Separation of the acetylcholinesterase-deficient red cells in paroxysmal ...... attack complex when it is initiated in fluid phase by cobra venom factor and are lysed.
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1986 67: 893-897
Separation of the acetylcholinesterase-deficient red cells in paroxysmal nocturnal hemoglobinuria FL Chow, SE Hall, WF Rosse and MJ Telen
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Separation
of the Acetyicholinesterase-Deficient Nocturnal Hemoglobinuria By
Blood uria
of
patients
(PNH)
most
with
lysis of
moderately
abnormal
(PNH
and
complement,
cells
(PNH PNH
markedly
We
have
devised
AROXYSMAL
sensitive
to
well
nocturnal
characterized
by red
the
normal
sensitivity
to
Study
a second
PNH
IlIb
lysis
than
to
other for
cells
action
by
membrane
are
is
some
III
may
than (15-
complement.”2
cells).
be moderately
These
populations
and
various
proportions
this
mixture
of cells
membrane In
defect
1960,
occur has
leading
Auditore
al
demonstrated cient;
that to the
the
normal
PNH
of the enzyme.6
by the protein
use of monoclonal immunologically
lacking how
in these these
separate
the
in
used
PNH
red
that are majority
the
that
cells
the
I cells
appeared
Recently,
Chow
the
activity
In the
present
been
used
cells,
were to
paper,
were have
definormal
a technique the
of
to
to complement
lysis
III
than
shown
Red
blood
cells.
Blood
from
cells
normal
donors
or
patients
with
(AE-l, AE-2, AE-3, and AE-4) made by mouse hybridomas against four different epitopes of the acetylcholinesterase molecule were the kind gift of Dr D. Fambrough9 in the form of purified lgG. In addition, AE-1 and AE-2 hybridoma cells were obtained from American Type Culture Collection and grown as ascites tumors in BALB/c mice. P3 (P3x63/Ag8), the immunoglobulin product of a murine myeboma cell line, was also grown as murine ascites tumors and used as an
Blood,
Vol 67, No 4 (April),
antibodies
1986:
of
to the
PNH
sensitive
to
lysed
PNH
identifica-
erythrocytes. complement
by
fluid-phase
II erythrocytes.
Inc.
to
monocbonal
the
reactions
of the
AE
mono-
action
and
Dacie.3
The total
the
proportions
and
compare ment
is the
CLS
used
(270
lents
of serum
determine
washed
of monocbonal protein
minutes.
The
100
and
tL,
[P3])
were
incubated
diluted
After
the
washing,
100 L
of 50 L
each
part
N-butyl
antibody
suspended
layered
blood
30
of
antimouse
for
30 minutes. 100
Eastman
zL
and
Triplicate
of phthalate
esters
Co,
Pittsburgh,
Kodak
Inc.
and centrifuged
Pa to
Rochester,
in a Beckman
Fullerton, Calif). The cells the tube, and the radioactivity
were was
counter.
cells
incubated
on a column. with
and
for
to a volume
(8 x l0 cpm).
Scientific
tubes
saturating of control
in
on a mixture
Fisher
a
of rabbit
temperature
scintillation
(AE-l
and
brought
SPA
To acetyl-
temperature
again
Instruments, and cutting
were
room
volume
of ‘25I-labeled
cells.
to the
dilution
at room
were
phthalate,
of red
108/mL)
cells
twice,
equal
microcentrifuge
Separation x
an
I :50)
in a well-type
counted
at
with
phthalate,
microfuge (Beckman removed by clamping
red
( I 00 zL) of a 5%
volumes blood
of
equiva-
cells.
bound
(or a comparable
washed
bis-2-ethylhexyl
in plastic
equal red
then
were
of the
to
dilution
milliliter
normal best
incubated
cells
with
(I .5 parts
cells,
cells were
to
used
by comple-
of the
of 50%
a
of Rosse
value
to lysis by the
antibodies
antibody
IgG (2 mg/mL incubated
lysis
normal
test
The
reciprocal
antibodies
monocbonal
of
the
red by
to 375 ML, but
same.
divided
to obtain
on normal
cholinesterase dilution
as
cells
of human
was reduced
populations
studies)
blood
determined (CLS)
the
cell
defined
monoclonal
which
suspension
volume remained
of different H,
was
bysis sensitivity
reaction
in these
to red A (SPA).
