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MEDICINE
REVIEW ARTICLE
Paroxysmal Nocturnal
Hemoglobinuria
Alexander Röth, Ulrich Dührsen
SUMMARY
Introduction: Paroxysmal nocturnal hemoglobinuria (PNH) is an acquired clonal disease of the
pluripotent hematopoetic stem cell, caused by a mutation of the PIG (phosphatidyl inositol
glycan) A gene. Methods: Review of PNH based on a selective, PubMed-based literature search.
Results: This mutation results in the clinical triad of hemolytic anemia, thrombophilia and
cytopenia. PNH can also be asymptomatic, and should be excluded where thrombosis affects
unusual sites or in the face of unexplained recurrent abdominal pain. Flow cytometry is the
standard for diagnosis and measurement of type and size of the PNH clone. Treatment is mainly
symptomatic. Low dose steroids can attenuate acute haemolytic exacerbation, but chronic use
is contraindicated. Allogenic bone marrow transplantation is the only curative option in case of
severe complications. A new treatment strategy is the inhibition of the terminal complement
cascade with a monoclonal antibody (eculizumab). As shown in the initial uncontrolled clinical
study, this appears to reduce complement mediated intravascular hemolysis, decrease the need
for transfusion and improve quality of life in patients with PNH. Recently a randomized controlled
trial has been published; N Engl J Med (2006; 355: 1233–43).
Dtsch Arztebl 2007; 104(4): A 192–7.
Key words: paroxysmal nocturnal hemoglobinuria, genetic mutation, therapy, monoclonal antibody
P
aroxysmal nocturnal hemoglobinuria is characterized by the classic triad of anemia,
thrombophilia and cytopenia. The extent of the individual symptoms is very variable,
and PNH can be relatively asymptomatic. In unclear cases of hemolysis, thrombosis or
cytopenia, it is therefore important to exclude PNH. PNH is an acquired clonal disease caused
by the somatic mutation of the gene coding for phosphatidyl inositol glycan (PIG) A in the
pluripotent hematopoetic stem cell. This leads to the destruction of the glucosyl phosphatidyl
inositol anchor (GPI anchor) hence causing reduction or complete absence of GPI anchored
surface proteins.
This clinical entity was first described in 1882 by Strübing. In 1911, this form of haemolytic
anemia was described in conjunction with the characteristic hemoglobinuria by Marchiafava
and Micheli, which gave rise to the eponymous name Marchiafava Micheli Syndrome. PNH is
a good example of progress in medicine. Based on the description of a syndrome, modern
investigations have in recent decades permitted the elucidation of its etiology and pathogenesis
and in turn the development and implementation of new treatments. This review article is based
on a literature search of PubMed. Individual articles were selected by currency and relevance.
No guidelines currently exist for the management of this disease, so the current recommendations
of the International PNH Interest Group were taken into account (1).
Epidemiology
PNH is a rare haematological disease, with and incidence of 1:100 000 to 1: 500 000 per
year. The true incidence may be higher, because the disease is under recognized. (2). The
age of peak incidence is between 25 and 45, with equal distribution between the sexes, and
no evidence of familial clustering, to date.
Pathogenesis
Molecular genetic background
The underlying defect in PNH is and acquired mutation of the PIG A gene on the X chromosome
of a pluripotent hematopoetic stem cell. The product of this gene, N-acetyl glucosaminyl
Klinik für Hämatologie, Zentrum für Innere Medizin, Universitätsklinikum Essen, Universität Duisburg-Essen (Dr. med. Röth,
Prof. Dr. med. Dührsen)
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Diagram 1
Structure of the GPI anchor. In PNH the
first step is disrupted due to a mutation
of the phosphatidyl inositol glycan A
protein, resulting in a partial or total
absence of the GPI anchor and relevant
anchored proteins. This can affect all
cell lineages.
transferase, is responsible for the biosynthesis of the GPI anchor protein, which is necessary
for the insertion of surface proteins into the cell membrane (diagram 1). Depending on the
type of mutation in the affected cell clone, the GPI anchor may be partly or wholly
dysfunctional. This distinguishes mutated cells from normal hematopoetic cells. The
relation between normal and GPI deficient cells is a measure of the relative size of the PNH
clone. The lacking expression of GPI anchored proteins leads in the affected cells to
compromized function, which is responsible for the various symptoms of PNH. In addition,
bone marrow changes appear to play an important role in allowing the further expansion of
the PNH clone. (3, 4).
