Cover Story

CONTINUING EDUCATION

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LEARNING OBJECTIVES

Upon completion of this article, the reader will be able to:

1. Discuss the progress of transfusion medicine since 1900.
2. List the three most common causes of transfusion-related deaths.
3. Discuss two advances in decreasing adverse events from blood transfusion.
4. Discuss two important findings that have changed blood-component transfusion practices.
5. Discuss a current blood substitute in terms of availability.
6. List one future application of cell therapy.

Transfusion medicine today: Mission accomplished?

By William B. Lockwood, PhD, MD

Although animal-to-human blood transfusions had been performed for several thousand years, and human-to-human transfusions were attempted in the 14th and 18th centuries, it was not until 1818 that a British obstetrician, James Blundell, reported the first successful human-to-human transfusion.¹ In 1900, with the discovery of the ABO blood group by Nobel Prize winner Karl Landsteiner,² the field of "transfusion medicine" was to evolve into today’s multifaceted science. The practice of transfusion medicine now encompasses blood collection, testing, manufacturing, storage, and distribution; traditional blood transfusion and advanced cellular therapies; recombinant technology, blood substitutes, and engineered tissues; improved transfusion safety; clinical medical practice specialty; and regulatory oversight (see Figure 1).

Landmark research findings have preceded the practice of blood transfusion today; and more research is needed in order to provide the best, safest, and most cost-effective blood and cellular therapies to patients requiring these treatments. How are we doing today? What innovations lie ahead?

Blood collection

The most recent U.S. National Blood Collection and Utilization Report (NBCUS 2007) indicates that the number of allogeneic whole blood/red blood cell (WB/RBC) collections (16,174.000) before testing increased 5.4% from 2004 to 2006.³ The report also states that allogeneic RBC transfusions increased 3.3% in 2006 to 14,650,000. Apheresis RBC collections increased 96.4% in 2006 due to the U.S. Food and Drug Administration (FDA) approval and introduction of two-unit RBC collections by automated equipment. This technology will allow blood centers to provide an increased RBC inventory to hospitals and help prevent RBC shortages that have plagued RBC availability during the summer and holidays.

Platelet-unit availability is always a concern due to the five-day expiratory date. The report indicates that the number of available platelets in 2006 was 10,388,000, and the number of platelet transfusions was 9,092,000. Although the increase in collected and transfused platelets was not statistically significant, these data indicate that demand will outpace supply unless more platelet donations can be obtained (apheresis collections becoming the primary method of donation). The demand for plateletpheresis is due to increased bone-marrow transplantation procedures requiring platelet support, increased trauma cases, and an increase in hematological fragile surgical/medical/oncology patients. An increase in the current 5% of eligible blood donors who routinely donate blood in this country is emergently needed.

Figure 1. History of transfusion medicine since 1900

Blood safety

Transfusion of blood components is safer today than at any time. Advances in testing technology with the use of nucleic-acid testing,
or NAT, and the addition of West Nile virus and Chagas disease tests performed on donated blood has provided additional layers of safety from acquiring transfusion-transmitted diseases. The estimated risk of transfusion-transmitted diseases per unit of blood component transfused is shown in Table 1.

Table 1. Risk per unit transfused of selected transfusion-transmitted diseases.4

Febrile non-hemolytic transfusion reactions have steadily declined since the advent of leukoreduction (removal of white blood cells by filtration or automation). Leukoreduction also minimizes alloimmunization and immunomodulation events from blood- component transfusion. With the United States approaching 100% leukoreduction, decline in these adverse events will be realized.

Transfusion reactions have also seen a decline in several categories but an increase in others. Total transfusion-recipient deaths reported by the FDA declined in the FY 2007 (see Table 2). Decline was noted in ABO incompatibility but increased for transfusion-related acute lung injury (TRALI). This increase, most likely, relates to improved scientific knowledge and education about the signs/symptoms of this entity.

