Transfusion Medicine, 2000, 10, 291303

A comparison of biochemical and functional alterations of rat and

human erythrocytes stored in CPDA-1 for 29 days: implications
for animal models of transfusion
M. S. d'Almeida, J. Jagger, M. Duggan, M. White, C. Ellis and I. H. Chin-Yee

Department of Hematology,

London Health Sciences Centre, the AC Burton Vascular Biology Laboratory and the Department of Pharmacology and Toxicology, University of
Western Ontario, London, Ontario, Canada
Received 13 March 2000; accepted for publication 26 June 2000

S U M M A R Y . Animal models of transfusion are employed

in many research areas yet little is known about the
storage-related changes occurring in the blood used in
these studies. This study assessed storage-related
changes in red blood cell (RBC) biochemistry, function
and membrane deformability in rat and human packed
RBCs when stored in CPDA-1 at 4 8C over a 4-week
period. Human blood from five volunteers and five bags
of rat RBC concentrates (five donor rats per bag) were
collected and stored at 4 8C. RBC function was assessed
by post-transfusion viability and the ability to regenerate adenosine triphosphate (ATP) and 2,3-diphosphoglycerate (DPG) when treated with a rejuvenation
solution. Membrane deformability was determined by a
micropipette aspiration technique. ATP in rat RBCs
declined more rapidly than human RBCs; after 1 week
rat ATP fell to the same level as human cells after
4 weeks of storage (rat, 22 ^ 02 mmol g21 Hb;
human, 25 ^ 03 mmol g21 Hb). Baseline DPG
concentrations were similar in rat and human RBCs
(162 ^ 23 mmol g21 Hb and 137 ^ 24 mmol g21

Hb) and declined very rapidly in both species. Human

RBCs fully regenerated ATP and DPG when treated
with a rejuvenation solution after 4 weeks of storage.
Rat RBCs regenerated ATP but not DPG. Posttransfusion viability in rat cells was 79%, 26% and
5% after 1, 2 and 4 weeks of storage, respectively. In
rats, decreased membrane deformability became significant (2 54%) after 7 days. Human RBC deformability
decreased significantly by 34% after 4 weeks of storage.
The rejuvenation solution restored RBC deformability
to control levels in both species. Our results indicate
that rat RBCs stored for 1 week in CPDA-1 develop a
storage lesion similar to that of human RBCs stored for
4 weeks and underscores significant species-specific
differences in the structure and metabolism of these
cells.

Animal models play an important role in preclinical

studies of blood transfusion and blood substitutes.
Recent clinical studies suggesting a lack of efficacy of
stored blood transfusions (Dietrich et al., 1990; Marik &
Sibbald, 1993; Hebert et al., 1999) have led to a renewed
interest in storage-related changes and their functional
implications. To further investigate this, a rodent model
was developed in which systemic oxygen consumption
is directly measured as a marker of improved tissue
oxygenation and function to assess the efficacy of red

blood cell (RBC) transfusion. Using this model it was

found that transfusion of rat RBCs stored in a citrate,
phosphate, dextrose adenine solution (CPDA-1) for
28 days was less effective than RBCs stored for 1
5 days in restoring depressed oxygen consumption
brought about by haemodilution-induced decreases in
oxygen delivery (Fitzgerald et al., 1997).
Despite the use of preservation solutions, storage of
blood inevitably results in a series of alterations in the
erythrocyte leading to irreversible damage to the cell
and reduced erythrocyte function and survival following
transfusion. These changes are collectively referred to as
the storage lesion. As a consequence, ex vivo storage of
RBCs could influence the ability of the RBC to deliver

Correspondence: Ian Chin-Yee, Department of Hematology, London

Health Sciences Centre, 800 Commissioners Road East, London,
Ontario, Canada N6A 4G5. Tel.: 11 519 685 8300 ext 55192; fax: 11
519 685 8477; e-mail: ian.chinyee@lhsc.on.ca
q 2000 Blackwell Science

Key words: 2,3 diphosphoglycerate, adenosine triphosphate, membrane deformability, post-transfusion viability, storage lesion.

291

292

M. S. d'Almeida et al.

oxygen in a form readily utilizable by tissue. Animal

models of transfusion allow direct measurement of
oxygen delivery/utilization and provide a potential
means to evaluate the physiological consequences of
storage-related RBC changes. The clinical relevance of
the animal model of transfusion therefore requires an
elucidation of the storage-related changes in animal
blood to determine if they are similar to the human RBC
storage lesion.
Although there is significant information regarding
the changes that occur in human cells during storage,
little is known about the changes occurring in rat RBCs
under similar conditions. Consequently, it is not known
whether rat RBCs stored for 28 days are truly representative of human RBCs stored for similar durations. It
was our intention therefore to determine the changes
occurring in rat RBCs during storage and how closely
they parallel those of stored human RBCs. Our
objectives in this investigation were to determine
selective biochemical changes that occur in rat and
human RBCs stored in CPDA-1 at 4 8C over a 29-day
period and to assess the 24-h post-transfusion survival of
rat RBCs at various points during the storage period.
Using a micropipette aspiration technique, we also
determined the changes in rat and human RBC
membrane deformability. RBCs were then treated with
a rejuvenation solution (phosphate, inosine, pyruvate
and adenine) to determine the ability of stored RBCs to
regenerate ATP, DPG and to assess the effect on
membrane deformability.
METHODS AND MATERIALS
All studies carried out in this investigation were
approved by the University Council on Animal Care
for the University of Western Ontario.
Blood harvesting
Male SpragueDawley rats (. 500 g) were used to
obtain blood. Rats were anaesthetized with pentobarbital
(65 mg mL21, i.p. injection, 01 mL pentobarbital per
100 g body weight). Following a midline laparotomy,
the abdominal aorta was exposed and punctured with a
20 gauge veno-puncture needle unit. Blood was drawn
into a 20-mL syringe containing 3 mL of CPDA-1
(Medsep Corporation). Approximately 1520 mL of
blood can be obtained from a single rat. The blood from
five rats was transferred into a 150-mL blood collection
bag (Medsep Corporation, Canada).
A total of 25 rats were bled to make five separate
units of stored blood. Harvested blood was centrifuged
within 1560 min of phlebotomy at 3600 relative
centrifugal force (rcf) for 5 min to separate plasma

