Article

Intravenous infusion of Hb‐vesicles (artificial oxygen carriers) after repetitive blood exchange with a series of plasma expanders (water‐soluble biopolymers) in a rat model

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  • Saitama-city-hospital
Article

Intravenous infusion of Hb‐vesicles (artificial oxygen carriers) after repetitive blood exchange with a series of plasma expanders (water‐soluble biopolymers) in a rat model

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Abstract

Hemoglobin-vesicles (HbV) are artificial oxygen carriers developed for use as a transfusion alternative. The extremely high concentration of the HbV suspension (solutes, ca. 16 g/dl; volume fraction, ca. 40 vol%) provides a sufficient oxygen carrying capacity to maintain oxygen metabolism. A suspension of HbV has no colloid osmotic pressure (COP). Consequently, a combination of a plasma expander is necessary for a massive dose of HbV. Clinically available plasma expanders include hydroxyethyl starch (HES), modified gelatin (MFG), or recombinant human serum albumin (rHSA). Our previous studies confirmed that these water-soluble biopolymers interact with HbV to induce flocculation of HbV reversibly by depletion interaction, especially with MFG and high molecular weight HES. It remains unknown whether such flocculate formation in blood might affect animal's hemodynamics. Using a rat model, we tested infusion of a series of plasma expander to maintain the blood volume (level of blood exchange led to 60%) at repeated hemorrhages and the subsequent infusion of HbV (20 ml/kg, 36% of blood volume). All rats survived for 4 hr after the infusion of HbV; hemodynamic and respiratory functions were preserved, indicating that the flocculation does not induce capillary embolism. Blood exchange with rHSA and subsequent infusion of HbV showed more stable systemic parameters because of the longer retention of rHSA in blood than other plasma substitutes, indicating that rHSA is suitable for combination with HbV in this experimental model. Copyright © 2011 John Wiley & Sons, Ltd.

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... This is the basic protocol of RBC transfusion at massive hemorrhage, minimizing the usage of allogeneic transfusion. In the case of HbV, it is possible to follow the protocol and to inject HbV in place of RBC [21], or inject HbV from the beginning of resuscitation. HbV can be mixed with a plasma expander before injection to provide a physiologically appropriate colloid osmotic pressure to the HbV suspension. ...
... However, both RBC and HbV suspended in saline possess no colloid osmotic pressure. (20)(21)(22)(23)(24)(25) Torr) at higher Hb concentrations. However, HbV as well as RBC shows no COP. ...
... Wistar rats 90% blood exchange with HbV suspended in HSA showed stable hemodynamics [22] Syrian golden hamsters 80% blood exchange with HbV suspended in HSA showed stable hemodynamics and microvascular responses [23,24] Wistar rats 40% blood exchange with HbV suspended in rHSA, and 14 days observation [15] Wistar rats 60% blood exchange with a plasma expander (high Mw HES, low Mw HES, MFG, or rHSA) and subsequent injection of HbV (20 mL/kg) [21] Hemorrhagic shock Wistar rats 50% blood withdrawal and resuscitation, 6 h observation [25] Japanese white rabbits Twice of 40% blood withdrawal and resuscitation [26] New Zealand white rabbits ...
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Hemoglobin (Hb) is the most abundant protein in whole blood. This fact implies that the oxygen binding and releasing function of Hb is the most vital for sustaining life. All Hb is compartmentalized in red blood cells (RBCs) with corpuscular Hb concentration of about 35 g/dL, covered with a thin biomembrane. In spite of its abundance, Hb sometimes shows toxicity once it is leaked from RBCs. The shielding effect of the RBC membrane is physiologically important. Based on this structural importance, we have studied artificial red cells (Hb vesicles, HbV) as artificial oxygen carriers, which encapsulate a purified and concentrated Hb solution in phospholipid vesicles, mimicking the cellular structure of RBCs. Our academic research consortium has clarified the safety and efficacy of this HbV, aiming at clinical applications. Because of some superior characteristics to those of RBCs, HbV has the potential for use not only as a transfusion alternative but also for oxygen and carbon monoxide therapeutics, perfusate for transplant organs, and photosensitizer. In this review paper, such potential applications are summarized.
