Download Concentration in pH 6.5 Citrate-Plasma

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Brief
Report
Factor
VIII (AHF)
Concentration
pH 6.5 Citrate-Plasma
By
S A RESULT
of
ulants
on the
is the
anticoagulant
ACD
platelets
which
vided
as
many
are
platelet
S. MomusoN
FmiNcIs
recent
survival
investigations
of transfused
into
for
required
platelet
The
the
can
suggested
be
most
6.5.
Many
laboratories
the
treatment
on Factor
ACD
might
VIII
find
it convenient
hemophilia.
) concentration
( AHF
\IETHODS
The
Factor
housma
VIII
assay’
by
as modified
volumes
Ten
of
whole
After
and
blood
the
separation
I. The
PPP was partitioned
the other
contained
ACD;
samples
the
were
fromil
7.0
2.
was
felt
separately
l)lmflering
the
the
that
of
as
platelet
with
at pH
plasma
for
of
additional
effect
of Langdell,
three
VIII
ranged
reduced
pH
normal
by
different
the
Wagner
volunteers
activity
at
procedures
before
from
6.3
to
into
centrifuging
were
tubes.
One tube
of Veronal
buffer
use
and
1.5
in
Brink.
volumes
1800-1900
g.
of
for
30
used:
contained
(pH 7.3).
0.3 nil.
Aliquots
and
after
storage
6.5
while
the
buffered
at
-20
might
affect
additional
of these
C.
The
p11
plasma
but
the
used
British
Medical
and
as
(pH
on
Research
6.3-6.5).
the
the
Council’s
contained
view
of
of
ranged
the
assay
then
returned
to
) was
7.0-7.2
standing
assayed.
VIII
three
was
at
The
technic
Haematology
in
determined
of
of approximately
Experimental
equal
temperature
was
cryoprecipitation
portions
an
treated
room
pH
system
this possibility
as described
Pool
2 ml.
Research
in
Unit,
England.
in
the
it
( pH
After
Factor
into
Londoa,
assay
plasma
and
partitioned
School,
assertions
reflecting
based
was
to
control
plasma
was
PPP
Medical
opinions
citrate
prior
The
“acidified”
The
hospital
construed
additional
immediately
erythrocytes.
method
the
Mary’s
amid/or
any effect on Factor
VIII. In an attempt
to circumvent
of erythrocytes
was utilized.
The
PPP
was treated
the piasmiia was again
separated
the incul)ation
with ervthrocytes.
after
Another
Time
be
of PPP,
from
obtained
into two plastic
the same volume
paragraph,
Shannon.’5”#{176}
Fronz
St.
making
investigated.
metho(l
collected
was
Factor
fresh-washed
and
3.
the
from
capacity
the same nianner
for 15 minutes
and
of
pro-
MATERIALS
one-stage
(PPP)
ACD
added
preceding
volume
before
for
with
that
to 7.2.
It
quite
in
assayed
samples
quantities
Macpherson.’4
were
plasma
The
plasma
(PRP)
ACD-plasma
this
the
was
AND
the
was
Hardistv
ACD#{176}.Platelet-poor
nuinutes.
used
to store
Therefore,
anticoag-
emerged
for
require
reduction
of pH of the platelet
rich
acid citrate;
consequently,
a by-product
is fresh
of
has
conveniently
technics
concentrates
additional
of classical
effect
fact
transfusion.
clinically
concentrates.12
the
platelets,1”2
of choice
usually
in
this
U.
paper
are
S. Navy
the
author’s
Departnuent
own
or
time
and
are
Naval
not
to
Service
at
large’.
First
FliAxcis
search
submitted
S.
Council’s
Jan.
18, 1966;
NIORRISON,
Experimental
accepted
LCDR,
NI.C.,
for publication
Haematology
School,
London.
#{176}ACDNIH Formula
A ( 0.8 per
per cent hydrous
dextrose).
cent
Visiting
U.S.N.:
Research
citric
acid,
April 25, 1966.
Investigator,
Unit,
2.2
per
St.
cent
British
Mary’s
sodium
Medical
Hospital
citrate,
Re-
Medical
and
2.45
479
BLOOD,
VOL.
