The effects of non-leukoreduced red blood cell transfusions

on microcirculation in mixed surgical patients

Banu Ayhan
a,c,,1
, Koray Yuruk
b,1
, Sophia Koene
b,1
, Altan Sahin
c,1
, Can Ince
a,b,1
, Ulku Aypar
c,1
a
Department of Intensive Care, Erasmus Medical Center, Rotterdam, The Netherlands
b
Department of Translational Physiology, Academic Medical Center, University of Amsterdam, Amsterdam, The Netherlands
c
Department of Anesthesiology and Reanimation, Hacettepe University, Faculty of Medicine, Ankara, Turkey
a r t i c l e i n f o
Article history:
Received 22 July 2012
Received in revised form 9 September 2012
Accepted 10 January 2013
Keywords:
Blood transfusion
RBC storage
Microcirculation
OPS imaging
Surgery
a b s t r a c t
Background: The impact of the storage process on oxygen-carrying properties of red blood
cells and the efcacy of red blood cell (RBC) transfusions concerning tissue oxygenation
remain an issue of debate in transfusion medicine. Storage time and leukocyte content
probably interact since longer storage duration is thought to cause greater accumulation
of leukocyte-derived cytokines and red blood cell injury.
Objectives: The aim of this study was to investigate the effects of storage and the efcacy of
fresh (stored for less than 1 week) versus aged (stored for more than 3 weeks) non-
leukoreduced RBC transfusions on sublingual microvascular density and ow in mixed
surgical patients.
Methods: Eighteen surgical patients were included in this study. Patients were randomly
assignedintotwogroups receivingfresh(GroupA) andaged(GroupB) RBCtransfusions. Sub-
lingual microcirculatory functional capillary density (FCD) and microvascular ow index
(MFI) were assessed using orthogonal polarization spectral (OPS) imaging. Measurements
and collection of blood samples were performed after induction of general anesthesia, before
RBC transfusion and 30 min after the RBC transfusion ended.
Results: In both groups RBC transfusions caused an increase in hemoglobin concentration
(p < 0.001). RBCtransfusions increased FCDinGroupA(p < 0.001), while FCDremained unaf-
fected in Group B. Changes in MFI following RBC transfusion in both groups remained unal-
tered.
Conclusions: Fresh non-leukoreduced RBC transfusions but not RBCs stored for more than
3 weeks, were effective in improving microciruculatory perfusion by elevating the number
of perfused microvessels in mixed surgical patients.
2013 Elsevier Ltd. All rights reserved.
1. Introduction
The objective of perioperative red blood cell transfu-
sions (RBC) is to improve the oxygen-carrying capacity of
blood by increasing the number of red blood cells (RBCs)
and to deliver oxygen to tissues. The benecial effects of
RBC transfusion is being questioned as well. RBC transit
time in the capillaries, local distribution of blood ow, cap-
illary heterogeneity, and position of the oxygen dissocia-
tion curve, are all important for the transport of oxygen
to the cell within the tissues [1,2]. In addition, the results
of an increasing number of studies demonstrated that
while RBC transfusions elevate the oxygen content of
blood, transfusions of stored blood products did not im-
prove impaired tissue oxygenation [37]. Nevertheless, de-
spite the possibility of the use of different storage methods
1473-0502/$ - see front matter 2013 Elsevier Ltd. All rights reserved.
http://dx.doi.org/10.1016/j.transci.2013.01.016

Corresponding author. Address: Department of Anesthesiology and

Reanimation, Hacettepe University, Faculty of Medicine, 06100 Shhye,
Ankara, Turkey. Tel.: +90 312 3051207, mobile: +90 532 2969290;
fax: +90 312 310 96 00.
E-mail address: banu.ayhan@gmail.com (B. Ayhan).
1
These authors are contributed equally to this work.
Transfusion and Apheresis Science 49 (2013) 212222
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and liquids alternatives, storage-dependent alterations of
RBCs cause a decrease on the oxygen-carrying properties
of RBCs. During storage, RBCs undergo a number of
changes that generally consist of two major alterations in
biomechanical (cellular membrane, morphology and
phospholipid content) and biochemical (2,3-diphosphogly-
cerate (2,3-DPG) and ATP concentrations) [812]. Although
some in vitro studies showed that these storage-dependent
changes in RBCs occur between 7 and 21 days [13,14],
other clinical studies did not nd any relationship between
storage time and patient outcome [1517].
Several studies argue that storage time and/or contam-
inating intracellular materials from leukocytes in the
packed RBC products may increase transfusion-related
complications resulting in patient morbidity and mortality
[18,19]. Storage time and white blood cell content can be
detrimental to packed RBC products; lengthy storage times
may cause accumulation of leukocyte-derived cytokines
producing injury to packed RBCs. Anniss and Sparrow
[20] observed that storage time and leukocyte burden of
RBC increase the number and strength of adhesion of RBCs
to vascular endothelium.
