This document discusses blood substitutes and their development. It covers: - The functions of blood and the challenges in meeting demand for blood transfusions. - The types of blood substitutes including plasma expanders and red blood cell substitutes like hemoglobin-based oxygen carriers. - The ideal properties of blood substitutes and examples of products in development or approved, including challenges faced. - Perfluorocarbon-based products and hemoglobin-based products, discussing sources, modifications made and examples of some products.

1. BLOOD SUBSTITUTES
IN THERAPY
Dr Shahid A Saache
Dept. of Pharmacology,
BJ GMC, Pune

2. Composition of blood

3. Major functions of blood
Respiration
Nutrition
Excretion
Coagulation
Maintenance of normal
acid base balance in the
body
Regulation of body
temperature by the
distribution of body heat
Regulation of water
balance through the
effects of blood on the
exchange of water
between the circulating
fluid and the tissue fluid
Defence against infection
Transport of hormones
and regulation of
metabolism
Transport of metabolites

4. A blood substitute is a substance used to mimic and fulfil
some functions of biological blood.
It aims to provide an alternative to blood transfusion,
which is transferring blood or blood-based products from
one person into another.

6. The Challenge!

7. The Challenge
• 38,000
The number of blood donations needed by India every day!
• 4 Crore
No. of units- every year our nation requires out of which
only a meagre 40 Lakh units of blood are available
• 1 million
New people are diagnosed with cancer each year
• 4
% people in the eligible population of India donates blood

8. Need for blood substitute
Shortage: A very important limitation especially during
disasters, wars, emergencies, when it is needed most.
Short shelf life: This leads to a large amount of wastage of
blood.
Rare blood groups: This can be a problem, especially in under
populated areas or in a situation where there is a large
demand for blood.
Immunological incompatibility.
Disease transmission: HIV, HBV, HCV to name some of the
most common pathogens.

9. Historical aspects

10. 1667 - Jean-Baptiste Denys performed the 1st documented
blood transfusion on a 15 yrs. anemic boy with sheep
blood.
pt. survived, but subsequent transfusions with sheep blood
failed
1900’s – WWII and Vietnam war ignited the search for
blood substitutes - hemoglobin solutions and synthetic O2
carriers
1980’s – the search for alternative blood substitutes further
fueled by the discovery of HIV and Hep C being transmitted
through transfusions

11. Ideal blood substitute
Oxygen carrying capacity, equalling 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

12. Types of blood substitute:
Can serve as
– Plasma volume expanders or
– Replicate the oxygen carrying function of natural blood
1. Plasma Expanders: These are compounds, which are either
entirely synthetic or processed from natural proteins that serve
as infusion solutions which expand intravascular volume.
2. RBC Substitutes:
Modified Haemoglobins - these are essentially human
haemoglobins extracted from outdated blood.
Perflurocarbons - Synthetic organic compounds that can take
over perfusion

13. Plasma Expanders:
Crystalloids:
normal saline, dextrose and
ringer lactate.
Can serve as volume
substitutes and maintain
plasma osmolarity.
Colloid:
plasma expanders
encompass a wide range of
substances.
These compounds hold
water and actually expand
volume over the amount
infused.

14. Gelatin:
These are modified gelatin polymers, which were among the
earliest used colloid expanders, but are now being phased out
due to an inherent risk of anaphylaxis.
Dextran:
This is a newer generation of colloid expander which uniquely
not only expands plasma volume, but also serves to decrease
blood viscosity, thereby improving perfusion.

15. Hydroxyethyl Starch (HES):
This is a polymerized form of plant starch which is structurally
similar to glycogen and is therefore the safest of the plasma
expanders.
Albumins and Purified Protein Derivatives:
These are prepared by fractionating albumin and other proteins
from pooled human blood and then sterilizing and processing
the albumin so that it is iso-osmolar with plasma. These carry the
greatest risk of anaphylaxis.

16. Uses of plasma expanders
Both crystalloids and colloids have a specific role in overall
management of hypovolemia & are life saving products for
acute emergency situations requiring resuscitation.
They are usually used prior to blood transfusion, because
no processing or testing of the patient is required for
transfusion of expanders and hence can be infused
immediately.

17. Future directions:
Dextrans are currently enjoying a large share of research
focus due to potential applications, related to their ability
to coat blood cells and prevent thrombus formation.
This makes them particularly useful in vascular surgery,
such as intravascular stent placement or surgery for DVT.
Also, coating of erythrocytes causes mutual repulsion
between erythrocytes, and this improves
microcirculation.
This property has led to investigations of a potential role
for dextrans in plastic surgery to improve perfusion and
thereby hasten healing of skin grafts.

