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Principles
Extraction
By
of Blood
Separation
and Component
in a Disposable
Continuous-Flow
Single-Stage
Channel
P. Hester,
Robert M. Kellogg,
Kenneth
B. McCredie,
Jeane
A single-stage
disposable
that
for
provides
cyte
by
collection
separation
channel
leukocyte
and
and
seal
has
been
channel;
granulo-
continuous-flow
(CFCS)
Alfred P. Mulzet,
and Emil J Freireich
cell
designed
emphasis
deficiencies
collect
of
components.
volume
resides
donor,
on
collected,
the
blood
The
used
and
systems
and
of the
variables
forces
in a CFCS
component
system,
and
(3)
what
the
are
cellular
those
con-
of
the
cells
response
to
in terms
The
variability
variability
the efficiency
the
Corporation
specific
used
variable
character.
day-to-day
agents,
to provide
some
of the response
that
for
in each
of the
procedure.
has
designed
disposable
seals,
to extract
yields
over those
variability,
which
standardization
of whole blood
realistically
in collection
to centrifugal
allows
in the
way
of
collection.
speeds,
procedure
influenced
time,
the
body
CFCS,
surface
(4)
total
blood
a stepwise
continuous-flow
NCI-IBM
or Aminco
area;
the University
Department
and
yield;
Abbreviations:
cell separator,
From
(4)
that
procedures
quite
performing
IBM
This article
describes
(I ) the principles
single-stage
channel;
(2) its performance
BSA,
the
(5) posttransfuin patients;
and
replacement
toward
blood
pathways,
consisting
of separation
channels
and ceramic
leukocytes
by continuous-flow
cell separation
(CFCS).
The design
objectives
were
( I ) to increase
the total cellular
currently
obtained
by CFCS,
(2) to minimize
operator-related
strongly
influences
total yield, and
by expanding
scientific
understanding
extracted;
integrity
component
the operator
group
which
is
extraction;
final yield;
response
components
quantity,
for collection,
biomedical
of
procedural
in hematologic
differences
between
donors,
nonuniform
response
to leukocytosis-inducing
technology
by
and
functional
attention
granulocyte
cellular
efficiency
and
(6)
mechanism
collected.
directed
Currently,
the
R. Kruger,
accumulates
tribute
to the
sion increment
specific
has
the
donor
IBM Corporation.
This paper describes
(1 ) the separation
characteristics
of whole
blood as it responds
to varying
gravitational (G) forces and flow rates through
the
physiologic
coat
(3)
by
the
I NCREASED
(2)
buffy
Victor
TBV,
of Developmental
cell
blood
(3)
ACD,
Therapeutics.
IFPP,
acid
those
Center.
positioning
dextrose;
HES,
ACD:whole
blood
Anderson
Hospital
M.D.
Houston,
donor
regression
interface
citrate
ACD:WB,
Cancer
and the operation
of the
of flow rates,
centrifuge
multiple
separator;
volume;
System
processed;
forward
Celltrifuge;
total
of Texas
of separation
over a range
Texas,
and
the
IBM
variables
that
analysis
that
port;
BCS,
hydroxyethyl
blood
starch;
dilution.
and
Tumor
Institute,
Corporation,
Endicott,
N.Y.
Supported
Bethesda.
in
Md.
Submitted
Address
D.
M.
254
Houston,
1979
by
by Grune
Grants
by the IBM
October
reprint
Anderson
Bertner,
0
part
and
26. 1978;
requests
accepted
to Jeane
Hospital
an4
Texas
77030.
cc
CA!
Stratton,
1520
Corporation,
Tumor
Inc.
and
CA19806
Endicott,
March
P. Hester,
Institute.
from
the
National
Institute
of
Health,
N.Y.
28, /979.
M.D..
The
University
Department
of
of Texas
Developmental
System
Cancer
Therapeutics.
Center,
6723
0006-4971/79/5402-0027$02.00/O
Blood,
Vol. 54, No.
1 (July), 1979
From www.bloodjournal.org by guest on June 17, 2017. For personal use only.
DISPOSABLE
provides
CHANNELS
FOR WBC
statistical
studies
of
correlation
cellular
responses.
circulating
COLLECTION
and
ranking
components;
and
Evidence
for mobilization
pool is demonstrated.
An
matical
expression
predicts,
with
to the
255
of
what
Principles
(5)
storage
function
and
increment
pools
that
is derived
donor
AND
in vitro
recipient
from
formula
the yield
MATERIALS
Channel Description,
cells
between
accuracy,
variables;
posttransfusion
empiric
relationship
reasonable
of the
(6)
outside
the
gives mathe-
procedure
should
variables
be for each
that
donor.
METHODS
of Separation
and
Operation
Description
The
that
single-stage
channel
is attached
from
the
to the
face
seal
RBC-plasma.
Three
ports
that
the
inner
from
the
barrier,
The
blood,
line
to the
of the
plasma
are
of the
the
barrier
plastic
tube
in Fig.
