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Platelet
Storage
at 22#{176}C:Effect
of
of Agitation
on Morphology,
Viability,
and Function
In Vitro
Type
By Stein
Recovery
let
in vivo
morphology,
Holme,
after
51Cr
and
platelet
Kalpana
labeling,
Vaidja,
was
1 012
in
between
platelets/liter.
bags
(PVC)
72
kept
The
constructed
bags
tal shaker
during
or
HERE
nique
No
were
at
placed
a ferris
1 .6
thrombin
stored
22#{176}Cfor
on
wheel
a horizon-
for
significant
agitation
to
changes
Murphy
but
had
the
same
viability
and
morphology
as PC in bags constructed
of
PVC. Maintenance
of normal
platelet
morphology
as determined
by phase-contrast
microscopy,
extent
of shape
change
response, and the size distribution
according
x
polyvinylchloride
(PE)
were
storage.
and
PC
of
or polyethylene
hr. The
T
0.8
Scott
pH or platelet
count were observed
during
storage.
PC stored on the wheel
showed
moderate
loss of viability
and a marked
deterioration
of platelet
morphology
and
aggregation
compared
to the shaker.
PC
stored on the shaker
in bags made
of PE
showed
better aggregation
with ADP and
plate-
aggregation
were
studied
with
platelet
concentrates
(PC) stored
for transfusion
under
carefully controlled
conditions.
PC were
prepared
to a final volume
of 50 ml from
whole
blood anticoagulated
with citratephosphate-dextrose
(CPD).
The platelet
count
and
in
the
Coulter
recovery
Counter
correlated
with
in vivo.
IS at present
considerable
controversy
for storing
platelet
concentrates
(PC)
concerning
the
for transfusion.’
optimal
Some
techof the
disappointing
results
obtained
with storage
at 22#{176}C(room
temperature)
may
result
from
inadequate
attention
to technical
detail.
For example,
some
form
ofgentle
agitation
is required.
In addition,
we have emphasized
the importance
of
extreme
preventing
changes
changes
in
result
from
unfavorable
the PC and the surrounding
tween
volume
and
platelet
count
of the
constructed
of materials
study we will show that
is itself
an important
if storage
pH
conditions
air and
PC
and
variable
in the
may
is to be successful.2
exchange
be prevented
by agitating
with increased
with maintenance
in vitro.
Previous
studies
have
phology
during
storage
for
the
permeability
of constant
maintenance
of
These
CO2 and 02
by controlling
PC
within
bethe
containers
for gas exchange.
In this
pH the type of agitation
of platelet
viability
and
func-
tion
falls below
6.0
lets takes
place,
PC prepared
From
the
Center,
for
Supported
A ddress
1015
©
Blood,
for
1-lematologic
28,
t’3 NIH
Grants
WalnutSt.,
1978
by Grune
& Stratton.
Vol. 52, No. 2 (August),
Thomas
accepted
HL
11047
requests:
Dr.
Presbyterian-
University
Research,
1977,
Philadelphia.
normal
discoid
of viability.
April
and
Stein
CA
of
Jefferson
14,
morpH
of the platetransfusion.2
upon
of
Medical
University,
Pennsylvania
School.
Philadelphia,
and
Medical
the
Carde:a
Pa.
1978.
12397
Holme.
University
Pennsylvania
and
Carde:a
Pa.
19107.
Inc.
0006-4971/78/5202-0418$02.00/0
1978
If
or stored
at 4#{176}C
show
only spherical
in vivo.39
In this study
we examined
the
Laboratory,
of Medicine.
October
reprint
maintenance
of
for maintenance
above
7.6 a disc-to-sphere
transformation
in marked
loss of recovery
in vivo
Research
Department
Submitted
that
essential
EDTA
as anticoagulant
have reduced
survival
Hematology
the
Foundation
or rises
resulting
with
also
and
platelets
indicated
may be
b.t’ NIH
Contract
Foundation
NHL
for
1-70-2212.
Hematologic
Research,
425
From www.bloodjournal.org by guest on June 18, 2017. For personal use only.
426
HOLME
relationship
after
between
maintenance
under
infusion
temperature
was
anticoagulant.
To do this
phologic
we
as measured
size
distribution
maintenance
methods
platelets.
by
of normal
morphology
pH
did
not
citrate-phosphate-dextrose
developed
of the
discoid
where
of storage
22#{176}C,and
integrity
response
platelet
of normal
conditions
that
We
discoid
quantitatively
shape
that
as judged
extent
of
and the
Counter
by
with viability.
Marked
decrease
in function
in vitro
age.8”#{176}’4In this study
we will show
that
the
gregating
agents during
storage
at 22#{176}C
varies
was
used
reflected
the
studies
Coulter
a
viability
significantly,
(CPD)
found
light
transmission
according
to
and
vary
ET AL.
as
the
mor-
shape
change
dispersion
correlated
phase-contrast
of the
with
microscopy
and
MATERIALS
PC
were
prepared
to a final
used as primary
anticoagulant.