Hemolysis
Coombs negative hemolysis and hemoglobinuria are the key clinical features of PNH,
and are caused by deficient binding of the complement regulating membrane factors CD
55 (DAF - decay accelerating factor) and CD 59 (MIRL - membrane inhibitor of reactive
hemolysis), via the missing GPI anchor on erythrocytes. Under normal circumstances,
these protect erythrocytes from the deposition of activated autologous complement
components and complement mediated cell lysis. The extent of hemolysis depends on the
size of the PNH clone and therefore the number of affected cells. Untreated, the hemolysis
usually becomes chronic, with episodes of hemolytic crisis or paroxysms with classic,
coca-cola coloured morning urine (illustration). It is not usually life threatening and can
be treated effectively with erythrocyte concentrates. These crises can be precipitated by
strenuous physical effort, infection, and surgery, but also by the administration of
contrast media (2, 5).
Thrombophilia
Venous thromboses, in particular intra abdominal (splenic vein, portal artery, hepatic vein
thromboses [Budd Chiari syndrome]) or cerebral, are a further key feature of PNH. Around
half of all PNH patients develop a thrombosis during the course of the disease; around a
third die of these (6, 7). The exact cause of the thrombotic tendency is as yet unclear. It is
assumed that GPI deficient platelets are more readily activated by complement than normal
platelets. This leads to a higher prothrombinase activity and to thrombus formation.
Intravascular hemolysis may also lead to direct activation of the clotting cascade (8, 9). The
diagnosis of PNH should therefore be excluded in young patients with thrombosis in
unusual locations, in particular.
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Illustration: Hemoglobinuria is a cardinal symptom of PNH. The classic dark morning urine gives the condition its name. However, the underlying condition is chronic hemolysis. The dark colouration of the morning urine comes from concentration overnight, and disappears over the
course of the day.
Cytopenia
Many PNH patients show an initial cytopenia, which can range from an isolated sub clinical
reduction in a single cell lineage, to severe aplastic anemia. (6). Both the cytopenia and the
lack of functionally important surface proteins on granulocytes, monocytes and
lymphocytes compromise the immune system. In addition, patients with aplastic anemia
often develop a secondary PNH (10).
Clinical picture
Diagnosis and differential diagnosis
The diagnosis of PNH is often difficult because the initial symptoms are often multifaceted
and because hemoglobinuria is absent in around 25% of cases for the duration of the disease.
Further clinical features useful in diagnosis are listed in the table.
It is assumed that many of the symptoms of PNH, such as dysphagia, abdominal pain,
back pain, headache and erective dysfunction are caused by compromised smooth muscle
activity due to the binding of nitric oxide (NO) to free haemoglobin. These symptoms
typically occur during haemolytic crises (11, 12). Flow cytometry is the established
investigation of choice in making a diagnosis of PNH. (13, 14). This allows the missing
GPI anchored surface proteins to be detected in all hematopoetic cell lineages. Cells
completely lacking surface proteins (type 3 cells) can be distinguished from cells with
only a partial loss of surface proteins (type 2 cells). Cells with normal expression are
known as type 1 cells (diagram 2).
The minimal diagnostic requirement is the absence of at least two different GPI anchored
proteins on at least two cell lineages. The size of the PNH clone as a proportion of hematopoetic function can best be determined via the proportion of GPI deficient granulocytes,
because this is unaffected by hemolysis or transfusion.
A new diagnostic approach uses the capability of the toxin aerolysin, produced by
the bacterium Aeromonas hydrophilia, to bind directly to the GPI anchor. Labelling with
a fluorochrome (FLAER, fluorescent aerolysin) allows the GPI anchor to be detected and
measured directly, rather than via the indirect measure of the bound surface protein (15).
Other diagnostic tests detecting increased sensitivity of PNH erythrocytes to complement
mediated hemolysis (sugar water test, acid hemolysis test [Ham test]) are less specific and
sensitive than flow cytometry. They can sometimes be helpful in screening, but are not
sufficient alone to make a diagnosis of PNH. A further diagnostic option is the demonstration
of the mutation in the PIG A gene, using molecular biological techniques. This is not used
routinely due to the large use of technical resources it requires.
