Stroma-free Hemoglobin Solution Research Articles

Use of the Labscale Tangential Flow Diafiltrator to Remove Tetrameric Hemoglobin from Polyhemoglobin, Purify Hemolysate, and Concentrate Polyhemoglobin

The Labscale tangential flow diafiltration system in conjunction with polyethersulfone Biomax filters (both purchased from Millipore Inc.) were used in the following studies: (1) A crucial step in the preparation of polyhemoglobin (polyHb) is the removal of tetrameric hemoglobin (Hb), which can cause adverse side-effects. The efficiency for this depends on the integrity of the 100kDa filters. Those with lower integrity were not effective whereas those with a higher integrity of 0.075ml/min were more effective. A filter with an integrity of 0.075ml/min can reduce the initial 11.2% tetrameric Hb concentration to 1.3% using a flow rate of 0.2ml/min. Higher flow rate of 2.2ml/min was not as effective. (2) Filtration with a 100kDa filter was quick and efficient in separating stroma-free hemoglobin solutions from cellular debris from hemolysate. (3) Both 5kDa and 100kDa filters are efficient in concentrating hemoglobin solutions.

VIRUS INACTIVATION IN HEMOGLOBIN SOLUTION BY HEAT TREATMENT

To increase the safety of stroma-free hemoglobin solution (SFH) as an artificial oxygen carrier source, we investigated the effect of heat treatment on virus inactivation in hemoglobin solution. The hemoglobin solution spiked with vesicular stomatitis virus (VSV) was treated at 60 degrees C for 1 hr under either an air or CO atmosphere. VSV was inactivated at >5.8 log10 and >6.0 log10 under the air and CO atmosphere, respectively. Although the methemoglobin rate increased after the heat treatment under the air atmosphere, no methemoglobin formation was observed by the treatment under the CO atmosphere. Isoelectric focusing analysis revealed the denaturation of hemoglobin after the heat treatment under the air, while hemoglobin banding was not altered in the carbonylated condition. Some protein bands other than hemoglobin were weakened or disappeared on SDS-PAGE after the heat treatment under both conditions. In addition, the hemoglobin concentration in the SFH was higher after the heat treatment than before the treatment. These findings indicate that the heat treatment under the CO atmosphere inactivates viruses without hemoglobin denaturation, and hence, this method is a promising approach to prepare a safer SFH as artificial oxygen carriers.

Tissue perfusion and oxygenation with blood substitutes

As an alternative to transfusion of red blood cells, intravenously (iv) administered artificial oxygen (O 2) carriers are intended to increase the reduced O 2 carrying capacity of blood in the case of acute severe anemia, i.e. hemorrhagic shock or extreme normovolemic hemodilution (ANH). Actually, two groups of artificial O 2 carriers are investigated: ultrapurified, stroma-free hemoglobin solutions (SFH) of human or bovine origin and synthetically produced perfluorocarbons (PFC). SFH may be administered in large amounts and are suitable for 1:1 replacement of blood losses in case of hemorrhage as well as for isovolemic exchange of blood during ANH. In both situations SFH solutions effectively restore (hemorrhagic shock) and maintain (extreme ANH) tissue oxygenation despite extremely low hematocrit values. The vasopressor property of the isolated Hb molecule leads to a species-dependent (rodent>pig>human) increase in systemic and pulmonary vascular resistance, but leaves overall distribution of cardiac output uninfluenced. Due to the particulate nature of PFC emulsions, iv administration has to be restricted to small doses (3–4.5 ml/kg body weight for the actually investigated 60% w/v perflubron emulsion) in order to avoid overload of the reticuloendothelial system. Thus PFC emulsions are unsuitable for isovolemic blood replacement in hemorrhagic shock or ANH. Low-dose iv PFC administration in already hemodiluted subjects, however, creates an additional margin of safety to guarantee adequate tissue oxygenation which allows for further, extreme ANH, without risking tissue hypoxia.

