ORIGINAL ARTICLE Red blood cell transfusion and immune function in critically ill children: a prospective observational study Jennifer A. Muszynski,1,2 Elfaridah Frazier,3 Ryan Nofziger,4 Jyotsna Nateri,2 Lisa Hanson-Huber,2 Lisa Steele,2 Kathleen Nicol,5 Philip C. Spinella,3 and Mark W. Hall1,2 for the Pediatric Critical Care Blood Research Network (Blood Net) subgroup of the Pediatric Acute Lung Injury and Sepsis Investigators (PALISI)
BACKGROUND: Our previous in vitro work showed that stored red blood cells (RBCs) increasingly suppress markers of innate immune function with increased storage time. This multicenter prospective observational study tests the hypothesis that a single RBC transfusion in critically ill children is associated with immune suppression as a function of storage time. STUDY DESIGN AND METHODS: Blood samples were taken immediately before and 24 (±6) hours after a single RBC transfusion ordered as part of routine care. Innate and adaptive immune function was assessed by ex vivo whole blood stimulation with lipopolysaccharide (LPS) and phytohemagglutinin, respectively. Monocyte HLA-DR expression, regulatory T cells, plasma interleukin (IL)-6, and IL-8 levels were also measured. RESULTS: Thirty-one transfused critically ill children and eight healthy controls were studied. Critically ill subjects had lower pretransfusion LPS-induced tumor necrosis factor-α production capacity compared to healthy controls, indicating innate immune suppression (p < 0.0002). Those who received RBCs stored for not more than 21 days demonstrated recovery of innate immune function (p = 0.02) and decreased plasma IL-6 levels (p = 0.002) over time compared to children transfused with older blood, who showed persistence of systemic inflammation and innate immune suppression. RBC storage time was not associated with changes in adaptive immune function. CONCLUSION: In this pilot cohort of critically ill children, transfusion with older prestorage leukoreduced RBCs was associated with persistence of innate immune suppression and systemic inflammation. This was not seen with fresher RBCs. RBC transfusion had no short-term association with adaptive immune function. Further studies are warranted to confirm these findings in a larger cohort of patients.
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ed blood cell (RBC) transfusion in critically ill patients is known to be associated with increased risks of nosocomial infection, suggesting a relationship between RBC transfusion and impaired host defense, although a causal link between RBC transfusion and immune function has not been clearly established.1-7 Acquired suppression of innate (e.g., monocyte) and adaptive (e.g., lymphocyte) immune function are increasingly recognized as complications of critical illness and are independently associated with increased risks of adverse outcomes.8-13 Immune function in the intensive care unit (ICU) can be measured by quantifying the ability of immune cells to produce cytokines in
ABBREVIATIONS: ICU = intensive care unit; LPS = lipopolysaccharide; PHA = phytohemagglutinin; PICU = pediatric intensive care unit; Treg = regulatory T cells. From the 1Pediatric Critical Care Medicine, Department of Pediatrics, and 5Pathology, Nationwide Children’s Hospital, and 2 The Research Institute at Nationwide Children’s Hospital, Columbus, Ohio; 3Pediatrics, Division of Critical Care Medicine, Washington University, St Louis, Missouri; and 4Critical Care Medicine, Department of Pediatrics, Akron Children’s Hospital, Akron, Ohio. Address reprint requests to: Jennifer A. Muszynski, MD, Critical Care Medicine, Nationwide Children’s Hospital, 700 Children’s Drive, Columbus, OH 43205; e-mail: [email protected]. This study was funded by K12HD043372 from the National Institute of Child Health and Development (JAM), The Research Institute at Nationwide Children’s Hospital (JAM, MWH), and Washington University School of Medicine Pediatric Critical Care Translational Research Program (PCS). Received for publication August 1, 2014; revision received August 25, 2014, and accepted August 29, 2014. doi: 10.1111/trf.12896 © 2014 AABB TRANSFUSION **;**:**-**. Volume **, ** **
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response to ex vivo stimulation. We have previously demonstrated that a markedly reduced ability to produce the proinflammatory cytokine tumor necrosis factor (TNF)-α upon ex vivo stimulation of whole blood with lipopolysaccharide (LPS) is significantly associated with increased risks of mortality and nosocomial infection in critically ill children.8,10,11,13 Similarly, we and others have demonstrated impaired lymphocyte responsiveness to ex vivo stimulation in adults and children with sepsis-related adverse outcomes.14,15 It should be noted that critically ill patients frequently demonstrate some degree of immune suppression during the acute phase of illness, but most recover their immune function over several days. Immune recovery is typically associated with recovery from critical illness while persistent immune suppression is associated with adverse outcomes.16,17 Mechanisms of persistent immune suppression in the ICU are unclear, but they may include RBC transfusion. Retrospective studies have suggested that transfusion with RBCs with longer storage duration places critically ill patients at particularly high risk for secondary infection and mortality.18-22 We have previously shown that among transfused critically injured children, receipt of RBCs stored for more than 14 days was associated with decreased innate immune function over time compared to those who received fresher RBCs.13 Further, in vitro studies conducted by our laboratory and others suggest that stored RBCs, particularly of longer storage duration, directly suppress innate immune cell function.23-26 The immunologic impact of a single RBC transfusion in critically ill children is unknown. We therefore designed a multicenter prospective, observational pilot study to test the hypothesis that a single transfusion of older RBCs would be associated with decreased innate and adaptive immune function over time compared to transfusion with fresher RBCs.
