rev bras hematol hemoter. 2 0 1 5;3 7(2):90–97
Revista Brasileira de Hematologia e Hemoterapia
Brazilian Journal of Hematology and Hemotherapy
www.rbhh.org
Original article
Paroxysmal nocturnal hemoglobinuria clone in 103
Brazilian patients: diagnosis and classification
Ana Paula de Azambuja ∗ , Mariester Malvezzi, Marco Antonio Bitencourt,
Michel Michels Oliveira, Larissa Alessandra Medeiros, Ricardo Pasquini
Universidade Federal do Paraná (UFPR), Curitiba, PR, Brazil
a r t i c l e
i n f o
a b s t r a c t
Article history:
Background: Paroxysmal nocturnal hemoglobinuria is an acquired chronic hemolytic ane-
Received 14 May 2014
mia, which often manifests as peripheral blood cytopenias and thrombosis.
Accepted 26 November 2014
Objective: The aim of this study is to describe a Brazilian population of paroxysmal nocturnal
Available online 30 January 2015
hemoglobinuria patients.
Keywords:
spectively reviewed and the clinical presentation, thrombosis, survival, and clone size were
Methods: One hundred and three paroxysmal nocturnal hemoglobinuria cases were retroHemoglobinuria
assessed. Diagnosis was established by flow cytometry.
Paroxysmal
Results: Fifty-two male and 51 female patients with a median age of 24.1 years (5.5–62 years)
Bone marrow diseases
were studied. Clinical symptoms included hemoglobinuria (18.4%), infection (46.6%) and
Flow cytometry
thrombosis (16.5%), and 80.6% had pancytopenia. Patients were classified as classic paroxysmal nocturnal hemoglobinuria (10), paroxysmal nocturnal hemoglobinuria with aplastic
anemia (39), and paroxysmal nocturnal hemoglobinuria with subclinical features and aplastic anemia (54). There were significant differences in terms of median age, size of clone,
clinical symptoms, and peripheral blood cell counts between the three subcategories. The
clone size in erythrocytes and granulocytes were respectively 0.04% (range: 0–18%) and 7.3%
(range: 0.3–68.7%) in patients with subclinical features and aplastic anemia, 15.8% (range:
0–99.7%) and 63.0% (range: 1.7–99.8%) in patients with aplastic anemia alone, and 82.2%
(range: 0–99.85%) and 98.0% (81.3–100.0%) in Classic disease. Statistical differences were
identified for platelets (p-value = 0.001), lactate dehydrogenase (p-value = 0.002) and the clone
size (p-value < 0.001) in patients who suffered thrombotic events compared to those who did
not. Overall survival was 81.7%, with patients with subclinical features and aplastic anemia
having lower overall survival (76.5%).
Conclusion: This retrospective review of 103 patients over an 11-year period represents the
largest collection of paroxysmal nocturnal hemoglobinuria cases from a single center in
Brazil. Flow cytometry showed that a larger clone was associated with classical symptoms
and increased risk of thrombosis, even in patients with bone marrow failure, whereas a
smaller clone was associated with bone marrow aplasia.
© 2015 Associação Brasileira de Hematologia, Hemoterapia e Terapia Celular. Published
by Elsevier Editora Ltda. All rights reserved.
Corresponding author at: Hospital de Clínicas, Universidade Federal do Paraná (UFPR), Rua Flavio Dallegrave, 1580, apto 31B, 80045-315
Alto da XV, Curitiba, PR, Brazil.
E-mail address: apazamb@gmail.com (A.P. de Azambuja).
http://dx.doi.org/10.1016/j.bjhh.2015.01.001
1516-8484/© 2015 Associação Brasileira de Hematologia, Hemoterapia e Terapia Celular. Published by Elsevier Editora Ltda. All rights
reserved.
