Paroxysmal Nocturnal

Hemoglobinuria
Updated: Jun 29, 2016
Author: Emmanuel C Besa, MD; Chief Editor: Koyamangalath Krishnan,
MD, FRCP,
Background
Paroxysmal nocturnal hemoglobinuria (PNH) is a rare, chronic,
debilitating disorder that most frequently presents in early adulthood and
usually continuous throughout the life of the patient. PNH results in the
death of approximately 50% of affected individuals due to thrombotic
complications and, until recently, had no specific therapy. The name of
the disorder is a descriptive term for the clinical consequence of red
blood cell (RBC) breakdown with release of hemoglobin into the urine,
which manifests most prominently as dark-colored urine in the morning
(see image below).

This series of containers

holds urine of a patient with paroxysmal nocturnal hemoglobinuria,
showing the episodic nature of the dark urine (hemoglobinuria) during
intravascular hemolysis, usually occurring at night. Early morning urine
is cola-colored. This may occur at different times of the day and vary
from patient to patient. Permission to use this image has been granted
by the American Society of Hematology Slide Bank, 3rd edition.
View Media Gallery
The term "nocturnal" refers to the belief that hemolysis is triggered by
acidosis during sleep and activates complement to hemolyze an
unprotected and abnormal RBC membrane. However, this observation
was later disproved. Hemolysis has been shown to occur throughout the
day and is not actually paroxysmal, but the concentration of urine
overnight produces the dramatic change in color.
PNH has been referred to as "the great impersonator" because of the
variety of symptoms observed during its initial manifestation and course.
This variety reflects the contributions of the following three underlying
pathophysiologic events[1, 2, 3, 4] :
 An acquired intracorpuscular hemolytic anemiadue to the
abnormal susceptibility of the RBC membrane to the hemolytic
activity of complement
Thromboses in large vessels, such as hepatic, abdominal,
cerebral, and subdermal veins
A deficiency in hematopoiesis that may be mild or severe, such as
pancytopenia in an aplastic anemia state
The triad of hemolytic anemia, pancytopenia, and thrombosis makes
PNH a unique clinical syndrome.
For patient education information, see Anemia.
Pathophysiology
Paroxysmal nocturnal hemoglobinuria (PNH) was previously classified
as purely an acquired hemolytic anemia due to a hematopoietic stem
cell mutation defect. This classification was abandoned because of the
observation that surface proteins were missing not only in the RBC
membrane but also in all blood cells, including the platelet and white
cells.
The common denominator in the disease, a biochemical defect, appears
to be a genetic mutation leading to the inability to synthesize the
glycosyl-phosphatidylinositol (GPI) anchor that binds these proteins to
cell membranes. [4, 5, 6] The corresponding
gene PIGA (phosphatidylinositol glycan class A) in the X chromosome
can have several mutations, from deletions to point mutations.[7]
Due to its location on the X chromosome, and X inactivation in female
somatic cells, only one mutation is required in either males or females to
abolish the expression of GPI-linked proteins. Most type II PNH cells
(total lack of GPI-linked protein) are due to a frame shift mutation
occurring in the early hematopoietic progenitor cells, resulting in the
same mutation in all blood cell lines.
The essential group of membrane proteins that are lacking in all
hematopoietic cells are called complement-regulating surface proteins,
including the decay-accelerating factor (DAF), or CD55 [8] ; homologous
restriction factor (HRF), or C8 binding protein; and membrane inhibitor
of reactive lysis (MIRL), or CD59. [9] All of these proteins interact with
complement proteins, particularly C3b and C4b, dissociate the
convertase complexes of the classic and alternative pathways, and halt
the amplification of the activation process.
The absence of these regulating proteins results in uncontrolled
amplification of the complement system. This leads to intravascular
destruction of the RBC membrane, to varying degrees
Breakdown of RBC membranes by complement leads to the release of
hemoglobin into the circulation. Hemoglobin is bound to haptoglobin for
efficient clearance from the circulation. After saturating the haptoglobin,
free hemoglobin circulates and binds irreversibly with nitric oxide (NO),
depleting NO levels in peripheral blood. Because NO regulates smooth
muscle tone, depletion of NO levels leads to smooth muscle contraction
with consequent vasoconstriction, constriction of the gut, and pulmonary
hypertension. Resultant symptoms may include the following:
Abdominal pain
Bloating
Back pain
Headaches
Esophageal spasms
Erectile dysfunction
Fatigue
Progressive chronic renal failure can occur after several years of
hemoglobinuria from the acute tubulonecrosis effects of heme and iron
(pigment nephropathy), decreased renal perfusion from renal vein
thrombosis, and tubular obstruction with pigment casts. Patients with
PNH experience a high incidence (40%) of thrombotic events (mostly
venous) in large vessels (cerebral, hepatic, portal, mesenteric, splenic,
and renal veins) and, most recently recognized, arterial thrombosis.
The pathophysiology of thrombophilia in PNH is not fully understood,
but the increased incidence that occurs during hemolytic episodes
suggest a direct relationship with the hemolytic process. Increased
procoagulant and fibrinolytic activity, suggesting increased fibrin
generation and turnover, increased plasma levels of urokinase-type
plasminogen activator and platelets deficient in GPI-linked proteins
activated by complement, have been implicated. However, none of
these identified platelet and coagulation abnormalities can fully explain
the hypercoagulable state in PNH.
Bone marrow failure is present in all patients with PNH, even when
peripheral blood counts are normal and the bone marrow is
hypercellular. The degree of marrow failure may vary from severe
aplastic anemia and show evidence of a decreased number of
hematopoietic stem cells, possibly due to similar destruction by
complement, but the cause or causes are still poorly understood.