MATERIALS AND METHODS Setting Subjects were enrolled from one of two participating centers. Nationwide Children’s Hospital and St Louis Children’s Hospital are both academic, free-standing children’s hospitals. The pediatric intensive care unit (PICU) at Nationwide Children’s hospital is a 40-bed, quaternary care, mixed medical–surgical unit with more than 2200 admissions annually. The PICU at St Louis Children’s Hospital is a 46-bed mixed medical–surgical unit with approximately 2700 admissions per year. The study was approved by the institutional review boards at Nationwide Children’s Hospital and at St Louis Children’s Hospital.
Subjects After informed consent and, where appropriate, assent was obtained, children (age 21 days (n = 10) 19 (3-92) 15 (8-112) 6 (29) 3 (30)
17 (55) 2 (6) 7 (23) 5 (16) 24 (77) 12 (39) 14 (45)
10 (48) 1 (5) 6 (29) 4 (19) 15 (71) 7 (33) 10 (48)
1.6 (1-4) 7 (4-13) 11 (1.5-11.5) 68 (17-232) 7.7 (7.1-8.1) 15 (12-17) 8 (26) 17 (8-27)
1.4 (0.8-4) 7 (3-12) 11 (1-16) 76 (17-232) 7.7 (7.1-8.5) 15 (11-19) 6 (29) 14 (7-17)
7 (70) 1 (10) 1 (10) 1 (10) 9 (90) 5 (50) 4 (40) 2.8 (1-4) 8 (6-15) 10.5 (2-14.5) 50 (14-471) 7.8 (6.9-8.5) 13 (11-17) 2 (20) 30 (27-33)
p value 0.4 1
0.4 0.4 1 0.4 0.4 0.8 0.7 1 0.6 1 21
Before transfusion 76 (17-232) 50 (14-471)
IL-6 (pg/mL) After transfusion 26 (12-127) 57 (15-122)
p value 0.002 0.9
Before transfusion 16 (11-70) 12 (6-29)
IL-8 (pg/mL) After transfusion 17 (7-35) 17 (6-91)
p value 0.4 0.2
* Data are reported as median (interquartile range).
Adaptive immune function Adaptive immune function was assessed by PHA-induced cytokine production capacity. For all critically ill subjects, pretransfusion ex vivo PHA-induced IFN-γ production was significantly lower compared to healthy controls (236 [152-352] pg/mL vs. 535 [419-1027] pg/mL, p = 0.001). Pretransfusion ex vivo PHA-induced IL-10 production tended to be lower than healthy controls (156 [60-270] pg/mL vs. 246 [169-338] pg/mL, p = 0.07). Pretransfusion ex vivo PHA-induced IL-2 and IL-4 were not significantly
different from healthy controls (31 [16-105] pg/mL vs. 31 [19-475] pg/mL, p = 0.6; and 2.9 [2.1-5.3] pg/mL vs. 2.5 [2.1-2.8] pg/mL, p = 0.3, respectively). Among all transfused subjects, ex vivo PHA-induced IFN-γ production improved over time while ex vivo PHA-induced production of other cytokines did not differ significantly between pre- and posttransfusion samples (Fig. 3A). Comparing children transfused with older versus fresher RBCs, significant differences in adaptive immune function over time were not detected (Fig. 3B). In the cohort who Volume **, ** **
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suggest a relationship between the storage duration of a single transfused p = 0.9; Mann-Whitney p = 0.01; Mann-Whitney 400 150 unit of RBCs and in vivo immune suppression in critically ill children. This study used a highly standard300 ized assay to quantify innate immune 100 function by measuring whole blood ex 200 vivo LPS-induced TNF-α production capacity. This assay has been success100 fully employed in single- and multicenter studies of pediatric critical illness 50 ≤ 21 > 21 > 21 days ≤ 21 days across multiple diagnoses including (N = 16) (N = 6) (N=6) (N=16) sepsis, influenza infection, respiratory RBC Storage Duration (days) RBC Storage Duration (days) syncytial virus infection, traumatic injury, and status after cardiopulmoFig. 2. (A) Among children undergoing flow cytometric analysis, monocyte antigennary bypass.8,10,11,13,34,35 In each of these presenting capacity, as measured by HLA-DR expression, did not significantly studies, lower ex vivo TNF-α production change after transfusion regardless of RBC storage duration, while receipt of fresher capacity was consistently associated blood was associated with increased ex vivo LPS-induced TNF-α production capacity with increased risks of nosocomial in this group of subjects (B). Boxes represent median and interquartile range. infection and/or death. This is not Whiskers represent 10th to 90th percentile. strictly a pediatric phenomenon, as persistently low innate immune function has been associated with adverse outcomes in critically ill underwent flow cytometric analysis, the pretransadults as well.36-38 It is worth noting that the majority of fusion percentage of immunosuppressive Treg among CD4+ T cells (%Treg) was similar to healthy controls patients in our cohort were transfused in the acute phase (5.2% [3%-8.4%] vs. 6.3% [5.2%-6.5%], p = 0.6). Among of illness, with most posttransfusion samples being drawn transfused subjects overall, %Treg was not signion ICU Days 2 to 5. This is a time when we would expect to ficantly different after transfusion compared to presee evidence of recovery of innate immune suppression in transfusion values (6% [3.8%-7.6%] vs. 5.2% [3%-8.4%], most patients. Children who received older RBCs failed to p = 0.3). There was a trend toward an increase in %Treg demonstrate this increase in innate immune function posttransfusion in children transfused with RBCs stored compared to children who received fresher RBCs. These for more than 21 days, which did not reach significance results are similar to those of our previous study of criti(Fig. 4). A difference in ex vivo PHA-induced IL-10 produccally injured children in whom receipt of RBCs stored for tion (which would suggest a Treg-dominant state) was not more than 14 days was associated with a failure to improve seen in this cohort. innate immune function over time during the first week postinjury.13 Our inability to demonstrate an immune effect at the 14-day cutoff in this study may have been DISCUSSION related to sample size or to other differences between the two patient populations. In this multicenter, prospective observational study, a Previous reports have quantified circulating plasma single transfusion with prestorage leukoreduced RBCs cytokines before and after RBC transfusion. In Keir and stored for more than 21 days was associated with impaired colleagues,39 28 nonseptic preterm infants between 2 and recovery