The microcirculatory network is an essential hemody-
namic compartment and plays a crucial role in the interac-
tion between blood and tissues. Thus, monitoring of the
microcirculation might be of great importance during
transfusion to determine the effects of RBC transfusions
on the microcirculation. Orthogonal polarization spectral
(OPS) imaging, a non-invasive imaging technique that is
utilized to monitor tissue microcirculation, is used for the
monitoring assessment and quantication of sublingual
microvascular capillary density and blood ow at the
bed-side [21]. There are only a few clinical studies, all in
septic patients, that have investigated the effects of blood
transfusions on the microcirculation [22,23].
Based on these observations we hypothesized that dif-
ferent storage times of non-leukoreduced RBC products
may inuence peripheral tissue perfusion dynamics and
it can be detectable especially on the microcirculatory le-
vel. The aim of this study was to examine the efcacy of
fresh (stored for less than 1 week) versus aged (stored for
more than 3 weeks) non-leukoreduced RBC transfusions
on sublingual microvascular density and ow
intraoperatively.
2. Material and methods
2.1. Study design and patient selection
The institutional ethics committee approved the proto-
col and informed consent was from each patient. This
study was design prospective, randomized and double-
blind. Participants eligible for inclusion in this study were
patients indicated to undergo major surgical procedures
requiring intraoperative blood transfusion, the absence of
iatrogenic bleeding, and transfusion of non-leukoreduced
RBC suspensions from different storage time; less than
7 days and more than 21 days. Patients with hemodynamic
instability prior to the operation, use of medications that
cause metabolic and acidbase balance defects, steroids,
antioxidant agents, drug allergies, and candidates with
alcohol or -drug addictions were excluded. Preoperative
laboratory ndings of participants revealed no abnormal
biochemical or hematological conditions and stable hemo-
dynamic parameters. All measurements including systemic
hemodynamic parameters, blood gas samples, and OPS
imaging were performed at the same designated time
points.
2.2. Randomization
Patients were randomly assigned to two groups; either
receiving fresh blood stored for less than 7 days (Group A)
or aged blood stored for more than 21 days (Group B).
Randomization assignment of patients to Group A and
Group B was prepared with a list of random numbers gen-
erated by computer software. The investigator who was
analyzed OPS images and anesthesiologist who adminis-
tered the blood transfusion to patients were blinded to
the age of the blood being administered.
2.3. Preparation of packed red blood cells and transfusion
practice
Packed red blood cell units were supplied from a regio-
nal blood center. Whole blood is collected into citrate
phosphatedextrose anticoagulant and separated by
centrifugation into red cell, platelet, and plasma compo-
nents. Non-leukoreduced red cells are ultimately trans-
ferred into a pack containing an additive solution and
stored at 4 2 C. The currently used additive solution is
a trehaloseadenineglucosemannitol (TAGM) solution.
All donations were non-leukoreduced at the time of initial
component preparation, plasma depleted, and suspended
in TAGM (trehaloseadenineglucosemannitol). Both
fresh blood and aged blood (maximum allowed storage
time of 35 days) are used in daily practice in Hacettepe
University, Medical Faculty Hospitals.
Observations were performed during standard transfu-
sion practice. Measurements and collection of blood sam-
ples were performed after induction of general
anaesthesia, before transfusion and 30 min after the blood
transfusion ended. The administration of blood transfusion
required approximately 60 min of infusion time. The red
cell units were warmed to 37 C during infusion using a
blood warmer. Blood samples were drawn at regular inter-
vals from an arterial catheter. The frequency, volume, and
age of the transfused blood was recorded for each patient.
The stored blood products used in this study met both
national and international criteria and guidelines for
routine clinical use [24].
2.4. Anesthesia management
The day before surgery, patients were preanesthetically
evaluated. All patients were premedicated with 5 mg diaz-
epam orally, 1 h before the surgery. In the operating room;
electrocardiogram (ECG), pulse oximetry and end-tidal CO
2
and invasive arterial pressure (20-gauge cannula, right
radial artery, internal volume 0.2 ml, BD oSvitch, Faraday
Road, Swindon, UK) were monitored. Anesthesia was
B. Ayhan et al. / Transfusion and Apheresis Science 49 (2013) 212222 213
intravenously induced with 12 mg kg
1
propofol (INN,
marketed as Diprivan by AstraZeneca), 1 mcg kg
1
fentanyl
citrate (Janssen-Cilag) and orotracheal intubation was per-
formed after iv administration of vecuronium bromide
(Organon, Turkey, marketed as Norcuron) 0.1 mg kg
1
.