Venous thrombosis usually manifests as a sudden catastrophic
complication, with severe abdominal pain and rapidly enlarging liver and
ascites (Budd-Chiari syndrome). This thrombosis may be due to a lack
of CD59 on platelet membranes that induces platelet aggregation and is
highly thrombogenic, particularly in the venous system.
Deficient hematopoiesis may occur due to diminished blood cell
production with a hypoplastic bone marrow; thus, patients have a 10-
20% chance of developing aplastic anemia in their course, and patients
known to have aplastic anemia eventually develop PNH in 5% of
cases. [7, 10] The nature of the pathogenetic link between these two
diseases is still unknown.
Pathogenesis
The clinical sequelae of intravascular hemolysis and extracellular
plasma hemoglobin (Hb) are discussed.
RBCs release free Hb into the plasma, which is rapidly dimerized and
bound by serum protein haptoglobin and rapidly removed by
macrophages, which then degrades it after endocytosis. Since
haptoglobin is not recycled, large amounts of free Hb can deplete the
body supply, leaving the excess Hb free in the plasma.
When the capacity to manage and degrade free Hb during acute or
chronic hemolysis is reached, levels of Hb and heme increase in the
plasma and urine. Plasma Hb has the ability to scavenge nitric oxide
(NO), resulting in rapid consumption of NO and clinical sequelae of NO
depletion.
NO plays a major role in vascular homeostasis and has been shown to
be a critical regulator of basal and stress-mediated smooth muscle
relaxation and vasomotor tone, endothelial adhesion, and platelet
activation and aggregation. Thus, clinical consequences of excessive
cell-free plasma Hb levels during intravascular hemolysis or the
administration of Hb preparations include dystonias involving the
gastrointestinal, cardiovascular, pulmonary, and urogenital systems, as
well as clotting disorders. Many of the clinical sequelae of intravascular
hemolysis in a prototypic hemolytic disease, PNH, are readily explained
by Hb-mediated NO scavenging.
Paroxysms or episodes of symptoms occur during sudden and marked
increases in the rate of intravascular hemolysis, and these episodes can
be precipitated by infections, drugs, or trauma, or they can occur
spontaneously. During paroxysms, PNH patients exhibit symptoms
consistent with smooth muscle perturbation through the release of Hb
and NO scavenging, including abdominal pain, esophageal spasms,
and erectile dysfunction.
Thrombosis is the most common cause of death in 50% of PNH patients
and is attributed to venous thrombosis. The most frequent types of
thrombosis in this study included hepatic, pulmonary, deep, cerebral,
and superficial veins, as well as the inferior vena cava. Interestingly,
there is a close correlation between thrombosis and a large PNH clone,
and clone size correlates with hemolytic rates.
NO plays an important role in the maintenance of normal platelet
functions through the down-regulation of platelet aggregation and
adhesion and the regulation of molecules in the coagulation cascade.
Accordingly, the long-term consumption of NO by plasma Hb has been
implicated in the formation of clots in PNH patients. [11]
Etiology
For some time, paroxysmal nocturnal hemoglobinuria (PNH) has been
known to result from somatic mutations in the PIGA gene, which
encodes phosphatidylinositol glycan class A (PIGA). These mutations
result in hematopoietic stem cells that are deficient in glycosyl-
phosphatidylinositol anchor protein (GPI-AP). Nonmalignant clonal
expansion of one or several of these stem cells leads to clinical PNH.
Shen et al have identified additional somatic mutations associated with
PNH. In addition to mutations in PIGA, mutations were found in genes
known to be involved in myeloid neoplasm pathogenesis,
including TET2, SUZ12, U2AF1, andJAK2. Clonal analysis indicated
that these additional mutations arose either as a subclone within
thePIGA-mutant population or had occurred prior toPIGA mutation. [12]
The clinical pathology in PNH may actually be an epiphenomenon
resulting from an adaptive response to injury, such as an immune attack
on hematopoietic stem cells.
In PNH, the peripheral blood and bone marrow is a mosaic composed of
GPI-AP+ and GPI-AP cells; with GPI-AP, cells can be derived from
multiple mutant stem cells. The GPI-AP mutant cells may appear to
dominate hematopoiesis in PNH by providing a proliferative advantage
under some pathologic conditions. For example, if damage to stem cells
causing bone marrow failure is mediated through a GPI-linked surface
molecule, the PNH cells lacking these molecules will survive. The close
association of PNH with aplastic anemia and myelodysplastic syndrome
(MDS) suggests that the selection process arises as a consequence of
this specific type of bone marrow injury.
Epidemiology
Paroxysmal nocturnal hemoglobinuria (PNH) is an uncommon disorder
of unknown frequency both in the United States and worldwide. There is
little information on the incidence of PNH, but the rate is estimated to be
5-10 times less than that of aplastic anemia; thus, PNH is a rare
disease.
Attempts to get a more accurate incidence and to learn more about its
natural course are currently in progress under the auspices of the PNH
Registry. This is a comprehensive, observational, multinational effort to
document the clinical outcomes in the treatment of patients with PNH.
It has been suggested that, like aplastic anemia, PNH may be more
frequent in Southeast Asia and in the Far East.
Mortality/Morbidity
The disease process of PNH is insidious and has a chronic course, with
a median survival of about 10.3 years. Morbidity depends on the
variable expressions of hemolysis, bone marrow failure, and
thrombophilia that define the severity and clinical course of the disease.
A study of the first 1610 patients enrolled in the International PNH
Registrya worldwide, observational, noninterventional studyfound
that overall, 16% of patients had a history of thrombotic events and 14%
a history of impaired renal function. Frequently reported symptoms
included the following [13] :
Fatigue (80%)
Dyspnea (64%)
Hemoglobinuria (62%)
Abdominal pain (44%)
Chest pain (33%)
Patients also reported impairment in quality of life from their disease,
with 17% stating that they were not working or were working less
because of PNH.
