Vox Sanguinis (2020) 115, 606–616

© 2020 International Society of Blood Transfusion

REVIEW ARTICLE DOI: 10.1111/vox.12971

Washed red cells: theory and practice

Rebecca Cardigan,1 Helen V. New,2 Hazel Tinegate3 & Stephen Thomas4
1
Department of Haematology, NHS Blood & Transplant, University of Cambridge, Cambridge, UK
2
Department of Haematology, NHS Blood & Transplant, Imperial College London, London, UK
3
NHS Blood & Transplant, Newcastle, UK
4
NHS Blood & Transplant, London, UK

Washing of red cells is sometimes performed to reduce allergic reactions due to

Received: 29 August 2019,
revised 27 May 2020,
accepted 9 June 2020,
published online 7 July 2020
contaminating plasma proteins or to reduce the concentration of potassium accu-
mulating in the supernatant of red cells during storage as an alternative to trans-
fusion of fresher red cells in patients at risk of hyperkalaemia. There are a
variety of methods for washing red cells, and the laboratory data suggest that
variables such as age of red cell before washing, washing method and solution,
storage medium and length of storage time after washing can all effect the final
red cell quality. Studies suggest that washing removes 90–95% plasma, but the
proportion of units below 0
05 mg/dl IgA (equivalent to IgA deficient) is variable
dependent upon methods used. Although potassium levels are reduced immedi-
ately following washing, the rate of leakage following subsequent storage is
method-dependent, requiring careful consideration of shelf life if potassium
reduction is the goal. Other markers of red cell quality such as haemolysis are
negatively impacted by washing so a reduced shelf life compared to standard red
cells is appropriate, especially following irradiation. Data from animal models
and clinical studies on possible additional benefits of washing, such as reduced
lung or kidney injury, are mixed, ranging from some benefit to some harm, and
further studies are warranted.
Key words: blood component production, red cell components, transfusion reac-
tions, washing.

injury following large volume transfusion. However, there

Introduction
Currently, the main use of washed red cells is to prevent
allergic reactions in patients who have severe reactions to
standard red cells, [1] although washing is sometimes
used as a method for reducing potassium content of red
cells for susceptible patients [2]. Methods for washing red
cells vary and are known to impact on markers of red cell
quality following washing. The age of red cells prior to
washing and length of storage after washing are also
known to be key variables determining product quality.
Further, there is renewed international interest in under-
standing whether washing of red cells might have addi-
tional clinical benefits such as a reduction in organ
is a lack of review articles on this topic. This review
therefore describes clinical indications for their transfu-
sion, current methods and standards for production, and
laboratory and clinical data on their quality and safety.
The review focuses on the washing of blood prepared for
transfusion (either in a blood transfusion laboratory or
using a cell saver machine) and does not include litera-
ture on the washing of intra-operatively salvaged blood.
We searched PubMed without any restriction on the date
of publication for studies up to May 2020 for the terms
[washed], [washing], [red cell], [transfusion], [allergic reac-
tion], [IgA deficiency]. We also scanned the reference list of
papers identified for further studies considered relevant.

Current indications for washed red cells

Correspondence: Rebecca Cardigan, NHS Blood & Transplant, Long
Road, Cambridge, CB2 OPT, UK The main indication for washed red cells is for patients
E-mail: rebecca.cardigan@nhsbt.nhs.uk that have repeated severe reactions to standard red cells