I . Four
tubes
and
input
interface
They
are
or a mixture
collection
The
separated
outer
extracted
and
by the
RBC
port
port (IFPP)
from
can
wall
of
chamber.
packed
positioning
Plasma
the
plasma
port
chamber.
the
chamber.
and
chloride
as shown
RBC,
the
and
along
recombined
packed
collection
coat
channel.
bottom
flow around
the
buffy
polyvinyl
loop,
between
within
The
a closed
coat,
buffy
of blood
positions
center
top
RBC
and
disposable
( 1 ) The
chamber
(2)
so that
Current
single-stage
blood
of rotation.
The
only
the relative
toward
position
pass
each
are
other
around
by a
the
of the chamber.
barrier
Downstream
IFPP.
flow
collection
rates
of packed
as anticoagulated
the
collection
in terms
blood
are
chamber.
flow
during
the
and
blood
flow
from
current
is in a circle
by the design
CFCS
around
the
of the collection
defined
control
of the
to obtain
the
channel
separation,
(PF)
is parallel
separation
direct
plasma
enters
Centrifugal
factor
is stabilized
under
RBC
whole
of a packing
ways
the
is required.
i.e.,
and
i.e.,
the channel
attention
devices,
position
in two important
differs
is circumferential,
within
operator
flow
extraction
channel
separation
interface
axial
occurs
be characterized
separation
during
minimal
are
interface
Separation
direction
flow
The
CFCS
rotation.
vary
the
the
and
RBC
radial
rectangular
to form
of Separation
new
devices.
from
wall,
Principles
for whole
of the chamber.
to lie on
semirigid
chamber
communication
different
extends
along
passages
wall
postioned
barrier
axis
have
of an extruded
collection
is no direct
the outermost
radially
and
provide
There
exit
reaches
consists
input
the desired
and
which
flows
allows
to the
operator,
axis
who
of
must
component.
in a counterclockwise
leukocyte
collection,
can
as:
Gts
(I)
PF7
where
G
distance
For
separation
-
through
the
acceleration,
which
channel
used
in this
-
I
sedimentation
time
takes
work,
the
of separation,
-
s
sedimentation
rate
of blood,
and
d -
place.
separation
time,
t,
is expressed as:
Al
(2)
A=..
where
-
A
channel
Substitution
area,
of (2)
-
I
into
channel
length,
and
Q =
G
The
channel
area
properties
controllable.
Thus,
of separation
separation
length
(A),
of
the
the degree
acceleration,
should
different
geometric
packing
factor
equivalent
(I),
and
system
and
of separation
G, to flow
be obtained
design,
flow
rate
through
the channel.
Al
(7)
PF
geometrical
mean
(1) gives:
rate,
at different
acceptable
to 580
RPM/40
yields
distance
are
through
fixed.
which
in the single-stage
Q,
through
G and
of 3.0
mI/mm
sedimentation
Sedimentation
the
Q.
channel
channel.
In early
x i0
flow
work
cells/liter
rate.
The
is
(s)
takes
variable
can be characterized
If this
with
ratio
this
of blood
combination
is held
system
processed
(d)
place
and
not
by the
constant,
using
were
of centrifuge
are
directly
ratio
similar
channels
of
found
at a
speed
and
From www.bloodjournal.org by guest on June 17, 2017. For personal use only.
256
HESTER
.Vhiii
S
w”tw
13ioaj
ET
AL.
P..
t.(,.#{149}
dil
:“
i
ll
.-
cJ
=-,
!
-----
A
Who
iPort
a’,)
Oood
Input Tube
.j 1:-i
Ps
Fig. 1 .
flow
rate,
provide
which
maintains
comparable
yields.
CsU
Single-stage
that
constant
Port
channel
ratio,
and cross-sectional
indicated
that
view.
650/50,
720/60,
and
820/80
should
Operation
Anticoagulated
As
the
system
whole
fills
approximately
250
stabilized
interface
blood
with
ml ofblood
is related
is drawn
blood,
into
separation
have
been
to the physical
the channel
occurs,
processed,
position
by extracting
and
plasma
RBC-plasma
of the I FPP.
priming
replaces
saline
saline
through
in
appear
in the IFPP.
The location
of this
the
the
IFPP.
IFPP.
After
The downstream
interface,
in turn,
From www.bloodjournal.org by guest on June 17, 2017. For personal use only.
DISPOSABLE
forces
CHANNELS
the
interface
collection
dense
relates
on the
upstream
design
RBC
moves
radially
inward.
location
can
be controlled
platelet
buffy
WBC
collect
processed)
this
the
coat
forms
Collection
on
(1 100-1600)
Donor
the
of the
to current
than
that
to take
of the
offset
between
and
packing
WBC
collect
coat
line.
for optimal
both
port.
through
the
(500-1000
rate
ml
of packed
collection
collection
(580-820
interface
A leukocyte-
occurred
platelet
a
interface
extracted
has
WBC
continues,
downstream
extraction
platelet
so that
upstream
RBC
are
RBC
the
collection
the
and
components
of the
optimal
granulocyte
and
the
Simultaneous
but
processing
through
Controlling
collection.
location
As
upstream
RBC
interface,
radial
coat.
channel,
the
of packed
procedures,
same
collect
sufficient
buffy
the
of a buffy
when
BCS
required
wall
rate
upstream
into
makeup
compared
outside
of radial
begins
barrier
visualization
extraction
the
257
of the
not
on the
amount
RBC
cellular
system
side
by the
port.
