Immunologic Effects of Packed red blood Cell Transfusion in critically ill children (IMPACT) J Muszynski, A Frazier, P Spinella, MW Hall BLOOD NET October, 2012
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Immune function in the ICU
Ex vivo TNFα response (pg/ml)
• Reduction in innate immune function is associated with nosocomial infection and death in critically ill adults and children 1600
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Day 3
Day 7
Monneret et al. Int Care Med 2006
• The etiology of immunoparalysis is likely multifactorial • RBC transfusion may contribute….. ………………..……………………………………………………………………………………………………………………………………..
Hall et al. Immunoparalysis and nosocomial infection in children with multiple organ dysfunction syndrome. Intensive Care Med (37)2011: 525-532. Monneret et al. Persisting low monocyte human leukocyte antigen-DR expression predicts mortality in septic shock. Intensive Care Med (32)2006: 1175-1183.
Red cell transfusion in the ICU • Up to 49% of children in the PICU for > 48 hours receive a red cell transfusion • RBC transfusion has been identified as an independent risk factor for mortality in adults with trauma, adults with ARDS, and critically ill children RBC transfusion is associated with increased risk of nosocomial infection Taylor et al. Crit Care Med. (34)2006:2302-2308. ………………..……………………………………………………………………………………………………………………………………..
Bateman, et al. Am J Resp Crit Care (178)2008: 26-33. Malone et al. J Trauma. (54)2003: 898-907. Kneyber et al. Intensive Care Med. (33)2007: 1414-1422. Taylor et al. Crit Care Med. (34)2006:2302-2308.
Red cell storage age • Adult trauma patients: storage age of transfused blood independent risk factor for development of infection • Adult cardiopulmonary bypass patients: receipt of blood stored > 14 days associated with increased rates of sepsis and mortality Are RBC with longer storage times immunosuppressive? ………………..……………………………………………………………………………………………………………………………………..
Koch et al. Duration of red-cell storage and complications after cardiac surgery. The New England Journal of Medicine. (358)2008: 1229-1239. Offner et al Increased rate of infection associated with transfusion of old blood after severe injury. Arch Surg (137)2002: 711-716.
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RBC storage time (days)
LPS-induced IL10 production (% of control)
LPS-induced TNFα production (% of control)
Are RBC with longer storage times immunosuppressive? 200 150 100 50 0
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RBC storage time (days)
Cytokine production from monocytes exposed to RBC in transwells then stimulated with LPS RBCs with greater storage time were immunosuppressive
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Muszynski, et al. Immunosuppressive effects of red blood cells on monocytes are related to both storage age and storage solution. Transfusion, 2012
IMPACT Multi-center, prospective, observational feasibility study Hypothesis: • A single PRBC transfusion will be associated with decreased function of the innate and adaptive immune system • The degree of immunosuppression will be related to PRBC storage time. Inclusion: Age < 18 yrs; Admission to PICU at NCH or St. Louis Children’s; catheter in place for painless blood draws Exclusion: Prior transfusion (w/in 1 month); WBC count less than 1,000; transplant recipient
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Methods • •
Blood sample drawn before (within 2 hours) and again 18-30 hours after red cell transfusion Immune function measured by ex vivo stimulated cytokine response and flow cytometry
PHA and LPS stimulation: PHA LPS
50 µl whole blood
4 hr at 37°C
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Methods Flow Cytometry: Stained with antibody panel: CD 14 (monocyte) HLADR Regulatory T cells CD 4 CD 25 CD 127
Red cells lysed and samples washed Flow data acquired locally
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Results: Patients • • • • • •
26 children enrolled to date Data from first 22 children presented here Median age: 52 [4.3 – 99] months Median time from ICU admit to transfusion: 2 [0.9 – 4.2] days Median PRISMIII at the time of transfusion: 7.5 [5.3 - 13.8] Median Hgb at the time of transfusion: 7.9 [7.3– 8.9]
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Ex vivo LPS-induced TNFα production (% of pre transfusion)
Red cell storage age and innate immune function p=0.02; ANOVA 200
150
100
50
pre-transfusion
• Receipt of red cells stored for greater than 21 days is associated with a failure to improve innate immune function over time
post-transfusion
Storage age ≤ 21 d (n = 15) Storage age > 21 d (n = 7)
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Ex vivo LPS-induced TNFα production (% of pre transfusion)
Or put another way … • AOB > 21 days associated with 4 – fold increased risk of failure to improve innate immune function RR 4.2 [1.01 – 18]
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Storage age of blood
40
• Looking at only those patients who had suppressed innate immune function pretransfusion [TNF production capacity < 250 pg/ml (red) or < 500 pg/ml (yellow)]:
• Effect may be more pronounced • Sample is size small ………………..……………………………………………………………………………………………………………………………………..