Initial diagnostic work up should include a full blood count and differential blood count,
reticulocyte count, and measurement of the serum lactate dehydrogenase, (LDH), indirect
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TABLE
Clinical symptoms in diagnosis
Symptoms
Anemia
Frequency
(percent)
35
Hemoglobinuria
26
Hemorrhages
18
Aplastic anemia
13
Gastrointestinal symptoms
10
Hemolytic anemia and jaundice
9
Iron deficiency anemia
6
Thromboembolic disease
6
Infections
5
Neurological symptoms
4
Modified from Dacie et al. (2)
bilirubin and haptoglobin. Bone marrow aspiration with cytology, cytogenetics and
histology is also recommended (1).
Classification of PNH
PNH is divided into a number of groups, depending on clinical presentation and course (1):
> Classical PNH: intravascular hemolysis (reticulocytosis, raised LDH and indirect
bilirubin, reduced haptoglobin) with no evidence of another bone marrow disease
> PNH in connection with another bone marrow disorder (aplastic anemia/myelodysplastic syndrome [MDS]): signs of hemolysis and additional or pre-existing bone marrow
disease. Cytogenetic analysis is essential here to exclude or detect aplastic anemia, MDS or
another myelopathy (such as myelofibrosis)
> Sub clinical PNH (PNH-sc): no evidence of hemolysis. Sensitive diagnostic techniques
reveal very small GPI deficient populations. This often arises in conjunction with other
systemic haematological disease, such as aplastic anemia or MDS, where it is of great
therapeutic and prognostic significance. The detection of sub clinical PNH in aplastic anemia
is associated with a good response to immunosuppressive treatment and better prognosis.
Treatment
PNH is a benign, chronic condition. Poor prognostic features include: thrombosis, pancytopenia,
MDS or acute leukaemia, age over 55, thrombocytopenia at the time of diagnosis, or the
need for extensive treatment (6). Median survival in affected individuals is 10 to 15 years.
Spontaneous remission occurs in the long term, in 15 percent of patients (6). These data
should be borne in mind in planning treatment.
Supportive measures
The mainstay of symptomatic treatment is transfusion of red cell concentrates cleaned of
leucocytes. The decision to prescribe these should take into account symptoms such as
weakness or dyspnea of effort, clinical examination findings, and the speed of decline in
haemoglobin levels. Theoretical concerns that residual donor plasma in transfused concentrates
might precipitate haemolytic crisis have not been confirmed (16). Only where there is an
absolute or relative lack of erythropoietin should the administration of recombinant
erythropoietin be considered. Under these circumstances, however, blood results must be
closely monitored since there is a risk of haemolytic crisis due to increased production of
GPI deficient erythrocytes. Iron should be given orally, as patients often lose iron and have
low ferritin levels due to the hemoglobinuria and hemosiderinuria. Folic acid (5mg/kg/day)
should be given in view of the increased cell turnover in the bone marrow. Infections
should be treated early with antibiotics, as they can cause haemolytic crises via activation
of the complement system (1).
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Diagram 2
Flowcytometric diagnosis of PNH. Flow cytometry is the diagnostic gold standard in PNH.
The individual cell populations are distinguished by differences in size (forward scatter) and
granulation (side scatter). Using fluorochrome labelled antibodies cell surface antigens can
be detected. The example given in this histogram is the expression of CD55 on granulocytes
(R1). Many cells lack CD55 partially (type 2 cells) or completely (type 3 cells). Only a small
number of cells show normal expression.
Bouts of abdominal pain should be treated symptomatically with plentiful fluids and
adequate analgesia up to and including opiates if needed. The differential diagnosis of
mesenteric vein thrombosis should be borne in mind, and the possible need for surgical
intervention considered.
Steroids
Steroid treatment is controversial, both for chronic hemolysis and for acute crises (17–19).
Treatment with steroids is purely empirical, and no randomized trials exist to support their
use. Some patients however appear to benefit rapidly from steroid treatment (0.25 to
1 mg/kg/day prednisolone. The rapid effect of this treatment suggests that it works via
suppression of the complement system. It may be that haemolytic crises are primarily
attenuated by steroid administration. Brief steroid treatment may therefore influence a crisis
positively while avoiding the complications of long term steroid use. Long term treatment
of paroxysmal nocturnal hemoglobinuria with steroids is contraindicated (1).
Anticoagulation
Confirmed thrombosis must be treated with lifelong warfarin. Warfarin is also recommended
for primary prevention in patients with more than 50 % GPI deficient granulocytes and no
contraindications to warfarin. A retrospective study suggested that these patients have an
approximately eight fold thrombosis risk over ten years (44 % compared with 6 %) (20).
