Red Cell Storage & Aging of Blood

Alan Tinmouth, MD MSc

University of Ottawa Centre for Transfusion Research, Ottawa Health Research Institute and the Ottawa Hospital

November 2009

Objectives

1. Understand the changes that occur during prolonged storage of red blood cells.

2. Review the clinical studies evaluating the effects of transfusing older stored red blood cells.

3. Review the design of studies evaluating the age of blood.

Purpose of an RBC transfusion

Increase O2 delivery and consumption.

Increase hemoglobin levels.

Decrease morbidity and mortality.

RBC transfusions and risk of death

Marik and Corwin, CCM 2008;36:2667

Adverse Effects Associated with Transfusion

FeverNeutrophilia

FlushingProinflammatory

Capillary leakTRALI / ARDS

MOF

Other adverse effects of leukocytes

Thrombosis

Impaired O2 deliveryAcidosis

K+, Na+, NH4+Hypothermia

GlucosePlasticisers

Jaundice

Thrombosis? ARDS

RES BlockadeMicrovascular Pathology

HypotensionFlushingAnxiety

GIT SymptomsPain

Proinflammatory

PLASMACleavage / activation of

Plasma proteins

BUFFY COAT

RED CELLS

1. Impaired RBC survival2. Reduced efficacy3. Adverse effects

Chemical,Metabolic

&Physical

HaemolysisBillirubin

LDHIron

Cytokines

KininsComplement

Histimine

Microaggregates

Procoagulants

Red blood cell storage lesionBiomechanical Changes• Membrane Phospholipid

Loss• Membrane Phospholipid

Re-distribution• Protein oxidation• Lipid Peroxidation

Biochemical changes• 2,3 DPG Depletion• ATP Depletion• Calcium• Metabolic Modulation• Loss of Nitric Oxide

Consequences of Biochemical and BioMechanical Changes in Stored RBCs• Left shift of oxygen-

hemoglobin dissociation curve

• Loss of red blood cell deformability

• Increased RBC aggregation• Increased RBC adhesion to

endothelial cells• Release of hypercoagulable

microvessicles• Increased NO scavenging• Accumulation of cytokinesTinmouth. Transfusion 2006

• 2,3 DPG depleted in first week of storage• Restoration occurs within 24-72 hrs of transfusion.

Changes in 2,3 DPG levels following transfusion

Heaton et al. Brit J Haem 71; 131-136, 1989

Adhesion of Stored RBCs to Endothelial Cells

• Under continuous flow, non-leukocyte reduced RBCsstored for 28 days in AS-3 have increased adherence to human umbilical vein endothelial monolayers

• Pre-storage leukoreduction abbrogates the increased adhesion observed with storage

Luk C et al. Transfusion 43: 151-156, 2002

RBC Rheologic Properties in Storage

63.9 ± 14.0*29.9 ± 4.0*46.8 ± 6.7*4236.7 ± 7.9*21.9 ± 5.0*40.6 ± 3.4*3570.5 ± 13.2*17.2 ± 4.1*35.2 ± 1.6*2851.8 ± 23.3*15.7 ± 3.3*30.6 ± 3.02170.9 ± 20.5*14.7 ± 2.6*27.9 ± 1.9*14114.7 ± 7.68.4 ± 1.613.6 ± 1.77118.9 ± 9.47.0 ± 1.614.0 ± 1.75

Deformability IndexIrreversibly ∆ RBCs (%)Reversibly ∆ RBCs (%)Days

Day 5 Day 14 Day 42

Berezina LT et al. J of Surg Res 102: 6-12, 2002

S-Nitrosohemoglobin and stored RBCs

• Blood flow to tissues is regulated by O2 content of blood.• Hypoxemia results in NO release by RBCs that leads to vasodilation• SNO hemoglobin rapidly depleted during RBC storage but rapidly

repleted with rejuvenation / posttransfusionReynolds et al. PNAS 2007; 104: 17059.

Iron accumulation and cytokine production

• Approx 25% of old RBCs do not survive 24 hrs

Mouse model of transfusion of aged RBCs1. Iron deposition in liver and spleen2. Increased non transferrin bound iron3. Accumulation of cytokines (IL-6 and TNF)

• With transfusion of RBC concentrates and washed RBCs but not RBC supernatant

• Increased susceptibility to bacterial infection• Cytokine accumulation negated by iron

chelationHod et al. AABB Annual Meeting 2009

Clinical studies …

14

RBC age and clinical outcomes: Observational studies

Increased thrombosis and mortality202ICUSpinella 2009Increased mortality with |3+ units1813TraumaWeinberg

No difference in survival or LOS stay2732Cardiac SxVan der Watering 2006No increase nosocomial infections2085ICUTaylor 2006

