Topics in Bioengineering, Fall 2010 1

Red Cell Blood Substitutes

Erica Chin

Abstract- Blood transfusions are routinely used to treat Microvascular perfusion is also dependent on blood viscosity
victims experiencing hemorrhagic shock. However, patients [12, 16]. Together, perfusion pressure and blood viscosity
often face the threat of life-threatening reactions to these determine blood flow which is essential to the delivery of
blood transfusions, which may also carry diseases from oxygen and nutrients and the removal of waste, which can
donors. Additionally, current blood supply will not be able to accumulate to create lethal toxicity levels [12, 14]. In fact,
sustain the current demand. As such, a possible solution to studies have found that the maintenance of functional
the inadequacies of the current system for allogeneic blood capillary density (FCD), or the number of capillaries with
transfusions would be the development of a blood substitute. blood flow per unit surface of tissue, determines survival
Currently, perfluorocarbon and hemoglobin based oxygen after hemorrhagic shock independent of oxygen delivery [12,
carriers are the blood substitutes furthest in development. 14-15].
Consequently, restoring circulating volume is the primary
However, due to side effects discovered in clinical trials, no
goal to treat hemorrhagic shock. Although the ideal volume
blood substitutes are currently approved in the U.S. restitution fluid, or plasma expander, has yet to be
ascertained, current practices use colloidal solutions, such as
I. INTRODUCTION albumin, and crystalloids, such as Ringers lactate or saline
[4]. For example, the Advanced Trauma Life Support
A. Necessity of Blood Transfusions guidelines recommend using crystalloid solutions, while the
American College of Surgeons recommends using albumin
Class
after trauma [4, 17].
Parameter I II III IV
Blood loss <750 750– 1500– >2000 2) Oxygen Delivery
(mL) 1500 2000 Impact of Insufficient Oxygen- In addition to lowered organ
Blood loss <15% 15–30% 30–40% >40% perfusion pressure and viscosity, a sudden loss of blood
(%) volume also lowers cardiac output. Since the amount of
Pulse rate <100 >100 >120 >140 oxygen delivered to tissues depends on cardiac output and
(beats/min) arterial oxygen content, a drop in circulating blood volume
Blood Normal Decreas Decreas Decreas results in decreased oxygen delivery [4]. When the total
oxygen concentration drops to a critical level (i.e. 2 mmHg
Pressure ed ed ed and 7 mmHg for the interstitial and intravascular partial
Respiratory 14–20 20–30 30–40 >35 pressure of oxygen respectively 1), cells switch to anaerobic
rate respiration [15]. However, as anaerobic metabolism yields
(breaths/min much less ATP, when ATP levels become insufficient to
) sustain membrane-associated ion transport pumps and other
CNS Normal Anxious Confuse Lethargi essential cellular functions, cellular swelling and acidosis
symptoms d c result- ultimately causing cell death, as shown in Figure 1
[4].
Table 1: Symptoms of hemorrhage classes. Modified from [4]

Traumatic injury is the third leading cause of death in the

United States [10]. There are four classes of hemorrhaging,
as defined by the Committee on Trauma. These four classes
are defined by the amount of blood lost relative to the total
body weight, as shown in Table 1. Class I hemorrhaging,
which may occur during blood donation, does not lead to
symptoms of shock, where tissue perfusion is insufficient for
aerobic metabolism [4]. However, severe hemorrhaging,
which encompasses classes III and IV, can lead to
hemorrhagic shock, the main contributing factor to fatalities
in trauma patients [11-13]. Current strategies, or resuscitation
methods, to combat the effects of hemorrhagic shock first
require the restoration of circulating blood volume followed
by the restoration of oxygen delivery [14].
1) Blood Volume Restoration
Severe hemorrhaging results in lowered organ perfusion
pressure and lowered viscosity- which is determined by the
concentration and molecular size of the solute [4, 15]. 1
The normal partial pressure of oxygen in the lungs is 100 mmHg and 40
mm Hg in the body [19].
Topics in Bioengineering, Fall 2010 2