28,
No.
3
(SEPTEMBER),
1966
From www.bloodjournal.org by guest on August 9, 2017. For personal use only.
480
S.
FRANGJ.S
small plastic
0.2 nil., was
containers.
To the first was added
0.2
added
to the second
and third
samples.
-20
C. The
first
ice”
mixture
2000
stored
and
and
second
were
samples
were
allowed
to
then
rapidly
thaw
ml. more
ACD,
while
Verona!
buffer,
The third
sample
was then stored
at
frozen
taken
out of the freezer,
samples
was
thawed
resuspended
at room
in 2.2
ml.
by immersion
into
an alcohol
at
were
centrifuged
at
plasma
the cold-precipitate
to be done,
all three
samples
was
were
overnight
g for 20 minutes.
After pouring
off the supernatant
at -20
C. for 1 to 10 days. When
the assay was
two
MORRISON
temperature,
4
and
C.
and
the
cold-precipitate
in
“dry
the
first
buffer.
RESULTS
the
1. When
the
sample
54 per
cent
pH
of
plasma
was
with
the
the
differed
fractions
by less
which
52 per
cent)
were
tion.
There
was
were
cold-precipitated,
of additional
than
0.1 unit.
thawed
0.2
control
Factor
stored
however,
of each
a pH
sample
( average
96 per
approximating
pH
been
were
ACD
cent).
of all
three
of the
(average
after
paired
samplasma
cent
immediately
the
sample
samples
40 to 60 per
C.
separated,
the
additional
concentration
was
having
themi
these
preassay
additional
from
with
between
pair
differ
of
( average
without
When
-20
estimate
cent
and
not
VIII
at
the
per
samples
final
at 4 C. overnight
sample
50
units.
the
the
The
difference,
and
did
of
of the
of assay,
erythrocytes
ACD
technic,
one
ACD
to fresh
cent
in the
no
of the
more
per
than
of that
concentration
concentration
cryoprecipitate
time
approximately
additional
by
VIII
at the
was
returned
ACD
Factor
to be 90 to 100
3. With
differed
ACD
VIII
sample
additional
ples
Factor
the
without
found
samples
additional
the
assayed,
was
of the
the
) of the
ACD.
2. When
the
pH
with
separa-
samples
processed
which
in the
presence
6.5.
DiscussioN
reduction
A
in plasma
acidified
plasma
tion.7 However,
pH
and/or
given.
salt
In
the
“acidified”
pH
It is well
reaction
known
by
buffer
alters
activity.
assay.
pH
the
the
Factor
However,
Alternatively,
fibrmnogen
pH
the
assay
of the
conditions
pendent
of the
question.
lower
of the
VIII
to assay.
the
salt
concentration
the
reflect
The
rule
out
per
not
not
the
seemi
assay
of the
when
found
to
unin
reduced
pH
the
the
a definitive
be
reduction
influence
of
It
diluted
that
physiologic
under
removal
the
of the
as the
to
is greatly
possibility
at
in
reaction.17
in a reversible
allow
of
assay
was a reflectiomi
mixture
as well
seen
or
cent
salt concentrato readjust
the
method
and
known
results
dilution
do
30
AHF
was
reversed
is also
data
the
on
(Plasma
so as to result
the
was
determined
assayed.
improved
to
thrombin-fibrinogen
was
is not
25
effect
VIII
assay
incubatiomi
not
molecule
change.
difference
Factor
actual
does
reduction
and
this
being
the
nor
prior
system
) This
may
pH
levels
plasma
assay.
assay,
cami influence
the
pH
of
to altered
pH or
attempt
was made
a comparable
However,
that
the
reaction,17
mental
this
of
the
during
seen.
that
therefore,
However,
to
here
to physiologic
mixture
affected
pH
was
concentration
prior
reported
readjusted
appeared,
lower
pH.
VIII
recently
ascribed
stated
whether
an
concentration
studies
plasma
was
Factor
has been
it was not
thrombin-
given
citrate
in
during
experi-
ion
conclusion
mdcon
From www.bloodjournal.org by guest on August 9, 2017. For personal use only.
