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Markedly High Population of Affected Reticulocytes Negative
for Decay-Accelerating Factor and CD59 in Paroxysmal
Nocturnal Hemoglobinuria
By Norihiro Iwamoto, Tatsuya Kawaguchi, Shoichi Nagakura, Michihiro Hidaka, Kentaro Horikawa,
Tadashi Kagimoto, Kiyoshi Takatsuki, and Hideki Nakakuma
Paroxysmal nocturnal hemoglobinuria (PNH) blood cells lackcytes. Moreover, the populationof affected erythrocytes became obviously low in patients who received transfusions
glycosylphosphatidylinositol-anchoredmembrane proteins
such as decay-accelerating factor(DAF) and CD59. This lack
and sufferedfrom hemolytic precipitation, whereas
the popis of diagnostic valuein PNH. Because reticulocytes in PNH
ulation of affected reticulocyteswas unchanged. The persistently high population of affected reticulocytes, despite cyare not yet well characterized, we analyzed reticulocytes
tolytic exclusionandaninherentlyshort
lifetime, might
obtained from 12 patients with PNH andfrom 5 healthy volunteers bytwo-color flow cytometry with a membrane-perpossibly be explained by relative reticulocytosiscaused by
meable fluorescentdye, thiazole orange, to identify reticuloan anemia-induced feedback stimulation of erythropoiesis
in PNH.Thus,affectedreticulocytescould
be areliable
cytes and monoclonal antibodies
to DAF and CD59. Healthy
marker for the diagnosis of PNH and for the evaluation of
individuals hadno affected cells. In all patients, the population of affected reticulocytesnegativefor DAF and CD59 was
erythropoiesis by PNHstem cell.
0 7995 by The American Societyof Hematology.
markedly higher than the population of affected erythro-
T
HEMOLECULAR MECHANISM for the increased
susceptibility of paroxysmal nocturnal hemoglobinuria
(PNH) blood cells to complement has recently been clarified.' An impaired transfer of N-acetylglucosamine to phosphatidylinositol leads to a synthetic defect in glycosylphosphatidylinositol (GPI),2.3 which is used by complement
regulatory membrane proteins, decay-accelerating factor
(DAF), and CD59, when they covalently attach to the plasma
rnemb~ane.~
The lack of these proteins causes an abnormal
sensitivity of blood cells to complement and leads to the
intravascular cytolysis that is characteristic of PNH. Accordingly, the presence of affected blood cells that lack GPIanchored membrane proteins has diagnostic ~a1ue.I.~
In fact,
flow cytometry, which detects affected erythrocytes, gives
comparable results to conventional hemolysis tests such as
the sugar-water test and Ham's acidifiedserum test and
has been applied practically to the clinical diagnosis of
PNH.5"0 Moreover, recent reports point to the value of flow
cytometric detection of affected granulocytes for the early
diagnosis of developing PNH.9.10An analysis of affected
erythrocytes is indispensable for the evaluation of intravascular hemolysis, because the population of affected erythrocytes changes in association with hemolytic exclusion. On
the other hand, another type of erythroid cells, reticulocytes,
often shows persistently high population and exceeds leukocytes in population because of anemia-induced feedback
stimulation of erythropoiesis in PNH." Moreover, reticulocytes are simply identified by flow cytometry of whole blood
cells.12"'In the present study, we suggest the diagnostic
value of performing flow cytometry of reticulocytes by
showing that the population of affected reticulocytes in PNH
is not only distinctively high under clinically stable conditions, but also is persistently high even under conditions of
massive hemolytic precipitation and blood transfusion.
MATERIALS AND METHODS
Patients. PNH was diagnosed on the basis of Coombs-negative
intravascular hemolysis and the presence of complement-sensitive
erythrocytes lacking DAF and CD59. The clinical findings on 12
patients withPNH are presented in Table 1. Patient no. 1 hadan
inherited deficiency oftheninth component of complement (C9)
and never experienced spontaneous massive hemolysis nor required
transfusion.16 Inher peripheral blood, the population of affected cells
was extremely high. Patients no. 2 through 9 infrequently required
blood transfusions. Patients no. 10 through 12, whoshowedmild
hemolysis, rarely required transfusions.