COLLECTION
and
forms
The
to introduce
controls
WBC
to channel
of packed
layer
FOR
RBC
is enhanced
requires
in
higher
RPM
RPM).
Selection
Donors
were
randomly
sis procedures
on the
were
evaluated
were
treated
by the
with
received
donors
prior
selected
from
NCI-IBM
the daily
2990,
usual
list of normal
Aminco
screening
procedures,
etiocholanolone,
10
mg,
and
and
and
scheduled
10-I
consent
forms
M30
signed.
mg,
intramuscularly.
to leukapheresis.
leukaphere-
separators.
were
2.0
2 hr prior
to undergo
Haemonetics
dexamethasone,
8.0 mg, orally,
dexamethasone,
donors
Celltrifuge,
Donors
Seventy-six
Ten
donors
Fifty-one
donors
received
no
medication.
Donor
CBC
parameters
with
studied
differential
prothrombin
time
ionized
calcium
protocols
being
included
and
(PT),
(Ca
partial
+
)
+
carried
pre-
platelets
and
all
postprocedure
donors.
thromboplastin
levels.
out
for
Cells
in the
time
collected
pressure,
donors
(PTT),
were
Department
blood
Some
were
Lee-White
transfused
temperature,
studied
for
clotting
of Developmental
rate,
chemistries,
serum
citrate,
time,
to 1 25 patients
pulse
SMA
entered
and
on replacement
Therapeutics.
Procedure
Hydroxyethyl
dilution
procedure
mean
was
starch,
was
used
time
RPM
500
was
142
of 720
with
ACD
of 8.8
(range,
78-200)
with
580-820).
Donor
measurement.
Bag
Samples
bag
collected
for determination
are
converted
to milliliter
and
platelets
RBC
Statistical
a mean
flow
for each
by ACD-A
was
insufficient
carried
out
(range,
rate
of 60 mI/mm
flow
rate
at a
combination
I .6-1
occurred,
mean
40-80)
speed
at 50 ml/min,
bleeding
rates
at 40 mI/mm.
ACD:WB
4.4-10);
(range,
centrifuge
I .6 sq m were processed
<
When
was
in a 1:13
processed
usually
.9 sq m at
related
to
x lOll
of leukocyte,
volume,
and
platelet,
total
yields
and
are
RBC
concentration.
expressed
Collection
as leukocytes
x l0,
and
cells.
Methods
Statistical
Data
of blood
Data
were
weights
followed
liters
selection
Donors
60 mI/mm,
and > 1.9 sq m at 80 mI/mm.
some limitation
in venous
access,
processing
Collection
concentrate
A mean
mm
(range,
on BSA-TBV
based
ml 6%,
for all procedures.
analysis
Corporation
Student’s
t test
between
more
possible
linear
In Vitro
of the
Cyber
was
than
used
two
data
was
performed
1 72 computer
for
comparison
groups,
combinations
Cell Function
and
by
the
Statistical
of
means
the
Scheffe
test
of group
means,
not just
Department
Package
between
was
two
applied.
pair-wise
of Biostatistics,
for
the
Social
groups.
This
method
using
Sciences
For
comparison
allows
a Control
program.
The
of
means
examination
of all
comparisons.
Studies
Granulocytes
The
test
formazan
Baehner
(1.6
method
produced
and
x
concentration
106
selected
Nathan’2
PMN),
(1:9
for
by conversion
was
NBT
dilution).
granulocyte
of nitro-blue
standardized
concentration
Spectrophotometric
phagocytic
function
was
a quantitative
measurement
of
(NBT)
dye.
A modification
of the
of
tetrazolium
relative
(0.5%),
to
polymorphonuclear
incubation
measurements
cell
time,
latex
at
510
(PMN)
particle
nm
concentration
size
were
method
(0.8
performed
MI).
and
on
a
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258
HESTER
Gilford
flow-through
spectrophotometer,
concentration/NBT
Granulocyte
morphology
on 1000 cells
tion
and
was
results
time
concentration/reaction
examined
in a Wright-stained
were
expressed
at 10-mm
and
graded
preparation
as
intervals
for cytoplasmic
of collected
micrograms
from
ET AL.
formazan/PMN
5 to 60 mm.
integrity
and
nuclear
configura-
cells.
Lymphocytes
Specimens
membranes
The
rate
were
examined
of viable
cells,
of DNA
lymphocyte
individual
and
was
consisted
(PHA)
the
trypan
excluded
blue
as measured
carried
out
exclusion
test
and
nonviable
cells,
by pulse
labeling
dye,
according
of a set of unstimulated
and
expressed
Red
synthesis,
culture,
glutinin
by
which
streptolysin
as cpm/l.5
control
0 (SLO).
x l0
lymphocytes.
pre-
and
to the
with
method
determine
which
percent
took
intact
up the
and
was
on serial
et al.3
cultures
of 3H-TdR
surface
dye.
thymidine(TdR)-3H
of Thurman
cultures
Incorporation
to
incubated
Cultures
with
measured
days
for
of
each
phytohemag-
on days
2 through
7
Cells
Aliquots
from
of donor
0.85%
blood
to 0.30%.
spectrophotometer.