(Hemoflex
from
and
bags
(2)
l0
in air,2
The
allow
from
gas
the
is identical
(Model
F).16
switch,
D = 64.
morphology
mated
platelets
in
as
counting
the
probability
window
51Cr
into
(Fig.
at
The
recovery
the original
Previous
when
the
of discoid
was
perature
I).
in
A straight
and
shape
and
with
from
shown
shape
aggregation
autologous
the
30,
was
size
frequency
divided
of
slope
day
all
response
after
by
phlebotomy)
storage,
plasma
to
a
were
number
Viability
was
were
labeled
(PPP)
platelet
some
to
count
of
0.3
it rep-
particles
on
with
log-normal
that
window
the
84. 1 3,,
as
with
and
SICr
x 10I2/liter.
as
cumulative
percentage
reinfused
may
be
maximal
reversed
recovery
taken.
at
at
defined
measured
were
dis-
number
was
undisturbed
by
windows.
because
plotted
precautions
(stored
were
obtained
of
of platelets
allow
for
a
platelets
l00
at
as
ob-
chosen
deviation)
window
classified
were
the
esti-
appearance
indicating
as
platelet
forms
consecutive
the
be
cases,
that
disc-to-sphere
transformation
change
is removed.19’20
In order
platelet-poor
of
60
determined
line.
disc
percentages
standard
the
current
was
to
and
in
was
Counter
ellipsoid
arbitrarily
50,
Coulter
aperture
platelet
seven
The
gensystem
:
Otherwise,
considered
a rack
heat
(this
hundred
or
on
of
One
than
(geometric
the
A
(2)
of
Counter
obtained
platelet
studies.
described
ferris
wheel
a platform
by
percentage
PRP.
40,
of
of storage,
Coulter
and
t
survival.
PC stored
for 3 days
volunteer
as previously
described.2
studies
have
factor
inducing
diluted
line
the
edge-on.
fresh
20,
as
cycles/mm
the
6 was
Ill.),
atmosphere
placed
completion
circular
greater
normal
13,
90
and
spherical.
with
dispersion
reflects
or
window
for
Median
dispersion
vivo
normal
above
absorption
the
viability
viewed
size
prevent
attenuation,
and
thick,
Grove,
controlled
were
was
At
generally
when
10,
cumulative
50’,,
a
with
numbers
used.
was
(PE)
inches
an
containers
to
were:
discoid
in
were
microscopy15
distributions
plateau
frequency,
number
frequency.’
sizes
the
log-normal.17”8
cumulative
50(o
with
window
paper
were
platelets
agitation
and
as
appearance
size
of
CPD
in air only.
the
and
microscopy
0.003
Morton
used:
(I)
a motor-driven
5 cycles/mm),
and
air
was
presenting
of
of
tube
that
polyethylene
cm,
stored
atmosphere
Counter
classified
Platelet
of particles
than
tnibutions
were
readily,
percentage
Coulter
aggregometer,
over
a rodlike
Hanker9).
were
out
(shaker):
and
(I)
9 x 22
bags
phase-contrast
oil-phase
fields
PE
CO2
speed
except
Laboratories,
carried
the
aperture
Counter,
midpoint
and
the
in
was
the
described2
containers:
measuring
Fenwal
surrounding
by
of
kept
Mich.)
by Slichter
by
and
greater
shaker,
observed
spherical.
the
the
SO-Mm
rolled
number
resented
with
determined
Coulter
face-on
Arbor,
standard
A
random
it
classified
size
settings
to
when
served
The
Ill.),
of
of agitators
were
(rate
of agitation,
Ann
described
was
was
prior
disc
one
count
types
from
22#{176}C,and
of the
previously
containers
two types
studies2
exchange
motor
to the
platelet
storage,
storage
previous
as
in two
PL-l46
PC
been
reported
after
storof responses
in vitro
to agthe conditions
of storage.
METHODS
Chicago,
thick.
in PVC
(Eberbach,
more
crated
PC
of
shaker
inches
ml
stored
(plastic
storage
During
used
in
horizontal
0.015
while
of 50
were
Carbide,
(PVC)
temperature
previously.2
(wheel)
as
The
Union
12 x 15 cm,
CO2
to
PC
polyvinylchlonide
measuring
volume
AND
has
loss
with
The
room
This
PC
temsus-
From www.bloodjournal.org by guest on June 18, 2017. For personal use only.
PLATELET
STORAGE
AT 22#{176}C
427
>-
0
0
z
Ui
20
Ui
40
L1
60’
_I
C
parentheses.