In several large studies, the main cause of death in patients with PNH
was venous thrombosis, followed by complications of bone marrow
failure; however, spontaneous long-term remission or leukemic
transformation of the PNH clone has been reported and well
documented.
The median survival after diagnosis was 10 years in a series of 80
consecutive patients seen at the Hammersmith Hospital in London who
were treated with supportive measures, such as oral anticoagulant
therapy after an established thrombosis, and transfusions. [14] Sixty
patients died; of 48 patients whose cause of death was known, 28 died
from venous thrombosis or hemorrhage. Thirty-one individuals (39%)
had one or more episodes of venous thrombosis during their
illness. [14] No leukemic transformations occurred in this series.
Twenty-two of the 80 patients (28%) survived for 25 years. [14] Of the 35
patients who survived for 10 years or more, 12 had spontaneous clinical
recovery at which time no PNH-affected cells were found among the
RBCs or neutrophils during their prolonged remission, but a few PNH-
affected lymphocytes were detectable in 3 of 4 patients tested. [14]
Race
The differences of PNH) among races were shown in a study that
compared 176 American patients seen at Duke University and 209
patients from Japan. [15] White American patients were younger with
significantly more classic symptoms of the disease, including
thrombosis, hemoglobinuria, and infection, whereas Asian patients were
older with more marrow aplasia and a smaller PNH clone. Survival
analysis showed a similar death rate in each group, although the causes
of death were different, with more thrombotic deaths seen in the
American patients. Japanese patients had a longer mean survival time
(32.1 vs 19.4 y), but Kaplan-Meier survival curves were not significantly
different. [15]
Other geographic ethnic differences were observed in the thrombosis
incidence in 64 patients with classic PNH. [16] The investigators found
that African Americans (n = 11) and Latin Americans (n = 8) had a higher
risk or rate of thrombosis by Cox regression analysis and that this had
an impact on length of survival compared with other patients (n = 45).
Sex- and Age-related Demographics
Men and women are affected equally with PNH, and no familial
tendencies exist.
PNH may occur at any age, from children (10%) as young as 2 years to
adults as old as 83 years, but it is frequently found in adults, with a
median age at the time of diagnosis of 42 years (range, 16-75 y) from a
series in England of 80 consecutive patients.[14] In childhood through
adolescence, patients with PNH presented with more of the primary
features of aplastic anemia than the healthy adult population. Other
complications, such as infections and thrombosis, occurred with equal
frequency in all age groups.
History
The classic manifestation of paroxysmal nocturnal hemoglobinuria
(PNH) is dark urine during the night with partial clearing during the day
(see image below); however, hemoglobinuria may occur every day in
severe cases; more frequently, in episodes lasting 3-10 days; or, in
some cases, not at all.
 This series of containers
holds urine of a patient with paroxysmal nocturnal hemoglobinuria,
showing the episodic nature of the dark urine (hemoglobinuria) during
intravascular hemolysis, usually occurring at night. Early morning urine
is cola-colored. This may occur at different times of the day and vary
from patient to patient. Permission to use this image has been granted
by the American Society of Hematology Slide Bank, 3rd edition.
View Media Gallery
A working classification has been developed for PNH that includes all
the variations in the presentation, clinical manifestations, and natural
history among PNH patients. PNH can present as any of the following
three syndromes or sets of symptoms:
Classic PNH
PNH in the setting of another specified bone marrow disorder (eg,
PNH/aplastic anemia or PNH/refractory anemia- myelodysplastic
syndrome [MDS])
Subclinical PNH (PNH-sc)
Hemolytic anemia is usually in the form of intravascular hemolysis. The
most common presentation is the presence of anemia associated with
dark cola-colored urine that is a manifestation of hemoglobinuria. The
latter may be confused with hematuria, and erroneous treatment could
be given for urosepsis.
Hemosiderin is nearly always present in the urine sediment and can
accumulate in the kidneys, which shows up on magnetic resonance
images (MRI) or computed tomography (CT) scans. Elevated
reticulocyte count and serum lactate dehydrogenase (LDH) level with a
low serum haptoglobin level in the absence of hepatosplenomegaly are
the hallmarks of intravascular hemolysis. The bone marrow is usually
markedly erythroid, with decreased or absent iron stores, depending on
how long the patient has been losing iron in the urine.
Thrombosis involves the venous system, and it typically occurs in
unusual veins, namely the hepatic, abdominal, cerebral, and subdermal
veins. The tendency of patients with PNH to suffer thrombosis has been
recognized as a major part of the syndrome and interpreted as a very
bad prognostic sign and the most common cause of death in PNH.
About 30-40% of patients of European origin have serious thrombosis at
some time; for unexplained reasons, only 5-10% of patients of East
Asian (Chinese, Japanese, and Thai) or Mexican origin develop this
complication. [17, 18]
The reason for this propensity is not entirely clear. Intravascular
hemolysis may provide altered membrane surfaces upon which
coagulation may be initiated. More likely, it is the effect of the activation
of complement on platelets and perhaps endothelial cells. In platelets,
the deposition of C9 complexes on the surface stimulates their removal
by vesiculation; these vesicles are very thrombogenic. Because PNH
platelets lack the mechanism for down-regulating C9 deposition (ie,
CD59), even a minimal stimulus from activated complement results in a
greatly increased production of these vesicles.[19]
Hepatic vein thrombosis results in Budd-Chiari syndrome, which
manifests as a sudden and catastrophic event characterized by
jaundice, abdominal pain, a rapidly enlarging liver, and accumulation of
ascitic fluid. This syndrome may be severe and lead to vascular collapse
and death, or it can be slow and insidious, leading to hepatic failure.
