Predicting intraoperative transfusion volumes of blood products in cardiovascular surgery: a retrospective study.

It is well known that the older the age of blood, the higher the supernatant potassium concentration (K+) of red blood cell (RBC) units. Other factors known to increase potassium during RBC transfusions, such as rate of transfusion, irradiation, and type of additive used, are well-described risk factors for hyperkalemia. When patients experience an increase in K+ concentration, the increase is often transient due to the redistribution of the potassium load. However, if a massive amount of blood is transfused, the stored RBC units may contain a sufficient amount of potassium to result in hyperkalemia. In this clinical scenario, transfusion-associated hyperkalemic cardiac arrest (TAHCA) may occur. The recent attention surrounding these reactions resulted in an advisory from the Society of Pediatric Anesthesia and its patient safety organization, Wake up Safe.1 In this issue of TRANSFUSION, a review article by Lee and colleagues2 identifies those risk factors and potential preventive measures by reviewing the published literature on TAHCA in a pediatric population. There is wide variation of RBC transfusion practice for both neonatal and pediatric populations. Spinella and colleagues3 conducted a survey in 2008 and 2009 to determine current practices in US and Canadian hospitals. Policies developed to prevent hyperkalemic reactions involve using fresher blood, RBC washing policies, and RBC irradiation. Based on the survey, the use of fresh blood was largely based on patient condition, such as cardiac surgery patients of all ages (46.5%), as opposed to the anticipation of massive transfusion (11.6%). Aside from neonatal exchange, the most common patient conditions for washing blood were patients undergoing neonatal cardiac surgery or cardiac surgery patients of all ages. Again, massive transfusion was not the most common indication for washing. The majority of the centers responded that blood was irradiated at the time of issue (64.5%), thus mitigating some of the concerns for the increase of potassium in an irradiated unit. In the current TRANSFUSION review, the authors take on the ambitious task of reviewing the prospective and retrospective published literature in the area of TAHCA associated with massive transfusion and find that the literature is quite limited. As an alternative to a meta-analysis, they conducted a review and detailed analysis of case reports and clinical studies of pediatric transfusion–associated hyperkalemia. Lee and colleagues reviewed 15 articles, nine case reports, and 13 clinical trials, although not all the clinical trials involved massive transfusions and five of the trials were routine and small-volume transfusions. Of the remaining eight that were not registries, six clinical studies that involved massive transfusions are described. Of interest, the authors note that some patients received smaller-volume transfusions that resulted in TAHCA. In one study, the common factors for TAHCA in patients who received rapid transfusion included acidosis, hyperglycemia, hypocalcemia, and hypothermia at the time of arrest. One important finding of this review is the identification of these metabolic abnormalities and methods of transfusion leading to TAHCA, which are just as important as developing blood bank protocols for product modification and age of units. For many transfusion services, procedures and protocols are set up for patient-specific conditions, but may not include specific measures to mitigate all risks. It is convenient to refer to these situations as "planned emergencies." An example of this process may be a massive transfusion protocol. However, this type of protocol may even require more detail to identify subpopulations with unique factors for additional complications, such as TAHCA. Where we struggle in transfusion services is the identification and preparation for these "unplanned emergencies," where complications such as hyperkalemia are potential problems during transfusion, but have not been previously anticipated. In these cases, there may not be time for processing steps, such as washing blood, so other strategies would be required. Lee and colleagues provide a summary of these measures to reduce TAHCA in pediatric patients. The first measure identified, "anticipate and replace blood loss before significant hemodynamic compromise occurs," is an excellent example of the planned emergency. If clinicians can identify surgical procedures with known complication rates for massive transfusion and TAHCA, it may be possible to keep "fresher" units reserved for a patient. In our institution, craniofacial surgical patients were identified as a high risk for massive transfusions, so those patients have been placed preemptively on a fresher RBC unit protocol. In addition, recent publications examining real-time maximum order blood schedules, using computerized anesthesia records as a tool to identify trends in transfusion based on diagnostic categories, may assist in identifying patients at higher risk.4 This knowledge will enable the physician to communicate concerns before surgery, so even if blood is not initially issued, a conversation can take place to know what is available, how long washing or plasma reduction would take, and the agreement to issue the fresher units first, with older units "on hold" in the event of a massive bleed. An example of this scenario is described in the review to illustrate how this measure may be achieved in practical terms. Lee and colleagues identified other measures to reduce TAHCA through their literature review, which included responsibilities of the clinician or anesthesiologist performing the transfusion. The size of the catheter, that is, larger bore, and peripheral transfusion over central lines may help mitigate the risk of TAHCA. At our institution, we also emphasize the rate of transfusion as a critical precaution that can be controlled even in patients with a central line. Along with this concept is the avoidance of rapid infusers in higher-risk patients. Pediatric transfusion medicine specialists, such as Strauss, have also described infusion rate as a critical factor in reducing the risk of the hyperkalemia in pediatric patients. In an earlier TRANSFUSION editorial, Strauss5 calculated potassium load based on different pediatric clinical scenarios. He recommended that if RBCs are required for unexpected massive bleeding, infusion rate be given at 0.5 mL/kg/min to avoid hyperkalemic reactions. Strauss also emphasized that guidelines should be developed to anticipate these situations. Other recommendations may include checking and treating electrolyte abnormalities frequently. Like any drug given in the operating room, RBC transfusions have the potential to change the patients' electrolytes. These effects may be exacerbated by the use of drugs used to anesthetize the patient. Understanding the effects of different drugs given, along with careful monitoring of electrocardiogram (ECG) and electrolytes, will allow the anesthesia team to expeditiously intervene before the untoward outcome of a cardiac arrest. The majority of cases of TAHCA occur in the perioperative setting. To mitigate the risk of TAHCA, it is important for the anesthesiologist to identify at-risk patients, as well as prepare the perioperative team and blood bank for the possibility of massive transfusions. Individual hospitals should utilize their perioperative electronic