Standard
‘
and
little
PC
For
was
pH
pH
were
by
were
albumin
was
gel
(Sigma
ranged
with
with
with
7.4
storage
on
and
all
The
pH
buffer
to
PC
2B
buffer
30
NUMBER
prior
to
were
column
of the
light-transmission
all
in
platelets
was
7.6.
because
PPP.
were
by
dextrose
platelet
studies
autologous
described
0.l’
buffer
a
diluted
as
5060
the
in
Mich.)
containing
obtain
40
comparable
Detroit,
a Sepharose
Mo.).
the
hr
was
Davis,
Tyrode
I
and
20
Lages
and
The
et
0.35
gel
suspension
separated
filtered
with
al.21
bovine
platelets
a count
of
x 1&2/liter.
Shape
change
response
Payton
Dual
scnibed
previously.
Platelet
added
and
Channel
change
(diluting
a maximal
were
quantitated
(SC)
given
by
b/a.
independent
Complete
extent
a.
ratio,
of the
settings
response
of response
in
was
(Fig.
sec
ml
later,
a
by
PCS,
in
A tracing
the
time
using
as
de-
)
was
10
in
a
the
trans-
was
shape
the
added,
change
is
platelets,
of
tracing
reflected
light
MM)
transmission
light-transmission
oscillations
of
of
light
5 mM
increase
Extent
t.
inherent
the
without
an
transformation
and
studies
Canada)
concentration
concentration
the
disc-to-sphere
observing
(final
producing
(final
b,
Scarborough,
0. 1 ml
ADP
aggregometer
light-transmission
2):
EDTA
,
transmission,
assured
reached.
).
of 0.9
reflects
of the
the
until
presence
was
PCS.
maximal
of
spherical
only.22
Platelet
aggregation
(Fig.
2B).
respectively.
The
increase
in
aggregating
agent
was
measured.
various
platelet
donors
count
of
respective
the
among
various
various
PCS
flects
IS
which
was
2A
by
Associates,
as follows
(Fig.
light
measured
(Payton
suspension
Then,
decrease
This
SC
platelets
stirred
effect),
were
Module
response
to a continuously
mission
aggregation
Aggregation
They
shape
giving
the
response
samples
the
PC
13
WINDOW
37#{176}Cfor
to
(Parke,
St. Louis,
diluted
at
7.2
thrombin
calcium-free
Chemical,
and
kept
during
of
SHAKER
.
#{176}
10
from
observed
filtration
eluted
collected
0.3
aerated
PCS
studies
plasma
Platelets
the
change
aggregation
from
well
of
PC
4.3Mm3
Latex particle
pension
(PCS)
WHEEL
‘#{176}
at
The
(I.81)
PC
PE
90
with
volume
of 4.3 pm3
peaks
window
number
marked
by arrow.
studies.
-0
6---
Fig.
1.
Size
analysis,
Coulter
Counter
(see
Materials
and
Methods). Typical
results for fresh PC and
for
PC stored
3 days on wheel
and
shaker.
Calculated
dispersions
in
FRESH
(1.74)
(2.20)
S
0-
80
PPP
platelet
differ
same
control
values
platelet
dilution
In
also
to
it is questionable
“degree
PCS
those
of
of serial
(in
dilutions
(PCS
mV)
(Fig.
equivalent
to
count,
2C).
the
differences
in
Thus
or
As
adjusted
sec
after
had
scattering
when
not
the
was
in
and
PCS
then
converted
into
light
transmission
D
produced
‘,
a
properties
of
light
transmission
re-
evaluation
the
the
from
transmissions
of
of
in autologous
against
of
PCS
light
transmission
plotted
20 mV,
standardized
to
light
transmission
adsorbance
in
for
2 and
addition
that
were
in
increase
light
to
the
observed
inherent
standard
compare
were
the
a given
nonaggregated
percent)
D
were
75
we
light
a primary
to
of the
increase
PPP
the
studies
themselves.
chose
and
even
when
all of them
due
not
only
to variations
whether
instead
PCS
D during
preliminary
aggregation.”
we
from
transmission
This
was
populations
suspensions
mission
but
signals
transmission
differed
in light
of 0.3 x l0’2/liter.
of various
with
The
light
the
PPP.
respective
giving
by
aggregation
aggregated
Dilutions
light-trans-
the
the
percentage
aggregation.
of
From www.bloodjournal.org by guest on June 18, 2017. For personal use only.
HOLME
428
A
ET Al.
C
EDTA
z
0
U)
b
0’
-C,)
(I)
z
4
At
4
C
a-
B
Pcs1
2(
D
COUNT
0!
Ifl
,o
100
2mV
80
z
60
0
U)
v)
w
D’
4O
-U)
4
I.-
20
I8mV
ADP
UM
log
Fig. 2.
Determination
of extent
of platelet
shape
change
response
and rate of aggregation
(see Materials
and Methods).