FACrOR
VIII
The
IN
experiment
hemophiliac.
buffering
in vivo
these
Factor
iii
481
PLASMA
the
the
Under
reductiomi
tional
CiTRATE
utilizing
to simulate
atteni1)t
capacity
events
experimental
VIII
of erythrocytes
attendant
upon
conditions
concentration
after
was
infusion
there
exposure
was
to,
a simple
of plasma
no
or
into
a
significant
storage
addi-
in,
ACD.
The
and
recent
its
development
application
in
forward
times
Factor
The
tional
ACD.
with
activity
The
the
The
plasma,
precipitated
VIII
on
also
of
product
presence
indicate
protein
the
additional
VIII
step
to
he
the
means
of
the assay
ACD
had
without
ACD
30
In
only
as a
factors
in
precipitated
of Factor
VIII
large
basis.
of additional
samples
that
method
a
is reported
quantitative
plasma
Factor
represent
method
was
used
the reduced
pH
as
the
cryoprecipitation
end
a
in the
as
results
of cryoprecipitation
banking1#{176} may
procurement.
frozen
plasma
Factor
same
blood
the cryoprecipitatiomi
excess
citrate
and
the
system.
technic
the
general
VIII
as
study
eliminating
of
to
potemit
as
presemit
fere
6.5
H
does
the
addi-
not
inter-
concentration.
SUMMARY
A
by-product
posed
of
methods,
a useful
source
unaltered
incubation
same
Factor
of
Factor
that,
assay
the
after
system.
Factor
acidified
plasma
untreated
plasma.
found
producto
undo
6,5.
secundari
recentemente
Viste
activitate
que
iste
to
il esseva
post incubation
he mesme
activitate
citrato,
su
pH
he
as
le addition
essayage.
preparate
preparate
Iste
de
de
ab
plasma
ab plasma
“acidified”
acidificate
to
cent.
However,
plasma
had
the
plasma
is probably
This
prepared
conclusion
was supported
by cryoprecipitation.
as
in
as
as
de
un
esseva
es
per
de
plasma
a
In
the
from
from
utile
fonte
de
con
plachettas
fresc
de 6,5 es
de Factor
supportate
Factor
eliminate
como
de
non
minus
VIII
alte
que
de
iste
l)la51’’It
cento.
plasma
post
citratate
In tal
preparatos,
in
systerna
le
in Ic precipitatos
in
a
Tamemi,
habeva
observationes
factores
Factor
pH
VIII,
probabilemente
VIII
que
per
see-
a un
nonalterate
cryoprecipitation.
le concentration
esseva
in
prepared
concentratos
comparation
de un pH
como
fonte
preparate
effective
factors
prepared
precipitates
es citratate
In
plasma
efficace
nontractate.
per
he activitate
amontava
a 54 pro
erythrocytos
fresc,
he “acidificate”
VIII como le preparato
de controlo.
que
be
INTERLINGUA
esser
in illo.
e le pH
trovate
54
pro-
might
Compared
are eliminated
in the
precipitates
high
methodos
VIII
citrato
Esseva
pH
IN
conclusion
Factor
6.5
preparation
poterea
un
minus
ordinari.
concentratos
plasma
trovate
COIl
eluite
de factor
Isto suggestiona
infusion
non
plasma
del
investigate
recently
plasma
investigated.
the
ACD-plasma.
concentrates
proponite
esseva
this
to be
erythrocytes
SuI\fAnIo
Un
was
found
ACD
and
concentration
VIII
was
by
Because
control.
infusion,
additional
concentrates,
6.5.
activity
was
fresh
as the
VIII
as ordinary
on Factor
VIII
preparatiomis
platelet
at pH
activity
washed
activity
suggests
of
VIII,
the
with
VIII
source
of Factor
by observations
such
preparation
citrate-plasma
citrate-plasma,
after
This
the
is fresh
precipitatos
con
de
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482
FRANCIS
S.
MORRISON
ACKNOWLEDGMENTS
The
in
author
the
is indebted
preparation
to Professor
of
this
P. L. Mollison
paper,
and
to
Miss
and
Dr.
