Effects of a 50% Exchange Transfusion in Rats with Hemoglobin Based Oxygen Carrier Solutions

1. Effects of a 50% Exchange Transfusion in Rats with Hemoglobin Based Oxygen Carrier Solutions Ramon Bonegio, Robert Fuhro, Gina Ragno, C. Robert Valeri Naval Blood Research Laboratory, Boston University School of Medicine, Boston, MA This work was supported by the U.S. Navy (Office of Naval Research Contract N00014-02-1-0923). The opinions or assertions contained herein are those of the authors and are not to be construed as official or reflecting the views of the Navy Department or Naval Service at large.

2. BACKGROUND The ideal surgical resuscitation solution remains elusive. Blood products are difficult to store, require anticoagulation and become immunologically active during storage; crystalloid solutions rapidly leave the intravascular space thus maintaining intravascular volume poorly and contributing to tissue edema; while some colloidal solutions have been associated with worsened mortality in critically ill patients (Alderson, Schierhout et al. 2000). We have developed an isovolemic transfusion model in rats. Using this model to mimic blood loss and replacement during surgery, we recently compared the effects of large volume exchange with human serum albumin (HSA), hetastarch, unmodified human hemoglobin, diaspirin-crosslinked human hemoglobin, and O-raffinose crosslinked human hemoglobin (Lieberthal, Fuhro et al. 2002). In comparison to fresh heparinized autologous blood, HSA and hetastarch were ineffective in maintaining blood pressure and renal function. In contrast, the human hemoglobin solutions maintained blood pressure by inducing some degree of systemic vasoconstriction. The exchange transfusion with whole blood was most closely reproduced in this study by the administration of o-raffinose crosslinked human hemoglobin. In this study we utilize a 50% exchange transfusion in rats to compare and contrast the effects of large volume fluid replacement with two different hemoglobin based oxygen carrier (HBOC) solutions; O-raffinose crosslinked human HBOC (HemolinkTM) and glutaraldehyde-crosslinked bovine HBOC (HemopureTM).

3. STUDY DESIGN AND METHODS To mimic blood loss during surgery, acute hemorrhage with isovolemic replacement was induced in rats weighing 275-330g by removing and replacing 15 ml of blood/300 g of body weight in 2ml fractions over a 15-minute period. This resulted in the replacement of approximately 50% of the rat’s total blood volume. The rats were randomly assigned to receive one of four replacement solutions: 1) fresh autologous whole blood collected into heparin, 2) pentastarch, 3) HemolinkTM, or 4) HemopureTM. HemolinkTM was deoxygenated and stored at 4°C in a volume of 250ml with a hemoglobin concentration of 10gm%. HemopureTM was deoxygenated and stored at room temperature in a volume of 250 ml with a hemoglobin concentration of 13 gm%. The two HBOC solutions had similar osmolalities and oxygen affinities (Table 1). The pentastarch solution (10 gm%) was diluted with 0.9% NaCl to achieve an oncotic pressure similar to that of the HBOC solutions (Table 1). The effects of transfusion with the two different HBOC solutions were compared to fresh heparinized autologous whole blood and pentastarch. The primary end points of the study were survival and the effects of volume exchange on systemic hemodynamics during the five days after exchange transfusion. Weight gain during this period served as an indicator of well-being.

4. Hemodynamic effects were assessed at four time points (1hr, 1,2,and 5 days) after the exchange by measuring cardiac output and blood pressure. Glomerular filtration rate (GFR) was estimated by inulin clearance. In addition, plasma and urinary hemoglobin levels were measured during the 5 days following the 50% exchange transfusion. In addition, to determine if the heme present in the HBOC stimulated erythropoiesis, we compared the recovery of the hematocrit in rats exchange transfused with HBOC solutions to that of rats that received pentastarch. Data is shown as mean ± SEM and p values determined using ANOVA with Bonferroni correction for multiple comparisons.

5. RESULTS There was no mortality in the 167 rats exchange transfused during this study. In keeping with our targeted exchange of 50% of intra-vascular volume, the hematocrit values decreased by >50% in all animals that received the HBOCs and pentastarch solutions (figure 1). The weight gain is reported in Figure 2. One hour after exchange transfusion, animals that received HBOCs maintained blood pressure, systemic vascular resistance and GFR with values comparable to those in animals that received autologous blood (figure 3 and 4). In contrast, Pentastach replacement was associated with a decreased systemic vascular resistance, blood pressure and GFR one hour after transfusion (figure 3 and 4). Despite this early difference, there was no significant difference in any of these parameters at later time points. Plasma hemoglobin levels during the 48 hour post-infusion period in rats infused with HemopureTM were significantly higher than the plasma hemoglobin levels in rats infused with HemolinkTM (Figure 5a). Urine hemoglobin levels during the 48 hour post-infusion period in rats infused with HemopureTMwere significantly reduced compared to rats infused with HemolinkTM (Figure 5b). The hematocrit recovered rapidly in all groups and this recovery was not accelerated in animals that received heme rich HBOC solutions (figure 1).

6. CONCLUSIONS Hemoglobin based oxygen carrying solutions (Hemolink™ and Hemopure™) are effective replacement solutions for large volume blood loss that occurs during surgery. Animals in all groups survived the 50% exchange transfusion and recovered to gain weight during the ensuing 5 days. Hemolink™ and Hemopure™ maintained blood pressure, systemic vascular resistance and cardiac output in a range similar to that achieved by autologous blood in the hour after exchange transfusion. In contrast, pentastarch failed to maintain hemodynamic stability and animals that received pentastrach developed reversible renal dysfunction. HBOC solutions did not accelerate erythropoiesis.

