2. PNH
• Hematopoietic stem cell disorder affecting both sex.PNH is diagnosed mostly
during 4th to 5th decade but also encountered in childhood and old age.The disease
has no familial tendency
• 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
4. 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
5. • 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’ base
•
7. CD 55
• 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
8. 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 s
11. PNH PHENOTYPES
• 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
12.
13. • 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 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
17. LABORATORY INVESTIGATION
• 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
18. • Blood picture –
Anaemia of varying variety (mostly severe),
Polychromasia (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.
19. • 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
20. • Serum Bilirubin – Increased.
• Serum LDH – Increased(Most important surrogate marker for
intravascular hemolysis).
• Iron stores are often reduced.
• Serum haptoglobin – Decreased
21. • 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
22. • 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 (NH4CL)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
23. • 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
24. • 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.
25.
26. DIFFERENTIAL DIAGNOSIS
• •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
27. (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
28. • 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
30. PAROXYSOMAL COLD HEMOGLOBINURIA
• 1st hemolytic anemia to be described.
• Donath-Landsteiner antibody is a hemolysin that binds to RBC at low
temperature and fixes compliment,when RBC returns to warm central
body core they are destroyed by complement lysis.
• PCH Ab are polyclonal IgG directed against RBC P antigen.
• D-L Ab occurs in 3 clinical syndromes
1)C/c PCH in sccociation with latent/cong.syphilis
2)A/c transient PCh after infectious illness
3)C/c idiopathic PCH