Blood substitutes: Haemoglobin therapeutics in clinical practice

Early approaches to the development of oxygen carriers involved the use of stroma-free hemoglobin solutions. These solutions did not require blood typing or crossmatching and could be stored for long periods. In addition, a variety of methods have been developed in chemically modifying and stabilizing the hemoglobin molecule. Several hemoglobin therapeutics are now in clinical trials as temporary alternatives to blood or as therapeutic agents for ischemia. The various hemoglobin products under development are derived from three principal sources: human, bovine and genetically engineered hemoglobin. Diaspirin cross-linked hemoglobin (DCLHb), administered at doses ranging from approximately 20-1000 ml, has been investigated in a number of clinical trials in patients undergoing orthopedic, abdominal aortic repair, major abdominal surgery, cardiac surgery and in critically ill patients with septic shock. In several studies, DCLHb was effective in avoiding the transfusion. However, Baxter Healthcare Corporation (Chicago, Illinois, USA) stopped the development of DCLHb after two unsuccessful trials in trauma patients. Bovine polymerized hemoglobin has also been extensively studied. Several phase II and phase III trials have been performed with this product in hemorrhagic surgery, cardiac surgery and vascular surgery, but data have not yet been published. Hemoglobin therapeutics could provide an important new option as an alternative to blood transfusion. Furthermore, they may be able to provide an immediate on-site replacement for traumatic blood loss, prevent global ischemia and organ failure, treat focal ischemia, and provide effective hemodynamic support for septic shock-induced hypotension.

Solid-phase extraction of phospholipids from hemoglobin solutions using Empore styrene–divinylbenzene disks

Styrene–divinylbenzene Empore disks were investigated for the extraction of phospholipids from red blood cells or aqueous solutions of hemoglobin as a means to reduce the time and solvent use required in sample preparation. Red blood cells are the source for hemoglobin used in the preparation of a hemoglobin-based oxygen carrier which is being developed to replace blood in transfusion therapy. Phospholipids are a major component of the membrane of red blood cells, and are toxic when administered directly into the vasculature. Sensitive analytical methods are required to detect phospholipids to ensure that concentrations in purified hemoglobin are well below toxic levels. This requires isolation from large volumes of purified hemoglobin solutions. The method described utilizes Empore disks to extract phospholipids from 30 ml of stroma free Hb preparations. Phosphatidylethanolamine, phosphatidylinositol, phosphatidylcholine and sphingomyelin were recovered with an average of 92% yield. The recovery of phosphatidylserine was 65%. The use of solvent and time required for sample preparation were reduced by an average of 80% relative to liquid–liquid extraction. The capacity of the 47-mm disk for the total of five phospholipids exceeds 0.3 mg. The method has been used for quantitation of phospholipids in red blood cells and stroma free hemoglobin solutions.

Isovolemic hemodilution with a bovine hemoglobin-based oxygen carrier: Effects on hemodynamics and oxygen transport in comparison with a nonoxygen-carrying volume substitute

Objective : Stroma-free hemoglobin solutions have been shown to maintain oxygen transport in the absence of red blood cells. This study was designed to investigate the impact of such solutions on hemodynamics and oxygen transport during progressive isovolemic hemodilution within and even beyond a clinically relevant range of hematocrit values. Design : Prospective, randomized experimental study comparing a bovine hemoglobin-based oxygen carrier (bHBOC) with a conventional nonoxygen-carrying volume substitute (hydroxyethyl starch [HES]). Setting : Animal laboratory of a university cardiovascular research center. Participants : Splenectomized full-grown foxhounds, anesthetized with pentobarbital and piritramid. Interventions : Twelve splecnectomized foxhounds were anesthetized and mechanically ventilated. Catheters were placed for hemodilution, arterial and venous blood sampling, and hemodynamic measurements. The baseline hematocrit (Hct) value was adjused to 0.35 by an initial isovolemic exchange of blood for identical volumes of HES (10% HES 200/0.5). Thereafter, the hematocrit was progressively reduced by isovolemic hemodilution using either HES ( n = 6) or bHBOC ( n = 6). Measurements and Main Results : Hemodynamic and laboratory parameters of oxygen transport were measured at Hct values of 0.30, 0.20, and 0.10. Oxygen content was directly estiamted using an oxygen-specific fuel cell. Arterial oxygen content at an Hct value of 0.10 nearly doubled in bHBOC-treated dogs as compared with HES-diluted animals ( p < 0.001). This gain in oxygen-carrying capacity was completely negated by a decrase in cardiac output (−32% Hct 0.35 v Hct 0.30; p < 0.001) immediately on the first infusion of bovine hemoglobin. Thus, oxygen delivery was significantly lower as compared with HES-treated dogs at Hct 0.30 and 0.20, but remained stable at a level of 60% of baseline until Hct was 0.10. Both the pulmonary and the systemic vascular resistances increased. Conclusions : Isovolemic hemodilution with bHBOC did not improve systemic oxygen delivery in comparison with a nonoxygen-carrying diluent (HES) in a range of Hct values down to 0.10. Unchanged mixed venous lactate levels and stable oxygen consumption indicate sufficiently maintained oxygen delivery. This might become advantageous in patients who are unable to adequately increase cardiac output during hemodilution.