18. Problems
Anaplylaxis: This remains the chief drawback with plasma
expanders. It is particularly prevalent with gelatins and albumins
while HES and Dextrans have a very low reported incidence.
Disease transmission: The incidence of transmission of disease is
negligible when compared with blood, but cases have been
reported.
Nephrotoxicity: It is reported especially with dextrans.
Volume overload and electrolyte imbalance: Can be prevented by
careful titration of transfused amount and monitoring.

19. Chemicals……..

20. PFC based blood substitutesFC)
PFC are biologically inert
materials that can dissolve
about 50 times more oxygen
than blood plasma.
They are relatively inexpensive
to produce and can be made
devoid of any biological
materials.
Emulsion particles are 0.2
micron in diameter → Can
perfuse smallest capillaries,
where no RBC flow.

21. Not soluble in water, which means to get them to work
they must be combined with emulsions.
Now a day’s most of the PFBOCs are mixtures of
perfluorocarbons with emulsifying agent
They have the ability to carry much less oxygen than
haemoglobin based products.
This means that significantly more PFC must be used.
One product of this type has been approved for use by
FDA, but it has not been commercially successful because
the amount needed to provide a benefit is too high.

23. 0
1
2
3
4
5
6
7
100 200 300 400 500 600 700
PFC 2.4g/dl
PFC 1.6g/dl
Plasma dissolved O2
PaO2 mmHg
O2Contentml/100ml Dissolved Oxygen Content in Blood

24. Advantages of PFC emulsions
Do not react with oxygen.
Inexpensive
Allow easy transportation of the oxygen to the body.
They allow increased solubility of oxygen in plasma.
minimize the effects of factors like pH and temperature in
blood circulation.

25. Disadvantages of perfluorocarbons (PFC)
emulsions
Often causes flu-like symptoms.
Unable to remain mixed as aqueous solutions – thus, they
must be prepared as emulsions for use in patients.
A decrease in blood platelet count.
PFC products cannot be used by the human body, and
must be discarded.

26. Hemoglobin-based products
PFCs absorb oxygen passively, patients must breathe at a
linear rate to ensure oxygenation of tissues.
The problem with Fluosal-DA was that they dissolve less
oxygen than pure liquids.

27. Examples of PFC based products
Oxygent Currently approved for Phase II Trials in US and Europe,
developed to ↓ need for donor blood during surgery.
Done well overall in most clinical trials, but recently, a
cardiac surgery study found participants to be slightly
more likely to suffer if treated with Oxygent rather than
by standard care.
Oxycyte Currently approved for Phase II-b Trials in the US.
Targeted as an oxygen therapeutic, with successful
small scale open label human trials treating traumatic
brain injury.
PHER-O2 In research

28. Perftoran Approved in Russian and Mexico. Distributed by KEM
Laboratory (Mexico).
Its infusion alleviates symptoms of ischemia at different
types of occlusion vessels disease, improves grafting in
plastic surgery, diminishes inflammation and prevents
rejection of transplants, inhibits retro-virus infection
development.
Local Perftoran applications are able to accelerate wounds
and ulcers healing.
Fluosol-
DA
withdrawn in 1994 due to usage complexity, limited
clinical benefit and complications
SMACC Gold 14

29. Carriers……..
O2

30. Hemoglobin Based Oxygen Carriers

31. Haemoglobin A
Tetrameric structure
α2β2
4 subunits
Heme moiety +
polypeptide
SMACC Gold 14

32. Haemoglobin Based
Oxygen Carrier

33. Sources of Haemoglobin

34. Oxygen Dissociation Curves
0
20
40
60
80
100
120
0 10 20 30 40 50 60 70 80 90 100 110 120 130 140 150
Partial Pressure of Oxygen PaO2 (mmHg)
OxygenSaturation(%)
RBC Hb
Stroma Free Hb0
HBOC
Hemospan P50 = 10
Hemolink P50 = 34
Hemopure P50 = 36
PolyHeme P50 = 26

35. Advantages of HBOCBOCS)-
Available in much larger quantities.
Can be stored for long durations.
Can be administered rapidly without typing or cross
matching blood types.
Can be sterilized via pasteurization.

36. Disadvantages of HBOCRS (HBOCS)
Short half-life
Disrupts certain physiological structures, especially the
gastrointestinal tract and normal red blood cell
haemoglobin.
They release free radicals into the body
Availability and cost

37. SMACC Gold 14
Nanobiotechnology

38. α α
β β
Haemoglobin Modifications
Antioxidant
Enzymes
Encapsulation

39. SMACC Gold 14

41. Vasoconstriction
Nitric oxide scavenging
Molecular size
Endothelin
Viscosity
Oxygen affinity

43. Other Concerns
SMACC Gold 14
Free radical
generation
Iron
deposition
macrophage
function
Antigenicity

44. Examples of HBOCs
Hemopure
Polymerised
bovine Hb
Currently approved for Phase III trials in US and was more
widely approved in South Africa. Many safety measures
are taken to render free of pathogens, including herd
control and monitoring.
Oxyglobin Currently approved for veterinary use in US & Europe.
Oxyglobin solution is the first & only oxygen
therapeutic to be both US FDA and European Commission
approved for veterinary use. Oxyglobin has been used
primarily for blood transfusions and for treatment of
anaemia in dogs.