Mary
P. Barkhan
Stevenson
for helpful
for
advice
capable
technical
assistance.
REFERENCES
1. Kissmeyer-Nielsen,
B.: Platelets
2.
F.,
and
Madsen,
C.
in blood
stored
in untreated
and siliconised
glass bottles
and plastic bags. II. Survival
studies.
J. Clin. Path. 14:630,
1961.
Aster,
R. H.,
and
Jandi,
J. H.: Platelet
sequestration
in
man.
I. Methods.
J.
Clin. Invest.
3. Morrison,
F.
effect
of
43:843,
1964.
S..
Baldini,
and
M.:
anticoagulants
survival.
Blood
25:612,
The
1965
6. Cohen,
P., Watrouse,
P., and
F. H.: Glycerol
preservation
concentrates
blood.
(Second
Conf.
Ridge,
7.
Cohen,
July,
P.,
F. H.:
on Blood
Cooley,
M.
of
ACD.
1965.
H.,
8.
Eng.
and
ACD
Abraharnsen,
EDTA
and
A.
F.:
The
Cr5’
survival
of
platelets.
Scand.
labelled
J.
brief
review
Sang.
(In press.)
Langdehl,
1.
Chiu,
J.
A., Sullivan,
S.: Transfusion
M.
of
P.,
and
platelet
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J.
tests.
Hardisty,
R.
D.,
Wagner,
K.
M.:
factor
Lab.
NI.,
and
Blood
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practical
of
recovery
platelets.
26:732, 1965.
Morrison,
F. S. : Platelet
a
aspects.
R.
Effect
on
Vox
H.,
of
one-stage
Clin.
Med.
and
anticlot-
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J.
Macpherson,
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7:215,
1962.
Pool, J. C., and Shannon,
372,
1965
(Abstract).
A. E.: Production
of high-potency
concentrates
of
antihernophilic
globulin
in a closedbag
system:
assay
in vitro
and
in
vivo.
New
Eng.
J.
Med.
273:1443,
1965.
17.
Biggs,
man
9. Shively,
on
human
16.
of
2:52,
incompatibility
transfused
Diath.
blood
Haernat.
prepared
in
acidified
27:449,
1966.
Effect
of anticoagulant
Pool, J. C., and Shannon,
A. E.: Simple
production
of high potency
antihernophilic
globulin
(A.HG)
concentrates
in
a closed
bag system.
Transfusion
5:
and
1965.
ABO
E. J.:
platelet
15.
of
effect
on the recovery
5:
A one stage
Factor
VIII
(antihemophilic
globulin)
assay
and its use on
venous
and capillary
plasma.
Thromb.
Gardner,
yields
acidified
Transfusion
Flatow,
F. A., Jr., and Freireich,
The increased
effectiveness
of
ting
1953.
Oak
derived
from
with EDTA
and
J. Med. 273:845,
from
plasma.
(Abstract).
Brinkhous,
hernophihic
III. Corn-
radioactivity
New
13.
(Abstract)
Platelets,
preservation.
platelet
concentrates
blood anticoagulated
12.
14.
1964).
Platelet
parison
1965
25:608,
1965
and
(Ab-
Gardner,
rich
concentrates
plasma.
Blood
1 1. Aster,
R. H.:
of platefrom ACD
derived
Blood
10.
of
stract) (Second
Conf. on Blood Platelets, Oak Ridge, July, 1964).
4. Morrison,
F. S., and Baldini,
M.: The
favorable
effect of ACD on the viability
of
fresh
and
stored
human
platelets.
Vox Sang.
(In press.)
5. Morrison,
F. S.: The effect of filters on
the efficiency
of platelet
transfusion
Transfusion.
(In press)
let
378,
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on
prepared
concentrates
platelet
R.,
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31.
and
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Coagulation,
F. A. Davis,
R.
3rd
1962,
C.:
Hu-
Ed.
Phil-
pp.
30-
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1966 28: 479-482
Brief Report: Factor VIII (AHF) Concentration in pH 6.5 Citrate-Plasma
FRANCIS S. MORRISON
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