The sensitivity
complement
materials
required of
attached protein
tests.
of
complement
sensitivity
serum
antibody
sensitivity
lytic
was obtained as affinityMalvern, Pa. This was
staphylococcal
lysis
the
of the
one
for
AE-2)
equal at
Washed
volumes
red
of each
a dilution
that
cells mono-
saturated
all
METHODS
108/mL.
Monoclonal
with
modification
the
PNH was stored under sterile conditions in Alsever’s solution.8 At the time of use, the erythrocytes were washed in phosphate-buffered saline (PBS), pH 7.4, and adjusted to a concentration of 2 x Antibodies.
& Stratton,
of
are
unlike
of radiolabeled
Complement cells
(1
AND
less but
(IgG 1) control
to react
by bindng
clonal MATERIALS
are cells
led
IlIb.
by use
chromatography.
has
PNH
to mouse IgG made in rabbits from Cappel Laboratories,
IgG
purified
NY)
PNH. We population
PNH
Grune
specific
aliquots
complement-
of patients with a fourth variant
a subpopulation
the was
we demonstrate
to devise
populations
lila
populations
affinity
antibodies.
ascitic
et al demonstrated
from
less sensitive III cells.
abnormally
that
by
mixed cell
subtype.
PNH
Binding
of the
to the enzyme that of the enzyme
complement-sensitive
slightly of PNH
It is
by complement.
that
antibodies characteristic
have
blood
PNH.
to analyze
of complement
red cells in the blood this technique to define
insensitive
have
cells.7
facts
found
action
amounts
lysis
more may be (PNH
was variably deficient on the with PNH.5 Kunstling and Rosse
it was
lytic
with
it difficult
cell
erythrocytes
Antibody
combinations
patients
made
to increased et
some normal
from and
of complement.
isotype
This
to fourfold) and than
in various
in different that
enzyme acetylcholinesterase red blood cells of patients sensitive
(two-
normal (PNH II cells), to 25-fold) more sensitive
J. Telen
Ill cells
a further
1986
clonal
abnormally
hemolytic action of complement are present in the circulation. Three degrees of sensitivity to complement have been described3’4: some of the red cells may be normal (PNH I sensitive markedly
of
activation S
abnormality is usually present in some but not all red cells; thus, populations of red cells with different sensitivity to the
cells),
II and
in Paroxysmal
pure.
(PNH)
that
of
lysis
isolating
hemoglobinuria blood
hemolytic
as
markedly exhibit
Marilyn
of purified
tion
or
and
Cells
antibodies
population
Ill cells
sensitivity
method
PNH
monoclonal
II cells)
a
intact
hemoglobin-
II and
as
F. Rosse,
of
increased
respectively.
defects.
Wendell
populations
and
Ill cells).
E. Hall,
or more
with I cells)
abnormal cells
moderately
P
two
population (PNH
Sharon
nocturnal
contains
by complement
Chow,
paroxysmal
often
erythrocytes-one
Feng-Lan
Red
pp 893-897
From
the
Department
of
Medical
Research,
Division
of Hematology-Oncology.
University
Beijing,
Biochemistry, People’s
Chinese
Republic Department
of
Center,
Durham,
NC.
Submitted
May
2. 1985;
accepted
Oct 21. 1985.
Supported
in part
Lung
and
This Duke
work
Address
Institute,
National
was performed
University
University ©
Blood
Medical
reprint Medical
I 986 by Grune
requests
I ROl
AM31379
Institutes
in the Myrtle Center,
Durham,
from
Bell
Lane
J. Telen. 27710.
& Stratton,
Duke
Heart,
Laboratories
at
NC.