Anesthesia was maintained with sevourane (Abbott Lab-
oratories Limited, Berkshire, SL6 4XE, UK) 2%, O
2
, and
N
2
O at a total gas ow of 6 L min
1
using a semi-closed cir-
cle system with a soda lime canister. Vecuronium bromide
was used for muscle relaxation during surgery. Ventilation
was controlled to maintain carbon dioxide tension be-
tween 35 and 40 mmHg. Two peripheral venous (18 G
and 16 G) and a bladder catheters were inserted. Percuta-
neous arterial blood oxygen saturation, end tidal CO
2
,
and body temperatures were monitored during the opera-
tion. During surgery the patients received crystalloids, col-
loids, fresh frozen plasma, and blood to provide uid
maintenance from blood loss. Patients were warmed by a
hot air blanket. The ambient temperature in the operating
theater room was set at 22 C. End of surgery, residual neu-
romuscular blockade was antagonized using neostigmine
methylsulfate (ADEKA, Samsun, Turkey) 1.5 mg and atro-
pine sulfate (American Regent, Inc., Shirly, NY 11967)
0.5 mg and all patients (with the exception of cardiovascu-
lar surgery patients) were extubated and transferred to the
post-anesthesia care unit.
In cardiovascular surgery patients, anesthesia induction
was performed with using 0.4 mg kg
1
etomidate (USAN,
INN, BAN) (marketed as Amidate), 0.1 mg kg
1
vecuro-
nium bromide, 1 mcg kg
1
fentanyl citrate. After induction,
a 9.5 F three-lumen central venous catheter (Multicath,
Vygon, Ecouen, France) was introduced via into the right
internal jugular vein, and a 20-gauge cannula (internal vol-
ume 0.2 ml, BD oSvitch, Faraday Road, Swindon, UK) via
the right radial artery, and two peripheral venous lines
(18 G and 16 G) were inserted. Anesthesia was maintained
with sevourane 2% and O
2
. Remifentanil (GlaxoSmithK-
line and Abbott as Ultiva) infusion was administered to
all patients before and after CPB in a dose of 0.025
0.05 mg kg
1
min
1
. During CPB, remifentanil was infused
0.025 mg kg
1
min
1
and sevourane was given with a
vaporizer, inserted into the oxygenators gas supply line.
Standart CPB procedures were performed [(Blood cardio-
plegia, mild hypothermia (28 C), membran oxygenator
(COBE Cardiovascular, Inc., Colorado, USA) (Sarns 9000 Per-
fusion System, 3M Health Care Group, Michigan, USA),
non-pulsatil ow 2.4 min
1
m
2
, perfusion pressure 50
60 mmHg)]. Anticoagulation was established with intrave-
nous heparin (3 mg kg
1
) given 15 min before initiation of
CPB. Target activated clotting time was 440 s. Hematocrit
concentrations were maintained above 22%. At the end of
the CPB, anticoagulation was antaganized with protamine
sulfate to achieve a normal activated clotting time. At the
end of the surgical procedure, CBP was weaned off slowly
until adequate core body temperatures returned to normal.
After decannulation, the chest was closed. Patients were
transferred to the cardiovascular surgery intensive care
unit and they were kept sedated with a continuous infu-
sion of 0.3 mcg kg
1
min
1
remifentanil. At the achieve-
ment of hemodynamically stabilization, bleeding control
and normothermia, the patients were weaned from the
mechanical ventilation and extubated.
2.5. OPS imaging
Sublingual microcirculatory functional capillary density
and perfusion were monitored using OPS (MicroVision
Medical, Amsterdam, The Netherlands) imaging. OPS de-
vice is embodied in a hand-held instrument. OPS imaging
illuminates tissues with polarized green light and mea-
sures the reected light from the tissue surface after lter-
ing out the polarized portion of the reected light. Thus,
surface reections are ltered out and images can be ob-
served of the microcirculation below the surface structures
without transillumination. In OPS imagine method, the
hemoglobin is used as a contrast agent, so as to red blood
cells are imaged as dark moving globules against a white/
grayish background. The vessel walls are not visualized di-
rectly, although pale contours can be identied depending
on the presence of erythrocytes in the vascular lumen [21].
After the removal of saliva and other secretions using
gauze, an OPS probe was covered by a sterile disposable
cap, the OPS device was gently applied without any
pressure on the sublingual mucosal at approximately
1.54 cm from the tip of the tongue. Five video image se-
quences of 20 s each from different adjacent areas were
recorded using a computer (SONY Video Walkman GV-D
1000E, Sony, Tokyo, Japan) and a video card (MicroVideo;
Pinnacle Systems, Mountain Views, CA) and stored under
a random number for ofine analysis. Every effort was
made to avoid movement and pressure artifacts to ensure
accurate microcirculation image quality. Images stored
on DVI tape were captured in 510 s video clips in DV-
AVI le format. OPS images were analyzed for functional
capillary density which is dened as the length of red
blood cell-perfused capillaries per observation area (FCD;
mm capillary/mm
2
tissue) and microvascular ow index
(MFI; AU), providing an index for microcirculatory blood
ow velocity (based on determining the predominant type
of ow was scored as being (absent = 0, intermittent = 1,
sluggish = 2, and continuous = 3) and these nal score rep-
resents the averaged values of the four, was analyzed
semiquantitatively in small- (diameter <25 mm) and med-
ium-sized vessels (25 mm < diameter < 100 mm) as
described previously [25,26]. OPS images were analyzed
by using a computer software package (Automated Vascu-
lar Analysis Software (AVA) (Microvision Medical BV,
Amsterdam, The Netherlands).
