Paroxysmal nocturnal hematuria

Paroxysmal Nocturnal
Hemoglobinuria
Aseem Jain
Department of Pathology

PNH
• Hematopoietic stem cell disorder.
• PNH arises as a result of nonmalignant clonal expansion of
one or several hematopoietic stem cells that have acquired
a somatic mutation of the X-chromosome gene PIGA
(phosphatidylinositol glycan class A).
• As a consequence of mutant PIGA, progeny of affected
stem cells (erythrocytes, granulocytes, monocytes,
platelets, and lymphocytes) are deficient in all glycosyl
phosphatidylinositol-anchored proteins (GPI-APs) that are
normally expressed on hematopoietic cells

Erythrocyte membrane protein deficiencies

CD55
• Also known as decay accelerating factor.
• First identified by Hoffmann in 1969.
• 70-kD protein,
• Inhibits the formation and stability of the C3 convertases
of complement.

CD59
• Also known as membrane inhibitor of reactive lysis
(MIRL), protectin, homologous restriction factor, and
membrane attack complex inhibitory factor.
• CD59 inhibits the formation of membrane attack
complex.
• Of the two complement regulatory proteins, MIRL is
more important than DAF in protecting cells from
complement-mediated lysis in vivo.

• The mutant gene, called PIGA (Xp22.1), is required for the
synthesis of a specific type of transmembranous glycolipid
anchor, phosphatidylinositol glycan (PIG).
• Without the membrane anchor, these "PIG-tailed" proteins
cannot be expressed on the surface of cells.
• The affected proteins include several that limit the spontaneous
activation of complement on the surface of cells.
• As a result, PIG-deficient precursors give rise to red cells that
are inordinately sensitive to the lytic activity of complement.
• Studies have shown that the half-life of complement-sensitive
PNH cells is only 6 days (compared to 60 days for normal
erythrocytes).

• A defining feature of PNH is ‘Phenotypic Mosaicism’ based
on sensitivity of the erythrocytes to complement-mediated
lysis.
• This remarkable characteristic was first clearly elucidated
by Rosse and Dacie in 1966 and Rosse further refined the
analysis in 1973.
• Using an in vitro test that quantitates the sensitivity of
erythrocytes to complement-mediated lysis (the
complement lysis sensitivity assay), three phenotypes of
PNH erythrocytes were identified.

•Most common phenotype – PNH I and PNH III cells.
•Least common phenotype – PNH I and PNH II cells.

• The intensity of the hemolytic component of the disease is
related to the size of the PNH III population.
• As a rule, visibly apparent hemoglobinuria is absent or mild
when PNH III erythrocytes constitute <20% of the red cell
population.
• Episodes of gross hemoglobinuria occur when the PNH III
population ranges from 20 to 50% of the population,
• Constant hemoglobinuria is associated with >50% PNH III
erythrocytes.
• PNH II cells, even when present in high proportions, are
associated with minimal or no visible hemoglobinuria.

Clinical features
1. Symptoms of anemia
2. Hemoglobinuria (25% patients)
3. Episodic hemolysis
4. Thromboembolic complications
5. Renal abnormalities
6. Dysphagia
7. Male impotence/Erectile dysfunction
8. Infections

Basic evaluation of PNH
1. CBC
2. Urine examination
3. Reticulocyte count
4. Serum LDH
5. Serum Bilirubin
6. Iron stores
7. Bone marrow aspiration and biopsy
8. Ham test
9. Sucrose hemolysis test
10. Flow cytometry

• Blood picture –
oAnaemia of varying variety (mostly severe),
oPolychromasia (nRBCs)
oLeucopenia (mod) due to reduction in neutrophils ,
oThrombocytopenia (mild).
• Reticulocyte count -
Increased (lesser comparing degree of anaemia).
• The plasma may be golden brown, reflecting the
presence of increased levels of unconjugated bilirubin,
hemoglobin, and methemalbumin.