7. REFERENCES • 1. Alderson, P., G. Schierhout, et al. (2000). "Colloids versus crystalloids for fluid resuscitation in critically ill patients." Cochrane Database Syst Rev(2): CD000567. • Lieberthal, W., R. Fuhro, et al. (2002). "Comparison of the • effects of a 50% exchange-transfusion with albumin, hetastarch, and • modified hemoglobin solutions.” Shock 17(1):61-9.

8. TABLE 1 COMPARISON OF THE SOLUTIONS USED TO PERFORM THE 50% EXCHANGE TRANSFUSION IN THE RATS HGB ONCOTIC PRESSURE p50 (gm/dl) (mm Hg) (mm Hg) Hemolink™ 10 21 34 Hemopure™ 13 21 31 Pentastarch* 10 >30 N/A *After Dilution with 0.9% NaCl 6.7 22 N/A

9. Figure 1: Hematocrit recovery after 50% exchange transfusion 0.60 0.50 0.40 Blood Pentastarch Hematocrit (%) 0.30 Hemolink™ Hemopure™ 0.20 0.10 0.00 0.04 1 2 5 Days after exchange transfusion Figure 1: Hematocrit during the five day recovery following 50% exchange transfusion Figure 1: In keeping with the targeted 50% exchange transfusion rats receiving red cell free replacement solutions show an initial drop in hematocrit that slightly exceeds 50%. All groups demonstrate rapid recovery of hematocrit in keeping with efficient erythropoiesis. HBOC solutions did not accelerate hematocrit recovery when compared to pentastarch treated animals.

10. Figure 2: Weight gain after 50% exchange transfusion 40.0 30.0 20.0 Blood 10.0 Pentastarch Weight gain (g) 0.0 Hemolink™ -10.0 Hemopure™ -20.0 -30.0 -40.0 0 1 2 5 Days after exchange transfusion Figure 2: Weight gain during the five day recovery following 50% exchange transfusion Figure 2: Weight gain is a good measure of well-being in rats. All animals gained weight over the 5 days following exchange transfusion.

11. Figure 3A: Cardiac output and Blood Pressure 1 1 hour after 50% exchange transfusion 140 120 100 80 Mean arterial pressure Cardiac output (ml/min) and MAP (mmHg) Cardiac output 60 40 20 0 Figure 3: Blood pressure, cardiac output and systemic vascular resistance after exchange transfusion Figure 3A: Cardiac output and intra-arterial mean arterial pressure were maintained in a range similar to that achieved by autologous heparinized blood in those animals that received Hemolink™ or Hemopure™. Pentastarch failed to maintain BP in the first hour after 50% exchange transfusion. ♣ Blood Pentastarch Hemolink Hemopure

12. Figure 3B: Systemic Vascular Resistance after 50% exchange transfusion 1.10 1.00 0.90 Blood Pentastarch 0.80 SVR Hemolink™ 0.70 Hemopure™ 0.60 0.50 0.04 1 2 5 Days post exchange transfusion ♣ Figure 3B: Systemic vascular resistance was well maintained after exchange transfusion in those animals that received autologous blood or either of the HBOCs. Pentastarch transfusion failed to maintain SVR after exchange transfusion. ♣ p<0.05 vs autologous whole blood

13. Figure 4: Glomerular Filtration Rate after 50% exchange transfusion 4.00 3.50 3.00 Blood 2.50 GFR (ml/min/300g) Pentastarch 2.00 Hemolink™ 1.50 Hemopure™ 1.00 0.50 0.00 0.04 1 2 5 Days after exchange transfusion Figure 4: Glomerular Filtration Rate after 50% exchange transfusion ♣ Glomerular filtration rate (GFR) estimated by inulin clearance during the 5 days following 50% exchange transfusion. Replacement of blood with Hemolink™ or Hemopure™ did not alter renal function despite frank hemoglobinuria (figure 5). Pentastarch was less effective in maintaining renal function early post transfusion. Animals did however recover normal renal function within 1 day of the pentastarch exchange. ♣ p<0.05 vs autologous heparinized whole blood

14. Figure 5A: Plasma hemoglobin levels following 50% exchange transfusion 8 7 6 5 HemolinkTM 4 HemopureTM 3 2 1 0 0.04 1 2 5 Days after exchange transfusion Figure 5: Plasma hemoglobin concentrations and hourly urine hemoglobin loss after 50% exchange transfusion with HBOC solutions (Hemolink™ and Hemopure™) Figure 5A: Plasma hemoglobin concentrations following 50% exchange with HBOC solutions. Note the initial higher hemoglobin concentration of the Hemopure™ solution that results from the higher hemoglobin content of this replacement solution. This concentration difference is maintained over the first two days.

15. Figure 5B: Urinary hemoglobin loss following 50% exchange transfusion with HBOC solutions 0.0035 0.003 0.0025 0.002 HemolinkTM 0.0015 HemopureTM 0.001 0.0005 0 -0.0005 0.04 1 2 5 Days after 50% exchange transfusion n Figure 5B: Urine hemoglobin following 50% exchange with HBOC solutions. Despite the higher plasma hemoglobin concentration in animals that received Hemopure™, this group had a lower urinary hemoglobin excretion rate suggesting that Hemopure™ contains less tetrameric hemoglobin. Much of the transfused hemoglobin leaves the circulation but is not excreted in the urine during the first five day post transfusion suggesting that the hemoglobin is going into the extravascular space.