Baseline measurements were performed after induction
of general anesthesia and repeated measurements were
performed before and 30 min after the completion of RBC
transfusion.
2.6. Statistical analysis
A study by Jhanji et al. [27] had shown that the decrease
in perfused capillary density from a mean of 5.7 to a nadir
of 3.8 mm
1
in patients (n = 25) after major abdominal sur-
gery was associated with the development of postopera-
tive complications. Hence, we calculated an anticipated
214 B. Ayhan et al. / Transfusion and Apheresis Science 49 (2013) 212222
effects size (Cohens d = 1.27) from their results and the
minimum sample size of 9 patients per group was required
in our study to detect a minimum clinically signicant dif-
ference of 2 units in perfused capillary density (FCD; mm
capillary/mm
2
tissue) between the within group changes
of the two groups with 80% power and 5% type I error level.
All data analysis was performed using GraphPad Prism
version 5.0 for Windows (GraphPad Software Inc., La Jolla,
California, USA). Normal distribution of the data sets was
conrmed using KolmogorovSmirnov/ShapiroWilks
normality test. All data sets were presented as mean SD.
Comparative analysis between FCD data sets was per-
formed using a one-way analysis of variance (ANOVA) for
repeated measurements with a Bonferroni post hoc test
and MFI was evaluated using non-parametric statistic test.
Differences between data sets with a p-value of <0.05 were
considered statistically signicant.
3. Results
3.1. Patients characteristics
Patient demographic characteristics are summarized in
Table 1. Eighteen adult (7 male, 11 female), ASA physical
status IIII patients scheduled for elective major surgery
ovarian cancer (n: 8), coronary artery bypass grafting
(CABG) (n: 3), hip fracture (n: 2), osteosarcoma (n: 1), cer-
vix cancer (n: 1), chondrosarcoma (n: 1), abdominal aneu-
rysm (n: 1), and mitral valve replacement (n: 1) were
admitted to this study.
No signicant differences between Group A (<7 days)
(n: 9) and Group B (>21 days) (n: 9) were identied with
respect to the demographic characteristics and operative
variables (p > 0.05). The volume of RBC transfusions in
Group A was 752 515 ml and storage on average was
3 2 days. The volume of RBC transfusions in Group B
was 712 188 ml and storage on average was 24 2 days.
No patients received autologous and leukoreduced banked
blood. No transfusion-related adverse reactions were ob-
served during study.
3.2. Hemodynamic and microcirculatory parameters during
surgery
In Group A, a signicant decrease in systemic hemoglo-
bin content at from 10.8 1 to 8.1 1.1 g dL
1
(p < 0.001)
and in Group B from 10.9 1.3 to 8.7 1.8 g dL
1
(p < 0.01) was observed when compared with values of
those after anesthesia induction and the values of those
before transfusion (Fig. 1). Systemic Hct levels decreased
in Group A from 32.5 3.6% to 26.3 2.3% and in Group
B from 33.6 4.6% to 27.6 3.3% in all patients at the be-
fore transfusion period when compared to after induction
of anesthesia period (p > 0.05) (Table 2). Mean arterial
pressure decreased in Group A from 94.5 17 mmHg to
83.6 11.2 mmHg and in Group B from 82.7 19.3 mmHg
to 74.3 14.7 mmHg (p > 0.05). Changes in heart rate were
in Group A from 79.1 16.3 to 77.5 14.8 bpm and in
Group B from 78.4 13.8 to 79.2 17.5 bpm (p > 0.05) (Ta-
ble 2). There were no differences in Hb, Hct, MAP and heart
rate values between the two groups of major surgery pa-
tients (p > 0.05) (Table 2).
At the microcirculatory level, FCD decreased from
14.9 0.5 to 10.7 1.3 mm capillary/mm
2
tissue in Group
A (p < 0.001) and from 15 1.4 to 10.9 1.9 mm capil-
lary/mm
2
tissue in Group B (p < 0.001) during surgery at
the before transfusion period (Fig. 2). Alterations in MFI
in Group A were in both small sized vessels from
2.65 0.2 AU to 2.39 0.38 AU (p < 0.01) and medium
sized vessels from 2.94 0.08 AU to 2.85 0.15 AU
(p > 0.05). Alterations in MFI in Group B were in both small
vessels from 2.88 0.1 AU to 2.72 0.23 AU (p < 0.05) and
medium sized vessels from 2.96 0.07 AU to
2.90 0.12 AU (p > 0.05) (Figs. 3a and 3b).