... Moreover, HbVs are much smaller than RBCs (250 nm vs. 8000 nm), but they recreate the functions of RBC that have been confirmed through numerous animal experiments to test their effectiveness as a resuscitative fluid for massive hemorrhage, hemodilution, and as a prime candidate for cardiopulmonary bypass [15][16][17] (Table 1). Other characteristics resembling those of RBCs are the following: (i) the colloid osmotic pressure of the HbV suspension is zero at [Hb] = 10 g/dL, and it must be co-injected with or suspended in a plasma substitute such as albumin and hydroxyethyl starch [29,30]; (ii) the viscosity of an HbV suspension is adjustable to that of blood (3-4 cP) [31]; (iii) HbVs are finally captured by the reticuloendothelial system (RES), and the components are degraded and excreted promptly [18,32,33]. The HbV particle itself is not eliminated through glomeruli [34]; (iv) co-encapsulation of PLP as an allosteric effector, instead of 2,3-diphosphoglyceric acid, to regulate oxygen affinity ( Fig. 2) [23,35]; and (v) no hemolysis occurs during circulation and the lipid bilayer membrane prevents direct contact of Hb and vasculature. ...
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Chapter
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Hemoglobin vesicles (HbV) as red cell substitutes were prepared from a purified carbonylhemoglobin (HbCO) solution and a lipid mixture composed of phospholipids, cholesterol, and alpha-tocopherol. The diameter was controlled to 251 +/- 87 nm using an extrusion method; the vesicles penetrated through the membrane filters with regulated pore sizes. After the ligand exchanging reaction (HbCO-->HbO2), the oxygen affinity (P50) of HbV was 32 Torr, which was controlled with the coencapsulation of pyridoxal 5'-phosphate. The rate of metHb formation in HbV was nonenzymatically reduced with the coencapsulation of DL-homocysteine. The Hb concentration of the HbV suspension, which was dispersed in a phosphate buffered saline solution (pH 7.4), was controlled at 10 g/dL. At this concentration, the total lipid concentration was 6.2 g/dL and the viscosity, 2.6 cP (230 s-1), was lower than that of the blood (4.4 cP). The HbV suspension showed a typical non-Newtonian flow for a particle dispersion and agreed well with the Casson model. The viscosity at shear rates lower than 23 s-1 showed a maximum with increasing the mixing ratio of human blood, plasma, or albumin, while no maximum was observed for the mixture with washed red blood cells. The aggregates of HbV are formed by interaction with plasma proteins, including albumin, while the aggregates reversibly dissociate at higher shear rate.
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Water-soluble polymers such as dextran and polyethylene glycol are known to induce aggregation and size growth of phospholipid vesicles. The present study addresses the dependence of these processes on vesicle size and concentration, polymer molecular weight, temperature, and compartmentalization of the vesicles and polymers, using static and dynamic light scattering. Increasing the molecular weight of the polymers resulted in a reduction of the concentration of polymer needed for induction of aggregation of small unilamellar vesicles. The aggregation was fully reversible (by dilution), within a few seconds, up to a polymer concentration of at least 20 wt %. At relatively low phosphatidylcholine (PC) concentrations (up to approximately 1 mM), increasing the PC concentration resulted in faster kinetics of aggregation and reduced the threshold concentration of polymer required for rapid aggregation (CA). At higher PC concentrations, CA was only slightly dependent on the concentration of PC and was approximately equal to the overlapping concentration of the polymer (C*). The extent of aggregation was similar at 37 and 4 degrees C. Aggregation of large unilamellar vesicles required a lower polymer concentration, probably because aggregation occurs in a secondary minimum (without surface contact). In contrast to experiments in which the polymers were added directly to the vesicles, dialysis of the vesicles against polymer-containing solutions did not induce aggregation. Based on this result, it appears that exclusion of polymer from the hydration sphere of vesicles and the consequent depletion of polymer molecules from clusters of aggregated vesicles play the central role in the induction of reversible vesicle aggregation. The results of all the other experiments are consistent with this conclusion.