from cells. Plasma was removed to obtain a packed cell

haematocrit of approximately 75%. RBCs were stored in
the hospital blood bank refrigerators at 4 8C for the
duration of the study. Prior to pooling rat blood, crossmatching of the five donor rats was carried out to ensure
RBC compatibility. A blood sample from each of the
five donors was individually centrifuged at 1200 rcf and
the plasma drawn off. A 5% suspension of red cells from
the rats were reacted with plasma from one of the other
rats. Each sample was centrifuged at 1200 rcf for 30 s
and assessed microscopically under 10 magnification
for immediate agglutination. The samples were then
incubated for 30 min at 37 8C, centrifuged for 30 s at
1200 rcf and observed microscopically for any further
agglutination. No evidence of agglutination was
observed at any point in this analysis.
Human whole blood from five healthy male volunteers was harvested from a brachial vein using a 20gauge needle and 60-mL syringe with 9 mL of CPDA-1.
Once the blood was obtained, it was processed in an
identical manner to that of the rat blood.
Analysis of the RBCs was carried out immediately
after plasma removal, 24 h post-harvesting and then on
days 8, 15, 22 and 29 of storage.
Storage-related biochemical changes in rat
and human RBCs
Samples of the packed rat and human RBCs were
diluted 1 : 1 with physiological saline and were
analysed in our clinical biochemistry laboratories for
the following: total haemoglobin concentration, haematocrit, supernatant potassium, lactate dehydrogenase
(LDH) and lactate concentrations (Beckman Synchron
CX7 Analyser, Beckman-Coulter, USA) and pH (NovaStat Analyser, Nova Biomedical, USA).
The organic phosphates ATP and DPG were
measured using spectrophotometric assays based on
the oxidation of NADH to NAD1 (Sigma Chemical Co.,
Canada). The lower limits of detection for the ATP and
DPG assays are 5 mmol dL21 and 02 mmol mL21,
respectively, and are well below our measured ranges.
Treatment of stored RBCs with rejuvenation solution
To assess the ability of stored RBCs to regenerate DPG
and ATP, stored human and rat RBCs were treated
with a commercially available rejuvenating solution
containing 0550 g sodium pyruvate, 134 g inosine,
0034 adenine, 0500 g dibasic sodium phosphate and
0200 g monobasic sodium phosphate (Rejuvesol,
Encyte Laboratories, Inc., Massachusetts, USA). Under
normal conditions, a 50-mL bottle of rejuvenation
solution is added to one unit of stored blood (250 mL).
Volumes of blood and rejuvenation solution were scaled
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Storage lesion in rat and human red cells

down to meet our requirements maintaining a similar
ratio of Rejuvesol to blood (1 : 5).
The RBC/rejuvenation solution was incubated in a
37 8C water bath and gently agitated for 60 min. Cells
were inverted after 30 min of incubation. Cells were
then washed and centrifuged three times with physiological saline. Following the last centrifugation, the
supernatant was drawn off, leaving a packed RBC
solution with a haematocrit of approximately 70%. This
procedure was carried out for each of the bags of stored
rat RBCs starting on day 1 of storage and again on days
8, 15 and 29.
ATP and DPG measurements were obtained from the
rejuvenated cells at each time period according to the
techniques noted above.
24 h post-transfusion viability of rat RBCs
(a) Preparation of 51Cr-labelled rat RBCs. RBCs were
tested on days 7, 15 and 29 of storage. Just prior to the
study, RBCs were labelled with 51Cr using 0102
mCi kg21 body weight according to the procedure of
Moroff et al. (1984a). In brief, using sterile technique,
25 mL of packed RBCs were withdrawn from each of
the five bags of donor RBCs, placed in a sterile
centrifuge tube and allowed to sit at room temperature
for 15 min. Cells were then incubated with 51Cr label at
room temperature for 30 min. Nonlabelling 51Cr was
washed from the RBC solution with 5 mL of
physiological saline and centrifuged at 1000g for
10 min. Labelled RBCs were washed and centrifuged
three times. Following the last centrifugation, the
supernatant was drawn off leaving a packed RBC
solution.
(b) Preparation of recipient rats. Twenty-four hours
prior to assessment of post-transfusion RBC survival,
five rats (male, SpragueDawley, 350400 g) were
anaesthetized with pentobarbital (i.p injections
625 mg mL21, 01 mL per 100 g body weight) and
instrumented with indwelling cannulas in the carotid
artery (PE 50) and jugular vein (silastic 063 mm inner
diameter, 12 mm outer diameter). Cannulas were
tunnelled subcutaneously to exit at the back of the
neck in the interscapular region and attached to a swivel
device. Skin incisions were closed with 2-0 silk.
Cannulas were perfused with heparinized saline
solution at a rate of approximately 2 mL h21 to
maintain patency.
(c) Assessment of post-transfusion survival. The day
following surgery, RBCs were prepared as described
above. Approximately 05 mL of labelled RBCs from
one incubation tube was injected into the jugular vein of
q 2000 Blackwell Science Ltd, Transfusion Medicine, 10, 291303

