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Blood First Edition Paper, prepublished online December 23, 2013; DOI 10.1182/blood-2013-11-539353
Washing older blood units before transfusion reduces plasma iron and improves
outcomes in experimental canine pneumonia
Irene Cortés-Puch,1 Dong Wang,1 Junfeng Sun,1 Steven B. Solomon,1 Kenneth E. Remy,1
Melinda Fernandez,1 Jing Feng,1 Tamir Kanias,2 Landon Bellavia,3 Derek Sinchar,2 Andreas
Perlegas,3 Michael A. Solomon,1,4 Walter E. Kelley,5 Mark A. Popovsky,6 Mark T.
Gladwin,2,7 Daniel B. Kim-Shapiro,3 Harvey G. Klein,8 and Charles Natanson1
1
Critical Care Medicine Department, Clinical Center, NIH, Bethesda, Maryland, USA;
2
Vascular Medicine Institute, University of Pittsburgh, Pittsburgh, PA, 15213; 3Department
of Physics and the Translational Science Center, Wake Forest University, Winston-Salem,
NC 27109; 4Cardiovascular and Pulmonary Branch, National Heart, Lung and Blood
Institute, NIH, Bethesda, MD 5Oklahoma Blood Institute, Oklahoma City, OK 73104;
6
Haemonetics Corporation, Braintree, MA 02184; 7Department of Medicine, Division of
Pulmonary, Allergy and Critical Care Medicine, University of Pittsburgh School of
Medicine, Pittsburgh, PA, 15213; 8Department of Transfusion Medicine, NIH, Bethesda,
Maryland, USA
Address correspondence to Irene Cortés-Puch M.D., Critical Care Medicine Department,
NIH Clinical Center, 10 Center Drive, Room 2C145, NIH, Bethesda, Maryland 20892. Tel:
301-496-5196; Fax: 301-480-5493; email: [email protected]
Running head: Washing older stored transfused blood improves outcomes.
Copyright © 2013 American Society of Hematology
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Scientific Category: Transfusion Medicine
Key Points:
•
Washing older blood before transfusion reduces plasma iron, improving outcomes
from established infection in canines.
•
In contrast, washing fresh blood before transfusion increases in vivo plasma cellfree hemoglobin release, worsening outcomes.
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ABSTRACT
In a randomized controlled blinded trial, two-year-old purpose-bred beagles (n=24)
with S. aureus pneumonia, were exchanged-transfused with either 7- or 42-day-old washed
or unwashed canine universal donor blood (80 ml/kg in 4 divided doses). Washing red
cells (RBC) before transfusion had a significantly different effect on canine survival,
multiple organ injury, plasma iron and cell-free hemoglobin (CFH) levels depending on the
age of stored blood (all, P< 0.05 for interactions). Washing older units of blood improved
survival rates, shock score, lung injury, cardiac performance and liver function, and
reduced levels of non-transferrin bound iron (NTBI) and plasma labile iron (PLI) In
contrast, washing fresh blood worsened all these same clinical parameters and increased
CFH levels. Our data indicate that transfusion of fresh blood, which results in less
hemolysis, CFH and iron release, is less toxic than transfusion of older blood in critically ill
infected subjects. However, washing older blood prevented elevations in plasma circulating
iron and improved survival and multiple organ injury in animals with an established
pulmonary infection. Our data suggest that fresh blood should not be washed routinely
because in a setting of established infection, washed RBC are prone to release CFH, and
result in worsened clinical outcomes.
