Among critically ill patients, red blood cell (RBC) transfusion is often prescribed for anemia in the absence of active or recent bleeding. The failure of RBC transfusion to improve physiological parameters and clinical outcomes in this setting may be explained by current understanding of the relationship between the RBCs and the microcirculation. It is now evident that the circulating RBCs contribute to microcirculatory hypoxic vasodilation by regulated nitric oxide (NO)-dependent vasodilation, thereby facilitating delivery of oxygen to oxygen-deprived tissue. The structural and functional changes in RBCs during storage, collectively known as the storage lesion, result in circulating RBCs that may not function as expected after transfusion. In recent years, there has been a significant focus on the dysfunctional interaction between stored RBCs and the microcirculation, with emphasis on understanding the mechanisms that drive erythrocyte NO-mediated vasodilation. The development of technology that allows noninvasive observation of the microcirculation in humans has allowed for direct observation of the microcirculation immediately before and after RBC transfusion. The current understanding of RBC NO-mediated vasodilation and the results of direct observation of the microcirculation in the setting of RBC transfusion are reviewed.
Introduction
For the treatment of hemorrhagic shock, the benefit of allogeneic red blood cell (RBC) transfusion is indisputable. Such patients are at the threshold of impending circulatory collapse and death, and replacement of shed blood with stored RBCs partially restores both intravascular volume and critical oxygen delivery until the site of bleeding is controlled. Until a viable “blood substitute” capable of delivering oxygenation to tissues is available, RBC transfusion will remain the primary treatment for acute blood loss anemia. RBC transfusion, however, is often prescribed in the absence of active bleeding. Moderate anemia often accompanies shock states unrelated to active bleeding, such as septic shock or systemic inflammatory response syndrome (traumatic shock) after injury, and it is not uncommon for physicians to transfuse RBCs in this setting to treat moderate anemia with the goal of improving oxygen delivery and ultimately oxygen uptake at the cellular level.
Such clinical practice contradicts a relatively large body of research that demonstrates that transfusion in this setting does not improve oxygen uptake . Similarly, many studies have suggested that RBC transfusion can, in fact, be harmful rather than beneficial. RBC transfusion has been demonstrated to be an independent predictor of mortality, infection, and multiorgan failure . In the landmark multiinstitutional TRICC trial, wherein nonbleeding patients were randomized to a liberal (goal hemoglobin 10–12 g/dL) versus restrictive (goal hemoglobin 7–9 g/dL) transfusion practice, no survival benefit was observed in the liberal transfusion group .
The failure of RBC transfusion to improve physiological parameters and clinical outcomes in such settings may be explained by current understanding of the relationship between the RBC and the microcirculation. In particular, it is evident that the circulating RBCs contribute to microcirculatory hypoxic vasodilation by regulated nitric oxide (NO)-dependent vasodilation, thereby facilitating delivery of oxygen to oxygen-deprived tissue. The structural and functional changes in RBCs during storage, collectively known as the storage lesion, result in circulating RBCs that may not function as expected after transfusion. Although recent randomized trials failed to demonstrate any differences in outcome associated with the transfusion of relatively fresh versus relatively older RBCs , it is well known that the functional and morphological degradation of RBCs occurs relatively early and progressively during storage, and multiple studies have demonstrated that stored RBCs fail to promote hypoxic vasodilation .
In recent years, there has been a significant focus on the dysfunctional interaction between stored RBCs and the microcirculation, with emphasis on understanding the mechanisms that drive erythrocyte NO-mediated vasodilation. The development of technology that allows noninvasive observation of the microcirculation in humans has allowed for direct observation of the microcirculation immediately before and after RBC transfusion. In this overview, the current understanding of RBC NO-mediated vasodilation and the results of direct observation of the microcirculation in the setting of RBC transfusion are reviewed.