606

[1]. The rationale is that plasma proteins in the
supernatant of the red cell in combination with recipi-
ent factors may be responsible for causing these reac-
tions, and that washing removes the majority of
plasma.
Guidelines recommend washed red cells for transfusion
of patients who have experienced repeated moderate or
severe reactions, regardless of immunoglobulin (Ig) A sta-
tus, and also for a small number of patients who are defi-
cient in IgA who have experienced a similar transfusion
reaction, and where red cells from IgA deficient donors
are unavailable [1,3]. If transfusion of such patients is
urgent such that washed red cells cannot be sourced in
time, standard red cells in additive solution would be the
alternative, with direct patient monitoring in a clinical
area with resuscitation facilities [1]. There is little evi-
dence to suggest that washed red cells should be trans-
fused to IgA-deficient patients in the absence of previous
reactions [4–6].
Some countries also wash red cells to reduce isoagglu-
tinins in transfusions following paediatric ABO mis-
matched cardiac transplant, based on the Toronto
protocol [7]. In addition, washing or ultrafiltration of red
cells is used by some as a means to reduce the potassium
load in red cell components for patients who may be at
risk of hyperkalemia, as an alternative/in addition to
using shorter shelf life standard red cells, [2,8] although
this remains controversial [9]. The level of reduction
afforded is determined by the method of washing, how
long cells are stored for afterwards and whether they
have been irradiated.
There are a number of clinical scenarios in which
washed red cells are no longer considered necessary.
There has been variation in clinical practice in the trans-
fusion management of neonates and children with T acti-
vation, with some clinicians using washed cellular
components in this situation and in atypical haemolytic
uraemic syndrome. However, there is no evidence to sup-
port the use of washed cells in this setting when standard
red cells are suspended in additive solution with little
residual plasma [10]. Washed red cells are no longer indi-
cated in transfusion management of paroxysmal noctur-
nal haemoglobinuria [11,12].
Washed red cells have also been transfused in the UK
to children awaiting renal transplantation as it was con-
sidered that this may afford a greater degree of leuco-
cyte removal than leucocyte depletion alone and hence
reduce HLA sensitisation. A recent study has suggested
that using washed leucodepleted red cells does not fur-
ther reduce patient HLA sensitisation rates due to the
limited effect of washing on residual leucocyte levels
[13].
© 2020 International Society of Blood Transfusion
Vox Sanguinis (2020) 115, 606–616
Washing of red cells 607
Clinical benefit in prevention of febrile or
allergic transfusion reactions
There are very few studies of the benefits of washed red
cells in preventing febrile or allergic transfusion reac-
tions, despite this practice being widely accepted. The
majority of studied on washed cells for transfusion focus
on platelets.
A prospective, non-randomised study, of washed versus
standard red cells (assumed to be non-leucocyte depleted),
found a significant reduction in rates of all types of reac-
tions, primarily febrile and allergic, (8/3799, 0
21% vs.
31/6359, 0
49%, P < 0
03) [14]. In the study, red cells
were washed with one litre of saline and one litre of sal-
ine with 0
2% dextrose in an COBE 2991 cell processor.
However, interpretation is difficult as 43/3799 ‘reactions’
were seen in the washed group, and 76 in the standard
group (1
1% vs. 1
2%) but many of these ‘reactions’ were
discounted on further analysis for reasons which are
unclear. Patients were allocated to either of these compo-
nents dependant on the availability of the component and
the orders of the requesting physician, and therefore, the
patient groups could have had different characteristics.
No adverse effects, other than the acute transfusion reac-
tions studied, were described in either group.
In a retrospective cohort study, Tobian demonstrated a
reaction rate of 64/8048 (0
8%) in 179 patients who
received standard, leucoreduced red cells and platelets.
Within this group, 39 patients who experienced reactions
to platelets had a higher reaction rate to red cells at 2
7%
(33 reactions in 1236 units transfused) [15]. Patients
within this group who went on to have further red cell
transfusions received red cells washed with saline in a
COBE 2991 processor. They experienced a subsequent
reaction rate to washed red cells of 0
3% (P < 0
001). The
authors were also able to show that patients who reacted
to red cells also had a higher reaction rate to unmanipu-
lated platelets (7
0%, 82/1168) compared to the other
individuals in the study P < 0
05. Allergic predisposition
in the recipient has been shown to be the most important
contributory factor to allergic transfusion reactions
[14,15].
In a study from Canada, the rate of acute reactions to
red cells transfused to b thalassaemia patients, most hav-
ing previously had reactions, was very low (occurring
after 0
15% of units transfused), and not different
between those washed using the COBE2991 device or the
Haemonetics ACP215 device [16]. However, the latter was
associated with fewer red cell transfusions.
The majority of reactions to red cells reported to the
UK Haemovigilance scheme SHOT are febrile in nature.
From 2010–2014 in England, there were 9614 washed red
608 R. Cardigan et al.
cell issues only 2 of which were reported to SHOT as