health record to identify the types of cases that have historically required massive transfusion (more than 70 mL/kg blood within a 24-hr period or greater than 35 mL/kg within a 3-hr period), as defined by the review by Lee and coworkers. Preoperative labs including electrolytes and hemoglobin should be obtained for these higher-risk pediatric patients. A large-bore peripheral IV (greater than 23 gauge in pediatrics) should be started and utilized for massive transfusion preferentially to central access. Ideally, the blood bank should select units that are fresh or washed (preferably within 6 hr of irradiation); however, this recommendation is often not easily accomplished in large-volume clinical centers. During massive transfusion, the anesthesia team has multiple patient care issues to identify and treat simultaneously, although care should be taken to check arterial blood gasses, electrolytes, and glucose to diminish the possibility of TAHCA. In addition, potassium levels can become rapidly elevated if the surgical procedure predisposes the patient to a period of hypoperfusion of a large vascular bed with subsequent reperfusion. Patients who are undergoing surgery and losing large amounts of blood are often hyperglycemic, hypovolemic, and acidotic, conditions that predispose to hyperkalemia. ECGs should be monitored for early signs of hyperkalemia including peaked T-waves, prolongation of the PR interval, and widening of the QRS complex. For these "unplanned emergencies" the anesthesia team should have treatments readily available, including dextrose-insulin and bicarbonate to shift potassium back into the cells and calcium as a physiologic antagonist and be mindful of hypovolemia and decreased cardiac output, which can both increase the possibility of hyperkalemia. It is prudent clinical practice to manage TAHCA not as an unanticipated complication, but as an expected complication that may occur with any given patient in high-risk situations. The case reports and literature surveyed in this review support that premise. There are patients who undergo TAHCA despite receiving fresher units, smaller-volume transfusions, or transfusions through a peripheral line. Therefore, it is important to recognize that hyperkalemia may occur at any time during surgery so that physicians have proper training and preparation to handle these events. Much like we identify patients at risk for other transfusion reactions and attempt to provide pretransfusion medication or mitigating steps, identifying patients at risk for a hyperkalemic reaction and/or arrest is important. One way to identify these patients is through the preanesthesia evaluation. A recent Practice Advisory for Preanesthesia Evaluation reviews preoperative laboratory tests that should be performed before surgery.6 Of interest, in asymptomatic or nonselected patients, abnormal potassium concentrations were reported in 0.2% to 16% of patients. However, for selected high-risk patients, abnormal potassium concentrations were reported in 2.9% to 71% of patients. These data may suggest that the anticipated transfusion population is a selected population that requires special consideration, especially with abnormal potassium concentrations reported before surgery. The cited Anesthesia Advisory concludes that perioperative therapies, that is, transfusions, endocrine disorders, risk of renal and liver dysfunction, and use of certain medications and alternative therapies, justifies careful perioperative serum chemistry monitoring. Lee and colleagues summarize the pediatric case reports and clinical studies of TAHCA in two convenient tables. The unrecorded data in many of the case reports and clinical trials lead to additional uncertainty about the contribution of the RBC units to the reactions in question. In attempts to underscore the importance of these types of reactions, the Society for Pediatric Anesthesia began collecting information about hyperkalemic reactions leading to arrests. After review of the case reports, the Pediatric Anesthesia Quality Improvement Initiative published an Advisory Report with recommendations for RBC products. Again, similar to the case reports and clinical trials in the current review by Lee and coworkers the collection of data was not standardized, making it difficult to evaluate each reaction to determine what role, if any, the RBC transfusions played. Overall, recommendations in the Advisory Report included the use of "fresh" blood in cases where massive transfusion is anticipated. The advisory defines fresh as 7 days or less, but that definition was not based on conclusive scientific evidence. Other recommendations include transfusing as soon as possible after irradiation and washing older or irradiated units before transfusion. Some challenges for meeting these requirements involve the fact that not every institution has an irradiator and/or cell washer on site, making the timing of these processing steps before transfusion less controllable. In response to the Wake up Safe Advisory, the AABB issued a statement addressing some concerns for the universality of some of the recommendations and precautions to developing guidance based on limited and inconclusive evidence.7 Evaluation of TAHCA reactions requires both input from the blood bank and clinical team to make the best decision moving forward for care of patients where major bleeding and hyperkalemic arrests may be predicted. Unilateral decisions made by either group, without discussion of the data and their implications for clinical practice, may lead to erroneous conclusions resulting in policy and procedures that are not medically indicated and/or adequate to mitigate these reactions. As one example, clinicians may begin to measure the K+ concentration in stored blood using nonvalidated instruments that are not approved for measuring K+ in stored RBCs. A further concern is that these erroneous K+ values may lead clinicians to label RBC units as either "good or bad," thus potentially resulting in the unnecessary wastage of RBCs. Clear communication, in addition to procedures and policies, is required, so as not to delay care in an emergent situation in a massively bleeding patient, where concerns of TAHCA are one of many complications during lifesaving measures. It sounds cliché to state, "We need more studies and data" to understand the risk factors associated with TAHCA and pediatric patients undergoing massive transfusion. However, more complete information will assist with guidance for policies and procedures that are medically indicated and within expectations that can be met with the current blood supply in hospital transfusion services.8 We applaud the efforts of Lee and colleagues for a comprehensive attempt to summarize the current literature with regard to these reactions and to bring our attention not only to the K+ load in the stored blood, but the low cardiac output state contributing to these reactions. In summary, the following risk factors were identified as contributors for TAHCA: longer storage age of the RBC product, speed and volume of RBC products transfused, age and size of patient, method of transfusion, and presence of comorbidities. The authors conclude that newer guidance to reduce TAHCA should be developed for the small subset of pediatric patients identified at risk. However, they also emphasize that other pediatric patients, without the cited risk factors, may continue to safely receive routine blood components of any age without concern of TAHCA. None.