Plasma
Results
hemoglobin
method
was
used4
postprocedure
Hemoglobin
are
by
freed
given
benzidine
on donor
were
from
as percent
specimens
RBC
to decreasing
was
hemolysis/NaC1
reagents
blood
exposed
lysed
was
carried
pre-
and
concentrations
measured
on
of saline,
a Gilford
flow-through
concentration.
out
for
1 8 prodecures.
Cyanmethemoglobin
when
reagents
postprocedure
benzidine
became
unavailable.
RESULTS
Performance
The mean total cellular
yields,
standard
1 Mean
collect
volume
was 1 70 ml (range,
deviation,
70-306)
.
concentration
of
(range
1 30-2260),
(Hct)
and ranges
with mean
202 x i03 WBC/,l
(range,
70-379),
693
and 556 x i03 RBC/.cl,
which
represented
Variables
Flow
and
Rate
platelets/al
hematocrit
The
four
RPM
centrifuge
characteristics
speeds
already
experiments
and
described.
documented
that
flow
rates
While
the
were
determined
the equations
WBC
yields
increased
RPM,
dropped
from
data support
80%
blood
G forces.
Donor
Table
Total (n
-
1 30)
to 85%, seen at 580-720
separation
principles
precounts,
1 . Total
bag
Cell Yields
RPM,
in that
collection
From
±
SD
wBC
x i09
33.0
±
11.6
PMN
x i09
26.7
±
10.2
±
2.3
Lymph
Mono
x i09
with
centrifuge
yields,
speed
at
was low (r = .243), the
0.003).
While
total
WBC
in that the percent
PMN
and
Leuk ocyte
at 820 RPM.
These
collection
requires
RBC
contamination
Channels
Range
13.7-63.6
9.0-55.4
0.85-13.7
2.0
±
2.0
x 10”
1.1
±
0.6
0.2-3.3
RBC x 10”
1.1
±
0.5
0.1-2.1
Plate
x i09
4.4
to separation
comparable
to 70%-75%
optimal
PMN
volume,
Single-Stage
Cell Yield
relative
predicted
as shown
in Fig. 2. Although
the correlation
differences
in yield
were statistically
significant
(p =
yield increased,
a subtle
shift in differential
was noted,
low
x i03
a mean
of 4.4%.
Procedure
820
are shown
in Table
collect
bag cellular
0-8.8
From www.bloodjournal.org by guest on June 17, 2017. For personal use only.
DISPOSABLE
Fig.
function
2.
were
CHANNELS
comparable
by
unpublished
was
a
in yield
Increasing
related
flow
in flow
for each
donor
and
donor
blood
volume
rates
avoids
to centrifuge
rates
reduces
represents
first
inappropriate
and
speed
donor
time
efforts
calcium
citrate
infusion
were
not
required
to
to individu-
depletion,
rates
which
(Hester
JP,
data).
Contamination
There
was a negative
correlation
a trend
toward
higher
bag Hct
and
WBC
mean
Hct
precounts
The highly
donor
20
Bag
x
lO
of4.O%
>
PMN,
and
between
(mean
in donors
yields
1 2,OOO/il.
significant
precount.
the packed
of4O
RBC contamination
4.8%)
and lower mean
with
precounts
x lO WBC
and
and yield. There
yields,
24 x iO
<lO,000/i.il,
compared
32 x lO PMN
in donors
to a
with
The differences
in bag Hct were not statistically
significant.
differences
in total yields
(Scheffe)
related
to differences
However,
no gain
in yield
a wide
range
can
be expected
by collecting
deep
in
within
red cell layer.
Volume
In current
specific
technology,
collect
volume
correlated
with
research
period
procedures
that
yield.
Research
positive
correlation
(p
changes
Variability
procedures
influenced
as
259
inix
variables.
WBC-PMN.
RBC
COLLECTION
yields
so that
by these
collect
alize
WBC
Leukocyte/granulocyte
of centrifuge
speed.
influenced
is
FOR
=
Donor
The
highly
0.0001),
nor
a range
of collect
of volumes
volumes
has
been
is obtained.
identified
that
Neither
a
could
be
optimal
yields.
Volumes
collected
during
the earlier
part of the
were based
on a mean
volume
of 169 ml obtained
in 980 BCS
indicated
a low but positive
correlation
between
collect
volume
and
on this channel
has indicated
that
as a single
variable
there
is
(r = .37) between
collect
volume
and yield that is significant
but
volume
contribution
to yield
diminishes
beyond
300
ml.
Variables
concentration
correlated
of WBC
(r
=
.607;
p
and
=
PMN
0.001)
with
in the
yield.
peripheral
Figure
blood
3 shows
of the
this
donor
relationship.
is
From www.bloodjournal.org by guest on June 17, 2017. For personal use only.
HESTER
260
LE
NONE
XAME
THA
SC)L
DE XAME
Unstimulated
donors
have
L
Fig.
lowest
to
granulocyte
Yields
shift
differed
At
bowls
PMN
p
had
donors
0.01)
=
as a function
concentrations
donors
(NCI-IBM:Aminco)
were
obtained
the
are
of
in
that
of the
Multiple
Variable
statistical
(yield)
reusable
Stepwise
bowl,
as seen
in precounts
Forward
to match
both donor
and volume
collected,
in Fig.