Table 2. FDA transfusion fatalities for FY05-07.5

Also noted in this report is the decline of reported transfusion fatalities associated with platelets collected by apheresis. Bacterial contamination of blood collections has seen peaks and troughs since collection of plasma started during World War I (WWI) and continued in massive volumes in WWII with the use of glass bottles (see Figure 2). Glass bottles, besides being breakable, required an air vent for flow, which made these containers an "open system," facilitating the entrance of bacteria into the container. With the introduction of plastic blood containers in the late 1950s, a closed system was maintained for whole-blood units. The use of plastic blood containers, however, allowed for the introduction of blood "component" manufacturing and whole-blood-derived platelets stored at room temperature then became a source of increased bacterial contamination. Many of these contaminants were from the skin flora, and several safety measures were recently introduced to decrease platelet bacterial contamination.

Figure 2. Blood collection set used in 20th century⁶

In May 2004, the AABB (American Association of Blood Banks) required all AABB-accredited facilities to "have methods to limit and detect bacterial contamination in all platelet components."⁷ Collection facilities and hospital-transfusion services began using several manual techniques (pH, glucose level, platelet swirling) to detect possible bacterial growth. Increased use of culture-based methods introduced by many facilities for both whole-blood-derived components and apheresis platelets is also noted. The FDA also licensed several automated devices to allow for "quality control" (QC) of platelet components — the platelet-collection process does not demonstrate a higher contamination rate that the predefined rate for the device.⁸ The FDA, as of this writing, has not licensed any of these devices as a "release test" — the platelet component being released/issued does not contain bacteria.

Another advance in eliminating bacterial contamination of whole-blood-derived components is the use of a blood-diversion pouch (see Figure 3). During whole-blood collection the first several milliliters of blood which contains a probable bacteria-laden skin-plug from the venipucture is diverted into a small blood container prior to the filling of the main collection bag. This collection process has resulted in up to 50% reduction of contamination of whole-blood components.⁹

Figure 3. Blood-collection diversion pouch used to decrease bacterial contamination and allow collection of donor testing samples.

A major risk to transfusion safety is mislabeled specimens, wrong blood in collection tubes (WBIT), and transfusion to the wrong patient. These errors in pre-analytical and post-analytical hospital procedures have not abated during the entire history of laboratory medicine. The Joint Commission issued Patient Safety Guidelines in 2005, trying to spearhead hospitals to decrease patient-specimen mislabels and WBIT by requiring the use of two patient identifiers (not a room number) Transfusion services have required patient-identity verification by the transfusionist and a witness for many years. Mistransfusions continue today, however, and alternate processes are being implemented to ensure a safe blood transfusion.

Bar coding is becoming a standard technology in laboratory practice for sample identification from bedside to analytical testing. Hospitals have been slow to implement bar coding on patient wristbands due in most cases to the cost of such technology. Bar-code technology (machine-readable) includes a variety of devices — one-dimensional and two-dimensional bar codes, as well as radio frequency identification, or RFID, tags. This technology is also expanding to other areas of hospital patient care requiring proper patient identification (medication given via "smart" infusion pumps).¹⁰ When a can of beans purchased in most grocery stores is more accurately, consistently, and correctly identified than a blood transfusion recipient, has not the time arrived for investing in this new technology to prevent possible deaths related to a mistransfusion?

Blood-transfusion practices

Over the past 20 years, researchers have investigated the appropriate indicators for blood-component transfusion. With the blood supply being both volatile in availability and not at "zero-risk," change in patient-transfusion practices is providing optimal patient care with lower risk. Several seminal publications have demonstrated that physicians "over transfuse" blood components and may even increase the morbidity and mortality rates of transfusion recipients.

A multicenter randomized control trial (RCT) of transfusion thresholds in critically ill patients in intensive-care units demonstrated that a liberal RBC-transfusion protocol (maintaining the hemoglobin level between 10 g/dL and 12 g/dL) resulted in a 5% higher mortality rate when compared to a restrictive RBC protocol (maintaining hemoglobin levels between 7 g/dL and 9 g/dL).¹¹ Although such a restrictive hemoglobin "transfusion trigger" may not be appropriate for all patients, less is better.

An RCT evaluating the optimal threshold for allogeneic platelet transfusions for prophylactic treatment of patients with acute myelogenous leukemia demonstrated that a lower platelet "transfusion trigger" (10,000/µL) resulted in no more bleeding episodes than those patients transfused at higher levels (20,000/µL).¹² Again, in this study population, less is better.