of innate immune function. Relationships between RBC storage duration and changes in adaptive 6 weeks of age demonstrated small, but significant immune function were less apparent. increases in circulating proinflammatory cytokines IL-1β, RBC storage duration has been associated with IL-8, TNF-α, and monocyte chemoattractant protein-1 2 adverse outcomes including nosocomial infection, deep to 4 hours after a single RBC transfusion. Infants in this vein thrombosis, mortality, and organ dysfunction across study were not stratified by RBC storage duration. By cona variety of critically ill adult and pediatric patient trast, two adult studies evaluated relationships between populations.18-22,30-33 While increasing attention is being RBC storage duration and plasma cytokines. Both studies failed to detect significant differences in changes in paid to the RBC storage lesion, little is known about the plasma cytokines immediately after a single transfusion impact of RBC storage on immune function in critically with fresher compared to older RBCs.40,41 However, circuill patients. We have previously demonstrated that leukoreduced RBCs directly suppress monocytes in vitro lating plasma cytokines may not reflect the functional as a function of RBC storage duration.25 This study is in capacity of the immune system. In this study, transfusion with older RBCs was associated with a failure to resolve agreement with our in vitro results and is the first to 6
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Fig. 3. (A) Ex vivo PHA-induced cytokine production capacity for IFN-γ was lower in critically ill subjects compared to healthy controls and improved over time. No differences in other cytokines were seen. Dashed lines and shaded areas represent median values and interquartile ranges for healthy controls. **p < 0.01 before versus after transfusion values. (B) Significant differences in posttransfusion ex vivo PHAinduced cytokine production were not seen when comparing children transfused with fresher or older RBCs. Boxes represent median and interquartile range. Whiskers represent 10th to 90th percentile.
systemic inflammation as evidenced by persistent elevations in plasma IL-6 levels. In the context of simultaneous improvement in innate immune function, we speculate that the reduction in plasma IL-6 levels seen in children transfused with fresher RBCs may reflect resolution of systemic inflammation and a return toward immunologic homeostasis. In addition to innate immune function, suppression of adaptive immune function has also been associated with increased risks of nosocomial infection and mortality in critically ill adults and children.9,12,14 While a recent in vitro study demonstrated that exosomes isolated from stored RBCs enhance T-cell proliferation, other reports show suppressed T-cell proliferation after exposure to stored
RBCs.42-44 In this study, we evaluated adaptive immune function using whole blood ex vivo PHA-induced cytokine production. In contrast to innate immune function, we were unable to detect significant relationships between RBC storage duration and adaptive immune function in transfused children. This may represent a lack of immunosuppressive effect of older RBCs on adaptive immune function in patients, but it is also possible that the timing of our 24-hour posttransfusion samples may have been too early to see an effect on lymphocytes. We chose this window, however, to limit the confounding effects of other ICU therapies on posttransfusion immune function. Our study has several important limitations. First, because sample processing for functional assessments of innate and adaptive immunity needed to be performed in real time, we were limited to nonurgent transfusions occurring during the day. This limited the number of patients available for study. We were, however, able to demonstrate the feasibility of consenting and enrolling critically ill children before transfusion and performing functional immune assays in a multicenter setting. Second, the number of patients with severe pretransfusion immune suppression was low. Our data suggest that relationships between RBC storage duration and immune function may be stronger in patients with a greater degree of baseline immune suppression. It will be important to confirm these findings in a larger cohort of severely critically ill children with more longitudinal sampling, allowing for more detailed evaluation of these relationships over time. Third, to capture a representative sample of transfused critically ill children, we chose to include transfused patients in the PICU with any diagnosis. This resulted in a high degree of baseline variability in diagnoses, severities of illness, and immune function. Despite this, we were able to detect an association between RBC storage duration and persistence of innate immune suppression with a single RBC transfusion. Larger studies with sample sizes large enough to permit multivariable analyses to control for clinical confounders, including severity of illness and timing of transfusion, are still needed. Finally, although our data suggest an RBC storage duration threshold of 21 days, this threshold may not apply to all patients. This too should be the subject of future investigations. This study represents the most comprehensive evaluation to date of immune function in transfused critically ill children and supports the hypothesis that transfusion with older RBCs, even when leukoreduced before storage, may be associated with the persistence of innate immune suppression over time. Additional studies are needed to confirm these results in a larger cohort of patients; to evaluate relationships between RBC storage duration, immune function, and clinical outcomes; and to determine mechanisms of immunologic effects of RBC transfusion in critically ill patients. Volume **, ** **
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mechanisms of immunoparalysis after cardiopulmonary bypass. J Thorac Cardiovasc Surg 2012;143:1160-6 e1. 9. Felmet KA, Hall MW, Clark RS, et al.