(A) Platelet
shape change
response.
Extent
of shape
change
given
by b/a,
where
a is diluting
effect
of 0.1-mI
EDTA
addition.
(B)
Platelet
aggregation.
Increase in light
transmission
during
75 5cc, D, was measured.
(C) Light
transmission
(in mV)
measured
for serial
dilutions
of PCS in autologous
PPP. D is then
converted
to D’ , giving
percentage
of platelet
dilution
equivalent
to increase
in light
transmission
produced
by aggregation.
(D) Rate of aggregation,
D’,
measured
at four to six different
ADP concentrations
versus log concentration
of ADP, giving
a logarithmic
dose-response
curve.
ED50 estimated
as
concentration
of ADP ( .iM ) producing
50% of maximal
response.
This
additional
donor
to
mean
SD
of
44
get
during
give
an
Maximal
response
increase
in
centration
the
of
ADP,
concentration
Osmotic
Informed
local
response
of
Human
Department
was
giving
reversal
response
consent
was
Investigation
of Health,
units),
the
was
obtained
Rate
was
of
character
mean
of
a fivefold
the
of
aggregation
freshly
D
‘
results
made
was
PC,
6 1,
50
D
‘
Committees
normal
in accord
and
Welfare.
was
curve
ofa
maximal
as described
all
deteriorating
four
with
from
D
had
a
with
an
six
different
in ADP
plotted
a
smaller
(Fig.
concentration
against
2D).
response
ADP
concenfailed
logarithmic
ED
was
to
con-
estimated
as
response.
by Valeni
donors
aggregation
to
increase
dose-response
from
the
with
aggregation
measured
Education,
of
With
the
determined
producing
obtained
while
when
a logarithmic
zM)
variability
standardization.
measured.
aggregation
was
the
this
about
response.
ofADP(in
by
chart
information
rate
because
reduced
(arbitrary
maximal
additional
justified
greatly
13 (SD)
±
storage,
trations.
seemed
was
of 7, when
To
the
effort
donor
and
et al.23
participants
assurance
filed
after
with
approval
and
approved
of
the
by
From www.bloodjournal.org by guest on June 18, 2017. For personal use only.
PLATELET
STORAGE
AT
429
22#{176}C
RESULTS
The
freshly
of 7.0-7.2.
made
We
PC
did
had
not
a platelet
observe
storage.
The pH ofthe
PC after
were no significant
differences
conditions
of 0.8-1.6
count
any
significant
3 days
in the
ofstorage
final pH
lO’2/liter
x
change
and
in platelet
ranged
for PC
count
from
stored
a pH
during
6.8 to 7.4. There
under
the four
studied.
Table
I shows
percentage
PC stored
for 72 hr under
in percentage
crease
recovery
varying
recovery
in vivo
conditions.
in vivo
was
and
A
survival
t
statistically
observed
after
of
fresh
PC
significant
and
de-
PC stored
on
storage.
the shaker
yielded
recovery
results
superior
to the wheel
when
the results
for
PE and
PVC
on the same
agitator
were combined.
When
PE was compared
with PVC either
on the shaker
or on the wheel,
no significant
differences
were
seen.
For
no significant
differences
were observed
in PE on the shaker
versus
the wheel,
in the same
donor
so that
comparison
survival
could
be
analysis
nificant
in vivo
(p
within
the
(p
relation
and
made
showed
differences
the
(p
the
recovery
for
the platelets
from
others.
The
percentage
shape
change
periment,
on
vivo
some
the
and
shaker
survival
individuals
of
platelets
found
it was
Table
volunteer
(Fig.
that
1 . Recovery
are
that
the
In Vivo,
and
Recovery
i,
better
storage
type
than
disc-to-sphere
of
and
that
those
of
transformation
could
Maximal
0.86
=
and
by the extent
In a preliminary
change
ADP for Fresh and
(%)
for
microscopy
aggregometer.
of shape
Survival,
EDyj With
suited
Rate
Stored
be used
of
ex-
to reflect
of Aggregation
PC
0
t (days)
EDy,
±
3 (10)
4.1 ± 0.1 (10)
61
±
2 (15)
PE
44
±
3 (7)
3.7
32
±
4 (22)
PVC
44±4(5)
3-day
This
the result
on the wheel:
r
respectively.
This
suggests
underwent
oil-phase
with the
extent
t
and
53
Fresh
3).
of
was estimated
by
response
as observed
storage
during
<
result
in
between
0.94
individual
the shaker
most
clearly.
Statistically
sigwith
the paired
i test:
percentage
recovery
survival
(p < 0.05),
maximal
rate
of aggregation
0.01).
For individual
donors,
there
was a high
cor-
t
ED50
same
superiority
were obtained
0.01),
<
0.005),
<
using
an unpaired
six pairs
of studies
of the two
methods
t
test.