See article on page 1093–1101, in this issue

There has been an international decline in the demand for red blood cell (RBC) units. In Australia, there has been a 21% reduction in demand between 2012 and 2015. In contrast, the demand for the "universal" group O D- RBC units is in fact proportionally increasing. The clinical use of the entire O D- RBC distribution for a 5-week period throughout Australia was reviewed. Fate data on each unit issued (n = 9733) were collected that included the indication and urgency of transfusion, reason for discard, component age, and patient demographics. A total of 74% of audit forms were returned (n = 7143). The national discard rate of issued units was 7.9%. A total of 6387 units were transfused into an estimated total of 3008 patients (55% males) with median patient age of 67 years and median RBC age of 21 days. Forty-seven percent were transfused to group O D- patients. A total of 17.4% were chosen for specific phenotype requirements, 24.5% of units were transfused close to expiry, and 24.5% were transfused into patients of other ABO groups. The data appear broadly representative of the current transfusion and inventory management practices surrounding the use of group O D- RBC units. Strategies to reduce O D RBC demand include reevaluation of inventory holdings particularly at smaller centers, increasing the panel of phenotyped RBC units across all ABO groups, more regular rotation of units between hospitals to minimize time expiry, and continuing education for promoting transfusion of ABO-identical RBC units.

O Rh(D)- red blood cell (RBC) units can generally be transfused to most patients regardless of their ABO blood type and are frequently used during emergency situations. Detailed usage patterns of O Rh(D)- RBC units in obstetric populations have not been well characterised. With the introduction of patient blood management guidelines, historical usage patterns are important for providing comparative data. To determine how the use of O Rh(D)- RBC units in pregnant women differs between hospitals of different sizes and obstetric capabilities prior to patient blood management guidelines. Data from 67 New South Wales public hospital blood banks were linked with hospital and perinatal databases to identify RBC transfusions during pregnancy, birth and postnatally between July 2006 and December 2010. RBC transfusions were divided into O Rh(D)- or other blood types. Hospitals were classified according to birth volume, obstetric capability and location, with transfusions classified by timing and diagnosis. Of the 12078 RBC units transfused into pregnant women, 1062 (8.8%) were O Rh(D)-. Higher use of O Rh(D)- RBC units was seen in antenatal transfusions, preterm deliveries and in regional or smaller hospitals. There was wide variation in rates of O Rh(D)- RBC transfusion among hospitals. The rate of O Rh(D)- RBC unit use in obstetrics was lower during the period assessed than the nationally reported usage. It is encouraging that O Rh(D)- RBCs were more commonly used in emergency or specialised situations, or in facilities where holding a large blood inventory is not feasible.