Fresh frozen plasma components (FFP thawed, thawed plasma, cryoprecipitate-reduced plasma, cryoprecipitate) transfusions have also been over utilized. Although a 1.9% decrease in FFP plasma components was noted in 2006 from 2004 transfusions,³ inappropriate FFP transfusions continue. The most common reason to prophylactically transfuse FFP is because of a minimally elevated prothrombin time (PT) and international normalized ratio (INR).^13,14 Due to the physiologic exponential rate curve between percent of factor concentration vs. mildly elevated PT/INR, larger volumes of FFP will not significantly improve the PT/INR values. Also, mildly elevated PT/INR (<1.5 times the upper limit of a laboratory reference range) cannot predict bleeding, including procedures such as minimally invasive liver biopsy or catheter insertions. The risk-benefit ratio is high for these blood components (i.e., TRALI), so less is better.

The new "kid on the block" in transfusion medicine is cellular therapy. Hematopoietic progenitor cell collection/HPC (peripheral and cord blood) and transfusion is the newest modality in bone-marrow transplantation (BMT). A 25% increase in apheresis HPC collections and 208% increase in cord-blood collections were noted between 2004 and 2006.³ Although a few patient diseases require the traditional bone-marrow-collected HPC, most BMT programs are utilizing apheresis and cord HPC collections/transfusions as their primary transplant support. In this area, more is better.

Future-transfusion-medicine innovations

What future technology awaits the transfusion-medicine community? Progress is being made in two important areas of routine blood transfusion: 1) pathogen inactivation and 2) blood substitutes. Pathogen inactivation (PI) techniques are varied depending on the component under investigation (cellular, plasma). PI has the potential to eradicate all currently known transfusion-transmitted viruses and bacteria, and even those not yet discovered.¹⁵ The success of such technology would also eliminate the need for bacterial detection, infectious-disease marker testing, and cellular- component irradiation. On the negative side, however, are the toxicities, neoantigenicities, efficacy of PI component transfusion in certain recipient populations (premature infants, neonates, pregnant patients), and cost. Although this is an exciting time in PI, caution is required based on the risk/benefit to society.

Blood substitutes have been slower to reach inclusion in the armamentarium of transfusion medicine. Only one product (PolyHeme, Northfield Laboratories, Evanston, IL), a human hemoglobin-based polymerized oxygen carrier (HBOC), is nearing submission to the FDA for a biologic license application.¹⁶ As with most of the HBOC products, this product is to be used for temporary oxygen delivery until the patient is stabilized and routine RBCs are available for transfusion. With a shelf-life of 12 months and 50 g of modified hemoglobin, the potential for use in critical-care emergencies holds promise. Many of the HBOC trials have been discontinued due to untoward organ toxicities.

Future developments in tissue engineering (ex vivo expansion), hopefully, will allow for use of various cells lines (e.g., liver, neuronal, cardiac valves, muscle, blood) to be available to specifically replace non-functioning tissue and treat diseases that consist now only of supportive-treatment modalities. New cellular-component storage mediums are also being evaluated to prolong the shelf life of stored donor blood and provide enhanced transfusion efficacy for the recipient. Will the blood donor become non-existent? Not in our grandchildrens’ lifetimes at the earliest. But that day will come. Stay tuned.

William B. Lockwood, PhD, MD, is a clinical professor in the Department of Pathology and Laboratory Medicine at the University of Louisville in Kentucky. He also is director of Transfusion Services and Tissue/Bone Bank at the University of Louisville Hospital; and director, Transfusion Services, Tissue/Bone Bank and Coagulation Laboratory at the Norton Hospital-Kosair Children’s Hospital, also in Louisville.