Abdominal vein thrombosis presents with upper abdominal pain, or it
can occur anywhere in the abdomen, lasting 1-6 days. It can lead to
bowel infarction in severe cases.
Cerebral vein thrombosis can range from the mildest form to a severe
headache, depending on which veins are involved. The sagittal vein is
commonly affected, which can give rise to papilledema and
pseudotumorcerebri.
Dermal vein thrombosis manifests as raised, painful, red nodules in the
skin affecting large areas, such as the entire back, which subsides
within a few weeks, usually without necrosis. In cases that do result in
necrosis, skin grafting may be necessary.
Deficient hematopoiesis usually presents withanemia despite the
presence of an erythroid marrow with suboptimal reticulocytosis. In
some cases, neutropenia and thrombocytopenia can occur in a
hypoplastic bone marrow similar to aplastic anemia (aplastic episodes).
Other symptoms of PNH include esophageal spasms that occur in the
morning and, like the dark-colored urine, clear up later in the day. In
males, erectile dysfunction can occur concomitant with hemoglobinuria,
the cause of which is unknown.
Physical
Most commonly, in patients with paroxysmal nocturnal hemoglobinuria
(PNH), pallor suggests anemia; fever suggests infections; and bleeding,
such as mucosal bleeding or skin ecchymoses, suggests
thrombocytopenia similar to that in aplastic anemia. Other physical
examination findings may include the following:
Hepatomegaly and ascites in the presence of Budd-Chiari
syndrome
Splenomegaly in the presence of splenic vein thrombosis \
Absent bowel sounds in the presence of bowel necrosis
Papilledema in the presence of cerebral vein thrombosis
Skin nodules that are red and painful in the presence of dermal
vein thrombosis
Diagnostic Considerations
Patients should be tested at least once for paroxysmal nocturnal hemoglobinuria
(PNH) if they have any of the following [3] :
Evidence of hemoglobinuria
Unexplained hemolysis (increased lactate dehydrogenase [LDH] level,
absent haptoglobin)
Abdominal or cerebral vein thrombosis
Thrombocytopenia, macrocytosis, or signs of hemolysis
Patients who should be tested repeatedly for PNH (at least every 6 mo or
annually) include the following:
All patients with known PNH, to monitor for clonal changes
All patients with aplastic anemia (including post bone marrow
transplantation if cytopenias recur)
Patients with myelodysplastic syndrome(MDS), although this remains
controversial
A review of 282 Korean patients with PNH by Kim et al identified 24 patients with
both PNH and severe aplastic anemia (ie, with at least two of the following three
criteria: hemoglobin 8 g/dL; absolute neutrophil count (ANC) <0.5 10 9/L;
platelet count <20109/L). Compared with patients with classic PNH, the
patients with PNH and severe aplastic anemia had a significantly lower median
granulocyte PNH clone size (26.7 vs. 51.0%, P = 0.021), a lower incidence of
lactate dehydrogenase levels 1.5 times the upper limit of normal (52.9 vs.
80.0%, P = 0.049), and significantly lower survival. Given those differences,
these authors suggest that identifying this subgroup at the time of diagnosis may
be important. [20]
Differential Diagnoses
Hemolytic Anemia
Mesenteric Artery Ischemia
Mesenteric Artery Thrombosis
Paroxysmal Cold Hemoglobinuria
Portal Vein Obstruction
Renal Vein Thrombosis Imaging
Approach Considerations
In addition to a complete blood cell count, the principal studies used to
establish the diagnosis of paroxysmal nocturnal hemoglobinuria (PNH)
are flow cytometry of peripheral blood and bone marrow analysis. Flow
cytometry measures the percentage of cells that are deficient in the
glycosyl phosphatidylinositolanchored proteins (GPI-APs) and
identifies discrete populations with different degrees of deficiency.
Because of the missing GPI-APs, red blood cells (RBCs) and other cells
in patients with PNH lack DAF (CD55) and MIRL (CD59), which regulate
complement.
Bone marrow examination will differentiate classic PNH from PNH that
develops in the setting of other bone marrow disorders. In addition,
bone marrow examination will identify an erythroid and hyperplastic
bone marrow during the hemolytic phase or a hypoplastic bone marrow
in the aplastic phase.
Imaging studies are indicated in patients with venous thrombosis.
Laboratory Studies
The tests involved in establishing the diagnosis of paroxysmal nocturnal
hemoglobinuria (PNH) demonstrate the presence of red blood cells
(RBCs) that are exceptionally sensitive to the hemolytic action of
complement. These tests include the following:
Flow cytometry
Acidified serum lysis and Ham test
Complement lysis sensitivity test
Sugar water or sucrose lysis test
Most laboratories no longer perform the Ham test or the sugar water
test.
Flow cytometry
The state-of-the-art laboratory test is flow cytometry of the patient's
blood to detect CD59 (MIRL), a glycoprotein, and CD55 (DAF) in
regulation of complement action. Absence or reduced expression of
both CD59 and CD55 on RBCs is diagnostic of PNH.
The use of flow cytometry in PNH differs from many applications in that
the diagnosis depends upon demonstrating the absence of relevant
antigens. In this context, it is important that at least two GPI-linked
antigens are studied to exclude rare congenital deficiencies of single
antigens (CD55 and CD59) and polymorphism with individual antigens
(CD16), which render them undetectable by some monoclonal antibody
clones.