• Urine –
o When the rate of blood destruction is increased, the
urine contains increased amounts of urobilinogen.
o In addition, intravascular hemolysis leads to depletion of
serum haptoglobin, which results in the continuous
presence of hemoglobin in the glomerular filtrate in the
kidney.
o The cells of the proximal convoluted tubules that
reabsorb much of the hemoglobin become heavily laden
with iron.
o The excretion of this iron in the form of granules gives
rise to hemosiderinuria.

• Serum Bilirubin – Increased.
• Serum LDH – Increased(Most important surrogate
marker for intravascular hemolysis).
• Iron stores are often reduced.
• Serum haptoglobin – Decreased.

• Bone marrow aspirate and biopsy are done to distinguish
PNH from PNH in setting of another bone marrow failure
syndrome.
o Normoblastic hyperplasia is the characteristic finding.
o As many as 50% of the nucleated cells may be
normoblasts, but only occasionally megaloblastic
changes are evident.
o The number of megakaryocytes may be decreased.
o When pancytopenia is evident, hypoplastic marrow may
be observed.

Acidified Serum Test (Ham Test 1939)
o From 1937 to 1939, Ham and Dingle made the seminal
observations that connected the hemolysis to
complement.
o Those studies demonstrated that the abnormal cells are
hemolyzed when incubated in acidified serum and that
the hemolysis is complement dependent.
o The lysis of the defective cells in acidified serum (a
process that activates the alternative pathway of
complement) became the standard technique for the
diagnosis of PNH, and appropriately, the assay is called
the Ham test.

• Sucrose Hemolysis Test (1970)-
o 10% sucrose provides low ionic strength which promotes
complement binding resulting in lysis of patient’s RBCs.
o May be positive in megaloblastic anemia, autoimmune
hemolytic anemia, others.
o Less specific than Ham test.
• While these tests are sensitive and specific when
properly performed and relatively simple in both theory
and practice, their accuracy is strongly operator
dependent.
• This problem is compounded by the fact that the tests
are usually performed on a sporadic basis in most clinical
laboratories because the diagnosis of PNH is entertained
relatively uncommonly.

• Flow cytometry –
o By analyzing expression of GPI-AP on hematopoietic cells
using monoclonal antibodies and flow cytometry, the
abnormal cells can be readily identified.
o The simplest method is to analyze expression of MIRL (CD59)
on erythrocytes.
o Because it is normally present in relatively high density, red
cells with either complete or partial deficiency of MIRL are
easily distinguished from normal.
o Analysis of erythrocyte DAF (CD55) expression is also
informative.

Differential diagnoses
• The diagnosis of PNH must be considered in any patient
who has the following:
(a) signs and symptoms of intravascular hemolysis
(manifested by an abnormally high LDH) of undefined
cause (i.e., Coombs negative) with or without
macroscopic hemoglobinuria often accompanied by iron
deficiency;
b) pancytopenia in association with hemolysis, whether or
not the marrow is cellular,

(c) venous thrombosis affecting unusual sites, especially intra-
abdominal, cerebral or dermal locations usually accompanied
by evidence of hemolysis;
(d) unexplained recurrent bouts of abdominal pain, low
backache, or headache in the presence of chronic hemolysis;
and
(e) Budd-Chiari syndrome

• PNH must be differentiated from
o antibody-mediated hemolytic anemias, especially
paroxysmal cold hemoglobinuria.
o cold agglutinin syndrome,
o and from HEMPAS (hereditary erythroblastic
multinuclearity with a positive acidified serum lysis test
or congenital dyserythropoietic anemia type II).
• Patients with aplastic anemia and the refractory anemia
variant of MDS should undergo screening for PNH-sc at
diagnosis and yearly thereafter.

References
• Williams hematology 8th edition,
• Wintrobe’s clinical hematology 12th edition,
• Robbins 7th edition.

Paroxysmal nocturnal hematuria

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