ObjectivesIn Australia, the demand for group O D− red blood cell (RBC) units is proportionally increasing in contrast with an overall reduction in RBC demand. A survey was conducted to understand the use of group O D− RBC units. Aggregated national data from this study have been reported. However, Australia has large geographical and jurisdictional differences in blood product funding arrangements, transfusion and inventory management practices. This region specific analysis was conducted to understand these differences in the use of group O D− RBC units.DesignThe fate of each O D− RBC unit issued during a 5‐week period (n = 9733) was analysed including urgency of transfusion, product age, patient demographics or reason for discard. The data were divided to represent five regions representing 1‐2 states and/or territories across Australia.ResultsRegions 1 and 5 had the lowest number of O D− RBC units issued per 1000 population. Region 4 had the lowest percentage of discard (3·8%) compared with the national average of 7·9%. Region 4 also had the lowest percentage (44·0%) of O D− RBC units transfused into nongroup O D‐ recipients compared with the national average of 52·2%.ConclusionsThe differences in usage of group O D− RBC units among regions were not statistically significant. However, possible region specific practices, such as the presence of a well‐developed blood rotation programme, could impact O D− RBC demand. Shared learning across all regions could assist in ameliorating the increasing national O D− RBC demand in Australia.

Red blood cell (RBC) units in hypothermic storage degrade over time, commonly known as the RBC storage lesion. These older RBC units can cause adverse clinical effects when transfused, as older RBCs in the unit lyse and release cell-free haemoglobin (Hb), a potent vasodilator that can elicit vasoconstriction, systemic hypertension and oxidative tissue injury after transfusion. In this study, we examined a novel method of washing ex vivo stored single RBC units to remove accumulated cellular waste, specifically cell-free Hb, using tangential flow filtration (TFF) driven by a centrifugal pump. The TFF RBC washing system was run under hypothermic conditions at 4°C, at a constant system volume with 0.9wt% saline as the wash solution. The RBC washing process was conducted on 10 separate RBC units. For this proof-of-concept study, RBC units were expired at the time of washing (60-70 days old). Cell-free Hb was quantified by UV-visible absorbance spectroscopy and analysed via the Winterbourn equations. Pre- and post-wash RBC samples were analysed by Hemox Analyser, Coulter counter and Brookfield rheometer. The RBC volume fraction in solution was measured throughout the wash process by standard haematocrit (HCT) analysis. No substantial decrease in the HCT was observed during the TFF RBC washing process. However, there was a significant decrease in RBC concentration in the first half of the TFF RBC wash process, with no significant change in RBC concentration during the second half of the TFF cell wash process with an 87% overall cell recovery compared with the total number of cells before initiation of cell washing. Utilization of the extinction coefficients and characteristic peaks of each Hb species potentially present in solution was quantified by Winterbourn analysis on retentate and permeate samples for each diacycle to quantify Hb concentration during the washing process. Significant cell-free Hb reduction was observed within the first four diacycles with a starting cell-free Hb concentration in the RBC unit of 0.105 mM, which plateaus to a constant Hb concentration of 0.01 mM or a total extracellular Hb mass of 0.2g in the resultant washed unit. The oxygen equilibrium curve showed a significant decrease in P50 between the initial and final RBC sample cell wash with an initial P50 of 15.6 ± 1.8 mm Hg and a final P50 of 14 ± 1.62 mm Hg. Cooperativity increased after washing from an initial Hill coefficient of 2.37 ± 0.19 compared with a final value of 2.52 ± 0.12. Overall, this study investigated the proof-of-concept use of TFF for washing single RBC units with an emphasis on the removal of cell-free Hb from the unit. Compared with traditional cell washing procedures, the designed system was able to more efficiently remove extracellular Hb but resulted in longer wash times. For a more complete investigation of the TFF RBC washing process, further work should be done to investigate the effects of RBC unit storage after washing. The designed system is lightweight and transportable with the ability to maintain sterility between uses, providing a potential option for bedside ex vivo transfusion in clinical applications.

Rh-Positive Fresh Frozen Plasma (FFP) Therapy Can Induce Cross-Reactive Immune Response to Red Blood Cell (RBC) Self-Antigen in Rh-Negative Patients Having Existing Alloimmunization to Rh-Antigen D

A Trauma Transfusion Pathway Decreases Coagulopathy without Increasing Blood Product Utilization

An increased risk of complications, including death, has been associated with stored red blood cell (RBC) units in observational studies but not in randomized trials. We aimed to evaluate for volume-dependent effects attributable to length of RBC storage in a secondary analysis of the Age of Blood Evaluation (ABLE) trial. In the 2510 critically ill adults from the ABLE trial randomized to receive RBC units stored not more than 7 days or the oldest compatible RBC units, we estimated the hazard ratio (HR) for death by intensive care unit (ICU) and hospital discharge and by days 28, 90, and 180, within subgroups defined by the number of RBC units received. Extended Cox proportional hazards regression was used to model the HR. A volume-dependent effect of storage age on survival was present for death by 90 and 180 days, but not earlier endpoints. The HR for death by 90 days was 0.55 (95% confidence interval [CI], 0.11-0.98, fresh vs standard) after transfusion of 6 RBC units but 1.45 (95% CI, 1.06-1.98) after transfusion of 1 RBC unit. In this exploratory analysis, volume-dependent effects related to RBC storage were documented in the ABLE trial. The harms associated with small volumes of fresh RBC units and large volumes of older RBC units should be further explored.