associated with reactions. The incidence of reactions to
washed and standard red cells during this five year period
was similar: for washed red cells at 0
21 per 1000 issues
(95% CI: 0
06 to 7
6 per 1000) and for standard red cells
at 0
13 per 1000 issues (95% CI: 0
12 to 0
13)–based on
10 500 000 standard red cell issues and 1342 reactions
(personal communication, Hazel Tinegate). No incidents
of reactions to washed red cells have been quoted in the
US National Haemovigilance Module, the Australian Hae-
movigilance report 2009-11, the Netherlands TRIP report
2012, the Canadian Haemovigilance report (TTISS) 2006-
12 or the ANSM (previously AFSSAPS) French haemovig-
ilance report 2009.
Overall, there are limited data demonstrating the effi-
cacy of washing red cells in reducing acute transfusion
reactions. The data of Tobian et al suggest a 10-fold
decrease in rates of reactions in individuals susceptible to
reactions to platelets and red cells, but haemovigilance
data suggest that rates of reactions to washed red cells
are similar to standard red cells, although the denomina-
tor is low for washed components.
Clinical benefits for other indications
Red cell transfusion management for patients undergoing
cardiac surgery for congenital heart disease has been the
subject of a Cochrane review [17]. This review included
two studies of washed cells and one of washed red cells
as part of a cardiac bypass prime. The study by Cholette
randomised children undergoing cardiac bypass to receive
either standard prestorage leucoreduced red cells and pla-
telets or washed red cells and platelets and demonstrated
a statistically significant reduction in inflammatory mark-
ers but no significant reduction in mortality or transfu-
sion requirements between groups [18]. Mean storage age
was 16
1 days prior to washing for washed and 17
6 days
for unwashed red cells. Hosking examined the use of
washed red cells to reduce accumulated blood glucose in
the prime and showed that washing reduced perioperative
hyperglycaemia [19]. Swindell et al. [8] studied the effect
on potassium levels of using pre-washed red cells for the
prime in infants and neonates (frequent practice in infant
cardiac surgery to reduce risks of hyperkalaemia) and
found a significant reduction in potassium levels. Two
patients receiving unwashed red cells and high potassium
levels at the start of cardiopulmonary bypass had ventric-
ular fibrillation, but the study was too small for conclu-
sions on clinical significance [2]. The Cochrane review
suggested no conclusion could be drawn from these stud-
ies [17]. Kier et al. have performed a Cochrane review of
studies of washed versus unwashed red cells transfused to
treat the anaemia of prematurity in neonates [20]. They
concluded that there is insufficient data to support or
refute the use of washed RBCs to prevent the develop-
ment of significant neonatal morbidities or mortality.
A recent study examined whether washing of 21-day-
old red cells at the point of transfusion could attenuate
organ injury following cardiac surgery and transfusion
[21]. The study did not demonstrate a benefit of washing,
and there was an increase in cell-free haemoglobin con-
centrations in washed red cell supernatants compared to
standard. It was underpowered as recruitment was termi-
nated early.
Therefore, data on the clinical benefits of washed red
cells, other than reducing allergic reactions, are mixed
and it is difficult to draw any firm conclusions.
Methods of production and standards
There are various methods employed to wash red cells for
transfusion. These include manual washing where the red
cell unit is centrifuged, the supernatant removed and
resuspended in a non-plasma solution. The latter may be
saline or an approved red cell additive solution. Several
commercial devices have been developed designed for use
in blood centres/banks to automate the washing proce-
dure, such as the COBE2991 and ACP215 device. The
method of washing, the number of wash cycles and
choice of washing and resuspension media differ between
countries. These factors in combination have a major role
in determining an appropriate shelf life for the compo-
nent, based on its clinical use. Current guidelines for
washed red cells from the UK, Council of Europe and
AABB are given in Table 1. In addition to these methods
which are mainly used by Blood Banks, it is evident that
there may be some use of cell salvage devices to wash
red cells prior to use in cardiopulmonary bypass [2,22].
The most recent survey of international practice was
reported in 2015 by Thomas & Cardigan based on 30
respondents on the production and use of washed compo-
nents [23]. Eight centres reported using manual methods,
but the majority of respondents [23] use automated meth-
ods – 15 use the COBE 2991, nine use the Haemonetics
ACP215, and some use both.
The reported mean age at the time of washing was
7
4 days (n = 9) although some regulations permit wash-
ing at any time up to 42 days, and most using the
ACP215 limit the age of the red cells to ≤14 days prior to
washing. A range of wash solutions were reported to be
in use: 19 services wash in saline, 10 use saline with glu-
cose (the recommended ACP215 wash solution), and three
wash with a red cell additive solution (note that some use
more than one wash solution). Following washing, 17 ser-
vices suspend the red cells in saline, five in saline with
glucose, and eight in additive solution (Table 2).
© 2020 International Society of Blood Transfusion
Vox Sanguinis (2020) 115, 606–616
 Washing of red cells 609

