Like as the waves make toward the pebbled shore, So do our minutes hasten to their end1
John Milton wrote that he was not yet mature at the age of 23 years.2 Human red cell life is a bit different, with a maturation time of approximately 8 to 9 days, and an in vivo circulation span of approximately 115 days.3,4 Red blood cells can survive for several weeks in liquid storage at 4°C,5 depending on the storage medium,6 and apparently indefinitely when frozen at −80°C.7
In this issue of Anesthesia & Analgesia, Brown et al.8 looked at a single-center database, examining the relationship between the duration of liquid storage of transfused red cells and “delirium,” after cardiac surgery. Using case controls, their primary outcome measure did not support their hypothesis: they were unable to find a difference in postcardiac surgery delirium between patients who received red cells stored exclusively for 14 days or less and those who received red cells stored exclusively for >14 days. In their secondary analyses, they noted a statistically significant but relatively small increase in the incidence of delirium in patients transfused with red cells of an average storage duration of greater than 14 or 21 days compared with red cells of lesser average storage duration. Both these latter associations were weak, with lower 95% confidence intervals (CIs) of the odds ratios of 1.01 and 1.02, respectively. Figure 2 of the report appears to show that the lower 95% CI of the odds ratio for the relationship between average duration of red cell storage and delirium does not exceed the upper 95% CI limit of the plateau of lack of relationship until average storage duration exceeds approximately 34 days. The confidence limits are wide at very long durations of storage, at least in part owing to relatively few patients who received those red cells. Brown et al.8 are appropriately cautious in stating that this work is not definitive.
Database analyses should not be regarded as definitive. The various statistical methods used may reduce but cannot eliminate bias. Such analyses may appropriately suggest hypotheses for testing in prospective randomized trials but should not replace such trials. The substantial uncertainties that are inherent to all database analyses have been seen in important transfusion studies. Two of the most prominent randomized trials in transfusion medicine9,10 failed to confirm the associations noted in their previous database analyses11,12 that generated the hypotheses tested by those randomized trials.
For some hypotheses, randomized trials may not be possible or feasible (also, see below). In those circumstances, retrospective or database analyses may be the best information possible. Several database analyses have examined the relationship between duration of red cell liquid storage and adverse outcomes, with disparate results. Perhaps the controversial single-center database analysis purporting to demonstrate such an adverse relationship after cardiac surgery13 was the most influential in spawning several expensive and resource-intensive prospective randomized trials, despite the lack of such a finding in a much larger well-conducted database analysis.14 One prospective randomized trial has already reported its results, also failing to find such a relationship.15 The other trials should finish soon (or perhaps will have been by the time of publication of this editorial), with the possibility of decreasing confusion for this issue, although the differences in experimental design and study populations may have the opposite effect. However, it does not appear that any of these trials have an a priori hypothesis related to delirium. Thus, these trials are not likely to answer the hypothesis generated by the work of Brown et al.8 Furthermore, it would seem unlikely that further large clinical trials will be undertaken to confirm or refute the findings reported by Brown et al.8 Consequently, for the foreseeable future, this report may be the best information we will have.
Some issues surrounding those raised by Brown et al.8 deserve examination. The report leaves unanswered what is the best dependent variable by which to judge the clinical effect of duration of storage: red cells of an exclusive storage duration, of an average storage duration, or the single oldest red cell unit transfused. These dependent variables are not interchangeable; they test somewhat different putative pathophysiological etiologies and hypotheses and importantly examine differing populations with different quantities of transfusion.16 Thus, the specific measure must be decided a priori and not post hoc. The issue is further complicated by the great variability among red cell units,17 because each unit is from a unique donor and individually is not required to pass biochemical, efficacy, or quality control standards.