References

1. Greenwalt TJ. The short history of transfusion medicine. Transfusion. 1997;37:550-563.
2. Lansteiner K. Zur Kenntnis der antifermentativen, lytischen and agglutinierenden Wirkungen des Blutserums und der Lymph. Zbl Balk. 1900;27:367.
3. U.S. Department of Health and Human Services. The 2007 Nationwide Blood Collection and Utilization Survey Report. Washington, DC: DHHS, 2008.
4. Bihl F, Castelli D, Marincola F, Dodd RY, Brander C. Transfusion-transmitted infections. J Transl Med. 2007;5:25 Available at http://www.translational-medicine.com/contents/5/1/25 . Accessed on December 8, 2008.
5. Fatalities Reported to FDA Following Blood Collection and Transfusion. Annual Summary for year 2007. Center for Biologics Evaluation and Research, Bethesda, MD. Available at http://www.fda.gov/cber/blood/fatal07.htm . Accessed on December 8, 2008.
6. Blood transfusion set, ca 1900. Oregon Health Sciences Historical Collections & Archives. Available at http://content.ohsu.edu . Accessed on December 8, 2008.
7. Price TH, ed. Standards for Blood Banks and Transfusion Services (ed 25). Bethesda, MD: AABB; 2008; p. 11.
8. Kaufman RM. Platelets: testing, dosing and the storage lesion-recent advances. Hematology. (Am Soc Hematol Educ Program). 2006:492-496.
9. McDonald CP, Roy A, Mahajan P, et al. Relative values of the intervention of diversion and improved donor-arm disinfection to reduce the bacterial risk from blood transfusions. Vox Sang. 2004;86(3):178-182.
10. Dzik WH. Technology for enhanced transfusion safety. Hematology. (Am Soc Hematol Educ Program). 2005;476-482.
11. Hebert PC, Wells G, Blajchman MA: A multicenter randomized controlled trial of transfusion requirements in critical care. NEJM. 1999;340:409-417.
12. Rebulla P, Finazzi G, Marangoni F, et al. The threshold for prophylactic platelet transfusions in adults with acute myeloid leukemia. NEJM. 1997;337:1870-1875.
13. Triulzi DJ. The art of plasma transfusion therapy. [editorial]. Transfusion. 2006;46:1268-1270.
14. Abdel-Wahab OI, Healy B, Dzik WH. Effect of fresh-frozen plasma transfusion on prothrombin time and bleeding in patients with mild coagulation abnormalities. Transfusion. 2006;46:1279-85.
15. Webert KE, Cserti CM, Hannon J, et al. Proceedings of a consensus conference: pathogen inactivation-making decisions about new technologies. Transfus Med Rev. 2008;28:1-34.
16. PolyHeme® Multicenter Phase III Trial. Available at http://www.northfieldlabs.com/polyheme.html . Accessed December 8, 2008.

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Plus

Nitric-oxide bioactivity depletion:
An added storage lesion in banked blood

By Faon Rodriguez, MS, and Diana Ramirez, MS

There is more to a blood transfusion than to increase the oxygen-carrying capacity of red-blood cells (RBCs). Blood transfusions should be able to enhance vasodilation, open blood vessels, and increase blood flow to hypoxic tissue. Blood transfusions should also help improve RBC rheology to facilitate flexible transport of RBCs through tiny capillaries. If, however, blood vessels vasoconstrict or if the RBCs become rigid, then the blood transfusion may be less effective.

An effective blood transfusion is one that not only raises the hematocrit to the normal range but also enhances arterial relaxation and vasodilation. In man, the enzyme that produces nitric oxide (NO) from L-arginine is called nitric-oxide synthase The vasodilator gas, nitric oxide, is carried by hemoglobin in the form of S-nitrosothiol (SNO) (see Figure 1 online). Nitric oxide released from S-nitrosothiol helps relax smooth muscle surrounding the tubules of arteries, arterioles, and metarterioles (see Figure 2 online). When smooth muscle relaxes, the arterial blood vessels expand, leading to vasodilation. Thus, wide-open vessels improve the blood flow from arteries to capillaries, making the transport of oxygen to tissue more efficient. Ultimately, in the mitochondria, oxygen contributes to produce adenosine triphosphate (ATP) from adenosine diphosphate (ADP) by electron-transport and oxidative phosphorylation. ATP is used as an energy source by tissue. Blood transfusions should be characterized by how well RBCs facilitate nitric-oxide bioactivity to open blood vessels, rather than by how much oxygen they can deliver to tissue.