Standard and high-sensitivity flow cytometric procedures for detecting
PNH cells are now available. [21] For routine analysis and diagnosis of
suspected PNH, the standard test is sufficient. This test can detect 1%
or more PNH cells, bu; most laboratories report only 10% or more as a
positive result. High-sensitivity analysis (in which as little as 0.01% PNH
cells can be detected) may be helpful in aplastic anemia patients, who
may eventually develop PNH, and possibly in those with hypoplastic
myelodysplasia syndrome (MDS), to predict responses to
immunosuppressive therapy.
Fluorescent aerolysin
A more accurate alternative reagent for PNH screening and PNH clone
measuresment is the bacterial toxin aerolysin, which binds to RBCs via
GPI anchor and initiates hemolysis. A modified, nonhemolytic form of a
fluorescently labeled molecule has been developed that can detect PNH
cells to a level of 0.5% (fluorescently labeled inactive toxin aerolysin
[FLAER] binding of peripheral blood granulocytes). The advantage of
this assay is that it can detect the clone in all hematopoietic cell
lineages in one assay.
This is the most specific test for PNH, as FLAER binds the GPI anchor
specifically. So the lack of FLAER binding to granulocytes (measured by
flow cytometry) is sufficient for the diagnosis of PNH. The disadvantage
of the test is in measuring binding in the absence of adequate
granulocytessuch as in severe aplastic anemia, when the number of
circulating granulocytes is extremely low.
Immmunotyping
Peripheral blood is the most suitable specimen for immunophenotyping
for PNH, and it is important to screen both RBCs and granulocytes,
because RBC transfusions are common among these patients and
granulocytes may not be present in severe hypoplastic anemia patients.
Studies have shown that the size of the PNH clone correlates with the
risk for venous thrombosis. Patients with less than 50% PNH
granulocytes seldom develop thrombosis, whereas patients with larger
clone sizes appear to be at great risk and will require anticoagulation.
Acidified serum lysis and Ham test
If performed properly, acidified serum lysis and the Ham test (from
Thomas Hale Ham) are reliable ways to diagnose PNH (see image
below). Dr. Ham demonstrated that the RBCs in PNH were lysed by
complement when normal serum was acidified or activated by
alloantibodies.
 The Ham test (acidified
serum lysis) establishes the diagnosis of paroxysmal nocturnal
hemoglobinuria (PNH), demonstrating a characteristic abnormality of
PNH red blood cells by acidified fresh normal serum. Here is a PNH
patient's (Pt) red blood cells lysed by normal serum at room temperature
(RT) and at 37C compared with normal red cells (no hemolysis)
(control [C]). Heated serum at 56C inactivates complement and
prevents hemolysis in PNH cells. Permission to use this image has
been granted by the American Society of Hematology Slide Bank, 3rd
edition.
View Media Gallery
The serum pH is lowered to about 6.2 and the magnesium level is
adjusted to 0.005 mol/L to achieve maximum sensitivity. The cells that
are hemolyzed are the sensitive cells, and those that remain intact are
normal cells, indicating 2-3 subpopulations of RBCs in the circulation.
A false-positive test result is seen in congenital dyserythropoietic
anemia, type II (hereditary erythroblastic multinuclearity with positive
acidified serum tests [HEMPAS]). These patients have a negative
sucrose hemolysis ("sugar water test") result. Some normal serum can
give a false-negative Ham test result; thus, the sucrose water test is
more sensitive but less specific for paroxysmal nocturnal
hemoglobinuria (PNH).
Complement lysis sensitivity test
The complement lysis sensitivity test of Rosse and Dacie is a more
precise method for diagnosing PNH. RBCs are sensitized with a potent
lytic anti-I antigen and hemolyzed with limiting amounts of normal serum
as a source of complement. [22, 23, 24]This demonstrates threee groups of
RBCs in patients with PNH, including the following:
PNH I cells are normal in sensitivity to complement
PNH II cells are moderately more sensitive to complement than
normal cells
PNH III cells are markedly sensitive to complement, requiring one
fifteenth to one twentieth of the amount of complement for an equal
degree of lysis; this group of cells is increased in patients with more
severe PNH, and it is associated with a mean life span of 10-15
days
Sugar water or sucrose lysis test
The sugar water or sucrose lysis test uses the ionic strength of serum
that is reduced by adding an iso-osmotic solution of sucrose, which then
activates the classic complement pathway, and complement-sensitive
cells are lysed. This test is less specific but more sensitive for PNH than
the Ham test, because some RBCs hemolyze from autoimmune
hemolytic anemias, leukemia, and aplastic anemia to a minor degree.
Although the tests are inexpensive and simple to perform, they are more
labor intensive and less sensitive due to the short half-life of circulating
PNH RBCs.
Other tests for intravascular hemolysis
Other tests to demonstrate intravascular hemolysis include the
following:
Elevated serum lactate dehydrogenase (LDH)
Elevated reticulocyte count
Low-to-absent serum haptoglobin
Hemoglobinuria and hemosiderinuria The presence of
hemolysis may be intermittent and can be missed easily, depending
on when the tests are performed
Imaging Studies
Thromboses of major veins are best evaluated by radiographic means.
Investigate hepatic vein thrombosis with a routine technetium-99m
(99m Tc) colloid scan of the liver and spleen. This study often reveals
diminished function in all portions of the liver except the caudate lobe,
which is spared because it is drained by the inferior vena cava rather
than the hepatic vein. A magnetic resonance imaging (MRI) study or
ultrasonogram can demonstrate the cessation of flow through the
hepatic vein or by injection or use of a dye to demonstrate a thrombus in
the vein.
MRI with contrast may demonstrate sagittal vein thrombosis.
Other Tests
PIG-A gene mutation analysis is still limited to research laboratories
and, although very specific, is still not diagnostic for paroxysmal
nocturnal hemoglobinuria (PNH).