Brown et al.8 examined neurocognitive dysfunction, which is a not an uncommon in-hospital diagnosis,18 especially after both cardiac and noncardiac major surgery.19 Acute severe anemia results in cognitive function impairment20 that can be reversed with transfusion.20 If anemia and some transfusions might produce similar symptoms, assigning an etiology is difficult, especially in a database analysis, and without the prospective use of a standardized test, as in the case of Brown et al.8 The problem of postoperative delirium has been approached from several perspectives, but not many have been related to transfusion. An exception is the recent, ancillary study of the FOCUS trial that failed to note a difference of association of delirium with liberal versus restrictive transfusion paradigms after hip fracture repair in patients aged 50 years or more, with the restrictive group having received fewer transfusions.18 It appears that Brown et al.8 are the first to raise the possibility of delirium being associated with the storage duration of red cells.
Thus, Brown et al.8 raise an issue of potential importance related to the safety and perhaps efficacy (because possible findings proposed to be related to red blood cell units stored for longer periods, and symptoms of untreated anemia, as would occur if the transfused red blood cell were not efficacious, might be similar) of red cell products. One may wonder how issues of safety and efficacy of red cells continue to appear regarding such an old, well-established biologic. Determination of efficacy and safety of whole blood and red cell preparations is an interesting and exceedingly complex issue. It may be surprising to some that these biologics have not undergone the standard Food and Drug Administration (FDA) process of approval based on data from randomized trials demonstrating efficacy and safety.
The regulatory basis of the FDA is built on the Biologics Control Act of 1902, the Food, Drug, and Cosmetic Act of 1938, and the subsequent amendments, especially the Kefauver-Harris Amendment of 1962 (with the addition of a mandate of demonstration of efficacy for a marketed indication), and the Public Health Service Act of 1944. The Drug Efficacy Study Implementation program was instituted to classify all pre-1962 drugs as effective, ineffective, or needing further study. After the Division of Biological Standards of the National Institutes of Health was transferred to the FDA in 1972, it was announced in August 1972 that it would review all biologics licensed before July 1, 1972, to ensure safety and efficacy, while acknowledging that adequate and well-controlled studies are not always feasible.21 A panel of individuals external to the FDA (5 in blood banking, 1 surgeon, and 1 anesthesiologist) met from June 1974 to November 1979, and provided an extensive report, examining published and submitted information, and “common experience,” recommending that whole blood and packed red cells be classified as effective and safe.22 By the time of publication of the report in December 1985, the transfusion-transmitted virus study had been completed, finding a 10% incidence of posttransfusion hepatitis23; the etiologic agent of acquired immunodeficiency syndrome (AIDS) had been discovered24,25; and the association of AIDS with transfusion of blood products had been reported.26,27 The incidences of transmission by transfusion of human immunodeficiency virus and hepatitis C virus are so low now (< 1:106) as to require mathematical modeling techniques for estimation.28 However, transmission by transfusion of many other pathogens,29 such as babesia,30 has been identified and undoubtedly others will emerge.31 Other adverse consequences added to the potential for disease transmission include immunomodulation and transfusion associated lung injury.32,33 There are many biochemical changes with storage duration of red blood cells,34–36 but prospective randomized studies in patients have failed to find that storage age of transfused red blood cells altered immune response as assessed by an incidence of infection15 or by biochemical markers of immunologic status.37 A prospective randomized study in volunteers noted subtle pulmonary function changes with transfusion of red blood cells that did not differ between fresh red blood cells and those stored for 21 days;38 and a prospective randomized clinical trial did not detect overt transfusion-associated lung injury related to storage age of red cells.37 However, in a severe test of this hypothesis, recent well-conducted laboratory studies found that dogs with a substantial pulmonary inoculation of S aureus sufficient to produce pneumonia and septic shock, but not a lesser dose, had increased alveolar to arterial PO2 difference, lung injury, and mortality when exchanged-transfused with red cells stored for 42 days compared with similar dogs exchange-transfused with red cells stored for 7 days.39,40 No changes were noted in similarly transfused animals without septic pneumonia. If these findings in canine septic pneumonia pertain to humans, it could help explain some disparate results in clinical trials in the intensive care unit41,42 and would need to be considered in future experimental designs in such patients. It is important to note that safety is relative, because no therapy is without risk and should always be weighed against a potential benefit (efficacy) it may have and the risk: benefit of other potential therapies, as well as the risk of the untreated disorder, in a specific clinical circumstance.
