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Background: Human polymerized hemoglobin (PolyHeme, Northfield Laboratories) is a universally compatible oxygen carrier developed to treat life-threatening anemia. This multicenter phase III trial was the first US study to assess survival of patients resuscitated with a hemoglobin-based oxygen carrier starting at the scene of injury., Study Design: Injured patients with a systolic blood pressure

Background: Acute chest syndrome (ACS) is a complication of sickle cell disease that can cause significant morbidity. Transfusion therapy has been shown to significantly increase oxygenation in patients with ACS and RBC exchange is considered the standard of care in patients at high risk of respiratory failure., Case Report: A patient with ACS and several high-risk features, including thrombocytopenia, profound anemia, bilateral pulmonary infiltrates, staphylococcal sepsis, and pulmonary embolism is presented. The patient refused transfusion on religious grounds and received 12 units of human polymerized Hb solution (poly SFH-P injection, PolyHeme, Northfield Laboratories) over the course of 13 days. The patient's respiratory status improved and she was discharged home without receiving RBC transfusions., Conclusion: This is the first reported case that describes the use of PolyHeme in a patient with sickle cell disease, ACS, and sepsis. This therapy is thought to have been lifesaving for this patient.

Background: Human polymerized hemoglobin (PolyHeme, Northfield Laboratories, Evanston, IL) is a universally compatible, immediately available, disease-free, oxygen-carrying resuscitative fluid being developed as a red cell substitute for use in urgent blood loss. PolyHeme should be particularly useful when red cells may be temporarily unavailable. This article assesses survival at life-threatening RBC hemoglobin concentration ([Hb]) in massively bleeding patients who do not receive red cells., Study Design: There were 171 patients who received rapid infusion of 1 to 20 units (1,000 g, 10 L) of PolyHeme in lieu of red cells as initial oxygen-carrying replacement in trauma and urgent surgery. The protocol simulated the unavailability of red cells, and the progressive fall in RBC [Hb] in bleeding patients was quantified. Thirty-day mortality was compared with a historical control group of 300 surgical patients who refused red cells on religious grounds., Results: A total of 171 patients received rapid infusion of 1 to 2 units (n = 45), 3 to 4 units (n = 45), 5 to 9 units (n = 47), or 10 to 20 units (n = 34) of PolyHeme. Forty patients had a nadir RBC [Hb] < or = 3 g/dL (mean, 1.5 +/- 0.7 g/dL). But total [Hb] was adequately maintained (mean, 6.8 +/- 1.2 g/dL) because of plasma [Hb] added by PolyHeme. The 30-day mortality was 25.0% (10/40 patients) compared with 64.5% (20/31 patients) in historical control patients at these RBC [Hb] levels., Conclusions: PolyHeme increases survival at life-threatening RBC [Hb] by maintaining total [Hb] in the absence of red cell transfusion. PolyHeme should be useful in the early treatment of urgent blood loss and resolve the dilemma of unavailability of red cells.

Background: Human polymerized hemoglobin (PolyHeme) is a universally compatible, disease-free, oxygen-carrying resuscitative fluid. This is the first prospective, randomized trial to compare directly the therapeutic benefit of PolyHeme with that of allogeneic red blood cells (RBCs) in the treatment of acute blood loss., Study Design: Forty-four trauma patients (33 male, 11 female) aged 19-75 years with an average Injury Severity Score (ISS) score of 21+/-10 were randomized to receive red cells (n = 23) or up to 6 U (300 g) of PolyHeme (n = 21) as their initial blood replacement after trauma and during emergent operations., Results: There were no serious or unexpected adverse events related to PolyHeme. The PolyHeme infusion of 4.4+/-2.0 units (mean +/- SD) resulted in a plasma [Hb] of 3.9+/-1.3 g/dL, which accounted for 40% of the total circulating [Hb]. There was no difference in total [Hb] between the groups before infusion (10.4+/-2.3 g/dL control vs. 9.4+/-1.9 g/dL experimental). At end-infusion the experimental RBC [Hb] fell to 5.8+/-2.8 g/dL vs. 10.6+/-1.8 g/dL (p < 0.05) in the control, although the total [Hb] was not different between the groups or from pre-infusion. The total number of allogeneic red cell transfusions for the control and experimental groups was 10.4+/-4.2 units vs. 6.8+/-3.9 units (p < 0.05) through day 1, and 11.3+/-4.1 units vs. 7.8 +/-4.2 units (p = 0.06) through day 3., Conclusions: PolyHeme is safe in acute blood loss, maintains total [Hb] in lieu of red cells despite the marked fall in RBC [Hb], and reduces the use of allogeneic blood. PolyHeme appears to be a clinically useful blood substitute.