Recombinant Factor VIIa Treatment of Severe Bleeding in Cardiac Surgery Patients: A Retrospective Analysis of Dosing, Efficacy, and Safety Outcomes

Most patients transfused red blood cells in elective surgery receive small volumes of blood, which is likely to be discretionary and avoidable. We investigated the outcomes of patients who received a single unit of packed red blood cells during their hospital admission for an elective surgical procedure when compared to those not transfused. This retrospective cohort study included elective surgical admissions to 4 hospitals in Western Australia over a 6-year period. Participants were included if they were at least 18 years of age and were admitted for elective surgery between July 2014 and June 2020. We compared outcomes of patients who had received 1 unit of red blood cells to patients who had not been transfused. To balance differences in patient characteristics, we weighted our multivariable regression models using the inverse probability of treatment. In addition to propensity score weighting, our multivariable regression models adjusted for hemoglobin level, surgical procedure, patient age, gender, comorbidities, and the transfusion of fresh-frozen plasma or platelets. Outcomes studied were hospital-acquired infection, hospital length of stay, and all-cause emergency readmissions within 28 days. Overall, 767 (3.2%) patients received a transfusion of 1 unit of red blood cells throughout their admission. In the propensity score weighted analysis, the transfusion of a single unit of red blood cells was associated with higher odds of hospital-acquired infection (odds ratio, 3.94; 95% confidence interval [CI], 2.99-5.20; P < .001). Patients who received 1 unit of red blood cells throughout their admission were more likely to have a longer hospital stay (rate ratio, 1.57; 95% CI, 1.51-1.63; P < .001) and had 1.42 (95% CI, 1.20-1.69; P < .001) times higher odds of 28-day readmission. These results suggest that avoidance of even small volumes of packed red blood cells may prevent adverse clinical outcomes. This may encourage hospital administrators to implement strategies to avoid the transfusion of even small volumes of red blood cells by applying patient blood management practices.