The panel cited above judged that “effectiveness has been documented at least by common experience. Thus, the issue of effectiveness has been a lesser consideration when adequate data are lacking…”22 Indeed, how could one design and perform an ethical randomized, controlled, clinical trial in humans to test efficacy of red cells, that is, the ability to reverse anemia-induced signs or symptoms? What could be an ethical comparator when red cell efficacy is acknowledged by “common experience,” laboratory studies, and volunteer studies,20,43,44 and a small study in healthy volunteers showed that both fresh and liquid-stored red cells are efficacious?44 Thus, the Center for Biologics Evaluation and Research (CBER)/FDA is left with an exceedingly difficult task of documenting red cell efficacy, while at the same time not impairing the availability of an extraordinarily valuable clinical therapy.
The FDA regulates blood banks and collection facilities. However, as the 1985 panel report noted, blood components do not have just a few large manufacturers with protocols, but a myriad of facilities, making a thorough review of all the products of every “manufacturer” impossible. Consequently, the FDA (CBER) focuses their attention on processes at these facilities (also inspected by the AABB [formerly known as the American Association of Blood Banks] and the College of American Pathologists). For example, when a new storage medium is proposed CBER requires in vitro assessment of the biochemical composition of stored blood or red blood cells; a maximum permitted red cell hemolysis of 1%; and as a surrogate of effectiveness, in vivo recovery and survival, reasoning that if transfused red cells survive (at least a mean of 75% at 24 hours, with a standard deviation (SD) of 9% or less; that is, a lower 95% confidence interval of at least 70% of units having at least 75% recovery at 24 hours), then red blood cell efficacy is assumed.
However, CBER/FDA applies different standards to what one might call “processed” red cells or plasma, such as when treated with pathogen reduction technology or in the case of plasma, freeze- or spray-drying. In those cases, there is the curious circumstance of a test article evaluated against the accepted “product” that has never undergone such testing.
Nevertheless, some question the immediate efficacy of liquid-stored red blood cells, doubting their ability to off-load oxygen. Red cell concentration of 2,3-diphosphoglycerate (2,3-DPG) decreases with duration of liquid storage45,46 falling to very low levels by 14 days and to depleted concentrations at 21 days.46,47 The affinity of hemoglobin for oxygen varies inversely with 2,3-DPG concentrations,44,48,49 with P50 decreasing from the normal value of 26.650 to 18.5 mm·Hg at 20 days stored in acid-citrate-phosphate51 and 15.0 mm·Hg by 21 days stored in citrate-phosphate-dextrose.44 Valtis and Kennedy51 appear to have been the first to discover the decreased P50 of liquid-stored blood (long before the cause was elucidated) and to question the immediate efficacy of transfused liquid-stored red cells, suggesting that the increased affinity would not allow for adequate release of oxygen by hemoglobin. P50 is restored by reestablishment of 2,3-DPG concentrations that begins within a few hours but is not complete for 1 to 2 days after transfusion.46,47,51–53 However, P50 is a standardized in vitro value that describes a physical-chemical property of the interaction of hemoglobin and oxygen. The decreased affinity of hemoglobin for oxygen caused by acidosis (Bohr effect)54,55 has led some to suggest that the in vivo effect might be minimized by the accumulated acid during storage within the red cell.56,57 We previously calculated that in contradistinction to the true P50, the in vivo “functional P50” (that is, the in vivo PO2 at which hemoglobin is 50% saturated with oxygen) of red cells stored for 3 weeks was 33 mm·Hg,44 thus, possibly explaining why stored and fresh red cells are equally effective in reversing anemia-induced cognitive deficits.44 The decreased 2,3-DPG concentration actually might be beneficial. If the in vitro (true) P50 were unchanged (that is, without a decreased 2,3-DPG), when considering the substantial red cell acidosis and base-deficit, I calculate that at a normal PaO2 of 90 mm·Hg, in vivo hemoglobin saturation would be approximately 84%.