Durham,
NC
the
the
of Health.
to Dr Marilyn
Center,
of
and
of Medicine,
Medical
by Grant
Institute China;
Box
3387.
Duke
Inc.
0006-4971/86/6704-0007$03.00/0
893
From bloodjournal.hematologylibrary.org by guest on July 10, 2011. For personal use only.
894
CHOW
for 30 minutes
sites
at room
temperature
on a rocker
platform.
They
were washed by centrifugation, suspended to the original volume, and incubated with an equal volume of rabbit antimouse IgG ( I :50) for 30 minutes at room temperature on a rocker. After two washes, they were pipetted onto the top of the separation column. The separation column consisted of 10 mL of Sepharose 6MB to which
SPA
Fine
had
been
Chemicals,
gravity
and
bovine
serum
After 0.2
albumin
rate
the
column
PBS
was
buffer
rate
with
0.1 mol/L
stored
at
(0.05%
w/v)
adjusted
until
no
column
(usually
about
to 3 to 4 mL/min
50
again
0.1
NaCI.
mob/L
studies.
cells; using
anti-IgG extremely
As
AE-4 AE-2
The
until
To coated cells
acetylcholinesterase
were
enzyme
the
antiacetylcholinesterase
specifically
retained
and
that
had
monoclonal on
the
column
under
conditions.
test cells from
the
ability
from a normal
and rabbit antimouse were reacted with
of the
those donor
not
column coated
were
IgG; type the control
to separate by antibody,
coated 0 cells protein,
with from P3.
antibodytype
AE-l
plus
A red AE-2
a normal donor The cells were
no further
buffer,
pH
washed
with
thoroughly buffer
shown
50 of
with
AE-2.
this
for the chromatographic
6MB-SPA flow
(80%
1 . Binding
Antibodies
this
the monoclonal
similar and
were
not
same
10%).
±
of SPA
eluted cells
way
were This
to Normal
with
conditions. radiolabeled
of
mixture
combination
of
was
cells.
AE-l
used
When
and on the
during ebuted
or
sensitized
indicated
that
Antibody’
P3
the red
first blood
by Monoclonal
Rabbit
Antimouse
(cpm
lgG
SPA
Bound)
AE-1
5,491
AE-2
5,777
AE-3
5,115
AE-4
the
with
during
and
(slow
during
eluted
Erythrocytes
nor-
AE-2 and Sepharose
the first
were
‘25l-Labeled
1,254 +
AE-2
7,319
AE-1
+
AE-3
6,612
AE-1
+ AE-4
6,077
AE-2
+
AE-3
6,557
AE-2
+ AE-4
6,037
AE-3
+
AE-4
6,098
P3 control A11 antibodies
an in
combinations of the
blood
sensitized
to Acetylcholinesterase
AE-1
rabbit SPA
experiments. red
5% of the
Cells
in the
phase
reason,
no cells ±
phase.
of radiolabeled
azide
,
I
case
cells sensitized with were chromatographed
80%
treated
found
in the
ofnormal
column,
rapid
PBS and
sodium
per cell, indicating No great increase
separation
Chromatography
phase;
equiv-
3, contain-
all bound via of radiolabeled
under antibody
was
except
For
mal red blood rabbit antimouse
Table
AE-3 amount
molecules antigen.
SPA
antibodies
flow)
volume
IgG (Amersham Corp, Arlington Heights, maximum binding of the radiolabeled
of radiolabeled and
in
and same
bound less monocbonal
was ten to low density
monocbonal
Table
cells
minutes).
(ten-bed
phosphate
AE-l, AE-2, IgG about the
F(ab’)2 antimouse Ill) showed that
(slow)
antibodies these
the with
pH 6.0.
