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A Method
for
Separating
and
Normal
By
T
HE
EFFECTS
donors
Hirsch,
by
H.
ALLEN
and,
in the
presence
the effect
patients;
complete
These
studies
of platelet-rich
of active
of direct
he noted
by a more
lasting
well
normalized.
intact platelets
recently
been
for
ion-exchange
resin are used,
on
the
use
centrifugation.
the
The
preparation
no
volves
change
clinical
studies
concentrates
have
platelet-rich
polycythemic
A
plasma
effects
have
suspension
blood,
this
of leukocytes
providing
agent.
complete
and
From
the
This
work
the
was
of
authors
used
terest
and
the
this
and
3,
desire
Miss
study.
accepted
acknowledge
encouragement.
Cross,
They
following
They
which
may
does
of
a
which
used
for
thus
in-
now
widely
of these
the
platelet
transfusion
be obtained
grants
both
of
Lenox
from
the
of
Chicago.
April
excellent-
technical
thank
provided
t-he
the
course
693
the
New
facilities
to
I)r.
of this
the
Hospital,
sedithe
presence
will
of
permit
These
platelets.
New
York,
Research
N.
V.
Foundation
3, 1952.
assistance
York
for
George
work.
fresh
in
centrifugation.
lull
t-he
to
in
agent
Hematology
publication
from
erythrocyte
present
for obtaining
for
wish
occur
prolonged
method
Cytology,
by
not
surface-active
following
are especially indebted
throughout
platelets
following
Foundation,
Luz Ortiz.
Red
of the
in part
1952;
to
transfusions
agglutination
addition
Experimental
Research
American
in
for
March
Turadian
basis
supported
Leukemia
that
described
and
of the platelets
the
Laboratory
Submitted
The
the
resuspension
are
differential
been
collection
of an agent
which
accelerates
will contain
most
of the platelets
platelet
Furthermore,
observations
indicated
to those
and
of
which
blood.8
citrated
blood
by the addition
ment-at-ion.9
This
suspension
whole
for blood
have
Cronkite,7
has
blood,
large
columns
and
which
ACD
procedure
and
anticoagulant,
a met-hod
from
the
similar
bene-
procedures
in which
Brecher
as an
describes
in
Two
of Freeman,6
of Dillard,
concentrates
significant
used.
Preliminary
that
Na2
report
of platelet
pronounced
beyond
the time
during
Duke5
had previously
blood.
normal
from
that
of Sequesterene
present
or less
of whole
blood
given
to 3 thromboplatelet
count-,
lasting
about
two days,
purpose:
and
polycythemic
hemorrhages
for three
days.
desirability
of a method
for obtaining
of functionally
this
from
have been followed
by changes
bleeding
time
and capillary
frato normal,
for varying
periods
of
bleeding,
relief
from
spontaneous
have
suggested
the
described
blood
purpura
have been studied
and
Gardner,2
Stefanini
and
diathesis,
been temporarily
transfusions
a rise in the
from
M.S.
BURNETT,
with thrombocytopenic
Dameshek,’
Hirsch
numbers
depends
LEE
Stefanini
et al.4 Such transfusions
consumption,
clot retraction,
normal,
and in many
instances
effect
on the hemorrhagic
the platelet
count
had
and
AND
and
ficial
which
Platelets
Blood
M.D.
of transfusions
time;
described
cytopenic
Human
MINOR,
given
to patients
Favre-Gilly
and
Chatterjea,3
in prothrombin
gility
toward
Concentrating
of
Regional
the
L.
collection
Rohdenhurg
Mrs.
Charlotte
Blood
of
Program
the
for
1)100(1
his
in-
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694
SEPARATING
AND
CONCENTRATING
PLATELETS
METHOD
Apparatus
The
apparatus
round
bottom,
to
all
fit
consists
500
ml.
bottles;
of
the
following:
; sedimenting
tubing,
blood
bottles,
8”
lengths,
700
fitted
plasma
ml.
(e.g.
with
centrifuge;
Cutter
needle
centrifuge
Saftiflasks)
adapters;
bottles,
; serum
stoppers
aspirating
needles,
8”
long.
All
glassware
DC
cone,
is
made
and
200,
up
at
fixed
100
C.
or
on
kitchen
oven.
siliconed
simple
and
are
then
air
drying
surface
This
before
The
drained
coating
filled
solvent
with
for
for
baking
through
25 to
Any
dextran
fraction
of Dow
solution
of
the
30
2 to
liquid
and
by
The
at
and
silisilicone
the
needles
for
30 mm-
heating
4 hours.