For it is not enough to possess a good mind; the most important thing is to apply it correctly. Whether transfusion of "older" blood is as beneficial as transfusion of "fresher" blood is a topic of active debate. Considering the sheer volume of recent publications including some excellent reviews2,3 and a meta-analysis4 on the issue, one may wonder why there is a need for yet another editorial5,6 addressing one more observational study on the subject.7 Distinctively, this editorial strives to 1) examine how a well-conducted observational study can intelligently inform a scientific debate, 2) demonstrate the benefit of evaluating an issue from a variety of angles using different study methods, and 3) illustrate how research in transfusion medicine can impact and promote scientific knowledge in other fields. Several studies have reported an association between transfusion of older blood and highly clinically significant outcomes, including an increase in length of hospital stay, postoperative infections, prolonged mechanical ventilation, multiple organ failure, and mortality (see Lelubre et al.3 and Zimrin and Hess2 for in-depth reviews of the literature). Two mechanisms could be conjectured to explain these potential detrimental outcomes if a "cause-and-effect" relationship between duration of red blood cell (RBC) storage and morbidity/mortality is present; note that this is purely speculative since such a causal link has not been demonstrated. One hypothesis proposes that the RBC storage lesion defects that occur in stored blood components cause immunomodulatory and inflammatory complications in transfusion recipients including changes in vasoregulation. The second hypothesis, which could coexist with the first, proposes that susceptibility factors predispose certain patient populations to the clinical-pathologic side effects of older RBC transfusion. This problem if present (meaning if a causal relationship exists) would represent a major public health impediment considering the 5 million US patients who, every year, rely on RBC transfusions for their care. A shortened RBC storage time would have a major impact on the availability of blood. Blood banks would need to adapt recruitment practices to ensure a steady supply of fresher cells and/or develop storage procedures that prevented the development of lesions which contribute to significant adverse clinical outcomes. Faced with this specter, it behooves us to evaluate this problem in a scientifically rigorous and rational manner. Scientific objectivity is crucial because the consequences of misinterpreting the data are significant. As discussed, incorrectly concluding that blood storage duration is causally related to increased morbidity and mortality would severely impact blood availability and put patient care at risk. On the other hand, erroneously concluding that storage duration does not impact morbidity and mortality, if indeed it does, would have appalling consequences. Just under 400 years ago, Descartes put forward four methods in Part II of the Discourse1 which, to this day, can guide our approach to research and scientific discoveries. In his words, "the first [method] was never to accept anything as true that I did not incontrovertibly know to be so; that is to say, carefully to avoid both prejudice and premature conclusions; . . . "; "the second [method] was to divide all the difficulties under examination into as many parts as possible, and as many as were required to solve them in the best way"; "the third [method] was to conduct my thoughts in a given order, beginning with the simplest and most easily understood objects, and gradually ascending, . . . to the knowledge of the most complex"; and "the last [method] was to undertake such complete enumerations and such general surveys that I would be sure to have left nothing out." In other terms, we should refrain from jumping to conclusions when there is insufficient evidence (sounds familiar?); evaluate the issue from different perspectives using several study methods to collect a comprehensive set of data points; resolve simplest issues first; be comprehensive and systematic in our approach; check for consistency; double check our results; and use our "good sense," that is, "the power of judging correctly and of distinguishing the true from the false"1 when conducting data analysis and interpreting results (as a side note, some philosophers argue that truth is never attainable). Armed with these principles and the desire to fund research that will most rapidly advance our understanding of this issue, the National Heart, Lung, and Blood Institute (NHLBI) undertook a review of the studies conducted in this area starting with the clinical literature. We concluded that it is extremely difficult to interpret existing data. Many of the studies are observational in nature and suffer from lack of adjustment, a small sample size, and/or the inability to generalize results (only a few small randomized clinical trials [RCTs] have been conducted so far). In particular, the frequently observed imbalance in baseline characteristics of the comparison groups renders adjustment for confounders (characteristics that are associated with both the outcome and the main independent variable) a critical requirement for interpretation. For example, the "number of transfusions," which is linked to both severity of disease and increased RBC storage duration, is a major confounder in all RBC storage analyses.8 It is therefore crucial to carefully scrutinize these reports to evaluate how data were analyzed and interpreted and note that adjustment can still be problematic because not all confounders may be known and the assumptions underlying the statistical method that was used may not hold. Some studies suggest that transfusion of older blood is associated with increased morbidity and/or mortality while others suggest a lack of association or even the converse.2-4 A cause-and-effect relationship was not demonstrated and equipoise was present—meaning that there is genuine uncertainty as to whether transfusing fresher blood is more, less, or equally beneficial as transfusion of older blood. These observations begged for the conduct of studies evaluating comparable patient groups (e.g., RCTs) and, possibly, observational studies that were sufficiently powered and adequately analyzed to better inform the debate. NHLBI decided to pursue support of a RCT using the Transfusion Medicine and Hemostasis Clinical Trials (TMH) Network infrastructure (see below) and an observational study which is the subject of the report by Edgren and colleagues7 in this issue of TRANSFUSION. Using the Scandinavian Donations and Transfusions (SCANDAT) database, a powerful tool that merges data from population and migration, death and inpatient care, and blood donations and transfusion registries in Sweden and Denmark, Edgren and colleagues conducted a retrospective cohort study aimed at evaluating mortality among transfused patients receiving RBC units stored in a saline-adenosine-glucose-mannitol solution for 0 to 9 days (25.5% of transfusion episodes), 10 to 19 days (34.7%), 20 to 29 days (15.7%), or 30 to 42 days (8.1%); 15.9% of the transfusion episodes involved RBC units of mixed ages. They included data from more than 350,000 patients, 15 to 90 years old, who were transfused with RBCs between 1995 and 2002. Data on a little more than 400,000 transfusion episodes were analyzed using a variety of sophisticated methods and adjusted, at a minimum, for number of transfusions, age, sex, blood group, calendar period, season, weekday, hospital, and transfusion indication. Several other subanalyses were conducted to evaluate cause-specific risks of death as well as the effect of leukoreduction. This study evaluated a robust outcome (mortality) and included a large number of patient transfusion exposures allowing for adjustment of major known confounders. There was unbiased ascertainment of both exposure and outcome because the data were compiled from national registers. The investigators evaluated the data through a variety of approaches and still reached similar conclusions: that the 1-week risk of death after transfusion did not significantly differ between recipients of RBCs stored for different periods. Conversely, there was a significant increase in the 2-year mortality risk in recipients of 30- to 42-day-stored RBCs (5% increase) and mixed-age (3% increase) RBCs compared to the risk of recipients receiving RBCs stored for 10 to 19 days. The hazard ratios for patients receiving blood stored for 0 to 9 days and 20 to 29 days were 1.01 and 0.99, respectively (neither value was significant). Observing a significant increase in risk of death at 2 years but not at 1 week for recipients of 30- to 42-day-old or mixed-age blood is contrary to the expectation that the effects of an acute harmful event usually decrease over time. This observation, along with the absence of a clear dose-response effect and not finding that any particular cause(s) of death explained the excess risk, led the authors to attribute these findings to residual confounding by severity of disease, for which they were not able to directly adjust. One may also wonder if these two significant findings could just have occurred by chance considering the number of comparisons made. However, adjustment for multiple comparisons can be problematic and the authors would probably have been criticized for using such an adjustment to "get rid of" significant associations. Subset analyses did not reveal significant associations. In particular, looking at