Validation of the heat treatment step used in the production of diaspirin crosslinked hemoglobin (DCLHb) for viral inactivation--effect of crosslinking.

Two experiments were performed to assess viral inactivation during the crosslinking and heat treatment steps of the DCLHb manufacturing process. Stroma free hemoglobin (SFHb) collected from a large scale manufacturing lot was tested in a 1:680 scaled down system in which the key parameters used in the manufacturing process were replicated. In the first study Porcine Parvovirus (PPV), a non-enveloped virus, was used to assess inactivation, while in the second study Bovine Viral Diarrhea Virus (BVDV), an enveloped virus, was utilized. In both experiments, the SFHb solution was deoxygenated and an aliquot of virus suspension was added. To initiate the crosslinking reaction, a solution of bis (3,5-dibromosalicyl) fumarate (DBBF) in HEPES buffer was added to the test solution. In both experiments the reaction times and the degree of crosslinking were normal. After crosslinking, the reaction mixtures were heated to 74 +/- 1 degrees C over 30 minutes, held at 74 +/- 1 degrees C for 90 minutes, and cooled to less than 10 degrees C over 30 minutes. In each experiment the degree of crosslinking of final product was 100% and yield of hemoglobin recovery was normal. Samples were removed prior to crosslinking, after crosslinking and before, during and after heat treatment for determination of virus titer and evaluation of key process parameters. The results from these experiments were consistent with those obtained from the full scale manufacturing process for the deoxygenation, crosslinking and the heat treatment step during the production of DCLHb. The results of virus assays showed that crosslinking has no effect on viruses and their subsequent inactivation by heat treatment.

Ferrrous hemoglobin scavenging of endothelium derived nitric oxide is a principal mechanism for hemoglobin mediated vasoactivities in isolated rat thoracic aorta.

The exact mechanism of hemoglobin (Hb) associated vasoconstriction has not been elucidated. We investigated this problem using isolated superfused rat aortic rings with intact endothelium. Human stroma-free hemoglobin solution (SFH) at 2uM reversed vaso relaxation induced by 33uM acetylcholine (Ach). Further, pretreatment with 4uM SFH inhibited Ach(333uM) induced dilation. The SFH induced contraction was reversible by glyceryltrinitrate (GTN), a nitric oxide (NO) donor. Preincubation with a NO synthase inhibitor nitro-L-arginine-methyl ester (NAME, 0.4nM) caused almost complete inhibition of the Hb vasoactivity. Unlike SFH (ferrous oxyHb), prenitrosylated SFH (HbNO) or ferric Hb derivatives (e.g., metHb, HbCN) did not elicit vasoconstriction. The presence of 2uM SFH did not significantly reduce the vasodilatory effectiveness of endothelium independent vasodilators isoproteranol (ISO) and papaverine (PPV). These results suggest that a primary mechanism for Hb vasoconstrictor activity is ferrous Hb scavenging of endothelium derived NO, a signal for guanylate cyclase-cGMP mediated smooth muscle relaxation. Additionally, it appears that the Hb induced vasoactivities may be modulated with NO independent vasodilators.