293

one of the recipient rats. The labelled RBCs were

flushed into the rat with 1 mL of saline. In total, five
labelled RBC samples were injected in to five rats.
Arterial blood samples were obtained at 25, 5, 75, 10,
125, 15, 30 and 60 min and again at 24 h posttransfusion to determine the elimination of the labelled
RBCs and the 24-h post-transfusion survival. Duplicate
025-mL aliquots of blood samples were drawn to
determine the level of 51Cr label in the sample. Samples
were counted after the 24-h sample was obtained
(Wallac Wizard 1480, Automatic Gamma Counter,
Finland).
The data were plotted as sampling time vs. net counts
per minute (cpm) of the sample (where net cpm cpm
of sample 2 background cpm). Data were extrapolated
back to t 0 to obtain the theoretical total cpm injected
into the animal.
Post-transfusion survival at any time point was
calculated according to the single label method as
follows:
Net cpm of sample at t x/Hct
4 net cpm of sample at t 0/Hct
Measurement of RBC membrane deformability
Rat cells were tested immediately after centrifugation on
the day of collection and on days 3, 5, 7 and 9 of storage.
Human cells were tested immediately after centrifugation on the day of collection and then on a weekly basis
thereafter.
(a) Preparation of micropipettes. capillary tubes
(15  100 mm; KIMAX-51, Kimble Products, USA)
were soaked in a solution of 95% ethanol for at least
48 h and oven-dried for 30 min. The capillary tubes
were pulled to a final micropipette diameter of 15 mm.
Pipettes were placed in a micropipette manipulator
(details below) and soaked in a concentrated albumin
solution to eliminate electrostatic interactions between
the micropipette and the red blood cell membrane during
the assay of membrane deformability.
The manual nature of the micropipette manufacturing
process leads to small variations in the final pipette tip
diameter (^ 01 mm). Since membrane displacement is
a function of the tip geometry, a separate micropipette
was used for each individual experiment. Consequently,
results of these studies represent the change in the values
of the membrane displacement rather than absolute
values.
(b) Assay of red blood cell membrane deformability via
micropipette aspiration. The micropipette aspiration set-up

294

M. S. d'Almeida et al.
chamber. The chamber was affixed to an inverted
microscope stage where the membrane deformability of
the contained red blood cells was assayed. Temperature
in the chamber was maintained at 37 8C. RBCs were
aspirated sequentially into the micropipette with an
aspiration pressure of 2 3 mmH2O. After a 30-s
equilibration period, on-line measurement was made of
the membrane displacement into the micropipette.
Membrane displacements were normalized to the mean
value of the fresh control measurements (individual
RBC membrane displacement measurement/mean of
fresh control RBC membrane displacement).
Separate aliquots of stored blood were treated with
the rejuvenation solution for the membrane deformability study. All procedures were the same as described
above except that cells were not washed following the
incubation period.
Statistical analysis
All values are reported as the mean ^ SD. Statistical
analysis within species was by one-way repeated
measures anova with Bonferroni post hoc analysis.
Comparisons between rat and human data was by t-test.

Fig. 1. Time-related changes in adenosine triphosphate

(ATP) and 2,3-diphosphoglycerate (DPG) concentrations in
rat and human erythrocytes stored in CPDA-1 at 4 8C over a
29-day period. Fresh refers to erythrocytes tested within 2 h
of collection and after centrifugation to remove plasma.
Statistical analysis between successive time points with
species was by one-way anova (*P , 005) and Bonferroni
post hoc analysis. Statistical analysis between rat and human
data at each time point was by t-test (#P , 005).

consisted of the following: an inverted microscope with

100 dry objective lens (Nikon 64131, Japan) and a
100-W halogen light source (Philips 7023, Germany), a
thermostatically controlled and optically transparent
chamber for placement of the RBCs during assay, a
micromanipulator for mounting and controlling the
positioning of the micropipette, and an inchworm
controller (PZ-550, Burleigh Instruments, USA) for
determining the relative height of the fluid reservoir to
the micropipette tip. The video capture system consisted
of a video camera (AVC 3260, Sony, Japan), a video
monitor (WV-BM 1400, Panasonic, USA) and a video
digitizer (SNAPPY, Play Incorporated, USA). The video
digitizer was computer-controlled and on-line
measurement of the membrane displacement was made
using a commercial software package (Sigma Scan Pro,
Jandel Scientific, USA).
Blood samples (100 mL) were diluted in 50 mL
phosphate buffered saline (300 mOsm) and placed in the

RESULTS
Changes in biochemical parameters
The results of the ATP and 2,3-DPG measurements for
the human and rat RBCs are depicted in Fig. 1.
Figure 1(a) depicts the time-related changes in rat and
human ATP levels. Immediately following collection,
rat (n 25 donors in five bags) and human (n 5)
ATP levels were 23 ^ 02 mmol g21 Hb and
39 ^ 05 mmol g21 Hb, respectively. After 24 h, ATP
in rat and human cells increased to 38 ^ 03 mmol g21
Hb and 45 ^ 02 mmol g21 Hb, respectively. ATP
concentrations in rat cells then underwent a progressive
and rapid decline towards zero and by day 29 had
decreased by 85% from the baseline level. ATP levels in
human cells remained steady from day 1 to day 15 of
storage after which a gradual decline was observed. By
day 29 ATP levels in human cells had dropped by
approximately 35% from the baseline level. Comparisons between rat and human cells indicated that ATP
levels in human RBCs were significantly higher than rat
ATP levels at each time point.
Baseline DPG levels were not significantly different
between rat and human RBC after collection
(162 ^ 23 mmol g21 Hb and 137 ^ 24 mmol g21
Hb, respectively). Unlike the changes in rat and human
ATP levels, DPG levels declined rapidly; 24 h post
collection, DPG levels had declined by 18% and 17%
for rat and human cells, respectively. By day 8 DPG
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Storage lesion in rat and human red cells