Table 1 Current guidelines relating to washed red cells

UK [24] Council of Europe [25] AABB [49]

Frequency of Testing All units All units ‘Prepared by a method known

Volume Within locally specified volume range To be defined for the system to ensure that the red cells
used are washed with a volume of
Haemoglobin ≥40 g/unit ≥40 g/unit compatible solution that will
Haematocrit 0
50–0
70 0
50–0
70 remove almost all of the
Haemolysis (at end of storage) None stated < 0
8% of red cell mass plasma. Shelf life of 24 h
Residual protein/Protein <0
5 g/unit <0
5 g/unit following washing
Content of Final Supernatant
Max age of red cells prior to Not stated Not stated
washing
Shelf life following washing Max 24 h in saline or as validated in AS Open system < 24 h Closed as
(currently max 14 days) validated
Shelf life once irradiated In saline 24 h In AS 5 days on day of Not stated
production or 48 h if irradiated later in shelf
life

Table 2 Results of international survey on methods of red cell washing

Manual ACP215 COBE2991

n = 9 (30%) n = 8 (27%) n = 15 (30%)

Optimal Additive Optimal Additive

Saline Solution Saline Saline + glucose Solution Saline Saline + glucose

Wash solution 7 (23%) 2 (7%) 3 (10%) 5 (17%) 0 10 (34%) 5 (17%)

Storage solution 7 (23%) 2 (7%) 2 (7%) 1 (3%) 5 (17%) 9 (30%) 6 (20%)
Shelf life 1 x0h 1 x 5 days 2 x 24 h 1 x 24 h 1 x 24 h 9 x 24 h 6 x 24 h
1 x6h 1 x 28 days 1 x 42 days 1 x 7 days
3 x 24 h 3 x 14 days
1 x 48 h
1 x 42 days

Data are taken from Thomas & Cardigan[23].

Data given are the number of respondents out of a total of 30 that wash red cells, and as a percentage of the total.
Some centres use more than one method; hence, data do not add up to 100%.
Number of blood services reporting use of solutions for washing and/or storage of red cells, and the resulting shelf life.

The COBE 2991 is an open processing system, and the
shelf life following washing is therefore 24 h in all cases.
With the closed system of the Haemonetics ACP215, a
maximal shelf life of 14 days was assigned following
washing in all cases except one, which retained the
42 days shelf life of the original red cell component
(Table 2).
Effectiveness of washing in removing plasma
Studies that have made a measurement before/after wash-
ing to assess the efficacy of plasma removal have
employed different tests: total protein, albumin or IgA,
often a surrogate for measuring the specific proteins
triggering the transfusion reaction. In theory, the estimate
of plasma reduction should be the same for all tests, but
this is not necessarily the case. Measurement of total pro-
tein in the supernatant of red cells will not only reflect
plasma carryover, but also be influenced by leakage of
proteins from red cells due to the washing process itself
or due to storage, most notably following haemolysis of
red cells. Therefore, it is likely that measurement of total
protein, as currently required by UK [24] and European
Guidelines [25] shown in Table 1, overestimates the
amount of plasma in the final component. Red cells in
additive solution typically contain 5–10% plasma in a
volume of supernatant of approximately 120 ml [26]. At
an average total protein level in plasma of 60 g/l, the