30 minutes
washings
Corning
the
solution
is evaporated
by
last
application
cent
temperature
apparatus
will
are
the
room
The
A 2 per
bottles
and
at
of the
use.
effective.
tetrachloride.
by
the
are
is
,
it ; they
in
utes
cs.
in carbon
immersed
then
needles
350
silicone
300
C.
film
F.)
(572
is
in
a
sterilizations.
Reagents
of
Dextran
solution,
240,000t
or
pared
by
aid
of
cent.
(intrinsic
dissolving
heat.
to each
6 per
above
6 Gm.
It
is then
sedimenting
0.37
viscosity
of dextran
passed
substance
through
bottle.
These
per
a “Seitz”
units
are
having
higher)
or
may
100
ml.
filter,
autoclaved,
an
average
be
used.
of
molecular
The
pyrogen-free
and
60 ml.
and
may
of
weight
solution
is pre-
saline
the
with
filtrate
be stored
at
are
room
the
added
tempera-
ture.
This
reagent
is a polysaccharide
erythrocyte
weight
of the
Triton
parts
The
dextran’#{176};
solution,
are
room
for
added
saline
to
each
of
reason,
This
at
up
rate
this
cent.
2 per
of pyrogen-free
tion
built
sedimentation.
from
a high
molecules.
is
molecular
is prepared
by
a temperature
centrifuge
glucose
sedimentation
weight
diluting
These
50 and
units
are
is used
to
to
the
fraction
2 parts
between
bottle.
It
proportional
molecular
is selected.
of Triton
WR-1339
60 C. Twenty
autoclaved,
accelerate
ml.
and
with
of this
may
be
98
solu-
stored
at
temperature.
This
used
reagent
to
low
a non-ionic
the
permits
them
toxicity”
and
thereby
its
is
prevent
surface-active
agglutination
to
he
agent,
of
platelets
resuspended
because
it
is
an
alkyl
brought
as
discrete
aryl
polyether
together
by
elements.
It
alcohol.
It
centrifugation,
was
is
and
selected
because
of
nonhemolytic.’2
Procedure
Sterile
precautions
Four
for
or
more
transfusion.
sentially
of
blood
of
clots,
vein;
tle
suction
As
soon
by
gravity
within
a very
of
used
any
in
the
blood
preparation
group,
in standard
three
of blood
and
the
the
bottle
the
Care
final
wide-bore
into
ACD
hours.
lower
sharp
necessary;
possible
as
bottle
suction,
with
The
supplies
the
authors
blood
As
Triton
determined
collection,
contains
tip
trap
two
of
the
60 ml.
needle
in
erythrocvtes.
hours.
are
of
of
after
which
tends
to
for about
supplies
t
been
be
of
since
the
platelet
final
concentrates
concentrate
is es-
bottles.
should
yield
of
which
should
be maintained
mixed
each
pint
is
with
need
taken
to
platelets.
needle
kept
These
be
not
be siliconized
avoid
the
formation
Venepuncture
passed
directly
at
the
should
into
a uniform
ACD
the
rate,
solution
by
be
lumen
using
of
gen-
swirling
at
intervals.
menting
*
may
invariably
flow
if
frequent
for
these
the
throughout.
have
erythrocytes.
processed
with
the
s?hich
ture
is
for
performed
donors
is collected
if the blood
observed
of blood
The
free
The
are
Pints
grateful
several
dextran
The
to
Pharmacia
fractions;
of
the
blood
ultracentrifugation
blood
solution.
solution,
thus
is then
and
method.
to
is transferred
This
to
Inc.,
Rohm
and
to
a 700
ml.
is accomplished
avoiding
allowed
Laboratories,
WR-1339.
by
of
dextran
the
sediment
270
Haas
formation
at
Park
Co.,
by
Avenue,
of
room
sedi.
gentle
froth,
tempera-
New
Philadelphia,
York,
for
From www.bloodjournal.org by guest on June 15, 2017. For personal use only.
ALLEN
The
supernatant
1)01 1 Ic
which
inversion
several
The
The
fluid
al
(0111
formed
elements
eel I-free
fluid
physiologic
sali
shaki
vigorous
The
so
tIC
to
present
wit
llott
intl
aI
to
this
rat
e(1 suspensiolls
the
erthrocytes
.
C
coot
2(XX)
r.p.nl.
ol)t
I ,rieflv
at
low
and
t he
rifuge
xed
flu
the
iii
nil.
V
of
se(limellt
1w
aic
by
1 hems romhine(1.
7 to
for
while
sc(lifllCIit.
non-pyrogenic
resUsl)CIldCd
manner
e.g..