Recently, Jonathan S. Stamler, MD, et al, and Timothy J. McMahon, MD, PhD, et al, at Duke University Center (DUMC), have shown how nitric-oxide bioactivity helps RBCs ferry oxygen to tissues by opening tiny vessels during hypoxic vasodilation.^1,2 They also report that when RBCs leave the body during blood-banking donations, nitric-oxide bioactivity and S-nitrosohemoglobin in RBCs begin breaking down almost immediately. This type of storage lesion continues during the shelf life of banked blood. The depletion of nitric-oxide bioactivity, along with the decrease in concentration of 2,3-diphosphoglycerate, or 2,3-DPG, both affect vasodilation, compromising oxygen delivery to tissues. Thus, it does not matter how much oxygen hemoglobin carries; if the blood vessels do not relax and open, oxygen cannot be delivered effectively to tissue. Therefore, the unimpeded flow of blood throughout the blood-vessel bed is vital for the efficient transport of oxygen.

The primary function of the cardiovascular system is to supply oxygen to tissues and organs in the body RBC transfusions are commonly used to treat anemia, including euvolemic patients with congestive heart failure, to increase oxygen delivery to hypoxic tissue. The RBC functions as an O₂ sensor contributing to the regulation of blood flow and oxygen delivery, by releasing nitric oxide, depending on the oxygenation state of hemoglobin. RBCs depleted of nitric-oxide bioactivity do not always improve oxygen delivery during blood transfusions. There are recent concerns about the benefit of blood transfusions to critically ill patients due to immunomodulation and storage lesions found in banked blood.³ Blood transfusions can lead to vasoconstriction, congesting the flow of oxygen in narrow passageways in the cardiovascular system — damaging the very heart tissue that the blood was transfused to help.

Blood transfusions that lack nitric-oxide bioactivity may become associated with ischemia, a dangerous drop in blood flow. Investigators are suggesting that re-nitrosylation — adding a purified aqueous solution of nitric-oxide gas into banked blood before transfusions — can raise the concentration of S-nitrosohemoglobin in vitro which, in turn, can amplify vasodilation in vivo. In fact, Dr. Stamler and the anesthesiology and biochemistry teams at DUMC have demonstrated that canine coronary-blood flow was greater during the infusion of rejuvenated re-nitrosylated RBCs than during infusion of S-nitrosothiol-depleted RBCs. This article reviews the literature on depletion of nitric-oxide bioactivity in banked blood affecting vasodilation, blood flow, and oxygen transport to tissue, and elaborates on current research about the rejuvenation of donated banked blood by adding nitric oxide prior to transfusion.

Surprises at the cellular level happen all the time. Nitric oxide or nitrogen monoxide is a formidable toxin commonly found in nature as a gas and as a component of air pollution. For instance, it is a pollutant produced by automobile exhaust fumes and by power plants. Oxygen from the air and nitrogen combine at combustion temperatures or in the presence of electrical energy to form nitric oxide. Nitric oxide is produced by many cells in the body; however, its production by the vascular endothelium — the innermost cell layer of blood vessels — is particularly important in the regulation of blood flow. Today, we know that nitric oxide plays a function in the cardiovascular system, the immune system, and in the central and peripheral nervous systems. Furthermore, this simple gas, nitric oxide, affects a variety of complex biological processes, including blood-pressure homeostasis, platelet aggregation, and transmission of signals by the nervous system.⁴ NO also plays a key role in the activation of macrophages and cellular defenses against microbial pathogens. It is a major pathophysiological mediator of inflammation and host-defense mechanisms.