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Vox Sanguinis (2020) 115, 606–616
610 R. Cardigan et al.
amount of total protein per unit would be expected to be
0
6 g/unit prior to washing. Therefore, a limit of 0
5 g/
unit (as is the case in UK and EU Guidelines, Table 1) in
a washed component does not appear to be a meaningful
or particularly stringent measure of washing efficacy.
Measurement of albumin or IgA concentration is more
specific and may give a more accurate estimate than total
protein of the amount of plasma removed by washing.
Early studies on manually washed RBC suggested that
washing reduces supernatant protein from 0
85 to 0
21 g/
unit and IgA from 15
08 to 0
13 mg/unit, supporting the
hypothesis that total protein measurements do not accu-
rately reflect the efficacy of washing [27]. A recent study
showed that residual IgA levels in manually washed units
tended to be lower than those washed using the ACP215
device and that the reduction in total protein (80%) was
lower than that for albumin (93%) or IgA (99%) [28].
The data on residual levels of IgA in washed red cells
produced using the ACP215 in the latter study (average
0
064 mg/dl (range 0
03-1
04, n = 15)) are consistent
with previous reports using the ACP215 method where
mean residual IgA levels are reported as being 0
03–
0
05 mg/dl [29]. Hansen et al. [30] assessed residual IgA
levels in red cells washed using the ACP215 and found
that 13/20 units from bottom and top processing (BAT)
but 0/20 units from top and top (TAT) processing had
levels of IgA below 0
05 mg/dl–the level below which the
Canadian Blood Service define donors as having severe
IgA deficiency [31]. In a further study, only 24/26 BAT
red cell units but 0/9 TAT red cell units washed using the
ACP215 device had IgA levels <0
05 mg/dl [32]. However,
if red cells underwent a second wash procedure then all
units whether from BAT or TAT were reliably depleted to
<0
05 mg/dL IgA [32]. They also noted that the level of
residual IgA appeared to be related to the pre-wash vol-
ume of red cells, although TAT red cells are also likely to
contain higher residual plasma levels than those from
BAT.
It is not clear what level of plasma removal is required
to prevent transfusion reactions in susceptible recipients,
including those with IgA deficiency. For IgA-deficient
patients with a history of transfusion reactions, AABB
[33] and the American Red Cross Rare Donor Programme
[34] stipulate that the level of IgA should be <0
05 mg/dl
(<0
0005 g/l). The rationale is based on a single study
which determined the amount of IgA that could be safely
transfused to six IgA-deficient recipients without causing
a reaction [35]. Both blood services in Canada also use
0
05 mg/dl as an upper limit for IgA in this situation
[31,36]. There have been no recent studies that would
confirm that this is an appropriate level.
Given the data above, it seems irrational to use total
protein as a measure of the efficiency of the washing
process in removing protein as it reflects a combination
of haemolysis and plasma content of red cells. Measure-
ment of a plasma protein such as albumin or IgA may be
more appropriate to assess the efficiency of plasma
removal, particularly if the component is required for an
IgA-deficient patient with transfusion reactions, but more
data are needed in order to inform their inclusion in
guidelines.
Laboratory data on the quality of washed red
cells
A summary of laboratory studies is given in Table 3.
Manual methods
Weisbach and colleagues assessed washing of 2- to 6-
day-old or 11- to 15-day-old red cells using two wash
cycles of 250 ml in either saline, SAGM or 5% albumin
followed by storage in the same medium for 6 h [37].
They observed a 15% loss of total haemoglobin and a
decrease in free haemoglobin and supernatant potassium
following washing which did not then alter over storage
for six hours (in any medium). ATP increased following
washing and did not decrease thereafter, whereas there
was a decrease in 2,3 DPG of 15–35% for 2- to 6-day-old
RBC, and 30–40% for 11- to 15-day-old cells following
washing. As a consequence of the loss of 2,3 DPG, the
authors suggest that red cells for massive transfusion to
neonates should be no more than 14 days old when
washed. Others have reported that washing of red cells
reduces the effect of the supernatant from red cells in
models of endothelial activation [38].
Automated methods
This COBE 2991 device is used more widely in the USA
than Europe. Harm et al. [23] demonstrated that washing
of 8- to 10-day-old non-LD red cells in AS-5 using the
COBE 2991 device and storage for up to 24 h resulted in
an increase in haemolysis (to approximately 1%) com-
pared to 8- to 10-day-old standard red cells (0
25%).
O’Leary et al. [21]demonstrated that leakage of potassium
and haemolysis following washing increased at a greater
rate using the COBE 2991 to wash red cells compared
with the Fresenius CAT device. Of note, the extracellular
potassium concentration reached or exceeded pre-wash
levels 24 h after washing using the COBE 2991 device.
The authors point out that keeping these cells for their
maximal permitted shelf life of 24 h following washing
will obviate any benefit of washing in terms of reducing
potassium load – a practice used by some centres interna-
tionally for patients at risk of hyperkalemia (such as
© 2020 International Society of Blood Transfusion
Vox Sanguinis (2020) 115, 606–616
 Table 3 Laboratory studies on washed red cells

Whole blood BAT Storage Washing Storage

storage or Anti- medium method & medium Days stored
conditions TAT coagulant n= LD? pre-wash Day of wash solution post-wash post-wash Key findings Study

N/A apheresis red cells CPD-50 30 (10 each Y SAGM 6, 14 or 21 ACP215 SAGM 14 Mean residual IgA levels approx 0
03– [29]
(MCS+) arm) 0
9% saline, 0
2% 0
05 mg/dl. K + levels approx
dextrose 15 mmol/l 14 days post-wash in all
groups. ATP 14 days post-wash
approx 50% of pre-wash values.

Vox Sanguinis (2020) 115, 606–616

Haemolysis increases post-wash with
age of RBC pre-wash, average ≤ 0
3%
day 14.
Ambient BAT CPD 24 (6 on Y SAGM 1, 7,14 or 21 ACP215 SAGM or AS-3 42 Quality of red cells dependent upon [40]