295

Table 1. Summary of biochemical changes in rat and human RBCs stored in CPDA-1 over a 28-day period

Days
stored
Rat
fresh
1
8
15
22
29
Human
fresh
1
8
15
22
29

K1
(mmol L21)

LDH
(U L21)

pH
(units)

Lactate
(mmol L21)

ATP
(mmol g21 Hb)

DPG
(mmol g21 Hb)

38
66
250
376
466
520

^
^
^
^
^
^

07#
04*#
13*#
13*#
11*#
14*#

358
892
2114
4209
6756
9724

^
^
^
^
^
^

69#
92#
174*#
688*#
212*#
1325*#

704
698
670
670
669
664

^
^
^
^
^
^

005
001*
002*
004*
004*#
002*#

26
46
198
256
300
328

^
^
^
^
^
^

04#
03#
08*#
18*#
18*#
06*#

243
378
224
125
068
039

^
^
^
^
^
^

020
025*
023
008*
004*
004*

1615
1297
157
101
098
051

^
^
^
^
^
^

233
268*
020*
073*
019*
018*

23
39
136
207
291
325

^
^
^
^
^
^

02
04
16*
22*
22*
19*

56
79
148
327
734
1154

^
^
^
^
^
^

20
14
38
77*
128*
186*

714
710
677
671
660
650

^
^
^
^
^
^

007#
004#
004*#
003*#
003*
002*

16
29
135
180
260
301

^
^
^
^
^
^

02
02
02*
11*
08*
12*

39
45
46
42
36
25

^
^
^
^
^
^

05#
02*#
02*#
04#
02*#
03*#

137
114
46
13
08
12

^
^
^
^
^
^

239
13
10*#
04*
07*#
03*

K1, potassium; LD, lactate dehydrogenase; ATP, adenosine triphosphate; DPG, 2,3-diphosphogylcerate. *P , 005 within species by one-way
anova compared with baseline levels; #P , 005 human vs. rat levels, indicates larger value.

levels had fallen by 90% in the rat and 60% in human

cells. DPG levels decreased by more than 90% from
baseline levels for rat and human cells by day 15 and
remained at these levels for the remainder of the study.
Comparison between rat and human data indicated that
DPG levels were not significantly different at the
various time points, except for at day 8 and day 29.
Table 1 details the changes for LDH, K1, pH and
lactate over the 29-day storage period. LDH is an

Fig. 2. Effect of the rejuvenation solution (PIPA) on the

concentration of 2,3-diphosphoglycerate (DPG) and
adenosine triphosphate (ATP) in human erythrocytes after
29 days of storage. See Methods for the rejuvenation
procedure. Rejuvenation of human erythrocytes significantly
increased DPG and ATP levels to normal or supranormal
levels (*P , 005).
q 2000 Blackwell Science Ltd, Transfusion Medicine, 10, 291303

indicator of the degree of cellular destruction; pH and

lactate reflect the metabolic condition of the cells.
In general, the rat and human data indicate that
significant time-related increases from baseline levels
occurred for the LDH, K1 and lactate starting from day
8 and for each successive time point. Exceptions to this
occurred for rat K1 levels which were significantly
elevated at day 1 and onward and for human LDH which
did not become significantly elevated from baseline
lines until after day 8 of storage. There was a
progressive and statistically significant decline in pH
from baseline levels over the entire storage period for rat
and human cells with the greatest fall in pH occurring
within the first 8 days.
When rat and human data were compared, small but
statistically significant differences were found at the
various time points for lactate and pH (Table 1). In the
case of K1, the levels measured in the rat cells were
approximately 15- to two-fold larger than that found in
human cells. By the end of the study period, K1
concentrations had increased by approximately 14-fold
in the rat and human cells. The concentration of K1
from the rat cells was significantly higher than that of
human levels at every time point.
Plasma LDH levels in rat cells were initially six-fold
larger than that of the human cells. As the storage
duration increased, the difference between rat and
human remained significantly elevated. With time, rat
and human blood both exhibited large increases in LDH
concentration. By the end of the study period rat and
human LDH levels were 27- and 21-fold higher than

296

M. S. d'Almeida et al.
their respective baseline levels. LDH levels from rat
cells were significantly higher than that of human cells
at every time point.
These data indicate that rat and human RBCs stored in
CPDA-1 under go similar biochemical changes with time,
although the changes occur more rapidly in rat RBCs. Rat
cells also tend to exhibit a larger degree of change than the
human cells for most parameters. The data also indicate
that both human and rat RBCs lose cellular integrity and
undergo haemolysis, with rat cells appearing to be more
susceptible to storage-related damage.
Regeneration of erythrocyte ATP and DPG

Fig. 3. Effect of rejuvenation solution (PIPA) on (a)

adenosine triphosphate (ATP) and (b) 2,3-diphosphoglycerate
(DPG) concentrations in rat erythrocytes at various time
points during the storage period. ATP levels significantly
increased from the respective, nonrejuvenated, time-matched
control at each time point tested (*P , 005). DPG levels
did not increase when following rejuvenation at any time
point tested. At the 2- and 29-day time points, DPG levels
were significantly decreased compared with the respective,
nonrejuvenated, time-matched control (*P , 005).