Regression
method
by which
single
can be ranked
statistically
donor
and
total
yields.
highest
precounts
intermediate.
All
and
in which
a mean
of 18.5 x
980 procedures.
Three-hundred
BCS procedures
were analyzed
each level of donor
precount
exceeds
The
variable
(Scheffe,
yield
yield,
and
three
and
the
in
the
granulocytosis
by pretreatment
of the donor
is also
reflected
fraction
of the total yield from treated
donors.
obtained
in the single-stage
channel
exceed
our mean
performance
the reusable
13.8
x lO
concurrent
volumes.
significantly
Total
and precount.
WBC-PMN
Etiocholanolone-dexamethasone-treated
total yields.
Values
for dexamethasone-treated
groups
3.
stimulation
‘.
the
AL.
fliASt.E
#{163}TlDU.OLt..CL.
:L7
ET
with
lO WBC
and
and
fifty
precount
and bag
the performance
4.
Analysis
variables
in order
80
BAG
contributing
to a dependent
of their
relationship
to each
VOLUMES
COLLECTION
‘125-175ML
#{149}225-275vit.
Ell
SINGLE
STAGE
60
20
6-8
Fig. 4.
Leukocyte
donor precount
and bag
yield
as
volumes.
related
to
5-10
10-12
DONOR
LEUKOCYTE
121
PRECOUNT
S x103/uL
1L-16
>16
From www.bloodjournal.org by guest on June 17, 2017. For personal use only.
DISPOSABLE
CHANNELS
Table
2.
FOR
WBC
Regression
Analysis
Multiple
Variable
for Variables
r
Contrib
Simple
uting
to Granuloc
r
r
yte
Yield
Square
Sign.ficance
.853
.853
.728
<.0001
RPM
.911
358
.831
.004
DonorHb
.935
.696
.875
.025
Flow
.943
.059
.889
NS
BagRBC
.947
-.227
.897
NS
.949
-
.902
NS
.905
NS
.002
.908
NS
.321
.915
NS
time
Recirculation
.95 1
Blood processed
.953
Bagvolume
.956
other
and
to
regression
this
the
precount
are
0.0001),
(p
<
are statistically
in flow rate,
Differences
processed,
and
ble ranking.
male
methasone
Hb
donors
variables
this
time.
is a
forward
contributing
stepwise
to PMN
in Table
speed
(p
did
not
and
donor
had
higher
a greater
contribute
122
2. The
=
0.004),
attain
statistical
precount
leukocytosis
to the
yield
were
variable
analyzed
in this
blood
multivaria-
linked
and
relate
values
and
etiocholanolone-dexa-
in males
dependently
by
donor
(Hb)
in PMN
collection.
time,
recirculation,
significance
are
hemoglobin
multiple
yield
analysis
indicates
that
and donor
hemoglobin
the most significant
variables
RBC
content,
procedure
volume
induced
two
Efficiency
bag
Donor
that
variables
summarized
centrifuge
and
0.025)
=
variable
Nine
.299
.
-
dependent
analysis.
method
fact
261
Precount
Procedure
(p
COLLECTION
than
primarily
females.
to the
Whether
or independently
these
is not
known
been
based
at
of Extraction
Previous
blood
the
to total
drop
in donor
leukocyte
WBC
channel
and
liters
expressing
of blood
of
counts,
the
In this
study,
the
leukocyte
the
the
through
was carried
leaving
of
mean
passing
efficiency.
and
9.0
and
cells
entering
efficiency
arithmetic
processed
yield
extraction
of
estimates
multiplying
Although
processed,
mean
assessment
by serially
WBC
and
measuring
counts
postprocedure
obtained
postcount
by
as a ratio
there
does
of the
have
the
efficiency
system.
arithmetic
out
and
extraction
the
channel.
extraction
precount
not
an
revealed
present
that
16
114
12
i10
Fig. 5.
Cell depletion
during
collection
single-stage
channel.
leukocyte
st.ooo(Lusts)
rocsssn
33%
reflect
efficiency
preprocedure,
cell
overall
accurately
the WBC
on
total
of actual
was
extraction
the
the
of the
in blood
at 3.0,
a linear
fall
6.0,
in
From www.bloodjournal.org by guest on June 17, 2017. For personal use only.
262
ET AL.
HESTER
donor
WBC does not occur during
reduction
in donor
WBC
during
of only 4%
Instantaneous
lines,
being
is seen between
measurements
however,
extracted
demonstrated
through
extraction
efficiency
following
completion
outside
the
comparing
x
WBC
The
to about
of HES.
This
the
of cells cleared
from
which
was collected.
number
to that
regression
was
occurring.
Granulocyte
analysis
yield
of cells
indicates
appears
available
a given
with
to be most
to be collected
equation
predicts
donor.
volume.
VB
can
infused
pools
be seen
(donor
channel,
are sufficiently
of donor
by
pre-post
a log
The
equation
ratio
characterized
to donor
could
count.