∗
rev bras hematol hemoter. 2 0 1 5;3 7(2):90–97
Introduction
Methods
Paroxysmal nocturnal hemoglobinuria (PNH) is a rare,
acquired, stem cell disorder characterized by hemolytic
anemia, bone marrow failure, and an acquired thrombophilic state.1–4 Manifestations of the disease are related
to complement-mediated intravascular hemolysis due to the
lack of glycosyl phosphatidylinositol-anchored complement
regulatory proteins (GPI-AP), CD55 and CD59 on red blood
cells.5,6 Patients with PNH may present not only with a
wide range of clinical manifestations such as weakness,
pallor, and asthenia due to hemolysis, but also abdominal
pain, dysphagia, or pulmonary hypertension.2–4 Thrombosis,
often occurring at unusual sites, is a major life-threatening
risk for patients with PNH.1–4 Ten-year risk of thrombosis has been associated with the PNH clone, as patients
with large PNH clones (>50%) had 44% of 10-year risk compared with 5.8% in patients with small clones.7 A frequent
association between PNH and aplastic anemia (AA) has
been described, with two potential patterns of evolution:
progressive marrow failure in patients without detectable
PNH clones or AA in patients in whom a PNH clone is
detected.8–10
Diagnosis of PNH has improved over the years with the
adoption of modern technologies. Two decades ago the Ham
test, which is based on increased sensitivity of PNH-affected
red blood cells to complement-mediated lysis, was used.5,11
Today, the Ham test has usually been substituted by the
more sensitive, informative and less cumbersome flow cytometric assay (FCM), which uses antibodies directed against
the GPI-AP.11–14 PNH cells are characterized by GPI-AP deficiency on the cell surface due to an acquired mutation of
the phosphatidylinositol glycan-class A (PIGA) gene in one or
more hematopoietic stem cells.6,11,12 The development of
FCM-based testing has allowed the detection of small PNH
clones, which would otherwise not be evident.13,15 PNH clones
are also detected in the setting of bone marrow failure, and
about 40–50% of AA patients have a PNH clone detected at
the time of diagnosis.8,15,16 The mechanism by which the
expansion of PNH cells occurs in AA remains unclear; one
hypothesis is that PNH cells have a proliferative advantage
over non-PNH cells by an immune selection mechanism.9,10
The presence of a PNH clone has been reported to be
predictive of a response to immune suppression in AA15
but other authors did not observe this finding.16 For optimum management, the contribution of both hemolysis and
marrow failure to the complex anemia of PNH should be
determined.1
The objective of this study was to assess the clinical presentation of PNH patients at the time of diagnosis, as well
as report complications, such as thrombosis, survival, difference between subcategories and clinical significance of
the PNH clone size. Patients were assigned to one of the
three subcategories, namely Classic PNH, PNH/AA syndrome,
and subclinical PNH (PNH-sc/AA) to explore the differences
between these categories. Furthermore, the size of PNH clone
was evaluated in the entire cohort and in each subcategory
to assess whether the size of PNH clones was associated with
some of the clinical features of PNH.
Patients and study design
91
One hundred and three PNH clone cases referred to a tertiary
medical center in Brazil from December 1999 through December 2011 were retrospectively reviewed. A total of 398 patients
were screened for the PNH clone using a FCM assay.5,14 The
diagnosis of PNH was established by detecting a GPI-AP deficient clone greater than 0.1%, with at least two cell lineages
showing GPI deficient populations. The study included 103
patients who had demonstrated the presence of a PNH clone
and had available clinical data. The date of PNH diagnosis was based on the first positive FCM analysis. Patients
with co-morbid AA were subclassified as severe or non-severe
according to published criteria.17,18
Multiparameter flow cytometry
The diagnosis of PNH was established by the detection of an
unequivocal positive PNH clone by multicolor FCM assay5,11
using a FACSCalibur® cytometer (BD Biosciences, San Jose,
USA) and CellQuest Pro software (BD Biosciences, San Jose,
USA). The proteins studied were CD55 and CD59 on red cells,
neutrophils and monocytes; CD16, CD24 and CD66b on neutrophils and CD14 on monocytes. PNH clones were defined by
the presence of GPI-AP deficient cells at a frequency greater
than 0.1% of neutrophils, monocytes and red cells, and the
proportion of GPI-AP deficient cells (clone size) was defined by
the highest level of these cells lacking GPI-anchored proteins.
Debris was thresholded out, and at least 50,000 events
in leukocyte tubes and 20,000 events in red cell tubes were
collected and analyzed using Paint-a-gate® (BD Biosciences,
San Jose, USA) or InfinicytTM (Cytognos, Salamanca, Spain)
software (for samples tested after January 2009). Red cells
and granulocytes were identified based on forward and side
scatter, and by staining with CD41a FITC and CD45 PercP,
respectively. The gates used to define GPI negative populations
were established by using normal red cells and granulocytes
as controls.
Subcategories of paroxysmal nocturnal hemoglobinuria
patients at diagnosis
Patients were assigned to one of the three subcategories based
on the recently proposed PNH working clinical classification1 :
(I) The Classic PNH subcategory included patients with clinical and laboratory evidence of intravascular hemolysis
(such as hemoglobinuria, hemoglobinemia, and elevated
LDH and bilirubin) but no evidence of bone marrow failure;
(II) Patients in the PNH/AA subcategory were defined by the
presence or a history of bone marrow failure in conjunction with clinical and laboratory evidence of intravascular
hemolysis;
(III) Patients in the subclinical PNH (PNH-sc/AA) subcategory
were identified by the presence of bone marrow failure
but without clinical or laboratory evidence of hemolysis.