,
(PIll
are
packed
Fort
in t his
spee(1
ml.
bottle
30 ninutes.
now
leukocytes
a 500
t he
of
is.
and
ained
5
for
remai
material,
into
cist
are
10 ml.
residual
ri fuged
is cent
Ic is aspirated
The
.
lout
695
BURNETT
suspension
in
i I only
addesl
down
bring
LEE
at
coneent
are
The
AND
(.eiltrifug(o(1
initially
suspension
as
then
is aspirated
tig.
pooled
r.p.m.,
ml
an(1
times
MINOR
(‘Itell sedimnentimig
. of
Tn I 015 sol1
in
20
I15
H.
11) mi
leaving
itt
at
es
nearly
S00
all
the
;-
#{149}
#{149}
iia
#{149}II
#{149}
.,t\
.
‘
.J
p;..
“
‘
.
lP#{149}b1Is
.#{149}‘
4f
:; :
_
4.’
I
$
I
#{149}#{149}
#{149}
Should
r.p.m.
platelet
platelet
30
.,‘.
#{149}
fluid;
a volume
suspension
should
agglutination.
been
Similarly,
*
fluid.
All
the
platelet
supernatant
followed
in
this
1w
solution
he
with
platelets
counts
way.
may
have
been
ready
(X
1000)
then
is
for
platelets
of Triton
mixed
,.
suspension
is
of the
he
#{149}
.‘b
concentrate
Concentrations
obtained
.‘
1.-Platelet
concentrate
may
‘S
..‘“
.:..‘
.,
concentration
minutes
I
#{149},
.
“,
The
platelet
further
for
of residual
have
the
#{149}
#{149} ,
#{149}s
‘
FIG.
suspension.
$
e1’.’
NM1
C-
#{149}
in
,
ti,i*
#{149}
.0.Z.
and
.
‘i4’
,.qW-.
possible
#{149}
%_
:.
platelets
..‘..#{149}..:‘
5.
.‘
‘
-
0!
‘i:
.
-
ft
t’.
‘‘
1)e desired,
resuspension
equivalent
the
latter
of discrete
aspirated
completely
as
as
transfusion.
prior
platelets
a second
of
the
to
10 per
to
cent
sediment
cent-
ion
a small
of the
centrifugation
exceeding
rifugat
in
at
volume
so
20 million
2000
volume
as to
ier
of the
prevent
Cu.
mn)
*
he
subjected
made
to
1w the
repeated
direct
with
washings
method,
using
saline
Rees-Eckers
to
remove
diluting
From www.bloodjournal.org by guest on June 15, 2017. For personal use only.
696
SEPA
residual
and
l)IaSma,
tion
is addled
Base(1
pared
to
an
analysis
four
pints
OIl
AND
negligible
prior
from
RATING
losses
each
will
PLATELETS
occur
l)rovi(ling
a small
the
composition
volume
of
Triton
solu-
centrifugation.
40
of
of
CONCENTRATING
concentrates,
blood
this
by
procedure
is as
of a platelet
follows
(within
transfusion
a range
pre-
of
±
25 per
cent):
Total
Total
l)latelets
volume
Physiologic
350
billion
200
ml.
ml.
saline
160
Plasma
21 ml.
ACD
solution
Dextran
Triton
About
SO l)C
recovered,
cent
concentrated
discrete,
andI
Studies
Platelet
final
stain
Platelet
on
ten
times.
Concentrates:
They
Wright’s
Effect
present
appear
stain
containing
2000
r.p.m.
for
Eight-tenths
first
the
was
third,
and
according
9
incubated
hour
prepared
and
drawn
blood
are
they
are
and
in
to
Prolhrombin
Con-
Thrombocytopenic
the
Blood.
procedure
order
described;
to lower
the
in
instances
into
from
2 hours,
ml.
added
and
the
plasma
was
of this plasma
0.1
ml.
after clotting
additional
15 minutes.
an
inspected,
occurred
tubes
ranged
control
were
absence
of retraction.
a wooden
applicator,
of the
good clot
concentrates
instances
obtained
these
times
were
prothrombin
studies
are
To
and
calcium
chloride
They
were
then
and
the
degree
and
determined
method
recorded
the
sera
on
of Quick,’4
in
incubated
the
sera
using
and
rabbit
by adsorption
with
the determinations
on
containing
10 per cent
table
1. In
all instances
retract-ion
was observed
in the recalcified
plasmas
to which
the platelet
IIa(1 1)een a(lded;
no retraction
occurred
in the control
tubes.