3.3. Effect of RBC transfusion on hemodynamic and
microcirculatory parameters
At the macrocirculatory level, in both Group A and B,
RBC transfusion caused a signicant increase in systemic
hemoglobin concentrations. Systemic Hb content in-
Table 1
Patient characteristics and transfusion data.
Surgical patients Group A (n: 9) Group B (n: 9) Signicance
Demographics
Age (years) 54 19 52 15 ns
Gender (Male:Female) 3:6 4:5
Body surface area (m
2
) 1.83 0.5 1.80 0.2
Diagnosis
Ovary cancer 3 5
Coranary artery bypass grafting 2 1
Hip fracture 1 1
Osteosarcoma 2
Cervix carcinoma 1
Abdominal aneurysm 1
Mitral valve replacement 1
Transfusion data
RBC volume/patient (ml) 752 515 712 188 ns
RBC unit storage time (days) 3 2 24 2
All data, except the gender and diagnosis, are presented as mean SD. ns: Non-signicance.
B. Ayhan et al. / Transfusion and Apheresis Science 49 (2013) 212222 215
creased in Group A from 8.1 1.1 to 9.8 1.2 g dL
1
(p < 0.001) and in Group B from 8.7 1.8 to
10.9 1.4 g dL
1
(p < 0.001). Hb values were different be-
tween the two groups after the transfusion (p < 0.05)
(Fig. 1). Hematocrit increased in Group A from
26.3 2.3% to 31.4 2.6% and in Group B from 27.6 3.3%
to 33.3 2.9% (p > 0.05) (Table 2). After RBC transfusion,
MAP increased from 83.6 11.2 to 84.8 13.1 mmHg in
Group A (p > 0.05) and in Group B from 74.3 14.7 to
81.7 15.4 mmHg (p > 0.05). Heart rate increased from
77.5 14.8 to 82 12 bpm in Group A (p > 0.05) and de-
creased from 79.2 17.5 to 77.5 16.4 bpm in Group B
(p > 0.05). There were no differences between the two
groups with regards to Hct, heart rate and MAP values
(p > 0.05) (Table 2).
At the microcirculatory level, RBC transfusion resulted
in increased FCD from 10.7 1.3 to 14.3 2 mm capillary/
mm
2
tissue (p < 0.001) in Group A, while FCD was changed
from 10.7 1.3 to 10.9 1.04 mm capillary/mm
2
tissue in
Group B (p > 0.05). FCD values were different between
the two groups after the transfusion (p < 0.001) (Fig. 2).
Alterations in MFI following RBC transfusion in Group A
were in both small blood vessels from 2.39 0.38 AU to
2.42 0.25 AU (p > 0.05) and medium sized blood vessels
from 2.85 0.15 AU to 2.82 0.15 AU (p > 0.05). Altera-
tions in MFI in Group B were in both small sized blood ves-
sels from 2.72 0.23 AU to 2.52 0.20 AU (p > 0.05) and
medium sized blood vessels from 2.90 0.12 AU to
2.84 0.20 AU (p > 0.05). There was no difference between
the two groups in MFI values after the transfusion
(p > 0.05) (Figs. 3a and 3b).
4. Discussion
The principal nding of our study was that non-leuko-
reduced blood transfusions alter microcirculatory hemody-
namics as observed in the sublingual area in patients
during major surgery. Even though systemic Hb concentra-
tions increased after non-leukoreduced RBC transfusions in
both groups of patients, sublingual FCD was improved
30 min after the completion of fresh non-leukoreduced
blood transfusions. Aged non-leukoreduced RBC transfu-
sions did not correct sublingual FCD. RBCs stored for less
than 7 days was superior to aged packed RBCs in restoring
sublingual microcirculation. In short, aged RBC transfu-
sions (stored for >21 days) did not improve microcirculato-
ry capillary density as anticipated.
When the RBCs volume of the blood is reduced, the oxy-
gen-carrying capacity of blood decreases and the oxygen
transport to the tissues might get impaired. It has been
showed that systemic oxygen delivery and oxygenation
of the skeletal muscle and internal organs can be protected
until a systemic hematocrit of around 20% or acute blood
loss of up to 30% of circulating volume can often be treated
[24]. Therefore, most intensivist and anesthesiologists
manipulated the oxygen transport by using uid, isotropic
and vasoactive treatments rather than blood transfusion in
both critically ill and perioperative patients. Reliance on
hemoglobin alone is not valid in this setting. The adequacy
of oxygen delivery must be assessed in individual patients,
particularly in patients with limited cardiac reserve. Antic-
ipated degree and rate of blood loss, the effect of body tem-
perature or drugs/anesthetics on oxygen consumption,
All data are presented as meanSD. TX: Transfusion
*p<0.001: When compared to baseline and before TX values in Group A (within group analysis)
**p<0.001:When compared to before and after TX values in Group A (within group analysis)
#p<0.01: When compared to baseline and before TX values in Group B (within group analysis)
##p<0.001: When compared to before TX and after TX values in Group B (within group analysis)
p<0.05: After TX, between the groups
Fig. 1. Changes in hemoglobin values between time points.