Although the efficacy of hemoglobin-based oxygen carriers was established more than 60 years ago, all prior clinical trials have demonstrated significant toxicity characterized by renal dysfunction, gastrointestinal distress, and systemic vasoconstriction. The mechanisms of these toxicities now appear to be understood. Tetrameric forms of the hemoglobin molecule extravasate from the circulation and interact with endothelium-derived relaxing factor, leading to unopposed vasoconstriction. Although numerous efforts are under way to chemically modify the native tetramer, it is likely that all tetrameric forms of the hemoglobin molecule will continue to extravasate. We have focused on developing a polymerized form of hemoglobin that is virtually free of unreacted tetramer. The development and characterization of this polymerized pyridoxylated hemoglobin solution (Poly SFH-P) is described. Clinical trials have been completed successfully in volunteers and are now under way to assess the safety and efficacy of Poly SFH-P as a clinically useful red blood cell substitute for treatment of acute blood loss in the setting of trauma and surgery.

Although the efficacy of hemoglobin-based oxygen carriers was established more than 60 years ago, all prior clinical trials have demonstrated significant toxicity characterized by renal dysfunction, gastrointestinal distress, and systemic vasoconstriction. The mechanisms of these toxicities now appear to be understood. Tetrameric forms of the hemoglobin molecule extravasate from the circulation and interact with endothelial derived relaxing factor, leading to unopposed vasoconstriction. Although numerous efforts are underway to chemically modify the native tetramer, it is likely that all tetrameric forms of the hemoglobin molecule will continue to extravasate. We have focused on developing a polymerized form of hemoglobin that is virtually free of unreacted tetramer. The development and characterization of this polymerized pyridoxylated hemoglobin solution (Poly SFH-P) is described. Clinical trials have been completed successfully in volunteers, and are now underway to assess the safety and efficacy of Poly SFH-P as a clinically useful red cell substitute in the treatment of acute blood loss in the setting of trauma and surgery.

Attempts to develop a hemoglobin-based red cell substitute have spanned many decades, but no clinically useful product has been produced to date. The issues preventing clinical application primarily are ones of safety--not efficacy. Numerous animal studies have documented the efficacy of SFH. Although effective, the solution has limitations that have caused concern. Oncotic considerations limit the concentration of the infusate SFH to 6 to 8 g/dL, or half-normal. Owing to the loss of organic phosphate modulators of P50, such as 2,3-DPG, the P50 of SFH is typically between 12 and 14 mm Hg, which is also half the normal value. And finally, the intravascular half-life of SFH is too short, ranging only from 2 to 6 hr. Polymerization provides a means of correcting these limitations. The high oxygen affinity can be greatly diminished by covalent binding of pyridoxal-5'-phosphate to the N-terminal of the chains. Colloid osmotic pressure exerted by a protein solution is proportional to the number of discrete colloid particles. Through polymerization, the number of colloid particles is reduced, leading to a decrease in COP. Data show that this can be achieved in a reproducible fashion. The rate at which COP diminishes determines the yield of polymeric species, as well as their molecular weight distribution. Polymerization can be controlled to result in a yield of 75% to 85% polymers with a molecular weight distribution of 128 to 400 kd. The number average and the weight average molecular weights indicate that the large proportion of polymers represent the cross linking of two tetramers. The data that reflect the interaction of oxygen with poly-SFH-P indicate that the oxygen carrying function of hemoglobin has not been significantly altered by the chemical modifications. The binding coefficient of oxygen is unchanged. As anticipated, there is a loss of cooperativity (diminished Hill coefficient) between the hemoglobin chains, suggesting structural restrictions in the polymeric species because of cross linking. A reduced alkaline Bohr effect is the expected result, and data confirm this. Finally, some increase in oxygen affinity is to be expected with polymerization. This is indeed the case, although the P50 of poly-SFH-P is comparable to banked blood (18 to 22 mm Hg). To be clinically useful, a modified hemoglobin solution requires a reasonable shelf-life.(ABSTRACT TRUNCATED AT 400 WORDS)