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and multiple organ failure. J Immunol 2005;174:3765-72.
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Fig. 4. (A) There was a trend toward increase in percentage of Treg after transfusion in children transfused with older RBCs. (B) Ex vivo PHA-induced IL-10 production capacity was not different based on RBC storage age in the cohort of children who underwent flow cytometric analysis. Boxes represent median and interquartile range. Whiskers represent 10th to 90th percentile.
10. Hall MW, Geyer SM, Guo CY, et al. Innate immune function and mortality in critically ill children with influenza: a multicenter study. Crit Care Med 2013;41:224-36. 11. Hall MW, Knatz NL, Vetterly C, et al. Immunoparalysis and nosocomial infection in children with multiple organ dysfunction syndrome. Intensive Care Med 2011;37:525-32. 12. Hotchkiss RS, Tinsley KW, Swanson PE,
CONFLICT OF INTEREST The authors have disclosed no conflicts of interest.
REFERENCES 1. Acker SN, Partrick DA, Ross JT, et al. Blood component transfusion increases the risk of death in children with traumatic brain injury. J Trauma Acute Care Surg 2014;76: 1082-7; discussion 1087-8. 2. Claridge JA, Sawyer RG, Schulman AM, et al. Blood transfusions correlate with infections in trauma patients in a dose-dependent manner. Am Surg 2002;68:566-72. 3. Malone DL, Dunne J, Tracy JK, et al. Blood transfusion, independent of shock severity, is associated with worse outcome in trauma. J Trauma 2003;54:898-905; discussion 905-7. 4. Paone G, Likosky DS, Brewer R, et al. Transfusion of 1 and 2 units of red blood cells is associated with increased morbidity and mortality. Ann Thorac Surg 2014;97:87-93; discussion 93-4. 5. Rachoin JS, Daher R, Schorr C, et al. Microbiology, time course and clinical characteristics of infection in critically ill patients receiving packed red blood cell transfusion. Vox Sang 2009;97:294-302. 6. Rohde JM, Dimcheff DE, Blumberg N, et al. Health careassociated infection after red blood cell transfusion: a systematic review and meta-analysis. JAMA 2014;311: 1317-26. 7. Shorr AF, Jackson WL, Kelly KM, et al. Transfusion practice and blood stream infections in critically ill patients. Chest 2005;127:1722-8. 8
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et al. Sepsis-induced apoptosis causes progressive profound depletion of B and CD4+ T lymphocytes in humans. J Immunol 2001;166:6952-63. 13. Muszynski JA, Nofziger R, Greathouse K, et al. Innate immune function predicts the development of nosocomial infection in critically injured children. Shock 2014;42: 313-21. 14. Boomer JS, To K, Chang KC, et al. Immunosuppression in patients who die of sepsis and multiple organ failure. JAMA 2011;306:2594-605. 15. Muszynski JA, Nofziger R, Greathouse K, et al. Early adaptive immune suppression in children with septic shock: a prospective observational study. Crit Care 2014;18: R145. 16. Hotchkiss RS, Monneret G, Payen D. Sepsis-induced immunosuppression: from cellular dysfunctions to immunotherapy. Nat Rev Immunol 2013;13: 862-74. 17. Volk HD, Reinke P, Docke WD. Clinical aspects: from systemic inflammation to “immunoparalysis.” Chem Immunol 2000;74:162-77. 18. Koch CG, Li L, Sessler DI, et al. Duration of red-cell storage and complications after cardiac surgery. N Engl J Med 2008;358:1229-39. 19. Manlhiot C, McCrindle BW, Menjak IB, et al. Longer blood storage is associated with suboptimal outcomes in highrisk pediatric cardiac surgery. Ann Thorac Surg 2012;93: 1563-9. 20. Offner PJ, Moore EE, Biffl WL, et al. Increased rate of infection associated with transfusion of old blood after severe injury. Arch Surg 2002;137:711-6; discussion 716-7. 21. Ranucci M, Carlucci C, Isgro G, et al. Duration of red blood cell storage and outcomes in pediatric cardiac surgery: an
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association found for pump prime blood. Crit Care 2009; 13:R207. 22. Weinberg JA, McGwin G, Jr, Vandromme MJ, et al. Duration of red cell storage influences mortality after trauma. J Trauma 2010;69:1427-31; discussion 1431-2. 23. Biedler AE, Schneider SO, Seyfert U, et al. Impact of alloantigens and storage-associated factors on stimulated cytokine response in an in vitro model of blood transfusion. Anesthesiology 2002;97:1102-9. 24. Karam O, Tucci M, Toledano BJ, et al. Length of storage and in vitro immunomodulation induced by prestorage leukoreduced red blood cells. Transfusion 2009;49:2326-34. 25. Muszynski J, Nateri J, Nicol K, et al. Immunosuppressive
critically ill children: a prospective observational study. Crit Care 2010;14:R57. 34. Hall MW, Gavrilin MA, Knatz NL, et al. Monocyte mRNA phenotype and adverse outcomes from pediatric multiple organ dysfunction syndrome. Pediatr Res 2007;62:597-603. 35. Mella C, Suarez-Arrabal MC, Lopez S, et al. Innate immune dysfunction is associated with enhanced disease severity in infants with severe respiratory syncytial virus bronchiolitis. J Infect Dis 2013;207:564-73. 36. Docke WD, Randow F, Syrbe U, et al. Monocyte deactivation in septic patients: restoration by IFN-gamma treatment. Nat Med 1997;3:678-81. 37. Monneret G, Lepape A, Voirin N, et al. Persisting low
effects of red blood cells on monocytes are related to both
monocyte human leukocyte antigen-DR expression pre-
storage time and storage solution. Transfusion 2012;52:
dicts mortality in septic shock. Intensive Care Med 2006;
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26. Mynster T. Effects of red cell storage and lysis on in vitro cytokine release. Transfus Apher Sci 2001;25:17-23.
