Artificial Blood: A Current Review

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Mr. Parag A. Kulkarni

Mr. Parag A. Kulkarni

Since the 17th century, blood transfusions have been attempted to
offset blood loss from trauma and childbirth, or as a therapeutic modality during
leeching or bloodletting. Until the identification of isoagglutinating antibodies,
however, transfusions were fraught with significant early complications. These
early complications sparked interest in using hemoglobin as an oxygen carrier
in plasma. Early trials of these solutions proved disastrous as well, with significant
immediate complications resulting from infusions of stroma-free human hemoglobin
solutions.1 Artificial blood is a product made to act as a substitute
for blood for the transportation of oxygen and carbon dioxide throughout the
body. The most promising blood products under development as blood substitutes
are perflourocarbons and haemoglobin based oxygen carriers. PFCs are long chain
compounds similar to Teflon having oxygen carrying capacity. The haemoglobin
based oxygen carrier’s works on haemoglobin’s unique oxygen binding
capacity and the lack of blood type antigen. The delivery of oxygen by the two
distinctly different classes of oxygen carriers has both benefits and risks
which are unique to its class. If all the research that is being put into blood
substitutes and synthetic blood products turns to be a success, then these can
possibly serve as an alternative to eliminate the side effects associated with
blood transfusions.

Introduction

Since the 17th century, blood transfusions
have been attempted to offset blood loss from trauma and childbirth, or as a
therapeutic modality during leeching or bloodletting. Until the identification
of isoagglutinating antibodies, however, transfusions were fraught with
significant early complications. These early complications sparked interest in
using hemoglobin as an oxygen carrier in plasma. Early trials of these
solutions proved disastrous as well, with significant immediate complications
resulting from infusions of stroma-free human hemoglobin solutions.1
These complications were most often acute renal failure thought to be the
result of direct hemoglobin nephrotoxicity.2

Artificial blood is a product made to act as a substitute for red blood cells. While true blood serves many different functions, artificial blood is designed for the sole purpose of
transporting oxygen and carbon dioxide throughout the body. Depending on the
type of artificial blood, it can be produced in different ways using synthetic
production, chemical isolation, or recombinant biochemical technology.
Development of the first blood substitutes dates back to the early 1600s, and
the search for the ideal blood substitute continues. Various manufacturers have
products in clinical trials; however, no truly safe and effective artificial
blood product is currently marketed. It is anticipated that when an artificial
blood product is available, it will have annual sales of over $7.6 billion in
the United States alone.10

Background

Blood is a special type of connective tissue that is composed

Blood is a special type of connective tissue that is composed of

1. White cells:-white cell  are responsible
for the immune defense. They seek out invading organisms or materials and minimize
their effect in the body.

2.Red cells: - red cells create the bright
red color. These cells are responsible for the transportation of oxygen and
carbon dioxide throughout the body

3.Platelets: - platelets are small fragment
of cell that clump together and stick to inner surface of vessels and prevent
leakage.

4.Plasma: - plasma is the extra cellular
material made up of water, salts, and various proteins that, along with
platelets, encourages blood to clot. Proteins in the plasma react with air and
harden to prevent further bleeding.

History

There has been a need for blood replacements for as
long as patients have been bleeding to death because of a serious injury.
According to medical folklore, the ancient Incas were responsible for the first
recorded blood transfusions. No real progress was made in the development of a
blood substitute until 1616, when William Harvey described how blood is
circulated throughout the body. In the years to follow, medical practitioners
tried numerous substances such as beer, urine, milk, plant resins, and sheep
blood as a substitute for blood. They had hoped that changing a person's blood
could have different beneficial effects such as curing diseases or even
changing a personality. The first successful human blood transfusions were done
in 1667. Unfortunately, the practice was halted because patients who received
subsequent transfusions died.

Of the different materials that were tried as blood
substitutes over the years, only a few met with minimal success. Milk was one
of the first of these materials. In 1854, patients were injected with milk to
treat Asiatic cholera. Physicians believed that the milk helped regenerate
white blood cells. In fact, enough of the patients given milk as a blood
substitute seemed to improve that it was concluded to be a safe and legitimate
blood replacement procedure. However, many practitioners remained skeptical so
milk injections never found widespread appeal. It was soon discarded and
forgotten as a blood replacement.

Another potential substitute was salt or saline
solutions. In experiments done on frogs, scientists found that they could keep
frogs alive for some time if they removed all their blood and replaced it with
a saline solution. These results were a little misleading, however, because it
was later determined that frogs could survive for a short time without any
blood circulation at all. After much research, saline was developed as a plasma
volume expander.1

Other materials that were tried during the 1800s include
hemoglobin and animal plasma. In 1868, researchers found that solutions
containing hemoglobin isolated from red blood cells could be used as blood
replacements. In 1871, they also examined the use of animal plasma and blood as
a substitute for human blood. Both of these approaches were hampered by
significant technological problems. First, scientists found it difficult to
isolate a large volume of hemoglobin. Second, animal products contained many
materials that were toxic to humans. Removing these toxins was a challenge
during the nineteenth century2

A significant breakthrough in the development of
artificial blood came in 1883 with the creation of Ringer's solution—a solution
composed of sodium, potassium, and calcium salts. In research using part of a frog's
heart, scientists found that the heart could be kept beating by applying the
solution. Using Ringer’s solution could restore this eventually led to findings
that the reduction in blood pressure caused by a loss of blood volume. This
product evolved into a human product when lactate was added. While it is still
used today as a blood-volume expander, Ringer's solution does not replace the
action of red blood cells so it is not a true blood substitute.3

Using blood samples from his colleagues,! he separated
the blood's cells from its serum, and suspended the red blood cells in a saline
solution. He then mixed each individual's serum with a sample grom every cell
suspension. Clotting occurred in some cares; in others there was no clotting.
Land Steiner determined that human beings could be separated into blood groups
according to the capacity of their red cells to clot in the presence of
different serums. He named his blood classification groups A, B, and O. A
fourth group AB, was discovered the following year. The result of this work was
that patient and donor could be blood-typed beforehand, making blood
transfusion a safe and routine medical practice. This discovery ultimately
earned Landsteiner the 1930 Nobel Prize in physiology or medicine.3