For storage
were carried
out
1
2.8 ± 0.3 (15)
storage
Shaker
PE
44
PVC
+
±
±
0.3 (7)
3.9±0.1
2 (12)
3.8
±
0.2 (12)
6.7
30
2.1 (22)
±
11.2±2.5(10)
25±3(10)
(5)
±
2 (32)
8.9
1.6 (32)
±
Wheel
PE
37 ±
6 (7)
3.5
±
0.3 (7)
8
±
3 (1 1)
-
PVC
33
±
4 (12)
4.3
±
0.3 (9)
7
±
2 (9)
-
34
±
3 (19)
3.9
±
0.2 (16)
7
±
2 (20)
-
of
studies
in parentheses.
PE
Mean
1 SEM;
±
recovery
wheel
PVC
+
in viva,
(p
<
number
Fresh
0.025).
PC vs 3-day-stored
Maximal
0.005); PE + PVC, shaker
Fresh PC vs 3-day-stored
for ED
could
be obtained
rate
vs PE
+
PE + PVC,
with
of
PE +
Statistically
PVC,
aggregation,
(p
PVC, wheel
shaker
0
(p
PC on the wheel,
<
‘ ,
significant
(p
shaker
Fresh
<
0.05);
differences
PE
PC vs 3-day-stored
+
PVC,
(Student’s
shaker
PE + PVC,
0.005);
PE, shaker
vs PVC, shaker
0.005);
PE, shaker vs PVC, shaker (p
<
since many
of the PC showed
vs PE
shaker
t test),
+
PVC,
(p
<
( p < 0.05).
ED50:
< 0.05).
No results
no aggregation.
From www.bloodjournal.org by guest on June 18, 2017. For personal use only.
430
HOIME
RECOVERY
Tl/2
D’
Fig. 3.
Recovery
in vivo, t survival,
maximal
for PC stored for 3 days in PE bags. Lines connect
and wheel.
In two studies
with PE on the wheel,
PC showed
no aggregation.
the
(Fig.
of
percentage
4).
This
was
between
percentage
the extent
of shape
in the size distribution
probability
paper.
discoid
platelets
confirmed
in
in a given
penior
(Fig.
The
5A).
for
the
results
shaker
as
for
opposed
all
to
EDso
sample
when
we
of
platelet-rich
found
a high
PC as estimated
by phase
(Fig.
5B). Figure
1 shows
PC stored
for 72 hr when
days
showed
an increase
Again,
there was a good
correlation
between
scopic
estimate
of percentage
of discs
(Fig.
correlation
was better.
The osmotic
reversal
tion
four
the
Al.
rate of aggregation
with ADP, and ED50 for ADP
studies carried
out on same individual
on shaker
no ED50 results could
be obtained
because
the
practice
of discs in stored
change
response
of platelets
in
PC stored
for 3
ET
this measurement
5C),
although
reaction
showed
measurements
microscopy
and
typical
changes
plotted
on login dispersion.
and the microthe shape
change
the least correla-
were
( percentage
wheel
plasma
correlation
significantly
of
discs,
p
su<
0.05;
z
100
0
Fig.
change
60
i
4.
Extent
of
shape
with ADP and percent.
age of discoid platelets.
PC that
4
I
V
.-
I
2
;i
V
I
2
sic
S
20
showed
only
Stored
spherical
platelets
was
mixed
fresh PRP in various
with
ratios.
Platelet
count
was
0.3 x
10’2/Iiter.
Extent
of
shape
change
response
measured
as
described
in Fig. 2A.
From www.bloodjournal.org by guest on June 18, 2017. For personal use only.
PLATELET
STORAGE
AT
I80
0
22#{176}C
431
r #{176}O.5l
r
0.82
::
u:;:
#{163}
20
0
0
O.67
r
#{176}
:
.:
00
-,
A
B
,
0
25
50
OSMOTIC
75
0.25
EXT
0.50
0.75
SHAPE
2 2
SHAKER
WHEEL
#{163}PVC,
SHAKER
CHANGE
2.0
1.8
DISPERSION,
COULTER
fO
Fig. 5.
Platelet
morphologic
integrity
as determined
by observations
quantitatively
reflected
by osmotic
reversal
reaction,
extent
of shape
with
Coulter
Counter.
All three of the latter
measurements
correlated
centage
of discs by phase microscopy
as reflected
by correlation
coefficients
osmotic
OPE,
#{149}
PE,
C
00
REVERSAL
0
‘
reversal,
p
0.05;
<
shape
of
extent
change,
by phase microscopy
and
change,
and dispersion
significantly
with
perr.
p
0.01;
<
dispersion,
0.01).
p <
There
was
a good
found
between
in vivo.
Median
creased
during
2%
PC
for
of shape
change
(Fig.