A polymerized pyridoxylated hemoglobin solution (Poly SFH-P) has been prepared with a normal [Hb] of 14 g/dL, a normal COP of 20 to 25 torr, a P50 of 16 to 20 torr, and a plasma T1/2 of 40 to 46 hours. Animals underwent a total exchange transfusion with Poly SFH-P to assess its ability to support hemodynamics and oxygen transport in the absence of red cells. All animals survived the exchange transfusion. Mean arterial pressure (MAP), heart rate (HR), cardiac output (CO), and oxygen consumption (VO2) remained at baseline values at zero hematocrit after the exchange. The final plasma [Hb] at Hct less than 1% was 9.7 +/- 0.4 g/dL. These results are significantly better than previous data with unmodified tetrameric hemoglobin solution (SFH). Poly SFH-P supports life in the absence of red cells. In contrast to SFH, Poly SFH-P achieves a nearly normal [Hb], a longer T1/2, and maintains baseline hemodynamics and oxygen consumption at zero hematocrit. These results document that Poly SFH-P is an effective oxygen carrier that offers greater potential than previous products as a clinically useful red cell substitute.

Hemoglobin solutions are undergoing clinical trials as erythrocyte substitutes. Some of these solutions have higher O2 affinities compared with normal erythrocyte hemoglobin. Also, they appear to interact with endothelial-derived smooth muscle relaxation. The purpose of this study was to evaluate the nature and limits of compensation to acute normovolemic anemia in the erythrocyte-free primate maintained with a hemoglobin solution. The experimental group consisted of six anesthetized paralyzed adult baboons (Papio anubis) that were exchange transfused (ET) with a pyridoxylated polymerized hemoglobin solution [hemoglobin concentration [( Hb]) = 14 g/dl, O2 half-saturation pressure of hemoglobin (P50) = 19.6 Torr] until a hematocrit less than 1% was achieved. They underwent a second ET with Dextran-70 until [Hb] = 1 g/dl. A control group (n = 6) underwent an ET with Dextran-70 until [Hb] = 1 g/dl. Both groups maintained O2 consumption (VO2) until [Hb] = 3 g/dl. Both groups were stable until [Hb] less than 1 g/dl, and both groups increased their cardiac output. The relation between VO2 and O2 delivery was similar for both groups. In vivo P50 and mixed venous O2 tension were significantly lower in the experimental group. The nature and limits of compensation to diminished O2 delivery due to anemia were similar in the two groups.

Hemoglobin solutions have potential as temporary red cell substitutes. Their efficacy has been demonstrated by their ability to maintain life-supporting levels of O2 consumption and CO2 production in animals virtually free of red cells. They do not exhibit major toxic effects, but transient alterations in renal function remain a concern. Cardiac output does not increase in the face of acute anemia associated with isovolemic exchange transfusion with hemoglobin solution. However, cardiac reserve does not diminish significantly after infusion of hemoglobin solution. Alterations in production techniques may lead to a polymerized hemoglobin solution that has a hemoglobin concentration and P50 close to fresh whole blood. Though this development is encouraging, it is clear that no perfect red cell substitute currently is available.