patients undergoing coronary artery bypass graft (CABG) surgery, the hazard ratios were similar, 1.09 and 1.10 (both nonsignificant) among patients receiving either blood stored for less than 10 days or blood stored for 30 to 42 days, respectively. The results of this well-conducted observational study are reassuring in the sense that 1) these investigators did not detect associations of the magnitude observed in other studies such as the study by Koch and colleagues9 and 2) the two significant findings can be reasonably explained by residual confounding and/or a multiple comparison effect. However, uncertainty still remains. What if these findings really reflect a cause-and-effect relationship (although it would be difficult to explain biologically why recipients of mixed-age blood would be at higher risk)? What if the subset analyses were insufficiently powered and patients undergoing CABG are at greater risk of complications if they receive fresh (<10 days) or old (>30 days) RBC units rather than blood that has been stored for 10 to 29 days? Clearly, RCTs are needed in a setting of equipoise because the ramifications for patient care and blood banking practices are profound. While RCTs can provide the strongest clinical evidence of whether RBC storage duration does or does not affect morbidity and mortality (the random allocation of patients to treatment arms provides for balance of baseline characteristics between comparison groups), RCTs are logistically difficult, lengthy, and expensive to conduct. Four large RCTs are now planned or under way in three different patient populations, highlighting the importance of this issue. The Age of Blood Evaluation (ABLE) study10 supported by the Canadian Institutes of Health Research (CIHR) is randomizing about 2500 intensive care unit patients at 25 or more Canadian sites to receive, if transfused, either less than 8-day RBCs or standard-issue RBCs (2-42 days); their primary outcome is 90-day all-cause mortality. The Age of Red Blood Cells in Premature Infants (ARIPI),11 also funded by CIHR, plans to randomize 450 premature infants (≤1250 g) to receive either less than 8-day RBCs or 2- to 42-day RBC aliquots (the standard practice is to take aliquots from the same unit of RBCs until its expiration); the primary endpoint is a 90-day composite measure of all-cause mortality and organ dysfunction. The Red Cell Storage Duration and Outcomes in Cardiac Surgery, which is being conducted at one center, the Cleveland Clinic (Ohio), is randomizing cardiac surgery patients who are 18 years and older to receive, if transfused, either less than 14- or more than 20-day RBC transfusions; their target is 2800 patients and their primary outcome is postoperative morbidity.12 Finally, the NHLBI TMH Network Red Cell Storage Duration Study (RECESS) currently plans to randomize approximately 1800 cardiac surgery patients at an estimated 15 centers across the United States to receive either 10-day-or-less or 21-day-or-more RBC units if transfused. Patients randomized to receive RBCs stored for 21 days or more will receive units from the hospital's existing blood bank inventory using the standard inventory management practice of releasing the oldest RBC units first. The primary outcome for RECESS is the change in the composite multiple organ dysfunction score (MODS) from baseline.13 Although results will not be known for several years, these clinical trials will be key to our understanding of whether the RBC storage lesion produces a clinically important phenomenon. Let us now turn our attention to the RBC storage lesion itself.14,15 What biologic and/or pathologic mechanisms could underlie some of the adverse clinical findings observed in transfused recipients? Can RBC products be optimized to increase oxygen delivery? The latter question should be considered independently of the first. Indeed, since about 1 in 60 individuals receive RBC transfusions in the United States every year, transfusion therapies should not only be safe but, importantly, as effective as possible. While some elements of the RBC storage lesion are well known (e.g., increase in free hemoglobin [Hb] and potassium; decrease in pH, adenosine triphosphate, and 2,3-diphosphoglycerate; and increased RBC morphologic changes leading to loss of deformability),14,15 less is known about other components such as the shed microparticles (MPs), RBC-dependent vasoregulatory compounds, and immune and inflammatory mediators that may be accumulating during storage. Further, surprisingly little current research has been performed to evaluate the effects of these elements on host cells, the vessel wall, and tissue microoxygenation. To begin rectifying this gap in knowledge, NHLBI recently established a targeted research program titled "Immunomodulatory, Inflammatory, and Vasoregulatory Properties of Transfused Red Blood Cell Units as a Function of Preparation and Storage." Eight teams of investigators around the country are conducting basic and translational research including animal models and/or basic human physiologic studies aimed at further characterizing the storage lesion and understanding the interaction between the storage lesion components and recipient/host cells such as endothelial and hematopoietic cells. As shown in Table 1, several of these teams (Gladwin and Kim-Shapiro; Patel, Barnum, and Weinberg; Roback; and Stamler) propose that transfusion damage from stored blood can be traced to suboptimal tissue oxygenation engendered by a loss of proper control of blood flow. These investigators will evaluate the role of nitric oxide (NO), nitrite, ATP, and/or S-nitrosohemoglobin (SNO-Hb) in this context. For example, consideration will be given to whether loss of vasoregulation could be secondary to increased levels of Hb and MPs in the plasma which scavenge NO and inhibit beneficial NO signaling and/or due to a loss of RBC-mediated hypoxic vasodilation (see Table 1 for each group-specific aim). Blumberg and Phipps will investigate the RBC units' supernatant including Hb, MPs, and fats and its effect (as well as that of RBCs) on patients' platelets (PLTs). Jy's group will explore the role of MPs in transfused products by studying their potential procoagulant, proinflammatory, immunosuppressive, and endothelial activities. Norris' team will focus on the potential inflammatory and immune effects of stored RBCs by characterizing immune and inflammatory mediators in fresher and older RBC units, evaluating RBC-endothelial cell adhesion as a function of RBC storage, determining if novel storage solutions or pathogen reduction can ameliorate the "storage lesion," and determining how RBC unit storage affects immunomodulation and inflammation in transfusion recipients enrolled in ABLE. Finally, the team led by Spitalnik will evaluate whether transfusion of older stored RBCs can produce a proinflammatory cytokine response due to delivery of substantial amounts of Hb iron to the monocyte-macrophage (reticuloendothelial) system and its consequent pro-oxidant effects (see Table 1 for additional descriptions of each of these eight projects). This research is expected to significantly increase our understanding of the RBC storage lesion and its potential clinical effects, as well as optimize methods and further development of assays to detect biomarkers of clinical relevance. New storage solutions could be developed. For example, identification of components in RBC units that may be potentially harmful to a specific patient population could lead to either development of improved RBC therapies that do not contain the harmful component or personalization of RBC therapy with transfusion of biomarker-negative units only in the patient population(s) at risk. Further studies may identify elements that are missing or in suboptimal concentrations because of preparation, manipulations, and storage time; RBC units may benefit from addition of such elements during component manufacturing or storage or before infusion. This multifaceted research could also have important ramifications for other fields. There are many examples of how research in transfusion medicine has positively impacted other scientific domains. For example, research conducted on infectious (e.g., human immunodeficiency virus, hepatitis B and C, West Nile virus) and immunologic complications of transfusions (e.g., transfusion-related acute lung injury) has not only helped advance the field of transfusion medicine by improving the safety of the blood supply, but has also been hypothesis generating. Thus, this work has fostered additional research to understand the pathogenesis of a wide variety of infectious and noninfectious diseases, including underlying host genetic and immunologic mechanisms involved in responding to and controlling infections and allogeneic immunologic challenges. The latter studies are critical to advancing the development of vaccines, for example. Similarly, we can anticipate that research on NO bioavailability, blood flow, tissue microoxygenation, interactions with the endothelium, PLT biology, immune and inflammatory modulators, and iron metabolism will be highly informative to the fields of vascular biology, hematology, immunology, and cellular therapies. It is hoped that this systematic and rational approach will lead to a better understanding of the RBC storage lesion and its clinical effects as well as to the development of enhanced transfusion therapies and strategies. These discoveries will help us achieve our mission, which is to improve and optimize patient care. The author declares no conflict of interest.