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Poly(ethylene glycol) (PEG5000)-conjugated phosphatidylethanolamine was introduced onto the surface of hemoglobin vesicles (HbV); phospholipid vesicles encapsulating concentrated Hb (d = 0.257 +/- 0.087 micron; P50 = 32 Torr). The obtained PEG-modified HbV (HbV-PEG) was studied for use as a red cell substitute from the viewpoint of rheology, surface properties, and hemodynamics. The viscosity of the unmodified HbV suspended in saline ([Hb] = 10 g/dL) was 2.6 cP (shear rate = 358 s-1, 37 degrees C), less than that of human blood (4 cP). However, when suspended in a 5 g/dL albumin solution (HbV/ albumin), it increased to 8 cP due to the molecular interaction between albumin and vesicles, and the viscosity increased with decreasing shear rate, e.g., 37 cP at 0.58 s-1. As for the HbV-PEG/albumin, on the other hand, the viscosity was 3.5 cP at 358 s-1 and was comparable with that of human blood. Optical microscopy showed formless flocculated aggregates of the unmodified HbV, while no aggregates were confirmed for the HbV-PEG. The steric hindrance of PEG chains seemed to be effective in preventing intervesicular access and the resulting aggregation. To estimate the flow profiles in the capillaries, the suspensions were allowed to penetrate through isopore membrane filters (pore size = 0.4-8 microns, cf. capillary diameter = 4-10 microns). The penetration rate of the HbV-PEG/albumin was higher than that of the unmodified HbV/albumin due to the suppression of aggregation, whereas both of them were significantly higher than that of human blood due to the smaller size of vesicles than RBC. Ninety percent exchange transfusion was performed with the HbV-PEG/albumin or HbV/albumin in anesthetized Wistar rats (n = 6). The blood flow in the abdominal aorta increased 1.5 times, and the total peripheral resistance decreased in the HbV-PEG/albumin-administered group in comparison with the HbV/albumin group. As for the blood gas parameters, the base excess and pH remained at higher levels in the HbV-PEG/albumin group, and the O2 tension in mixed venous blood for the HbV-PEG/albumin group tended to be maintained at a higher level than that for the HbV/albumin group. Thus, the PEG modification of HbV reduced the viscosity by the suppression of aggregation and resulted in prompt blood circulation in vivo.
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A series of hemoglobin (Hb)-based O(2) carriers, acellular and cellular types, were synthesized and their physicochemical characteristics were compared. The acellular type includes intramolecularly cross-linked Hb (XLHb), polyoxyethylene (POE)-conjugated pyridoxalated Hb (POE-PLP-Hb), hydroxyethylstarch-conjugated Hb (HES-XLHb), and glutaraldehyde-polymerized XLHb (Poly-XLHb). The cellular type is Hb-vesicles (HbV) of which the surface is modified with POE (POE-HbV). Their particle diameters are 7 +/- 2, 22 +/- 2, 47 +/- 17, 68 +/- 24, and 224 +/- 76 nm, respectively, thus all the materials penetrate across membrane filters with 0.4 microm pore size, though only the POE-HbV cannot penetrate across the filter with 0.2 microm pore size. These characteristics of permeability are important to consider an optimal particle size in microcirculation in vivo. POE-PLP-Hb ([Hb] = 5 g/dL) showed viscosity of 6.1 cP at 332 s(-1) and colloid osmotic pressure (COP) of 70.2 Torr, which are beyond the physiological conditions (human blood, viscosity = 3-4 cP, COP = ca. 25 Torr). XLHb and Poly-XLHb showed viscosities of 1.0 and 1.5 cp, respectively, which are significantly lower than that of blood. COP of POE-HbV is regulated to 20 Torr in 5% human serum albumin (HSA). HES-XLHb and POE-HbV/HSA showed comparable viscosity with human blood. Microscopic observation of human red blood cells (RBC) after mixing blood with POE-PLP-Hb or HES-XLHb disclosed aggregates of RBC, a kind of sludge, indicating a strong interaction with RBC, which is anticipated to modify peripheral blood flow in vivo. On the other hand, XLHb and POE-HbV showed no rouleaux or aggregates of RBC. The acellular Hbs (P(50) = 14-32 Torr) have their specific O(2) affinities determined by their structures, while that of the cellular POE-HbV is regulated by coencapsulating an appropriate amount of an allosteric effector (e.g., P(50) = 18, 32 Torr). These differences in physicochemical characteristics between the acellular and cellular types indicate the advantages of the cellular type from the physiological points of view.