216 B. Ayhan et al. / Transfusion and Apheresis Science 49 (2013) 212222
mixed venous O
2
levels and O
2
extraction ratios may be
helpful in assessing tissue oxygenation during periopera-
tive period. American Society of Anesthesiologists Task
Force have been proposed: transfusion is rarely indicated
when the hemoglobin level is above 10 g dL
1
and is
almost always indicated in patients when the hemoglobin
level is less than 6 g dL
1
. Patients may be transfused at a
hemoglobin >8 g dL
1
, when there are signs of inadequate
oxygen delivery including; tachycardia, hypotension,
myocardial ischemia and hypoxemia [24,28,29].
There were no differences in MAP and heart rate values
but, FCD decreased in both groups of major surgery pa-
tients before the blood transfusion in our study. Because,
microvascular oxygen delivery cannot be predicted from
global haemodynamic measurements and adequate tissue
perfusion i.e. microcirculation rather than maintenance of
macrocirculation parameters (arterial pressure, HR, central
venous pressure, CO, PCWP, etc.) [26,30,31]. Therefore,
functional capillaries have been accorded increasing
importance. In the view of these evidences, we evaluated
the effects of non-leukoreduced red blood cell transfusions
on the microcirculation in mixed surgical patients using
OPS with microcirculation parameters.
However, most investigators showed that anaemia is
associated with adverse outcome at the admission of trau-
ma, most of studies have been reported an association be-
tween increased risk of mortality, morbidity and
perioperative transfusions, with a large proportion of
deaths occurring within 30 days in cardiac surgery patients
[32]. Moreover, in another a large clinical study it was
showed that a more restrictive RBC transfusions strategy
in critically ill patients resulted lower rates of hospital
morbidity and mortality. In this study (the TRICC study),
the authors used restrictive (transfusion trigger of 7 g dL
1
and target Hb of 79 g dL
1
) and liberal (transfusion trigger
of 10 g dL
1
and target Hb of 1012 g dL
1
) transfusion
strategy [33]. We selected patients undergoing major sur-
gery because these procedures are typically associated
with both an increased mortality rate and increased likeli-
hood of multiple RBCs transfusions. In the present study,
our pre-transfusion mean Hb concentrations 8.3 g dL
1
which is similar when compared to the indicated universal
blood transfusion threshold because in the CRIT study [34],
the mean pre-transfusion Hb concentration was found to
be 8.6 g dL
1
and it is similar to the TRICC [33] and ABC
studies [35]. During this study, our post-transfusion Hb
concentration ranged from 8.1 g dL
1
to 9.8 g dL
1
for fresh
blood and from 8.7 g dL
1
to 10.9 g dL
1
for old blood.
Under standard conditions, RBC units can be stored for
up to 42 days at 16 C [36]. Blood units are used for trans-
fusion an average on day 16 of storage in EU [37] or on day
21 in United States [34]. In our university hospital where
this study was performed whole blood is collected into cit-
ratephosphatedextrose anticoagulant containers and
separated by centrifugation into red cell, platelet, and plas-
ma components. Non-leukoreduced red cells are ultimately
transferred into a pack containing an additive solution and
stored at 4 2 C. The currently used packaging additive
solution is trehaloseadenineglucosemannitol (TAGM),
which, in Turkey, is licensed for the storage of RBCs for up
to 35 days.
Trehalose is a non-reducing sugar and crucial for stabi-
lizing biomembranes. Crowe et al. [3841] have been stud-
ied the benecial effects of trehalose in preserving
biological material. Many research groups investigated
various techniques with trehalose. Satpathy et al. [42]
developed one method to introducing trehalose into hu-
man RBCs. They have demonstrated that trehalose exerted
osmotic protection on RBCs and also incubation of RBCs in
a hypertonic trehalose solution results can be removed by
washing and resuspending the RBCs in an iso-osmotic
medium. These results provide an important step in long-
term preservation of RBCs with trehalose. Tozok et al.
[43] investigated the effects of trehalose-loading and
freeze-drying RBCs on hemolysis, and RBC structure and
functionality. They have been demonstrated that hemoly-
sis was minimized by freeze-drying RBCs in lyophilization
with trehalose and freeze-dried RBCs had similar effects of
superoxide dismutase, catalase, ATP, and a similar hemo-
globin structure to fresh RBCs. But they had lower levels
of 2,3-diphosphoglycerate (2,3-DPG). Kanias and Acker
[44] showed contradictory results regarding the process
of trehalose-loading. They demonstrated that trehalose is
correlated with high osmotic pressure, which had minor
effects during incubation at 4 C, but seemed to have exac-
erbated the severity of cellular injury at 37 C, as measured
Table 2
Systemic hemodynamic parameters.