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INTRODUCTION
Transfusion of older stored canine universal donor blood in a canine model of
experimental S. aureus pneumonia results in markedly increased lung injury and mortality
rates.1 Transfusion with older blood is also associated with increased levels of cell-free
hemoglobin (CFH), transferrin bound iron (TBI), non-transferrin bound iron (NTBI) and
plasma labile iron (PLI). NTBI represents iron excess bound to proteins that do not
normally handle circulating iron and PLI is the toxic iron moiety in plasma. Whereas
increased nitric oxide scavenging by CFH causing vasoconstriction and vascular injury and
increased available iron promoting bacterial growth represent two candidate mechanisms of
injury, multiple other biological changes have been documented with increasing blood
storage interval.2, 3 Some of these changes involve the release into the plasma of
biologically active proteins, microvesicles, potassium, acid, and plasticizer, all of which can
be reduced by means of standard red cell (RBC) washing procedures.4-10 The clinical
effect(s) of washing on the red cell storage lesion has not been studied.
Red cell washing has long been performed to reduce potassium levels in stored
blood transfused to neonates, debris from red cells recovered during surgery, cryoprotectant
glycerol from cryopreserved red cells, and plasma proteins from blood intended for patients
who have been sensitized to those proteins.11-13 Automated cells washers capable of
removing more than 90% of plasma solute have been available for more than 40 years.
Washing has been shown to improve the in vitro characteristics of stored red cells.4-10, 14-17
Earlier generations of cells washers were labor intensive and considered “open systems,”
which limited the shelf-life of washed cells to 24 hours. Current microprocessor-driven,
closed-system cell-washers can be programmed to provide extensive cell washing and an
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approved shelf-life of 14 days. These instruments effectively reduce supernatant potassium
and plasma proteins while maintaining low levels of hemolysis and acceptable in vivo red
cell survival.5, 8, 9
Transfusion of older units of RBCs reportedly increases morbidity and mortality in
a variety of clinical situations.18 Currently, five randomized controlled trials are being
conducted in Canada, the US, and Australia to confirm or refute these reports.19-23
Strategies to improve the quality of stored RBC by limiting the storage period and licensing
improved preservative solutions have already been introduced in the US and in Europe.24
Washing blood is another practical approach to improving red cell quality by removing
substances that accumulate progressively during the six weeks of refrigerated storage.
We conducted a blinded randomized controlled study of RBC washing in our canine
model of transfusion injury. We hypothesized that washing older units of blood prior to
transfusion would improve clinical outcomes by removing CFH and iron, whereas washing
fresher units would have no effect on outcome. We found that washing improved clinical
outcomes of canines receiving older blood transfusions but unexpectedly worsened
morbidity and mortality in animals that received fresh, washed blood.
MATERIALS AND METHODS
Study design
Synopsis
Twenty-four purpose-bred beagles (12 to 28 months old, 9 to 12.5 kg) were studied using a
2x2 factorial study design comparing transfusion of 42 vs. 7 day old stored canine
universal donor blood that was 1) washed using a commercially available automated blood
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cell processing system just prior to transfusion or 2) not washed. Four animals each study
week for 6 sequential weeks were given an intrabronchial challenge of S. aureus (1.5 x 109
CFU/kg). Four hours later, animals were randomized to undergo an exchange-transfusion
(80 ml/kg) in four divided doses (20 mL/kg) given sequentially over 3 h each with either
washed or unwashed old or washed or unwashed fresh universal donor canine blood.
Except for the four different treatments of blood used for transfusion, animals were treated
identically. All animals were given standard intensive care ancillary therapies including
mechanical ventilation, fluids, vasopressors, and antibiotics. The animals were
continuously monitored and cared for by a clinician or trained technician blinded to animal
treatment groups throughout the 96 h study. All studies were approved by the National
Institutes of Health Clinical Center Institutional Animal Care and Use Committee.