Liver function and morphology after resuscitation from severe hemorrhagic shock with hemoglobin solutions or autologous blood

To test the effects of three hemoglobin solutions on liver function and hepatic morphology after resuscitation from severe hemorrhagic shock. Prospective study. Laboratory. Thirty-three beagle dogs. Hemorrhagic shock was induced in anesthetized dogs by removal of blood at a rate of 2 mL/kg/min until systolic blood pressure (BP) reached 50 mm Hg. BP was maintained at this level 2 hrs by further withdrawing 5 to 10 mL aliquots whenever BP increased > 50 mm Hg. Resuscitation was then initiated with autologous whole blood (n = 7), 4% pyridoxalated-hemoglobin-polyoxyethylene conjugate (4% PHP [n = 6]), 8% pyridoxalated-hemoglobin-polyoxyethylene conjugate (8% PHP [n = 9], or 8% stroma-free hemoglobin (n = 7). Four dogs were managed identically but were not resuscitated. Gross necropsy and histologic examination of the liver were performed on all dogs after 7 days, or earlier if death occurred. In vitro interferences of PHP and stroma-free hemoglobin with liver function tests were determined and recommendations for interpretation of results from blood samples containing PHP and stroma-free hemoglobin were made. Blood was collected before, during, and after resuscitation from hemorrhagic shock. The dogs were then awakened and survivors were monitored daily with blood sampling until they were killed and necropsy was performed. After 7 days, the survival rate following hemorrhagic shock was 100% for whole blood and 4% PHP, 86% for stroma-free hemoglobin, and 33% for 8% PHP. Of the resuscitated dogs not surviving 7 days, all but one died within 27 hrs from coagulopathy. All dogs not resuscitated died within 1.75 hrs after 2 hrs of shock. Bilirubin, alkaline phosphatase, and lactic dehydrogenase concentrations could not be measured due to interferences of stroma-free hemoglobin and PHP. Aspartate (AST) and alanine (ALT) aminotransferase concentrations could be measured after dilution to overcome the interferences. Significant increases in AST and ALT values in all groups 24 hrs after resuscitation were attributed to hypoxic hepatocellular damage associated with the severity of the shock model rather than to the resuscitation fluid. Liver histology showed no changes attributed to toxic damage of hepatocytes in dogs resuscitated with stroma-free hemoglobin or PHP. However, changes, were less severe in dogs resuscitated with 4% PHP than in other groups. Morphologic studies at necropsy and liver function tests in dogs receiving hemoglobin solutions, compared with autologous blood, support the conclusion that the PHP and stroma-free hemoglobin solutions tested did not produce hepatic toxicity when used as resuscitation fluids in this model of severe shock.

Blood Substitutes

Blood Substitutes

Resuscitation from severe hemorrhage

The potential to be successfully resuscitation from severe traumatic hemorrhagic shock is not only limited by the "golden 1 hr", but also by the "brass (or platinum) 10 mins" for combat casualties and civilian trauma victims with traumatic exsanguination. One research challenge is to determine how best to prevent cardiac arrest during severe hemorrhage, before control of bleeding is possible. Another research challenge is to determine the critical limits of, and optimal treatments for, protracted hemorrhagic hypotension, in order to prevent "delayed" multiple organ failure after hemostasis and all-out resuscitation. Animal research is shifting from the use of unrealistic, pressure-controlled, hemorrhagic shock models and partially realistic, volume-controlled hemorrhagic shock models to more realistic, uncontrolled hemorrhagic shock outcome models. Animal outcome models of combined trauma and shock are needed; a challenge is to find a humane and clinically realistic long-term method for analgesia that does not interfere with cardiovascular responses. Clinical potentials in need of research are shifting from normotensive to hypotensive (limited) fluid resuscitation with plasma substitutes. Topics include optimal temperature, fluid composition, analgesia, and pharmacotherapy. Hypotensive fluid resuscitation in uncontrolled hemorrhagic shock with the addition of moderate resuscitative (28 degrees to 32 degrees C) hypothermia looks promising in the laboratory. Regarding the composition of the resuscitation fluid, despite encouraging results with new preparations of stroma-free hemoglobin and hypertonic salt solutions with colloid, searches for the optimal combination of oxygen-carrying blood substitute, colloid, and electrolyte solution for limited fluid resuscitation with the smallest volume should continue. For titrating treatment of shock, blood lactate concentrations are of questionable value although metabolic acidemia seems helpful for prognostication. Development of devices for early noninvasive monitoring of multiple parameters in the field is indicated. Molecular research applies more to protracted hypovolemic shock followed by the systemic inflammatory response syndrome or septic shock, which were not the major topics of this discussion.