Group A (n: 9) Group B (n: 9)
After induction of
anaesthesia
Before
transfusion
After
transfusion
After induction of
anaesthesia
Before
transfusion
After
transfusion
Systolic pressure (mm/Hg) 126.8 24.4 106.3 13.6 115.8 24.9 112.6 24.2 95 20.7 106.4 14.7
Diastolic pressure (mm/Hg) 73.4 14.7 66.8 9.7 67.1 9.5 67.7 17.2 59.1 11.3 67.6 12.5
Mean arterial pressure (mm/
Hg)
94.5 17 83.6 11.2 84.8 13.1 82.7 19.3 74.3 14.7 81.7 15.4
Heart rate (bpm) 79.1 16.3 77.5 14.8 82 12 78.4 13.8 79.2 17.5 77.5 16.4
Saturation (%) 97 2 98 1 99 1 98 2 98 2 98 1
Hct (%) 32.5 3.6 26.3 2.3 31.4 2.6 33.6 4.6 27.6 3.3 33.3 2.9
All data are presented as mean SD.

p > 0.05: When compared to after induction of anesthesia and before transfusion values (within group analysis).

p > 0.05: When compared to before and after transfusion values (within group analysis).
#
p > 0.05: Between the groups.
B. Ayhan et al. / Transfusion and Apheresis Science 49 (2013) 212222 217
by higher levels of hemolysis, methemoglobin and lipid
peroxidation. In our daily practice, during preparation of
blood, RBCs were tested for osmotic fragility by our insti-
tute blood bank because of these effects of TGAM solution.
Therefore, we thought that using RBCs in TGAM additive
solution could not be inuenced our study results.
All data are presented as meanSD. TX: Transfusion
*p<0.001: When compared to baseline and before TX values in Group A (within group analysis)
**p<0.001:When compared to before and after TX values in Group A (within group analysis)
#p<0.001: When compared to baseline and before TX values in Group B (within group analysis)
##p>0.01: When compared to before and after TX values in Group B (within group analysis)
p<0.001: After TX, between the groups
Fig. 2. Changes in FCD values between time points.
All data are presented as meanSD. TX: Transfusion
*p<0.01: When compared to baseline and before TX values in Group A (within group analysis)
**p>0.05:When compared to before and after TX values in Group A (within group analysis)
#p<0.05: When compared to baseline and before TX values in Group B (within group analysis)
##p>0.05: When compared to before TX and after TX values in Group B (within group analysis)
p>0.05: After TX, between the groups
Fig. 3a. Changes in MFI values in small-sized vessels between time points.
218 B. Ayhan et al. / Transfusion and Apheresis Science 49 (2013) 212222
We compared blood stored for more than 21 days
against blood stored for less than 7 days in our study; ret-
rospective analysis has determined that a number of units
older than 21 days is an independent risk factor for multi-
ple organ failure [45].
There are conicting data on whether transfusion of old
or fresh RBCs increases tissue oxygenation because accu-
mulating evidence is showing that there can be a negative
relationship between the storage time and RBCs function.
Van Bommel et al. [46], using a rat haemorrhagic shock
model, evaluated the effect of RBC storage lesion on oxy-
gen delivery. They have found that transfusion of RBCs
stored for 28 days did not restore the microcirculatory oxy-
genation. Chin-Yee et al. [47] have also observed that old
(7 days storage) RBCs adhered to the vessel walls to signif-
icantly greater degree than the fresher cells (less than 24 h
of storage). Raat et al. [13] used human blood for transfu-
sion and they showed that old blood (56 weeks storage)
resulted in a 25% decrease in rats intestinal microvascular
oxygenation after transfusion, and it was not observed
with fresh blood (26 days). Collins and Stechenberg [48]
have studied the effect of the exchange transfusion of
90% of the original RBC mass in rats with blood stored
1 day or 1420 days to nal Hct of 36% (normal), 28%
(moderate anemia) and 17% (severe anemia). They have
found that survival being lower only for the rats transfused
with old RBCs at low Hct. In our study, although there were
no differences both pre-transfusion and post-transfusion
Hct levels in the between two groups, in the fresh blood
group, blood transfusion increased FCD and FCD remained
unaffected in the old blood group. And also, we found that
Hb values were different between the two groups after the
transfusion. In the old blood group, while post-transfusion
Hb levels were higher than the other group, FCD was not
affected. Windsant et al. [49] have demonstrated that pro-
longed storage signicantly increased free Hb concentra-
tions transiently and nitric oxide (NO) consumption.
Development of hemolysis during storage might contrib-
ute to release of free hemoglobin and a nitric oxide (NO)
scavenger. This may impair microcirculatory perfusion
after transfusion with old blood. Therefore, in our study,
FCD could not affected from blood transfusion in the old
blood group.