Figure 1: As circulating blood volume decreases, total oxygen hepatitis B and C viruses, human immunodeficiency virus
delivery also decreases. When over 30% of total blood volume is lost, (HIV), human T cell lymphotropic virus (HTLV), and
a critical point in total oxygen delivery is reached. At this point, cells syphilis [20-21]. Donor interviews and blood tests, such as
begin using anaerobic metabolism to generate ATP. When ATP nucleic acid testing, has dramatically reduced the risk of
supply is insufficient to meet cell demands, cell death occurs. infection in ABT recipients to 1 in 250,000 – 500,000 for
Modified from [4]
hepatitis B, 1 in every 1,600,000 for hepatitis C, and 1 in
Hemoglobin- Arterial oxygen content depends on the
every 1,800,000 for HIV [21-22]. At the same time, the
concentration of hemoglobin in the blood and the degree to
prevention of some infections, such as malaria, Chagas
which hemoglobin is saturated with oxygen [4]. Hemoglobin disease (also known as American trypanosomiasis), West
is a 64 kDa protein found in red blood cells, as shown in Nile virus (WNV), and severe acute respiratory syndrome
Figure 2. (SARS), is completely dependent on the veracity of donor
interviews. Additionally, these blood donor interviews reduce
an already shrinking pool of blood donors [21]. No standards
are available to detect and screen against prion diseases, such
as the fatal neurodegenerative disease variant Creutzfeldt-
Jakob disease (vCJD) [21-22]. In other words, current safety
measures do not completely prevent the transmission of
blood borne pathogens; overall, infectious diseases account
for 14.4% of transfusion-related deaths reported to the US
FDA since 2006 [23-24].
2) Noninfectious Dangers
Transfusion reactions pose a greater threat to ABT
recipients than infectious diseases [25]. One common
example of noninfectious complications is an acute
hemolytic transfusion reaction (HTR), or the destruction of
donor red blood cells by the recipient’s immune system
within 24 hours [24]. Acute HTR occurs due to an ABO
incompatible transfusion- the leading direct cause of
Figure 2: Structure of hemoglobin. Adult hemoglobin is composed of
4 polypeptides- specifically two α polypeptides and two β transfusion-related death Chen [24, 26-27]. The rate of
polypeptides. Modified from [2] mislabeled blood samples in the US is 1,000 to 10,000 times
greater than the risk of transfusion-transmitted viral
Each of the protein’s four polypeptide subunits attaches to a infection, with the risk of fatality under one in 1.5 million
heme molecule. Heme molecules are ring structures that transfusions [20]. Other noninfectious dangers include
reversibly bind to oxygen [18]. As the oxygen-transporter in transfusion-related acute lung injury, graft-versus-host
blood, hemoglobin’s binding affinity to oxygen rises with the disease, and anaphylactic reactions [20, 24, 28].
partial pressure of oxygen (pO2) in the environment in an
sigmoidal relationshipTherefore,
Blood Transfusions. [19]. the goal of transfusing red C.Blood Shortage
blood cells is to raise the hemoglobin concentration in the There is a projected shortage of 3 to 4 million units of
blood and thus the delivery of oxygen to tissues. During the blood per year in the US by 2020 due to blood’s limited shelf
last forty years, the standard treatment for trauma victims life and shrinking donor pool [29]. This estimated shortage
requires blood transfusion when circulating blood does not consider the impact of pandemics or wars.
hemoglobin has fallen between 6 and 8 g/dL, a value that is The number of suitable blood donors is shrinking. As
lower in young individuals and higher for the elderly. Blood potential blood-borne diseases are identified, restrictions are
raised, disqualifying potential donors from giving blood [24].
transfusions are also standard when 2L of blood volume
At the same time, given that about two thirds of all
expanders does not appear to affect the patient’s blood
transfusions in the US are associated with surgery, an
pressure [4]. However, many complications are associated increasing older population places increasing demand for
with allogeneic blood transfusion. blood [18].
B. Dangers of Allogeneic Blood Transfusion Blood, when refrigerated, has a shelf life of 42 days [29].
During this time, many changes occur including increased
Although there have been many advancements in
concentrations of lactate and potassium ions from 1 to 15
screening processes to improve the safety of allogeneic blood mM and 4 to 20 mM in three weeks respectively. At the
transfusions (ABT), there are still many hazardous infectious same time concentrations of glucose and ATP are reduced
and noninfectious complications. These complications can [25]. In fact, the pH drops from 7.4 to 6.9 within a few hours,
lead to death or longer, and more expensive, ICU stays [7]. which lowers hemoglobin’s affinity to oxygen [3]. Perhaps
1) Infectious Hazards the most alarming change is the reduction of 2,3-
Although blood screening has dramatically reduced the diphosphoglycerate (DPG), which lowers hemoglobin’s
chances of acquiring diseases from blood transfusion, the risk binding affinity to oxygen, allowing the release and delivery