Patient Demographics
Pt age (months) Female gender, n (%) PRISM III* Plasma IL-‐6* Transfusion Time from ICU admit to txf (days) Volume of transfusion (ml/kg) PaSent hemoglobin* Storage age of blood
Storage age of Storage age of blood ≤ 21 D blood > 21 D n = 15 n = 7 p 50 [2-‐110] 88 [7-‐168] 0.4 5 (33) 2 (29) 1 7 [3-‐13] 9 [6-‐18] 0.3 76 [23-‐159] 88 [31-‐735] 0.5 1.6 [0.6-‐4.6] 2.9 [1.2-‐3.5] 0.5 15.9 [14-‐18.4] 12.7 [10-‐13.6] 0.07 7.8 [7.4-‐9] 7.9 [6.9-‐9.5] 0.8 7 [6-‐17] 29 [27-‐33] 0.0002
Patient characteristics were similar between groups
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250 200 150 100 50 AOB > 21 D
AOB ≤ 21 D
300 250 200 150 100 50 AOB > 21 D
AOB ≤ 21 D
Ex vivo PHA-induced IL-4 production (% pre-transfusion)
300
Ex vivo PHA-induced IL-10 production (% pre-transfusion)
Ex vivo PHA-induced IFN γ production (% pre-transfusion)
Ex vivo PHA-induced IL-2 production (% pre-transfusion)
Adaptive immune function: PHA stimulation data 200
150
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50 AOB > 21 D
AOB ≤ 21 D
• No significant differences seen in PHA-stimulated cytokine production (marker of T cell function) • Red cell storage age may not be associated with change in T cell function • Or, 24 hours may be too short a time frame to detect a difference
250 200 150 100 50 AOB > 21 D
AOB ≤ 21 D
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Flow cytometry data 140
Regulatory T cells (% pre-transfusion)
Monocyte HLADR (% pre-transfusion)
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Post
AOB > 21 days (n=4) AOB ≤ 21 days (n=9)
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post
AOB > 21 days (n=4) AOB ≤ 21 days (n=9)
No significant differences seen in monocyte HLA-DR expression or %regulatory t cells by flow cytometry
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Challenge: Multi-center flow cytometry
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Challenge: Multi-center flow cytometry
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Potential solution: Lysing solution
Antibody stain mix
15 minutes RT dark
- 80°C
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Multi-center flow cytometry staining with central data acquisition • Single sample stained by lysed whole blood method, fixed and stored at 4C (top • or stained, lysed (no wash) then stored at -80C (bottom) • Both samples acquired three days later ………………..……………………………………………………………………………………………………………………………………..
Challenge: Patient enrollment 25
N u m b e r o f p a t ie n ts
• To address this challenge: • Daily screening of ICU census • Consent and enrollment for children with risk of blood transfusion during ICU stay • Intubated patients • Septic shock
20 15 10 5 0 Nov Dec
P ts tra n s fu s e d
Jan
Feb
A pr
P ts tra n s fu s e d 7 a m -3 p m
M ay
Jun
P ts tra n s fu s e d W e e k d a y 7 a m -3 p m
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Challenge: Patient heterogeneity
Storage age of blood ≤ 21 D n = 15
Storage age of blood > 21 D n = 7
p
Diagnosis n, (%) Sepsis/sepSc shock 0 3 (43) Trauma 6 (40) 1 (14) CHD or cardiomyopathy 4 (27) 1 (14) BronchioliSs 3 (20) 1 (14) Cardiac arrest 2 (13) 0 Center 0.4 NaSonwide Children’s 8 (53) 2 (29) St Louis Children’s 7 (47) 5 (71) ………………..……………………………………………………………………………………………………………………………………..
Solutions • Enroll more patients through multi-center studies • Target specific patient populations Contact us if you can help
[email protected] [email protected]
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Acknowledgements Immune Surveillance Laboratory at Nationwide Children’s: • Jyotsna Nateri, MS • Lisa Hanson, BS • Kristin Greathouse, MS, BSN • Lisa Steele, BSN, CCRN • Ryan Nofziger, MD • Mark Hall, MD
St. Louis Children’s at Washington University: • Ayu Frazier • Phil Spinella Pediatric Blood Research Group (BLOOD NET) of PALISI Dr. Muszynski is supported by K12HD043372 from the National Institute of Child Health and Development, and the Research Institute at Nationwide Children’s
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Red cell storage age • Adult trauma patients: storage age of transfused blood independent risk factor for development of infection • Adult cardiopulmonary bypass patients: receipt of blood stored > 14 days associated with increased rates of sepsis and mortality Are RBC with longer storage times immunosuppressive? ………………..……………………………………………………………………………………………………………………………………..
Koch et al. Duration of red-cell storage and complications after cardiac surgery. The New England Journal of Medicine. (358)2008: 1229-1239. Offner et al Increased rate of infection associated with transfusion of old blood after severe injury. Arch Surg (137)2002: 711-716.