6). The
the
three
and
highest
osmotic
tests
the
l7
shaker.
±
The
2
(±
1 SEM)
differences
in
vitro,
reversal
correlation
the Coulter
Counter
dispersion
and
the
platelet
size as reflected
by the median
storage:
on
between
correlation
Counter
dispersion,
extent
the viability
in vivo results
for
were
PC
percentage
window
on
Coulter
reaction,
(r = 0.83)
the
recovery
number
de-
wheel,
statistically
and
was
1l
significant
±
(p
<
80
t#{176}038
0
PE,
r#{176}06I
0
>-
60
PE.
A
0
0
A
SHAKER
PVC,SHAKER
#{149}
#{149}:
#{149}
40
#{163}*
00
#{149}
0
#{149}.
a
S.
0
#{149} #{149}
O
0
0
#{149}
#{163}
#{149}#{149}
0
0
0
#{176}
,
.
#{149}
Ui
(.5
r’0.83
WHEEL
0
0
0
0
20
,4:1
0
I
I
25
50
OSMOTIC
I
75
REVERSAL
,B
100
.
0.25
EXT.
0.5
SHAPE
0.75
CHANGE
.
2.2
DISPERSION,
2.0
1.8
COULTER
0/0
Fig. 6.
Correlation
between
three
tests in vitro:
osmotic
reversal,
and dispersion
with Coulter
Counter,
and percentage
recovery
in vivo.
viability,
with dispersion
having
highest
correlation
coefficient
r.
extent
of shape
change
All tests correlated
with
From www.bloodjournal.org by guest on June 18, 2017. For personal use only.
432
HOIME
Table
2.
Rate
of Aggregation
With
bin Aft er Gel
Throm
Filtration
3-day
Thrambin
.
with
35±12
19±11
61±9
59±3
52±6
62±9
number
Significant
median
size
were
and
correlated
in
59±4
of
studies
superior
PVC
was
recovery
the
also
survival.
t
wheel
conditions
p
0.01
<
4±4
container
found
and,
48,
had
significantly
the wheel
The
obtained
container.
marked
on the
rate
Even
the
percentage
None
=
there
to
found
recovery
to
by the
ED
results,
when
in
data
the
decrease
the
of
studies
in
tests
vitro
in vivo
institution
in
for
PC
reported
of
the
for
studies
on platelet
aggregation
shaker
showed
the best
rate ofaggregation
was
a 2.3-fold
greater
concentra-
needed
to obtain
5O
of maximal
PC stored
in PVC on the shaker
compared
with
PE,
while
storage
on
effect
on the aggregation
response
in either
optimal
and identical
conditions
of storage,
there
was
a
the aggregation
results.
For example,
PC stored
in PE
a mean
of 32
±
l6
(SD)
(22 studies)
for maximal
under
compared
to 6l)
7
±
(15
studies)
for
At present,
we have no explanation
for such variation.
Aggregation
studies
with thrombin
showed
results
similar
(Table
2). Aggregation
responses
were superior
after
storage
opposed
to the wheel.
During
storage
of platelets
in PE bags
sensitivity
obtained
was
deleterious
variability
of
shaker
showed
of aggregation,
results
difference
0.57).
prior
in log dose)
was
with fresh
platelets.
aggregation
a marked
22±5
aggregation
(r
comparative
done.
were
as shown
poorer
had
18±8
significant
Marked
influence
of the various
conditions
of storage
was observed
(Table
1, Fig. 3). PC stored
in PE on the
maintenance
ofaggregation
response
to ADP.
Maximal
reduced
18±11
between
in vivo
No
were
tion of ADP
(0.40 increase
response
when
compared
(5)
5±5
17±8
the
No statistically
.
PVC
agitator.
percentage
on
these storage
in vitro.
In either
to the wheel,
correlation
with
PE (5)
55±5
in parentheses.
in PE or PVC with either
0.05).
stored
(6)
70±8
storage
vitro
PVC
0.5
PC on the shaker
between
Wheel
PE (6)
0.1
1 SEM;
±
PC
.
PC (8)
1.0
Mean
Stored
Storage
Shaker
No Storage,
(U/mi)
of Fres h and
ET Al.
was
thrombin
was no significant
decreased
change
when
in maximal
compared
rate
with
freshly
made
PC.
to those
for ADP
on the shaker
as
on the shaker
the
fresh
platelets,
but
of aggregation.
DISCUSSION
In this
equivalent
storage
stored
study
we
and
that
in
vitro
quality
microscopically
and
to show
technique
attempted
not
may
be
all
methods
an
important
to a horizontal
shaker.