Blood transfusion research did not move forward until
scientists developed a better understanding of the role of blood and the issues
surrounding its function in the body. During World War I, a gum-saline solution
containing galactoso-gluconic acid was used to extend plasma. If the
concentration, pH, and temperature were adjusted, this material could be
designed to match the viscosity of whole blood, allowing physicians to use less
plasma. In the 1920s, studies suggested that this gum solution had some
negative health effects. By the 1930s, the use of this material had
significantly diminished. World War II reignited an interest in the research of
blood and blood substitutes. Plasma donated from humans was commonly 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 1947.1

In 1966, experiments with mice suggested a new type of
blood substitute, perfluorochemicals (PFC). These are long chain polymers
similar to Teflon. It was found that mice could survive even after being
immersed in PFC. This gave scientists the idea to use PFC as a blood thinner.
In 1968, the idea was tested on rats. The rat's blood was completely removed
and replaced with a PFC emulsion. The animals lived for a few hours and
recovered fully after their blood was replaced.6

However, the established blood bank system worked so
well research on blood substitutes waned. 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 and other synthetic oxygen carriers. Research in this area was
further fueled in 1986 when it was discovered that HIV and hepatitis could be
transmitted via blood transfusions. During this era of blood substitute
research in the 1960s, Dr. Leland Clark began experimenting with a class of
compounds known as perfluorocarbons. Oxygen has approximately 100 times greater
solubility in perfluorocarbon solutions than in plasma. As a result, the amount
of oxygen dissolved in plasma may be sufficient to sustain life, without the
need for RBC-contained hemoglobin to provide additional oxygen. The hydrophobic
nature of these compounds necessitated further development of perfluorocarbon
emulsions prior to considering these compounds for use as a plasma oxygen carrier.

The use of Pluronic 64 as an emulsifying agent for
perfluorocarbons enabled the production of Fluosol by the Green Cross
Corporation of Japan. Clinical trials with this perfluorocarbon, however, were
disappointing. Fluosol was present only in low concentrations in the emulsion,
and Pluronic 64 caused rare but significant complications when the emulsion was
infused intravenously. Further development of emulsion technologies resulted in
the production of compounds, which utilized smaller chain perfluorocarbon
molecules to more effectively emulsify the perfluorocarbons, allowing higher
concentrations of active agent in the emulsion and thus higher oxygen carrying
capabilities. The improved stability of the newer emulsions are vastly superior
to the first generations of perfluorocarbons; current emulsions can be stored
at 4°C for extended periods of time (months) without appreciable degradation of
activity.15

Design

Ideal blood substitute

Blood substitutes or synthetic blood are currently
labeled as "oxygen carriers". This is because they are unable to
mimic many of the other functions of blood; they do not contain cells,
antibodies, or coagulation factors. Their main function is to replacing lost
blood volume and oxygen carrying capacity.

The ideal blood substitute could be defined by the
following terms:

  • 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

Advanced Technology

There are two significantly different products that are
under development as blood substitutes. They differ primarily in the way that
they carry oxygen. One is based on PFC, while the other is a hemoglobin-based
product.

1) Perfluorocarbons (PFC)

Perfluorocarbon emulsions

Perfluorocarbon emulsions (PFCE) are one of the two major
classes of oxygen therapeutics currently on
the market. They are composed of liquid perfluorocarbon emulsified in water
and salt. Due to the PFC's inability to remain mixed with aqueous solutions,
they must be prepared as emulsions before being used in patients. The PFCE
particles are spherical, averaging about .2 microns in diameter, with a
perfluorocarbon core and a thin egg yolk phospholipids surfactant as a coating.

PFC is 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. This eliminates
the real possibility of spreading an infectious disease via a blood transfusion.
From a technological standpoint, they have two significant hurdles to overcome
before they can be utilized as artificial blood. First, they are not soluble in
water, which means to get them to work they must be combined with
emulsifiers—fatty compounds called lipids that are able to suspend tiny
particles of perfluorochemicals in the blood. Second, they have the ability to
carry much less oxygen than hemoglobin-based products. This means that
significantly more PFC must be used. The Federal Drug Administration (FDA) has
approved one product of this type for use, but it has not been commercially
successful because the amount needed to provide a benefit is too high. Improved
PFC emulsions are being developed but have yet to reach the market. Perfluorocarbon
Emulsions

In 1989, Fluosol ®, a PFCE produced by Green Cross Corp. of Osaka , Japan ,
became the first of its kind to receive FDA approval. It was approved for use
in coronary balloon angioplasty procedures. Unfortunat
ely, due to a number of problems with the product such as low PFC content (20%/voume)
and the necessity for a labor-intensive preparation among others, Fluosol ®
was discontinued in 1994.16

Osaka

After the initial excitement regarding Fluosol,
subsequent small studies demonstrated no benefit from Fluosol infusions in
patients with profound anemia.4,5 With colloid solutions as a
comparator, Fluosol did not improve indirect measures of oxygenation. However,
Fluosol continued to be available for infusion as an oxygen carrier during
high-risk percutaneous transluminal angioplasty procedures until early 1993,
when the Food and Drug Administration rescinded approval for this indication for
the emulsion.

New emulsions have been developed which utilize
emulsifying agents similar to the primary compound. In particular, perflubron
(perfluorooctyl bromide) has been developed as a stable emulsion safe for
intravenous infusion by the addition of small amounts of perfluorodecyl bromide
as an emulsifying agent; the emulsion is then buffered with egg yolk
phospholipids. The resulting emulsion has a calculated oxygen carrying
capacity, which is approximately three fold the amount of oxygen carrying capacity
of the earlier Fluosol solutions.16

Perflubron oxygen carrying capacity is directly related
to the oxygen partial pressure (Figure 2). In this regard, perflubron oxygen
delivery is predictable; direct diffusion of oxygen is the mechanism by which
oxygen is off-loaded to peripheral tissues. Theoretically, oxygen delivered by
diffusion may be more available, and more readily off-loaded from the
bloodstream, than hemoglobin-delivered oxygen. However, no data have been
produced which support this premise.4

image

image

Figur The oxygen content of
perfluorocarbon emulsions obeys Henry's Law of partial pressures; the amount
of oxygen dissolved in a perfluorocarbon solution is directly related to the
partial pressure of oxygen to which the solution is exposed.

Figure A comparison of the amount
of oxygen dissolved in normal plasma and two clinically achievable plasma
concentrations of perflubron.