Regeneration of ATP and DPG in human RBCs has been

previously studied (Valeri et al., 1984). Consequently, we
only assessed human cells at the end of the study period
(29 days of storage). The results for the regeneration of
ATP and DPG indicate that human RBCs are capable of
synthesizing DPG and ATP to normal and/or supranormal levels after 29 days of storage (Fig. 2).
Rat RBCs were treated with the rejuvenation solution
on days 2, 8, 15 and 29 of storage. The results for ATP
and DPG are represented in Fig. 3. At each time point
the ATP concentration in the RBCs treated with the
regeneration solution was significantly higher than the
untreated cells. Except for the day 1 time point 2,
absolute levels of regenerated ATP did not reach
baseline concentrations.
Rat cells did not regenerate DPG (Fig. 3b). RBCs
treated with the rejuvenation solution had DPG
concentrations approximately equal to or less than the
nonrejuvenated control.
Post-transfusion viability of rat blood
According to the American Association of Blood Bank
(1999), banked blood must have a 24-h post-transfusion
survival rate of greater than 75%. Our results indicate
that after 7 days of storage in CPDA-1, 1 h and 24 h
post-transfusion viability (PTV) were 88 ^ 8% and
79 ^ 12% (Fig. 4). After 15 days of storage, the 1-h
PTV was 79 ^ 13% whereas, after 24 h, RBC survival
was reduced to 26 ^ 13%. By 29 days of storage, 1-h
and 24-h PTV were 51 ^ 7 and 5 ^ 1%, respectively.
The 24-h PTV at each of the successive time points was
significantly reduced from the PTV for rat blood
determined after 1 week.

Fig. 4. Results of the 24-h post-transfusion viability of 51Crlabelled rat erythrocytes when tested after 8, 15 and 29 days
of storage in CPDA-1. The percentage viability at days 15
and 29 are significantly reduced compared with viability
after 7 days of storage (*P , 005, one-way anova).

Rat RBC membrane deformability measurements

RBC membrane displacement was used as an index of
RBC membrane deformability. Figures 5 and 6 are
histograms depicting the normalized RBC membrane
displacement measured over the storage period for rat
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Storage lesion in rat and human red cells

297

Fig. 5. Histograms of storage-related changes in rat RBC membrane displacement measured by micropipette aspiration
technique. The abscissa represents membrane displacements normalized to the mean (broken vertical line) of freshly collected
RBCs and the ordinate represents the proportion of cells with a given displacement. Control and test RBCs were measured at
each time point with the same pipette to control for differences in pipette diameter. Rat RBCs demonstrated a storage-dependent
decrease in the mean membrane displacement (solid vertical line) compared with control cells and an increase in the proportion
of cells with reduced membrane displacements. A large subpopulation of nondeformable cells can be seen developing by day 7.

and human RBCs. In these figures, values , 100

correspond to cells with reduced membrane displacements, i.e. cells that are less deformable, relative to the
control cells. Values . 100 correspond to cells with
increased membrane displacement, i.e. cells that are
more deformable, compared to the control cells.

Freshly collected rat cells (day 1, n 160 cells

analysed) were characterized by a normal distribution
around a normalized mean displacement of 100 ^ 009
(Fig. 5). On day 3 of storage (n 190 cells analysed)
no significant changes in deformability were observed
(mean displacement, 102 ^ 014, i.e. 2% increase in

Fig. 6. Histograms of storage-related changes in human RBC membrane displacement measured by micropipette aspiration
technique. The abscissa represents membrane displacements normalized to the mean of freshly collected RBCs (broken vertical
line) and the ordinate represents the proportion of cells with a given displacement. Human RBCs demonstrated a storagedependent decrease in the mean membrane displacement (solid vertical line) compared with control cells.
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M. S. d'Almeida et al.

Fig. 7. Histograms of (a) rat and (b) human membrane

displacement measured after treatment with a rejuvenation
solution. Rats were incubated with the solution after 1 week
of storage and human cells after 4 weeks of storage.
Following incubation, mean membrane displacements for rat
(n 200) and human (n 100) RBCs (solid vertical line)
were not significantly different from fresh control values
(broken vertical line). Membrane displacements returned to a
normal distribution around the mean value for the rat cells.

membrane displacement compared to control). Measurements on day 5 (n 100 cells analysed) indicated that
the distribution of membrane displacements was
widening and the mean displacement was 079 ^ 019
(21% decrease in membrane deformability). By day 7
(n 200 cells analysed), the mean membrane displacement was significantly reduced to 046 ^ 035 (54%
decrease in membrane deformability). The distribution
of membrane displacements also indicated that a large
subpopulation of RBCs (approximately 30%) had
developed in which membrane displacement could not
be measured with the 3 mmH2O negative pressure. Cells
did, however, remain intact during the measurement. Rat
cells could not be measured on day 9 due to membrane
rupturing upon aspiration into the pipette because of
increased membrane fragility.
On day 1, human cells (n 100 cells analysed) were
also characterized by a normal distribution around a

