Sloan et al., over 15 years ago, tested the diaspirin cross-linked hemoglobin (DCLHb), a purified and chemically modified human Hb solution (HemAssist®, 10 g/dL diaspirin cross-linked human hemoglobin in balanced electrolytes solution) [21]. Their randomized multicenter study had the primary objective of reducing 28-day mortality for hemorrhagic shock trauma patients. The study design included the addition of 500–1,000 mL DCLHb to standard treatment during initial fluid resuscitation. In the 58 treated patients, death rate was higher than in the 53 controls (46 % vs. 17 %; p = 0.003). It is likely that DCLHb might have worsened outcomes by scavenging nitric oxide (NO) with worsening of hemorrhage and reduction of tissue perfusion due to vasoconstriction. Nitric oxide, an endothelial-derived relaxing factor, is a strong heme ligand, and its reduction results in systemic and pulmonary vasoconstriction, decrease in blood flow, release of proinflammatory mediators, and loss of platelet inactivation, predisposing conditions for vascular thrombosis and hemorrhage [17, 22] (Table 11.3). Nitric oxide scavenging causing microvascular vasoconstriction and reduction in functional capillary density is the major side effect for many of the HBOCs (Table 11.3). Endothelin-1, a strong vasoconstrictor produced by endothelial cells, has also been suggested to be involved in vasoconstrictor effects of HBOCs [27] together with sensitization of α-receptors [28].

In 2003, a randomized controlled study was performed by Kerner et al. [29] in trauma patients with hypovolemic shock. The study population was sorted into the standard care group (n = 62) or into the HemAssist® group (1,000 mL) (n = 53) during transport from the scene of trauma to the hospital and until definitive control of bleeding source. The trial was interrupted prematurely for futility after an interim evaluation. In fact, no difference in either 5- or 28-day organ failure or mortality between the two groups was found.

PolyHeme® (hemoglobin glutamer-256 [human]; polymerized hemoglobin, pyridoxylated; Table 11.2) was produced starting from human purified Hb, then pyridoxylated (to decrease the O₂ affinity), and polymerized with glutaraldehyde. In 1998, Gould et al. [30] first compared, in a prospective randomized trial, the therapeutic benefit of PolyHeme® with that of allogeneic RBCs in the treatment of acute blood loss in 44 trauma patients. PolyHeme® was designed to avoid the vasoconstriction issues observed with tetrameric Hb preparations, probably due to endothelial extravasation of the molecules and binding of NO. The patients were randomized to receive either RBCs (n = 23) or up to 6 U (300 g) of PolyHeme® (n = 21) as their initial blood replacement after trauma and during emergent operations. The first results were encouraging since no serious or unexpected adverse events were related to PolyHeme®, which maintained total Hb concentration, despite the marked fall in RBCs Hb concentration. This led to reduction in the use of allogeneic blood [30]. In 2002, the same group of authors performed a study in massively bleeding trauma and urgent surgery [31]. A total of 171 patients received a rapid infusion of 1–20 units (1,000 g, 10 L) of PolyHeme® instead of RBCs as initial oxygen-carrying replacement, simulating the unavailability of RBCs. Forty patients had a nadir RBC [Hb] ≤3 g/dL. However, total [Hb] was adequately maintained because of plasma [Hb] added by PolyHeme®. The 30-day mortality (25 %) was compared with a similar historical group (64.5 %; p < 0.05). On the basis of these results, the authors concluded that PolyHeme® should be useful in the early treatment of urgent blood loss and resolve the dilemma of unavailability of red cells. These first encouraging results led to a multicenter phase III trial performed in 2009 in the United States [32]. The study was designed to assess survival of patients resuscitated with PolyHeme® starting at the scene of injury. The patients were randomized to receive either up to 6 U of PolyHeme® during the first 12 h post-injury before receiving blood or crystalloids. After 714 patients were enrolled and randomized, 30-day mortality was higher in the PolyHeme® arm than in the crystalloid arm (13.4 % vs. 9.6 %), although this difference was not statistically significant. The incidence of multiple organ failure was similar (7.4 % vs. 5.5 % in PolyHeme® and controls, respectively). Total adverse events instead were higher in intervention vs. control group (93 % vs. 88 %; p = 0.04); this was similar to serious adverse event, including myocardial infarction (MI) (40 % vs. 35 %; p = 0.12).

Hemospan® (Table 11.2) is an oxygenated, polyethylene glycol-modified hemoglobin: it showed some promising results in clinical trials [15, 23]. Olofsson et al. conducted a safety phase II study in patients undergoing major orthopedic surgery. The authors compared Ringer’s lactate with Hemospan® given before the induction of anesthesia in doses ranging from 200 to 1,000 mL. Hemospan® mildly elevated hepatic enzymes and lipase and was associated with less hypotension and more bradycardic events. Nausea was more common in the patients receiving Hemospan®, without correlation with the dose [23]. A “Phase III Study of Hemospan® to Prevent Hypotension in Hip Arthroplasty” has been completed, but the results have never been published [33]. Moreover, due to the lack of investor interest, this product is not currently used in clinic [34].

In the mid-1990s, recombinant technology for hemoglobin production (use of E. coli transfected with human hemoglobin genes; rHb1.1, Optro®) gave some promising results [35]. Nevertheless, when tested in animal models, vasoconstriction due to NO scavenging and increase in amylase and lipase levels led to project abandonment [35]. Further modification of rHb 1.1 (rHb 2.0), which aimed at mitigating the vascular response [24], did not reach the desired objective, and consequently, due to the hemodynamic side effects, synthesis of recombinant product was discontinued [36].