38. Volk HD, Reinke P, Krausch D, et al. Monocyte deactivation—rationale for a new therapeutic strategy in
27. Leteurtre S, Martinot A, Duhamel A, et al. Validation of the paediatric logistic organ dysfunction (PELOD) score: prospective, observational, multicentre study. Lancet 2003;
sepsis. Intensive Care Med 1996;22(Suppl 4):S474-81. 39. Keir AK, McPhee AJ, Andersen CC, et al. Plasma cytokines and markers of endothelial activation increase after packed
362:192-7. 28. Pollack MM, Patel KM, Ruttimann UE. PRISM III: an updated Pediatric Risk of Mortality score. Crit Care Med 1996;24:743-52. 29. van de Watering L. Pitfalls in the current published observational literature on the effects of red blood cell storage. Transfusion 2011;51:1847-54. 30. Gauvin F, Spinella PC, Lacroix J, et al. Association between length of storage of transfused red blood cells and multiple organ dysfunction syndrome in pediatric intensive care patients. Transfusion 2010;50:1902-13. 31. Spinella PC, Carroll CL, Staff I, et al. Duration of red blood cell storage is associated with increased incidence of deep vein thrombosis and in hospital mortality in patients with traumatic injuries. Crit Care 2009;13:R151. 32. Zallen G, Offner PJ, Moore EE, et al. Age of transfused blood is an independent risk factor for postinjury multiple organ failure. Am J Surg 1999;178:570-2. 33. Karam O, Tucci M, Bateman ST, et al. Association between length of storage of red blood cell units and outcome of
red blood cell transfusion in the preterm infant. Pediatr Res 2013;73:75-9. 40. Kor DJ, Kashyap R, Weiskopf RB, et al. Fresh red blood cell transfusion and short-term pulmonary, immunologic, and coagulation status: a randomized clinical trial. Am J Respir Crit Care Med 2012;185:842-50. 41. Lebiedz P, Glasmeyer S, Hilker E, et al. Influence of red blood cell storage time on clinical course and cytokine profile in septic shock patients. Transfus Med Hemother 2012;39:271-6. 42. Bernard A, Meier C, Ward M, et al. Packed red blood cells suppress T-cell proliferation through a process involving cell-cell contact. J Trauma 2010;69:320-9. 43. Danesh A, Inglis HC, Jackman RP, et al. Exosomes from red blood cell units bind to monocytes and induce proinflammatory cytokines, boosting T-cell responses in vitro. Blood 2014;123:687-96. 44. Long K, Meier C, Ward M, et al. Immunologic profiles of red blood cells using in vitro models of transfusion. J Surg Res 2013;184:567-71.
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BACKGROUND: Our previous in vitro work showed that stored red blood cells (RBCs) increasingly suppress markers of innate immune function with increased storage time. This multicenter prospective observational study tests the hypothesis that a single RBC transfusion in critically ill children is associated with immune suppression as a function of storage time. STUDY DESIGN AND METHODS: Blood samples were taken immediately before and 24 (±6) hours after a single RBC transfusion ordered as part of routine care. Innate and adaptive immune function was assessed by ex vivo whole blood stimulation with lipopolysaccharide (LPS) and phytohemagglutinin, respectively. Monocyte HLA-DR expression, regulatory T cells, plasma interleukin (IL)-6, and IL-8 levels were also measured. RESULTS: Thirty-one transfused critically ill children and eight healthy controls were studied. Critically ill subjects had lower pretransfusion LPS-induced tumor necrosis factor-α production capacity compared to healthy controls, indicating innate immune suppression (p < 0.0002). Those who received RBCs stored for not more than 21 days demonstrated recovery of innate immune function (p = 0.02) and decreased plasma IL-6 levels (p = 0.002) over time compared to children transfused with older blood, who showed persistence of systemic inflammation and innate immune suppression. RBC storage time was not associated with changes in adaptive immune function. CONCLUSION: In this pilot cohort of critically ill children, transfusion with older prestorage leukoreduced RBCs was associated with persistence of innate immune suppression and systemic inflammation. This was not seen with fresher RBCs. RBC transfusion had no short-term association with adaptive immune function. Further studies are warranted to confirm these findings in a larger cohort of patients.
