LECTURE
The unusual properties of effective blood substitutes
Amy G. Tsai and Marcos Intaglietta
Department of Bioengineering, University of California, San Diego, La Jolla, CA, USA(Received for publication on October 19, 2001)

Abstract. Blood substitutes or oxygen carrying plasma expanders were originally formulated to simulate the transport properties of blood, particularly oxygen carrying capacity, viscosity, p50, and colloid
osmotic pressure, under the hypothesis that blood is the most desirable ﬂuid in volume restitution.
However, changes introduced into the organism during hemorrhage adversely affect microvascularfunction due to reﬂex vasoconstriction which causes the fall of functional capillary density, and lowers
tissue oxygenation, conditions that are not universally reversed with retransfusion of blood. The restoration of microvascular function is seldom complete upon retransfusion of blood. New formulationsof hemoglobin molecules in solutions whose oncotic pressure is in the range of 60–100 mmHg, p50 isabout 5 mmHg, viscosity 3–4 cP, and oxygen carrying capacity in the range of 4–7 g/dl equivalent hemoglobin deliver better microvascular function after resuscitation when compared to whole blood andoxygen carrying plasma expanders with transport properties similar to those of blood. The improved
performance is in part due to the increased plasma viscosity which increases capillary transmuralpressure which reverses capillary collapse induced during low perfusion pressures. High oncotic pressure reinforces this effect, since it brings more ﬂuid into the circulation. Microvascular transport studiesof the effects of resuscitation in shock show that functional capillary density is the primary determinantof survival, thus maintenance of an open and fully perfused microcirculation is more critical thaninsuring oxygen supply, since closed capillaries lead to the accumulation of slowly diffusing byproductsof metabolism which ultimately become toxic. The required combination of properties can be achievedby conjugating hemoglobin and polyethylene glycol. Resuscitation ﬂuids based on hemoglobin containing vesicles may provide the next level of functional improvement in the formulation of volume
restitution ﬂuids since their biophysical properties can be speciﬁcally controlled through the inclusionof specialized compounds into the vesicles, and the formulation of the suspending medium. (Keio J Med
51 (1): 17–20, March 2002)
Key words: blood substitutes, hemoglobin, plasma viscosity, functional capillary density

presumed beneﬁcial modiﬁcation was introduced when
it was found that some of the oxygen carriers basedon modiﬁed hemoglobins exhibited high p50’s, a characteristic that was rationalized to facilitate oxygen
unloading and tissue oxygenation. In the course of therather extended period of development nitric oxide(NO) was found to be a fundamental element in cardiovascular regulation. As acellular modiﬁed molecularhemoglobin became the oxygen carrier of choice, it became increasingly apparent that this material is vasoactive, causing hypertension, which was determined to
be deleterious in resuscitation. Since hemoglobin scavenges NO leading to vasoconstriction there is to theThe development of artiﬁcial blood, blood substitutes or oxygen carrying plasma expanders (OCPEs)
from the onset had as a goal to reproduce the transportcharacteristic of blood, with some modiﬁcations aimed
at improving its ability to deliver oxygen derived fromthe perception that lowered viscosity would improveblood ﬂow and the facilitation of oxygen release would
be beneﬁcial. On this basis oxygen carrying capacityand oncotic pressure were prescribed to be within therange found in blood, while the experience with hemodilution suggested that improvements in transport
would be obtained by lowering blood viscosity to values
signiﬁcantly below those of whole blood. An additional

Presented by Marcos Intaglietta at the 1226th Meeting of the Keio Medical Society in Tokyo, September 6, 2001
Reprint requests to: Dr. Marcos Intaglietta, Department of Bioengineering, UCSD, 9500 Gilman Dr. La Jolla, CA 92092-0412, USA, phone
1 858 534 4275, fax 1 858 459 8698, e-mail: mintagli@ucsd.edu
17

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Tsai AG and Intaglietta M: Effective blood substitutes