Binding
and
had
reacted
to about
glycine-HCI
It was then
4#{176}C in 0.1
antibodies antimouse
AE-l
by 1%
further
by thorough
mol/L
RESULTS
binding
packed
containing
was
maintained
was regenerated
washing
to red Studies
Pharmacia
use. the flow
was
increased
from
that
been
eluted.
The column alents)
each
rate
from
was then
cells were
ing
before flow
The
with
of the cells,
this
(obtained
NJ).
thoroughly
application be eluted
flow
attached
Piscataway, washed
mL/min;
could
covalently
cells
ET AL
389
used in saturating antimouse
Ig.
amounts,
as determined
by binding
Fig 1 . Separation of the populations of PNH cells by affinity chromatography. After incubation with monoclonal antibodies to acetylcholinesterase and rabbit antimouse antibody. PNH red cells were applied to a column containing Sepharose 6MB to which SPA was covalently attached. The PNH I cells bearing acetylcholinesterase were retarded (upper band). whereas those lacking the enzyme (PNH III cells) passed through the column (lower band).
From bloodjournal.hematologylibrary.org by guest on July 10, 2011. For personal use only.
SEPARATION
OF PNH
RED
CELL
895
POPULATIONS
Table
2.
Complement
Sensitivity
of Affinity-Purified
Red Blood
:
Analysis
chromatographed
with
only
lack
of agglutination
A cells
type
were
during
blood
III cells)
of
cells
of the
were
treated
the column
into
tion
from
eluted
PNH
elution process, phase. When these
cells
red
with
way,
there
populations
column
anti-A.
of rapid
blood
I cells and
of the two (Fig
(PNH was
eluted
column,
with they
were
C).
(Fig
1): one
first
(slow)
eluted cells
during the were tested
rapid
flow
further
cells
were enriched
curve D), indicating that the did not remain on the column with
but
If the
were
65
36
35
19
58
65
31
35
17
W.M.
28
49
67
51
18
J.G.
44
69
38
31
O.P.
42
100
27
-
ED.
47
100
24
-
-
J.B.
37
100
37
-
-
E.G.
61
100
41
-
-
15
100
41
-
-
M.F.
60
100
27
-
-
>95
C.K. CLS
for
passed PNH
I cells
a normal
the
on
elution elution
phase. phase
of
III
use
containing
population
straight
line
Fig 3. Analysis of the sensitivity to complement lysis of PNH I and PNH II cells separated by affinity chromatography. The analysis of CLS of the unseparated cells is shown in solid symbols; 66% of the cells were PNH 1 #{149} and 34% were PNH II. The analysis of CLS of the cells eluted from the column by slow flow rate is shown in open symbols; these cells were entirely PNH II cells. The CIS of the PNH II cells was not changed by passage through the column. Lysis of 50% of the purified PNH II cells occurred at approximately 29-mL equivalents of serum. whereas lysis of 50% of the PNH II cells within the unseparated cell suspension occurred at approximately 35-mi equivalents of serum.
dilution
1 mL of which in a total
PNH
and
passed
also
eluted
were eluted II cells (Fig
of
would
volume
II
of 7.5
cells
PNH III cells on the column
were
through
the
during
the
slow
during 3).
the
slow
a subpopulation
of I 1 patients
in that (as
defined
sensitive cells four patients
the
in curve
appeared
who
of
were
PNH
defined
as
by conventional CLS by the aforementioned
demonstrated
2).
However,
a single in four
to be a second population sensitive than the PNH III
(Table to
analysis
B, Fig
2 and
consist
Fig
entirely
4).
The
cells
of one less these and
were
the
Thus,
slightly
these
I I
(Table 2). When the PNH III cells from were isolated by affinity chromatography activated of the PNH
of
of
of cells cells as
slightly
lysed by complement venom factor, 100% lysed.