Storage-dependent changes in RBCs and effects on no signaling
How RBCs modulate hypoxic vasodilation can be generalized into 2 mechanisms involving: (i) changes in how hemoglobin scavenges NO and inhibits NO signaling and (ii) how RBCs stimulate formation of NO and subsequent signaling. This balance between inhibitory and stimulatory effects has emerged as a key aspect of our understanding of vascular NO homeostasis mechanisms . The changes that occur in RBCs during storage dramatically change this balance toward to the inhibitory side through multiple mechanisms . With respect to inhibition of NO, the central reaction is that of oxy- or deoxyhemoglobin (where iron is in the ferrous oxidation state) with NO formed by endothelial nitric oxide synthase (eNOS). With cell-free hemoglobin, this reaction is rapid (rate constant ∼10 7 M −1 s −1 ), but is slowed significantly (rate constant ∼10 4 M −1 s −1 ) when hemoglobin is encapsulated within the RBC, a consequence of diffusion barriers created by the RBC membrane . This property of hemoglobin compartmentalization is critical in allowing eNOS-derived NO to affect local signaling.
With storage, however, several changes occur that result in RBCs that scavenge NO as quickly as cell-free Hb. The first change pertains to hemolysis, which increases with storage time. In fact, older stored RBCs (35–42 days old) may typically have 50–100 μM cell-free hemoglobin concentrations; transfusion with as little as 5 μM cell-free hemoglobin is sufficient to scavenge NO and cause hypertension . Second, storage results in the formation of microvesicles that contain hemoglobin, and in these microvesicles, the rate of NO scavenging by hemoglobin is the same as that by free hemoglobin . Finally, even without RBC destruction and hemolysis, during storage, RBCs become smaller and denser and have altered shape and surface area. This results in cells that scavenge NO faster than “normal” biconcave RBCs . In summary, during storage, there is a continuum of changes occurring in RBCs, from RBC shape to the formation of microvesicles and ultimately hemolysis, all of which lead to products that are more potent inhibitors of NO signaling ( Fig. 1 ). Indeed, hypertension, oxidative stress, and inflammation associated with acute loss of NO bioavailability are observed after transfusion with older stored RBCs compared with fresh RBCs .
On the other side of the balance is the ability of RBCs and hemoglobin to sense local oxygen tensions and link this to increasing NO bioavailability. This paradigm has been discussed in the context of hypoxic blood flow and specifically coupling of hemoglobin deoxygenation with NO formation mechanisms . Three mechanisms have been proposed, including nitrite reduction, S-nitrosohemoglobin, and ATP release and subsequent eNOS activation . Although biochemically distinct, these proposed pathways share the need for hemoglobin deoxygenation. During storage, loss of 2,3-BPG leads to RBCs with increased oxygen affinity, and thus immediately upon transfusion, there is likely a mismatch between appropriate deoxygenation and activation of NO-dependent vasodilation. In addition, stored RBCs oxidize nitrite (a substrate for the nitrite reduction pathway), providing an additional pathway for preventing NO formation .
Storage-dependent changes in RBCs and effects on no signaling
How RBCs modulate hypoxic vasodilation can be generalized into 2 mechanisms involving: (i) changes in how hemoglobin scavenges NO and inhibits NO signaling and (ii) how RBCs stimulate formation of NO and subsequent signaling. This balance between inhibitory and stimulatory effects has emerged as a key aspect of our understanding of vascular NO homeostasis mechanisms . The changes that occur in RBCs during storage dramatically change this balance toward to the inhibitory side through multiple mechanisms . With respect to inhibition of NO, the central reaction is that of oxy- or deoxyhemoglobin (where iron is in the ferrous oxidation state) with NO formed by endothelial nitric oxide synthase (eNOS). With cell-free hemoglobin, this reaction is rapid (rate constant ∼10 7 M −1 s −1 ), but is slowed significantly (rate constant ∼10 4 M −1 s −1 ) when hemoglobin is encapsulated within the RBC, a consequence of diffusion barriers created by the RBC membrane . This property of hemoglobin compartmentalization is critical in allowing eNOS-derived NO to affect local signaling.