Full study design
This model of pneumonia using S. aureus and performing exchange-transfusions was
previously described 1. Briefly, anesthetized animals day 0 were instrumented (external
jugular vein catheter, femoral artery catheter and urinary catheter) followed with placement
of a balloon tipped pulmonary arterial thermodilution catheter and a tracheostomy
performed as described.1, 25 Following these procedures, animals were weaned off
inhalation anesthesia and continuous sedation (fentanyl, midazolam, and medetomidine)
and mechanical ventilation via a tracheostomy tube were initiated. At time 0 h (T0), S.
aureus isolates were prepared and administered via bronchoscope into the right lower lobe
of the lung.25 The dose of S. aureus (1.5 x 109 CFU/kg suspended in 1ml of PBS), based
on previous studies produced 100% lethality with unwashed older stored canine blood and
30% mortality with fresher blood. The bacterial dose was determined
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spectrophotometrically and confirmed turbidometrically as previously described.1 At 4 h,
antibiotic treatment with Oxacillin (30 mg/kg IV) demonstrated to be effective against this
strain of S. aureus was initiated and administered every 4 h until 96 h or death. Following
antibiotic therapy at 4 h, an exchange transfusion was initiated with each animal,
randomized in a blinded fashion kept by the statistician (JS) to receive either: 7-day old
unwashed blood, 42-day old unwashed blood, 7-day old washed blood and 42-day old
washed blood. At 0 h and prior to each transfusion (4, 7, 10 and 13 h), intravascular
hemodynamic measures and blood samples were obtained including arterial blood gases
(ABG), complete blood counts (CBC), serum chemistries, plasma cell-free hemoglobin
(CFH), haptoglobin, non-transferrin bound iron (NTBI), plasma labile iron (PLI) and
transferrin-bound iron (TBI) (see Supplemental Material). All animals were provided
conventional intensive care unit support, including prophylaxis for gastrointestinal ulcer
(famotidine 1 mg/kg iv, every 12 h) and deep venous thrombosis (heparin 3000 IU, sq,
every 8 h) as well as procedures to prevent pressure ulcers (regular position change).
Mechanical ventilation, fluid support and vasopressor therapy were also employed and
titrated using physiological and laboratory endpoints using previously described algorithms
1, 25
(see Supplemental Methods, Mechanical Ventilation, Fluid and Vasopressor Support).
Animals alive at 96 h were considered survivors and euthanized while still sedated
(Beuthanol; 75 mg/kg IV).
A detailed description of the statistical methods and the cell washing procedure is
provided in the Supplemental Material.
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RESULTS
Survival
Washing RBC prior to exchange transfusion in animals with experimental S. aureus
pneumonia had a significantly different and opposite effect on survival rates depending on
the age of the stored blood (qualitative interaction, p=0.03, figure 1). In animals transfused
with 42-day stored blood, washing the RBC improved survival time. In contrast, with 7-day
old stored blood, washing the RBC worsened survival time.
Shock Reversal score and Lung Injury Score
The shock reversal score takes into account the level of vasopressor support
(norepinephrine) needed to maintain the mean arterial pressure at a preset normal level for
canines (80-100 mmHg).1 The lung injury score (LIS) takes into account five parameters
of lung function (mPAP, arterial alveolar oxygen gradient, plateau pressures, breathing
rates, and oxygen saturation based on pulse oximetry), and its use has been previously
validated to increase the ability to detect abnormalities in the lung.1 Similar to the effect on
survival, washing had significantly different and opposite effects on the level of shock
reversal score (48 h) and LIS (24 h) (qualitative interaction, p=0.04 and p=0.05
respectively, figure 2). Washing older RBCs improved the LIS and the shock reversal score
(i.e. less hypotension and vasopressor use and less severe lung injury respectively). In
contrast, at these same time points washing fresher RBCs worsened both the LIS and the
shock score. For completeness the effect of washing on the different components of the
shock and lung injury scores are provided in the Supplemental Material (Supplemental
Figures 1 and 2).
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Cardiac Performance
To investigate the cardiac response to these acute changes in lung injury with
washing (Figure 2 and Supplemental Figure 1) we examined the effect of washing older or
fresher RBCs on the components of cardiac output, heart rate, and stroke volume. Similar
significant qualitative interactions were found for heart rate and stroke volume at 24 h after
infection (p=0.009 and p=0.02 respectively, figure 3) as were found for survival, shock and
LIS scores. Washing of older red blood cells, lowered (normalized) heart rate and improved
stroke volume index. In contrast, at the same time point, washing fresher RBCs increased
the heart rate causing marked tachycardia while decreasing stroke volume index. Using
cardiac echo, the size of the right ventricle was estimated at 24 h in comparison to the left
ventricle in a 4 chambers view. Right ventricle dilation was considered present if the right
ventricle was greater than 2/3 the size of the left ventricle read blinded to allocation groups.