Systemic Responses to Sfhs-Infusion in Hemorrhaged Dogs

Anesthetized mongrel (weight range: 16-27 Kg) dogs were prepared for monitoring hemodynamics, blood flow distribution, plasma colloid osmotic pressure and renal functional parameters at various intervals. Removal of 35 ml/Kg blood resulted in marked drop and only partial spontaneous recovery in systemic and pulmonary arterial pressures, cardiac output and organ blood flows (> 50% flow-decrements occurred in kidney, spleen, heart, gut and pancreas); plasma colloid osmotic pressure as well as urine output and creatinine clearance also fell. Group I (n = 6) of dogs was transfused after 45 minutes of hypovolemia with their own anticoagulated blood, while Group II (n = 6) received an equal volume of unmodified 6% stromafree hemoglobin solution (SFHS). Comparison of the two groups' responses to resuscitation yielded some differences. There was a significant overshoot (30 mmHg) in systemic arterial blood pressure accompanied by bradycardia in Group II only. Cardiac output recovered in both groups but was less well sustained in Group II. Cerebral blood flow rose higher and hepatic arterial flow-increment was less in Group II than in Group I; the responses to resuscitation in the other organs were comparable. Colloid osmotic pressure decreased in Group I whereas it rose immediately after resuscitation in Group II, declining thereafter with a converging trend and 30 minutes thereafter the differences were not significant between the groups. Urine excretion and creatinine clearance recovered to comparable extents in both groups, but N-acetyl-beta-D-glucosaminidase (N.A.G.) excretion rose over 10-fold higher in Group II than in Group I. These experiments have defined the response of bled animals to resuscitation with unmodified, unpurified SFHS, when compared to resuscitation with whole blood, showing a less well sustained but adequate hemodynamic and renal functional recovery while revealing indications of early renal tubular cellular injury, providing baseline comparison for testing highly purified and modified hemoglobin solutions.

In vitro oxyhaemoglobin saturation measurements in haemoglobin solutions using fibreoptic pulmonary artery catheters

We compared in vitro oxyhaemoglobin saturations using two pulmonary artery catheters (catheter SO2), with oxyhaemoglobin saturations (SO2) measured by the IL282 co-oximeter and derived partial oxyhaemoglobin saturations (partial SO2) at different oxygen tensions (PO2) in six solutions: whole blood, 50:50 mixture of whole blood and Plasmalyte A (haemodiluted blood), 50:50 mixture of whole blood and 8% pyridoxylated haemoglobin-polyoxyethylene (PHP) conjugate (WB-PHP), 75:25 mixture of 8% PHP and Plasmalyte A solution (PHP66), 50:50 mixture of 8% PHP and Plasmalyte A solution (PHP44) and stroma-free haemoglobin solution (SFH). Calculated P50 values (PO2 vs SO2) were 3.79, 3.58, 3.49, 3.15, 3.04 and 2.07 kPa, respectively. However, if partial SO2 was used the curves were shifted to the left, reducing P50. Catheter SO2 correlated well with SO2 in whole blood (r2 > 0.99 for both catheters), haemodiluted blood (r2 > 0.98 for both catheters) and WB-PHP solution (r2 = 0.94 for both catheters). In PHP44 (r2 = 0.64 and r2 = 0.57), PHP66 (r2 = 0.40 for the Oximetrix and r2 = 0.25 for the Edwards catheter) and SFH solutions (r2 = 0.33 for the Oximetrix and r2 = 0.22 for the Edwards catheter) both catheters performed poorly. We conclude that mixed venous oxyhaemoglobin saturations measured by oximetric pulmonary artery catheters are inaccurate in the presence of haemoglobin solutions. For accuracy a multi-wavelength co-oximeter should be used if blood containing PHP or SFH is to be analysed.