Medical Care
According to current understanding of paroxysmal nocturnal
hemoglobinuria (PNH), the ideal treatment is to replace the defective
hematopoietic stem cell with a normal equivalent by stem cell
transplantation; however, this is not realistic for many patients, because
stem cell transplantation requires a histocompatible donor and is
associated with significant morbidity and mortality. This form of
treatment is reserved for severe cases of PNH with aplastic anemia or
transformation to leukemia, both of which are life-threatening
complications.
In 2007, eculizumab (Soliris), an anti-complement antibody targeting the
CD5 complement component, was approved by the US Food and Drug
Administration (FDA). Eculizumab alleviates the hemolysis associated
with PNH and its sequelae, dramatically improving symptoms, improving
quality of life, and eliminating complications of PNH. [8] Eculizumab does
not alter the underlying defect of the disease, however; thus, treatment
needs to continue life-long or until spontaneous remission, which
occurred only in a minority of patients (12 of 80 patients in one study [14] )
before the advent of eculizumab.
Treatment of bone marrow hypoplasia
Bone marrow hypoplasia is a serious cause of morbidity and mortality. It
is treated most effectively with bone marrow transplantation; however, if
there is no suitable donor available, antithymocyte globulin (ATG) has
been used in the treatment of aplastic anemia with considerable
success.
Anemia
The anemia of PNH may have three components: intravascular
hemolysis, inadequate erythropoiesis, and superimposed iron
deficiency(massive iron loss through hemoglobinuria). In view of
increased rate of erythropoiesis, give 5 mg/d of folic acid orally. Assess
iron stores with the use of the transferrin saturation index (TSI) and give
oral ferrous sulfate if the result is <20%. (Ferritin levels should not be
used for this purpose, as ferritin is an acute-phase reactant and levels
can be misleading.)
Determine steady-state hemoglobin levels after correction for iron
deficiency. When appropriate, transfuse packed red blood cells (RBCs)
with leukocytes depleted by filter. Washing RBCs is no longer
necessary, and use of irradiated blood products is recommended for
future stem cell transplantation.
Supportive care for severe anemia includes blood transfusion using
leuko-depleted packed RBCs to prevent alloimmunization. Development
of alloantibodies can be a problem with future transfusions because of
activation of complement and delayed hemolysis of transfused blood.
Iron
Replacement of nutritional iron, because of increased loss of iron from
the hemolysis and the 200-fold increase in iron urinary excretion, is
necessary to prevent development of iron deficiency. Iron replacement
can stimulate reticulocytosis that can trigger hemolysis by releasing a
new cohort of complement-sensitive cells. This process can be
prevented by adding prednisone during replacement therapy.
Stimulation of erythropoiesis using androgenic hormones has been
successful in patients with a moderate decrease in RBC production.
This has been replaced mainly by using recombinant erythropoietin
therapy.
Thromboembolism
Management of thrombotic complications follows standard principles,
including using heparin emergently, then maintenance therapy with the
use of an oral anticoagulant, such as warfarin. Sometimes, heparin can
exacerbate the thrombotic problem, possibly by activating complement.
This can be prevented using inhibitors of the cyclooxygenase system,
such as aspirin, ibuprofen, and sulfinpyrazone.
Primary prophylaxis of thromboembolism for patients with PNH has
been advocated. Whether this approach is safe and effective in all
patients with PNH remains controversial, however.
Corticosteroids
Modulation of complement is controlled poorly by high doses of
glucocorticoids. The usual adult dose of prednisone is 20-40 mg/d (0.3-
0.6 mg/kg/d) given daily during hemolysis and changed to alternate
days during remission. On this regimen, about 70% of adult patients
experience improvement in hemoglobin levels, but long-term therapy is
fraught with complications.
Eculizumab
The anticomplement agent eculizumab is a humanized monoclonal
antibody against terminal protein C5; it has been shown to be highly
effective in reducing intravascular hemolysis. [25,26] Eleven transfusion-
dependent patients with PNH were given intravenous (IV) eculizumab at
900 mg over 30 minutes every 2 weeks. The mean lactate
dehydrogenase (LDH) level decreased, transfusion requirements fell
from 2.1 units per patient per month to 0.0, and a global improvement in
quality of life occurred. [26] Long-term analysis showed that these
improvements can be maintained over 3 years, and erythropoietin can
overcome anemia due to bone marrow failure in patients on the drug.
The 5-year survival of patients with PNH prior to eculizumab therapy in
a cohort followed at Leeds Hospital in the United Kingdom was 66.8%.
With eculizumab therapy, 5-year survival improved to 95.5%, which is
not statistically different from age-matched controls in the general
population.[27]
Treatment breakthrough from complement control can occur in small
minority (10%) of patients due to an inadequate dosing schedule. The
eculizumab level must remain above 35 g/mL, but trough levels at 2
weeks may fall below this level and cause recurrence of hemolysis.
The recommended adjustment for patients whose eculizumab levels fall
into this category is to increase the dose to 900 mg every 12 days or
1200 mg every 2 weeks. Withdrawal hemolysis can occur by stopping
therapy for any reason, as accumulation of PNH RBC increases over
time by protecting type II and III PNH cells from destruction due to
therapy, which can potentially trigger a massive hemolysis.