Examining all available studies, animals receiving older unwashed red blood cells had
nominally more right ventricular dilation (3/4 [75%]) compared to washed cells (2/5 [40%],
and animals receiving washed fresher RBC had nominally more right ventricular dilatation
1/4 [25%] than those receiving unwashed fresh RBCs 1/5 [20%]. These data taken together
are most consistent with the notion that washing older RBCs either directly or indirectly
lessened severe lung injury lowering mPAP and right side afterload causing stroke volume
to increase and heart rate to decline or normalize. Washing fresher RBCs had the opposite
effect.
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Liver Function
To investigate the effect of washing on liver function, we examined serial mean
levels of ALT and total bilirubin. At 48 h after S. aureus challenge, we found, similar to the
survival, shock, LIS and cardiovascular data, a significantly different and opposite effect of
washing on ALT and total bilirubin levels depending on the age of transfused blood
(qualitative interaction, p=0.03 and p=0.02 for ALT and bilirubin respectively,
Supplemental Figure 3 A and B). This effect was mainly driven by a marked decrease with
washing of both markers of liver dysfunction in the animals transfused with older blood.
Serial mean plasma levels of non-transferrin-bound iron (NTBI), labile plasma iron (PLI)
and transferrin-bound iron (TBI).
We measured serial indicators of excess plasma iron after transfusion with assays
for NTBI and PLI. After 75% of the blood was transfused, at 13h after bacterial challenge,
we found a significantly different and opposite effect of washing red blood cells on plasma
iron levels depending on the age of blood storage (qualitative interaction, p=0.009 and
p=0.02 for NTBI and PLI respectively, Figure 4). Washing older RBCs just prior to
transfusion significantly lowered the plasma levels of both NTBI and PLI found after
transfusion, and washing fresher RBCs just prior to transfusion raised, if minimally, the
plasma levels after transfusion. TBI levels were significantly lower after fresh blood
transfusion compared to older blood, but there were no differences between washed and
unwashed blood of each age of blood group.
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Serial mean levels of plasma cell free hemoglobin (CFH) and haptoglobin
We also measured serial plasma CFH, the levels of which are known to be increased after
older stored blood transfusion and also to be increased with severe bacterial infections 26, 27
At 48 h after bacterial challenge and 34 h after transfusion, washing RBCs had a
significantly different and opposite effect on plasma levels of CFH depending on the age of
the stored blood (qualitative interaction, p=0.002). Washing older red blood cells decreased
CFH-plasma levels 34 h after transfusion (figure 5A) while washing fresher red blood cell
increased CFH levels at 34 h after transfusion (figure 5C). Plasma levels of haptoglobin, a
high-affinity hemoglobin scavenger, significantly decreased during transfusion in the 4
treatment groups and slowly recovered to reach near baseline values 10 to 34 h after
transfusion (figures 5B and D).
Other laboratory values
For completeness, commonly measured laboratory parameters are shown in
Supplemental Tables 1 and 2. None of these measurement show marked changes as a result
of red cell washing nor were any differences noted that might not be expected by chance.
In vitro studies – Effects of washing stored RBC units
To examine the changes occurring over a 6 week storage period of canine universal
donor blood, as well as the effects of washing, two units of canine stored RBCs were
washed each week during the storage period (12 bags of canine universal donor stored
blood DEA 1.1 ABRINT, Dixon, CA were used to complete this series of in vitro studies).