In all
the serum
prothrombin
times
of the recalcified
plasmas
to which
plate-
concentrates
plasma
in
in a
at
from 4+,
which
represented
restudies
on platelet-rich
normal
with
Prot-hrombin
adaptation
the
thromboplastin.15
Prothrombin-free
plasma,
obtained
barium
sulfate,
was used
as the source
of fibrinogen
in
serum,
and also as a diluent
in the preparation
of plasma
prothrombin.
results
A small
was then
into each of three
glass tubes.
to the second,
0.1 ml. saline,
lung
The
individuals;
removed.
placed
plasma,
had
to 0, which
represented
clots were theii removed
for
normal
0.2 molar
shaking.
plasma,
The
by
of
concentrate.
One-tenth
ml.
the tubes
mixed
1)y lateral
retraction
recorded.
The degree
cOmparal)le
to that
found
in
37 C.
blood
technic
were used.’3
the first syringe; a second
syringe
of clot
traction
plasmas
the
a two-syringe
ml. of blood
drawn
without
stasis
or frothing
and placed
1 ml. 0.2 molar
sodium
citrate.
The blood
was centrifuged
0.1 ml. platelet
to each,
and
at 37 C.
added
One
was
and
tube
was
equipment
blood
of
substituted
let
ACD
intact,
1).
Retraction
Plasma
in some
in the
morphologically
(fig.
on Clot
Recalcfied
were
prepared
was prolonged
plasma
siliconized
quantity
at
2 ml.
initially
count.
chilled,
was
6 ml.
2%
of platelets
s-itli
in Platelet-Poor
Platelet-poor
to
number
to
6%
solution,
normally
concentrates
cent rifugat-ion
platelet
the
total
eight
they
sumption
the
of the
11 ml.
solution,
containing
had
been
10 per
added
cent-
were
prothrombin.
greater
than
The
the
prothrombin
prothromhin
times
consumption
of
in
From www.bloodjournal.org by guest on June 15, 2017. For personal use only.
ALLEN
these
tubes
was
therefore
H.
MINOR
greater
than
trol tubes,
however,
were
only
times,
indicating
the abnormally
virtual
absence
of platelets
in
Similar
results
penic
patients
trates.
Definite
TABLE
was
tubes,
1.-Effect
to tubes
retraction
consistently
and
of
the
90 per
containing
was
observed
over
90 per
prothrombiii
Platelet
697
BURNETT
cent.
The
serum
small
volumes
after
one
cent-.
No
on
Platelet-Poor
Clot
Retraction
and
Clot Retraction
with added
I
\o
Plasma
1
445
0
2
785
0
Plasma
Time
Saline
0
Prothromhin
on
Plasma
‘ialine
17.7
13.3
0
0
940
0
-
Time
added
14.4
935
6
Prothrombin
with
17.6
3+
5
Consumption
Serum
32.0
0
-
Prothrombin
(seconds)
27.8
0
925
(‘onsumption
11.7
0
830
4
11.2
25.7
13.5
14.6
11.7
27.8
14.4
13.3
3+
11.3
26.5
15.7
15.1
0
3+
11.2
25.7
13.5
14.6
-
in
low.
12.1
0
3
occurred
2+
3+
3+
-
concen-
Plasma
Platelets
0
-
t-he
prothrombin
retraction
and
con-
thrombocyto-
abnormally
was
J?ecalcified
in the
prothrombin
caused
by
platelet
of
Prothrombin
Platelet
Concentrate
times
hour,
clot
consumption
Concentrates
in
LEE
slightly
greater
than
the plasma
low prothrombin
consumption
the specially
prepared
plasmas.
when
freshly drawn
blood
from
obtained
added
clot
consumption
control
were
was
AND
I
seconds)
Platelets
93
118
>180
129
So
>180
7
1135
0
0
4+
12.3
27.9
14.7
14.2
41
8
1210
0
12.3
27.9
14.7
14.2
>180)
12.3
27.9
14.7
14.2
12.3
27.9
14.7
14.2
>180
14.2
>180
9
1305
0
0
0
4+
4+
10
1340
0
0
4+
11
1460)
0
0
4+
12
1580
0
0
4+
Transfusions
of Platelet
Twenty-five
tients
were
transfusions
to a drug,
11.9
-
27.9
14.7
27.5
16.2
-
15.4
111
Concentrates
of platelet
who were
thrombocytopenic
5 cases
of thrombocytopenic
secondary
12.3
-
115
concentrates
have
been
and bleeding
when
first
purpura,
of which
4 were
Gantrisin’6;
leukemia.