Cell preparation. With informed consent, peripheral bloodwas
obtained from both 5 healthy volunteers and 12 patients with PNH
into a tube containing 3 mmoliL EDTA. Whole blood cells were
washed twice with phosphate-buffered saline (pH, 7.4) and used for
erythrocyte and reticulocyte analysis. Granulocytes were separated
from heparinized blood (20 mL) using dextran sulfate and Ficoll-
Table 1. Laboratory Data of 12 Patients With PNH
From the Laboratory of Biomembrane Research, the Second Department of Internal Medicine, Kumamoto University School of Medicine; and the College of Medical Science, Kumamoto University,
Kumamoto, Japan.
Submitted August 25, 1994; accepted November 22, 1994.
Supported in part by grants from the Ministry of Education, Science and Culture of Japan; Yamanouchi Foundation for Research
on Metabolic Disorders; and Ono Medical Foundation.
Address reprint requests to Hideki Nakakuma, MD, PhD, The
Second Department of Internal Medicine, Kumamoto University
School of Medicine, Honjo 1-1-1, Kumamoto 860, Japan.
The publication costsof this article were defrayedin part by page
charge payment. This article must therefore be hereby marked
"advertisement" in accordance with 18 U.S.C. section 1734 solely to
indicate this fact.
0 1995 by The American Society of Hematology.
0006-4971/95/8508-00I6$3.00/0
2228
Sex/Age
Leukocytes
Erythrocytes
No.
(vr)
l109/L)
1
F160
MI57
F128
MI17
F145
MI26
Ff7 1
MI82
M114
Mf7 1
3.3
6.0
4.0
5.1
3.6
3.0
3.6
3.4
2.9
6.1
2.3
3.0
Patient
2
3
4
5
6
7
8
9
10
11
12
F132
F/51
IIO'z/L)
Reticulocytes
(1O"IL)
LDH*
WL)
2.75
1.30
1.81
3.28
3.36
1.60
3.36
2.54
2.12
3.85
2.70
1.65
1.98
1.64
2.26
2.34
1.01
2.01
1.01
0.86
2.35
1.08
0.46
0.18
622
4,515
3,340
3,845
1,813
4,115
1,813
2,135
3,448
731
535
320
Sugar-Water
TesWHam's
Test
+l+
+l+
+l+
+l+
+l+
+l+
+l+
+l+
+l+
+I+
+l+
+l+
~~
* Lactate dehydrogenase activity in serum.
Blood, Vol 85,No 8 (April 15),
1995:
pp 2228-2232
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AFFECTEDRETICULOCYTES
2229
IN PNH
Fig 1. Reticulocyte
populations determined by flow cytometry with thiazole orange and by
conventional microscopic count
with newmethyleneblue.
[A)
Flow cytometriccount
Cells
+om
representative
a
with
thiazole
orange
healthycontrol; (B. C. and Dl
Microscopiccount with
from
cells
3 patients with PNH. methylene
new
blue ("h)
(x.)
Thiazole orange
1.l
5.5 28.1
0.8
5.4
Hypaque (Pharmacia, Uppsala, Sweden) as described previously.2
The purity of the isolated cells was confirmed by flow cytometry.
Chemicals. Mouse antihuman DAF monoclonal antibody
(MoAb; IgG,) was purchased from Wako Pure Chemical Industries
(Osaka, Japan), and mouse antihuman CD59 MoAb (IgG,) was a
gift from Dr Motowo Tomita (Showa University (Tokyo, Japan)."
Control
PNH
4
4
11.8
26.0
10.6
Mouse antihuman glycophorin MoAb (IgGJ was obtained from
Sigma Chemical Company (St Louis, MO). Phycoerythrin-conjugated goat antimouse IgG was obtained from Jackson Immunoresearch Laboratories, Inc (West Grove, PA). Thiazole orange reagent
(Retic-Count) was purchased from Becton Dickinson Immunocytometry Systems (San Jose, CA). Anti-CD13 MoAb (MCS-2, IgG,)
and fluorescein isothiocyanate-conjugated goat antimouse IgG were
obtained from Nichirei Corporation (Tokyo, Japan) and Zymed Laboratories Inc (San Francisco, CA), respectively. New methylene blue
for conventional reticulocyte staining was purchased from Muto
Pure Chemicals Corporation (Tokyo, Japan). CD13 is a marker for
granulocytes and monocytes.
