nocturnal hemoglobinuria Separation of the ...

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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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From bloodjournal.hematologylibrary.org by guest on July 10, 2011. For personal use only.

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.

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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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WF, Parker blood cells in paroxysmal 14:105, 1985 2.

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WF:

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OF

in paroxysmal

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mobogies

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