R
ed blood cell (RBC) transfusion in critically ill patients is known to be associated with increased risks of nosocomial infection, suggesting a relationship between RBC transfusion and impaired host defense, although a causal link between RBC transfusion and immune function has not been clearly established.1-7 Acquired suppression of innate (e.g., monocyte) and adaptive (e.g., lymphocyte) immune function are increasingly recognized as complications of critical illness and are independently associated with increased risks of adverse outcomes.8-13 Immune function in the intensive care unit (ICU) can be measured by quantifying the ability of immune cells to produce cytokines in
ABBREVIATIONS: ICU = intensive care unit; LPS = lipopolysaccharide; PHA = phytohemagglutinin; PICU = pediatric intensive care unit; Treg = regulatory T cells. From the 1Pediatric Critical Care Medicine, Department of Pediatrics, and 5Pathology, Nationwide Children’s Hospital, and 2 The Research Institute at Nationwide Children’s Hospital, Columbus, Ohio; 3Pediatrics, Division of Critical Care Medicine, Washington University, St Louis, Missouri; and 4Critical Care Medicine, Department of Pediatrics, Akron Children’s Hospital, Akron, Ohio. Address reprint requests to: Jennifer A. Muszynski, MD, Critical Care Medicine, Nationwide Children’s Hospital, 700 Children’s Drive, Columbus, OH 43205; e-mail: [email protected]. This study was funded by K12HD043372 from the National Institute of Child Health and Development (JAM), The Research Institute at Nationwide Children’s Hospital (JAM, MWH), and Washington University School of Medicine Pediatric Critical Care Translational Research Program (PCS). Received for publication August 1, 2014; revision received August 25, 2014, and accepted August 29, 2014. doi: 10.1111/trf.12896 © 2014 AABB TRANSFUSION **;**:**-**. Volume **, ** **
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response to ex vivo stimulation. We have previously demonstrated that a markedly reduced ability to produce the proinflammatory cytokine tumor necrosis factor (TNF)-α upon ex vivo stimulation of whole blood with lipopolysaccharide (LPS) is significantly associated with increased risks of mortality and nosocomial infection in critically ill children.8,10,11,13 Similarly, we and others have demonstrated impaired lymphocyte responsiveness to ex vivo stimulation in adults and children with sepsis-related adverse outcomes.14,15 It should be noted that critically ill patients frequently demonstrate some degree of immune suppression during the acute phase of illness, but most recover their immune function over several days. Immune recovery is typically associated with recovery from critical illness while persistent immune suppression is associated with adverse outcomes.16,17 Mechanisms of persistent immune suppression in the ICU are unclear, but they may include RBC transfusion. Retrospective studies have suggested that transfusion with RBCs with longer storage duration places critically ill patients at particularly high risk for secondary infection and mortality.18-22 We have previously shown that among transfused critically injured children, receipt of RBCs stored for more than 14 days was associated with decreased innate immune function over time compared to those who received fresher RBCs.13 Further, in vitro studies conducted by our laboratory and others suggest that stored RBCs, particularly of longer storage duration, directly suppress innate immune cell function.23-26 The immunologic impact of a single RBC transfusion in critically ill children is unknown. We therefore designed a multicenter prospective, observational pilot study to test the hypothesis that a single transfusion of older RBCs would be associated with decreased innate and adaptive immune function over time compared to transfusion with fresher RBCs.
MATERIALS AND METHODS Setting Subjects were enrolled from one of two participating centers. Nationwide Children’s Hospital and St Louis Children’s Hospital are both academic, free-standing children’s hospitals. The pediatric intensive care unit (PICU) at Nationwide Children’s hospital is a 40-bed, quaternary care, mixed medical–surgical unit with more than 2200 admissions annually. The PICU at St Louis Children’s Hospital is a 46-bed mixed medical–surgical unit with approximately 2700 admissions per year. The study was approved by the institutional review boards at Nationwide Children’s Hospital and at St Louis Children’s Hospital.
Subjects After informed consent and, where appropriate, assent was obtained, children (age 21 days (n = 10) 19 (3-92) 15 (8-112) 6 (29) 3 (30)
17 (55) 2 (6) 7 (23) 5 (16) 24 (77) 12 (39) 14 (45)
10 (48) 1 (5) 6 (29) 4 (19) 15 (71) 7 (33) 10 (48)
1.6 (1-4) 7 (4-13) 11 (1.5-11.5) 68 (17-232) 7.7 (7.1-8.1) 15 (12-17) 8 (26) 17 (8-27)
1.4 (0.8-4) 7 (3-12) 11 (1-16) 76 (17-232) 7.7 (7.1-8.5) 15 (11-19) 6 (29) 14 (7-17)
7 (70) 1 (10) 1 (10) 1 (10) 9 (90) 5 (50) 4 (40) 2.8 (1-4) 8 (6-15) 10.5 (2-14.5) 50 (14-471) 7.8 (6.9-8.5) 13 (11-17) 2 (20) 30 (27-33)
p value 0.4 1
0.4 0.4 1 0.4 0.4 0.8 0.7 1 0.6 1 21
Before transfusion 76 (17-232) 50 (14-471)
IL-6 (pg/mL) After transfusion 26 (12-127) 57 (15-122)
p value 0.002 0.9
Before transfusion 16 (11-70) 12 (6-29)
IL-8 (pg/mL) After transfusion 17 (7-35) 17 (6-91)
p value 0.4 0.2
* Data are reported as median (interquartile range).