RISK OF ANTI-D PRODUCTION IN SICKLE CELL DISEASE PATIENTS (SCD) WITH CONVENTIONAL D AND PARTIAL D TRANSFUSED WITH D+ RED BLOOD CELLS

Dengue virus (DENV) is a transfusion-transmissible arbovirus that threatens blood donor systems with approximately 200 million high-titer asymptomatic infections occurring annually. Here we investigated the viability of DENV during storage of donor-derived platelet (PLT) and red blood cell (RBC) units. While purified PLTs have been shown to generate viable DENV, RBCs are replication incompetent. Combined with different storage criteria, distinct virus persistence profiles were anticipated in PLT and RBC units. Mimicking the virus titer of asymptomatic donors, purified DENV was spiked (10(5) -10(6) infectious units/mL) into PLT or RBC units produced and stored according to blood bank operating procedures. DENV was measured by infectious plaque-forming assays and by quantitative reverse transcription-polymerase chain reaction. In both PLT (7 days, 20-24°C) and RBC (42 days, 1-6°C) units, infectious DENV persisted throughout storage despite logarithmic decay. In buffer alone, DENV infectivity was insignificant by Day 1 at 20 to 24°C or 14 days at 1 to 6°C. Infectious virus production was identified in stored PLT units using a translation inhibitor and supported by virus genome replication. Surprisingly, DENV was also produced in RBC units, implying the involvement of cells other than RBCs. Both virus propagation and effects independent of cell function mitigate the intrinsic lability of DENV. Nevertheless, the overall rapid storage decay suggests that aged PLT and RBC units may be safer. These data raise awareness to the possible persistence of other conceivably more robust RNA viruses during the storage of cellular blood products.

To determine whether red blood cell (RBC) or plasma transfusion is associated with the one-year survival rate variation previously detected in liver transplantation. A retrospective study of 206 consecutive liver transplantations was undertaken. Intraoperative transfusions of blood products were identified. Twenty-seven variables were studied using univariate and multivariate analyses to identify factors that were associated significantly with survival rate. For analysis of one-year survival, the cases were studied according to the transfused blood products. Patients were stratified according to the degree of RBC and plasma transfusion into four groups: more than four units of RBC, one to four units of RBC, plasma transfusion only, and no plasma or RBC transfusions. Patients received an average of 2.8 +/- 3.5 units of RBC and 4.1 +/- 4.1 units of plasma. Thirty-two percent of the patients did not receive any RBC transfusion and 19.4% did not receive any blood products. The one-year survival rate was 81.9% for all patients and 97.4% for patients without any transfusions. Of the 27 variables evaluated, only RBC and plasma transfusions were associated with significant decrease in the one-year survival rate, which was seen in the group who received only plasma (76.9%, P = 0.014) and the group who received more than four units of RBC (62.5%, P < 0.0001). Although we cannot demonstrate causality, our analysis shows that our one-year survival rate following liver transplantation decreased significantly with the intraoperative transfusion of any amount of plasma or more than four units of RBC.