Benefits Of Perfluorocarbon Oxygen Transport

Transport of oxygen as soluble gas in plasma is
radically different from hemoglobin-based oxygen transport. Although some
oxygen is normally dissolved in plasma, the amount typically constitutes less
than 1% of the total oxygen content in arterial blood, even with significant
anemia. By contrast, administration of perflubron can increase dissolved oxygen
to approximately 10-15% of the total arterial oxygen content, an increase from
the norm of two to three fold, depending on the partial pressure of oxygen
inspired (Figure 3).

There is evidence to suggest that diffusion of oxygen
does occur, and increased tissue oxygenation is the result. Studies on solid
tumor treatment with either chemotherapy or radiation therapy have demonstrated
enhanced tumor kill ratios when animals are pretreated with perflubron.
Diffusion of oxygen into the hypoxic core of these tumors, thus spurring these
"dormant" hypoxic tumor cells to divide, results in greater
sensitivity of these now dividing tumor cells to antimitotic agents, enhancing
their effectiveness.6 This theory now awaits clinical trials to
evaluate the efficacy of diffusion of oxygen into tissues.4

Problems With Perfluorocarbons

Perfluorocarbons are inert biologically. The molecules
are sequestered in the reticuloendothelial system, particularly in the Kupffer
cells of the liver and macrophages, and subsequently released back into the
plasma as a dissolved gas. The perfluorocarbon gas is then exhaled unchanged
and non-metabolized via the lungs. While previous perfluorocarbons had a
significant amount of retention in the reticuloendothelial system, current
generation perfluorocarbons such as perflubron have a retention time of
approximately one week. This allows effective elimination of perfluorocarbons
from the liver and spleen without the potential for significant organ
dysfunction.

However, despite the inert nature of perfluorocarbons,
sequestration in the reticuloendothelial system may result in subtle
consequences. Platelet count is known to decrease, presumably due to
opsonization of platelets by the perfluorocarbon and subsequent sequestration
and elimination by the reticuloendothelial system. Sufficient perfluoro-carbon
may also overwhelm the reticuloendothelial system, resulting in potential
infectious or other complications; however, this is only a theoretical concern,
as no increase in infectious complications has been noted in early clinical
trials.

The retention of perfluorocarbons does pose an
additional problem with respect to dosage. Perfluorocarbons are relatively
evanescent in the plasma, with a half life of approximately 3-4 hours in the
plasma phase. The reticuloendothelial system, however, has an approximate 3-5
day retention phase prior to exhalation of the perfluorocarbon. Therefore,
although extremely short-lived in the plasma phase, additional dosing of
perfluorocarbons may not be possible for several half-lives of the
tissue-reticuloendothelial terminal elimination, i.e., one to two weeks.
Thus perfluorocarbons become a single dose drug, with limitation of dosage due
to the capacity of the reticuloendothelial system to handle the plasma
elimination phase. At present, this limitation of dosing is theoretical, as no
clinical data exist to discern whether perfluorocarbon redosing results in
serious adverse effects; future studies and newer generation emulsions will
address this issue.

The dependence of perfluorocarbons on Henry's Law of
partial pressures allows the potential for increased oxygen availability. This
fact of oxygen delivery also limits the effective use of perfluorocarbons to
situations when the partial pressure of oxygen is supranormal, i.e.,
when the partial alveolar oxygen tension approaches 400 mmHg or greater.
This is impossible to attain without supplemental oxygen administration; an
effective partial oxygen pressure may be impossible with any maneuvers at
altitude. Even in the presence of supplemental oxygen and controlled
ventilation, patients with significant pulmonary disease may be unable to reach
partial pressures of oxygen to allow perfluorocarbon to function as an
effective oxygen carrier.4

Current Status Of Pfce Based Product

Current PFCE products are referred to as second
generation PFCE's and are marketed as oxygen therapeutics for patients at risk
of acute hypoxia resulting from transient anemia, blood loss or ischemia. They
use different PFC's and surfactants than the previous products. The PFCE
particles travel in the plasma near the vessel walls and between RBC's. The
largest plasma gaps between RBC's exist in the microcirculation so as a result,
PFCE's provide the most benefit to smaller vessels. Also, when there are local
areas of vasoconstriction or blockages of the vessels, some plasma can still
pass through and deliver PFCE's/oxygen to the tissues. Initially, they were
created in order to avoid or reduce the need for blood transfusions in the treatment
of trauma patients but as time went on, they began to be used in
cardiovascular, orthopedic, urologic and other general surgeries.

Alliance Pharmaceutical Corp.

image

Alliance Pharmaceutical Corp., with the help of Johnson and Johnson, is currently
working for FDA approval of Oxygent™. Oxygent™ is a second generation
PFCE with a median particle diameter of .16-.18 microns, an optimal storage
temperature of 2-8 °C, and a PFC content of 60%/voume. Data from numerous studies,
including a European Phase III study of 492 patients investigating the use of
Oxygent™ in general surgery, showed that the product reduced the need
for transfused blood. While a Phase III trial involving CABG in the US was halted
due to high stroke rates in both the experimental and control groups, Alliance
is hoping to initiate further Phase III studies involving general surgery.Alliance
has also patented a procedure called Augmented Acute Normovolemic Hemodilution
(Augmented-ANH), which is a technique that will further decrease the need
for blood transfusions in moderate and high blood loss surgical procedures.
Immediately prior to surgery, several pints of blood are removed from the patient
and Oxygent™ is used to replace the oxygen-carrying capacity of the missing
blood and following surgery, the removed blood is reinfused.

Augmented-ANH

Recently, the European Agency for the Evaluation of Medical Products (EMEA)
recommended that Alliance pursue an indication for Oxygent™ that does
not require direct comparison to allogeneic blood transfusions. Since the incidence
of serious adverse side effects associated with blood transfusions are so low,
it would be difficult to show that Oxygent™ is safe or safer than allogeneic
blood. Also recently, Alliance and its subsidiary PFC Therapeutics, LLC began
working with Double-Crane Pharmaceutical CO., Ltd. And LEO Pharma A/S to develop
and market Oxygent™ in The People's Republic of China (Double-Crane),
Europe and Canada (LEO).12

Synthetic Blood International, Inc.

Synthetic Blood International (SYBD) is currently
marketing its product Oxycyte™. Oxycyte is a second-generation PFCE similar to
Oxygent™ with a mean particle diameter of .19 microns, PFC content of
60%/volume and a typical pH of 7.1. When compared to other PFC products, data
from screening animal studies is providing a good safety profile for Oxycyte™.