© 2020 International Society of Blood Transfusion

storage each day, 0
9% saline, 0
2% day of storage pre-wash, how long
overnight 3 in each dextrose stored post-wash and storage medium
AS) used post-wash. Max of 14 days pre-
wash and 7 post-wash met their
acceptance criteria of < 0
8%
haemolysis in> 90% units, <0
29%
visual haemolysis, ATP ≥ 2
7 µmol/g
Hb and supernatant potassium levels
of ≤ 10
2 mmol/l.
Ambient BAT & CPD 18 Y SAGM 14 ACP215 SAGM 1,4,5 or 7 RBC were irradiated 1,4,5 or 7 days [30]
storage TAT 0
9% saline, 0
2% following washing and stored and
overnight dextrose tested up to 7 days post-wash.
Ambient TAT CPD 6 Y SAGM or 14 ACP215 0.9% SAGM or AS7 2,7,14,21,28,42 RCC were washed or irradiated on day [43]
storage AS-7 saline, 0.2% 14. Reduced haemolysis and red cell
overnight dextrose microparticles and increased ATP in
AS7 stored RCC suggests that storage
in AS7 may improve the quality of
RCC that have undergone secondary
processing in the form of washing or
irradiation.
Ambient BAT CPD 9 Y SAGM 14 ACP215 0.9% SAGM or Saline 5,7,11,14 RCC were washed with ACP or [28]
storage and saline, 0.2% manually using 0
9% saline, 0
2%
overnight TAT dextrose or dextrose or SAGM or saline and
Manual wash into stored in SAGM or saline. Overall, the
SAGM or Saline removal of plasma proteins was better
using manual methods and Hb loss
Washing of red cells 611
 Table 3 (Continued)

Whole blood BAT Storage Washing Storage

storage or Anti- medium method & medium Days stored
conditions TAT coagulant n= LD? pre-wash Day of wash solution post-wash post-wash Key findings Study

was lower in manually washed units

than in ACP215-washed units.
612 R. Cardigan et al.

NA NA NA 40 (5 each Y SAGM 2–6 or 11–15 Manual – two SAGM or Saline 6h Units washed and stored at 4oC: 15% [41]
group) washes SAGM or or 5% loss of total Hb, decrease in free Hb
Saline or 5% albumin and K + following washing which did
albumin not then alter over 6 h. ATP increased
following washing and did not
decrease thereafter. 15-35% decrease
in 2,3 DPG for 2- to 6-day-old RBC,
30–40% for 11- to 15-day-old cells.
Washing and storage at room temp
increased haemolysis and reduced 2,3
DPG on storage compared with
washing/storage at 4°C.
NA NA NA 20 washed N AS-5 8–10 COBE 2991 0
9% Saline? 1 Increase in haemolysis (to approx 1%) [50]
20 saline and susceptibility to mechanically
controls fragility following washing v controls
NA NA NA 24 8 Y AS-3 4 1) COBE 2991 Saline? 1 Potassium leakage and haemolysis [21]
2) Fresenius CATS following washing increased at a
0.9% saline greater rate using COBE 2991 than
Fresenius CAT. Using COBE
2991 K + levels reached or exceeded
pre-wash values 24 h after washing.
NA NA NA 20 in each NA AS-1 or 40-42 1) COBE 2991 Saline? 0 Small decrease in free Hb and red cell [39]
group AS-3 2) Haemonetics Cell microparticles immediately following
Saver Elite 0.9% washing using COBE 2991, whereas
saline cell saver Elite increased both. Neither
device resulted in an increase in
mechanical fragility of red cells or
change in ATP. 85–88% haemoglobin
recovery.

AS, additive solution; BAT, bottom and top processing; CPD, citrate phosphate dextrose; Hb, haemoglobin; LD, leucocyte depletion; NA, not applicable; RCC, red cell concentrate; SAGM, saline adenine glucose
mannitol; TAT, top and top processing.

