This document provides information on paroxysmal nocturnal hemoglobinuria (PNH). It describes PNH as an acquired hemolytic anemia caused by a somatic mutation in hematopoietic stem cells, rendering red blood cells susceptible to complement-mediated lysis. This results in intravascular hemolysis, thrombosis, and cytopenias. The document discusses the pathogenesis, clinical manifestations, diagnostic testing including flow cytometry, treatment including supportive care and the complement inhibitor eculizumab (Soliris), and risks and outcomes of PNH.

1. PNH
Paroxysmal nocturnal heamoglobinuria

2. PNH
PNH is an acquired chronic hemolytic anemia
which arises from a somatic mutation in a
hematopoietic stem cell.
This defect renders the red cells highly susceptible
to complement mediated lysis resulting in the
characteristic intravascular hemolysis.
In addition to intravascular hemolysis the patient
may
have thrombosis and cytopenias
This triad when present Is highly characteristic of
PNH

3. WHAT IS PNH?
• Paroxysmal – sudden
onset
• Nocturnal – occuring at
night (or early in morning
upon awakeneing)
• Hemoglobinuria
Despite the name, most patients do not present this way.

4. EPIDEMIOLOGY
• Rare disease -
• frequency less than 1 in 100 000
• Median age at diagnosis
• ~ 35 yrs
• PNH reported at extremes of age from 1 to 73
• Female:Male ratio = 1.2:1.0
• No increased risk of PNH in patient relatives
• Median Survival after diagnosis ~ 10-15 yrs

5. WHAT CAUSES PNH?
• The mutation in the PIG-A gene
located on the x-chromosom in a
hematopoietic stem cell leads to a
defect in the production of an anchor
protein that ties other proteins to the
cell surface.
• The lack of the GPI anchor protein
leads to a lack of many proteins on
the surface of affected blood cells.
• In PNH, the major two proteins lacking on
the surface of the red cells are CD59 and
CD55.
• These proteins are important in protecting the
red cells from complement.
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6. PATHOGENESIS - FUNCTIONAL CONSEQUENCES OF LACK
OF GPI LINKED PROTEINS
• CD55 and CD59 functions are well known
• CD55 (decay accelerating factor) inhibits the formation or
destabilizes complement C3 convertase (C4bC2a)
• CD59 (membrane inhibitor of reactive lysis) Protects the
membrane from attack by the C5-C9 complex
• Inherited absences of both proteins in humans have been
described
• Most inherited deficiencies of CD55 - no distinct clinical hemolytic
syndrome
• Inherited absence of CD59 - produces a clinical disease similar to
PNH with hemolysis and recurrent thrombotic events

7. WHAT CAUSES PNH?
HOW DO CELLS WITH A MUTATION TAKE OVER THE
BONE MARROW?
• Normal people may carry cells in their bone marrow with a PIG-
A mutation, usually at a very low level .
• In PNH, something allows the abnormal cells to become the
dominant cells and become the major population in the marrow.
• This “something” may be related to aplastic anemia,
• The cause of aplastic anemia (immune assault?)
may play a role in the development of PNH.

8. THE CLINICAL PICTURE OF PNH
• Hemolysis due to complement activation
• fatigue,headache , exhaustion due to anaemia
• Hemoglobinuria, kidney damage
• Nitric oxide trapping >> Esophageal spasm, abdominal pain, impotence,
fatigue?
• Thrombosis –
• Unusual sites of blood clots
• Bone marrow failure
• Decreased blood counts (cytopenias)

9. Chronic intravascular hemolysis
Chronic intravascular haemolysis in PNH is
probably explained by activation of Complement through the
‘alternative’ pathway, which goes on at a low rate all the time,
whereas the dramatic exacerbation of haemolysis that is
characteristically associated with intercurrent viral or bacterial
infection in patients with PNH is probably explained by brisk
activation of Complement by an antigen–antibody reaction (i.e.
through the ‘classical

10. TERMINAL COMPLEMENT ACTIVATION
RENDERS RBCS SUSCEPTIBLE TO LYSIS
Normal RBCs PNH RBC
Intact RBC
Complement
Activation
Lysed PNH RBCs and free hemoglobin in the plasma
Chronic Hemolysis
CD59

11. WHAT HAPPENS WHEN RED CELLS LYSE?
 The red cells are destroyed - anemia
 Hemoglobin is released into the plasma
 Some of the hemoglobin passes through the kidneys
and into the urine leading to the dark color of the urine
 Loss of iron
 May lead to kidney damage in the long run
 Free hemoglobin binds nitric oxide
 What is nitric oxide?

