PNH is an acquired chronic hemolytic anemia characterized by persistent intravascular hemolysis subject to recurrent exacerbations, in association with pancytopenia, and a distinct tendency to venous thrombosis. It occurs due to a mutation of the PIGA gene.

1. PAROXYSMAL NOCTURNAL
HEMOGLOBINURIA
Dr. Ajay Kumar Yadav
PGY3, Internal Medicine
2019/01/10
IOM-TUTH, Kathmndu

2. LAYOUT
• Introduction
• History
• Etio-pathogenesis
• Clinical presentation
• Diagnosis
• Management
• Newer therapies in pipeline

3. Introduction
• Acquired chronic hemolytic anemia characterized by persistent intravascular hemolysis
subject to recurrent exacerbations, in association with pancytopenia and a distinct
tendency to venous thrombosis
• Nonmalignant clonal expansion of one or several hematopoietic stem cells that have
acquired a somatic mutation of PIGA gene.
• PIGA gene
• Located on the X chromosome
• Encodes an enzyme that is an essential component of the complex biosynthetic
pathway that generates GPI
• The GPI moiety serves as a membrane anchor for more than 20 proteins of diverse
function that are normally expressed on hematopoietic cells
PIGA = Phosphatidyl Inositol Glycan–A
GPI = Glycosyl Phosphatidyl Inositol

4. Cont..
• Somatic mutation of PIGA gene  progeny of affected stem cells deficient in all GPI-anchored
proteins (GPI-APs)  deficiency of complement regulatory proteins CD55 (decay accelerating
factor [DAF]) and CD59 (membrane inhibitor of reactive lysis [MIRL])  complement-mediated
intravascular hemolysis.
• Element of BM failure is present in all pts.
• Frequently arises in a/w defined BM failure process, particularly AA and, to a lesser extent,
low-grade MDS
• PNH is characterized, in the classic case, by macroscopic hemoglobinuria
• Thrombocytopenia, leukopenia, and thrombosis involving unusual sites are other notable clinical
characteristics of PNH

5. History
•1882: Strübling -Hypothesis on the etiology of nocturnal hemoglobinuria: attributed it to acidosis that resulted from CO2 accumulation associated with sleep-related hypoventilation
•Marchiafava and Nazari (1911) and Micheli (1931) subsequently detailed the clinical characteristics of the disease; aka Marchiafava-micheli syndrome
•By 1953, at least 162 cases had been collected.

6. Etio-Pathogenesis
• Sensitivity to complement mediated lysis
• The chronic intravascular hemolysis that is the clinical hallmark of PNH is due
to the abnormal sensitivity of the RBCs to complement-mediated lysis.
• A defining feature is phenotypic mosaicism based on the sensitivity of the
RBCs to complement-mediated lysis – 1st
clearly elucidated by Rosse and
Dacie in 1966.

7. • Paroxysms of gross hemoglobinuria: a/w 20-50% of PNH III population (PNH clone
size of 60% to 90%)
• Constant hemoglobinuria: a/w >50% PNH III population (PNH clone size >90%)
• Half-life of complement-sensitive PNH cells is approximately 6 days.

8. Complement mediated lysis of PNH RBCs

9. Erythrocyte membrane protein deficiencies

10. Pathophysiology of PNH
Transmembrane proteins have three domains: an ectoplasmic domain (rectangle with horizontal
lines), a transmembrane domain (blue rectangle), and a cytoplasmic domain (red rectangle).
In contrast, GPI-anchored proteins lack the cytoplasmic and transmembrane domains. This class of
proteins is anchored to the cell by a GPI moiety consisting of phosphatidylinositol (brown
hexagon), glucosamine (green circle), and three mannose (yellow circles) residues

11. The PIGA gene product is essential for the transfer of the nucleotide sugar uridine diphosphate-N-
acetylglucosamine (UDP-GlcNAc) to phosphatidylinositol (PI) to form GlcNAc-PI, the first intermediate in the
synthesis of the GPI anchor. PIGA is located on the short arm of the X chromosome. Hematopoietic cells in PNH
are deficient in all proteins that are GPI-anchored because a somatic mutation in a hematopoietic stem cell
partially or completely inactivates the PIGA gene product. Consequently, the GPI moiety is not synthesized