Increased mortality.321Cardiac SxBasran 2006Increased pneumonia.897Cardiac SxLeal-Noval 2003

Increased LOS.86TraumaKeller 2002Increased infections.61TraumaOffner 2002

No change LOS or ventilation268Cardiac SxVamvakas 2000Increased risk of pneumonia416Cardiac SxVamvakas 1999Increased multi-organ failure63TraumaZallen 1999

Increased mortality31Septic ICU Purdy 1997Increased LOS698ICUMartin 1994

Increased mortality6002Cardiac SxKoch 2008No difference in morbidity/mortality670Cardiac SxYap 2008

OutcomesNPopulationStudy

Impossible to adjust for confounding

• Sicker patients = more transfusions

= older RBCs

• Temporal relationship of outcomes and

transfusions not always ascertained

The Question ?

Do older RBC units cause harm when transfused to critically ill patients?

Day 0 Day 42Day 14

The age of RBCs: one or two questions?

Is old blood harmful?

Is fresh blood beneficial?

Evaluating the age of RBCs“EFFECT OF OLD BLOOD”• Age of oldest units• Age greater than 14, 18, 21, 28, 30 days

– Number of units– Exposure to blood greater than this age– Exposure to only blood greater than this age

“EFFECT OF FRESH BLOOD”• Age of youngest unit

“INFLUENCE OF THE AGE OF BLOOD”• Mean/median age of RBCs transfused• Age as continuous variable

Effect of Older RBCs

Oldest unit of RBCs• Increased mortality with age of oldest unit in

Cardiac Sx• Increased pneumonia with age of older RBC unit in

Cardiac Sx• No increase in nosocomial infections in ICU patients• No difference in morbidity and mortality in Cardiac

Sx.• Increased pneumonia but no difference in length of

stay or survival in Cardiac Sx

Effect of Older RBCsRBCs > 14 days• Increased ICU stay in ICU patients• Increased ICU stay and infection in trauma pts• Increased hospital stay in trauma patients• Increased multiorgan failure in trauma pts• Increased mortality and morbidity in Cardiac Sx

RBCs > 18 or 21 days• No difference in length of stay or survival in Cardiac Sx• Increased multiorgan failure in trauma pts

RBCs > 28 or 30 days• No difference in morbidity and mortality in Cardiac Sx.• Increased DVT and mortality in ICU patients

Effect of Fresh RBCsFreshest unit of RBCs• No difference in length of stay or survival in

Cardiac Sx

Influence of age of RBCs• Increase hospital mortality associated with

increased median age of RBCs in Cardiac Sx.• Negative correlation with age of RBCs and survival

in ICU patients• No change in length of stay and survival with

greater mean age of RBCs in Cardiac Sx.• No change in morbidity and mortality with greater

mean age of RBCs in Cardiac Sx. • Increase risk of pneumonia with higher average age

of RBCs but no change in ventilation or length of stay in Cardiac Sx

Prospective Studies ….

Stored RBCs – physiological effects in Traumatic Brain Injury

• Design: prospective observational study• Population: 66 male non bleeding and hemodyamically

stable; GCS ≤ 8 and with hemoglobins < 95 g/L• Measurements: cerebral oxygen pressure (Pti02 baseline,

after transfusion, 1-6, 12, and 24 hours– Groups: blood stored for < 10, 10 – 14, 15 – 19, and > 19 days

Leal Noval, CCM, 2008

Efficacy of fresh and stored blood to reverse brain oxygenation defects

• 9 healthy volunteers• Acute normovolemic hemodilution to Hg 75 g/l then 50 g/l• Transfused autologous fresh or stored (23d) RBCs

Weiskopf et al. Anesthesiology 2006; 104: 911.

• 23 septic patients had no evidence of increased oxygen uptake following transfusion of 3 units of RBCs

• Transfusion of older blood (> 15 days) was associated with evidence for gastric mucosal ischemia

Stored RBCs - Effect in Sepsis

0 10 20 30 40

-0.28

-0.18

-0.08

0.02

0.12

0

r=-0.71p<0.001

Age of blood (days)

Max

imal

cha

nge

in p

Hi

Marik and Sibbald. JAMA 1993;269:3024, 1993.

RCTs examining Age of Blood in the critically ill

• RCT to determine whether age of blood alters gastric mucosal oxygenation or global oxygenation in 22 euvolemic and non hemorhaging critically ill patients

Inclusion criteria: HB < 90 g/L and need for ≥ 2 RBC transfusions

Intervention: Leukodepleted RBCs ≤ 5 days versus ≥ 20 days old

Measurements: Gastric pH and gastric to arterial CO2 gap during transfusion and hourly post transfusion

Results: No difference in pH and CO2 gap between groups using treatment by time analysis or comparing pre-post transfusion periods

Walsh et al. Crit Care Med 2004.