Flow cytomerry. Whole blood cells (2 X lo7) were incubated
with anti-DAF MoAb or anti-CD59 MoAb, then labeled with phycoerythrin-conjugated antimouse IgG and, finally, with thiazole orange reagent to identify reticulocytes.12"' The reticulocyte and erythrocyte population was collected by gating withthe forward-angle
versus 90"-angle scatter and confirmed by cell-specific markers, glycophorin and RNA.""' Nonspecific background staining was performed using preimmune mouse IgG and phosphate-buffered saline
instead of MoAb and thiazole orange, respectively. Granulocytes (1
X IO6) were incubated with anti-DAF MoAb, anti-CD59 MoAb, or
anti-CD13 MoAb, and then labeled with fluorescein isothiocyanateconjugated antimouse IgG. Cells were then examined with a flow
cytometry analyzer (FACScan; Becton Dickinson) as described previ~usly.~.*
LL
RESULTS
Flow cytometry of reticulocytes. Figure 1 shows the flow
cytometric counts of reticulocytes in the gated reticulocyte
and erythrocyte population using either thiazole orange or
conventional microscopic counts of reticulocytes on new
methylene blue-stained smears. The percent population was
determined by the number of reticulocytes per 10,000 erythrocytes in flow cytometry or per 1,000 erythrocytes in microscopic counting. In the end, the two methods provided nearly
identical reticulocyte populations. Because thiazole orange
is a membrane-permeable fluorophore that binds tightly to
nucleic acid and forms a fluorescent nucleotide-reagent complex, the reagent may stain leukocytes. However, in the gated
reticulocyte and erythrocyte population, virtually all cells
were positive for the erythroid cell marker glycophorin (Fig
2, upper panel). Among the glycophorin-positive cells, thiazole orange selectively stained reticulocytes. Reticulocytes
were, thus, clearly discriminated byflow cytometry using
this reagent, as described p r e v i o ~ s l y . ' ~In" healthy
~
controls,
two-color flow cytometry with boththiazole orange and anti-
1
Thiazole orange
Fig 2. Two-color flow cytometry with thiazole orange and M o b
to glycophorin, DAF, and CD59 on
the gated reticulocyte and erythrocyte population. Cells were obtained from patient no. 7 in Table 1
(PNH) and a healthy volunteer (Control).
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IWAMOTO ET AL
2230
2
1
cn
$
0
!
W
cn
-8a
0
0
c
m
d
4
3
5
6
LkkLkk
LLLLLL
cn
8
~~~~~~~
0
P
W
cn
g
0
m
a
0
B
W
t
Ll!JLLUULk
Fluorescence intensity (DAF)
Fig 3. DAF expression in reticulocytes and erythrocytes obtained from 12 patients with PNH and a representative of 5 healthy volunteers.
Panels no. 1 through 12 show cells from patients no. 1 through 12 as listed in Table 1. Panel C shows cells from a healthy control. Dashed
lines show background staining.
Table 2. Population of Affected Cells Negative for DAF and CD59
~~
Erythrocytes
Patient
No.
32
46
21
76
36
52
20
1
2
3
4
5
6
7
a29
9
10
DAF-
8895
12
65
85
81
29
69
17
56
8
22
3
3 1
Control
0
11
CD59-
89
48
75
48
27
31
40
Reticulocytes
OAF-
92
90
88
76
80
20
13
8
23
3
0
0
Values are shown as percentages.
Abbreviation: ND, not determined.
Granulocytes
CD59-
DAF-
CD59-
93
92
92
87
84
81
73
ND
ND
93
94
ND
ND
93
89
87
94
77
82
ND
ND
56
31
33
6
0
64
11
69
6
ND
6
0
ND
2
0
DAF MoAb showed both reticulocytes and erythrocytes to
be positive for DAF and showed reticulocytes to be strongly
positive for DAF (Fig 2, middle panel). In PNH (patient no.
7 in Table l), flow cytometry showed relative reticulocytosis
and two populations of reticulocytes positive and negative
for DAF, as well as mature erythrocytes. It is noteworthy
that the population of affected reticulocytes was higher than
that of affected erythrocytes. CD59 showed expression similar to DAF (Fig 2 , lower panel).