Adaptive immune function Adaptive immune function was assessed by PHA-induced cytokine production capacity. For all critically ill subjects, pretransfusion ex vivo PHA-induced IFN-γ production was significantly lower compared to healthy controls (236 [152-352] pg/mL vs. 535 [419-1027] pg/mL, p = 0.001). Pretransfusion ex vivo PHA-induced IL-10 production tended to be lower than healthy controls (156 [60-270] pg/mL vs. 246 [169-338] pg/mL, p = 0.07). Pretransfusion ex vivo PHA-induced IL-2 and IL-4 were not significantly
different from healthy controls (31 [16-105] pg/mL vs. 31 [19-475] pg/mL, p = 0.6; and 2.9 [2.1-5.3] pg/mL vs. 2.5 [2.1-2.8] pg/mL, p = 0.3, respectively). Among all transfused subjects, ex vivo PHA-induced IFN-γ production improved over time while ex vivo PHA-induced production of other cytokines did not differ significantly between pre- and posttransfusion samples (Fig. 3A). Comparing children transfused with older versus fresher RBCs, significant differences in adaptive immune function over time were not detected (Fig. 3B). In the cohort who Volume **, ** **
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suggest a relationship between the storage duration of a single transfused p = 0.9; Mann-Whitney p = 0.01; Mann-Whitney 400 150 unit of RBCs and in vivo immune suppression in critically ill children. This study used a highly standard300 ized assay to quantify innate immune 100 function by measuring whole blood ex 200 vivo LPS-induced TNF-α production capacity. This assay has been success100 fully employed in single- and multicenter studies of pediatric critical illness 50 ≤ 21 > 21 > 21 days ≤ 21 days across multiple diagnoses including (N = 16) (N = 6) (N=6) (N=16) sepsis, influenza infection, respiratory RBC Storage Duration (days) RBC Storage Duration (days) syncytial virus infection, traumatic injury, and status after cardiopulmoFig. 2. (A) Among children undergoing flow cytometric analysis, monocyte antigennary bypass.8,10,11,13,34,35 In each of these presenting capacity, as measured by HLA-DR expression, did not significantly studies, lower ex vivo TNF-α production change after transfusion regardless of RBC storage duration, while receipt of fresher capacity was consistently associated blood was associated with increased ex vivo LPS-induced TNF-α production capacity with increased risks of nosocomial in this group of subjects (B). Boxes represent median and interquartile range. infection and/or death. This is not Whiskers represent 10th to 90th percentile. strictly a pediatric phenomenon, as persistently low innate immune function has been associated with adverse outcomes in critically ill underwent flow cytometric analysis, the pretransadults as well.36-38 It is worth noting that the majority of fusion percentage of immunosuppressive Treg among CD4+ T cells (%Treg) was similar to healthy controls patients in our cohort were transfused in the acute phase (5.2% [3%-8.4%] vs. 6.3% [5.2%-6.5%], p = 0.6). Among of illness, with most posttransfusion samples being drawn transfused subjects overall, %Treg was not signion ICU Days 2 to 5. This is a time when we would expect to ficantly different after transfusion compared to presee evidence of recovery of innate immune suppression in transfusion values (6% [3.8%-7.6%] vs. 5.2% [3%-8.4%], most patients. Children who received older RBCs failed to p = 0.3). There was a trend toward an increase in %Treg demonstrate this increase in innate immune function posttransfusion in children transfused with RBCs stored compared to children who received fresher RBCs. These for more than 21 days, which did not reach significance results are similar to those of our previous study of criti(Fig. 4). A difference in ex vivo PHA-induced IL-10 produccally injured children in whom receipt of RBCs stored for tion (which would suggest a Treg-dominant state) was not more than 14 days was associated with a failure to improve seen in this cohort. innate immune function over time during the first week postinjury.13 Our inability to demonstrate an immune effect at the 14-day cutoff in this study may have been DISCUSSION related to sample size or to other differences between the two patient populations. In this multicenter, prospective observational study, a Previous reports have quantified circulating plasma single transfusion with prestorage leukoreduced RBCs cytokines before and after RBC transfusion. In Keir and stored for more than 21 days was associated with impaired colleagues,39 28 nonseptic preterm infants between 2 and recovery of innate immune function. Relationships between RBC storage duration and changes in adaptive 6 weeks of age demonstrated small, but significant immune function were less apparent. increases in circulating proinflammatory cytokines IL-1β, RBC storage duration has been associated with IL-8, TNF-α, and monocyte chemoattractant protein-1 2 adverse outcomes including nosocomial infection, deep to 4 hours after a single RBC transfusion. Infants in this vein thrombosis, mortality, and organ dysfunction across study were not stratified by RBC storage duration. By cona variety of critically ill adult and pediatric patient trast, two adult studies evaluated relationships between populations.18-22,30-33 While increasing attention is being RBC storage duration and plasma cytokines. Both studies failed to detect significant differences in changes in paid to the RBC storage lesion, little is known about the plasma cytokines immediately after a single transfusion impact of RBC storage on immune function in critically with fresher compared to older RBCs.40,41 However, circuill patients. We have previously demonstrated that leukoreduced RBCs directly suppress monocytes in vitro lating plasma cytokines may not reflect the functional as a function of RBC storage duration.25 This study is in capacity of the immune system. In this study, transfusion with older RBCs was associated with a failure to resolve agreement with our in vitro results and is the first to 6
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Fig. 3. (A) Ex vivo PHA-induced cytokine production capacity for IFN-γ was lower in critically ill subjects compared to healthy controls and improved over time. No differences in other cytokines were seen. Dashed lines and shaded areas represent median values and interquartile ranges for healthy controls. **p < 0.01 before versus after transfusion values. (B) Significant differences in posttransfusion ex vivo PHAinduced cytokine production were not seen when comparing children transfused with fresher or older RBCs. Boxes represent median and interquartile range. Whiskers represent 10th to 90th percentile.