Infection prophylaxis
Consequences of complement inhibition include an increased risk of
infections from Neisseria meningitides, as seen in inherited terminal
complement deficiency. [8] Before the administration of eculizumab, all
patients should be vaccinated with a serogroup B meningococcal
vaccine. [28]
Despite vaccination, patients may develop meningococcal septicemia
(not meningitis). Although this is rare, occurring at a rate of 0.5 cases
per 100-patient years, prophylactic antibiotics are recommended to
prevent this complication. One study used penicillin V, 500 mg twice
daily orally, or erythromycin 500 mg twice daily for patients intolerant to
penicillin. [29]
Effect on thromboembolic complications
Eculizumab treatment reduces the risk of clinical thromboembolism in
patients with PNH (the leading cause of death in PNH) and is
recommended for PNH patients with a history of prior
thromboembolism. [30] The rate of thrombotic complications prior to
eculizumab was 5.6 per 100 patient years; after eculizumab, it dropped
to 0.8 per 100 patient years.
In an international multi-institutional cooperative study involving 195
PNH patients, the thromboembolic (TE) event rate per 100 patient-years
with eculizumab treatment was 1.07, compared with 7.37 events
(P <0.001) prior to eculizumab treatment, a relative absolute reduction
of 85%. With equalization of duration of exposure before and during
treatment for each patient, TE events were reduced from 39 before
eculizumab to 3 during eculizumab (P<0.001). The TE event rate in
antithrombotic-treated patients (n = 103) was reduced from 10.61 to
0.62 events/100 patient-years with eculizumab treatment (P <0.001).
One study has documented elevated D-dimer levels in PNH patients
with a history of thrombosis. D-dimer levels decreased immediately after
initiation of eculizumab therapy. [31]
Continuation of anticoagulation in patients with PNH with a previous
thrombosis while on eculizumab is recommended, as stopping therapy
has not been studied. However, patients with no previous thrombosis
have discontinued warfarin after starting eculizumab, with no thrombotic
sequelae. [29, 32]
Eculizumab and renal dysfunction
Chronic hemosiderosis and/or microvascular thrombosis from PNH
causes kidney dysfunction at an incidence of 65% for renal dysfunction
or damage, defined by stages of chronic kidney disease (CKD), in a
large cohort of PNH patients. Eculizumab treatment was safe and well-
tolerated in patients with renal dysfunction or damage and resulted in
the likelihood of improvement as defined as categorical reduction in
CKD stage (P <0.001) compared with baseline and placebo (P = 0.04).
Improvement in renal function was more commonly seen in those with
less severe impairment. Improvements occurred quickly and were
sustained for at least 18 months of treatment. Administration of
eculizumab to patients with renal dysfunction or damage was well
tolerated and was usually associated with clinical improvement. [33]
Iron
Monitoring iron even if the patients no longer require transfusions is
recommended, because hemosiderinuria no longer occurs with
eculizumab, which is a protective mechanism in PNH to excrete iron.
Measuring serum ferritin is recommended and chelation therapy may be
necessary in patients with high levels.
Hematopoietic stem cell transplantation
Hematopoietic stem cell transplantation (HSCT) using allogeneic donors
is the only curative therapy for PNH. With the advent of eculizumab, the
indications for HSCT have changed. Clinical results from HSCT from
various programs in a rare disease are limited to small numbers of
patients. A retrospective analysis of the Italian BM transplantation group
in 26 patients with a median age of 32 years (22-60 y, range) with 23
HLA-identical donors (22 siblings, one unrelated) shows a transplant-
related mortality of 42%, 8% graft failure, and a 10-year survival
(disease-free) of 57% for all patients. [34] The mortality rate remains high,
so this form of therapy is reserved for those who are severely
hypoplastic and refractory to other forms of therapy.
The International Bone Marrow Transplant Registry (IBMTR) reported a
2-year survival probability of 56% in 48 recipients of HLA-identical
sibling transplants between 1978 and 1995. [35] Data using
nonmyeloablative conditioning and haploidentical donors was similar to
the identical donors, indicating some form of graft-versus-PNH effects.
Now that an effective, nontransplant therapy is available, the use of
allogeneic HSCT to treat PNH has decreased.
Before the introduction of eculizumab, PNH patients with severe
symptoms from classic PNH and patients with AA/PNH with peripheral
cytopenias meeting criteria for severe aplastic anemia were considered
good candidates for allogeneic bone marrow transplantation, especially
if a matched sibling donor was available.
With eculizumab for PNH, the indications for allogeneic HSCT in this
setting have changed. First, HSCT should not be offered as initial
therapy for most patients with classic PNH, given the high transplant-
related mortality, especially when using unrelated or mismatched
donors. Exceptions are PNH patients in countries where eculizumab is
not available. HSCT is also a reasonable option for patients who do not
have a good response to eculizumab therapy. Second, aplastic
anemia/PNH patients continue to be reasonable candidates for HSCT if
they have life-threatening cytopenias. [36]
Pregnancy
Pregnancy in patients with PNH poses very significant risks. There is a
very high risk of thrombotic complications for the expectant mother, as
well a risk of developing hypoplastic anemia. Maternal mortality in these
patients is approximately 20%, mostly from thrombosis and infections,
and risk of fetal loss is increased. Consequently, full anticoagulation with
low-molecular weight heparin (LMWH) is recommended for pregnant
women with PNH. Warfarin may be substituted after the first trimester.
The use of eculizumab in pregnancy has proved beneficial. [37] In a
review of 75 pregnancies in 61 women with PNH, Kelly and colleagues
reported a high rate of fetal survival and a low rate of maternal
complications. No maternal deaths occurred. There were three fetal
deaths (4%) and six first-trimester miscarriages (8%). During pregnancy,
patients demonstrated increased requirement of red blood cell
transfusions, and approximately half required an increase in the
eculizumab dosage. Ten hemorrhagic events occurred, and two
postpartum thrombotic events. Eculizumab was detected in some
infants' cord blood, but not in breast milk. [38]
Consultations
In centers that do not have a bone marrow transplantation program,
consultation and identification of possible donors should be undertaken
early.