We
storage
to the morphologic
to quantitate
changes
may
well documented
PC
that
transfusion
at 22#{176}C.Specifically,
had moderate
loss of viability
compared
during
These morphologic
First,
it has been
When
able
agitation
of platelets
for
on a ferris
wheel
function
of platelet
surfaces.24
were
are
agitated
these
changes
of
agitation
variable
we showed
and a marked
related
the
changes
storage,
the
that
PC
loss of
deterioration
we observed
objectively.
have
been induced
in two different
that
platelets
are activated
by
during
are
in the
platelets
are
ways.
foreign
likely
to
From www.bloodjournal.org by guest on June 18, 2017. For personal use only.
PLATELET
STORAGE
AT
22#{176}C
with
disc-to-sphere
the
and
constituents
collide
platelet
that the fluid
platelet-surface
433
walls of the
transformation
plastic
container.
of the platelets
may
be
dynamics
within
interactions.25’26
released.
This
with
interaction
extension
Various
platelets
from
the direction
of the container.
in greater
loss
platelets
are
interacting
of flow
of agitation.
On the shaker
Takano
et al.27 showed
that
the plastic
continuously
wall.
to greater
that platelet
to aggregating
As a practical
consideration,
lets than
the horizontal
shaker
forms
of agitation
used by Slichter
and
shear
stress
during
the
from
the tube
on the shaker,
on
to
storage
on the
and
high
walls.
keep-
the wheel,
the walls
resulting
is that
the
wheel.
It has
development
shear forces.28
be devices
better
for agitating
it. Our study
does
show
that
there
may
as we used
may be worse.
Since
Harker9
in obtaining
shown
the flow of the PC
for oscillatory
flow
wall collisions,
explanation
shape
change,
lysis,
release,
agents may take place under
method
excellent
a
on observed
of the PC are
With
PC
with
respect
This may lead to more platelet-plastic
of shape
and function.
Another
possible
subjected
been shown
refractoriness
with
changes
have
influence
patterns
in a tube there are forces
present
that drive particles
away
Similar
mechanisms
may be present
in PC with agitation
ing the
however,
may
lead to
pseudopods,
investigators
a system
exert a strong
In our study
the flow
clearly
different
in the two types
is to and fro in a linear
fashion.
of
we used is identical
results
with storage
of
plateother
to that
at 22#{176}C,
it would
seem unwise
to use other methods
until they have been proven
to be of
efficacy.
Considerable
variability
was found
from
donor
to donor
in
recovery
in vivo even under
the same
conditions
of storage
(Fig.
3). However,
there was a very good correlation
between
results
obtained
on the shaker
and
on the wheel in each individual
with the PE containers.
Therefore
differences
equivalent
between
donors
play
a significant
role
in studies
of this
type.
Paired
studies
be necessary
to detect
small differences
between
storage
techniques.
It has been reported
that
the uptake
of serotonin
and the osmotic
response
are useful tests in vitro
to predict
viability
in vivo.’4’23
It has
subjective
normal
viability
may
reversal
been our
impression,2
and Kunicki
et al.29 have
shown,
that
maintenance
discoid
morphology,
as judged
by phase
microscopy,
correlated
in vivo.
Our
studies
add further
confirmation
for this concept.
of
with
We
showed
that the degree of maintenance
of discoid
shape could
be quantitated
the shape
change
response
to ADP
and that shape
change
response
correlated
with viability.
Recently,
we have developed
a more
sensitive
and standardized
test
for measuring
on the
is based
spherical
platelets
the disc/sphere
ratio
in a platelet
difference
in optical
properties
of
when
stirred.
It does
not
depend
by
suspension.#{176} This method
suspensions
of discoid
and
on
the
capacity
to
respond
to ADP,
as does the shape
change
response.
The dispersion
of platelet
size distribution
appeared
to be the best measure
for predicting
viability
(Fig.
6). The observed
increase
in dispersion
after
storage suggests
an increased
proportion
of small
and
large
platelets,
actually
voltage
pulses,
registered
by the Coulter
Counter
as the platelets
pass through
the orifice.
The height
of the pulse is not only a function
of the size of the platelet but also of its shape and specific
resistance.3’
Disc-to-sphere
transformation
of an entire
population
with no change
in the
of platelets
dispersion.’7
causes
an increase
in the height
A possible
explanation
for our
of all pulses
results
is that
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HOLME
434
during
injurious
from
while
release
other
pulses
storage
a certain
of constituents,
platelets
become
recorded
by the
proportion
resulting
spherical
Coulter
of
the
platelets
in an increased
only,
resulting
become
number
in an
ET
Al.
smaller
of small
pulses,
increase
in large
Counter.
No test in vitro
adequately
documents
the viability
of stored
platelets.
However, in serial studies
of the multiple
variables
that might
affect
the outcome
of
storage
it is costly
and time-consuming
to perform
studies
in vivo each
step of
the way.
We propose
this battery
of studies
in vitro
for developmental
work.