In addition, red cell deformability decreases with duration of liquid storage,58 perhaps limiting red cell access through the microcirculation, although again, a study in volunteers using a sensitive, reproducible measure of cognitive function found that red blood cells stored for 3 weeks reversed anemia-induced cognitive deficits as well as did fresh red cells, implying successful access through the microcirculation of the brain with appropriate off-loading of oxygen, at least in normal adults.44 Clinical studies have not been definitive, perhaps owing to inadequate study design and to the difficulties of selecting and studying patients with demonstrated inadequate oxygen delivery, and assessing the clinical effects of minimally inadequate oxygen delivery. However, exchange transfusion leaving less than half of the native red cells, as in patients with sickle cell disease, has been without adverse events referable to inadequate oxygen delivery.59,60 Perhaps even more to the point is the patent survival, without impairment, of innumerable patients with blood loss and transfusion of liquid-stored red blood cells or whole blood, to the extent where residual native red cells alone would not have permitted survival. The data of Viele and Weiskopf61 and Carson et al.62 and as recently analyzed together63 indicate 100% patient mortality below a hemoglobin concentration of 1.3 g/dL. Depending on the initial hemoglobin concentration, reaching a hemoglobin concentration of 1.3 g/dL would require a maximum blood loss of approximately 2.2 to 2.4 blood volumes.64 Maintenance of a total hemoglobin concentration of 8 g/dL during this loss would require transfusion of 15 to 16 units of stored red cells, an uncommon, but certainly not extraordinarily rare clinical circumstance. For example, in 1971, Collins et al.65 reported 36 severe combat casualties, transfused with 6 to 66 units (average 15 and 29 units in rapidly transfused and large-volume transfusion groups) whole blood, with the preponderance having been stored between 2 and 3 weeks. Two died of exsanguinating hemorrhage, 1 other during transfusion; all others survived the immediate episode and all but 2 (who died of sepsis) survived long term. Similarly, a substantial number of patients have survived liver transplantation66 and complex spinal surgery67 with blood loss and transfusion of several blood volumes. More recently, Holcomb et al.68 reported 60% 30-day survival of 466 civilian casualties who received an average of 21 units red cells, and Stanworth et al.69 reported approximately 50% survival in trauma patients receiving more than 20 units of PRBCs. Maintenance of isovolemia with transfusion of 30 units blood (as in Collins et al.65 large-volume transfusion group) would produce a residual native hemoglobin concentration of <0.5 g/dL (a uniformly lethal concentration without oxygen-carrying supplementation).
“You can see a lot just by looking” responded Yogi Berra when queried “what do you see” for the New York Mets as he took over as manager in 1972 after the unexpected death of the great, beloved former Brooklyn Dodger Gil Hodges. John Milton became completely blind is his early 40s, with concern for his further abilities70 but later wrote (more correctly, dictated) the incomparable “Paradise Lost.”71 Brown et al.8 looked and may have seen something; it is hard to know. Clarity may be a long way off.
Name: Richard B. Weiskopf, MD, Professor Emeritus.
Contribution: The author wrote the manuscript.
Attestation: Richard B. Weiskopf approved the final manuscript.
Conflicts of Interest: The author has a relationship with or consults for the following companies and organizations that have an interest in erythrocyte transfusion: U.S. Food and Drug Administration; U.S. National Heart, Lung, and Blood Institute/National Institutes of Health; U.S. Department of Defense; CSLBehring; and TerumoBCT. The author was project/corp VP, Chief Medical Officer Biopharmaceuticals, and Executive Scientific Advisor at Novo Nordisk A/S 2005–2007. The author of this editorial had no involvement in the research or manuscript to which this editorial refers. No one from any of these organizations influenced or participated in the writing or had any knowledge of this editorial other than those acknowledged below.
This manuscript was handled by: Jerrold H. Levy, MD, FAHA, FCCM.
The author is grateful to many colleagues, including those in the FDA, for many conversations and expositions of their views, during many years before writing this editorial, related to some of the topics discussed. The author thanks Drs. H. Klein and T. Silverman for their review of a draft of this editorial and their helpful comments.
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