I and
to define
cells
patients, there appeared that were slightly less patient
16 of the
When the red cells lacking acetylcholinesterase in the CLS test, six of 1 1 were found to be of a
single
the column.
column
red
PNH I and were separated
previously
were
The cells that were pure PNH
The
column 2,
PNH
II cells
ofthe
for PNH
(Fig
of complement,
described
PNH
procedure. were tested
III cells reaction
reciprocal
cells of a population
previously
cells.
having testing
the
amount
the
CLS is 2.0 to 3.2 CLS H50 units.
cells
The
>95
-
by determining
of the sensitized
mL. Normal
popula-
through
contaminating PNH because of a specific
containing
column,
phase
-
is quantitated
as
were entirely adherent were on the
H
When
PNH
16 -
A.H.
treated
rapid elution in the CLS
enriched
retained
again
I and
CLS H,,., Units’
Percentage
73
type
a separation
the
populations
2, curve
The
H
J.P.
lyse 50%
When PNH
cells.
test, those that were not adherent to the column PNH III cells (Fig 2, line B). Those that were a mixture
sepa-
flow.
during
the other separated
Two
slow ebution phase, phase as shown by
phenotype
in this
two distinct
the
the this
the phase
common
the
the column.
during during
of the eluted
eluted
Chromatography
red
through
were evident 0 cells eluted
Percentage
PNH IlIb CLS Units
V.D.
serum
and
of Cells Lacking
PNH lIla
Percentage of PNH Ill in Whole Blood
L
Fig 2. Analysis of the sensitivity to complement lysis of PNH I and PNH Ill cells separated by affinity chromatography. The analysis of the CLS of the unseparated cells is shown in curve A; 50% of the cells were PNH I. and 50% were PNH III. The analysis of the CLS of the cells eluted from the column by slow flow rate is shown in curve B; these cells were entirely PNH Ill cells since the CLS curve forms a straight line. Results of the CIS of the cells retained on the column in the first passage are shown in curve C; the proportion of PNH I cells was increased from 50% to 83.3%. When these cells were rechromatographed. the proportion of PNH I cells in the retained cell population increased to 97.1 %. as shown by curve D.
rate populations
Ill
Acetylcholinesterase
Patient
mixed
PNH
Cells
in the fluid phase by cobra III cells from each patient less
complement-sensitive
Fig 4. Analysis of the sensitivity to complement lysis of PNH I. PNH lIla. and PNH IlIb cells separated by affinity chromatography. The analysis of CLS of the unseparated cells is shown in solid symbols; 50% of the cells were PNH I. and 50% were PNH Ills + PNH IlIb. The analysis of CLS of the cells eluted from the column by slow flow rate is shown in open symbols; 43% of these cells were PNH lIla cells. and 57% were PNH IlIb cells.
From bloodjournal.hematologylibrary.org by guest on July 10, 2011. For personal use only.
896
CHOW
PNH
III cells
did not resemble
We
have
named
these
the
more
complement-sensitive,
ten of 1 1 examples,
PNH
II cells
intermediate
cells common
PNH
lila
in this
PNH
respect.’#{176}
no
cells
and
Ilib
variant,
and
present
in
of the facts that of the membrane
them
susceptible
tions.
Thus,
depended it has
to the
in most
existence,
upon
patients
cannot
PNH
PNH
I or
complex
when
in
that
for the renders
PNH
I and
is the
small
populahave
present,
this
I cells
to be eluted
alone, agents.
without
the
III
cells
abnormal
II cells have been separated lysis.’4 PNH III, but not
tivity
of
bind
attack
abnormal
cells
venom
apparent.