to ABT recipients has not been eliminated; additionally, of oxygen to tissues [25, 29]. With the exception of rare
present tests cannot effectively detect new blood-borne blood types, exhaustive efforts to rejuvenate outdated RBCs
pathogens. Currently, all donated blood is screened for
 Topics in Bioengineering, Fall 2010
are often foregone in favor of discarding expired blood units
[3, 25].
D.Blood Substitutes
1) Necessity of a Blood Substitute
The creation of a substitute for the oxygen-carrying
capacities of red blood cells could eliminate the issue of
blood shortages and make blood transfusions safer.
Additionally, the creation of a blood substitute could allow
surgical procedures that require blood transfusions to take
place in rural and third world countries, and treatments to be
given to those who would normally refuse blood transfusions
on the basis of their religion or culture, as in the case of
Jehovah’s witnesses [30]. In fact, the sheer potential of blood
substitutes has been recognized- annual prospective sales for
blood substitute are at twelve billion dollars [29].
2) Design Requirements
The ideal blood substitute should be safe, convenient, and
economical. Safe blood substitutes should be pathogen-free,
non-toxic, and nonimmunogenic. For convenience’s sake,
blood substitutes should have universal compatibility, an
extended shelf life (compared to packed red blood cells), and
be easily administrated and transported. For wide-spread use,
blood substitutes should also be cost-effective [5, 29, 31].
3) Current Types
Presently, there are two types of artificial oxygen carriers, or
blood substitutes, undergoing clinical trials: hemoglobin
(Hb)-based oxygen carriers (HbOCs) and perfluorocarbon-
based oxygen carriers (PFCOCs).
II.PERFLUOROCARBON-BASED OXYGEN CARRIERS (PFCOCS)
Figure 3: Structural differences resulting from the substitution of
Figure 4: Characteristic oxygen release profile of native blood and
hydrogen with fluorine. Fluorocarbon chains (top) have a helical
PFCs. Oxygen dissociation in red blood cells has a sigmoidal behavior.,
carbon skeleton, while hydrocarbon chains (bottom) have a planar zig-
but linear in PFCs. Image modified from [7].
zag arrangement. Cross-sectional views of the chains are shown at the
right. Image modified from [1].
A. Construction of PFCOCs
1) Construction of Perfluorocarbons (PFCs)
Perfluorocarbons are synthetic molecules with a molecular
weight ranging between 450 and 500 Daltons [28]. They are
constructed from linear or cyclic hydrocarbon chains by
replacing hydrogens with halogens, such as fluorine or
bromide [7, 9, 28, 31-32]. . Since halogens have more
valence electrons then hydrogens, they have a denser electron
cloud and a larger volume. As a result, the carbon backbone
is forced into a helical instead of a planar zig-zag
configuration as shown in Figure 3 [1]. The impact of this
configuration change is the increased coverage, and thus
3
protection, of the carbon skeleton. Additionally, in this
helical configuration, fluorine’s strong attraction for
electrons causes the C-C bonds to shrink- raising the C-C
bond energies. Since single bonds between carbons and
fluorines are also very strong, no low energy orbitals are
readily available for reactions [1]. Consequently, the
substitution of hydrogens with fluorines results in a very
chemically stable and inert molecule [1, 5, 7, 28, 31].
2) Impact of Nonpolarity
PFCs are nonpolar due to fluorine’s low polarizability and
the symmetrical structure of fluorocarbons. This nonpolar
character contributes to PFCs’ ability to dissolve oxygen and
biological inertness [1]. However, since nonpolar substances
cannot dissolve in water, an emulsifier is needed for PFCs to
be used as an artificial oxygen carrier.
Oxygen Delivery Ability- The ability of perfluorocarbons
to deliver oxygen is rooted in its capacity to dissolve oxygen.
Since it is difficult to polarize fluorine, the electron cloud of
PFCs as a whole does not have many fluctuations in polarity.
As a result, not many van der Waals forces develop in
between PFCs. Given that nonpolar molecules are only held
together by van der Waals interactions, the intermolecular
forces in PFCs are low - allowing liquid PFCs to act like a
gas and dissolve other gases of low cohesivity [1].
Although PFCs can dissolve 100 times more oxygen than
blood plasma, its ability to carry oxygen is still much less
efficient than hemoglobin’s at alveolar pO2, as shown in
Figure 4 [31]. To improve oxygen-saturation in PFCs,
environmental pO2 is raised artificially by having patients
breath 70-100% oxygenated gas [28].
Necessity of Emulsions- Since PFCs are nonpolar, they
will not dissolve in water [9, 14, 31]. Although this property
results in desirable biological inertness, the inability to
dissolve in water prevents PFCs from being applied
intravenously [5, 7, 28, 33]. To resolve this issue, PFCs
emulsions are created by high shear homogenization of PFC
with a surfactant, as shown in Figure 5 [31]. In order for the
creation of a stable emulsion, PFC droplets should be within
0.1-0.2 μm [1].
 Topics in Bioengineering, Fall 2010 4