12. WHAT IS NITRIC OXIDE?
• A gas produced by the body to regulate smooth muscle cells.
• An increase in free nitric oxide causes smooth muscle cells to
relax. A decrease causes smooth cells to contract.
• Smooth muscle cells are in many tissue
• Blood vessel walls: ischemia, impotence
• Esophagus and GI tract: esophageal spasm, reflux, abdominal
pain

13. WHAT ABOUT THROMBOSIS (BLOOD CLOTS) IN
PNH?
• Blood clots are a presenting sign in 10-20% of
patients with PNH.
• Can occur in up to 40% of patients with PNH.
• Occur in unusual locations – veins of the liver
(Budd-Chiari syndrome), spleen, brain, and
skin.
• Associated with a very bad prognosis
Mesenteric / splenic
18%
Hepatic / portal
16%
PE
7%
Cerebral
6%
Superficial
4%
Arterial / CVA
14%
Arterial / MI
2% DVT: leg
18%
DVT: other
15%

14. Pathogenesis of thrombosis :
It could be due to
1.impaired fibrinolysis
2.hypercoagulability or hyperactivity of platelets
3. deficiency of CD59 on PNH platelet could lead to abnormal insertion of the
C5b–9 complex in the platelet membrane.
Although all of these three factors may play a role in producing a
thrombophilic state in PNH, it seems very likely that the primary cause
lies in the PNH platelets, which are abnormal precisely because they belong to
the PNH clone.

15. CLINICAL FEATURES - LONG TERM
• 25% of PNH patients survive >25 years - one half of these go on to spontaneous
remission
• Remission patients
• hematological values revert to normal
• no PHN rbcs or granulocytes detected
• PNH lymphocytes - still detected but no clinical consequence
• Higher incidence of acute leukemia (6%)
• “preleukemic condition”

16. CLINICAL FEATURES - RELATIONSHIP TO
APLASTIC ANEMIA (AA)
• AA described as pancytopenia with nonfunctioning bone marrow. Cytopenia in one or all
cell lineages also common to PNH
• High percentage of patients with AA develop clinical PNH or have lab evidence of PNH
abnormality at some point (52%)
• Supports the theory that bone marrow failure supports the abnormal PNH cells - more
later

17. WHEN PNH SHOULD BE SUSPECTED ?
1. Haemoglobinuria due to intravascular haemolysis
2. Haemolytic anaemia with a low platelet and/or low
neutrophil count
3. Haemolytic anaemia and recurrent abdominal pain with or
without blood in the stool
4. Hepatic vein thrombosis (Budd–Chiari syndrome)
5. Any venous thrombosis anywhere in young adult with low
platelet and/or the neutrophil counts
6. Idiopathic pancytopenia (IAA)

18. LABORATORY FINDINGS
• anaemia may be normocytic, but it is often macrocytic on account of an
absolute reticulocytosis, which may be quite high.
• If (MCV) is normal rather than high, there is probably
iron deficiency.
• morphology is not characteristic.
• Neutrophils may range from normal to below 1.0 × 109/L.
• The platelet may range from normal to below 20 × 109/L.
• The lymphocytes are usually normal.but there may be lymphopenia

19. LABORATORY FINDINGS
• The bilirubin level is usually only moderately increased in striking contrast to:
• (LDH) which is often in the thousands (U/L).
• Haptoglobin is markedly decreased.
• The serum iron and transferrin saturation may be decreased, indicating
secondary iron deficiency.
• Hemoglobinurea and hemosiderinurea
• The bone marrow aspirate will usually yield a cellular
marrow with erythroid hyperplasia..
• There may be MDS-like changes in one or more
cell lineages.

20. LABORATORY DIAGNOSIS OF PNH
Ham’s test
Acidified serum activates alternative complement pathway
resulting in lysis of patient’s rbcs still in use today and it is
highly reliable
• The Ham’s test gives no false-negative results;
Ham test can give rise to false-positive results only in
 congenital dyserythropoietic anaemia type II (but not
if the patient’s serum is used).
Autoimmune hemolytic anemia when patient’s red
cells are already coated with a lytic antibody.

21. FLOW CYTOMETRY
• Antigen expression is generally categorized into three antigen
density groups(using monoclonal antibodies to CD59 and CD55 )
• type I Normal Ag expression
• type II Intermediate Ag expression
• type III No Ag expression
• Patient samples that demonstrate cell populations with diminished or
absent GPI-linked proteins (Type II or III cells) with multiple
antibodies are considered to be consistent with PNH.
Should examine multiple lineages (ie red cells and granulocytes)
appreciable haemolytic anaemia never been detected in a patient in
whom less
than 5% red cells are of the PNH type.

22. TREATMENT OF PNH
• Supportive (hemolysis,thrombosis)
• Curative (bone marrow transplantation)

23. PNH – WHO NEEDS TO BE TREATED?
• Patients with blood clots
• Patients with symptomatic anemia from hemolysis
• Patients with severe bone marrow failure

24. HOW DO WE TREAT PNH -HEMOLYSIS
Transfusion
Iron, folic acid
Steroids
Eculizumab (Soliris)

25. WHAT DOES SOLIRIS DO?
• Quickly and effectively blocks complement activation at C5.
• Blocks hemolysis and related effects
• Stops hemoglobinuria
• Markedly reduces transfusion requirements
• Hemoglobin / hematocrit may not return to “normal”
• Reduces thrombotic events

26. WHAT SOLIRIS DOES NOT DO
• Probably does not help bone marrow failure (improve other low blood counts)
• Completely correct anemia

27. DOWNSIDE OF SOLIRIS TREATMENT
• Increased risk of meningococcal infections
• All patients must be vaccinated
• All patients educated on signs and symptoms of meningitis and what to do
• All patients given cards describing this
• Costy