A Post-graduate Credit Seminar On ARTIFICIAL BLOOD Speaker: Patel Mehul D. Reg. No. 04–VETMA–01194–2013 Course No. : VPY-691 Major Advisor Dr. A. Lateef Minor Advisor Dr. B.S. Chandel DEPARTMENT OF PHYSIOLOGY AND BIOCHEMISTRY
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. 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 INTRODUCTION
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. 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. 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. 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. 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. 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. 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. 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. 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. 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. 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. 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. 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. 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. 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. 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. 21 HISTORY OF ARTIFICIAL BLOOD
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. 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. 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. 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. 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. 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. 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. Perfluorocarbons 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. 31 Fluosol-DATM OxygentTM PerftoranTM
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. 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. Structure of PFC 34(Tao and Ghoroghchian, 2014)
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. 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. 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. 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. 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. 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. Hemoglobin-based oxygen carriers 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. 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. 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. HBOCs In search for an alternative to PFCs, considerable
efforts have been made in the development of acellular Hb based
oxygen carriers 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. 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. 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. (Mohankrishna et al., 2011)50 Stabilization of stroma free
Hb
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. 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. 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. 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. 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. 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. 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. 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. 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. Hemopure and oxyglobin 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. 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. 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. 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. 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. 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. 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. 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. 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. 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