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Each unit was sampled before and after washing. First, we examined the hemolysis rate
over time and the effect of washing on the hemolysis rate (Figure 6 I-II). Over the 6 week
storage period all the units maintained an hemolysis rate below 1% as required by the FDA
(Figure 6-I). There were no significant increments of hemolysis rate over time, with the
average hemolysis rate ranging between 0.3 and 0.4% over the whole 6-week storage
period (Figure 6-II). We then examined the RBC loss induced by washing (Figure 6-III).
Canine units lose more RBCs with washing as storage age increases (y=9.1 + 2.9x, p=0.03
for slope). 7-day old blood loses 12% and 42-day old blood 26% of the RBCs with
washing. We also investigated the biochemical effects of washing over the storage period
(Figure 6-IV, panels A to J). Washing decreased the levels of potassium, pCO2, lactate,
base deficit and glucose over the whole 6 week storage period. Minimal effects on pH,
which decreases over storage time, were found after washing the units.
An additional three units of canine blood were stored for six weeks and sampled
weekly for analysis of iron and CFH levels. Over the 6 week storage period, NTBI as well
as CFH levels increased significantly over time (p<0.001 for both trends, Figure 7 A-C).
Six additional canine RBC units were sampled at week 1 (n=3), before and after washing,
and week 6 (n=3), before and after washing. Washing efficiently lowered NTBI, PLI and
CFH levels at both extremes of the storage period (Figure 7 D-F).
DISCUSSION
We conducted a blinded randomized controlled trial to determine the effect of an
FDA-licensed washing procedure on canine universal donor blood used to examine the red
cell storage lesion in a transfusion model of experimental canine S. aureus pneumonia. We
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found a significant interaction for survival rates between washing and the age of blood
transfused. Transfusing washed 42-day-old stored blood improved survival rates when
compared with unwashed stored blood of the same age, whereas transfusing washed 7-dayold stored blood had an unexpectedly different and opposite effect that worsened survival
rates (Figure 1). Similar significant findings regarding multiple organ injury and
physiological abnormalities appear to corroborate the survival results. Transfusing 42-dayold washed stored blood lessened the degree of lung, and liver injury when compared to
unwashed controls, decreased vasopressor requirements (reversed shock) and improved
cardiac performance (Figure 2 and 3). In contrast, transfusing washed fresher blood had a
different and opposite effect that made these same parameters worse when compared with
unwashed controls.
Previous studies in this model have suggested that substances released into the
plasma both during red cell storage and as a result of hemolysis after transfusion are
responsible for the morbidity and mortality associated with stored red cells. Iron released
during transfusion can enhance tissue injury by promoting bacterial growth. Cell-free
hemoglobin released during storage and transfusion may cause injury both by scavenging
nitric oxide and by providing an additional source of iron. We used an automated cell
washing procedure to study the effects of removing these substances and determine whether
washing might provide a strategy to interdict the clinical changes seen in this model and
attributed to the red cell storage defect(s). We found significant interactions between
washing, the age of stored red cells, and the plasma levels of iron and CFH. Washing older
blood prevented significant elevations of plasma iron in the form of NTBI and PLI found
during and after transfusion of unwashed older blood (Figure 4). This effect was
significantly different when fresh blood was washed. Fresh blood washed or unwashed
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resulted in NTBI and PLI plasma levels that were very low throughout the study, and below
levels believed to be potentially injurious (0.20 µM). However, washing fresh blood
significantly raised levels of cell-free hemoglobin after transfusion compared to unwashed,
and this effect was significantly different and opposite from washing older blood (Figure
5).