Vox Sanguinis (2020) 115, 606–616

© 2020 International Society of Blood Transfusion
 Washing of red cells 613

neonatal cardiac surgery) [2]. Bennett-Guerrero et al com- Comparison of automated and manual methods
pared 40- to 42-day-old red cells immediately before and
Proffitt et al. [28] compared red cells washed manually in
after washing using the COBE2991 device or the Haemon-
either SAGM, saline or saline/dextrose to those washed
etics Elite Cell Saver device [39]. The authors noted a
using the ACP215 device. Red cells were washed on day 14
small decrease in free haemoglobin and red cell micropar-
of storage and stored for 14 days thereafter. The authors
ticles immediately following washing using the COBE
conclude that a maximal shelf life of red cells of 14 days
2991, whereas there was an increase in both parameters
pre-wash and 14 days post-wash is acceptable since levels
following washing with the Cell Saver Elite. Neither
of supernatant potassium are lower and ATP levels and
device resulted in an increase in fragility of red cells in
haemolysis are similar compared with those in standard red
response to mechanical stress.
cells in SAGM stored to their maximal shelf life (35 days).
Grabmer et al. [29] studied red cells stored in SAGM
Further, the study showed that the in vitro quality of man-
and washed on either day 6, 14 or 21 of storage using
ually washed red cells was improved compared to those
the ACP 215 device, and subsequently stored in SAGM
washed using the ACP215 device. Therefore, for washed
for 14 days. As expected, supernatant potassium levels
red cells stored in SAGM that are produced by sterile con-
were reduced following washing and increased again on
nection and closed systems, storage of manually washed
further storage. Levels were similar 14 days following
red cells could also be extended from 24 h to 14 days.
washing whether red cells were 6,14 or 21 days old when
washed and reached approximately 15 mmol/l, a level
below that found in red cell units prior to washing (30– Irradiation
50 mmol/L depending on age of red cells). At day 14 fol-
Red cells for patients at risk of developing transfusion-as-
lowing washing, ATP levels were approximately 50% of
sociated graft-versus-host disease (TA-GvHD) are irradi-
pre-wash values. Since the pre-wash values will differ
ated to render any contaminating lymphocytes non-
between each group due to age of red cells, and absolute
viable. A study has shown that using either the ACP215
values for ATP are not given, these are difficult to inter-
device or manual washing methods, significant numbers
pret. Haemolysis levels were slightly reduced following
of leucocytes remain following washing of units of red
washing and then increased to day 14, the extent of
cells spiked with a level of leucocytes that mimics failure
which was greater the older cells were prior to washing.
of the LD process [13]. Thus, there is no convincing scien-
Nonetheless, levels were well below the European level of
tific argument or data to suggest that the risk of TA-
0
8% at end of shelf life for all groups (average 0
3% for
GvHD from washed red cells is less than that of standard
day 21 washed RBC).
red cells. There is therefore a need to irradiate this com-
Hansen et al. [40] performed a study to assess optimal
ponent for at-risk patients.
pre- and post-storage times and conditions when using
Irradiation of red cells is a key determinant of red cell
the ACP215 device with SAGM red cells. They assessed
quality, whether washed or not.
red cells washed on day 1, 7, 14 or 21 of storage which
Hansen and colleagues assessed irradiation of SAGM
were then subsequently stored in SAGM or AS-3. They
red cells washed using the ACP215 device [30]. Based on
set acceptance criteria of <0
8% haemolysis in >90%
their previous work [26], they assessed red cells stored for
units, <0
29% visual haemolysis, ATP ≥ 2
7 µmol/g Hb
14 days prior to washing and irradiated (target 25Gy,
and supernatant potassium levels of ≤10
2 mmol/L. The
minimum 15Gy) on day 1,4,5 or 7 following washing.
latter was set to ensure that these remained below that of
Units that were irradiated on day 1 or 4 following wash-
a 5-day-old unit of red cells in SAGM, since they use
ing did not meet their requirement for >98% of units to
washed red cells if <5-day-old red cell is not available for
have haemolysis levels of <0
8% with >95% confidence if
‘K + sensitive patients’. They observed that the increase
stored for more than 48 h after irradiation. However,
in haemolysis following washing was dependent upon the
units irradiated at day 5 following washing and stored for
age of red cells prior to washing and that this was worse
48 h or less did meet their acceptance criteria. Units irra-
for units stored in SAGM compared with AS-3. However,
diated on day 7 were tested the same day, and therefore,
maintenance of ATP levels was worse in units stored in
no conclusions can be drawn regarding their storage.
AS-3 following washing. They concluded that for red
A study from the UK also assessed irradiation of
cells stored in SAGM, a 14-day maximum pre-wash per-
washed red cells [28]. Mean levels of haemolysis when
iod followed by maximum 7 day post-wash storage per-
red cells were irradiated on the day of washing (day 14)
iod met their acceptance criteria. The latter was
followed by storage for up to 5 days were similar to those
principally governed by supernatant potassium levels,
of standard red cells at end of storage, but increased if
since ATP and haemolysis requirements were met 14 days
stored for longer.
following washing.