In the 1800s, Alfred Nobel invented dynamite, in which one of the main components is the explosion-prone nitroglycerine. Centuries later, nitroglycerine is being used as a vasodilator by patients with chest pain, suffering with angina. Nitroglycerine is converted into nitric oxide in the bloodstream, relaxing the muscle lining of vessels to allow better blood flow.⁵ In the mid-1980s, scientists were surprised to find out how nitric oxide was being produced meticulously in human cells. Nitric oxide went from being an extraneous toxic and corrosive gas to becoming an ubiquitous elixir of life. In 1987, Salvador E. Moncada, MD, PhD, discovered the vital role of nitric oxide as a messenger in the relaxation of muscle.⁶ In 1991, a team headed by K. E. Andersson of Lund University Hospital in Sweden showed how nitric oxide was the principal neurotransmitter mediating erectile function.⁷ In 1992, Science published a cover story naming nitric oxide the molecule or the year. In 1998, Robert F. Furchgott, MD, from the State University of New York; Louis J. Ignarro, MD, from the University of California; and Ferid Murad, MD, PhD, from the University of Texas, were given the Nobel Prize in Physiology for their discoveries of nitric oxide as a signaling molecule in the cardiovascular system.⁸

Non-biological functions of NO

Nitric oxide should not be confused with a) nitrous oxide (HN2O), a general anesthetic; or b) nitrogen dioxide (NO2), which is another poisonous air pollutant; or c) nitric acid (HNO3). Nitric oxide is a very unstable free radical turning, within seconds, into univalent radicals of nitrate (NO3) in vivo and into nitrite (NO2) in vitro. Nitric oxide reacts with ozone in the air to form nitrogen dioxide (2NO + O2 ? 2N02).

The synthesis of NO from molecular nitrogen and oxygen ( N2 + O2 ? 2NO) requires elevated temperatures of greater than 1,000°C. Internal-combustion engines have increased the concentration of NO in the environment by automobile-exhaust fumes. The purpose of catalytic converters is to minimize nitric-oxide emissions by catalytic conversion to O2 and N2. Nitric oxide in the air can convert into nitric acid, which has been implicated in acid rain.

Biological functions of nitric oxide

Nitric oxide is a lipophilic radical that readily moves across permeable cell membranes via passive diffusion. Nitric oxide is one of the few gaseous particles with biological-signaling capabilities. NO is known as the endothelium-derived relaxing factor, or EDRF, and a liable free radical with a half-life of about three to five seconds. It is biosynthesized from L-arginine and oxygen to citrulline by several nitric-oxide synthases, or NOS, enzymes and by the reduction of inorganic nitrate. NO is known to be produced in bacteria but found to act differently in mammals as a signaling molecule. Produced by many types of cells including nerve cells and the endothelium, nitric oxide is regulated by biofeedback and by the ability of superoxide anion and superoxide dismutase to inactivate NO. Nitric oxide is also controlled by the "on-and-off redox switch" — the reduction/oxidation potential states of biochemical reactions.

Discussion

Banked-blood packed RBCs have had most of their leukocytes and plasma removed. Packed RBCs undergo rigorous testing before their use. The blood-banking industry does an extraordinary job of manufacturing a safe and effective product. In order to prevent transfusion-transmitted diseases, the Food and Drug Administration (FDA) mandates testing for viral markers including hepatitis B; human immunodeficiency virus 1,2, or HIV 1,2; human T-lymphocytotrophic virus 1,2, or HTLV-1,2; cytomegalovirus, or CMV; serologic test for syphilis, nucleic-acid testing, or NAT, for West Nile virus, and hepatitis C virus.⁹ In addition, in order to conserve red-cell survival and function, RBC units are treated with additive solutions containing sodium chloride, dextrose, adenine, monosodium phosphate, mannitol, sodium citrate, and citric acid. RBCs also contain anticoagulants like citrate-phosphate dextrose, or CPD; citrate-phosphate dextrose-dextrose, or CPD2D; or citric–phosphate dextrose-adenine, or CPDA-1. The 42-day expiration date of banked blood stored at 1°C to 6°C depends mainly on the type of additive solutions used including AdsolR (Fernwall, Lake Zurich, IL), NutricelR (Pall Life Sciences, Ann Arbor, MI), or OptisolR (Terumo, Somerset, NJ).¹⁰ RBCs collected using the Trima Accel Collection System (CaridianBCT, Lakewood, DO) also have a shelf life of 42 days.