Older blood, whether washed or not, produces much higher levels of cell-free
hemoglobin than fresher blood, washed or not. Red cells lose membrane during the storage
interval and are progressively more susceptible to lysis. We found that approximately 12 to
26% of the RBCs are lost during the washing process. Mechanical shear stress associated
with washing has been shown previously to cause a sublethal injury to RBCs and increased
in vivo hemolysis after transfusion with subsequent release of CFH and iron28, 29. Although
exposure to saline during the wash may contribute to increased cell fragility, the washed
cells are immediately resuspended in standard preservative solution. We postulate that the
“older” more fragile cells are lost during the washing process and the cells that remain are
more susceptible to lysis than before washing, especially after 42 days of storage. If this is
true, more “fragile” RBCs will be transfused after washing fresh blood and more hemolysis
should occur during the several hours after transfusion with washed cells than with
unwashed cells. After 42 days of storage more “very fragile” cells should be lysed and the
cellular debris washed away by the washing process. This is consistent with our current
observations and previous studies. Washing has the added benefit of removing ironcontaining proteins including CFH from the storage bag. Washing fresh RBCs should have
far less effect, since there are far fewer cells present with increased susceptibility to
hemolysis. However washing fresh red blood cells with saline solution introduces some
membrane damage making red cells more prone to in vivo hemolysis (as noted above) than
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are fresh unwashed cells. The effect overall is the opposite of that observed with older
blood, an increase in cell-free hemoglobin levels with washing but much lower levels than
seen with the unwashed blood that still contains very fragile but intact old red cells.
The opposite effects on outcome that we found with washing different ages of
stored blood can be explained by the observation that older stored blood has an increased
propensity to hemolyze and release iron in the form of NTBI and PLI, which is not seen
with fresher blood. Washing older blood removes this iron, which is no longer available
after transfusion to promote bacterial growth and worsen outcome. In addition, washing
older blood removes CFH and may also be beneficial because of removal of older cells that
are prone to in vivo hemolysis and can cause nitric oxide scavenging and vascular injury,
and potentially raise iron levels and promote bacterial growth. The effect of washing
fresher blood appears to be intermediate; washed fresh cells increase morbidity and
mortality when compared to unwashed fresh blood, similar to the effects of washed older
blood, and significantly less than unwashed old blood. The increased in vivo cell-free
hemoglobin with washing fresh blood may worsen outcome, either through scavenging
nitric oxide, causing vasoconstriction and vascular injury, or by providing an additional a
source of iron for promoting bacterial growth.
To examine the changes in RBC during storage, as well as the effects of washing,
we performed serial in vitro studies weekly on canine RBC units over a 6 week storage
interval. Canine stored blood shows no significant increases in hemolysis rate over the 6
weeks of storage and levels are always lower than 1%, and washing does not significantly
change the hemolysis rate in the storage bag (Figure 6-I-II). We found about 12% of 1week RBC and 26% of 6-weeks red blood cells are lost during washing (Figure 6-III).
Significantly more blood is lost with washing as stored blood ages, consistent with the
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notion that the older RBC are removed preferentially. Washing lowers the base deficit,
lactate, glucose, bicarbonate, and PCO2 levels in the storage bag over the 6 weeks, but has
little effect on pH of the storage solution, which decreases steadily over the 6-week storage
time (supplementary Figure 6-IV). Over a 6 week storage period, canine stored blood
accumulates CFH and NTBI significantly and progressively in the supernatant of the bags,
reaching a mean of 190 µM of CFH and of 8 µM of NTBI at the end of the 42-day period
(p < 0.0001, Figure 7 A-C). Washing efficiently lowers iron and CFH in the stored bags at
both extremes of the storage period (1 and 6 week, Figure 7 D-F).
Our study demonstrates that washing removes two sources of iron: 1) iron in the
form of NTBI as well as CFH, that accumulate over time in the storage bag,30-32 and 2)
older cells that are more likely to hemolyze in vivo after transfusion and release iron.
Several studies have previously shown that human stored blood also accumulates CFH and
NTBI over time, producing levels of CFH from 80 to 90 μM (26) and NTBI from 5 to 15
μM.31, 32 Our study of canine stored blood found that CFH and NTBI increased over the
storage period, reaching mean levels of CFH of 190 μM and of NBTI of 8 μM at 6 weeks.