92
rev bras hematol hemoter. 2 0 1 5;3 7(2):90–97
The criteria for bone marrow failure included bone marrow
hypoplasia (cellularity < 50%) and at least two of the following three laboratory abnormalities: hemoglobin level <12 g/dL,
absolute neutrophil count <1.50 × 109 /L, and platelet count
(PLT) <100 × 109 /L. Pancytopenia was considered when the
three hematopoietic lineages were affected. Haptoglobin values were not used in this study.
Statistical analysis
Overall survival (OS) was calculated from the date of diagnosis to the date of death or the date of last follow-up. Survival
analysis was performed using the Kaplan–Meier method. The
distributions of the presentation of characteristics were compared between the three subcategories and between Classic
PNH and PNH-sc/AA by the chi-squared or Fisher’s exact test as
necessary for categorical variables, and by the Kruskal–Wallis
(three subcategories) or Mann–Whitney (two groups) test for
continuous variables. The percentages of GPI-AP deficient red
blood cells and granulocytes were compared using Student’s
t-test. All statistical analyses were performed using the Statistica v.8 program. A p-value <0.05 was considered statistically
significant.
Ethical approval
The Ethics Committee of the Hospital de Clinicas, Universidade Federal do Paraná, Brazil approved the study.
Results
Patient characteristics
Of the 398 patients tested, 125 (31.4%) had a detectable PNH
population. Of these, 103 (51 male and 52 female), who had
clinical data available, were studied. The median age at presentation was 24.1 years (range: 5.5–62.0 years). There were
24 patients below the age of 18 years (range: 5.5–17.9 years;
median: 14.7). All these young patients were in the aplastic
groups (11 HPNsc/AA and 13 HPN/AA).
Hemoglobinuria was identified during monitoring in 43
(41.7%) patients, while it was the initial symptomatic manifestation in only 19 (18.4%) patients. The frequencies of
infections and bleeding were 46.6% and 47.1%, respectively.
Ninety-eight patients (95.1%) presented with asthenia, 33
(32.0%) with abdominal pain, and 14 (13.6%) with renal insufficiency; 17 (16.5%) developed thrombosis during monitoring
and 49 patients (47.6%) had documented hemolysis. Twentysix patients (25.2%) with aplasia at diagnosis developed the
PNH clone and hemolysis in a median of 2.35 years after diagnosis. Of these patients, eleven (10.7%) developed hemolysis
five years after the diagnosis of aplasia.
Peripheral blood abnormalities were present in 101
patients: 83 (80.6%) had pancytopenia, 12 (11.6%) anemia
and thrombocytopenia, and four (3.9%) had anemia and
leukopenia. The median hemoglobin level was 8.8 g/dL (range:
3.8–14.5 g/dL), the median absolute neutrophil count was
0.94 × 109 /L (range: 0.26–1.45 × 109 /L) and the median platelet
count was 25 × 109 /L (range: 2–294 × 109 /L). The median LDH
Table 1 – Patient characteristics (n = 103).
Characteristic
Gender, male – n (%)
Median age – years (range)
52 (50.5)
24.1 (5.5–62.0)
Clinical symptoms at presentation – n (%)
Asthenia/fatigue
Infection/fever
Hemoglobinuria
Bleeding (petechiae and ecchymosis)
Abdominal pain
Jaundice
Renal insufficiency
Thrombosis
Hemolysis
98 (95.1)
48 (46.6)
19 (18.4)
33 (47.1)
33 (32.0)
26 (25.2)
14 (13.6)
17 (16.5)
24 (23.3)
Peripheral blood abnormalities at presentation – n (%)
Anemia alone
Anemia and thrombocytopenia
Anemia and neutropenia
Pancytopenia
4 (3.9)
12 (11.6)
4 (3.9)
83 (80.6)
Bone marrow biopsy – n (%)
Aplasia (<5%)
Hypocellular bone marrow (6–49%)
Normo or hypercellular bone marrow (≥50%)
52 (50.5)
38 (36.9)
13 (12.6)
concentration was 328 U/L (range: 30–7970 U/L; normal range
190–240 U/L). Ninety patients (87.4%) had hypocellular bone
marrow.
Patients included in this study were divided into the subcategories of Classic PNH (10 patients), PNH/AA (39 patients),
and PNH-sc/AA (54 patients) based on the proposed PNH working clinical classification.1 The initial characteristics of the
subgroups are summarized in Table 1.