45. PolyHeme
Polymerised
pyridoxilated
HB
Unique human HBOC in development for the treatment
of urgent, large volume blood loss in trauma and surgical
settings. It is the only blood substitute that has
completed a Phase III trial. Also, free haemoglobin
can be taken up by the kidney, causing dysfunction and
failure, similar to a hemolytic transfusion reaction
Optro
Cross-linked
HB from
genetically
modified E.
coli
Phase II trials completed.

46. Hemospan
Conjugated
human Hb
Hemospan is currently in Phase II trials in the US. It is
produced in powder form, which can then be mixed
into liquid form and transfused immediately, regardless
of a patient’s blood type. This technology relies on
coupling with polyethylene glycol (PEG) to eliminate
the toxicity associated with free haemoglobin.
Hemolink
Cross-linked
human Hb
Phase 3 trials completed. Abandoned due to cardiac
toxicity.

47. Safety of HBOCs
1980 – 2008
70 trials
16 trials
5 products
n =3711

50. Lumbricus terrestris (earthworm) erythrocruorin (LtEc)
A naturally occurring extracellular
Hb with high molecular weight
(3.6 MDa), low autoxidation rate,
and limited NO dioxygenation
activity.
These properties make LtEc a
potential candidate for use as RBC
substitute
Can be safely transfused into
mice, rats, and hamsters without
eliciting major side effects.

51. Polyhemoglobin-Fibrinogen (polyHb-Fg)
A novel blood substitute that is an oxygen carrier with
platelet-like activity.
Formed by crosslinking fibrinogen to hemoglobin to form
polyhemoglobin-fibrinogen (polyHb-Fg).
In the in vitro experiments, PolyHb-Fg showed similar
clotting times as whole blood, whereas polyHb showed
significantly higher clotting times.

52. Hemopure, as with any oxygen carrying
therapeutic, carries a risk of being used
improperly as a performance
enhancement in sports.
on July 20, 2007, Michael
Rasmussen was accused by mountain
bike racer Whitney Richards of
attempted Hemopure smuggling for
potential later use in a doping
program.

53. Platelet substitutes
Infusible platelet membranes
Red cells with surface-bound fibrinogen
Liposome-based agents
Thrombosphere

54. Blood products grown from stem cells
RBCs may be grown either from embryonic stem cells or
hematopoietic progenitor cells.
in 2008, Lu et al reported success using embryonic stem
cells to grow mature erythrocytes on a large scale.
However, the cells produced show a phenotype more
consistent with fetal or embryonic erythrocytes than
adult blood cells.
Further research will be required to increase yields,
eliminate potentially tumorigenic nucleated cells, and
determine the half-life and immunogenicity of
erythrocytes grown from stem cells.

55. Other potential techniques
Dendrimers
Compromises of fluorocarbon
and hydrophilic moieties.
Compatibility with plasma is
due to discrete well‐defined
globular shapes, flexibility,
chemical stability low
cytotoxicity and hydrophilicity
of exterior makes it a major
step forward in the field of
blood substitutes e.g. Poly
amidoamino dendrimers
(PAMAM).

56. Biodegradable micelles
To enhance circulation times,
recombinant or polymerized
haemoglobin can be encapsulated
within micellar-forming
amphiphilic block copolymers
The hydrophobic core of polymer
micelle is able to solubilize the
similarly hydrophobic Hb protein,
while the water soluble corona
(polyethylene glycol) provides a
steric barrier to protein
absorption, and provides
protection from clearance by RES.

57. Placental umbilical cord blood
Cord blood collected aseptically from
the placenta after the birth of a healthy
baby can be used safely as a blood
substitute.
higher haemoglobin content and
growth factors than normal blood from
an adult, which has the potential to
benefit patients in varying diseases.
Respirocytes
Are hypothetical, microscopic, artificial
red blood cells that can emulate the
function of its organic counterpart with
increased efficiency.

58. A team of Romanian researchers
announced that they discovered a colorless
substance that can replace blood. The
substance is based on hemerythrin
,extracted from sea worms and it was
tested on mice with encouraging results.
October 31, 2013
1. Not featuring a superoxide ligand (unlike Hb), which
drastically reduces the reactivity towards NO, and
2. Exhibiting stability towards peroxide (unlike Hb).

60. The more you sweat in peace, the
less you bleed in war.
--Norman Schwarzkopf