systemic inflammation as evidenced by persistent elevations in plasma IL-6 levels. In the context of simultaneous improvement in innate immune function, we speculate that the reduction in plasma IL-6 levels seen in children transfused with fresher RBCs may reflect resolution of systemic inflammation and a return toward immunologic homeostasis. In addition to innate immune function, suppression of adaptive immune function has also been associated with increased risks of nosocomial infection and mortality in critically ill adults and children.9,12,14 While a recent in vitro study demonstrated that exosomes isolated from stored RBCs enhance T-cell proliferation, other reports show suppressed T-cell proliferation after exposure to stored
RBCs.42-44 In this study, we evaluated adaptive immune function using whole blood ex vivo PHA-induced cytokine production. In contrast to innate immune function, we were unable to detect significant relationships between RBC storage duration and adaptive immune function in transfused children. This may represent a lack of immunosuppressive effect of older RBCs on adaptive immune function in patients, but it is also possible that the timing of our 24-hour posttransfusion samples may have been too early to see an effect on lymphocytes. We chose this window, however, to limit the confounding effects of other ICU therapies on posttransfusion immune function. Our study has several important limitations. First, because sample processing for functional assessments of innate and adaptive immunity needed to be performed in real time, we were limited to nonurgent transfusions occurring during the day. This limited the number of patients available for study. We were, however, able to demonstrate the feasibility of consenting and enrolling critically ill children before transfusion and performing functional immune assays in a multicenter setting. Second, the number of patients with severe pretransfusion immune suppression was low. Our data suggest that relationships between RBC storage duration and immune function may be stronger in patients with a greater degree of baseline immune suppression. It will be important to confirm these findings in a larger cohort of severely critically ill children with more longitudinal sampling, allowing for more detailed evaluation of these relationships over time. Third, to capture a representative sample of transfused critically ill children, we chose to include transfused patients in the PICU with any diagnosis. This resulted in a high degree of baseline variability in diagnoses, severities of illness, and immune function. Despite this, we were able to detect an association between RBC storage duration and persistence of innate immune suppression with a single RBC transfusion. Larger studies with sample sizes large enough to permit multivariable analyses to control for clinical confounders, including severity of illness and timing of transfusion, are still needed. Finally, although our data suggest an RBC storage duration threshold of 21 days, this threshold may not apply to all patients. This too should be the subject of future investigations. This study represents the most comprehensive evaluation to date of immune function in transfused critically ill children and supports the hypothesis that transfusion with older RBCs, even when leukoreduced before storage, may be associated with the persistence of innate immune suppression over time. Additional studies are needed to confirm these results in a larger cohort of patients; to evaluate relationships between RBC storage duration, immune function, and clinical outcomes; and to determine mechanisms of immunologic effects of RBC transfusion in critically ill patients. Volume **, ** **
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mechanisms of immunoparalysis after cardiopulmonary bypass. J Thorac Cardiovasc Surg 2012;143:1160-6 e1. 9. Felmet KA, Hall MW, Clark RS, et al.
400 300
Prolonged lymphopenia, lymphoid depletion, and hypoprolactinemia in
200
children with nosocomial sepsis 100
and multiple organ failure. J Immunol 2005;174:3765-72.
0
≤ 21
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≤ 21
> 21
(n = 16)
(n = 6)
(n = 16)
(n = 6)
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Clinical implications and molecular
RBC Storage Duration (days)
Fig. 4. (A) There was a trend toward increase in percentage of Treg after transfusion in children transfused with older RBCs. (B) Ex vivo PHA-induced IL-10 production capacity was not different based on RBC storage age in the cohort of children who underwent flow cytometric analysis. Boxes represent median and interquartile range. Whiskers represent 10th to 90th percentile.
10. Hall MW, Geyer SM, Guo CY, et al. Innate immune function and mortality in critically ill children with influenza: a multicenter study. Crit Care Med 2013;41:224-36. 11. Hall MW, Knatz NL, Vetterly C, et al. Immunoparalysis and nosocomial infection in children with multiple organ dysfunction syndrome. Intensive Care Med 2011;37:525-32. 12. Hotchkiss RS, Tinsley KW, Swanson PE,
CONFLICT OF INTEREST The authors have disclosed no conflicts of interest.
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et al. Sepsis-induced apoptosis causes progressive profound depletion of B and CD4+ T lymphocytes in humans. J Immunol 2001;166:6952-63. 13. Muszynski JA, Nofziger R, Greathouse K, et al. Innate immune function predicts the development of nosocomial infection in critically injured children. Shock 2014;42: 313-21. 14. Boomer JS, To K, Chang KC, et al. Immunosuppression in patients who die of sepsis and multiple organ failure. JAMA 2011;306:2594-605. 15. Muszynski JA, Nofziger R, Greathouse K, et al. Early adaptive immune suppression in children with septic shock: a prospective observational study. Crit Care 2014;18: R145. 16. Hotchkiss RS, Monneret G, Payen D. Sepsis-induced immunosuppression: from cellular dysfunctions to immunotherapy. Nat Rev Immunol 2013;13: 862-74. 17. Volk HD, Reinke P, Docke WD. Clinical aspects: from systemic inflammation to “immunoparalysis.” Chem Immunol 2000;74:162-77. 18. Koch CG, Li L, Sessler DI, et al. Duration of red-cell storage and complications after cardiac surgery. N Engl J Med 2008;358:1229-39. 19. Manlhiot C, McCrindle BW, Menjak IB, et al. Longer blood storage is associated with suboptimal outcomes in highrisk pediatric cardiac surgery. Ann Thorac Surg 2012;93: 1563-9. 20. Offner PJ, Moore EE, Biffl WL, et al. Increased rate of infection associated with transfusion of old blood after severe injury. Arch Surg 2002;137:711-6; discussion 716-7. 21. Ranucci M, Carlucci C, Isgro G, et al. Duration of red blood cell storage and outcomes in pediatric cardiac surgery: an
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