Major
conclusions
It is clear that
varies
markedly
storage
on
can then be verified
the degree to which
with
the conditions
the
wheel
was
very
by study
in vivo.
platelet
aggregation
in vitro
deteriorates
of storage
at 22#{176}C.Aggregation
after
poor.
PC
stored
on
the
shaker
in
structed
of PE showed
superior
aggregation
The relevance
of function
studies
in vitro
response
to PC stored
to hemostatic
function
been
study,9
appropriately
questioned.
In a recent
Slichter
that the bleeding
time of thrombocytopenic
recipients
fresh PC and PC stored
for 3 days at 22#{176}C.If this
may
seem
firmed
irrelevant.
by Aster
However,
the
Slichter
et al.32 Furthermore,
and
let functions
in vivo. Finally,
there
may
which
the defects
in vitro are corrected.
con-
and
Harker
showed
was corrected
equally
by
is correct,
studies
in vitro
Harker
the bleeding
bags
in PVC bags.
in vivo has
time
studies
may
not
not
were
reflect
con-
all plate-
be a time interval
after
infusion
In bleeding
patients,
hemostasis
during
during
this interval
may
be critical.
Until
the nature
of the refractory
state
that
develops
after
storage
and the mechanisms
of recovery
in vivo
are better
understood,
it seems
wisest
to maintain
the function
of stored
platelets,
as measured
in vitro,
These
previous
as close to fresh platelets
as possible.
studies
were carried
out with blood
anticoagulated
experience
had
been with
acid-citrate-dextrose
with CPD,
whereas
(ACD).2
The major
problem
with storage
in PVC, rapidly falling pH in PC with high platelet
isjust
as significant
a problem
with PC from
CPD
blood
as it is for ACD
The use of plastics
such as PE that are permeable
to gases eliminates
storage
whether
during
know
in PC from both
it will be possible
equipment
will continue
or
such as the
the platelet
shaker.
count
external
gas
CPD and ACD
to incorporate
blood.
However,
such plastic
into
whether
the requirement
for an external
lO
when these plastics
are used with less traumatic
In any
in PC,
event,
it is clear
that
the type of agitator,
interact
atmosphere-all
to
we do not
plateletpheresis
CO2
forms
the
yet
atmosphere
of agitation
for a given
PC four
the type of container,
determine
count,
blood.
pH fall
success
variablesand
the
failure
of
or
storage.
REFERENCES
I.
TH
Murphy
(ed):
bosis,
5:
Platelet
Progress
vol
3.
in
New
transfusion,
Haemostasis
York,
in
and
Grune
&
Spaet
ThromStratton,
1976, p 289
2.
at
Murphy
46:209,
5,
in
Gardner
of
gas
FH:
Platelet
transport
maintenance
of
across
viability.
4.
plastic
ACD
Blood
Ill.
Comparison
of platelet
EDTA
storage
1975
3. Cohen
yields
J Med
22#{176}C:Role
containers
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ity
and
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273:845,
labelled
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from
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N
EngI
1965
Abrahamsen
on
of radioactiv-
concentrates
AF:
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the
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and
blood
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of
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survival
of
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Haematol
52, 1965
P, Cooley
MH,
Gardner
FH:
Plate-
5.
Morrison
FS,
Baldini
M:
The
favorable
and
2:
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STORAGE
effect
of
ACD
stored
human
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to
RH,
48
platelet
on
tenance
of
to
of
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viability-
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and
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human
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Hardemann
ofhuman
and
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CJL:
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G,
and
enumeration
AppI
Physiol
16.
BS,
Pathol
17.
Smith
graphical
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46:321,
19.
JM:
J
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G:
Am
Zucker
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of
subjected
RA:
quality
the
log-normal
analysis.
law
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in
man.
Blood
mann
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Bornelli
J: Reversible
altera-
produced
by anti-
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Blood
9:602,
1954
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stress.
Tuccelli
M,
of
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biohuman
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GA,
the
temperature.
5: A simple,
measurement
RH,
Becker
affecting
1975
of
38:214,
V, Pilwat
G,
Bechers
fields
on
Bioenerg
Becker
methods
Transfusion
GA,
of
quantita-
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shape.
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of external
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CW,
in
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Zimmerman
improve
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Lewis
changes
5, Murphy
F: Effects
32.
to
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morphology
LB.
JD:
study
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31
size
SG:
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in pulsabile
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15:414,
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Soc
Mason
1968
Ti,
A
Holme
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Am
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Sci 27:253,
to
JL:
1975
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30.
and
HL,
functional
Kunicki
Aster
1964
Platelet
MB,
and
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86:462,
Trans
through
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and
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From www.bloodjournal.org by guest on June 18, 2017. For personal use only.
1978 52: 425-435
Platelet storage at 22 degrees C: effect of type of agitation on morphology,
viability, and function in vitro
S Holme, K Vaidja and S Murphy
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