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Twopopulationsoferythroidcellprogenitorsinparoxysmalnocturnalhemoglobinuria

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1984 64: 847-851   

 B Rotoli, R Robledo, N Scarpato and L Luzzatto hemoglobinuriaTwo populations of erythroid cell progenitors in paroxysmal nocturnal

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Blood. Vol 64, No 4 (October), 1984: pp 847-851 847

Two Populations of Erythroid Cell Progenitors in Paroxysmal

Nocturnal Hemoglobinuria

By Bruno Rotoli, Renato Robledo, Nicola Scarpato, and Lucio Luzzatto

We have grown erythroid cell colonies from two patients

with paroxysmal nocturnal hemoglobinuria (PNH). At 1 1 to

1 3 days. individual bursts were picked and incubated for 24

hours with 3H-leucine in order to label total cell protein

(mainly hemoglobin). After appropriate washing. each

burst was subjected to a miniaturized acidified serum test.

and lysis was measured by the release of radioactivity. In

bursts from normal controls. lysis was 1 9% ± 1 3% SD. By

contrast. of 58 bursts from PNH patients. 1 4 had lysis

similar to that of controls (mean 1 5.4% ± 1 0.6%). while 44

P AROXYSMAL NOCTURNAL hemoglobinuria

(PNH) is a chronic hemolytic disorder defined

and identified by the acidified serum test.’ It is well

known that, in this test, only a fraction of the patient’s

red cells are lysed by virtue of their hypersensitivity to

activated complement, with the remaining cells being

not susceptible to lysis. On this basis, Dacie2 suggested

in 1962 that there are two distinct red cell populations

in the patients’ blood that can be referred to as

“normal” and “PNH.” This suggestion was corrob-

orated by the findings of (a) biphasic 51Cr-labeled red

cell survival curves3 and (b) biphasic (and sometimes

triphasic) cold antibody lysis curves.4 However, these

lines of evidence could still be regarded as circumstan-

tial, because the tests employed might have created

apparent discontinuities even within a single red cell

population. For instance, only a fraction of red cells

lyse in the autohemolysis test in hereditary spherocyto-

sis, and only a fraction of red cells are irreversibly

sickled in sickle cell anemia, even though all the cells

clearly belong to one and the same population. In

addition, it is not certain that the standard Ham test

correctly measures the fraction of PNH cells, because

the percentage of cells lysed varies, for instance, with

the ionic strength ofthe medium.5 Thus, one might still

surmise that the PNH abnormality arises as a result of

an extracorpuscular factor in a finite fraction of hemo-

poietic cells. More direct evidence for the monoclonal

origin of PNH cells came from the demonstration of

only one type of glucose-6-phosphate dehydrogenase

(G6PD) in the red cells lysed in acidified serum from

two female PNH patients who were heterozygous for

two different G6PD variants.6

In vitro cultures of erythroid cells now make it

possible to test the two-population model in another

direct way. This model predicts that erythroid cell-

progenitors, such as burst-forming units, erythroid

(BFU-E), must also be oftwo kinds: normal and PNH.

Each erythroid burst grown from a single cell would

had lysis ranging from 42.2% to 85.8% (mean

70.3% ± 10.4%). Colonies sensitive to acidified serum

were acetylcholinesterase (AchE) negative. whereas nor-

mal colonies were AchE-positive. Thus. based on two

independent criteria. a dual population of erythroid burst-

forming units (BFU-E) can be demonstrated in PNH. These

data confirm directly the somatic mutation model of the

pathogenesis of PNH. and by these methods the relative

sizes of the normal and the PNH cell populations can be

measured at the level of the erythroid cell precursors.

have to consist entirely of either normal or PNH cells.

Using two independent criteria, we show that this is

indeed the case.

MATERIALS AND METHODS

Patients and Controls

PNH was diagnosed on the basis of chronic hemolytic anemia

with hemosiderinuria and a positive Ham test. Patient I had never

been transfused; and patient 2 had been transfused with RBCs on

three occasions, the last transfusion having been given three months

before the culture experiments. Peripheral blood mononuclear cells

were obtained by venipuncture. In one patient, bone marrow cells

were also cultured when an aspirate was performed as part of the

patient’s initial hematologic assessment. Mononuclear cells from

peripheral blood of some of us served as normal controls.