Approximately 13.9 million units of blood are transfused to 4.8 million patients each year in the United States, and the basis for approved use is determined by meeting regulations during collection, processing, and storage.¹¹ Banked blood is a biological product that is under the scrutiny of many regulatory agencies and the scientific community. The AABB (American Association of Blood Banks) also publishes guidelines for a safe transfusion. Blood has both benefits and risks, and, therefore, should be evaluated in the same manner as medications. To make a better product, however, the industry has pursued the idea of introducing synthetic banked blood, but its success remains to be proven. Until then, investigators are proposing ways to improve the product that is already at hand today. The current interest in the literature is about nitric-oxide bioactivity found in the form of S-nitrosothiol, which is crucial for the delivery of oxygen to tissues. Nitric oxide is not only needed for RBCs to transport oxygen but also may be responsible for the flexibility of the RBCs. When nitric-oxide levels decrease, the RBCs become stiffer, making it more difficult for them to adapt their shape in order to travel through the tiny capillary spaces during the delivery of oxygen (see Figure 3 online).

Storage lesions include RBC rheology, the loss of shape, and flexibility,¹² the decrease in the concentration of molecular modulators of oxygen binding (e.g., 2,3-DPG), the decrease in nitric-oxide bioactivity, and the increase in RBC adhesiveness during prolonged storage. Storage lesions in banked blood have been found to be responsible for adverse outcomes, like those leading to increased mortality rates after blood transfusion. Alterations in RBC rheology and adhesion may exacerbate rather than correct ongoing ischemia and — at least, partly — account, for the adverse effects of blood transfusions.

At the Cleveland Clinic Foundation, Colleen G. Koch, MD, et al, examined data from 1998 to 2006 for patients who received RBC transfusions. A total of 2,872 patients received 8,802 units of blood that had been stored for 14 days or less ("fresh blood"); 3,130 patients received 10,782 units of blood that had been stored for over 14 days ("aged blood"). After cardiac-surgery patients who were transfused, "aged blood" had an increased risk of postoperative complications and reduced chance for survival.¹³

Nobel laureate Dr. Ignarro, in his book No More Heart Disease, indicates that the endothelial cells can get sabotaged by a variety of health conditions that compromise the production of nitric oxide. Some of the health conditions that add more stress to the blood vessels and that inflict endothelium-cell damage include high blood pressure; atherosclerosis; high blood-cholesterol levels; elevated blood glucose; high low-density lipoprotein, or LDL; and cigarette smoking. The endothelial cells produce nitric oxide to protect us from many diseases by regulating blood pressure and blood flow. The endothelial cells, however, have a much harder job producing nitric oxide in patients with hypertension, coronary heart disease, or stroke.¹⁴ Thus, patients with underlying cardiovascular disease who are in need of blood transfusions have a much bigger challenge to process nitric-oxide-depleted banked blood, especially if it is more than 14 days old.

Furthermore, patients with sickle-cell anemia have abnormal hemoglobin, which is needed to deliver oxygen and nitric oxide to tissue. Hemoglobin-S has a lower affinity for oxygen and, once deoxygenated, the RBCs become distorted or sickled. Hemoglobin-S does not transfer nitric oxide from heme to thiol as well as normal hemoglobin during S-nitrosohemoglobin conformation The symptoms of sickle-cell disease are attributed to the physical obstruction of blood vessels by distorted or sickled and rigid RBCs.¹⁵ Thus, sickle cells become fragile, demonstrate vasooclusion, and lead to hemolytic anemia. Consequently, sickle cells have added disadvantages pertaining to blood-vessel dilation when tissue experiences oxygen deficiency during hypoxemia. Sickle-cell patients who receive blood transfusions may have the transfused RBCs accumulate in their vascular system, impeding the free flow of blood and transportation of oxygen to tissue. Relieving the vasoconstriction and restoring nitric oxide to RBC membranes may help prevent the painful symptoms of sickle-cell disease. Thus, there is an opportunity for clinical trials on the therapeutic use of nitric oxide with sickle-cell patients.

There is growing interest to improve the safety of our blood supply. Investigators at DUMC have shown that the level of S-nitrosohemoglobin was reduced by 85% to 95% at storage days seven and 43 compared to day one. They also noted a deficiency in vasodilotary activity in banked blood when compared with "fresh blood." Banked blood used for transfusion still has some shortcomings, but researchers are now contemplating reducing some of the storage lesions by replenishing nitrosylation in banked blood before using it for transfusion.