normalized mean of 100 ^ 002 (Fig. 6). After 1 week

of storage, membrane deformability was not significantly affected (mean 098 ^ 002, n 80 cells
analysed) and after 2 weeks (n 80 cells analysed),
mean membrane deformability had increased slightly to
108 ^ 003, but was not statistically different from
fresh controls. By week 3 of storage, deformability
decreased significantly to a mean of 088 ^ 002
(n 100 cells analysed) and by 4 weeks mean deformability was significantly reduced to 066 ^ 002
(n 100 cells analysed). Unlike the rat cells that
developed a large subpopulation of very poorly
deformable cells, the distribution of displacements
maintained a relatively normal and narrow distribution
through out the storage period.
Treatment of the rat and human cells with the
rejuvenation solution after 1 and 4 weeks of storage,
respectively, completely restored membrane deformability to the same level as that of fresh control cells
(Fig. 7). As can be seen from Fig. 7, the mean
membrane deformability of rat and human cells returned
to 093 ^ 015 (n 200 cells analysed) and
094 ^ 002 (n 100 cells analysed). Furthermore,
the large subpopulation of very poorly deformable cells
present in the nonrejuvenated sample on day 7
completely disappeared, suggesting that these cells were
amenable to the rejuvenation solution.
DISCUSSION
Animal models of transfusion are relevant in a number
of research areas. Studies conducted in rat models are
used to investigate the effect of blood and blood
substitutes on the cardiovascular system. Furthermore,
assessment of transfusion efficacy as a therapeutic
modality in disease states such as sepsis and heart
failure is also conducted in rat models.
The objective of this investigation was to gain a better
understanding of the changes occurring in rat blood
during ex vivo storage to refine an animal model of
transfusion therapy. To this end, we assessed the
storage-related biochemical and biomechanical changes
in rat and human RBCs as well as functional aspects of
the RBCs. The results from the present study represent
the first concerted effort at characterizing storagerelated biochemical changes in rat blood stored in
CPDA-1 and comparison with those changes observed
in human cells.
Biochemical changes
We studied the effects of RBC storage in CPDA-1 on
ATP and DPG because of their bioenergetic and
functional significance in the red cell. Baseline and
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Storage lesion in rat and human red cells

storage-related changes in ATP and DPG from human
cells have been well documented (Valeri & Zaroulis,
1972; Moore et al., 1981; Dawson et al., 1984; Moroff
et al., 1984b). The declines in DPG and ATP in this
study for stored human blood are in complete agreement
with those reported in the literature. We also observed
that rat RBCs underwent similar biochemical changes to
those noted for the human cells.
When ATP and DPG levels were compared between
species, the most notable difference between rat and
human RBCs was the rate of ATP decline. Between weeks
1 and 2, ATP levels in rat RBCs fell to levels similar to that
of ATP in human RBCs stored for 4 weeks. Arturson &
Westman (1975) measured the changes in ATP and DPG
levels in stored rat blood and also observed that they
declined rapidly over the first 2 weeks of storage.
Whether this suggests that rat cells undergo structural
degradation faster or metabolize substrates at higher rates
is not clear. Based on studies using 59Fe-labelled rat
erythrocytes (Owen & Orvis, 1966), it was determined
that rat cells had a life span of approximately 54 days in
the circulation compared to 120 days for human cells and
may explain some of the time-related differences that we
observed between the rat and human cells.
Unlike the decline in ATP, the rate at which rat and
human DPG levels fell was very similar between both
species. In human and rat cells, DPG levels fell rapidly
by 18% within the first 24 h and by 1 week levels had
dropped 60% to 90%, respectively. The rate of decline
in the organic phosphates in rat and human RBCs
observed in our study is in agreement with that reported
by others (Bartlett & Barnet, 1960; Valeri & Zaroulis,
1972; Arturson & Westman, 1975; Moore et al., 1981;
Dawson et al., 1984; Moroff et al., 1984b).
Of note from our investigation was that ATP levels
initially rose from baseline levels over the first 24 h of
storage in both rat and human cells. This has been
reported previously for stored human cells stored in
ADSOL preservative (Valeri et al., 1988), CPDA-1 and
CPDA-2 (Moore et al., 1982; Moroff et al., 1984b), but
not for rats cells. In the presence of excess substrate
(CPDA-1), it appears that ATP production continues for
some time in rat and human RBCs under storage
conditions despite being processed and stored at 4 8C
where glycolytic activity is substantially reduced.
Regeneration of ATP and DPG
The rejuvenation studies were carried out to assess the
metabolic function of the RBC during storage. ATP and
DPG are products of the glycolytic pathway with DPG
produced by a branch of glycolysis, the Rapoport
Leubering shunt (Sohmer & Dawson, 1979). Regeneration of ATP and DPG by incubation of stored RBCs in a
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299

solution containing phosphate, inosine and pyruvate

(IPP) was first observed over 30 years ago (McManus &
Borgese, 1961). Since that time, various combinations of
phosphate, inosine, pyruvate, glucose and adenine have
been assessed for this purpose. In this investigation, we
used a solution consisting of phosphate, inosine,
pyruvate and adenine (PIPA). Aside from supplying
the substrates required to produce ATP and DPG, it is
also necessary for these compounds to pass through the
RBC membrane into the cytoplasm to supply the
glycolytic pathway. After 29 days of storage, human
RBCs demonstrated a full capacity to produce the
organic phosphates, indicating that the glycolytic
pathway in stored human RBCs is intact. These results
are in complete agreement with previously published
values for regenerated ATP and DPG in human RBCs
(Valeri et al., 1984).
By contrast, when incubated with the rejuvenation
solution, rat RBCs did not appear to produce DPG and
were only marginally capable of producing ATP
regardless of the duration of storage (Fig. 3). It is not
surprising that there was little or no production of ATP
and DPG in the second half of the storage period since
the viability of rat RBCs was significantly reduced
compared with human cells. Even early during storage
(, 8 days) in CPDA-1, rat RBCs had consistently
higher LDH levels than human cells, indicating some
degree of RBC lysis. Failure of early stored rat RBC to
rejuvenate to baseline or supranormal levels of ATP and
DPG suggests that CPDA-1 may not be optimal for rat
erythrocyte metabolism. The inability to synthesize
DPG with PIPA may also be explained by the findings
of Duhm (1974), who conducted a comparative study on
DPG production in the RBCs of various mammalian
species. Duhm observed significant species-specific
differences in the ability of RBCs to produce DPG
following incubation with a PIPA solution. While
human, mouse and rabbit RBCs readily manufactured
DPG, RBCs from pigs, dogs, cats, sheep and rats did not
produce this compound. In the case of the rat, Duhm
reported that membrane permeability to inosine was
poor and significantly less inosine entered the RBC
compared with human cells. Thus, the amount of
substrate for ATP and DPG was reduced compared to
human cells.
Another important finding reported by Duhm was that
the activity of phosphoglycerate kinase in rat RBCs was
very high. Phosphoglycerate kinase catalyses the
conversion of 1,3-diphosphoglycerate to 3-phosphoglycerate through which ATP is generated during glycolysis. Phosphoglycerate kinase also competes with
phosphoglycerate mutase for 1,3-diphosphoglycerate
which produces 2,3-diphosphoglycerate. As a consequence, the higher activity of phosphoglycerate kinase