Ervthroid Cell Cultures

Peripheral blood (10 to 30 mL) or bone marrow aspirate (1 mL)

was collected with preservative-free heparin (final concentration 10

U/mL). Undiluted blood was layered on an equal volume of

Lymphoprep (Nyegaard and Co. Oslo), and centrifuged at 400 g for

40 minutes at room temperature.7 The mononuclear cell layer was

collected and the cells washed twice in Iscove’s modified Dulbecco’s

medium (IMDM) (GIBCO, Grand Island, NY). The cells were then

resuspended in IMDM at a concentration of 5 x l06/mL for

peripheral blood and lO’/mL for bone marrow. Erythroid cell

cultures were set up by the methylcellulose technique,’ with minor

modifications. Briefly, the cell suspension was diluted 1:10 in a

mixture containing 0.8% methylcellulose (Methocel 4000 cps, Dow

Chemical Co. Midland, Mich), 30% fetal calf serum (Eurobio,

Paris), 1% bovine serum albumin (Sigma, St Louis) prepared

according to Tepperman et al,9 beta-mercaptoethanol l0�� mol/L

and erythropoietin (step III, Connaught, Stillwater, Pa) 2 U/mL,

From the Department ofllematology, Royal Postgraduate Med-

ical School, London; the International Institute of Genetics and

Biophysics, Naples; and the Section of Hematology. 2nd Medical

School, University of Naples.

Submitted Feb 2. 1984; accepted April 30. 1984.

Address reprint requests to Professor L. Luzzatto, Royal Post-

graduate Medical School, Ducane Rd. London W12 OHS,

England.

© I 984 by Grune & Stratton, Inc.

0006-4971/84/6404-0014$03.OO/O

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848 ROTOLI ET AL

Membrane acetylcholinesterase (AchE) activity was tested on

individual colonies by a modification of the method described by

and was then distributed in l-mL aliquots in 35 x 10 mm Falcon

culture dishes. Cultures were incubated at 37 #{176}Cin a humidified

atmosphere flushed with 5% CO2 in air.

Scoring and labeling of cultures was carried out on days I 1 to I 3.

Erythroid colonies were identified on the basis of their orange-red

color and their characteristic morphology. In the case of bone

marrow, we collected colonies produced from BFU-E (bursts) and

not those produced by erythroid colony-forming units (CFU-E).

Labeling of Cultures

In order to detect lysis of a small number of cells, 3H-leucine was

incorporated into erythroid cells at the time of maximal hemoglobin

synthesis by a technique similar to that described by Comi et al.’#{176}

Individual erythroid colonies (or bursts), consisting of between 500

and 3,000 cells, were identified on an inverted microscope. A single

colony was aspirated through a micropipette and transferred to the

bottom of a sterile plastic lO-mL conical test tube containing 10 zL

of a mixture of 0.8% methylcellulose in IMDM, 30% fetal calf

serum, erythropoietin 2 U/mL, and 10 �zL of high specific activity

(180 Ci/mmol) 3l-I-leucine (Amersham). In order to maximize the

incorporation of radioactivity, all components of this mixture were

dissolved in leucine-free IMDM. The appropriate amount of 3H-

leucine had been previously dried under a flow of nitrogen and then

dissolved directly in the incubation mixture. Incubation was carried

out in a humidified atmosphere at 37 #{176}Cfor 18 to 24 hours. At the

end of this period, 10 mL of phosphate-buffered saline containing

0.5% bovine serum albumin (BSA-PBS) was added to each tube,

together with S x 1 06 unlabeled red cells of the same blood group, to

act as carrier for the labeled burst. (The use of BSA-PBS as a

suspending medium for the washes was critical in order to avoid

unspecific lysis of labeled erythroid cells.) Cells were then washed

three times with 10 mL of BSA-PBS, resuspended in I mL of the

same solution, and transferred to a I .5-mL Eppendorf conical tube.