The median ages of PNH-sc/AA, PNH/AA and Classic
PNH patients were 25.1, 21.5 and 34.7 years, respectively (p-value = 0.016). The clinical symptoms of hemolysis,
hemoglobinuria and thrombosis were higher in the hemolytic
forms of PNH (PNH/AA and Classic PNH) than in PNH-sc/AA
(p-value < 0.001), whereas infection and fever were higher in
the PNH-sc/AA group (p-value < 0.001). The median numbers
of neutrophils and platelets were significantly lower in the
aplastic groups than in the Classic PNH group (p-value for both
<0.001). On the other hand, the Classic PNH group had higher
hemolytic markers, such as LDH (p-value = 0.001), reticulocyte
count (p-value <0.001) and bilirubin levels (p-value <0.001 –
Table 2).
Correlation of paroxysmal nocturnal hemoglobinuria clone
with clinical characteristics
FCM data showed that larger PNH clones were associated
with classical PNH and thrombotic events, while smaller PNH
clones were associated with bone marrow aplasia. At diagnosis PNH clone sizes in red cells, granulocytes and monocytes
were significantly different between the three clinical subcategories (p-value <0.001) for all the studied monoclonal
antibodies (CD55, CD59, CD14, CD16, CD24 and CD66b). The
median PNH clone percentages in red cells were 0.04% (range:
0–18.0%), 15.8% (range: 0–99.7%) and 82.2% (range: 0–99.85%)
in PNH-sc/AA, PNH/AA and Classic PNH, respectively (p-value
<0.001). Moreover, the median PNH clone percentages in
93
rev bras hematol hemoter. 2 0 1 5;3 7(2):90–97
Table 2 – PNH subgroup characteristics.
PNH category
Age
Asthenia/fatigue
Infection/fever
Hemoglobinuria
Thrombosis
Hemolysis
Total
(n = 103)
a – PNH-sc/AA
(n = 54)
24.1 (5.5–62.0)
98 (95.1%)
48 (46.6%)
43 (41.7%)
17 (16.5%)
49 (47.6%)
88 (38–145)
Hemoglobin (g/L)
2970 (1140–8800)
Leucocytes
(×103 /L)
ANC (×109 /L)
0.94 (0.26–4.95)
Platelets (×109 /L)
25 (2–294)
53.8 (2.4–437)
ARC (×109 /L)
LDH (U/L)
328.5 (30–7690)
Bilirubin (mg/dL)
0.9 (0.17–7.1)
Bone marrow
5% (0–100%)
cellularity
Flow cytometry
Neutrophil
clone size
(median)
Erythrocyte
clone size
(median)
25.2 (0.3–100)
2.2 (0–92.2)
b – PNH/AA
(n = 39)
c – Classic
PNH (n = 10)
25.1 (7.5–62.0)
51 (94.4%)
23 (42.6%)
4 (7.4%)
2 (3.7%)
0 (0%)
21.5 (5.5–49.4)
37 (94.9%)
25 (64.1%)
30 (76.9%)
9 (23.1%)
39 (100%)
34.7 (26.7–55.9)
10 (100%)
0 (0%)
9 (90%)
6 (60%)
10 (100%)
80 (38–132)
2675 (1140–8800)
94 (45–145)
89.5 (51–117)
3110 (1470–7780) 4490 (1600–6680)
0.825 (0.26–4.66)
20 (2–99)
35.4 (2.4–153)
234 (30–783)
0.7 (0.17–4.93)
5%
1.06 (0.59–4.95)
27 (3–137)
70.8 (3.2–214)
517 (187–4550)
1.06 (0.39–3.32)
10%
7.3 (0.3–68.7)
0.04 (0–18)
p-Valuea
(a × b × c)
p-Value
(a × b)
p-Value
(a × c)
p-Value
(b × c)
0.322
0.017
0.004
0.062a
0.008a
0.05
0.005
0.106
2.724 (0.16–3.90)
1815 (6–294)
240 (78–437)
1608 (328–7690)
2.54 (0.39–7.1)
70%
0.000a
<0.001a
<0.001a
0.001b
<0.001a
<0.001a
0.084
0.041
0.002
0.002
0.004
0.00
0.000
0.00
0.00
0.00
0.00
0.00
0.001
0.00
0.00
0.004
0.00
0.00
63 (1.7–99.8)
98 (81.3–100)
<0.001a
0.00
0.00
0.001
15.8 (0–99.7)
82.2% (0–99.85)
<0.001b
0.00
0.00
0.018
0.016
0.001
<0.001
<0.001
<0.001
a: PNH-sc/AA group; b: PNH/AA group; c: Classic PNH group.