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M. S. d'Almeida et al.

in the rat RBC may preferentially shunt 1,3-diphosphoglycerate to 3-phosphoglycerate, thereby possibly
explaining the lack of effect of the rejuvenation solution
on DPG production in rat RBCs. RBC metabolism is
also known to be influenced by pH (Hogman &
Meryman, 1999). Activation of enzyme diphosphoglycerate phosphatase at lower pH is primarily responsible
for loss of 2,3-DPG. The affect of pH and temperature
changes in CPDA-1 on specific rat RBC enzymes has
not been studied, but probably accounts for observed
species-specific differences in RBC integrity.

Post-transfusion viability of stored and 51Cr-labelled rat

RBCs
Units of stored RBCs contain a mixture of RBCs of
varying age, including cells that will be greater than
120 days when eventually transfused. As the duration of
storage increases, RBCs consume and deplete nutritional
stores in the preservative media and may be affected by
the damaging effects of cytokines released from costored leucocytes (Andreu, 1991). Thus, it is not
surprising that a population storage of altered transfused
cells will immediately be cleared from the circulation
upon transfusion. The therapeutic effectiveness of
transfused stored blood is determined, in part, by the
24-h PTV of the transfused cells. The established
standard for PTV has been arbitrarily set at 75%
(American Association of Blood Banks, 1999). We
used the single isotope technique proposed by Moroff
et al. (1984a). To account for the shorter circulation time
in rats compared with humans, we started blood
sampling at 25 min after we injected the labelled
RBCs, rather than 5 min after the injection. As a result,
we should have a reasonable estimate of the initial
amount of injected label, i.e. extrapolation to t 0 for
baseline radioactivity.
The results from the rat cells revealed large
differences compared with standards for human cells.
As can be seen in Fig. 4, rat blood stored for 1 week had
a PTV of approximately 80%, whereas by 15 and
29 days PTV had dropped to 26% and less than 5%,
respectively. The poor PTV we observed for the rat
RBCs was surprising since a previous study using rat
blood stored in CPDA-1 over a 28-day period reported a
PTV of 80% (Kovacs et al., 1993). It is not clear why
this discrepancy exists, and it is highly unlikely that any
differences in technique could be responsible for the
disparate results.
Furthermore, the biochemical data of rat RBCs during
storage also support a more rapid loss of cellular
integrity in keeping with a more rapid clearance
indicated by a shorter PTV. Rat RBCs stored for

approximately 1 week have a PTV equivalent to human

RBCs stored for 4 weeks.
It has been reported that rats possess the ABO blood
typing as in humans (Keeler, 1967). Consequently, it
may be argued that a potential exists for reactions upon
transfusion from unmatched donor blood and that this
may affect our PTV results. The cross-matching,
however, showed no evidence of major blood group
incompatibility. Furthermore, since rats were only
exposed once to transfused cells, an anamnestic immune
response would not have occurred. From our previous
experiences over several years of studying the cardiovascular and microvascular effects of blood transfusion
in our rodent model of transfusion efficacy, we have not
seen evidence of transfusion-related haemolytic reactions in any of our investigations. As a result, we do not
feel that donorrecipient transfusion incompatibility
interfered with the PTV analysis in this investigation
and that the consistent differences in PTV that we
observed were storage-related.
A possible reason for the more rapid decline in rat
PTV compared with reported values for human RBCs
may be related to the levels of ATP in the RBC. In
human RBCs, there is a poor correlation between ATP
and PTV except at very extreme levels of ATP depletion
(Dern et al., 1967). Total adenine nucleotide pool (ATP,
ADP, AMP) rather than ATP alone is likely to be a more
important determinant of PTV and thus isolated
measurements of ATP in rat cells may not accurately
predict PTV (Hogman & Meryman, 1999). ATP in
human cells, however, does not usually fall to the
extremely low levels observed in the rat cells. It is
therefore likely that the profound ATP depletion in rat
RBCs reflects total adenine nucleotide pool depletion
and may contribute to poor PTV. It has been postulated
that in human cells, ATP depletion may lead to
activation of other secondary mediators of injury such
as intracellular Ca21 and or the reduction in protein or
lipid kinase phosphorylation which may be important in
membrane integrity (Card, 1988).
Membrane deformability
In this investigation we used membrane displacement as
an index of membrane deformability. It is important to
note that only a small segment of the membrane is
aspirated into the pipette tip when using the aspiration
technique. Consequently, the effects of surface area to
volume ratios and cytoplasmic viscosity on overall RBC
deformability are largely eliminated and the results refer
to the membrane-specific changes that have occurred.
Our results indicate that both rat and human RBCs
undergo time-dependent decreases in membrane
deformability. This is consistent with the work of
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Storage lesion in rat and human red cells