A 50-�zL aliquot was counted. At this stage, bursts with high

radioactivity were split into two equal portions, and one of the

aliquots was used as control of the specificity of the Ham test (see

below). After centrifugation (4,000 rpm, five minutes, in a Beckman

Microfuge TI I), the supernatant was removed, and 50 �sL was

counted to ensure that the washing procedure had effectively

removed all unincorporated radioactivity.

Miniaturized Ham Test

The cell pellet from the above step was resuspended in 50 �tL of

fresh, ABO-compatible acidified (pH 6.5 to 7.0) serum and incu-

bated at 37 #{176}Cin a water bath for one hour. After centrifugation

(Microfuge, 4,000 rpm for five minutes), the supernatant was

collected; the pellet was hemolyzed with 50 �zL ofdistilled water, and

centrifuged again (4,000 rpm, five minutes). Radioactivity in both

supernatants was determined by counting in 6 mL of scintillation

liquid (Ready-Solv MP, Beckman) in a Beckman beta-counter

(model 7,000) and was expressed as dpm. Counting efficiency was

about 40%. Hemolysis in acidified serum was calculated for each

burst as the percentage of radioactivity in the supernatant serum

with respect to cytoplasm radioactivity (sum total of 3H released in

supernatant serum plus 3H released by lysis in distilled water). The

specificity of the miniaturized Ham test was checked by performing

tests with heat-inactivated (30 minutes at 56 #{176}C)acidified serum on

an aliquot of a burst, the other aliquot being processed with

non-heat-inactivated acidified serum.

AchE Staining

Perona et al.� A whole burst or part out (a subcolony) was picked at

days I I to I 3 and added to a drop of molten agar (1% in saline) at

40 #{176}C,in a well of a microtiter plate (Micro Test Plate, Nunc,

Roskilde, Denmark). After the agar had set, the well was filled with

staining mixture.’2 The plate was incubated at 37 #{176}Cin humidified

atmosphere for 24 to 48 hours. Upon examination under an inverted

microscope, positive colonies showed black staining, with the disap-

pearance of cell border and the presence of needle-like crystals,

whereas negative colonies showed well-preserved cells without any

crystals. Colonies from a normal subject served as positive controls.

Omission of acetylthiocoline from the staining mixture served as

negative control.

RESULTS

Both patients had reduced numbers of peripheral

blood BFU-Es. A quantity of S x IO� mononuclear

cells plated yielded 5.35 ± 1 .5 and 4.25 ± 1 .5 colonies

for the patients, compared to 20. 1 ± 1 .9 in normal

controls (P < .001).

Our main objective was to prove that some, but not

all, erythroid bursts from PNH patients had the PNH

abnormality. Because bursts derived from BFU-E con-

sist of only a few hundreds to several thousand cells, it

is not practical to observe lysis of one of them by

conventional spectrophotometric analysis of hemoglo-

bin release. However, if individual mature bursts are

incubated for 24 hours with a radioactive amino acid

(we have used 3H-leucine), this is incorporated into

newly synthesized cellular protein-mainly hemoglo-

bin. Cell lysis can then be measured with high sensitiv-

ity and accuracy by simply determining the proportion

of counts released into the medium. Erythroid colonies

at I I to I 3 days consist of mature erythrocytes and of

normoblasts, some of which, at least, are susceptible to

acid lysis.’3 Erythrocytes grown in vitro are much more

fragile than normal ones. However, once suitable con-

ditions are worked out (see Materials and Methods),

lysis in acidified serum of individual bursts from

normal peripheral blood BFU-Es is mostly below 25%,

and it never exceeds 40% (Fig I ). On the other hand,

when bursts from peripheral blood or bone marrow

BFU-Es of PNH patients are similarly tested, percent

lysis ranges from 2.1% to 85.8% (Fig I). The distribu-

tion of bursts according to this property is clearly

bimodal (Fig 2). One group of bursts from PNH

mononuclear cells is abnormal, and we regard them as

PN H bursts. Another group of bursts behaves in a way

that is indistinguishable from bursts from normal

blood, and we regard them as normal bursts from PNH

patients. If an aliquot of an abnormal burst is tested

with heat-inactivated serum, percent lysis falls within

the normal range, indicating that lysis is complement

dependent (Fig I). Representative data obtained on

individual colonies are shown in Table 1 . By choosing a

cut-off point of 40% lysis, the difference between

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NORMAL AND PNH ERYTHROID COLONIES IN PNH 849