We have previously reported on the efficacy of a 20% fluorocarbon emulsion (Fluosol-DA, 20%) as an acellular O2 carrier at an FIO2 = 1.0. We are concerned, however, about the potential O2 toxicity that may result from extended exposure to FIO2 = 1.0. The O2 content of the fluorocarbon phase is linearly related to both the FIO2 and the fluorocarbon concentration (Fct). It should therefore be possible to maintain the same O2 content by raising the Fct using a higher fluorocarbon concentration and lowering the FIO2. The purpose of this report is to assess the ability of a 35% fluorocarbon emulsion (Fluosol-DA, 35%) at an FIO2 = 0.6 to support hemodynamics and O2 transport. Five adult baboons were paralyzed, anesthetized, intubated, and mechanically ventilated at FIO2 = 0.6. An isovolemic total exchange transfusion (E.T.) with Fluosol-DA, 35% was performed. Measurements were made at Hct's of 20, 10, 5, and less than 2%. All animals survived the exchange. Total arterial O2 content fell from 17.4 +/- 0.7 to 3.3 +/- 0.2 vol% (p less than 0.01), and O2 delivery decreased from 21.8 +/- 2.2 to 5.1 +/- 0.7 cc/min-kg (p less than 0.01) during the exchange. There was no significant change in MAP, H.R., C.O., or VO2 during the exchange transfusion. Fluosol-DA, 35% maintains normal hemodynamics and O2 transport despite a marked fall in arterial O2 content and total O2 delivery. Fluosol-DA, 35% is thus an effective O2 carrier at the safe FIO2 of 0.6.

The fluorocarbon emulsion Fluosol-DA (20%) is an acellular O2 carrier that has potential as a red cell substitute. Clinical evaluation of efficacy requires knowledge of the ability of the perfluorochemical (pfc) phase to both load and unload O2. A method is described to measure the oxygen dissolved in the pfc and aqueous phases, and the oxygen chemically bound to hemoglobin, during use of this drug. These O2 contents are used to calculate the contribution of the pfc phase to total O2 delivery and O2 consumption (VO2) and the fractional extraction of O2 in the pfc phase. The technique requires a blood gas analyzer, a microhematocrit centrifuge, and a standard co-oximeter found in many blood gas laboratories. This technique appears to be simple and reliable, and should facilitate the evaluation of efficacy of fluorocarbon emulsions.

We have prepared a polymerized pyridoxylated hemoglobin solution (Poly SFH-P) with a normal [Hb] of 14 gm/dl and a normal COP of 20 torr. Although this normal [Hb] is a significant improvement over prior products, the P50 of 16-20 torr raises a concern about the ability of Poly SFH-P to effectively transport O2 in the presence of red cells with their normal P50 of 26 torr. This study quantitatively assessed the contribution of Poly SFH-P to total O2 delivery and consumption in the clinically relevant range of hematocrits. The results document that Poly SFH-P supports life at zero hematocrit, and makes significant contributions to total O2 delivery and consumption in the presence of red cells. Poly SFH-P permits a higher plasma [Hb], and has a longer intravascular persistence than any unpolymerized hemoglobin solution. Poly SFH-P is thus an effective O2 carrier, and offers greater potential than prior products as a clinically useful red cell substitute.

We assessed the safety and efficacy of Fluosol-DA as a red-cell substitute in acute anemia. Twenty-three surgical patients with blood loss and religious objections to receiving blood transfusions were evaluated. Fifteen moderately anemic patients with a mean hemoglobin level (+/- SE) of 7.2 +/- 0.5 g per deciliter had no evidence of a physiologic need for increased arterial oxygen content and did not receive Fluosol-DA. Eight severely anemic patients with a mean hemoglobin level of 3.0 +/- 0.4 g per deciliter met the criteria of need and received the drug until the physiologic need disappeared or a maximal dose of 40 ml per kilogram of body weight was reached. We observed no adverse reactions to Fluosol-DA. The average peak increment in arterial oxygen content with the drug was only 0.7 +/- 0.1 ml per deciliter. There were no appreciable beneficial effects of Fluosol-DA, perhaps because of the small increase in arterial oxygen content, the brief half-life of the drug (24.3 +/- 4.3 hours), and the limited total dose. Six of the eight patients receiving Fluosol-DA died. One of the survivors received red-cell transfusions against his wishes, under a court order, after his total Fluosol-DA dose. Fourteen of the 15 moderately anemic patients survived. The data in this select group of patients refusing blood products suggest that, after blood loss, Fluosol-DA is unnecessary in moderate anemia and ineffective in severe anemia.