The
precount
and
total
PMN
expression
yields
of what
value,
is:
we
procedural
implies
that
well
to give mathematical
accuracy
what
represents
donor
=
single-stage
related
YP=2CPVB1n[1
collect
was
were
This
storage
studied,
and additional
the yield.
It further
was derived
with reasonable
This
precount,
the
is a function
to what
was available
to be collected.
from least-squares
fit to the data.
The
donor
pool
significantly
be
=
for this
circulating
parameters
of yield.
relative
derived
C
ACD-A
from
evidence
the
variables
affect
should
where
NIH
mobilization
Further
that
and collection
predictability
volume.
An empiric
relationships,
which
for
entering
the channel
the infusion
of HES.
Yield
for 9 1 .5% of the yield by the
are not likely
to significantly
number
when
that
is a 28%
decrease
7% between
6.0 and 9.0 liters.
taken
on the input
and return
of cells
during
50%-55%
suggests
pool
separation,
extraction,
in this system
to allow
blood
these
that
60%-65%
collection
port
the
dropped
Equationfor
account
variables
and 6.0 liters,
and
of leukocyte
counts
circulating
TBV)
Predictive
3.0
leukapheresis,
as shown
in Fig. 5. There
the first 3.0 liters
processed,
but further
8.2,
was
to
(Yr)
collected
is a constant
(k)
+
blood
(1)
volume,
In
=
natural
log,
and
V
=
‘O
0
U)
a,
0
0
0,
0
0
a,
>0
a,
>
a)
U)
.0
0
20
Predicted
40
Yield
60
x
0’ Cells
80
Fig.
served
yields.
6.
Correlation
of obyields
to
predicted
From www.bloodjournal.org by guest on June 17, 2017. For personal use only.
DISPOSABLE
CHANNELS
Table
FOR
3.
COLLECTION
WBC
Posttransfusion
263
Corrected
Single-Stage
Granulocyte
Channel
x 1O/isl
Increments
Versus BCS
ABO
Compatible
Incompatible
Single-Stage
HLA
BCS
Single-Stage
B
Compatible
t-0
.813
.624
t-0
-
.288
t-lhr
.803
.652
t-lhr
-
.408
t-0
.830
.476
t-0
.556
.150
t-lhr
.736
.464
t-lhr
.550
.212
Incompatible
Figure
6 shows
yields.
It indicates
yield.
Less
than
the correlation
between
that most of the observed
10%
of the observed
and can be accounted
resultant
lower WBC
Recipient
were
receiving
tion
could
have
increment
PMN/il).
replacement
analysis
the
lower
predicted
predicted
standard
or nonuniform
receiving
with red
or prophylactic
expected,
bag
but
was
induction
cells and
deviation
sampling
with
PMN
chemotherapy.
All
platelet
components
replacement.
not documented.
The
Alloimmunizamean
after
transfusions
was
1050
PMN/l
corrected
for donor
body
surface
area
absolute
PMN
(range,
0-5000
and
total
cells
arranged
according
to HLA
and ABO compatibility
are shown
are compared
to those obtained
in an analysis
of 980 therapeutic
transfusions,
of 8 variables
statistically
obtained
revealed
with
ABO
significant
(p
contribution
the
reusable
0.005)
=
and
bowl
in which
HLA
(p
to posttransfusion
=
in
a regression
0.02)
made
the
4. The
19%
increments.
Data
Donor
drop
therapeutic
been
immediately
Increments
transfused
and
Table
3. These
Donor
lie outside
problems
were acute
leukemia
patients
been transfused
extensively
and
most
observed
yields
and the
yields
lie very close to the
Data
Recipients
previously
had
yields
for by procedural
concentrations.
our
hematologic
in Hb
data
is comparable
pre-
and
to that
dilution
with HES:ACD
and
tion in leukocyte
and platelet
greater
efficiency
Table
of the
4.
Donor
postprocedures
with
our
are
current
shown
BCS
sampling
for multiple
studies.
counts
exceeds
that for current
diposable
Data
blood
for Leukocyte
pathway
in Table
procedures6
Pre-
reflects
The 30%-33%
reducBCS and reflects
the
in extracting
Collection
and
cellular
compo-
and Postprocedure
Percent
Value
Pre
SD
Change
p Value
Hb(g/lOOml)
14.3
±
1.7
11.7
±
1.6
19%
0.0001
WBC(xlO3/izl)
11.5
±
3.4
8.0
±
2.0
31%
0.0001
9.4
±
2.9
6.6
±
2.0
30%
0.0001
0.038
PMN
(x 1034tl)
Lymph
Mono
Platelets
(x 1034il)
(x 103/zl)
(x 1034il)
Bboodvolume
‘Two-tailed
t test.
±
Post
±
SD
1.6
±
1.0
1.0
±
0.5
38%
0.3
±
0.4
0.2
±
0.2
34%
NS
277
±
64
2 15
±
52
33%
0.0001
4.6
±
0.6
-
-
From www.bloodjournal.org by guest on June 17, 2017. For personal use only.
264
HESTER
nents.