NORM% PMiImolysis

PNI4

NORM PNH PNHPS PS SM

Fig 1 . Acidified serum lysis of single erythroid colonies. Hori-zontal bars represent the mean for each group of data. Vertical

lines connect data from two aliquots of a single burst. NORM.

normal control; PNH. (patient 1 ); PB. peripheral blood BFU-Es; BM.bone marrow BFU-Es. � lysis in acidified serum. 0 lysis in macti-vated serum.

bursts from normal subjects (N = 1 2) and normal

bursts from PNH patients (N = 14) compared to

PNH bursts from PNH patients (N = 44) is highly

significant (x2 = 24.68; P < l0�).

The activity of AchE, a membrane enzyme, is

markedly reduced in most PNH patients.’4”5 Perona et

1 using cytochemical staining, have demonstrated

normal and AchE-deficient erythrocytes in the periph-

eral blood of PNH patients, suggesting that this

enzyme is a good marker ofthe PNH abnormality. We

have found that all erythroid colonies (23/23) from six

normal controls were strongly positive by AchE stain-

ing. In contrast, by testing single subcolonies from 25

different bursts from a PNH patient, we found 22 of

them to be negative (Fig 3A) and only three positive

(Fig 3B). The miniaturized Ham test, carried out on

the remaining subcolonies from the same 25 bursts,

showed that the three positive bursts all had lysis in the

normal range, whereas the 22 AchE-negative colonies

all had lysis above 45%. No differences were observed

between normal and abnormal cultures from PNH

patients in terms of size and morphology of colonies

and subcolonies, or in the degree of hemoglobiniza-

tion.

DISCUSSION

The finding of two kinds of erythroid cell bursts

cultured from PNH patients indicates that there are

two kinds of BFU-Es in their bone marrow and periph-

eral blood. Indeed, if the PNH abnormality were

acquired at random by a finite proportion of cells

during their differentiation from BFU-Es, then all

bursts would include both lysis-susceptible and normal

cells in approximately constant proportion, as each is

made up of a sufficiently large number of cells to make

statistical fluctuation negligible. In this case, bursts

from PNH patients would show a unimodal distribu-

tion, with an abnormally high modal value of lysis.

Instead, a bimodal distribution is observed (Fig 2), and

the modal value of lysis of one group of bursts is

normal. Thus, normal cells and PNI-I cells are clearly

predetermined at the BFU-E stage.

The evidence presented in this article, combined

with the G6PD studies referred to previously, supports

Dade’s concept2 of a clonal population of abnormal

erythroid progenitors, which has probably arisen by a

single somatic mutation, which generates the PNH

erythrocyte population of patients with PNH.

By the experimental procedures we have described,

it is possible to obtain a quantitative estimate of the

size of the PNH clone in PNH patients. This estimate

is probably much more accurate than that based on the

percentage of circulating RBCs that are lysed in the

Ham test, as with the latter, we may miss the fraction

of abnormal cells that has been already lysed in vivo

Fig 2. Bimodal distribution of susceptibility tolysis in acidified serum of erythroid colonies grown invitro from two patients with PNH (B) compared withuniformly low lysis of erythroid colonies for normal

controls (A).

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850 ROTOLI ET AL

Fig 3. Erythroid subcolonies from patient 2 stained for AchE.(Left) negative; (right) positive.