On April 18, 2005, SYBD announced that the first two of
sixty patients were enrolled in a Phase II trial for Oxycyte™ in the prevention
of tissue hypoxia in hip revision surgery. Currently, there are four US
clinical sites involved and SYBD hopes to eventually have six. Future Phase II
trials will involve the use of Oxycyte™ in CABG, and heart valve replacement
surgery, among others.

Sanguine Corp.

Sanguine Corp. is working towards the FDA approval of
the second-generation PFCE PHER-02 that was designed to overcome the
shortcomings of its predecessor: Fluosol ®. On April 25, 2005, Sanguine Corp.
announced that it is currently working to determine a final FDA indication for
PHER-02 so that any further research and clinical trials are geared towards the
fastest path to FDA approval.

Perftoran

The Russian based company Perftoran is currently
producing Perftoran, which is not a second-generation PFCE like the previous
products but rather; it is an improved first-generation PFCE. It was registered
in Russia in 1996 and is similar to Fluosol ® except that it uses a different
emulsifier which contributes to its lower incidence of side effects. The
product particles are .07 microns in diameter, allowing them to evade RES
macrophages to an extent and remain in the vasculature longer with fewer side
effects. It has a PFC content of 10%/volume, can be stored at 3 years at -4 ° C
- -18° C and two weeks at 4° C. Within two weeks before infusion, Perftoran
must be thawed at room temperature and subsequently stored at 4° C. Also, it
can be thawed and refrozen up to five times.

By the end of 2000, more than 2000 patients had
participated in clinical studies of Perftoran, 37% of which involved anemia,
hemoraghic and traumatic shock, 19% involved polytrauma and fat embolism, 13%
involved ischemic brain edema and transplantation, and 15% involved acute
ischemia.

2) Hemoglobin-based products

Understanding Hemoglobin

The structure of Hb was determined in 1959 by Max Perutz for which he was awarded
a Nobel Prize. Human Hb is a 64 kDa tetrameric protein comprised of two a subunits
and two ß-globin subunits that fold into compact quaternary structure (a2ß2).

image

Each a and ß subunit contain an iron-heme group that binds to oxygen
molecule allowing for transport. A fully saturated Hb molecule carries a maximum
of four oxygen molecules. Environmental conditions such as pO2, pH,
temperature, and pCO2 cause Hb to undergo conformational change from a high
oxygen affinity state to a lower oxygen affinity state. Such a transition is
also facilitated by the binding of an allosteric effector, 2,3-diphosphoglycerate
(DPG), causing a decrease in Hb oxygen affinity and facilitating oxygen offloading.
As oxygen is being unloaded CO2 binds to the globin chain, resulting in carbamino-Hb,
which is then transported to the lungs. However, only about 20% of the CO2 is
transported in the blood. The rest of the CO2 is transported in the form of
bicarbonates. Local conditions in the lungs including higher pO2, higher
pH, and lower temperature, cause Hb to shift back to the higher oxygen affinity
state and dissociate with DPG. Such a transition favors CO2 release, which is
then exhaled. Although blood transfusion is effective, it is not without risks.
Allogeneic blood transfusion may cause fatal hemolytic reactions, transmit blood-borne
infectious agents, and compromise overall immune function. It is therefore highly
desirable to have an artificial oxygen carrying fluid that is readily available,
free of infectious agents, and can be used independent of the recipient blood
type. The idea of using purified Hb as possible universal substitute for red
blood cells has been around for almost a century due to Hb's unique oxygen binding
property and the lack of blood type antigen. In 1916, Hb was used in human subjects
in an attempt to treat anemia.

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However, such early attempt to use Hb-saline solution within the clinical setting failed due to renal toxicities. It was later determined that early Hb contained erythrocyte membrane stroma
lipids that were contaminated with end toxins, causing sever nephrotoxicity
in patients. Consequently, Hb solutions had to be prepared “free”
of stromal lipids and endotoxin in order to prevent nephrotoxicities. Two other
problems shortly emerged: stroma free Hb had too high an oxygen affinity and
too short of an intravascular half in order to be therapeutically useful. Hb
had too high of an oxygen affinity because 2,3-DPG normally present in erythrocytic
Hb was lost during the purification process. Such a high oxygen affinity did
not result in optimal oxygen off-loading in the tissues. In addition tetrameric
Hb (a 2 ß 2) dissociated into aß dimmers that were filtered by the kidneys and
excreted in the urine. Consequently, the HBOCs being clinically tested today
have been chemically or genetically “engineered” to produce desirable
oxygen offloading characteristics and an extended circulation half time in order
to become a therapeutically useful agent.10

image

Some of the ey approaches of hemoglobin oxygen
carriers as red blood cell substitutes are illustrated above. Once stroma-free
Hb are prepared from human or bovine red blood cells they must be chemically
stabilized in order to become therapeutically useful. (A,B) Tetrameric
stabilization is accomplished by intermolecular crosslinking between the two a
or ß subunits using a site-specific crosslinker. (C) The effective molecular
weight of Hb can be increased by conjugating it to polyethylene glycol. (D)
Polymerized Hb of molecular weights greater than the native Hb tetramer of 64
kDa may be produced through polyfunctional crosslinking agents. (E) Hb can also
be encapsulated into liposomes in order to recreate the natural properties of
red blood cells.10

a) Intramolecular cross-linking:

Preventing the Hb tetramer's dissociation is a major concern in order to suppress
renal filtration. Because the alpha/beta (a-ß) dimers are relatively stable,
the goal of intramolecular modification is to cross-link the two alpha (a-a)
or beta (ß-ß)

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subunits and stabilize the association of the two alpha/beta
(a-ß) dimers. The cross-linking not only prevents tetramer dissociation, but
also reduces the affinity of Hb for O 2 . The most popular cross-linkers currently
used are DBBF and nor-2-formylpyridoxal 5-phosphate (NFPLP).10

b) Polymerization:

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.