Heparins can also be used in paroxysmal nocturnal hemoglobinuria.
Immunosuppressive treatment
Immunosuppression is not recommended for treatment of the haemolytic activity alone. It
should only be used in the context of clinical trials investigating the treatment of possible
underlying bone marrow diseases underlying pancytopenia (such as aplastic anemia or
MDS).
Bone marrow transplantation
The only potentially curative treatment for PNH is allogenic bone marrow transplantation
(BMT) or peripheral blood stem cell transplantation. Because of the long median survival
time and the possibility of spontaneous remission, bone marrow transplantation should
currently be reserved for severe cases of aplastic anemia, severe haemolytic crises or a marked
thrombotic tendency. Because long term survival following bone marrow transplantation in
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Diagram 3
Mechanism of action of eculizumab: inhibition of the terminal complement pathway. The
activation of the complement system leads to the formation of a membrane attack complex
and thereby to cell lysis. The splitting of C5 is the key trigger for the terminal complement
cascade. This activation can be blocked by the anti C5 antibody eciluzumab; reproduced from
(11). Hematology Am Soc Hematol Educ Program 2004; 48-62, by kind permission: American
Society of Hematology.
non malignant conditions is poor (around 56%) the indication should be tightly drawn.
(21). The possibility of regimes with reduced conditioning and therefore reduced toxicity
related to chemotherapy and whole body irradiation of the recipient in preparation for
transplant, are currently under investigation. In this regime, donor cells are intended to
eradicate the recipient's hematopoetic cells, including the PNH cells. (22).
Complement inhibition using eculizumab
A possible new treatment option is the blocking of the complement system using a monoclonal
antibody, which is currently undergoing phase 3 trials in over 180 patients (TRIUMPH:
transfusion reduction efficacy and safety clinical investigation, randomised, multi center,
double blind, placebo controlled, using eculizumab in paroxysmal nocturnal hemoglobinuria,
SHEPHERD: safety in haemolytic PNH patients treated with eculizumab: a multi center
open label research design study).
The antibody eculizumab binds complement factor C5 prevents its splitting and blocks
the ensuing activation of the terminal complement system (diagram 3). Eculizumab almost
completely prevents intravascular hemolysis and hemoglobinuria. An early uncontrolled
study in 11 patients showed a clear fall in LDH levels from 3,111 ± 598 U/L to 594 ± 32
U/L during treatment (p = 0.002).
Transfusion requirements were reduced by 70 % from a median of 1.8 bags a month to 0
(p = 0.003), and hemoglobinuria by 96 % (p = 0.001). Quality of life was also improved
significantly. In addition, typical symptoms related to the binding of nitric oxide (NO) to
free hemoglobin, such as dysphagia, abdominal pain and erectile dysfunction, were also
improved (12, 23). The continuation of the initial study and the studies currently in progress
show that long term treatment for more than four years with eculizumab is effective and
well tolerated (24, 25).
No serious complications have so far been reported, and patients have complained so far
only of non-specific side effects such as headache or back pain. The results of the
TRIUMPH trial were recently published (N Engl J Med 2006: 355: 1233–43). It is unclear
whether this treatment also influences thrombotic risk. The cost of this treatment is in the
same region as that of other treatments with monoclonal antibodies.
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PNH registry and self help organizations
Because of the rarity of the condition, the International PNH Interest Group has set up a
worldwide database collecting detailed epidemiological information about PNH patients,
with a view to generating new insights and treatments (www.pnhregistry.org). Further
information, in particular for affected individuals in Germany, can be found on the homepage
of the German PNH self help group (www.pnh-info.de).
Conflict of Interest Statement
Prof. Dührsen is taking part in trials of eculizumab with Alexion. Alexion has provided financial support to cover costs to the
University of Essen. Dr. Röth declares no conflict of interest in the terms of the International Committee of medical Journal editors.
Manuscript received on 2 May 2006, final version accepted on 27 Juli 2006.
Thanks
This review is dedicated to Prof. G. Brittinger on the occasion of his 75th birthday.
Translated from the original German by Dr. Sandra Goldbeck-Wood.
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Corresponding Author
Dr. med. Alexander Röth
Klinik für Hämatologie, Zentrum für Innere Medizin,
Universitätsklinikum Essen
Universität Duisburg-Essen
Hufelandstrasse 55
45122 Essen, Germany
[email protected]
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