present a signiﬁcant effort aimed at modifying the hemoglobin molecule so that its afﬁnity for NO is reduced.
Thus at present an optimal OCPE would have the following properties: Oxygen carrying capacity equivalent
to 10–14 g/dl hemoglobin, p50 > 30 mmHg, viscosity
1 cP, oncotic pressure @ 25 mmHg, and low NO binding.
Developments in the understanding of microvascularand systemic physiology show that all of these parameter speciﬁcation are inappropriate for the formulationof an OCPE. Furthermore products that include thesespeciﬁcations are potentially dangerous, which has beenshown in practice, and/or impractical in terms of economic considerations. The following discussion analyzes each of the assumptions presently incorporated inOCPEs being developed or in clinical trials.
Oxygen Carrying Capacity
Measurements in the microcirculation utilizing thetechnique of phosphorescence quenching (Torres &Intaglietta, 1993)1 show that when hemodilution is carried out to a total remaining hemoglobin content in red
blood cells of 5.6 g/dl, tissue oxygen is higher but notstatistically signiﬁcantly so than normal (Tsai, 2001),2thus conservatively one may take a total oxygen carrying capacity of 5 g/dl as an adequate blood oxygen
carrying capacity, a number that is well established in
the hemodilution literature (Messmer, et al., 1972).3 It
should be noted that even though experiments werecarried out in the hamster skin fold, acid base balance
was normal in these experiments indicating that theobservations reﬂected conditions in all the tissues. This
numerical value for the needed oxygen carrying capacity is reﬂected also by a simple calculation that relates
the whole body oxygen consumption and cardiac output, which yields a nearly identical number for theorganism at rest. Therefore in principle the oxygen
carrying capacity of an OCPE does not need to reproduce the value for normal blood and can be signiﬁcantly
lower.
The Viscosity of the Circulating Blood
Since oxygen carrying capacity is not the limiting
factor at a blood hemoglobin content that is much lower
than the blood transfusion trigger, the question arises
why signiﬁcantly higher minimum hemoglobin conditions are taken as the indication for restitution blood
oxygen carrying capacity with red blood cells. Analyzing the mechanical and transport conditions of the circulation at the transfusion trigger it becomes apparent
that at a hematocrit that is half of normal or lower,
blood viscosity is less than half of that in the circulation.
Given a notably reduced peripheral resistance the or-ganism attempts to compensate by reﬂex vasoconstriction, a response that becomes increasingly prominent
with further drops of hematocrit (and therefore viscosity). Vasoconstriction accompanied by low blood
viscosity depressurizes the capillaries causing them tocollapse, thus lowering functional capillary density(Lindbom and Arfors, 1985).4
Functional capillary density is a critical parameter intissue survival, as shown by the study of Kerger, et al.,
1996,5 who found that this is the only functional anatomical event that separates surviving from non surviving animals subjected to hemorrhagic shock. Conversely there was no difference in tissue oxygenation
between these groups. Consequently the clinical evidence calling for the transfusion of blood is determined
in no small part by capillary collapse, which causes theaccumulation of products of metabolism in the tissue. It
should be evident that further diluting blood beyond
the transfusion trigger with any low viscosity plasma
expander including oxygen carriers, further aggravates
this process. When the plasma expander is also a vasoconstrictor such as in the case of aa cross linked hemoglobin the organism is placed in a precarious condition.
Reversal capillary collapse in extreme hemodilution
cab be obtained by increasing plasma viscosity as demonstrated in the study of Tsai, et al., 1998,6 who induced
hemodilution with dextran 70 kDa and continued unto
extreme hemodilution with dextran 500 kDa, achieving
a circulating plasma viscosity of 2.8 cp and a near normal functional capillary density. This condition was not
attained if extreme hemodilution was performed witheither dextran 70 kDa or the Biopure product Oxyglobin (Biopure Inc., Boston, MA), even though in thelater case total hemoglobin content was 6.7 g Hb/dl(Tsai, 2001).2 Figure 1 summarizes these ﬁndings. A
remarkable side effect of extreme hemodilution withhigh viscosity plasma is that blood ﬂow is signiﬁcantly
increased above control non-hemodiluted values,6 a
phenomenon attributable to the release of shear dependent generated endothelial relaxing factors (Frangos, et al., 1985; de Wit, et al., 1997).7,8
This analysis shows that a viscosity formulation
leading to high plasma values is a critical factor in resuscitation, since the administration of an OCPE ismade in conditions of extreme hemodilution, and giventhat there is no need for using these products prior toreaching the transfusion trigger.
The Appropriate Value for p50
Various cross linked or polymerized hemoglobinsthat have been developed as the oxygen carrier for
OCPEs have resulted in a high value of p50, which has
been incorporated unto the product under the assumption that it is beneﬁcial because it facilitates oxygen

Keio J Med 2002; 51 (1): 17–20

Fig. 1 Experimental ﬁndings in the microcirculation of the awake
hamster model during extreme hemodilution with low and high viscosity plasma expanders. Acute moderate hemodilution is inducedwith dextran 70 kDa (v) until 60% of the red cell concentration hasbeen replaced. Continuing exchange transfusions were made with different viscosity solutions and are presented as a function of hemoglobin. The solutions used were dextran 70 kDa (v), dextran 500 kDa(e), and OxyglobinTM (9) which have viscosities of 1.8, 6.4 and 2.8
cp. Both low visocisity solutions caused vasoconstriction, decreased
functional capillary density, and low perfusion. Conversely, if plasma
viscosity is restored by using 500 kDa dextran, hemodilution can becontinued to extreme values. In the situation microvascular function ismaintained, functional capillary density returns to normal, and tissue
perfusion increase above baseline.9