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The increase of the size of the oligomers is significant because the molecular weight of the molecule exceeds 500 kDa, compared to a
64.5 kDa unpolymerized Hb tetramers. The increase in size prevents the rapid
excretion of the molecule, prolonging the Hb plasma half-life. Unpolymerized
Hb tetramers have, however, the unfortunate result of generating excessive viscosity,
oncotic pressure, and O 2 affinity. Consequently, it is crucial to obtain high
polymerization yields in the manufacturing process. Otherwise, the unpolymerized
tetramers must be separated as not to create adverse reactions in the patients.
In conclusion, intravascular retention times of HBOCs can be increased by intermolecular
crosslinking of stabilized Hbs using crosslinker with poly-functional groups.10

c) Conjugation:

Conjugation of Hb is the covalent binding of Hb to a biocompatible polymer,
such as polysaccharide, in order to increase its overall size. Such a process
achieves

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similar improvements than those made using polymerization. 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. It
radius, for example, increases from 3 nm to 15 nm once pegylated with 6 PEG
chains. Human Hb conjugation with PEG appears to protect the molecule from renal
excretion. Conjugating Hb with a macromolecule.10 extends the intravascular
circulation time of a HBOC

d) Encapsulation:

The encapsulation of Hb is based on the idea of recreating the natural properties
of RBC without the presence of blood group antigens.

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Encapsulated
Hb is often referred to as “hemosome.” The process of involves the
encapsulation of Hb within lipid vesicles using a solution of phospholipids.
The encapsulation allows engineers to specify membrane properties of the vesicle.
The negatively charged lipids, for example, have demonstrated to limit the aggregation
between hemosomes. The alteration of the bilayer membrane may allow for the
better diffusion of O 2 and CO 2 .10

e) Recombinant Hemoglobin:

Genetic engineering is an alternative to chemically
modifying Hb. With advances in recombinant DNA technologies, specially modified
Hb may be produce from microorganisms, like E. coli or yeast.
Prestabilized recombinant human Hb, for example, was produce in E. coli using
an expression vector that contained tow mutant globin genes; one had a low
oxygen affinity and the other tandemly fused a-globins. Modifications have been
made to increase the tetramer's stability and decrease its affinity for O2.
Future genetic manipulations may also be able to solve other problems such as
the oxidation of Hb into metHb, reaction rate with NO, and short circulation
half-life.7

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Raw Materials

Depending on the type of artificial blood that is made,
various raw materials are used. Hemoglobin-based products can use either
isolated hemoglobin or synthetically produced hemoglobin.

To produce hemoglobin synthetically, manufacturers use
compounds known as amino acids. These are chemicals that plants and animals use
to create the proteins that are essential for life. There are 20 naturally
occurring amino acids that may be used to produce hemoglobin. All of the amino
acid molecules share certain chemical characteristics. They are made up of an
amino group, a carboxyl group, and a side chain. The nature of the side chain
differentiates the various amino acids. Hemoglobin synthesis also requires a
specific type of bacteria and all of the materials needed to incubate it. This
includes warm water, molasses, glucose, acetic acid, alcohols, urea, and liquid
ammonia.

For other types of hemoglobin-based artificial blood
products, the hemoglobin is isolated from human blood. It is typically obtained
from donated blood that has expired before it is used. Other sources of
hemoglobin come from spent animal blood. This hemoglobin is slightly different
from human hemoglobin and must be modified before being used.

The Manufacturing Process

The production of artificial blood can be done in a
variety of ways. For hemoglobin-based products, this involves isolation or
synthesization of hemoglobin, molecular modification then reconstitution in an
artificial blood formula. PFC products involve a polymerization reaction. A
method for the production of a synthetic hemoglobin-based product is outlined
below.10

Hemoglobin synthesis
  • 1 To obtain hemoglobin, a strain of E. coli bacteria that has the
    ability to produce human hemoglobin is used. Over the course of about
    three days, the protein is harvested and the bacteria are destroyed. To
    start the fermentation process, a sample of the pure bacteria culture is
    transferred to a test tube that contains all the nutrients necessary for
    growth. This initial inoculation causes the bacteria to multiply. When the
    population is great enough, they are transferred to a seed tank.
  • 2 A seed tank is a large stainless steel kettle that provides an ideal
    environment for growing bacteria. It is filled with warm water, food, and
    an ammonia source which are all required for the production of hemoglobin.
    Other growth factors such as vitamins, amino acids, and minor nutrients
    are also added. The bacterial solution inside the seed tank is constantly
    bathed with compressed air and mixed to keep it moving. When enough time
    has passed, the contents of the seed tank is pumped to the fermentation
    tank.
  • 3 The fermentation tank is a larger version of the seed tank. It is also filled
    with a growth media needed for the bacteria to grow and produce
    hemoglobin. Since pH control is vital for optimal growth, ammonia water is
    added to the tank as necessary. When enough hemoglobin has been produced,
    the tank is emptied so isolation can begin.
  • 4 Isolation begins with a centrifugal separator that isolates much of the
    hemoglobin. It can be further segregated and purified using fractional
    distillation. This standard

Hemoglobin synthesis

Once fermented, the hemoglobin is purified and then
mixed with water and other electrolytes to create useable artificial blood.

column separation method is based on the principle of
boiling a liquid to separate one or more components and utilizes vertical
structures called fractionating columns. From this column, the hemoglobin is
transferred to a final processing tank.

Final processing
  • 5 Here,it is mixed with water and other electrolytes to produce the artificial
    blood. The artificial blood can then be pasteurized and put into an
    appropriate packaging. The quality of compounds is checked regularly
    during the entire process. Particularly important are frequent checks made
    on the bacterial culture. Also, various physical and chemical properties
    of the finished product are checked such as pH, melting point, moisture
    content, etc. This method of production has been shown to be able to
    produce batches as large as 2,640 gal (10,000 L).10

Comparison of HBOCs to Transfused Red Blood Cells

The table below characterizes the major difference
between transfused red blood cells and infused HBOCs.D.11

Comparison of HBOCs to Transfused Red Blood Cells

Attribute

Infused HBOCs

Transfused Red Cells

Onset of action:

Immediate

2,3-DPG dependent

Oxygen affinity:

Red cell 2,3 DPG not required for oxygen release

Red cell 2,3 DPG required for oxygen release

Oxygen transport:

Red cells plus plasma

Red cells only

Risk of disease
transmission:

Sterile pharmaceutical; no leukocyte exposure

Risk minimize by improved donor selection; leukocyte
exposure

Storage:

Room temperature; no loss of efficacy

Refrigeration required; progressive loss of efficacy

Shelf life:

36 months

42 days

Compatibility:

Universal

Type-specific

Preparation:

Ready to use

Requires typing and cross-matching

Viscosity:

Low

High

Duration of action:

Maximum of 3 days

Estimated 60 to 90 days

Benefits and Challenges

Benefits:

The general benefits of HBOCs over transfused red blood
cells are summarized below:

  • No prior planning
  • Faster & better O 2 distribution
  • Ready to use
  • No waste
  • No equipment
  • Long shelf life
  • No refrigeration
  • Universally compatible
  • Immediately offloads oxygen
  • No 2,3-DPG
  • Can be use by Jehovah's Witnesses

Challenges:

The challenges associated with the development of HBOC
can be categorized into the following:

·Availability – Ironically while one of the primary reasons to develop
oxygen carriers is to have a readily available solution to ease the projected
future shortages in blood supply, some approaches to the development of HBOCs
face a similar supply challenge. It was estimated that over 70,000 kg of Hb
would be needed to replace 20% of RBC transfusion in the United States. This
presents a significant challenge to human HBOC products. While production of
human Hb by recombinant DNA could be a possible solution, it remains unclear
whether the technology could produce such massive quantities of Hb for future
demand. A study has estimated that a population of 100,000 transgenic pigs
would be required to stably produce up to 50% of human Hb.

·Short half-life – Outside a red blood cell, Hb dissociates into 32 kDa
dimmers and are freely filtered by the glomerulus resulting in severe renal
toxicity. Current HBOC products have chemically or genetically cross-linked Hb
chains resulting in 128 kDa or larger molecules that are not readily filtered
by the glomerulus, thus possessing a greatly increased half-life.

·Increased oxygen affinity – Hb in the plasma has a much
higher affinity for O 2 than it does within the context of a red blood cell.
The increased affinity for O2 is due to lack of 2,3-diphosphoglycerate (DPG) in
the plasma. Consequently the HbO2 dissociation curve shifts to the left, making
such a high-affinity Hb not an ideal oxygen delivery substance. However,
chemically cross-linking the Hb structure has the net effect of decreasing O 2
affinity and optimizing intracellular oxygen delivery.

·Vasoactive properties – One of the major challenges
facing the development of HBOCs is their effects on vasoactive properties. The
theories regarding the mechanism of action of the vasoconstrictive effect:

1) nitric oxide scavenging by Hb,

2) excess O 2 delivery to the peripheral tissues,

3) direct effect on peripheral nerves, and

4) the oxidative properties of HB.

·Soluble Hb, unlike Hb in RBCs, interacts with NO to form
metHb and NO-Hb. NO by definition is a potent endothelial vasorelaxant that
inhibits the conversion of proendothelin to the vasoconstrictor endothelin. In
the prevailing theory on vasoactive properties, the decrease in NO
concentration due to its reaction with Hb is responsible for vasoconstriction.
Alternative theories suggest that too much O 2 is delivered causing an
autoregulatory vasoconstrictor reflex. Yet another theory argues that oxidation
of soluble Hb can result in heme loss, free radical formation, loss of reactive
iron, and oxidation of lipids. Such reaction and products result in endothelial
stress causing vasoconstriction

Current Status Of Hboc Based Product

HBOCs represent by far the greatest number of products
under development in the field of blood substitutes. Most products are
currently in Phase III clinical trial which is the final step in safety and
efficacy data collection prior to submission for FDA approval.

Graph of Leading HBOCs

Graph of Leading HBOCs

Taken from Bing Lou Wong, Ph.D., Advantek Biologics Limited.11

Three forms of HBOCs are currently in advanced testing
stages of the regulatory approval process. One bovine hemoglobin-based solution
under development uses the natural low affinity of bovine hemoglobin for oxygen
as a major rationale for its use. In addition there are two human
hemoglobin-based solutions – one a polymer and the other an oligomeric solution
containing a polymeric compound and cross-linked hemoglobin tetramer. These
different solutions have different chemical properties as well as different
biologic activities. Characteristics of these HBOCs solutions are summarized in
the table below.11

Comparison of HBOCs:

 

Biopure

Northfield

Hemosol

Source:

Bovine Hgb

Expired Human RBCs

Expired Human RBCs

Shelf-life:

3 years

1 year

1 year

Storage:

Room Temperature

Refrigerated

Refrigerated/ Room Temperature

Half-life:

18-22 hours

24 hours

14 hours

Approved oxygen
therapeutics:

2

0

0

Product Name:

Hemopure & Oxyglobin

PolyHeme

Hemolink

Hemopure from Biopure, Cambridge , Ma.

Hemopure is a glutaldehyde cross-linked polymer of
bovine Hb in which two or more tetramers are covalently linked. Hemopure has
been reported to significantly reduce the need for RBCU transfusion undergoing
cardiac and aortic surgeries. In addition Hemopure was well tolerated as a
resuscitation fluid administered intraopertively. In 2001 Hemopure was approved
in South Africa for treatment of adult surgical patients who are acutely anemic
and in order to eliminate, reduce, or delay the need for allogeneic RBC
transfusion. In October of 2002, Biopure filed a biologic license application
(BLA) to the US Food and Drug Administration to market Hemopure in the USA for
similar indication in surgical patients than in South Africa . In August 2003, the
Food and Drug Administration issued Biopure a response letter requesting
additional information concerning Hemopure. Biopure is currently addressing the
FDA's questions regarding the product's safety and efficacy. In addition
Biopure is conducting additional FDA-requested animal studies. Biopure is also
trying to develop Hemopure for other potential medical applications such as in
trauma and cardioprotective agent in ischemic conditions.

Hemopure Clinical Trials

Hemopure Clinical Trials.jpg

Hemolink from Hemosol, Inc, Mississauga , Ontario ,Canada .

Hemolink is a polymerized human hemoglobin with
residual unpolymerized tetramers. It has completed Phase III trials in
cardiothoracic surgery in Canada and is currently ongoing Phase II studies in
the same population in the United States . The preliminary studies showed that
Hemolink was effective in reducing red blood cell unit (RBCU) requirement in
coronary artery bypass graft patients. In early 2003, Hemosol voluntarily
suspended a phase II cardiac surgery study when it discovered an imbalance in
the incidence of adverse cardiac events occurring in greater numbers in the
HemoLink treated group. After electing to terminate the study, Hemosol started
investigating the cause of the imbalance, which is thought to be associated
with a higher rate of diabetes in the Hemolink treated group.

PolyHeme from Northfield Laboratories, Inc., Evanston , Il .