Article
The stability of hemoglobin vesicles (HbV) as an oxygen infusion was tested during the storage for 1 year at 4, 23, and 40 degrees C. The surface of the HbV was modified with poly(ethylene glycol) (PEG), and the suspension was deoxygenated with nitrogen bubbling. The samples stored at 4 and 23 degrees C showed a stable dispersion state for 1 year, though the sample stored at 40 degrees C showed the precipitation and decomposition of vesicular components, a decrease in pH, and 4% leakage of total Hb after 1 year. The PEG chains on the vesicular surface stabilize the dispersion state and prevent the aggregation and fusion due to their steric hindrance. The original metHb content (ca. 3%) before the preservation gradually decreased to less than 1% in all the samples after 1 month due to the presence of homocysteine inside the vesicles which consumed the residual oxygen and gradually reduced the trace amount of metHb. The rate of metHb formation was strongly dependent on the partial pressure of oxygen, and no increase in metHb formation was observed due to the intrinsic stability of the deoxygenated Hb. Preservation at 4 and 23 degrees C slightly reduced P(50) (increased the oxygen affinity) from 38 Torr to 32 and 31 Torr, respectively. These results indicate the possibility that HbV suspension can be stored at room temperature for at least 1 year.
Article
Hemoglobin-vesicles (HbV) have been developed for use as artificial O(2) carriers in which a purified Hb solution is encapsulated within a phospholipid bilayer membrane. In this study, bovine Hb (BHb) was tested as a source of HbV instead of human Hb (HHb). We compared the preparation process and characteristics of BHbV with those of HHbV. The purification of BHb was effectively performed simply with an ultrafiltration system including a process for removing virus and scrapie reagent. The removal ratio of the phospholipid components of bovine red blood cells was over 99.99%, and the protein purity was over 99.9%. The deoxygenated and carbonylated BHb showed denaturation transition temperatures at 83 and 87 degrees C, respectively, which are higher than those of HHb (80 and 78 degrees C, respectively), and resistant to pasteurization (60 degrees C, 10 h). The purified BHb was concentrated to over 40 g/dl, and encapsulated in a phospholipid bilayer membrane to form BHbV with a diameter of about 280 nm. The O(2) affinity (P(50)) of the BHbV was regulated by coencapsulation of an appropriate amount of Cl(-) (as NaCl), which binds to BHb as an allosteric effector, in the range 16-28 Torr, comparable to human blood (P(50) = 28 Torr). This is quite simple in comparison with HHb which requires phosphate derivatives such as pyridoxal 5'-phosphate as a replacement for 2,3-diphoshoglyceric acid. The viscosity and colloid osmotic pressure of the BHbV when suspended in 5% human serum albumin are 3.5 cP and 20 Torr, respectively, comparable to those of human blood. In conclusion, BHb can be used as a source for the production of HbV, not only because of its abundance in the cattle industry, but also because of the physicochemical advantages of the purification process, thermal stability, and regulation of O(2) affinity in comparison with HHb.
Article
We are aiming to improve the encapsulation efficiency of proteins in a size-regulated phospholipid vesicle using an extrusion method. Mixed lipids (1,2-dipalmitoyl-sn-glycero-3-phosphatidylcholine (DPPC), cholesterol, 1,5-dipalmitoyl-l-glutamate-N-succinic acid (DPEA), and 1,2-distearoyl-sn-glycero-3-phosphoethanolamine-N-[monomethoxy poly(ethylene glycol) (5,000)] (PEG-DSPE) at a molar ratio of 5, 5, 1, and 0.033 were hydrated with a NaOH solution (7.6 mM) to obtain a polydispersed multilamellar vesicle dispersion (50 nm to 30 microm diameter). The polydispersed vesicles were converted to smaller vesicles having an average diameter of ca. 500 nm with a relatively narrow size distribution by freeze-thawing at a lipid concentration of 2 g dL(-)(1) and cooling rate of -140 degrees C min(-1). The lyophilized powder of the freeze-thawed vesicles was rehydrated into a concentrated protein solution (carbonyl hemoglobin solution, 40 g dL(-1)) and retained the size and size distribution of the original vesicles. The resulting vesicle dispersion smoothly permeated through the membrane filters during extrusion. The average permeation rate of the freeze-thawed vesicles was ca. 30 times faster than that of simple hydrated vesicles. During the extrusion process, proteins were encapsulated into the reconstructed vesicles with a diameter of 250 +/- 20 nm.