These dimers have a short circulation retention period since

they are quickly filtered out through the kidney tubules [26,
29, 36]. The dimers could then cause renal toxicity if they
accumulate and obstruct the kidney tubules [28].
Extracellular hemoglobin may also react with nitric oxide
[28, 37]. Produced by endothelial cells, nitric oxide (NO) can
behave as a paracrine signal, activating guanylyl cyclase in
smooth muscle cells. This enzyme catalyzes the production
of cGMP, a second messenger involved in vasodilation [3, 6,
19]. In other words, hemoglobin scavenging of NO can cause
vasoconstriction. Vasoconstriction can lead to lowered heart
Figure 5: Perfluorocarbon emulsions are produced by applying high- rate and cardiac output – which results in reduced tissue
shear homogenization to the mixture of PFCs and a surfactant. Image perfusion [35]. NO scavenging can also create
modified from [1]. methemoglobin, a form of hemoglobin that does not carry
oxygen, and which, at high levels, can cause tissue hypoxia
B. Current Potential [37]. Normally, red blood cell membranes act as a diffusional
barrier, discouraging such reactions. Additionally, the size of
1) Advantages
red blood cells would normally prevent the passage of cell
Although the raw material for the production for PFCs
free hemoglobin through inter-endothelial junctions; cell-free
may be expensive, mass production of PFCs is predicted to
Hb’s smaller size allows it to reach and react with NO, which
make PFCs a cheap alternative to allogeneic blood [5, 28].
normally does not diffuse very far [3, 19].
The synthetic nature of PFCs and the sterilization step during
the creation of PFC emulsions dramatically reduces the threat
of infectious transfusion-related dangers [28]. The latest PFC
products have a long shelf life of at least two years [5, 28]2.
2) Disadvantages
The disadvantages of PFCOCs lie with its adverse effects
and administration requirements.
The most common side effects of PFC emulsions involve
flu-like symptoms. These symptoms include facial flushing,
back-aches, and fevers [28, 34].
One of the desired goals for developing an artificial
oxygen carrier is so that medical operations that require
blood transfusions can take place in regions with
underdeveloped or nonexistent medical infrastructure.
PFCOCs’ high oxygen concentration requirement for the
patient limits the locations that PFCOCs can be used.
3) Current Status
No PFCOCs are currently approved for clinical use, or
clinical trials, in the US. However, Perftoran has been
approved for use in Russia since 1996. Perftoran is also used
in Mexico, while Oxygent is currently approved for use in
Figure 6: The dissociation of extracellular hemoglobin leads to adverse
South Africa [32]. side effects. NO scavenging results in vasoconstriction while the
oxidation of the dimers forms met-Hb. High levels of met-Hb can lead
III. HEMOGLOBIN-BASED OXYGEN CARRIERS (HBOCS) to tissue hypoxia [3].