PolyHeme is a glutaldehyde cross-linked polymer of human Hb in which two or
more tetramers are covalently linked. In order to replace 2,3-DPG, a pyridoxal
molecule is incorporated into each tetramer of PolyHeme to facilitate oxygen offloading.
Currently, PolyHeme is being tested in phase III pre-hospital trauma study. The study proposes to
demonstrate the safety and efficacy of PolyHeme in improving survival for
severely injured bleeding trauma patients. It is important to note the study is
being conducted under the informed consent waiver provision, meaning that
patients eligible for the study will be unable to provide informed consent.
Over 700 patients are being enrolled in the study from over 20 trauma centers
across the country. In a separate study PolyHeme has bee shown to be effective
in reducing mortality of patients with acute anemia. When compared to an anemic
control made up of individual who refused allogeneic RBC transfusion on
religious ground, the PolyHeme group had a lower mortality.

PolyHeme Ongoing Phase III Clinical Trial:

Scale:

20 Level I trauma centers

Size:

700+ patients expected

Control:

Standard of care

Endpoint

Improved Survival

Ethics:

Exception form informed consent

Clinical Utility Of Blood Substitutes

Current blood substitutes have been demonstrated to be
safe when administered in small quantities to volunteers. Both perfluorocarbon
and hemoglobin based oxygen carriers have undergone clinical trials designed to
determine the safety of these compounds when given to otherwise healthy
patients. These preliminary studies have shown that a clinical useful dose of a
blood substitute can be infused to patients. However, further information
regarding the effectiveness and clinical usefulness of these compounds is in
short supply at present.

The short plasma half-life of these compounds limits
the usefulness of blood substitutes to short periods of time. Ultimately, the
blood substitute will be sequestered or metabolized, and decreased oxygen
carrying capacity will reappear as the plasma oxygen carrying capacity
diminishes. Thus, if no longer acting agents are available, it is likely that
these blood substitutes will merely delay an allogeneic transfusion, rather
than avoiding exposure, when used in place of conventional allogeneic red blood
cell transfusions.

In order to effectively use these compounds, special
techniques should be considered. One technique which theoretically should
optimize blood substitute utility is acute normovolemic hemodilution.
Aggressive harvesting of potentially several units of autologous fresh whole
blood is possible when the solution to replace the harvested blood is capable
of transporting oxygen. Coupling of blood substitutes with acute normovolemic
hemodilution has been successful in small clinical trials; whether this mode of
using blood substitutes will result in substantial clinical and economic
benefits await larger clinical trials.

The Future

Currently, there are several companies working on the production of a safe
and effective artificial blood substitute. The various blood substitutes all
suffer from certain limitations. For example, most of the hemoglobin-based products
last no more than 20-30 hours in the body. This compares to transfusions of
whole blood that lasts 34 days. Also, these blood substitutes do not mimic the
blood's ability to fight diseases and clot. Consequently, the current artificial
blood technology will be limited to short-term blood replacement applications.
In the future, it is anticipated that new materials to carry oxygen in the body
will be found.

The Future.jpg

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Additionally, longer lasting products should be developed,
as well as products that perform the other functions of blood. Blood substitutes
have the potential of replacing allogeneically transfused blood as a therapy,
but will this happen? And if so, when? These are integral questions that need
answers, especially for all those companies pouring their resources into research
and production. It remains to be seen whether blood substitutes will be another
good idea that simply failed to be embraced by the medical community, either
by not living up to standards, or by failure to break through the traditions
of medical practice.

In the running for the blood race are two major categories that title themselves blood
oxygen carriers: perfluorocarbon emulsions (PFCEs) and hemoglobin based oxygen carriers (HBOCs).
Questioned in an email about the topic of blood substitutes, Dr. H. Kim stated that HBOCs seem more
promising than PFCs because there is currently an HBOC Phase III clinical trial
for use in traumatic hemorrhage. He believes that PFCs are having difficulty
establishing their safety and effectiveness. Our other expert Lelio M. Sarteschi, M. D., also believes that
HBOCs are winning the race. This may be because PFCs don't seem to have the
ability to replace blood, although data suggests they are beneficial because
they reduce the need for transfused blood.

With all the research being put into blood substitutes and the production of synthetic
blood, it seems as if there is no way these products could possibly fail.
However, it is important to note that while the products are created to
simulate human blood, they are not in fact human blood. This may seem a simple
statement, but it is because of this that FDA approval is hard to get: the
products must be able to demonstrate a higher or at least equal efficiency to
that of blood, with little or no side effects. And nature is a tough
competitor. Many of the blood substitutes in trials still have problems that
need to be solved before they will be adequately safe, first and foremost
increasing the oxygen carriage and discovering any side effects.

Transfused blood has reached a very high safety level in the United States. The incidence
of HIV infected blood has fallen to one in two million. However, there are
reasons to continue investigation into alternate therapies. There is now a
concern that prions may infect the blood supply and
quickly spread, causing an outbreak similar to that of HIV in the past. Also,
even though blood may be matched from donor to recipient, their are still
medical side effects that take place. Along with blood substitutes comes the
possibility to eliminate these side-effects. And there are still occasional
deathly clerical errors related to transfused blood. Blood substitutes could
eliminate all of these problems. In addition they would have a longer shelf
life and could be stored over a higher range of temperatures making them ideal
for storage depots in preparation for disaster relief of any kind. Finally,
because blood oxygen substitutes are expected to be different from blood by
nature, it is expected that after approval of several different types,
physicians will be able to choose which characteristics best suit their
patients' needs.

Another large factor in whether or not blood substitutes will become widely accepted is
the consumer base. Is there a market for synthetic blood products? One market
would be the military, which has long sought to add a universally compatible
blood equivalent to its repertoire to simplify emergency situations. Another
significantly smaller market is the Jehovah's Witnesses, who have become
advocates of what they call bloodless surgery. Blood substitutes would
be able to equally match their need, especially in the event of traumatic
injury, where blood cannot be saved. In addition, a growing number of
physicians have come to believe that patients should be able to choose their
own treatment, and research indicates that at least eighty percent of patients
when questioned, indicate that they would prefer not to have blood
transfusions.