Article
Hemoglobin-vesicle (HbV) has been developed to provide oxygen-carrying ability to plasma expanders. Its ability to restore the systemic condition after hemorrhagic shock was evaluated in anesthetized Wistar rats for 6 hrs after resuscitation. The HbV was suspended in 5 g/dL recombinant human serum albumin (HbV/rHSA) at an Hb concentration of 8.6 g/dL. Prospective, randomized, controlled trial. Department of Surgery, School of Medicine, Keio University. Forty male Wistar rats. The rats were anesthetized with 1.5% sevoflurane inhalation throughout the experiment. Polyethylene catheters were introduced through the right jugular vein into the right atrium for infusion and into the right common carotid artery for blood withdrawal and mean arterial pressure monitoring. Shock was induced by 50% blood withdrawal. The rats showed hypotension (mean arterial pressure = 32 +/- 10 mm Hg) and significant metabolic acidosis and hyperventilation. After 15 mins, they received HbV/rHSA, shed autologous blood (SAB), washed homologous red blood cells (wRBC) suspended in rHSA (wRBC/rHSA, [Hb] = 8.6 g/dL), or rHSA alone. The HbV/rHSA group restored mean arterial pressure to 93 +/- 8 mm Hg at 1 hr, similar to the SAB group (92 +/- 9 mm Hg), which was significantly higher compared with the rHSA (74 +/- 9 mm Hg) and wRBC/rHSA (79 +/- 8 mm Hg) groups. There was no remarkable difference in the blood gas variables between the resuscitated groups; however, two of eight rats in the rHSA group died before 6 hrs. After 6 hrs, the rHSA group showed significant ischemic changes in the right cerebral hemisphere relating to the ligation of the right carotid artery followed by cannulation, whereas the HbV/rHSA, SAB, and wRBC/rHSA groups showed less changes. HbV suspended in recombinant human serum albumin provides restoration from hemorrhagic shock that is comparable with that using shed autologous blood.
Article
This study clarifies the contribution to overall osmotic kinetics of colloid osmotic pressure (Pi) and the interaction of synthetic colloids with the membrane. Solutions (6%) of dextran with weight average molecular weight (MW(w)) 68 800 (DEX 70), dextran with MW(w) 40 000 (DEX 40), hydroxyethyl starch with MW(w) 70 000 (HES 70), gelatin with MW(w) 60 000 and albumin were tested. An osmotic flow cell fitted with membranes of molecular weight cutoff size 30 000 or 50 000 was used to measure time-dependent changes in Pi for each of these solutions. A linear viscoelastic model was fitted to the curve describing changes to Pi as a function of time. Values of total effective Pi for DEX 40 and DEX 70 were larger than those for HES 70, gelatin, and albumin. As an index of solute-solvent exchange rate at the membrane surface, these values were in the order DEX 40 > DEX 70, HES 70 > gelatin, albumin. The findings suggest that DEX 40 may be preferable for the temporary restoration of plasma volume because of a heightened initial osmotic force. In contrast, the osmotic force exerted by gelatin is slower to increase but is likely to be longer lasting in vivo as a result of the inhibition of gelatin from penetrating the capillary membrane due to its interaction with negatively charged groups in the endothelial glycocalyx.
Article
The methylotrophic yeast Pichia pastoris is well known as a host strain for the production of a variety of heterologous proteins. We have used P. pastoris for the production of recombinant human serum albumin (rHSA), for which we have developed efficient and specialized downstream processes. Results from structural analysis suggest that purified rHSA possesses an identical conformation to plasma derived human albumin (pdHA) and no difference from pdHA has been observed in neo-antigenicity. Host-cell-derived impurities (i.e. Pichia yeast component, DNA and mannan) have been evaluated in the purification process as well as in the drug substance and relevant specifications established. The efficacy and safety of rHSA have been tested in clinical studies and no difference from pdHA has been found in comparative study. Such studies have confirmed rHSA to have high efficacy with little or no adverse reaction.