Nevertheless,
red
cells
the
in fluid
membrane
phase
by cobra PNH factor
III cells in a and serum,
is not
under
investigation.
with
intact
the
of their
enriched
by
structure.
complement-
than
by centrifugation are
the
However,
proportion the
is always
contaminated
in the to
normal
technique the
normal these
described
separation
of the
in this
paper
abnormal
PNH
I cells
by
taking
abnormal
cells
lack
the
cells
I
protein
for the
Since the antibodies do not react with the abnormal
for the
first
from
the
in PNH
advantage
of the enzyme
fact
These
III cells
that
cells.
sensi-
Only
when
difference of these
CLS
cells
curves
to separate
the
become has
of
some
the abnormal
of decay-accelerating factor that normally controls the
the
C3
convertases
of
effects purified
of DAF and of the repletion of DAF cells.2#{176} The nature of the relationship
of the from
absence normal
between acetylchobinesterase is currently under investigation. the
use
of the
or partially
comple-
red cells we have
of these
cells
allow
lacking
in the
of PNH.’5”9 By the been able to demonstrate
deficiency
techniques
for the more
nature
ready
and
DAF
described
use
of the
deficiency
herein,
purified
acetyl-
of their
membrane
comparison
defect(s)
of the
cells
and
of different
patients. ACKNOWLEDGMENT
that
that are used in these cells, there is little or
cholinesterase.7
studies
allows
in the
cells.
complement-sensitive separated populations,
will
The
of the
is completely
By
and PNH
of
III
PNH
the
the
technique
activity
analysis
illustrated
identification
presence protein
rate
populations of PNH cells can be obtained from nearly all patients with PNH. This will markedly simplify the analysis
for
incomplete,
by the
and This
red
of
flow
of complement
did
of
and PNH
protein
of these
enrichment
of
separated
(probably
of denaturing
are
usual
is so antigen
normal
of PNH
the
This
low density
in the
analysis
populations
the
the
addition
in PNH
from
the
by increased
or the
in the
were
of
in this test
separations
different
clarified the interpretation patients. We have also used this
ment.’7
column
cells
detected
mixed
formation
reacts
subpopulation
cells for analysis of the (DAF), a membrane
are lysed by complement of complement compo-
the analysis
cells
Further,
red
a new
I and PNH by differential
cells. time
to define
not
populations that by the presence
cells
of such
copies
that allows
the
lysis.
of the antigen
It is also
in pH
of the so little
circulation is much greater in the peripheral blood.’5
PNI-I the
advantages
ability
among
from
to pres-
to acetylcholinesterase the sensitivity of the
of
cell).’6
are
the
to complement
antibody per
cells
in response
Further,
of copies
number
changes
abnormalities
on
cells
molecules7
has large
The
blood
the anti-I 600,000
were
by this process.’#{176}The lysed by cobra venom
of
than
are
interfere
Suspensions
which
they
that
may
against
cells
it is initiated
the one
the number
were
patients are of findings
comparisons
red the
The
of lysis
antibodies no effect
normal to
immunoproteins
be analyzed.
because
compared
greater
can
acetylcholinesterase
and
observations
sensitive cells can be obtained by differential centrifugation since the proportion of abnormal cells tends to be greater the young and hence less dense cells.3 This is because proportion of abnormal (PNH III) cells that are delivered cells
is probably
with structure.
the characteristics
made.
II,
membranes of the are contaminated
and
monocbonal IgG has
surviving non-PNH III cells can thus be purified. the surviving population(s) (usually PNH I cells)
is often
nents;
cells
of the
whole,
membrane
membrane
of the antimouse
In the past, this difficulty of cells of patients with
difficult
factor and are lysed mixture of cells are and the However,
cell
isolated
of
ence and
analyses
cells.”’3 However, such makes statistical analysis
be readily
PNH III cells
which
cells. the use
them
Previously, from PNH
of red
biochemical
of the
disruption activation
of complement
of abnormal
to define
ofabnormal their rarity from
of
the proportion
difficult
derived
action
of a mixture
results
pertinent to abnormal been minimized by porportions rare, and
lytic
patients,
the
been
has made it difficult to search defect in PNH red cells that
no
complement
cells.
DISCUSSION
One nature
contamination
ET AL
We wish to acknowledge the technical We wish to thank Dr Douglas Fambrough ies used
in these
assistance
of Mary
for providing
Bass.
the antibod-
studies.
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