Prothrombin
times
blood.
were
SMA
ture
and
time,
partial
normal,
confirming
screening
studies
elevation,
peripheral
pattern
similar
In Vitro
Function
thromboplastin
time,
that anticoagulation
disclosed
no significant
shaking,
edema,
chills,
related
to that
with
and
Lee-White
was limited
abnormalities.
or hypotension
occurred
in any
to volume
expansion
by HES:ACD,
BCS-M30
procedures
performed
ET
AL.
clotting
to extracorporeal
No temperadonor.
Headaches
were noted
in a
in our
laboratory.
Granulocytes
Phagocytic
produced
by
(active)
research
function
is intact,
as
nonstimulated
(resting)
by latex particles
for cells
prototype
(Apheresor),
differences
among
;zg formazan
after
Normal
groups.
a 30-mm
incubation
PMN
structure,
age
(0%
-
systems
noted
3%)
of smudge
currently
by our current
BCS
2997
are compared.
Nonstimulated
period.
as
related
on Wright-stained
cells
Fig. 7. Micrograms
and duplicate
cultures
cells
This
same
formazan
stimulated
reusable
There
produced
is the
of
bowl,
were
the
no
approximately
value
4
in 23 studies
from normal
blood. A rise to 16-19
tg formazan
granulocytes
are stimulated
by latex beads.
morphology,
was
in
collected
and
the
the three
ofperipheral
blood granulocytes
seen in all three groups
when
shown
PMN
can
to
smears
be seen
membrane
integrity
of collected
cells.
on slides
of cells
collected
and
A small
by any
is
nuclear
percentof these
in use.
Lymphocytes
Figure
8 shows the mean values
ofcontrol
and mitogen-stimulated
lone and/or
dexamethasone-treated
as
lymphocytes
sample
was
were
obtained
isolated
from
from
the
bag
(±
standard
deviation)
of 3H-TdR
incorporation
and SLO)
lymphocytes
from etiocholanodonors.
Each donor
served
as his own control,
(PHA
blood
peripheral
prior
to the
procedure,
collected.
PT-1JUCT1(T1
CRNtJL0CYW
cc?ssRIsnI
RESTING
OF
iai-srrwotiots
IIIIL:TIVE
2997
55
I
REMTIE5
Fig. 7.
Granulocyte
TD.t
function.
NBT
reduction.
and
a
From www.bloodjournal.org by guest on June 17, 2017. For personal use only.
DISPOSABLE
CHANNELS
FOR WBC
COLLECTION
PROTEIN
=
265
2t(v/v)
urcoous
sntu’i
U
200
CONTROL
PERIPI#{149}#{128}RAL
54.000
BM3 cOU..ECTS
tRtE
SLO
160
00
2
3
L
5
DAYS
Fig. 8.
2
7
In vitro
IN
lymphocyte
3
LI
CULTtSPE
transformation.
Erythrocytes
Standard
erythrocyte
osmotic
fragility
curves
were
constructed
and
demon-
strated
no significant
differences
in donor
peripheral
blood,
pre- and postprocedures,
for the IBM
2997 procedures
(n = 76) compared
to Aminco
(n = 36) or
Haemonetics
M30 (n = 8) procedures.
Plasma
hemoglobin
determinations
by the
cyanmethemoglobin
method
were
ml pre and I 1 72 mg/ I 00 ml post),
.
the seal
or outflow
parts
of the
2997.
unchanged
indicating
after
the
no injury
procedure
occurred
( 1 I 95 mg/ 100
to donor
blood in
.
From www.bloodjournal.org by guest on June 17, 2017. For personal use only.
266
HESTER
ET
AL.
DISCUSSION
blood
Nd-IBM
effective,
cell
reliable,
anticoagulant
separator
closed
could
undergo
cyte
in a CFCS
sis/granulocytosis;
a
injection
applied
leukapheresis.
1971,
Graw
medicating
donors
In 1974,
an
the
collection
confirmed
etiocholanolone
1 2 hr
with
cell
sedimenting
agent
reported
leukocytosis
an
and
intramuscular
precursor.
McCredie
of donors
continuous-flow
cell
available
and
undergoing
In
by
has
or thrombocytosis,
for
processing,
contribution
of
RBC-sedimenting
a statistically
significant
to the input line.
broad
of infected
stem cell),
application
in
neutropenic
patients.
cellular
depletion
for
and
for centrifugation
loop” circuit
with
or the
usefulness
the
and
separation
leukocytosis
cellular
constituents
to maximize
the
reported
demonstrated
of this agent
patients
becomes
granuloleukocyto-
following
steriod
et al.’4 both
of investigators
by the addition
all evolved
through
the technology
patient,
becomes
part of a “closed
volume
of compo-
to induce
plasma-expanding
collection
for management
(lymphocyte,
monocyte,
ing specific
In order
and
collection
to pretreatment
addition
to granulocyte
Mononuclear
collection
blood
an
blood
or steroids.
derivative
leukemic
provide
of
was modestly
improved
over untreated
controls.
that
total
leukocyte
yields
could
be enhanced
amylopectin
with
to
to increasing
red
kinetics,
occurring
Mischler
of
of the
in 1970
et al.’3 and
application
while
pretreatment
leukocyte
McCredie
properties.’5
Both groups
increase
in PMN
collected
donor
approximately
observation
with
1960s
a mixture
lymphocytes.
contribution
addition
a naturally
this
PMN
et al.’2
was
of
donors
of etiocholanolone,
Freireich”
The
was
study
in normal
the
which
processing
primarily
a significant
One
other
starch.
al.,’#{176}in
granulocytosis
HES,
system.
the
hydroxyethyl
Vogel
et
in
in
by Freireich
et al.9 in 1965,
produced
good
in vitro
in vivo separation
initially
involved
chronic
leukemia
and leukocyte
yields were
factors
subsequently
made
yields
developed
system
uninterrupted
nents was in progress.78
The first trials,
reported
recovery
of leukocytes.