Stem cell transplantation is a curative therapeutic option for paroxysmal
nocturnal hemoglobinuria (PNH). However, the risks of therapy must be
carefully weighed against factors related both to PNH and comorbid
conditions. Furthermore, the heterogeneous presentation of the
disease, its unpredictable course, and its association with bone marrow
failure conditions confound the decision process regarding
transplantation.
An analysis by the International PNH Interest Group reviewed data from
67 patients from single centers and from teo registry studies, with
special emphasis in eliminating duplication in patient reporting. [3] Results
included the following:
Of the seven patients transplanted from a twin syngeneic donor,
the four who had no conditioning therapy either failed to engraft or
relapsed after transplantation, indicating that a marrow ablative
conditioning is necessary before syngeneic transplantation.
In 47 of 67 patients, a human leukocyte antigen (HLA)-identical
sibling was used as the donor, 1 from a haploidentical family
member and 12 from an unrelated donor (matched unrelated donor
[MUD]).
In the only single-center study providing a Kaplan-Meier analysis,
overall survival at 5 years was 58 +/- 13%. This is less favorable
than the survival estimate of approximately 75% generated by
combining the data from the other reports.
Investigation is currently in progress regarding whether reduced-
intensity conditioning can improve the outcome.
Medication Summary
The drugs used in treatment of paroxysmal nocturnal hemoglobinuria
(PNH) include the following:
Eculizumab, to stop hemolysis
Recombinant erythropoietin or androgens, to stimulate
erythropoiesis
Anticoagulants, to treat thrombotic complications
Immunosuppressive agents, to stimulate hematopoiesis in the
aplastic phase
Androgens
Class Summary
Androgens are used to stimulate erythropoiesis by increasing
endogenous levels of erythropoietin and by enhancing the response of
precursor cells to the growth factor.
Attenuated androgens, such as danazol, are recommended for use in
women, as the attenuated androgen has fewer adverse virilizing and
masculinizing effects.
Oxymetholone (Anadrol-50)
View full drug information
Anabolic and androgenic derivative of testosterone in an oral
formulation.
Used to stimulate erythropoiesis by increasing endogenous levels of
erythropoietin and by enhancing the response of precursor cells to the
growth factor.
Stanozolol (Winstrol)
Anabolic and androgenic derivative of testosterone in an oral
formulation.
Danazol (Danocrine)
View full drug information
Synthetic steroid analogue, derived from ethisterone, with strong
antigonadotropic activity (inhibits LH and FSH) and weak androgenic
action without adverse virilizing and masculinizing effects. Increases
levels of C4 component of the complement. May push the resting
hematopoietic stem cells into cycle, making them more responsive to
differentiation by hematopoietic growth factors. May also stimulate
endogenous secretion of erythropoietin.
May impair clearance of immunoglobulin-coated platelets and
decreases autoantibody production.
Immunosuppressive Agents
Class Summary
Antithymocyte globulin is an antiserum to human T cells prepared from
horses or rabbits. [39] The mechanism of action of polyclonal
antilymphocyte preparations to suppress immune responses is not fully
understood.
Antithymocyte globulin; rabbit (Atgam)
View full drug information
Purified preparation of pasteurized polyclonal IgG obtained from rabbits
immunized against human thymocytes (T cells) for IV use. This
preparation has replaced the Upjohn preparation Atgam (horse serum)
and is considered an equivalent.
Complement Inhibitor
Class Summary
The C-5 inhibitor eculizumab has been designated as an orphan drug
for the treatment paroxysmal nocturnal hemoglobinuria (PNH).
Eculizumab (Soliris)
View full drug information
Orphan drug indicated for treatment of paroxysmal nocturnal
hemoglobinuria (PNH) to reduce hemolysis. Blocks complement-
mediated destruction of PNH red blood cells. Inhibits C-5 component of
complement system, thereby preventing final stages of complement
activation.
Dosage Forms & Strengths
injectable solution
10mg/mL
MORE...
Paroxysmal Nocturnal Hemoglobinuria
600 mg IV infusion over 35 minutes q7days for the first 4
weeks, THEN
900 mg (fifth dose) after 7 days, THEN
900 mg q14days thereafater
Further Outpatient Care
In many patients with paroxysmal nocturnal hemoglobinuria (PNH), the abnormal
clone may eventually disappear. This usually takes at least 5 years, and often as
long as 15-20 years. Reactivation of PNH in these patients has been observed
with acute infections. Patients with chronic anemia alone, without thrombotic
complications, can live relatively normal lives for many years.
Further Inpatient Care
In severe cases with of paroxysmal nocturnal hemoglobinuria (PNH) with an
aplastic phase, referral to a bone marrow transplantation center is indicated for
possible allogeneic bone marrow transplantation. Umbilical cord stem cell
transplantation from the patient's own cord blood, from a related donor, or from
the registry for HLAmatched unrelated donors may be an option in pediatric
patients.
Inpatient & Outpatient Medications
Outpatient RBC transfusion often is necessary to keep a patient with chronic
anemia and paroxysmal nocturnal hemoglobinuria (PNH) able to function and live
a normal life.
Complications
Primary care physicians should be aware of the thrombotic complications of
paroxysmal nocturnal hemoglobinuria (PNH) and how to diagnose them when
they occur.
Prognosis
The prognosis in patients with paroxysmal nocturnal hemoglobinuria (PNH) is
variable, depending on the severity of symptoms and the presence of
complications. An aplastic phase is a serious prognostic factor, because the
resulting pancytopenia and thrombosis of hepatic, abdominal, and cerebral veins
can have life-threatening consequences. Prophylactic anticoagulation has not
been shown to be of benefit because of a lack of data from a clinical trial setting.