Oxyradical Generation After Resuscitation of Hemorrhagic Shock with Blood or Stromafree Hemoglobin Solution

Hypovolemic states are characterized by inadequate tissue perfusion; when this state is reversed, the reintroduction of oxygen is accompanied by the excess generation of oxyradicals and these, in turn, may cause "reperfusion injury" in susceptible tissues. When hemoglobin solution is used to resuscitate the hypovolemic state, the generation of oxyradicals may be enhanced by catalytic means. The generation of oxyradicals was estimated in dogs subjected to the acute removal of 35 ml/Kg blood, and resuscitated 45 mins thereafter with an equal volume of either autologous blood (Group I, n = 6) or 6% stromafree hemoglobin solution (S.F.H.S.) (Group II, n = 6). Hepatic and pancreatic enzymes were measured in blood drawn at intervals. The hypovolemic state was characterized by profound hypotension which was reversed by resuscitation. Oxyradical generation in arterial blood samples, drawn at various times, was estimated by the generation of oxidation products (2,3- and 2,5-dihydroxybenzoic acid) of exogenously administered sodium salicylate, determined by HPLC in plasma samples extracted with diethyl ether. Salicylate oxidation products rose significantly above the baseline value in Group I dogs, whereas they rose 5-6-fold higher than the baseline values in those of Group II. The actual values attained and the increments were significantly (p < .05) greater in Group II than in Group I. In the group resuscitated with S.F.H.S., catalytically active iron concentration in plasma also rose 10-12-fold higher and was associated with spuriously elevated levels of gamma-glutamyl transferase due to interference with the assay. These findings are consistent with the hypothesis that blood-resuscitation of hypovolemic shock is accompanied by oxyradical generation of a modest degree; in contrast, S.F.H.S.-resuscitation introduces catalytically active iron and is accompanied by oxyradical generation of a significantly greater degree.

Function of isolated rabbit hearts perfused with erythrocyte suspension is not stable but improvement may be feasible with hemoglobin solution.

In the present study isolated rabbit hearts were perfused with erythrocyte suspensions (hematocrit 21.5 +/- 0.5%) or hemoglobin solutions according to Langendorff with a constant flow at 37 degrees C. In preliminary experiments three types of stroma-free hemoglobin were used: unmodified, but carefully purified, stroma-free hemoglobin (SFHb), HbNFPLP which is a chemically modified Hb molecule and polyHbNFPLP which is a polymer of HbNFPLP. In hearts perfused with erythrocyte suspensions left ventricular developed pressure and oxygen consumption decreased and perfusion pressure increased steadily from the beginning of the perfusion. Dark spots appeared on the surfaces of these hearts, which were the result of extravasation of erythrocytes. As a consequence capillaries probably became obstructed, leading to reduced cardiac function. Hearts perfused with stroma-free hemoglobin solutions showed an initial increase in left ventricular developed pressure after switching from Tyrode perfusion to perfusion with hemoglobin solutions. Left ventricular developed pressure and perfusion pressure were stable for about 2 hours in hearts perfused with SFHb and were reasonable for 2 hours when the heart was perfused with HbNFPLP or more than 4 hours with polyHbNFPLP. More extensive experiments with stroma-free hemoglobin solutions when these become available in sufficient quantities have, according to the results from preliminary experiments, the potential of showing good oxygen supply resulting in reasonable cardiac function.

Coagulation responses of human plasma after hemodilution with hemoglobin solution in-vitro.

To assess the potential for interference of Hb with the normal coagulation mechanism, we performed in-vitro hemodilution tests. Platelet rich plasma (PRP) was prepared from citrated blood samples of 5 normal volunteers diluted 3:1, 1:1, and 1:3 volume ratio with human stroma-free hemoglobin solution (SFH) or human albumin (HSA). Coagulation kinetics and clot strength were assessed with a thrombelastograph (TEG). Extrinsic and intrinsic coagulation factors were assessed measuring prothrombin time (PT) and activated partial thromboplastin time (aPTT) with an optical coagulation timer. Statistical significance was assessed using ANOVA and Neuman-Keuls tests at p < 0.05. At all dilutions, SFH diluted plasma showed significantly prolonged initial rate of clot formation compared to undiluted control or HSA (p < 0.05). However, there was no difference in formed clot strength between SFH and HSA. At high Hb concentrations Hb seems to interfere with the optical measurements of coagulation times (particularly aPTT). SFH appears to interfere with the initial phase coagulation mechanism in human plasma in-vitro; further study is needed to clarify the cause. In measuring coagulation times of plasma containing Hb a non-optical instrument should be considered.

Systemic hemodynamic and hepatic microvascular responses to a 33% blood volume exchange with whole blood, stroma-free hemoglobin, and oxypolyhemoglobin solutions.