Table 1 . Acidified Serum Test on Single Erythroid Cell Colonies From Normal Subj acts and PNH Patients (Repr esentative Data)

Source of BFU-E

A

3H Released inAcidified Serum

(dpm)

B

3H Released byHypotonic Lysis

(dpm)

Percent Lysis in

AcidifiedSerum

[A/(A + B)]

Type of

BFU-E

Normal PB

Exp 1 2,500 28.927 7.9 Normal

Exp2 27,174 421,428 6.0 Normal

PNH patient 1 PB

Burst 1 10.975 50,374 17.8 Normal

Burst2a’

2b

21,436

1.944

13,886

59.702

60.6

3.1PNH

Patient 1 BM

Burst 1 4.565 44.375 9.3 Normal

Burst2a

2b

21.781

1,081

5,986

35.049

78.4

2.9

PNH

Patient 2 PB

Burst 1 66.121 307,381 17.7 Normal

Burst2 33,961 8,005 80.9 PNH

‘After labeling, the cell suspension was divided into two aliquots. One aliquot (a) was processed as described in detail under Materials and Methods;

the other (b) was processed in the same way, except that the acidified serum had been previously heat-inactivated (30 minutes at 56 #{176}C).

PB, peripheral blood BFU-Es; BM, bone marrow BFU-Es.

and which may be quite substantial. In both patients

we have studied, lysis by the Ham test carried out on

the patient’s blood at the time of setting up the cultures

was about 40%. By testing erythroid colonies, we have

found in patient I 60% abnormal BFU-Es in peripheral

blood and 80% in the bone marrow; in patient 2, 88%

abnormal BFU-Es were found in peripheral blood.

These data are based on the assumption that the

plating efficiency for both normal and abnormal BFU-

Es from PNH patients is the same. We do not yet know

whether this assumption is correct. On the other hand,

we are aware that the high proportion of abnormal

BFU-Es may be due in part to shortage of normal

BFU-E, because a marked decrease in the overall

number of erythroid colonies from peripheral blood in

PNH patients has been found by ourselves’6 and

others.’7’8

Although AchE deficiency is thought to be a secon-

dary phenomenon in PNH, it is characteristically

associated with the PNH abnormality because the two

populations are seen cytochemically in PNH

patients,” and the complement-sensitive red cells,

physically separated by differential lysis, were found to

have practically no AchE activity.’9 Our finding of a

close correlation between acidified serum lysis and

AchE in individual erythroid colonies confirms that

this enzyme is a good marker for PNH. Again, both

AchE-positive and AchE-negative colonies are seen,

and the match between the two tests employed vali-

dates the specificity of both. Because AchE staining is

far less laborious than the miniaturized radioactive

Ham test, but it appears to be equally sensitive and

specific, it could be used as a convenient method to

quantitate abnormal BFU-Es in PNH patients.

After these experiments were completed, Dessypris

et aI2#{176}have reported independent evidence that abnor-

mal complement sensitivity can be demonstrated in

erythroid and myeloid progenitors in PNH. They have

found that erythroid colony numbers were reduced by

about 60% when mononuclear cells were incubated

with fresh serum and sucrose before plating. Our

results agree with those of Dessypris et al2#{176}in proving

that the PNH abnormality is already determined at

the BFU-E level. However, these authors have not

shown that the BFU-Es killed by fresh serum and

sucrose are actually the progenitors of PNH cells, and

in this respect, our findings are complementary to

theirs.

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NORMAL AND PNH ERYTHROID COLONIES IN PNH 851

14. Dc Sandre G, Ghiotto G: An enzymic disorder in the erythro-

The role in oncogenesis of somatic mutations makes

them most prominent in human pathology, but they

are also presumed to play a role in the physiologic

process of the generation of antibody diversity.2’ That

somatic mutation can be responsible for the production

of a veritable acquired mosaicism in a particular cell

population is proven by the examples of PNH and of

Tn polyagglutinability.22 PNH has a special relation-

ship to aplastic anaemia23 and to myeloproliferative

disorders,24 and it might be regarded as a preneoplastic

condition. Very recently, a defect in C3 convertase

decay-accelerating factor has been discovered in PNH

cells,25’26 but it is not certain whether this is the

primary mutated gene product. The availability of an

in vitro culture system for PNH erythroid cells,

reported here, may help to test this directly and to

explore PNH as a model system of disorders caused by

somatic mutation.

ACKNOWLEDGMENT

We thank Dr M. Clarke of St Helier Hospital, Carshalton,

Surrey, for providing blood samples from one of the patients, and the

patients themselves for their cooperation.

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