Blood Substitutes

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Developing a safe blood substitute has been a goal of medical
researchers for decades, promoted by the traumas of both
World Wars, as well as more recent wars in Asia and the Middle
East. Further efforts for development of blood substitutes are
mainly due to blood-borne infections, especially hepatitis B and
C, and HIV.
The two major classes of oxygen-carrying blood substitutes2
studied are the haemoglobin-based oxygen carriers and per-
fluorocarbon emulsions.
Haemoglobin-Based Oxygen Carriers (HBOCs)
HBOCs are solutions that contain haemoglobin from purified.
human, animal, or recombinant sources. HBOCs prepared from
various sources have been investigated; one such product is
available for veterinary use (bovine derived), and another for
human use in South Africa (bovine derived). While no HBOC
is anticipated to replace allogenic blood, a safe HBOC would
facilitate haemodynamic stabilization until blood is available,
and do so without concern for infectious agent transmission
or transfusion reactions. HBOCs benefit when blood is in short
supply or unavailable.
Haemoglobin provides oxygen-carrying capacity, transpor-
tation, and the modulation of other biochemical processes. The
haemoglobin normally contained in red blood cells (RBCs) is a
tetramer of two alpha and two beta polypeptide chains that
are bound to a central protoporphyrin ring. When the natural
haemoglobin molecule escapes RBCs, it rapidly dissociates into
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dimers composed of an alpha and a beta subunit that does not
transport oxygen well. Thus, it is only inside RBCs that the iron-
containing protoporphyrin ring (heme group) binds one oxygen
molecule causing conformational changes that further increases
the affinity of haemoglobin for added oxygen. This variable
affinity for oxygen is the basis of the oxygen-haemoglobin disso-
ciation curve, and alterations in oxygen delivery are modulated
by temperature, pH changes, or 2,3-diphosphoglycerate.
Haemoglobin solutions have a lower P50 (higher affinity)
of 12–14 mm Hg compared with normal values for RBCs of
~27 mm Hg, which compromises the liberation of oxygen to
the periphery. For therapeutic use, purified haemoglobins have
undergone multiple chemical modifications, including cross-
linking, conjugation and polymerization to other compounds
to change their physiochemical characteristics.
Polymerised haemoglobin (PolyHb), is haemoglobin pro-
duct cross-linked and polymerized with glutaraldehyde and
o-raffinose/pyridoxol molecules to increase its ability to deliver
oxygen and increase its duration of action in circulation. The
limited duration of efficacy is due to increased clearance of free
haemoglobin in circulation, and auto-oxidation to methaemo-
globin. PolyHb is still in trial phase.
Diaspirin, a product produced by US Army, is a cross-linked
haemoglobin (DCLHb). The cross-linking agent is bis (dibromo-
salicyl) fumarate (DBBF). This has shelf life of 9 months,
however, has side effects like intense vasoconstriction, reduced
cardiac output and increased vascular resistance. Because of
the adverse effects the production has been discontinued.
Although HBOCs cannot replace allogeneic red blood cells
completely, they may be used in special circumstances,
including instances of life-threatening haemorrhage, or when
allogeneic RBCs are not available. However, defining the
studies to allow these to be approved by regulatory agencies is
exceedingly difficult. One of the major problems in using
HBOCs in shock states is that free haemoglobin in solution
avidly binds nitric oxide, and thus may impair regional auto-
regulation of blood flow in major organ systems, causing
vasoconstriction and hypoperfusion. In preclinical and animal

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Essentials of Blood Banking and Transfusion Medicine
models, free haemoglobin is an important mechanism respon-
sible for organ injury.
Allogeneic RBCs and HBOCs are not equivalent; RBCs are
more effective oxygen-carrying agents. Although studies
demonstrate that HBOCs reduce allogeneic RBC transfusions
in elective surgical procedures, several units are required to
replace one unit of allogeneic blood. HBOCs have different side
effects; most commonly related to nitric oxide scavenging, their
side effect profile is a difficult problem. Given the widespread
civilian availability of allogeneic RBCs and the appearance of
excess mortality with HBOCs, many trials have been held,
suspended, or abandoned. There is no current FDA approval
for these agents.
Perfluorocarbon Emulsions (PFCs)
PFCs are a class of hydrocarbons produced by substitution of
single hydrogen of each carbon atom of benzene ring with
fluoride radical in the selected hydrocarbon base and have the
following properties.
1. These are chemically inert molecules, clear and colorless liquids.
2. They can dissolve many gases including O2 and CO2 gases.
3. They carry these gases all over the body.
4. They are not miscible and need to be emulsified for intra-
venous administration.
5. Their ability to carry O2 is directly proportion to their concen-
tration in blood, importantly to the partial pressure of oxygen
in blood.
6. These PFCs can flow in the microcirculation, can perfuse
even the tiniest capillary and augment oxygen delivery,
much more than what would be expected from an increase
in oxygen content alone.
7. All the oxygen carried by these PFCs is in a dissolved state,
resulting in a higher oxygen partial pressure, thus augment-
ing the driving pressure for diffusion of dissolved oxygen
into the tissues.
PFCs have great value in treatment of myocardial infarction,
thrombosis, atherosclerosis and vasculo-occlusive diseases
carrying oxygen beyond the occluded area.

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The first generation PFC is Fluosol (Green Cross) uses polo-
xamer type Pleuronic F-68 as an emulsifier. However, this
emulsifier can produce anaphylaxis and complement activation.
The second generation PFC is Oxygent, uses egg yolk phospho-
lipids as an emulsifier which is well tolerated and also contains
a stabilizer perflubrodec (Perfluorodecyl bromide-C10F21Br).
Recombinant Haemoglobin
This is cross-linked haemoglobin genetically produced in
organisms like E. coli or saccharomyces cerevisae yeast. This
also has adverse effects like vasoconstriction, GI distress, fever,
chills and backache, hence trials have been discontinued.
Role of Nanotechnology
Nanotechnology can be used to develop artificial haemoglobin.
Cross-linking haemoglobin into PolyHb of nano dimensional
structure, artificial cells can be prepared in the form of lipid vesicles
containing haemoglobin or haemoglobin containing bio-
degradable membrane. This technology is safe, does not cause
complement activation or adverse effects, but still in trial phase.
Artificial RBCs
Development of synthetic haemoglobin and combining with
functional enzymes similar to natural RBCs has been tried.
These RBCs have short life and an attempt to prepare artificial
RBCs is not successful.
Lipid Membrane Artificial Cells
Incorporation of polyethylene glycol (PEG) into lipid mem-
brane of artificial RBCs has increased circulation half time of
more than 30 hours. Biodegradable polymers, such as polylactic
acid (PLA) for the microencapsulation of Hb, enzymes and
other materials have been used in place of lipid membrane.
Haemoglobin nanocapsule prepared with PLA can survive for
longer time in circulation. Enzymes such as methaemoglobin
reductase have been incorporated into the membrane. This
membrane also allows ascorbic acid to pass inside the cell.
These efforts are to prevent methaemoglobin formation and
longer survival of the cells.