Replenishing bioactivity modulators in banked blood is nothing new. During the Vietnam era, United States Navy Physician C. Robert Valeri and N. M. Hirsch showed that ‘‘spiking’’ stored RBCs with diphosphate glycerate and ATP precursors led to significant improvements in cardiovascular function.¹⁶ Some establishments use rejuvenating solutions, like RejuvesolR (Cytosol Labs, Lenoir, NC) which contains pyruvate, inosine, phosphate, and adenine, to restore oxygen transport and improve post-transfusion survival of RBCs.

It is being suggested that adding nitric oxide to banked blood before its use could, theoretically, improve hemoglobin nitrosylation and the ability of nitric oxide in S-nitrosothiols to dilate and open blood vessels and, thus, prevent heart attacks and even death. Investigators have demonstrated that replenishing banked blood with nitric- oxide gas raises S-nitrosohemoglobin, or SNO-Hb, concentrations and restores the hypoxic vasodilatory activity of RBCs.

The RBC is more than a "passive bag" full of hemoglobin that transports oxygen. In fact, the RBC is a regulator of its own destination. The matching of oxygen supply with demand requires nitric-oxide bioactivity to increase blood flow in response to decreased levels of oxygen in tissue. Increasing the hematocrit into the normal range after a blood transfusion should be supplemented by increasing vasodilation, blood flow, and oxygen transport. This can be achieved by adding nitric oxide to banked blood prior to blood transfusions. Therefore, we support the new paradigm from Joseph Bonaventura, PhD, for testing nitric-oxide bioactivity in banked blood, once re-nitrosylation of banked blood becomes a reality.¹⁷ There exist opportunities to further investigate nitric-oxide bioactivity as suggested by Bonaventura which include testing of arterial and venous RBC S-nitrosohemoglobin as a diagnostic indicator for transfusion; assaying hemoglobin re-nitrosylation treatment of stored RBCs; verification of normalized RBC rheology before transfusions; and verification of normalized RBC vasoactivity prior to transfusion.

Allogenic, autologous, and directed blood transfusions are not scrutinized with a risk/benefit analysis common for all biologics. Furthermore, there are no regulations or clinical standards aimed at examining the clinical outcome of an effective blood transfusion in patients with respect to nitric-oxide bioactivity in vasodilation, blood flow, and oxygen transport to tissue. Consequently, an opportunity exists for clinical trials to evaluate the outcome and effects of transfusing improved re-nitrosylated banked-blood products to patients. Thus, further research is needed to measure the effectiveness of transfusions by testing the concentration of nitric oxide in the form of S-nitrosothiols or S-nitrosohemoglobin in the peripheral blood of patients who receive blood transfusions.

Adding soluble portions of nitric oxide to banked blood is in its infancy, but this seems to be more promising than the current results and developments seen with the manufacturing of a synthetic blood product. The addition of nitric oxide to banked blood eventually may need to undergo rigorous clinical trials, FDA approval, and re-evaluation of the current 42-day expiration date. Nonetheless, the growing concern of transfusing banked blood that is over 14 days old in patients undergoing cardiac surgeries may help expedite more research and diligent acceptance by the medical community and regulatory agencies. Re-nitrosylation of banked blood with nitric oxide is the most promising project undertaken by DUMC investigators to preserve more of our blood supply.

Faon Rodriguez, MS, is section supervisor at Florida Hospital, Celebration Health, FL, and Diana Ramirez, MS, is transfusion service supervisor at Osceola Regional Medical Center, Kissimmee, FL.

Acknowledgements: The authors want to thank these colleagues for their advice and comments after reading this manuscript: Patrick J. O’Sullivan, laboratory director, Florida Hospital, Orlando, FL; Pamela Hargrave-Thomas, laboratory director, Osceola Regional Medical Center, Kissimmee, FL; Theresa Palmer, assistant director, Florida Hospital, Kissimmee, FL; and Kathryn Pearson, assistant director; Gail S. Borysko, laboratory supervisor; and Sonaly Cosme, medical technologist — all from Florida Hospital, Celebration Health, FL.

References

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