RCTs examining Age of Blood in the critically ill

• Pilot RCT to examine feasibility of conducting large multi-centre age of blood trialPaitents:– 66 critically ill and cardiac surgical patients 4 Canadian

hospitalsIntervention:– Blood stored < 8d vs. standard issueResults:– Feasible to randomize pts to fresher and older RBCs

• Fresh arm – media 4 day old RBCs• Standard arm – median 19 day olds RBCs

– Fresh arm had non-significant higher mortality or serious complication

Hebert et al. Anesth Analg 2005.

Does the age of donated blood affect premature infants? The

ARIPI Trial.

Principal InvestigatorDean Fergusson

Age of RBCs in Premature Infants (ARIPI) trial

Objective:• To determine if RBCs < 7 days old decrease

major morbidity and mortality in neonates admitted to the NICU and requiring at least one unit of RBCs compared to current standard practice

• Current practice: dedicated unit from a single donor used until outdate (35/42 days)

Age of RBCs in Premature Infants (ARIPI) trial

Study Design: Randomized double-blind controlled clinical trial

Setting: 6 Canadian NICUsStudy Population: 450 LBW neonates (<1250 grams) who

require at least one red cell transfusionOutcomes: The primary outcome for this study will be a

composite measure of major neonatal morbidities:• NEC (≥ Grade 2 Bell’s)• ROP (≥ Stage 3)• BPD (oxygen dependency at 36 weeks post-menstrual age + total O2

duration of ≥ 28 days post-randomization)• IVH (Grade III/IV Papile)• Mortality

Age of BLood Evaluation (ABLE) Trial in the Resuscitation of Critically Ill Patients

Principal Investigators: Jacques LacroixDean FergussonPaul HebertAlan Tinmouth

Coming soon to

Manitoba

ABLE study

Double-blind, multicentre RCT:– 25 Canadian academic & community centres.– Intensive care units for adults.

Objective:– To compare the effect of transfusing ‘fresh’

RBCs (stored for less than 8 days) to standard issue red cells on mortality and morbidity

ABLE Inclusion / exclusion criteriaInclusion criteria: Patients who…

– ICU patients– Require at least 1 RBC unit transfusion,– Have cardiovascular dysfunction (vasopressors for

more than 30 minutes) – And an anticipated ventilation > 48 hrs

Exclusion criteria related to the patients:– Age less than 16 years of age.– Previous enrolment in this study.– Brain death or suspected brain death.– Require uncrossmatched blood– Difficulties with cross-match

ABLE study: basic design

Eligible patientsEligible patients

Outcome: 90 day mortality

Outcome: 90 day mortality

Experimental group: fresh blood ( < 8 days)

Control group: usual transfusion strategy (up to 42 days)

Only pre-storage leukocyte-reduced packed

RBC units will be used

ABLE OutcomesPrimary outcomes• 90 day mortality

Secondary outcomes• Severity of MODS• Mortality (28 days, ICU and hospital discharge)• Nosocomial infections. • Length of ICU and hospital stays. • Need for cardiovascular, respiratory and renal

support • Adverse events, acute reactions to RBC

transfusion.

ABLE study: Statistical analysis

Sample size - 2510 patients– Detect an absolute risk reduction of 5%

(assuming a baseline mortality of 25%) in all-cause 90-day mortality

Final analysis– Superiority– Intention-to-treat

RCTs evaluating the age of blood

Post op morbidity

RBCs > 21 dRBCs < 14 dCardiac SxOutcomes Consortium

Organ Dysfunction

RBCs ≥ 21 dRBCs ≤ 10 dCardiac SxRECESS

MortalityStandard age RBCs

RBCs < 8 dICUABLE

OutcomeControlInterventionPatientsStudy

Age of RBCs in Canada

Weighted mean age variations: inventories versus transfusions

10

15

20

25

1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30

weeks of observation

days

inventories transfusions

Conclusions

• RBCs undergo significant change during storage– May affect ability of stored RBCs to deliver O2

• Unclear if transfusion of older stored RBCsassociated with increased morbidity and mortality

• Prospective trials needed to evaluate effects of transfusion stored RBCs– Results may require change to RBC inventory

management

AcknowledgementWinnipeg ABLE TeamDr. Ryan ZarychanskiDr. Debra LaneWendy JanzSharon FloodTheresa Wiwchar

Principal Investigators• Jacques Lacroix• Paul Hebert• Dean Fergusson

Co-Investigators• Deborah Cooke• John Marshall• Alexis Turgeon• Lauralyn McIntyre• Jeannie Callum