ANC: absolute neutrophil count; ARC: absolute reticulocyte count; LDH: lactate dehydrogenase.
a
b
ANOVA p < 0.05.
Kruskal–Wallis, p < 0.05.
granulocytes were 7.3% (range: 0.3–68.7%), 63.0% (range:
1.7–99.8%) and 98.0% (range: 81.3–100.0%) in PNH-sc/AA,
PNH/AA and Classic PNH, respectively (p-value <0.001 –
Figures 1–3).
Thrombotic events
Seventeen patients presented with thrombotic events: six in
the Classic PNH group, nine in the PNH/AA group and two
in the PNH-sc/AA group. There were five cases of deep vein
thrombosis (DVT), five abdominal thromboses, five arterial
thromboses with ischemic stroke, one sinus venous thrombosis and one renal vein thrombosis. Two PNHsc/AA patients had
DVT despite the absence of hemolytic symptoms, and died.
Four of the PNH/AA patients died due to thrombotic events.
Regarding the PNH clone, clones larger than 50% were seen
in neutrophils of 13 (76.5%) patients (p-value <0.001), and in
erythrocytes of six (35.3%) patients (p-value = 0.005 – Table 3).
PNH clone percentages in flow cytometry
100
90
80
% median clone
70
60
50
40
30
20
10
0
Total (n=103)
PNHsc/AA (n=54)
AA/PNH (n=39)
Neutrophils %
25.2
7.3
63
Classic PNH (n=10)
98
Erytrocytes %
2.2
0.04
15.8
82.2
Figure 1 – Median percentages of paroxysmal nocturnal hemoglobinuria clone in erythrocyte and neutrophil clones.
94
rev bras hematol hemoter. 2 0 1 5;3 7(2):90–97
30
0
Erythrocytes
27
>0-1
24
>1-3
>3-5
21
>5-10
18
>10-20
15
>20-30
>30-40
12
>40-50
9
>50-60
6
>60-70
>70-80
3
>80-90
0
PNHsc/AA
PNH/AA
≥90
Clássic PNH
Figure 2 – Number of patients with predominantly small erythrocyte clones in paroxysmal nocturnal hemoglobinuria with
subclinical features and aplastic anemia, intermediate clones in paroxysmal nocturnal hemoglobinuria with aplastic
anemia and large clones in classic paroxysmal nocturnal hemoglobinuria.
27
Granulocytes
≤5
>5-10
24
>10-15
21
>15-20
18
>20-30
15
>30-40
12
>40-50
>50-60
9
>60-70
6
>70-80
>80-90
3
>90-100
0
PNHsc/AA
PNH/AA
Clássic PNH
Figure 3 – Number of patients with predominantly small granulocyte clones in paroxysmal nocturnal hemoglobinuria with
subclinical features and aplastic anemia, intermediate in paroxysmal nocturnal hemoglobinuria with aplastic anemia and
large in classic paroxysmal nocturnal hemoglobinuria.
The differences between patients with or without thrombotic events are shown in Table 4. There were statistically significant differences in the number of platelets
(p-value = 0.001), LDH (p-value = 0.002) and in median percentages of the PNH clone in neutrophils (p-value <0.001) and
erythrocytes (p-value = 0.008). The median neutrophil PNH
clone in thrombotic patients was 92.7% (range: 3.4–100%)
vs. 21.8% (range: 0.25–99.9%) in non-thrombotic patients.
The median erythrocyte PNH clone was 31.8% (range:
0.0–97.1%) vs. 1.2% (range: 0.0–92.2%) in thrombotic and nonthrombotic patients, respectively. The bone marrow cellularity, hemoglobin concentration, leukocyte count and absolute
reticulocyte count were similar between the two groups.
There were six deaths among patients with thrombotic
events (35.3%), and eight (9.3%) among the non-thrombotic
group.
Table 3 – Paroxysmal nocturnal hemoglobinuria neutrophil and erythrocyte clones in thrombotic patients.
PNH clone
<10%
Neutrophil clone size – n (%)
Erythrocyte clone size – n (%)
1 (5.9)
5 (29.4)
PNH clone
10–50%
3 (17.6)
6 (35.3)
PNH clone
≥50%
13 (76.5)
6 (35.3)
p-Value (2
test)
<0.001
0.005
95
rev bras hematol hemoter. 2 0 1 5;3 7(2):90–97
Table 4 – Characteristics of patients with and without thrombosis.