With storage, however, several changes occur that result in RBCs that scavenge NO as quickly as cell-free Hb. The first change pertains to hemolysis, which increases with storage time. In fact, older stored RBCs (35–42 days old) may typically have 50–100 μM cell-free hemoglobin concentrations; transfusion with as little as 5 μM cell-free hemoglobin is sufficient to scavenge NO and cause hypertension . Second, storage results in the formation of microvesicles that contain hemoglobin, and in these microvesicles, the rate of NO scavenging by hemoglobin is the same as that by free hemoglobin . Finally, even without RBC destruction and hemolysis, during storage, RBCs become smaller and denser and have altered shape and surface area. This results in cells that scavenge NO faster than “normal” biconcave RBCs . In summary, during storage, there is a continuum of changes occurring in RBCs, from RBC shape to the formation of microvesicles and ultimately hemolysis, all of which lead to products that are more potent inhibitors of NO signaling ( Fig. 1 ). Indeed, hypertension, oxidative stress, and inflammation associated with acute loss of NO bioavailability are observed after transfusion with older stored RBCs compared with fresh RBCs .
On the other side of the balance is the ability of RBCs and hemoglobin to sense local oxygen tensions and link this to increasing NO bioavailability. This paradigm has been discussed in the context of hypoxic blood flow and specifically coupling of hemoglobin deoxygenation with NO formation mechanisms . Three mechanisms have been proposed, including nitrite reduction, S-nitrosohemoglobin, and ATP release and subsequent eNOS activation . Although biochemically distinct, these proposed pathways share the need for hemoglobin deoxygenation. During storage, loss of 2,3-BPG leads to RBCs with increased oxygen affinity, and thus immediately upon transfusion, there is likely a mismatch between appropriate deoxygenation and activation of NO-dependent vasodilation. In addition, stored RBCs oxidize nitrite (a substrate for the nitrite reduction pathway), providing an additional pathway for preventing NO formation .
Direct observation of microcirculation during RBC transfusion
The development of orthogonal polarization spectral (OPS) microscopy, followed by the next-generation sidestream dark-field (SDF) imaging, has allowed for real-time visualization of the microcirculation, typically in the sublingual region when performed at the bedside. In addition, near-infrared spectrography (NIRS) has been used to measure tissue oxygenation saturation in muscle beds in a noninvasive manner (usually the musculature of the thenar eminence in humans). Using these modalities, several studies have demonstrated that the microcirculation is altered in sepsis, and persistence of microcirculatory derangement is associated with multiorgan failure and mortality. De Backer et al. used OPS imaging to compare the sublingual microcirculation between septic and nonseptic patients. In patients with severe sepsis, the density of all vessels was reduced relative to the nonseptic cohort, and these vessel density alterations were more severe in septic nonsurvivors . More recently, Edul et al. similarly compared patients with septic shock with healthy volunteers using SDF imaging paired with analytical software, and found that the sublingual microcirculation in patients with septic shock is characterized by hypoperfusion and flow heterogeneity relative to healthy controls .
In the setting of sepsis and other nonhemorrhagic states, observation of the microcirculation in response to RBC transfusion has been examined in several studies. Sakr et al. were among the first to evaluate the effect of RBC transfusion on microvascular perfusion in septic patients . Using OPS imaging, they identified that patients with relatively altered baseline microcirculatory perfusion had improvement in perfusion following transfusion. However, when the pretransfusion microcirculation appeared relatively normal, transfusion was followed by either no change or deterioration in microvascular perfusion. This negatively correlated response to transfusion related to baseline microcirculation has been observed in similar studies ( Fig. 2 ). Creteur et al. used NIRS to evaluate the effect of RBC transfusion on muscle tissue oxygenation consumption . They observed improvement in oxygen consumption among patients with altered microvascular reactivity at baseline, and deterioration in oxygen consumption among patients with preserved baseline microvascular reactivity.
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