The
patients,
Two
was
centrifuge
plasma
exchange
of whole blood.
The
the separator,
wherein
extracting,
exchanging,
acellular
fraction.
of continuous-flow
cell
have
donor,
or
his total
or replac-
separation,
it is
necessary
to understand
the response
of whole
blood
to centrifugal
forces
as it
passes
through
the channel,
as well as the donor
or patient
role in the processing.
The reponse
in this disposable
single-stage
channel
for granulocyte
collection
can
be summarized
At
low G
collected.
as follows.
forces,
with
Because
plasma.
This
sedimenting
of size
allows
and
optimal
optimal,
platelet
yields.
As G forces are increased,
leukocyte-rich
interface,
the plasma
to the interface
platelet
yield, but sacrifices
The
interface
collection.
superior
extraction
PMN
positioning
density,
agents,
the
granulocyte
collection
granulocytes
and
a larger
and
PMN
port
granulocytes
majority
are
number
provides
and
replaced
with
the
a semiautomatic
The barrier
allows
accumulation
to that obtained
by BCS.
be
are
left
adequate,
effectively
free
but
less
by mononuclear
of platelets
are extracted
yield.
may
of platelets
of the
are
brought
buffy
coat.
interface
buffy
coat
cells
down
This
in the
than
in the
out
of
increases
control
and
provides
for
From www.bloodjournal.org by guest on June 17, 2017. For personal use only.
DISPOSABLE
CHANNELS
The donor
manipulated.
FOR
contribution
Both donor
WBC
267
COLLECTION
is a biologic
stimulation
and
variable
HES were
reveal
unambiguously
the role of donor
pretreatment
Mobilization
of cells from storage
pools outside
make
a significant
leukocyte
contribution
depletion
to the
by leukapheresis,
Although
ADL-Cohn
large
numbers
fractionator,
Leukocyte
collection
of
the
final
was
leukocytes
peripheral
Leukocyte
yields
cannot
donor
blood
to yield.
volume)
The
data
channel
and
those
et al.
suggests
in terms
equation
between
procedural
as
by Bierman
were
level
that
variables
the same
of circulat-
a fixed
quantity
of
study
(donor
attempts
count
to
and
that
yields
40
contributed
x
l0
can be achieved-that
is, collect
volumes
in the order of 250 ml and donor
16,000/sl.
Finally,
there
is clinical
importance
in the capability
to deliver
larger
for replacement
tion of transfused
transfusion
to date
Applebaum
of neutropenic
PMN.
survival
study
yield
purposes.
PMN,
have
Outside
the short
left
et al.’7
dogs
blood
PMN
dose-response
patients
have
with
in which
survival
was
from
donors,
coupled
the problems
half-life
and
linked
with
concentration,
relationship
exposed
reported
septicemia.
inherent
inadequate
to
the
allow
for
WBC
counts
cell
doses
in immunologic
destruccell doses delivered
by
1 8 hr of inadequate
critical
importance
Vogler
et al.’8 have
with higher
increments.
a more
physiologically
should
necessary
#{149}16
phenomenon
collected)
in which
of
in 1961
is depletion
of collecting
(volume
a result
removed
on the
was maintained.
derived
in this
biologic
variables
circumstances
be
data
by CFCS.
appears
to
Mobilization,
of mobilization
is not known.
be standardized
The empiric
relationships
indicate
yield.
in PMN
collection
circulating
pools
(5-1 72 x 10)
blood
leukocyte
in the disposable
in each
procedure.
mathematically
the
suggested
is being observed.
Whether
the mechanism
ing cells or persistent
influence
of medication
cells
relate
that
can,
to some
extent,
utilized
in this study.
The
circulating
of cell dose in
reported
a clinical
Increasing
appropriate
the cellular
peripheral
more
accurate
characterization
successful
replacement
in
of the
neutropenic
patients.
CONCLUSION
This
standing
forces.
rates,
will
second-generation
of the characteristics
It allows
centrifuge
optimize
biologic
expression
CFCS
blood
of whole
some standardization
speed,
procedure
component
variability
of these
pathway
has expanded
our scientific
underblood separation
in response
to centrifugal
of procedure
bag volume,
time,
collection.
It allows
on component
relationships
by identifying
and interface
a range
positioning
us to understand
collection,
that
offers
and
it
guidelines
the
effect
of flow
that
of donor
permits
a mathematical
for realistic
component
collection.
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1979 54: 254-268
Principles of blood separation and component extraction in a disposable
continuous-flow single-stage channel
JP Hester, RM Kellogg, AP Mulzet, VR Kruger, KB McCredie and EJ Freireich
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