unloading and tissue oxygenation. The problem is thatthe partial pressure of oxygen in the microcirculation isregulated so that there is a signiﬁcant decrease in oxygen tension from the systemic circulation to the capillaries which typically have a pO2 of about 30 mmHg
as they branch from the arterioles. Since blood p50 is
28 mmHg (human, hamsters) half of the blood oxygenis delivered by arterioles in normal conditions, however
if p50 of the OCPEs is above this value as in the case of
Oxyglobin (p50 ¼ 40 mmHg) most of the oxygen inblood is delivered by the arterioles.
Current results on the partition of oxygen supply
between arterioles and capillaries shows that these vessels extract a signiﬁcant amount of oxygen from thecirculation while consuming a major portion of thisoxygen ﬂux (Tsai, et al., 1998)9 and this consumption isdirectly dependant on the oxygen availability in blood.
The net effect of this process is that if too much oxygen
is unloaded prior to arrival to the capillaries and thisoxygen is consumed by the arterioles, it does not reachthe tissue.
McCarthy, et al., 2001,10 identiﬁed the mechanism offacilitated diffusion as an additional process that has
the potential of over oxygenating the arteriolar wall.
The mechanism takes place at the blood tissue inter-19

face where oxygen traverses the red cell poor or freeplasma layer between the blood column and tissue. In
this space oxygen diffusion takes place by the gradientof pO2 which also determined the gradient of oxyhemoglobin (Nishide, et al., 1997).11 As a consequenceof the large amount of oxygen bound by hemoglobin, theﬂux of oxygen determined by the ﬂux oxy-hemoglobin
is similar for concentrations of hemoglobin that arerelevant for their use as an oxygen carrying plasma expander, therefore this ﬂux is also determined by the p50of the hemoglobin in solution and is increased if this has
a high value. Taking these factors into consideration theappropriate p50 for a noncellular, molecular hemoglobin based OCPE should be low in such a fashion thatoxygen is delivered only where it is need, i.e., the anoxic
portions of the tissue. This approach renders these formulations similar to therapeutic agents with targeted
drug delivery, where the agent in this case is oxygen,delivered only where it is required.
Colloid Osmotic Pressure
In the design of a solution for plasma expansion it
has usually been assumed that the colloid osmoticpressure should be similar to that of blood and in therange of 20–25 mmHg. However a variety of plasma
expanders have zero colloid osmotic pressure (saline,Ringer’s lactate) and the process of small volume resuscitation utilizes ﬂuids with very high osmotic properties. Resuscitation with non colloidal ﬂuids leads totissue edema and is not universally indicated. Conversely there are some advantages in using ﬂuids withhigh colloidal and osmotic pressures, since they cause
tissue ﬂuid to come in the vascular compartment. Of
particular interest is that most conditions of hemorrhage are associated with endothelial edema, which has
been demonstrated to be rapidly reversed upon the introduction of hyperosmotic and hyperoncotic ﬂuids(Mazzoni, et al., 1990).12 This effect is specially relevant
in view of the importance of maintaining functional
capillary density as a part of the process of recovery of
the injured organism.
Synthesis of an Effective Oxygen
Carrying Plasma Expander
Taking this collection of factors into consideration
for the design of an OCPE the ﬂuid that emerges has
fundamentally different properties than blood, since its
oxygen carrying capacity is low, about one third of normal, p50 is equally low and in the neighborhood of
5 mmHg, while viscosity, so that when introduced into
the circulation plasma, viscosity should be of the orderof 3 cP, and colloidal osmotic pressure is also high withvalues greater than 50 mmHg. A ﬂuid with these prop-

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Tsai AG and Intaglietta M: Effective blood substitutes

erties (Hemospan, Sangart Inc., San Diego) is achievedby conjugating hemoglobin with polyethylene glycol,and various formulations have been tested in both animal experiments and human trials with excellent results(Winslow, et al., 1998).13 Furthermore the NO scavenging characteristics do not appear to be relevant orsigniﬁcant since these ﬂuids have the same NO binding constant as other formulations that are vasoactive(Rolhfs, et al., 1998).14
These ﬂuids may in some cases be more effective
than blood because they are speciﬁcally designed tomaintain functional capillary density, which is as necessary as restoring tissue oxygenation for the recovery
from blood losses. This process is not intrinsic to bloodtransfusions, because blood does not increase plasma
viscosity, which is necessary for re-pressurizing thecapillaries.
In the foreseeable future OCPEs will be based onhuman hemoglobin therefore ﬂuids designed according
to the new paradigm are practical since their hemoglobin content is low and more than two units of bloodequivalent unit of resuscitation ﬂuid can be obtained
from one blood. Finally this low oxygen carrying
capacity is practical and safe because it is combined witha signiﬁcant improvement of microvascular function,
therefore these ﬂuids have also therapeutic properties
aimed at improving the microcirculation.
Acknowledgments: Research supported by grants form the UnitedStates Public Health Service, Heart, Lung and Blood Institute BRP
R24 HL-64395, RO1-HL 40696 and RO1-HL 62354, M. Intaglietta
Principal Investigator.

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