Haradin et al. (1969), who demonstrated a time-related
decrease in human red cell deformability using the
viscosity and filtration techniques. More recently, using
cell transit analysis, Friedlander et al. (1998) observed
that transit time of stored human RBCs decreased with
storage duration, suggesting a decrease in RBC deformability. Our investigation also demonstrated that rat
RBC deformability declined at a faster rate than the
human cells; rat RBC deformability decreased by 54%
after 1 week of storage compared with 34% in the
human cells after 4 weeks.
This study was not intended to determine the
mechanisms of decreased membrane deformability;
however, loss of deformation can occur by three
mechanisms: membrane stiffening, reduced surface area
to volume ratio and or increased internal viscosity (Card,
1988). It is also apparent that these membrane changes
are reversible since, in the present investigation,
incubation with a rejuvenation solution returned
membrane deformability to the same levels as that of
the fresh controls in rat and human cells. Improved
filterability and decreased viscosity of stored human
RBCs (Haradin et al., 1969) as well as a reversal of
echinocyte morphology to discocyte morphology has
also been demonstrated to occur in response to stored
red cell incubation with adenosine (Laczko et al., 1979).
It is also known that incubation of stored RBCs at 37 8C
can restore RBC morphology and deformability by
energy-dependent processes but this effect was not
studied independently from rejuvenation processes with
PIPA solutions.
Aside from being able to determine a mean value for
membrane displacement, one of the major advantages
that the micropipette technique has over filtration/
viscosity methods is the ability to obtain information
about individual cells. For example, the histograms in
Figs 57 depict the proportion of analysed cells
possessing a specific membrane displacement. As can
be seen in Fig. 5, the time-related changes in rat RBC
membrane displacement were characterized by an
increase in the population of cells possessing very poor
deformability, i.e. a widening of the frequency distribution to the left. This was most evident on day 7 in which
a large subpopulation of cells had little or no membrane
displacement upon micropipette aspiration. This is in
contrast to the human cells that maintained a normal
distribution of membrane deformability around the
mean, despite significant decreases in this value
(Fig. 6). From Fig. 7 we can also see that treatment
with the rejuvenation solution restored membrane
deformability in all cells rather than just a specific
population of cells. This is especially evident in the rat
cells since the large population of very poorly deformable cells was no longer present and membrane
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301

displacements returned to a normal and narrow distribution around the mean.

Implications
The influence of storage-related changes in DPG
concentrations on the ability of the RBC to deliver and
unload oxygen to the tissues has long been an issue of
investigation. One of the difficulties associated with
studying this is the great number and progressive nature
of the changes that occur with RBCs during storage.
Investigators have studied the effect of decreased DPG
levels or supranormal levels of this compound (Valeri
et al., 1975; Spector et al., 1977; Dennis et al., 1978),
but other changes occurring in the RBC or their potential
effects have rarely been considered. Consequently, in
light of our results, previous studies that examined the
cardiovascular consequences of stored blood transfusion
must be carefully reinterpreted. For example, in rats,
Fitzgerald et al. (1977) found that systemic oxygen
consumption did not improve following a transfusion of
rat RBCs stored for 28 days when it had been reduced
by severely limiting oxygen transport. Our results
indicate that rat cells stored for 28 days do not
accurately represent the condition of human RBCs
stored for 28 days and that the inability of the stored rat
RBCs to improve systemic oxygen consumption was
most likely due to loss of post-transfusion RBC
viability. It is important to note that while extrapolation
to a clinical scenario based on animal studies using
stored blood should be done cautiously, it does not
nullify the clinical findings by Dietrich et al. (1990) and
Marik & Sibbald (1993), which demonstrated that
transfusion of standard donor blood from hospital blood
banks did not improve oxygen consumption in the
critically ill. In the latter study, failure to improve tissue
oxygenation measured by gastric intramucosal pHi was
associated with prolonged duration of storage.
Our study has demonstrated that incubation of human
and rat RBCs with the rejuvenation solution improves
membrane deformability, but, unlike human cells, rat
RBCs regenerate ATP and not DPG upon this treatment.
Rejuvenation of rat RBCs stored for approximately
1 week should therefore produce a cell with restored
deformability, normal ATP concentration and a selective
DPG lesion. These results raise the possibility that
rejuvenated rat RBCs may represent a potential research
tool with which to critically assess the role of DPG, and
possibly membrane deformability on transfusion efficacy.
CONCLUSIONS
The primary objective of this study was to determine a
storage duration for rat RBCs that produces a storage

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M. S. d'Almeida et al.

lesion similar in extent to that of human RBCs near the

end of the shelf life. Rat RBCs undergo a more rapid
rate of deterioration under storage conditions compared
with human RBCs, leading to striking differences in
PTV and ATP depletion between rat and human RBCs.
This may be due to the shorter life span of roughly
60 days for rats compared with 120 days for human
cells. Rejuvenation of human and rat RBCs reverses
significant decreases in membrane deformability and
restores ATP and DPG concentrations in human cells,
but only ATP in rat cells. Based on these biochemical
and functional assessments, the results of our investigation indicate that storage of rat RBCs for 1 week in
CPDA-1 produces a storage lesion similar in extent to
human RBCs stored for 4 weeks.
ACKNOWLEDGMENTS
We thank Leslie Gray-Statchuk, Gary Morrissey, Janice
Walters, Kathy Copley and the staff of the LHSC
Central Biochemistry Laboratory and Blood Bank for
their assistance in this project. This research was
supported by a Canadian Blood Services Intramural
Grant and London Health Sciences Internal Grants.
M.S.d'A. is a recipient of a Canadian Red Cross Career
Development Fellowship.
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