The Debate on Nitric Oxide and RBC Storage
By Jay Pennington

Storage for just about anything has an inverse relationship with viability. Blood is certainly no exception, and AABB provides thorough guidelines to ensure storage keeps this delicate resource as viable as possible. But recent studies focused on Red Blood Cell (RBC) storage have raised questions about the risk/benefit ratio for transfusion, even for RBCs stored for any amount of time.

Could RBCs be rendered ineffective components for transfusion much earlier than believed, or even from the moment of collection from the donor? If so, do they bring more harm than help to the recipient, with all the attendant risks of infectious and noninfectious reactions?

When Proceedings of the National Academy of Sciences (PNAS) released an article last year titled “Evolution of Adverse Changes in Stored RBCs,” and a similar second article (both from Duke University), the media picked up the story, raising public concern about transfusion, although some scientists balked at the hypothesis put forth. Representatives for both sides of the issue came together to compare divergent conclusions from their studies on nitrogen compounds and RBC storage in the session “Hot Topic: Update on Red Blood Cell Storage” (9311-S).

RBCs and the Storage Lesion

Moderator David F. Stroncek, MD, from the Warren G. Magnuson Clinical Center (National Institutes of Health), introduced the hot topic by reviewing relevant foundational concepts such as the term “storage lesion.” Used in reference to RBCs, this term refers to a set of biochemical and biomechanical changes that may occur in RBC units as they age and that, some believe, increase the likelihood of transfusion-related adverse effects.

A variety of changes have typically been described as resulting in storage lesions, including the depletion of adenosine triphosphate (ATP) and 2,3-diphosphoglycerate in red cells, as well as reduced deformability and increased adhesion to endothelial cells. Some of these changes are thought to begin as soon as red cells are collected from the donor and to continue as RBC units sit in storage.

A concern accompanying the set of changes in the storage lesion is the possibility that the oxygen-delivery capability of red cells is diminished during RBC storage. However, the mechanisms and timing of all storage lesion effects are uncertain. The crux of the disagreement between the two speakers centered around the role, if any, of nitric oxide (NO) in the so-called storage lesion.

The SNO-Hb Hypothesis

First, Timothy McMahon, MD, PhD, from Duke University and Durham VA Medical Centers, and an author of the PNAS article named above, explained the findings of his 2007 study in his presentation “Adverse Functional Changes in Banked Red Cells: Role of NO Loss.”

Previous studies suggested that hemoglobin NO levels dropped dramatically upon removal of red cells from circulation. The hypothesis of McMahon’s group of researchers was that the most important factor in RBC storage lesions is the depletion of S-nitrosohemoglobin (SNO-Hb) and that release of NO by this substance is key in RBC-dependent vasodilation to regulate blood flow. McMahon conceded that there are multiple potential sources for the vasodilatory mechanism (ATP being one), but he postulated that the oxygen sensing capability of hemoglobin is activated through SNO-Hb, releasing NO according to the local need in surrounding tissue.

In RBC storage, SNO-Hb may be destabilized through oxidation, degradation or release into the storage medium. The study found that although deformability was lost gradually during storage, SNO-Hb levels and red-cell-dependent vasodilatory capacity decreased rapidly after collection. This loss could be responsible for aggravating a patient’s condition because after a transfusion of stored RBCs the blood  is not able to pass through capillaries.

The implications of the study, McMahon claimed, are that loss of SNO-Hb through storage lesions may cause RBCs to be not only ineffective in addressing anemia, but even harmful to the recipient, potentially contributing to increased incidence of transfusion-related adverse events such as pneumonia, infections and mortality. Citing the troubling array of complications resulting from transfusion as cause for considering the implications of the SNO-Hb model, McMahon concluded, “We can do better.”

The Nitrite Ion Hypothesis

Alan N. Schechter, MD, from the Molecular Medicine Branch of the National Institutes of Health, challenged McMahon’s claims, with a presentation titled “Science and Fantasy in the Study of Red Cells and Nitric Oxide.”

Schechter, who has worked for years studying sickle-cell disease, admitted he was initially hopeful that the SNO-Hb hypothesis might hold a key for addressing the blood vessel blockages that occur in sickle cell patients. However, while testing the SNO-Hb hypothesis, he and his colleagues became skeptical of it for lack of important supporting evidence. Moreover, he claimed, mounting evidence has led many scientists to believe that the importance of SNO-Hb in the process of vasodilation is minimal or even nonexistent. He summed up his rebuttal with the slogan, “The NO field warms up and the SNO-Hb hypothesis melts.”

Schechter and his colleagues have argued that nitrite ions may account for many of the properties ascribed to SNO-Hb by McMahon and others. Through studies measuring nitrite concentrations in RBC storage, Schechter found that NO reserves decrease slowly before leveling off. The time frame in those studies lines up with that of storage lesion effects as described earlier. “These changes could contribute to an NO-related red cell storage lesion that develops over weeks,” he said, “as could increased hemolysis (which rapidly destroys NO), but neither has yet been demonstrated.” The low levels of SNO-Hb, in contrast, are depleted within minutes of collection. This points away from SNO-Hb as the major source of a NO-related storage lesion, even if it contributed to hypoxic vasodilatation.

However, much is still unknown, and Schechter identified areas needing future study to test his hypotheses and clarify other aspects of the storage lesion:

·          Mechanisms for preservation of nitrite and NO bioactivity in stored RBCs.

·          Effects of red cells stored for different periods on canine NO-sensitive cardiovascular physiology and pathology (or that of other animals).

·          Clinical correlations of nitrite levels and short-term and long-term effects of RBC transfusions.

·          Examination of other parameters that may contribute to the storage lesion.

What does it all mean for the blood center and the transfusion service? In a response during the question-and-answer session, Stroncek’s statement summed up what can be concluded at this time about NO activity in stored erythrocytes: “There is not yet enough evidence to recommend reducing the shelf life of RBCs.”

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