After FDA approval, major restrictions in the area of blood substitutes are cost and
effectiveness. While cost may be an issue, Dr. Kim remarks that if we wait
another ten to twenty years, HBOCs should have become an effective therapy and
be approved by the FDA. At that point larger quantities may be produced, and
the cost will have been lowered. Also significant is the rising cost of
transfused blood. Because screening techniques are becoming more advanced, the
cost of screening one unit of transfused blood is also rising. However,
assuming HBOCs were licensed, 70 000 kilograms of hemoglobin would be required
to replace only twenty percent of the US red cell transfusions in one year, and
manufacturing of this large amount of raw materials presents a great challenge.
It is estimated that the price of one allogeneic unit of blood lies between 100
to 150 US dollars, whereas HBOC prices are estimated to range between 400 to
800 US dollars. Because of these and other factors there is
no simple way to predict to what scale blood substitutes will succeed as an
industry, even after FDA approval, although doctors seem enthusiastic. Blood
substitutes are not however expected to completely replace allogeneic
transfusions unless their spectrum of clinical application of blood oxygen
carriers is brought closer to the level of allogeneic blood. A current projection
estimates that a decrease of allogeneic red cell use of upt twenty percent may
occur once some products are FDA approved, and this may pose some economic
problems for blood clinics.

Reaching into the unforeseeable future, it is only possible to hope that eventually
blood substitutes will be able to cover worldwide shortages, and be cheap and
stable enough to be distributed in third world countries where much of the
allogeneic supplies are contaminated, or wherever else a need occurs.

Conclusion

Blood substitutes are currently undergoing preliminary
clinical trials to determine their safety. Two distinctly different classes of
oxygen carriers are being developed, each capable of transporting and
delivering oxygen to peripheral tissues. The delivery of oxygen by these two
methodologies may have both benefits and risks which are unique to its class.
Early clinical trials have been promising; however, effective use of these
blood substitutes may involve using them in conjunction with other techniques
such as normovolemic hemodilution to effectively reduce or eliminate the need
for transfusions in certain instances. However, this first generation of
clinically safe blood substitutes will not replace allogeneic blood
transfusions as a means of treating many types of anemia.

References

1. Amberson WR, Jennings JJ, Rhode CM. Clinical experience with
hemoglobin-saline solutions, American Journal of Physiology, vol-1, 1949 page no- 469-489.

2. Lee R, Atsumi N, Jacobs EEJ, Austen WG, Vlahakes GJ.
Ultrapure, stroma-free, polymerized bovine hemoglobin solution: evaluation of
renal toxicity, Journal of Surgical Research. Issue 47, 1989,page no-407-411.

3. Amberson W, Flexner J, Steggerda FR, et al. On the use of
Ringer-Locke solutions containing hemoglobin as a substitute for normal blood
in mammals, Journal of Cellular and Comparative Physiology, issue 5,1937, page no 359-382.

4. Tremper KK, Lapin R, Levine E, Friedman A, Shoemaker WC.
Hemodynamic and oxygen transport effects of a perfluorochemical blood
substitute, Fluosol-DA (20%). Critical Care Medicine, issue 8 1980, page no -738-741.

5. Tremper KK, Friedman AE, Levine EM, Lapin R, Camarillo D. The preoperative treatment of severely anemic patients with a perfluorochemical oxygen-transport fluid, Fluosol-DA, New England Journal of Medicine. Issue 307 1982, page no- 277-283.

6. Teicher B. Use of perfluorocarbon emulsions in cancer
therapy. In: Chang TMS, ed. Blood Substitutes and Oxygen Carriers. New
York: Marcel Dekker, Inc, 1993, page no- 640-647.

7. Hoffman SJ, Looker DL, Roehrich JM, et al. Expression of
fully functional tetrameric human hemoglobin in Escherichia coli. Proceedings
of the National Academy of Sciences, U.S ,issue 87,
 1990 ,page no 8521-8525.

8. Laver MB, Jackson E, Scherperel M, Tung C, Tung W, Radford
EP. Hemoglobin-O2 affinity regulation: DPG, monovalent anions, and hemoglobin
concentration. J Appl Physiology,  issue 44 1977, page no-  632-642.

9. White CT, Murray AJ, Smith DJ, Greene JR, Bolin RB. Synergistic toxicity of endotoxin and hemoglobin. Journal of Laboratory and Clinical Medicine, issue 108, 1986, page no- 132-137.

10.  Winslow, R. Hemoglobin-Based Red Cell Substitutes. Johns Hopkins University Press, 1992,page no-34-35

11.  Chang TM. Hemoglobin-based red blood cell substitutes. ARTIFICIAL ORGANS, issue Sep28 (9), 2004,page no- 789-94

12.  Winslow RM. Current status of blood substitute research: towards a new paradigm. J Intern Med, issue May 253 (5) 2004, page no- 508-517

13.  Kim HW, Greenburg AG. Artificial oxygen carriers as red blood cell substitutes: a selected review and current status. Artif Organs, issue Sep 28(9) 2004, page no 813-28.

14.  Kirschman, Ruth Ann BS, RN,
C, WHNP. “Finding Alternatives to Blood Transfusion.” Nursing, issue 34 vol 6,28 March 2005

15.  Kovelenov, A.Yu., B.O.Voitenkov, E.I.Mayevsky, and S.Yu. Pushkin. “ Perspectives of Using
Perfluorocarbon Compound in HIV-Infection (Abstract).” 3 rd Baltic Region
Conference “Together Against AIDS!”. Final Program issue 19 April 2005.

16.  Riess, Jean G. “Understanding
the Fundamentals of Perfluorocarbons and Perfluorocarbon Emulsions Relevant to
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About Authors:

Mr. Parag A. Kulkarni

Parag A. Kulkarni

Lecturer of Pharmaceutics. Padmashree Dr. D. Y. Patil College of Pharmacy,
Akurdi. Pune-411044. E-mail. kparag4@yahoo.com.

Mobile no. +919823296713 Office (020) 27656141, 27656237

Anuragsinh.D.Chauhan

Anuragsinh.D.Chauhan

B. Pharm Final Year.Padmashree Dr. D. Y. Patil College of Pharmacy, Akurdi.Pune-411044

Dhaval.N.Gajera

Dhaval.N.Gajera

B. Pharm Final Year, Padmashree Dr. D. Y. Patil College of Pharmacy, Akurdi.
Pune-411044

Pramod V. Kasture

Pramod V. Kasture

M. Pharm. P.h,. Principal and Professor of Pharmaceutics, Padmashree
Dr. D. Y. Patil College of Pharmacy, Akurdi. Pune-411044

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