12. Aplastic anemia and PNH
• Two groups:
• Without a preceding h/o AA (classic PNH)
• With an antecedent h/o AA who subsequently develop PNH (PNH/aplastic anemia).
• The time between the diagnosis of AA to the development of PNH may vary from a few months to several
years
• While 40% to 60% of pts with AA have small, subclinical populations of GPI-AP− hematopoietic cells at
diagnosis, only 10% to 15% subsequently develop clinically apparent PNH
• The basis of the relationship between PNH and aplastic anemia is speculative
• BM injury may play a central role in the development of PNH by providing the conditions that favor the
growth/survival of PIGA-mutant, GPI-AP-deficient stem cells

13. Leucocytes and platelets
• Deficiency of GPI-AP has been on neutrophils, monocytes, platelets, and
lymphocytes has been demonstrated in the peripheral blood.
• Identical PIGA mutations have been identified in neutrophils, monocytes, and
lymphocytes from the same patient.
• These studies indicate that the somatic mutation that gives rise to PNH affects a
hematopoietic stem cell.
• Most PNH pts have pancytopenia or either neutropenia or thrombocytopenia in
combination with anemia at some point during the course of their illness

14. Clinical presentation
• Onset: insidious onset, abrupt onset in 25% of cases
• Course: chronic
• Age of presentation: 4th
to 5th
decade of life (range: 16-75 yrs)
• Sex: Slight female preponderance
• No familial tendency
• Most commonly, pts. with classic PNH complain initially of malaise, lethargy, and asthenia.
• Yellowish discoloration of the skin (jaundice) and sclera (icterus) may be observed by astute family members.

15. Cont..
 Hemoglobinuria
• Although essentially all pts. with classic PNH have episodes of hemoglobinuria
sometime during their illness, presenting symptom in only 1/4th
of all pts.
• Urine is usually darkly discolored in the morning and clears gradually over the
course of the day
• Retention of CO2 causing a slight fall in plasma pH sufficient to activate the
alternative pathway of complement is a possible explanation, but this hypothesis
has been challenged.

17. Cont..
• Episodic hemolysis
• Irregular but recurrent exacerbations of hemolysis and hemoglobinuria.
• Paroxysms may be precipitated by a wide variety of events, including
infections (even minor ones), surgery, transfusions, iron supplementation,
vaccinations, and menstruation.

18. Cont..
 Thromboembolic complications
• Striking predisposition toward intravascular thromboses, especially within the venous
circulation.
• Intra-abdominal veins are most commonly affected.
• Hepatic venous thrombosis (Budd-Chiari syndrome) is a serious, potentially fatal complication
of PNH
• 15-30% of the pts with PNH has hepatic venous thrombosis.
• Cerebral vein and superficial dermal veins are also affected less often.

19. Cont..
 Renal abnormalities
• Both acute and chronic renal insufficiency occurs in patients with PNH.
• Acute renal insufficiency is a/w hemoglobinuric crises and may resolve without
residual damage.
• Patients with PNH may also have hematuria, proteinuria, hypertension, an inability to
concentrate their urine, or some combination of these abnormalities.
• Chronic renal insufficiency probably result from repeated thrombotic episodes
involving small venules.

20. • Dysphagia
• Painful or difficult swallowing.
• Often worse in the morning and appears to be exacerbated during hemolytic
episodes
• It has been proposed that the plasma free hemoglobin that is a consequence of
the chronic intravascular hemolysis acts as a sump for nitric oxide
• Male impotence
• Worse during hemolytic exacerbations
• Nitric oxide deficiency as a consequence of the sump effect of plasma free
hemoglobin

21. Natural history of PNH
• Without treatment, the median survival is estimated to be about 8–10
years.
• In the past, the mcc of death has been venous thrombosis, f/b infection
secondary to severe neutropenia and hemorrhage secondary to severe
thrombocytopenia.
• Rarely, 1–2% of all cases may terminate in acute myeloid leukemia.
• Full spontaneous recovery from PNH has been documented, albeit rarely.

22. Lab findings
 Blood examination
• Anemia: usually severe
• Usually macrocytic but may be microcytic hypochromic d/t IDA from chronic
and acute hemoglobinuria.
• Relative reticulocytosis may be marked, but the absolute reticulocyte count is
often lower. This discrepancy reflects underlying marrow dysfunction.
• Negative Coomb’s test
• Leucopenia: often severe in PNH/AA.
• Thrombocytopenia: moderate to sever degree

23. Cont..
• Plasma
• May be golden brown, reflecting the presence of increased levels of unconjugated bilirubin,
hemoglobin, and methemalbumin.
• Sr. haptoglobin - low and LDH - markedly elevated (in thousand)
• Unconjugated bilirubin: mildly or moderately elevated
• Urine
• Increased urobilonogen
• Hemosiderinuria
• Bone marrow
• Classic PNH: Cellular, with marked to massive erythroid hyperplasia
• At some stage, may become hypocellular or frankly aplastic.

