Paroxysmal NocturnalHemoglobinuria

Olufemi-Aworinde K.J MBBS, FMCPath FWACPSenior Lecturer/Consultant HaematologistBowen University, Iwo.

RED CELL MEMBRANE

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.

COMPLEMENT PATHWAY

MOLECULAR BASIS OF PNH

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.

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

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 PNH1.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 –

Anaemia of varying variety (mostly severe),

Polychromasia (nRBCs)

Leucopenia (mod) due to reduction in neutrophils ,

Thrombocytopenia (mild).

Reticulocyte count -Increased (lesser comparing degree of anaemia).

The plasma may be golden brown, reflecting thepresence of increased levels of unconjugated bilirubin,hemoglobin, and methemalbumin.

Urine –

When the rate of blood destruction is increased, the urine contains increased amounts of urobilinogen.

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.

The cells of the proximal convoluted tubules that reabsorb much of the hemoglobin become heavily laden with iron.

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.

Normoblastic hyperplasia is the characteristic finding. As many as 50% of the nucleated cells may be normoblasts, but only occasionally megaloblastic changes are evident.

The number of megakaryocytes may be decreased.

When pancytopenia is evident, hypoplastic marrow may be observed.

Acidified Serum Test (Ham Test 1939) Acidified Serum Test (Ham Test 1939)

From 1937 to 1939, Ham and Dingle made the seminal observations that connected the hemolysis to complement.

Those studies demonstrated that the abnormal cells are hemolyzed when incubated in acidified serum and that the hemolysis is complement dependent.

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 10% sucrose provides low ionic strength which

promotes complement binding resulting in lysis of patient’s RBCs.

May be positive in megaloblastic anemia, autoimmune hemolytic anemia, others. 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 By analyzing expression of GPI-AP on hematopoietic

cells using monoclonal antibodies and flow cytometry, the abnormal cells can be readily identified.

The simplest method is to analyze expression of MIRL (CD59) on erythrocytes. Because it is normally present in relatively high density, red cells with either complete or partial deficiency of MIRL are easily distinguished from normal.

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 intraabdominal, 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 antibody-mediated hemolytic anemias, especially paroxysmal cold hemoglobinuria. cold agglutinin syndrome, 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

Treatment of PNH today still mainly symptomatic

Blood transfusions used during periods of severe hemolysis

Bone marrow transplantation only available curative therapy Risky because Matching transplants not easily available

Medication Anticoagulation therapy indicated during venous

thrombotic events

Immunosuppressive chemotherapy

– When pancytopenia present

– Stimulation of hematopoiesis in aplastic phase

High doses of corticosteroids considered

beneficial

Androgens stimulate erythropoiesis

Complement inhibitor

– On March 16, 2007, FDA approved Soliris(eculizumab) for treatment of PNH

– Results of studies showed treatment with eculizumab produced dramatic reduction in hemolysis, and days of hemoglobinuria each month decreased