Article
Hemoglobin-vesicles (HbVs; diameter, 251 +/- 81 nm) are artificial O(2) carriers. Their efficacy for acute exchange transfusion has been characterized in animal models. However subsequent profiles of recovery involving the degradation of HbV in the reticuloendothelial system (RES) and hematopoiesis remain unknown. Isovolemic 40 percent exchange transfusion was performed in 60 male Wistar rats with HbV suspended in 5 g per dL recombinant human serum albumin (rHSA; HbV/rHSA, [Hb] = 8.6 g/dL), stored rat RBCs suspended in rHSA (sRBC/rHSA), or rHSA alone. Hematological and plasma biochemical analyses and histopathological examination focusing on the spleen were conducted for the subsequent 14 days. The reduced hematocrit (Hct) level (26%) for the HbV/rHSA and rHSA groups returned to its original level (43%) in 7 days. Plasma erythropoietin was elevated in all groups: the rHSA group showed the highest value on Day 1 (321 +/- 123 mIU/mL) relating to the anemic conditions (HbV/rHSA, 153 +/- 22; sRBC/rHSA, 63 +/- 7; baseline, 21 +/- 3). Simultaneously, splenomegaly occurred in all the groups as HbV/rHSA > rHSA > sRBC/rHSA. Histopathologically, the accumulated HbV in the spleen was undetectable by Day 14, but hemosiderin was deposited in slight quantities for both the HbV/rHSA and sRBC/rHSA groups. Considerable amounts of erythroblasts were apparent in the spleens of both the rHSA and the HbV/rHSA groups. HbVs were phagocytized and degraded in RES, a physiological compartment for the degradation of RBCs, and the elevated erythropoietic activity resulted in the complete recovery of Hct within 7 days in the rat model.
Article
Artificial oxygen carriers such as perfluorocarbon (PFC) emulsions have reached Phase III clinical trials as alternatives to homologous blood, but their rheologic effects have not been characterized. In this study, the rheologic effects of PFC emulsion in the presence of clinically used volume expanders were investigated. The effects of a new PFC emulsion (small droplet size with narrow size distribution) at two PFC concentrations (4 and 8 g/dL) on plasma and whole-blood viscosity in the presence of human albumin solution (HAS), hydroxyethyl starch (HES), or modified fluid gelatin (MFG) were investigated. Three hematocrit (Hct) levels were investigated: 30, 20, and 13 percent. Plasma, PFC emulsions, and whole-blood viscosity, with a Couette viscometer, and RBC elongation, with an ektacytometer, were measured for shear rates of 0.2 to 128 per second. The two PFC concentrations increased plasma and whole-blood viscosities. Viscosity values similar to physiologic ones (Hct level, 40%) were observed at: 1) Hct level of 13 percent, with 4 or 8 g per dL MFG-PFC; 2) Hct level of 20 percent, with 4 g per dL MFG-PFC; and 3) Hct level of 30 percent, with 4 g per dL HES-PFC and 4 and 8 g per dL HAS-PFC. RBC deformability was unchanged. It is concluded that this new PFC emulsion increases plasma and blood viscosity and that among the three studied volume expanders, the interaction with MFG can result in viscosity values above the physiologic one even at low Hct values. The possible consequences of the increased viscosity at low Hct values are discussed.
Article
Hemoglobin vesicles (HbV) or liposome-encapsulated Hbs are artificial oxygen carriers that have been developed for use as transfusion alternatives. The extremely high concentration of the HbV suspension (solutes, ca. 16 g/dL; volume fraction, ca. 40 vol %) gives it an oxygen-carrying capacity that is comparable to that of blood. The HbV suspension does not possess a colloid osmotic pressure. Therefore, HbV must be suspended in or co-injected with an aqueous solution of a plasma substitute (water-soluble polymer), which might interact with HbV. This article describes our study of the rheological properties of HbV suspended in a series of plasma substitute solutions of various molecular weights: recombinant human serum albumin (rHSA), dextran (DEX), modified fluid gelatin (MFG), and hydroxylethyl starch (HES). The HbV suspended in rHSA was nearly Newtonian. Other polymers-HES, DEX, and MFG-induced HbV flocculation, possibly by depletion interaction, and rendered the suspensions as non-Newtonian with a shear-thinning profile (10(-4)-10(3) s(-1)). These HbV suspensions showed a high storage modulus (G') because of the presence of flocculated HbV. However, HbV suspended in rHSA exhibited a very low G'. The viscosities of HbV suspended in DEX, MFG, and high-molecular-weight HES solutions responded quickly to rapid step changes in shear rates of 0.1-100 s(-1) and a return to 0.1 s(-1), indicating that flocculation is both rapid and reversible. Microscopically, the flow pattern of the flocculated HbV that perfused through microchannels (4.5 microm deep, 7 microm wide, 20 cmH2O applied pressure) showed no plugging. Furthermore, the time required for passage was simply proportional to the viscosity. Collectively, the HbV suspension viscosity was influenced by the presence of plasma substitutes. The HbV suspension provides a unique opportunity to manipulate rheological properties for various clinical applications in addition to its use as a transfusion alternative.