The possibility that iron might increase the risk of transfusion by promoting bacterial
growth, inducing inflammation, or both as has been proposed previously.33, 34 Although the
role of NTBI in promoting bacterial growth is well documented, its role in inflammation
and the effect of washing are less clear. Non-infectious murine models are contradictory;
some studies report that washed older blood induces a significantly lower inflammatory
cytokine response whereas others find that washing had no effect on this response.33, 35
Iron has also been shown to inhibit cellular immune function, mainly through monocyte
effector pathways 36, 37 and to impair neutrophil function 38, affecting the host´s response to
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bacteria and possibly worsening the infection and outcome in our study. In addition, iron
participates in the formation of toxic reactive oxygen species which contribute directly to
the pathogenesis of endothelial dysfunction and tissue injury and worsen the effect of
infection in our model.37, 39
Findings in this canine model should be extended to human transfusions with
caution. The hemolysis rate we found in the storage bags, although low (<1%), could
indicate that canine blood may not store as well as human blood. We have implicated two
possible mechanisms of toxicity. We cannot exclude the possibility that other changes that
occur in red cells during are equally important in producing the clinical outcomes. We used
a standardized model of lethal bacterial pneumonia. Results may vary in a different
infectious model, a non-infectious model or in a model of pneumonia that uses a different
organism. Lastly, although we tried to mimic as closely as possible standard human
intensive care and blood banking conditions, differences in clinical practice remain.
In summary, older blood releases iron during storage and after transfusion, has an
increased propensity for hemolysis with release of cell-free hemoglobin and iron, and
increases the risk of transfusion in subjects with established infection. In critically ill
subjects with infection, our study found a favorable risk-to-benefit ratio for washing older
blood. However, our data show that fresh blood is still superior to older blood, whether
washed or not, in critically ill subjects with infection; fresh blood results in less in vivo
hemolysis and less accumulation of CFH, NTBI and PLI after transfusion. We found
unexpectedly that fresh blood should be washed with caution in a setting of established
infection; RBC treated with extensive saline washing may undergo increased hemolysis and
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iron release and result increased morbidity and mortality when compared with unwashed
fresh blood controls.
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Acknowledgments
Intramural NIH funds and NIH external grants HL058091 and HL098032 were used to
support this study. The work by the authors was done as part of U.S. government-funded
research; however, the opinions expressed are not necessarily those of the National
Institutes of Health. Haemonetics® (Braintree, MA) provided the ACP215 Automated cell
processing system) as well as the disposable kits (RBC De-Glycerol Set 325 mL BMB
Ref.236) used throughout the experiments.
Authorship Contributions
I.C.-P. performed experiments and wrote the first draft of the manuscript; D.W. and S.B.S.
performed experiments and helped to write first draft of the manuscript; J.S.: study design
and statistical analysis; M.F., J.F. and K.E.R. performed experiments and laboratory
analysis; T.K., L.B., D.S. and A,P.: laboratory analysis; M.A.S. performed
echocardiograms; W.E.K. performed laboratory tests and set up assays; M.A.P. helped to
train in blood washing techniques; M.T.G.: laboratory support,; D.B.K.-S.: laboratory
support, edited the manuscript; H.G.K.: set up blood-banking procedures and editing of
manuscript; C.N.: study conception and design, wrote the first draft, and edited the
manuscript.
Conflicts of Interest
D.K.-S. and M.T.G. are listed as coauthors on a patent application on methods of
treating hemolysis and on patent applications related to development of blood substitutes
using recombinant human neuroglobin.
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Figure Legends
Figure 1. Survival curves. Kaplan-Meier plots over the 96 hours study comparing the four
types of transfused blood are represented. The different types of blood are represented by
different line patterns in the following way: dashed black line (42 day old unwashed
blood), solid black line (42 day old washed blood), solid grey line (7 day old washed
blood), and dashed grey line (7 day old unwashed blood). There is a qualitative interaction
between washing and the age of stored blood (p = 0.03).