Yes
No
p-Value (2 test)
10
8.8
3500
78.2
67.0
607
92.7%
31.8%
6/17 (35.3%)
5
6.5
2600
48.6
31.5
299
21.5%
1.2%
8/86 (9.3%)
0.166
0.682
0.663
0.046
0.001
0.002
<0.001
0.008
<0.001
Thrombosis
Bone marrow cellularity – %
Hemoglobin (g/L)
Leukocyte (×103 /L)
ARC (×109 /L)
Platelets (×109 /L)
LDH (U/L)
Neutrophil clone size (median) – %
Erythrocyte clone size (median) – %
Death – n (%)
LDH: lactate dehydrogenase; ARC: absolute reticulocyte count.
Survival and treatment
As a retrospective study, the cohort of patients received
different treatments depending on the physician and time
criteria. The majority of patients (81.9%) of aplastic groups
(PNH-sc/AA and PNH/AA) received cyclosporine A-based
immunosuppressive therapy with or without corticosteroids
some time during the follow-up, and 12 patients (12.7%)
received anti-human thymocyte immunoglobulin immunosuppressive therapy. Hematopoietic stem cell transplantation
(HSCT) was performed in 27 patients (28.7%), the majority of
whom were in the PNH-sc/AA group. Nine PNH/AA and two
Classic PNH patients received HSCT due to hemolytic symptoms. Eculizumab was introduced in 2010, and five patients
received this drug until the conclusion of the study.
The median follow-up period for all patients was 49.2
months (range: 2.4–310 months), with an OS of 81.7% at ten
years after diagnosis. There was a slight difference in survival
between PNH-sc/AA and the other two groups (76.5% vs. 87.9%;
p-value = 0.112). In total 14 patients died, nine in the PNHsc/AA group and five in the PNH/AA group. The causes of death
were sepsis (six patients), pulmonary thromboembolism (four
patients), mesenteric thrombosis (two patients), severe graftversus-host disease (GVHD) after HSCT (one patient) and
sudden death (one patient). No death was reported in the classical PNH group by the end of the follow-up period.
Discussion
PNH is a rare acquired disorder of hematopoietic stem
cells, which is characterized by a highly variable clinical
course, including intravascular hemolysis, bone marrow failure syndromes and thrombosis.1–3 The natural history of PNH
has been widely discussed in the literature in retrospective
series,3,4,8,9,16,19,20 which confirm the association between PNH
and bone marrow syndromes. The proportion of patients with
bone marrow hypoplasia who develop clinical or subclinical
PNH varies from 22%8 to 40%.16 Some studies that used Ham’s
test to diagnose PNH showed that 5–10% of patients with Classic PNH develop pancytopenia.2,4
The current study identified 103 patients with a PNH clone,
87.4% of whom had hypoplastic marrow at diagnosis. PNHassociated cytopenias were common and recurrent infections
and bleeding were seen in 46.6% and 47.1% of patients, respectively. The high prevalence of bone marrow failure may be
a result of an ascertainment bias explained by the fact that
the Hospital de Clinicas in Curitiba receives cases of aplasia
from many regions of Brazil. For the same reason the majority
of patients (81.9%) received immunosuppressive therapy and
28.7% received HSCT.
Patients included in this study were assigned to three
subcategories based on the proposed PNH working clinical
classification,1 namely Classic PNH, PNH/AA, and PNH-sc/AA.
As expected, the clinical symptoms of hemolysis, hemoglobinuria and thrombosis were higher in the hemolytic forms of
PNH (PNH/AA and Classic PNH) than in PNH-sc/AA, whereas
infection and fever were higher in the latter group. There
were significant differences in terms of peripheral blood cell
counts between the three subcategories, especially between
the Classic PNH and PNH-sc/AA subcategories. The median
PNH clone size was also significantly different between the
three clinical subcategories at diagnosis. On the other hand,
eight patients in the PNH/AA group presented with intense
hemolytic symptoms and PNH clones greater than 50%,
despite the hypocellular bone marrow, suggesting an overlap between the subgroups. This fact could be explained by
the heterogeneity of the sample and the disease, and by the
retrospective bias of this study.
PNH mainly presents as a disease of adults, but the median
age found in the current series was lower than in the literature
(24.1 vs. 34.2 years).2,3,19 This difference can be explained by
the proportion of aplastic patients who evolved with the PNH
clone (PNH-sc/AA) after a long period of time in our series.