Efforts to produce a clinically acceptable hemoglobin solution which could function as a temporary oxygen carrier have evolved through several stages. Beginning with the simple tetramer, we have lowered the affinity state by pyridoxylation and then polymerized the modified tetramer. This new substance, polyhemoglobin, has a normal oxygen capacity and a half-life of 38 hours. Issues of nephrotoxicity and immunocompetence after infusion remain to be defined.

The two acellular oxygen carriers currently being evaluated as red cell substitutes are hemoglobin solutions and fluorocarbon emulsions. We have shown that both products can maintain normal levels of oxygen consumption, CO2 production, and circulatory dynamics in primates in the virtual absence of the red blood cell. Although each solution thus satisfies the most important criteria for a red cell substitute, development continues with both products. The clinical trials with the fluorocarbons have been discontinued due to the lack of efficacy of Fluosol-DA--20% in the setting of acute blood loss. Our current hemoglobin preparation is a polymerized, pyridoxylated product that has a normal oxygen-carrying capacity. Clinical testing must await further evaluation of the safety and efficacy of this product. Alternative uses for both of these oxygen carriers continue to be explored, and may eventually be the area of their greatest utility in the clinical setting.

Development continues for a safe and effective hemoglobin solution to be used as a temporary red cell substitute. Our own efforts have progressed from the unmodified tetramer, to the pyridoxylated tetramer, to our current product--polymerized, pyridoxylated hemoglobin (Poly SFH-P). We recently completed two studies to evaluate the efficacy of Poly SFH-P. The results document that Poly SFH-P supports life in the absence of red cells while maintaining baseline hemodynamics and O2 consumption. In addition, Poly SFH-P achieves a near-normal plasma [Hb], and has a longer intravascular persistence than any unpolymerized product. Poly SFH-P thus is an improved red cell substitute for use in the clinical setting.

Stroma-free hemoglobin solutions have been the subject of extensive studies as potential acellular oxygen carriers. Oncotic pressure considerations limit the hemoglobin concentration of the solutions (6-8 g/dl) to one-half the normal whole blood values. Furthermore, the free hemoglobin has a short plasma half-life (2-4 hours). In principle, polymerization offers a means of normalizing the oxygen-carrying capacity as well as extending the plasma half-life. Pyridoxylation of hemoglobin prior to polymerization provides an acceptable P50. Pyridoxylated hemoglobin (14-16 g/dl) was polymerized with a 12.5 percent glutaraldehyde solution. Since the goal was to obtain a 15 g per dl solution iso-oncotic with plasma, the polymerization reaction was monitored by the drop in colloid osmotic pressure. The reaction was quenched with 1.3 M lysine when the colloid osmotic pressure reached normal values (20-25 torr). The polymerization yield was 80 percent, with molecular weights ranging from 120,000 to 600,000 Dalton. The polymerized hemoglobin had a binding coefficient of 1.32, a P50 of 16 torr, a Bohr coefficient of -0.12, and a Hill coefficient of 1.7. The viscosity of the solution was 4.5 centipoise. The methemoglobin levels were comparable to that of unpolymerized stroma-free hemoglobin. Polymerized hemoglobin solutions provide a normal oxygen-carrying capacity with a P50 comparable to that of unpolymerized stroma-free hemoglobin solutions.