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Vox Sanguinis (2020) 115, 606–616
614 R. Cardigan et al.
There is scant data on the effect of washing red cells
after they have been irradiated, but one study suggests
that if cells are not stored for long afterwards (several
hours), this may be acceptable [41].
Novel additive solutions
A pivotal study by Meryman in the 1990’s [42] exam-
ined storage of washed red cells in an experimental
hypotonic AS containing phosphate (ARC-8) compared
to storage of standard red cells in Adsol, both for
42 days. Low chloride in the suspending AS results in
the chloride shift, that is passive diffusion of chloride
out of the cells leading to a net loss of intracellular
chloride. To maintain electrical neutrality, hydroxyl
anions must diffuse into the cells in proportion to the
chloride loss, where they raise the intracellular pH. The
net result is that due to this change in intracellular pH
and the buffering effects of phosphate, laboratory mea-
sures such as haemolysis, ATP levels and 2,3 DPG are
considerably improved. Further, in a crossover study in
6 subjects, the 24-hour recovery of red cells washed and
stored in ARC-8 was improved compared with standard
red cells in Adsol [42]. Proffitt and colleagues investi-
gated storing washed red cells in AS-7-a hypotonic,
alkaline additive solution. Laboratory measures such as
haemolysis, ATP and potassium leakage were improved
when stored in AS-7 following washing using the
ACP215 processor [43]. This effect was observed whether
cells were stored in SAGM or AS-7 prior to washing,
but maximal benefit was seen when stored in AS-7 prior
to washing. This suggests that storing red cells in better
additives may help to protect them from the damage
that then occurs due to the washing process.
Taken together, the laboratory data suggest that the
method of washing, storage medium, age of cells at wash-
ing, length of storage following washing and irradiation
are all critical in determining an appropriate shelf life. If
the criteria of an acceptable shelf life are those which keep
haemolysis levels similar to those of standard red cells at
end of shelf life, then the studies cited suggest that for red
cells stored in SAGM washing up to day 14 of storage with
storage for a further 14 days appears acceptable. If red
cells are then irradiated, the shelf life should be reduced to
between 2–5 days depending on what point in shelf life
irradiation takes place. However, if washing is being used
as a method to reduce potassium levels for patients at risk
of hyperkalemia, then the shelf life may need to be further
reduced. Further, the quality of red cells is not the same
for all automated washing devices, and haemolysis and
potassium leakage following washing using the ACP215
device is lower than that using the COBE 2991 device and
may be even lower for manual washing methods.
Data from animal models
Valeri et al. [44] used a canine model of intra-operative
blood salvage where blood was subsequently washed
using a Haemonetics Cell Saver and then immediately
transfused. The recovery of red cells 24 h following trans-
fusion was 90%, and the red cells had a normal half-life
and oxygen transport function. Plasma haemoglobin
levels were slightly increased following transfusion.
Belizaire et al compared transfusion of fresh RBC or
15-day-old RBC that were washed (in AS-3 but not stored
thereafter) or unwashed in a mouse model of haemor-
rhagic shock [45]. Concentrations of pro-inflammatory
cytokines such as IL-1a, MIP1a and MIP-2 in the serum
of recipients were increased in animals that received 15-
day-old red cells compared with fresh, which was par-
tially attenuated by washing of red cells.
In a more recent study [46], dogs with S. aureus pneu-
monia were exchange transfused with either 7- or 42-
day-old washed or unwashed canine donor blood. Wash-
ing 42-day-old blood improved survival rates, lung injury
and liver function. However, when 7-day-old blood was
washed these outcomes were worsened, and serum-free
haemoglobin was also increased following transfusion.
These data suggest that washing fresh red cells may be
detrimental; however, it is not clear how these studies
relate to humans since canine and human red cells may
not store the same, that is a 42-day-old canine red cell
may not be equivalent to the same age human red cell.
In a porcine model of large volume transfusion in car-
diac surgery, washing of stored red cells did not reduce
lung injury, and increased endothelial activation and kid-
ney injury [47]. However, the latter study did demonstrate
a positive effect of rejuvenated and washed red cells in
reducing lung and kidney injury relative to 14-day-stored
red cells.
In a rodent model, in healthy animals, washing
increased the recovery of red cells following infusion and
decreased trapping of RBC in organs. This was not
observed in rats with pneumonia, [48] but in these ani-
mals washing reduced bacterial outgrowth and lung
injury.
The data from animal models are therefore mixed, with
some suggesting a benefit of washing, others showing no
benefit and some indicating worse outcomes. In part, this
may be dependent upon recipient factors such as infection.
Conclusion
There are many different methods for washing red cells.
The laboratory data summarised above demonstrate that
washing of red cells may not be a benign process. The
most accepted indication for washed red cells is in
© 2020 International Society of Blood Transfusion
Vox Sanguinis (2020) 115, 606–616
 Washing of red cells 615

prevention of severe or recurrent allergic reactions but
there have been surprisingly few clinical studies. Whether
washing of red cells has a beneficial effect in terms of
improving clinical outcome following surgery is contro-
versial. This emphasises the need to ensure washed red
cell components are only used where clearly indicated
until further data demonstrating wider clinical benefits
are obtained. Many factors such as choice of washing
methods, storage solutions and age of red cells before
and after washing impact on red cell quality. Therefore, it
is important that they are produced according to vali-
dated laboratory techniques with defined quality
parameters.
Given the data published to date, there is a need for
well-designed studies to better understand the potential
benefit of washed red cells. Further, production methods
that reduce the negative impact of washing on red cells
and that can be readily employed in routine production
departments would be a valuable goal.

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