Population of reticulocytes and erythrocytes negative for
DAF. Figure 3 shows the flow cytometric analyses of DAF
expression of reticulocytes and erythrocytes obtained from
12 patients with PNH and a representative of 5 healthy volunteers. Affected cells werenot detectable in the healthy
control (Fig 3, panel C ) .In all patients, reticulocytes showed
a markedly high population of affected cells as compared
with erythrocytes. Even in patients no. 11 and 12, whose
affected erythrocytes were, respectively, ambiguous and undetectable, affected reticulocytes were clearly and barely detected, respectively. CD59 showed similar expression to
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2231
AFFECTED RETICULOCYTES IN PNH
58
tG,Lt,L i;,
,
ticulocytes
Fig(AI
4. and
DAF
and
after
expression
fore
(B),transfusion
in beerythrocytes
rewith 800 rnL of washed erythrocytes (patient no. 21, hemolytic
precipitation (hemoglobin
decreased from 8.4 to 5.6 gldL; patient no. 41, or both hemolysis
and 400-rnL transfusion (Patient
no. 51. Patients no. 2, 4, and 5
correspond to those listed in Table l .
2
a
,
‘E
8
Patient
2
Patient
intensityFluorescence
Patient
4
5
(DAF)
DAF (data not shown). The population of affected reticulocytes, erythrocytes, and granulocytes from patients is summarized in Table 2. The population of affected reticulocytes
was as high as that of affected granulocytes.
Effects of hemolytic precipitation and transfusion on the
Figure 4 shows the
population of affectedreticulocytes.
DAF-expression of reticulocytes and erythrocytes before and
after transfusion with washed erythrocytes (patient no. 2),
massive hemolytic precipitation (patient no. 4),or hemolysis
and transfusion (patient no. 5). Hemolytic precipitation and
transfusion markedly decreased the population of affected
erythrocytes (from 32%to 18% in patient no.2,62% to
22% in patient no. 4, and 21% to 11% in patient no. 5 ) but
hadno effect on the population of affected reticulocytes
(from 92% to 88% in patient no. 2 and stable at 80% in
patients no. 4 and 5). After the in vitro hemolysis test (Ham’s
test) or in vitro dilution with healthy erythrocytes, the population of affected reticulocytes and affected erythrocytes simultaneously decreased to a similar extent (data not shown).
DAF is high in reticulocytes and becomes low in association
with erythrocyte maturation and aging in healthy individuals.”It is then conceivable that a portion of the affected
reticulocytes underwent complement-mediated hemolysis
and that the surviving reticulocytes differentiated to erythrocytes. Despite complement-mediated cytolytic exclusion of
the affected cells, the reticulocytes and granulocytes actually
showed comparably high populations of affected cells in all
patients. Because reticulocytes and granulocytes are known
to have inherently short lifetimes,2’~22
it is conceivable that
the population of affected cells in both cell lineages properly
reflects hematopoiesis by mutated PNH stem cell in bone
marrow. Flow cytometry of reticulocytes requires a smaller
blood sample (20 pL) and nopurification
procedures,
whereas granulocyte analysis requires an approximately 10mL bloodsample and appropriate isolation of the cells. Thus,
we conclude that affected reticulocytes are a potential probe
for PNH diagnosis and for the evaluation of hematopoiesis
by PNH stem cells.
DISCUSSION
ACKNOWLEDGMENT
We examined the expression of DAF and CD59 on reticulocytes from 12 patients with PNH by flow cytometry. As
with erythrocytes, reticulocytes show two populations of
cells positive and negative for the proteins. The population
of affected reticulocytes was markedly higher than that of
affected erythrocytes inall12 patients with PNH, and affected reticulocytes were detectable even in patients for
whom the presence of affected erythrocytes was equivocal.
Furthermore, the population of affected reticulocytes was
unchanged by hemolytic precipitation and transfusion,
whereas the population of affected erythrocytes became obviously low. Reticulocyte analysis could, thus, be of greater
value than erythrocyte analysis for PNH diagnosis.
The present findings are supported partly by previous reports that the reticulocyte-enriched fraction in PNH shows
a high population of affected cells,’*a low activity of acetylcholinesterase (which is a GPI-anchored membrane protein),” and a marked sensitivity to complement.” Our findings are also consistent with a report that the expression of
We thank Drs M. Kawakitaand T. Kanazawa in our laboratory
for their advice and help, and MS F. Inukai for secretarial assistance.
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From www.bloodjournal.org by guest on June 16, 2017. For personal use only.
1995 85: 2228-2232
Markedly high population of affected reticulocytes negative for
decay- accelerating factor and CD59 in paroxysmal nocturnal
hemoglobinuria
N Iwamoto, T Kawaguchi, S Nagakura, M Hidaka, K Horikawa, T Kagimoto, K Takatsuki and H
Nakakuma
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