Stroma-free hemoglobin (SFH) solution is an acellular oxygen carrier under development as a temporary red blood cell (rbc) substitute. SFH will be of value if it contributes to oxygen loading (delivery) and unloading (consumption). If there is no interaction between red cell hemoglobin and SFH, then the composite oxygen dissociation curve for whole blood (WB) can be written as: SWB = rSSFH + (1 - r) Srbc, where S is the fractional saturation, and r is the SFH/WB hemoglobin ratio. This composite whole-blood curve was compared with the calculated curve, and the root mean square difference was 0.9%. Direct and indirect methods of measuring oxygen concentration ([O2]) of whole blood or plasma were compared. The regression line was [O2]indirect = 0.995 [O2]direct + 0.07 (r = 0.995, N = 16, p less than .01). The adequacy of anaerobic separation of plasma was evaluated by measuring the changes in PO2 and PCO2. The PO2 increased by less than 2 torr and the PCO2 decreased by less than 1 torr during in vitro tests. Both the direct and indirect methodology yielded consistent [O2] values for each carrier.

Polymerized pyridoxylated hemoglobin provides a means of obtaining an acellular oxygen carrier that can be infused at a normal hemoglobin concentration, with a normal COP and is stable in vivo. We have also shown that rats can survive a total exchange transfusion with poly SFH-P (Sehgal et al, 1980). It thus become a prime candidate for further in vivo assessment of its oxygen transport characteristics.

Polymerization of pyridoxylated stroma free hemoglobin (poly SFH-P) yields a solution with a normal hemoglobin concentration, normal colloid osmotic pressure (COP), and a suitable P50. In the present study we compared its in vivo O2-carrying capacity and intravascular persistence with those of pyridoxylated hemoglobin (SFH-P), following a partial exchange transfusion (900 ml) in adult baboons (n = 4 in each group). Poly SFH-P in plasma had an O2-carrying capacity that was 77% greater than that achieved with SFH-P (8.0 versus 4.5 vol%). Furthermore, poly SFH-P provided 5.0 vol% at the end of 24 hours and 2.5 vol% at the end of 48 hours; in contrast SFH-P provided only 2.2 vol% at the end of 6 hours and none within 24 hours. A normal COP was maintained throughout the postexchange period with poly SFH-P despite a 68% increase in plasma protein concentration (from 7.1 to 11.9 gm/dl). The plasma P50 of poly SFH-P decreased from 16 torr immediately after exchange to 12 torr at the end of 48 hours. The in vivo rate of conversion to methemoglobin was similar with both solutions. Polymerized pyridoxylated hemoglobin is currently the only modification of hemoglobin solution that approximates the O2-carrying capacity of whole blood and can be infused without altering the plasma COP.

Stroma-free hemoglobin (SFH) and Fluosol-DA (FL-DA) are the two acellular oxygen carriers that represent potential red cell substitutes. Although both can support life at zero hematocrit, their effectiveness as O2 carriers at intermediate hematocrits has been unclear. In the presence of red cells, the efficacy of an acellular O2 carrier can be assessed by its contribution to O2 delivery and O2 consumption. This study evaluated the efficacy of SFH and FL-DA in the presence of red cells at hematocrits less than or equal to 10%. The results illustrate that SFH and FL-DA do contribute to O2 delivery and O2 consumption in the presence of red cells. The data indicate that SFH and FL-DA are both effective acellular O2 carriers.

In summary, we remain optimistic about the potential value of perfluorochemicals as acellular oxygen carriers. The results of our first patient show that at an FiO2 of 1.0, Fluosol-DA was not necessary. The oxygen content of the plasma alone was adequate, although the FiO2 was at an unsafe level. At the safe FiO2 of 0.6, however, the Fluosol-DA was needed, since the oxygen content of the plasma was inadequate. Fluosol-DA was thus necessary and effective. At an FiO2 = 0.6, the total of the oxygen content of plasma and the oxygen content of Fluosol-DA was adequate. Although these results are encouraging, the temporary nature of this benefit is an important limitation. We hope that the additional data from both our clinical study and the multicenter study will help resolve some of these questions in the future.

Efforts to produce a clinically acceptable hemoglobin solution that could function as a temporary oxygen carrier have evolved through several stages. Beginning with the simple tetramer, we have lowered the affinity state by pyridoxylation and then polymerized the modified tetramer. This new substance, polyhemoglobin, has a normal oxygen capacity and a half-life of 38 hours. Issues of nephrotoxicity and immunocompetence after infusion remain to be defined.