerized Hb PolyHeme TM Northfield Lab (USA) Glutaraldehyde, pyridoxal human Hb Discontinued Hemopure TM Biopure (USA) Glutaraldehyde bovine Hb Approved * Conjugated Hb Hemospan TM Sangart (USA) Maleimide PEG- human Hb Discontinued PEG-Hb TM Enzon (USA) PEG conjugated bovine Hb Discontinued PHP TM Apex (USA) Polyoxyethylene- conjugated human Hb Discontinued * Approved in South Africa for perioperative anemia; approved in USA and Europe for veterinary use under the name Oxyglobin. (Tao and Ghoroghchian, 2014)62
62. 62. Benefits of HBOCs No prior planning Faster & better O2 distribution Long shelf life No refrigeration Universally compatible Immediately offloads oxygen Ready to use (Mohankrishna et al., 2011; Singh et al., 2012)63
63. 63. Side effects of HBOCs Vasoactivity/ hypertension Gastrointestinal side effects Pancreatic and liver enzyme elevation Antigenicity Cardiac involvement Platelet aggregation Neurotoxicity Renal effects (Cole et al., 1997; Mohankrishna et al., 2011; Singh et al., 2012; Winslow , 2004) 64
64. 64. Advantages of Artificial blood No risk of infection Biological blood transfusion is the second largest source of HIV infections in Nigeria. In certain regions of southern Africa, it is believed that as much as 40% of the population has HIV/AIDS. A disease-free source of blood substitutes would be incredibly beneficial in these region (Shalini, 2012) Can be kept at room temperature and carry a shelf life of more than 1 year Rapid treatment of patients in trauma situations Medical care in the armed services would get benefit from artificial blood 65
65. 65. Artificial blood allows for immediate full capacity oxygen transport (Schimmeyer, 2002) An alternative for those patients that refuse blood transfusions for religious or cultural reasons. (Jani et al., 2012) Synthetic oxygen carriers may also show potential for cancer treatment, as their reduced size allows them to diffuse more effectively through poorly vasculated tumour tissue, increasing the effectiveness of treatments like photodynamic therapy and chemotherapy. (Won, 2005) Transfused blood is currently more cost effective, but there are reasons to believe this may change. For example, the cost of artificial blood may fall as manufacturing becomes refined. 66 Contd
66. 66. 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) 67 Other Promising Technique
67. 67. Artificial blood controversy Doctors abandoned the use of HemAssistTM in the United States, after patients who received the HBOC died more often than those who received donated blood. Haemoglobin-based blood substitutes may increase the odds of deaths and heart attacks. Sometimes, pharmaceutical companies may get trouble in proving that their oxygen carriers are effective, i.e. Northfield Laboratories Blood substitutes may be misused as performance-enhancing drugs. (Shalini, 2012) 68
68. 68. 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. Most of the initial attempts at synthesizing blood substitutes were not favorable because of significant adverse effects. 69 Conclusion
69. 69. 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. 70 Contd
70. 70. Thank you. 71