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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
Key words: 2,3 diphosphoglycerate, adenosine triphosphate, membrane deformability, post-transfusion viability, storage lesion.
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M. S. d'Almeida et al.
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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.
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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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.
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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. 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).
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.
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.
q 2000 Blackwell Science Ltd, Transfusion Medicine, 10, 291303
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M. S. d'Almeida et al.
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
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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.
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