A. Construction of HBOCs 2) Second Generation HBOC Strategies

Since hemoglobin is what is naturally used to transport
oxygen, initial development strategies for blood substitutes
involved the use of cell-free hemoglobin. The lack of a cell
would mean the absence of blood antigens that make blood-
typing and blood-matching necessary [28, 35]. However, it
was soon discovered, that simply using extracellular
hemoglobin led to many adverse effects, and modification of
See [5] and [9] for an extensive list and discussion of past and present
PFC products.
hemoglobin would be needed before hemoglobin could be
used as an oxygen carrier [26].
1) Extracellular Hb
Hemoglobin tetramers readily dissociate into αβ-dimers
outside of the red blood cell, as shown in Figure 6 [18].
2
Strategies aimed at preventing renal toxicity and
vasoconstriction are based on either artificially increasing the
size of the αβ dimers or preventing Hb dissociation in the
first place [3]. Increasing the size of HBOCs also increases
the viscosity of the HBOC solution; when viscosity of a
transfused fluid is low, the shear stress exerted on endothelial
cells lining vascular structures is low. Low shear stress is
correlated with lower NO production [6].
Current approaches to increasing the size of HBOCs include
modifying the surface of hemoglobin, cross-linking the
polypeptides in hemoglobin, polymerizing hemoglobin, and
encapsulating hemoglobin in liposomes, as shown in Figure 7
[3, 28-29, 33, 38]. Figure 8 shows the relative sizes of
hemoglobin that result from these strategies.
 Topics in Bioengineering, Fall 2010 5

3) Current Status
No HBOCs are approved for clinical use in the US and many
clinical trials have been placed on hold by the FDA due to
the observed side-effects [39]. In 2001, Hemopure was
approved for clinical use in South Africa by the Ministry of
Health [7].

IV. CONCLUSION
While the development of artificial oxygen carriers may
bring a solution to the threats of a diminishing blood supply
and transfusion-related dangers, the process of this
development has illuminated topics that needed, and still
need, greater understanding. In particular, these topics
include the toxicity mechanisms of HBOCs and PFCOCs [6,
33]. From a logistical standpoint, the investigation into
blood substitutes has also shown the need for increased
transparency from the FDA and companies; currently, the
FDA mandates that all material, submitted by a company
Figure 7: Four methods of improving molecular stability: (1) cross- during the development of a product, is to be treated as
linking between polypeptides, (b) polymerization, (c) modifying the confidential. Data from clinical trials is only provided
surface of hemoglobin – usually with polyethylene glycol (PEG), and through press releases and advisory committee presentations
(d) encapsulating hemoglobin in liposomes. Image modified from [3] [39]. Many details, such as the methods used in the trials, are
and [8]. often unavailable. This purposeful obscuration of safety and
effectiveness data has hampered the improvement of AOCs
to the frustration of many [39-41]

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