In 9 of the patients
effect lasting
from 1 to 5 days
2 patients
this effect followed
4 cases
of aplastic
anemia
there
was definite
clinical
following
a single platelet
subsequent
transfusions.
given
to
11 paThere
and
1
transfused.
idiopathic
and
2 cases
of acute
evidence
of a hemostatic
transfusion;
in the other
Concomitantly,
there was
a return
thrombin
fusions.
to normal,
or near normal,
of bleeding
time,
capillary
consumption.
One pyrogenic
reaction
occurred
in the
Thromboembolic
phenomena
have
not been
observed,
adverse
reactions.
fragility
and proseries of 25 transnor have
other
DISCUSSION
By
yield
means
large
of the
procedure
numbers
fluid.
It is thus
ment
of three
possible
or more
presented
of platelets
to transfuse
pints
above,
normal
concentrated
of blood
the
blood
in a small
equivalent
without
significant
can
volume
of the
total
effect
be processed
of physiologic
platelet
on the
complecirculating
to
From www.bloodjournal.org by guest on June 15, 2017. For personal use only.
698
SEPA
fluid
volume
of the
as indicated
t-io,i
platelets
be
effect
are
so obtained
flexible
when
the
erythrocytes
modification
retraction
according
of blood
Satisfactory
weight
active,
in
thrombocytopenic
for
to con-
the prepararecoveries
of
polyvinylpyrrolidone
used in laboratory
12 per cent of the
as
studies
platelets
to replace
initially
red cell concentrate
following
concentration
of the platelets,
erythrocytes
omitting
this
a platelet
in
compatible
with
those
may
l)e reduced
to one
clot
into
volumes
studies.
molecular
Tw’een
80 has been
these
agents,
about
functionally
and
transfused
to permit
high
are
consumption
when
to use with small
for laboratory
with
PLATELETS
are cent-rifuged
out with the residual
The latter
loss may be avoided
by
done
be
transfusion
sedimentation
platelets
blood
remain
with
the
no loss occurs
during
the
about
8 per cent
in the final step.
as may
The
on prothrombiii
obtained
agent;
With
ACI)
essentially
in
CONCENTRATING
is sufficiently
can
mentation,
AND
hemostatic
be adapted
concentrates
the sedimenting
Triton
W1-1339.
present
effect
their
It can
platelet
of
recipient.
their
by
vitro,
and
by
patients.
The J)rocedure
venience.
RATING
sediand
and leukocytes
centrifugation,
concentrate
prepared
of the recipient.
Similarly,
hour,
if the presence
of small
for
the time
numbers
of
of
erythrocytes
in the fiuial concentrate
It is probable
that
somewhat
higher
is of HO consequence.
recoveries
of platelets
by
siliconed
or plastic
containers.
Until
however,
it would
seem
convenient
collecting
containers
in
the
blood
become
most
instances
directly
into
widely
available,
to leave
unchanged
the
standard
might
technic
be obtained
more
of blood
such
collection.
SUMMARY
A method
is described
for
volumes
of normal
human
are removed
by sedimentation
are concentrated
which
permits
by
prolonged
centrifugation.
cent
of the total
platelet
are
discrete
penic
and
been
the
in
mt-act,
from
bottles.
dextran,
Erythrocytes
and platelets
of a surface-active
of physiological
fluid
pint
agent,
follow-
results
in the recovery
of about
80
of t-he blood.
The platelets
obtained
and
when
added
of platelet
followed
concentrates
ACD
weight
presence
volume
in a small
morphologically
have
of platelet
in standard
molecular
The procedure
complement
and clot retraction
blood.
Transfusions
patients
preparation
collect-ed
with high
centrifugation
resuspension
their
ing
per
sumption
cytopenic
the
blood
induce
normal
to platelet-poor
concentrates
by hemostatic
effects
prothrombin
plasma
given
to
lasting
con-
or thrombothrombOcyto-
from
1 to 5 days.
REFERENCES
E.
‘HIRSCH,
0.,
successful
2
AND
-
transfusion
M.
STEFANINI,
J.
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Clin.
H.:
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J. Clin.
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Investiga-
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in
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obtaining
interrelation
LEE
transfusion.
yields
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Federation
erythrocyte
AND
erythrocytes;
time
by
large
Soc.
L.:
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A method
-:
fluid.
Proc.
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acceleration
527,
G.
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AND
normal
AND
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AND
bleeding
114:
platelets.
of
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relieved
for obtaining
Science
G. H.
DILLARD,
-
the
of hemorrhagic
G.:
Method
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H.
A.
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induced
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in
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rabbit
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plate-
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1952 7: 693-699
A Method for Separating and Concentrating Platelets from Normal Human
Blood
ALLEN H. MINOR and LEE BURNETT
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