We know that decreased or remained FCD is related
with lowered perfusion pressure and observed with ische-
miareperfusion injury which has the complication of oxi-
dative stress [50]. On the other hand, Van der Lindan et al.
[51] demonstrated that fresh RBC transfusion and in-
creased blood ow (pump ow) are equally effective in
restoring tissue oxygenation in anaesthetized dogs under-
going cardiopulmonary bypass. These experimental stud-
ies conclude that fresh blood is a better resuscitation
uid than older [52,53]. But, unfortunately, experimental
studies in animal species have interspecies differences in
RBC metabolism and structure, therefore they do not fully
reproduce clinical conditions or replicate human
physiology.
Some of the prospective and retrospective studies that
evaluate the effect of RBC storage on patient outcomes in
cardiac surgery, trauma, and critical care [5457]. These
studies found mixed results with some showing worse out-
comes in patients who received older RBCs, whereas others
showed no difference. Kiraly et al. [58] have been exam-
ined the effect of the age of the blood transfused on the tis-
All data are presented as meanSD. TX: Transfusion.
*p>0.05: When compared to baseline and before TX values in Group A (within group analysis)
**p>0.05:When compared to before and after TX values in Group A (within group analysis)
#p>0.05: When compared to baseline and before TX values in Group B (within group analysis)
##p>0.05: When compared to before TX and after TX values in Group B (within group analysis)
p>0.05: After TX, between the groups
Fig. 3b. Changes in MFI values in medium-sized vessels between time points.
B. Ayhan et al. / Transfusion and Apheresis Science 49 (2013) 212222 219
sue oxygenation using near infrared spectroscopy in thirty-
two critically injured trauma patients. In this study, they
dened old blood as blood stored for 21 days or greater
and new blood as blood stored for less than 21 days.
They found that older RBCs decreased in peripheral tissue
oxygenation. On the other hand, Weiskopf et al. [59]
showed that erythrocytes stored for 3 weeks have similar
effects as erythrocytes stored for 3.5 h in reversing the
neurocognitive decit of acute anemia in nine healthy vol-
unteers donated. In addition most of the studies on blood
bankinginduced alterations in RBC ow properties have
shown that routine cold storage is associated with
reduction in RBC deformability [60], increased RBC ow,
elevation of their adherence to endothelial cells [61,62]
and increased aggregability [63]. Actually, if patients had
lower hematocrit, it may have improved microvascular
ow. In our study; while microvascular ow index (MFI)
remained unaffected in both groups, blood transfusion in-
creased FCD in only the fresh blood group. In addition,
there were no differences between the Hct values for both
groups. According to these results; RBC ow properties did
not effect from storage in our study.
We used RBCs that were prepared with the routine stor-
age procedures of our Faculty of University Blood Bank,
specically non-leukoreduced. Most studies have showed
that removal of white blood cells reduces storage-induced
damage to RBCs [6468]. The presence of leukocytes in
transfused blood may play an important role in quality of
transfused erythrocytes and responsible for the transmis-
sion of blood-borne infections. It is generally accepted that
universal leukocyte depletion include a reduction in the
incidence of non-hemolytic transfusion reactions, immu-
nosuppression [69], and mortality. On the other hand,
Weinberg et al. [57]. have been demonstrated that a mor-
tality association with older blood (>14 days) despite uni-
versal leukoreduction and leukoreduction on mortality is
doubtful. Nathens et al. [70] compared 1864 injured pa-
tients to receive leukoreduced versus standard non-leuko-
reduced transfusions during the hospitalization. They have
found that no difference in mortality or infectious morbid-
ity in the 268 patients. But, they did not evaluate the age of
transfused blood in their study design. Similarly, Englehart
et al. [71] showed that the use of leukoreduction blood
does not improve outcome in trauma patients. In our
study, we used non-leukoreduced blood transfusion in
our patients. To our study results, we thought that the
age of blood could be more important than the presence
of leukocytes in RBCs on microcirculation.
Our study has its limitations. First, we investigated a
relatively small number of patients so that we may have
missed minor differences attributable to measurements.
Second, we included patients who underwent different
kind of surgery that may have different microcirculation
properties. Lastly, we did not correlate microcirculation
parameters with any clinical outcome parameters like
long-term survival or morbidity and mortality rate in our
study.
In summary, we showed that the microvascular effects
of RBC transfusions are quite variable and are correlated
with the age of the transfused RBCs in major surgical pa-
tients. These effects cannot be predicted from systemic
hemodynamics or systemic blood parameters like Hb and
Hct variables. The risks and benets of RBC transfusion
should be assessed for every patient before transfusion
during major operation. These results will need to be
tested in selected populations under same kind of major
surgery that have similar microcirculation characteristics.
Acknowledgments
The authors would like to thank Academic Medical
Center in Amsterdam in Netherlands and Hacettepe Uni-
versity, Faculty of Medicine, Dept. of Anesthesiology and
Reanimation in Ankara in Turkey.
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