Figure 2. Mean (± SE) shock scores and lung injury score (LIS) at serial time points
(panels A-D). The shock score accounts for the level of vasopressor support
(norepinephrine) needed to maintain the mean arterial pressure at a preset normal level for
canines (mean 80 mmHg).1, 40, 41 In panels A and C the shock score is plotted over time (xaxis) for animals receiving 42-day old (panel A) or 7-day old blood (panel C). The LIS in a
previously published scoring system increases our ability to find the abnormalities in the
lungs which includes mean pulmonary artery pressure, alveolar-arterial oxygen gradient,
plateau pressure, O2 saturation and respiratory rate.1, 40, 41 In panels B and D the LIS is
plotted in the same fashion as the shock score for old and fresh blood. In each of these
panels, unwashed blood is represented by a solid line and washed blood by a dashed line.
The p-value in each panel reports differences between washed and unwashed blood (**)
and the presence of qualitative interactions between age of blood and washing (*).
Figure 3. Mean (± SE) heart rate and stroke volume index (SVI) at serial time points:
This figure uses the same format as Figure 2, except now heart rate (panels A and C) or
SVI (panels B and D) are plotted on the y-axis. Similarly, p-values are denoted by
asterisks in each panel and explained below the figures.
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Figure 4. Mean (± SE) levels of non-transferrin-bound iron (NTBI), plasma labile iron
(PLI) and transferrin-bound iron (TBI) at serial time points. The format is similar to Fig
2, except now plasma levels of NTBI (panels A and D), PLI (panels B and E) and TBI
(panels C and F) are plotted on the y-axis. P-values are denoted by asterisks and explained
below the figures.
Figure 5. Mean (± SE) levels of plasma cell-free hemoglobin (CFH) and plasma
haptoglobin at serial time points. The format is similar to Fig 2, except now plasma levels
of CFH (panels A and C) and haptoglobin (panels B and D) are plotted on the y-axis. Pvalues are denoted by asterisks and explained below the figures.
Figure 6. Hemolysis rate, red blood cells lose and biochemical changes with washing of
canine blood over 6 week storage period. Serial changes in stored blood components over
6 weeks. Serial values of (I) percent hemolysis rate (calculated by dividing the supernatant
hemoglobin levels by the total sample hemoglobin levels and multiplying by 100 minus the
hematocrit value); (II) the change in hemolysis rate with washing; (III) red blood cells loss
with washing and (IV A-J) biochemical changes with washing of canine blood of 1 through
6 weeks of storage period are shown. Two bags of canine universal donor leukorreduced
blood were sampled each week before and after washing. In panels I-III, each dark circle
represents an individual bag of ~250 g of packed red blood cells. In panel IV (A-J)
individual mean values (two bags) of the different biochemical parameters before washing
are represented as dark circles and after washing as white circles.
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Figure 7. Mean (± SE) levels of non-transferrin bound iron(NTBI), plasma labile iron
(PLI) and cell-free hemoglobin (CFH) and effect of washing in canine stored blood over
6 week storage period. Mean levels of NTBI (panel IA), PLI (panel IB) and CFH (panel
IC) sampled from three bags of canine universal donor leukorreduced blood are plotted
over time of storage. In panels II D-F mean levels of NTBI, PLI and CFH respectively are
represented before and after washing three additional bags of 1 week and 6 week stored
blood.
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Prepublished online December 23, 2013;
doi:10.1182/blood-2013-11-539353
Washing older blood units before transfusion reduces plasma iron and
improves outcomes in experimental canine pneumonia
Irene Cortés-Puch, Dong Wang, Junfeng Sun, Steven B. Solomon, Kenneth E. Remy, Melinda
Fernandez, Jing Feng, Tamir Kanias, Landon Bellavia, Derek Sinchar, Andreas Perlegas, Michael A.
Solomon, Walter E. Kelley, Mark A. Popovsky, Mark T. Gladwin, Daniel B. Kim-Shapiro, Harvey G. Klein
and Charles Natanson
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