However, if the median age of the Classic PNH group is considered, it is similar to other studies.4,16,20
In the last decade, the detection of GPI-AP deficient cells by
flow cytometry greatly increased the sensitivity of detecting a
PNH clone in both red blood cells and leukocytes, minimizing
the effects of red cell transfusions on estimating clone size.5,11
Since the introduction of FCM, quantitative and kinetic differences of GPI-AP deficient clones have been reported between
PNH patients with and without marrow failure. The literature suggests that the clinical manifestations due to chronic
hemolysis in PNH appear to be more common in patients
with large populations of cells deficient in GPI proteins.1,19–21
Pu et al. showed a direct relationship between the size of
PNH clone and the development of intravascular hemolysis
in patients with aplasia.19 In the current study large granulocyte PNH clones were found in Classic PNH (98.0%), medium
clones in PNH-AA (63.0%) and small PNH clones in PNHsc/AA (7.3%), suggesting that larger PNH clones (>50%) are
96
rev bras hematol hemoter. 2 0 1 5;3 7(2):90–97
associated with increased risks of hemoglobinuria and thrombosis, whereas small PNH clones (<50%) are associated with
bone marrow failure.
In this study, life-threatening thrombotic complications
were present in 16.5% of patients, with six deaths occurring in this group (35.3% vs. 9.3% in the non-thrombotic
group). All three subcategories had at least one episode of
thrombosis. Two PNHsc/AA patients had DVT despite the
absence of hemolytic symptoms, and died. Four of the PNH/AA
patients died due to thrombotic events. PNH clones larger than
50% were seen in the granulocytes of 13 (76.5%) thrombotic
patients, which is consistent with the literature that says that
a clone size greater than 50% is associated with increased risk
of thrombosis and the need of primary anticoagulation.7,21
In the current cohort the size of the PNH clone was greater
in patients with thrombosis for both neutrophils (92.7% vs.
21.5%) and erythrocytes (31.8% vs. 1.2%).
The OS ten years after diagnosis was 81.7% in this study,
which is slightly higher than other studies,20 which reported
77.6% at 10 years. Infection and thrombotic events were identified as the main causes of death. There were 14 deaths
(13.5%), 12 of which were attributable to PNH or aplasia; of
the deaths, six (42.8%) were secondary to thrombosis. With
regard to the disease subcategories, the long-term outcomes
were similar between patients in the hemolytic PNH subcategories, but slightly different in PNH-sc/AA group, probably due
to a high risk of infection. Further studies may shed insights
into the hypothesis that the PNH-sc/AA subcategory might be
a separate disease entity from Classic PNH.1,5,22
Conclusion
This retrospective study of 103 PNH patients over an 11-year
period represents the largest collection of such patients from
a single center in Brazil. The results confirmed the suggestion
that PNH is not a simple binary diagnosis and both flow cytometric characterization of GPI-AP expression on peripheral
blood cells and marrow analysis are required for comprehensive disease classification. FCM data from this study shows
that larger PNH clones are associated with classical PNH symptoms and increased risk of thrombosis, even in patients with
bone marrow failure, whereas smaller PNH clones are associated with bone marrow aplasia.
Conflicts of interest
The authors declare no conflicts of interest.
Acknowledgments
The authors wish to thank Rosana Inara Cattaneo, Miriam Perlingeiro Beltrame, Noely Silva and Leila Oliveira for technical
assistance with flow cytometric studies. Thanks to Dr. Adam
Campbell Smith for the English revision and to Dr. Alberto
Orfao for the scientific comments.
references
1. Parker C, Omine M, Richards S, Nishimura J, Bessler M, Ware
R, et al. Diagnosis and management of paroxysmal nocturnal
hemoglobinuria. Blood. 2005;106(12):3699–709.
2. Socié G, Mary JY, de Gramont A, Rio B, Leporrier M, Rose C,
et al. Paroxysmal nocturnal haemoglobinuria: long-term
follow-up and prognostic factors. French Society of
Haematology. Lancet. 1996;348(9027):573–7.
3. de Latour RP, Mary JY, Salanoubat C, Terriou L, Etienne G,
Mohty M, et al. Paroxysmal nocturnal hemoglobinuria.
Natural history of diseases subcategories. Blood.
2008;112(8):3099–106.
4. Hillmen P, Lewis SM, Bessler M, Luzzatto L, Dacie JV. Natural
history of paroxysmal nocturnal hemoglobinuria. N Engl J
Med. 1995;333(19):1253–8.
5. Borowitz MJ, Craig FE, Digiuseppe JA, Illingworth AJ, Rosse W,
Sutherland DR, et al. Guidelines for the diagnosis and
monitoring of paroxysmal nocturnal hemoglobinuria and
related disorders by flow cytometry. Cytometry B Clin Cytom.
2010;78(4):211–30.