Little is known about the microvascular effects of blood replacement solutions. This study was undertaken to develop an animal model suitable for studies of the microcirculatory effects of such solutions and to investigate microvascular responses to isovolemic transfusion with stroma-free hemoglobin (SFH), whole donor blood, or a new potential blood substitute solution containing oxypolyhemoglobin (OPH) as an oxygen carrier. Hamster livers were exposed and the microcirculation studied using intravital epifluorescent video microscopy. 33% blood volume replacement with SFH elevated systemic blood pressure by 25 Torr. Accompanying this increase in pressure was a 36% decrease in sinusoidal blood flow velocity and a 10% decrease in terminal hepatic venular diameters. Terminal portal venular diameters did not change. Decrease in liver sinusoidal perfusion was not due to neutrophil mediated injury, as myeloperoxidase activity in jejunum, liver, kidney, and lung remained unchanged. The reduction in perfusion was likely due to systemic vasoconstriction produced by SFH. In contrast, transfusion with whole blood did not change any of the measured parameters showing the excellent stability of the model. OPH transfused animals exhibited only a small 10 Torr transient increase in MAP 15 min post-transfusion. By 30 min MAP returned to the pre-infusion value. No significant changes were observed in either venular diameters or sinusoidal velocities in this group of animals. These results demonstrate suitability of this model for studies of the microcirculatory and hemodynamic effects of blood replacement solutions. Furthermore, OPH solution produced only minor transient disturbances in microvascular and systemic parameters.

Detection of erythrocyte membrane components in hemoglobin-based blood substitutes

Two methods for the detection of membrane components in human stroma-free hemoglobin solutions are described. The first is a phospholipid assay with a detection limit of 0.5–1 nmol phospholipid/ml hemoglobin-solution. For the detection of membrane proteins an immunoassay with a monoclonal antibody against glycophorin α was developed (detection limit 0.01% of the original amount). These methods were used to determine the purity of Hb solutions prepared in two different ways. Hb solutions prepared by filtration of red blood cells, gradually swollen in hypotonic buffer, contained 0.25% of the original amount of phospholipid and no detectable glycophorin α. For Hb solutions prepared in a similar way from red blood cells lysed in water, the values for phospholipid and glycophorin α were 2.5% and 0.06%, respectively. The determination of both glycophorin α and phospholipid gives a useful indication of the purity of Hb solutions.

Detection of membrane fragments in hemoglobin solutions.

Two methods for the detection of membrane components in human stroma-free hemoglobin (SFHb) solutions are described. The first method is a phospholipid assay with a detection limit of 0.5-1 nmol phospholipid/ml SFHb. For the detection of membrane proteins an immunoassay with a monoclonal antibody against glycophorin alpha was developed (detection limit 0.01% of the original amount). The determination of both glycophorin alpha and phospholipid yields useful information on the purity of SFHb solutions, as was shown by determination of the purity of two SFHb solutions prepared in different ways.

Systemic hemodynamic and renal effects of unmodified human SFHS in dogs.

Isovolumic exchange transfusion (25% of total estimated blood volume) was carried out in the anesthetized dogs using 9 g/dl of unmodified human stroma-free hemoglobin solution (SFHS). The objective was to determine the systemic hemodynamic, blood distribution and renal effects of SFHS over a 2-3 hour period post-exchange. At 30 minutes after the exchange, blood pressure rose from 114 +/- 117 to 133 +/- 22 mmHg, but this rise was not sustained thereafter. Mean pulmonary arterial blood pressure rose from 8 +/- 3 to 13 +/- 2 mmHg, and remained above the pre-exchange level up to 3 hours post-exchange. Cardiac output remained within normal limits. Significant flow-increments were seen at 30 minutes in heart, brain, liver, gut, and kidney, but these were also not sustained. A fall in glomerular filtration rate (GFR) occurred after the exchange and remained below the pre-exchange level. A reduction in urine flow at 150 minutes post-exchange was observed and was accompanied by a reduction in urinary electrolyte excretion. The findings suggest that the initial effects of the administration of unmodified stroma-free hemoglobin solution are those of peripheral vasoconstriction which does not appear to significantly restrict flow to the vital organs, such as heart and brain. Unmodified hemoglobin was found to cause a decrease in renal function.