24. Diagnostic tests
 Historical tests (until early 1990s)
• Ham's test (acidified serum lysis)
• Sucrose lysis test (sugar water test of Hartmann)
• Sensitive and specific when properly performed
• Accuracy is strongly operator-dependent.
• Flow cytometry: Gold standard
• GPI-AP-deficient populations that comprise >1% to 3% of the red cells can be
identified

25.  FLAER test
• Use fluorescent aerolysin (FLAER)
• Eploits the unique properties of the bacterial toxin aerolysin: binds directly to the GPI anchor.
• The primary advantage of this assay is that because it detects all GPI-APs, it is specific for
PNH.
• The primary disadvantage is that the FLAER reagent does not bind well to RBCs. Thus FLAER
cannot be used to characterize GPI-AP expression on erythrocytes.

26. D/D
 The diagnosis of PNH must be considered in any patient who has the following:
• S/S 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
• Pancytopenia in association with hemolysis
• Venous thrombosis affecting unusual sites, especially intra-abdominal, cerebral, or dermal locations
accompanied by evidence of hemolysis
• Unexplained recurrent bouts of abdominal pain, low backache, or headache in the presence of chronic
hemolysis
• Budd-Chiari syndrome.

27. MANAGEMENT

28. Classification of PNH
Category Rate of intravascular
hemolysis
Bone Marrow Flow Cytometry Befit from
Eculizumab
Classic Florid (markedly
abnormal LDH often
with episodic
macroscopic
hemoglobinuria
Hypocellular with
erythroid
hyperplasia and
normal or near-
normal
morphology
Large population (>
50%) of GPI-AP
deficient PMNs
Yes
PNH in the setting
of another BM
failure syndrome
Mild (often with
minimal
abnormalities of
biochemical markers
of hemolysis)
Evidence of
concomitant BM
failure syndrome
% of GPI-AP
deficient PMN is
usually relatively
small (25-50%)
Typically No, but
some pts have
clinically significant
hemolysis and
benefit from t/t
Sub-Clinical No clinical or
biochemical evidence
of intravascular
hemolysis
Evidence of a
concomitant BM
failure syndrome
Small (<1%) No
Based on recommendations of the International PNH interest Group

29. Management of PNH

31. T/t cont..
• Folic acid supplementation mandatory
• Iron supplementation as appropriate
• Long-term steroids not indicated, as there is no evidence that they have any effect on chronic hemolysis.
• Eculizumab
• Humanized monoclonal antibody; bind to complement C5.
• IV every 14 days
• Definitive cure for PNH is allogenic BMT
• Indications of BMT: Severe AA, MDS, Unresponsive to eculizumab.

32. T/t cont..
• PNH/AA syndrome
• IST with ATG and cyclosporine A
• Any pt. who has had venous thrombosis or who has a genetically
determined thrombophilic state in addition to PNH
• Regular anticoagulant prophylaxis

33. Complement therapies in development
• C5 inhibitors
• Ravulizumab is virtually identical to eculizumab but with a longer half-life;
the drug is administered IV every eight weeks after a brief induction period.
• An international, randomized, phase III trial of ravulizumab versus eculizumab
has been completed and shown to be non-inferior to eculizumab
• C1 esterase inhibitor
• C1INH inhibits the early stages of complement activation and associated
inflammatory proteases.

34. Cont..
• C3 inhibition
• The C3 component of complement is another potential target for inhibition, because both
intravascular and extravascular hemolysis depend on C3.
• An antibody against C3 was developed but abandoned due to the high concentration of C3 in plasma
• Antibodies, proteins, and small peptides that target C3 fragments are under investigation.
• Factor D inhibition
• Factor D is the only enzyme in the blood that can activate factor B and is a rate-limiting step in the
alternative pathway of complement.
• An oral agent, ACH-4471, that inhibits factor D, blocks PNH cell hemolysis, mitigates the
accumulation of C3 fragments on the surface of PNH cells, and also blocks the APC in in vitro models
of aHUS
• Human studies with this compound in PNH patients with suboptimal response to eculizumab are
underway

35. Reference
• Wintrobe’s Clinical Hematology 13th
Edition
• Harrison’s Medicine 19th
Edition
• UpToDate 2019

36. THANK YOU