6. Mortazavi Y, Merk B, McIntosh J, Marsh JC, Schrezenmeier H,
Rutherford TR, BIOMED II Pathophysiology and Treatment of
Aplastic Anaemia Study Group. The spectrum of PIG-A gene
mutations in aplastic anemia/paroxysmal nocturnal
hemoglobinuria (AA/PNH): a high incidence of multiple
mutations and evidence of a mutational hot spot. Blood.
2003;101(7):2833–41.
7. Hall C, Richards S, Hillmen P. Primary prophylaxis with
warfarin prevents thrombosis in paroxysmal nocturnal
hemoglobinuria (PNH). Blood. 2003;102(10):3587–91.
8. Dunn DE, Tanawattanacharoen P, Boccuni P, Nagakura S,
Green SW, Kirby MR, et al. Paroxysmal nocturnal
hemoglobinuria cells in patients with bone marrow failure
syndromes. Ann Intern Med. 1999;131(6):401–8.
9. Nakakuma H, Nagakura S, Iwamoto N, Kawaguchi T, Hidaka
M, Horikawa K, et al. Paroxysmal nocturnal hemoglobinuria
clone in bone marrow of patients with pancytopenia. Blood.
1995;85(5):1371–6.
10. Griscelli-Bennaceur A, Gluckman E, Scrobohaci ML, Jonveaux
P, Vu T, Bazarbachi A, et al. Aplastic anemia and paroxysmal
nocturnal hemoglobinuria: search for a pathogenetic link.
Blood. 1995;85(5):1354–63.
11. Hall SE, Rosse WF. The use of monoclonal antibodies and flow
cytometry in the diagnosis of paroxysmal nocturnal
hemoglobinuria. Blood. 1996;87(12):5332–40.
12. Piedras J, López-Karpovitch X. Flow cytometry analysis of
glycosylphosphatidyl-inositol-anchored proteins to assess
paroxysmal nocturnal hemoglobinuria clone size. Cytometry.
2000;42(4):234–8.
13. Hernándes-Campo PM, Almeida J, Sanchez ML, Malvezzi M,
Orfao A. Normal patterns of expression of
glycosylphosphatidylinositol-anchored proteins on different
subsets of peripheral blood cells: a frame of reference for the
diagnosis of paroxysmal nocturnal hemoglobinuria.
Cytometry B Clin Cytom. 2006;70(2):71–81.
14. Richards SJ, Rawstron AC, Hillmen P. Application of flow
cytometry to the diagnosis of paroxysmal nocturnal
hemoglobinuria. Cytometry. 2000;42(4):223–33.
15. Sugimori C, Chuhjo T, Feng X, Yamazaki H, Takami A,
Teramura M, et al. Minor population of CD55− CD59− blood
cells predicts response to immunosuppressive therapy and
prognosis in patients with aplastic anemia. Blood.
2006;107(4):1308–14.
16. Scheinberg P, Marte M, Nunez O, Young NS. Paroxysmal
nocturnal hemoglobinuria clones in severe aplastic anemia
rev bras hematol hemoter. 2 0 1 5;3 7(2):90–97
patients treated with horse anti-thymocyte globulin plus
cyclosporine. Haematologica. 2010;95(7):1075–80.
17. Camitta BM, Rappeport JM, Parkman R, Nathan DG. Selection
of patients for bone marrow transplantation in severe
aplastic anemia. Blood. 1975;45(3):355–63.
18. Swerdlow SH, Campo E, Harris NL, Jaffe ES, Pileri AS, Stein H,
et al., editors. WHO classification of tumors of
haematopoietic and lymphoid tissues. Lyon: IARC; 2008.
19. Pu JJ, Mukhina G, Wang H, Savage WJ, Brodsky RA. Natural
history of paroxysmal nocturnal hemoglobinuria clones in
patients presenting as aplastic anemia. Eur J Haematol.
2011;87(1):37–45.
97
20. Ge M, Li X, Shi J, Shao Y, Zheng Y. Clinical features and
prognostic factors of Asian patients with nocturnal
hemoglobinuria: results from a single center in China. Ann
Hematol. 2012;91(7):1121–8.
21. Kim JS, Lee JW, Yoon S-S, Lee JH, Jo DY, Jang JH, et al.
Association between elevated hemolysis at diagnosis and
early mortality and risk of thrombosis in paroxysmal
nocturnal hemoglobinuria (PNH) patients with cytopenia.
Blood. 2010;116:4241 [ASH Annual Meeting Abstracts].
22. Brodski R. Paroxysmal nocturnal hemoglobinuria: stem cells
and clonality. Hematol Am Soc Hematol Educ Progr.
2008:111–5.