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1. Paroxysmal Nocturnal Hemoglobinuria
AnupamSingh
Moderator- Dr Rashmi Kushwaha

2. History
Investigator Year Contribution
Gull 1866 Described nocturnal and paroxysmal nature of
intermittent hematuria in a young man
Strubing 1882 Distinguished PNH from paroxysmal cold
haemoglobinuria
Enneking 1925 Coined the name PNH
Ham 1937-39 Identified the role of complement in lysis of PNH red
cells. Developed the acidified serum test, also called
Ham test
Davitz 1986 Suggests defect in membrane protein anchoring system
responsible
Hall & Rosse 1996 Flow cytometry for dx of PNH

3. Introduction
• Non-malignant clonal expansion of one or several
hematopoietic stem cells that manifests with
hemolytic anemia, bone marrow failure, and
thrombosis.
• Intravascular hemolysis and hemoglobinuria that occur
chiefly at night while the patient is asleep.
• Partial or complete lack of GPI-linked proteins on a
population of cells of the hematopoietic system
• Mainly a disease of adults, although children and
adolescents also affected

4. • Many cases develop in people who have previously
been diagnosed with aplastic anemia or MDS.
• Thrombophilia is a major cause of morbidity and
mortality in PNH
• Sometimes referred to as Marchiafava-Micheli syndrome.
• PNH is rare, with an annual rate of 1-2 cases per million.

5. Pathophysiology
• Due to somatic mutations in pluripotent hemopoietic
stem cell of PIG-A gene (phosphatidylinositol glycan,
class A) located on X-chromosome.
• Lead to block in biosynthesis of
glycosylphosphatidylinositol (GPI) molecule.
• Some proteins attached to outer cell membrane by GPI
anchors.
• More than 100 membrane proteins are anchored by GPI.

6. •First identified by Hoffman
in 1969.
•70-kD protein
•Decay Accelerating Factor
(DAF)
•Activates the conversion of
C3b to C3d
•Membrane inhibitor of reactive
lysis (MIRL), 18-kD protein
•Inhibits the lysis of red cells from
the C5-C9 membrane attack
complex
•More important than DAF in
protecting cells from complement-
mediated lysis in vivo.
CD55
CD59

7. (A) Core structure of the GPI anchor. The inositol-phospholipid
(PI) is anchors into the lipid bilayer of the plasma membrane.
The glycan core consists of a molecule of N-glucosamine, 3
manose molecules (Man), and a molecule of ethanolamine
phosphate. The protein is covalently attached through an
amide bond to an ethanolamine on the terminal mannose.

8. (B) GPI anchor biosynthesis takes place in the
endoplasmic reticulum.

9. (C) eventually the GPI anchored protein is
transported to the plasma membrane.

11. Normal erythrocytes are protected against complement-mediated lysis primarily by CD55
and CD59. Deficiency of these GPI anchored complement regulatory proteins results in
complement activation on PNH erythrocytes.

12. Model of two-step hypothesis of paroxysmal nocturnal hemoglobinuria (PNH) pathophysiology.
Clonal expansion,
proliferation of the mutant cells
PIGA mutant cells are selected because
they have a growth advantage based on GPI-AP

13. Glycosyl Phosphatidylinositol–Anchored Proteins Deficient in Paroxysmal Nocturnal
Hemoglobinuria
• Protein
• Leukocyte alkaline phosphatase
• Acetylcholinesterase
• Decay accelerating factor (DAF, CD55)
• Membrane inhibitor of reactive lysis (MIRL, CD59)
• FcγRIIIb (CD16b)
• Lymphocyte function-associated antigen 3 (LFA-3, CD58)
• Endotoxin-binding protein (CD14)
• Campath 1 (CD52)
• 5′-ectonucleotidase (CD73)
• Urokinase plasminogen activator receptor (CD87)

14. • JMH-bearing protein (CD108)
• CD66b (formerly CD67)
• CD66c
• p50-80
• CD24
• CD48
• Thy-1 (CD90)
• CD109
• CD157
• GP500
• GP175
• Folate receptor
• Cellular prion protein

15. Normal red blood cells are
protected from complement
attack by a shield of
terminal complement
inhibitors (2,3)
Without this protective
complement inhibitor
shield, PNH red blood
cells are destroyed (2,3)
Intact RBC
Free Haemoglobin in
the Blood from Destroyed
PNH RBCs
Complement
Activation
Significant
Impact on
Morbidity
Significant
Impact on
Survival
Anaemia
Haemoglobinuria
Thrombosis
Fatigue
Renal Failure
Pulmonary
Hypertension
Erectile Dysfunction
Dyspnoea
Dysphagia
Abdominal Pain
PNH is a Progressive Disease of Chronic Haemolysis

16. 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
E
• Budd-Chiari syndrome
Diagnosis of PNH must be considered in any patient who has the following:

17. Major clinical manifestations & complications of PNH
Related to intravascular
hemolysis
Related to thrombophilia Related to bone marrow
failure
Anemia
Hemoglobinuria
Mild jaundice
Fatigue
Chronic renal failure
Cholelithiasis
Venous thrombosis:
Budd chiari syndrome
Splenic vein thrombosis
Renal vein thrombosis
Portal hypertension
Cerebral vein
thrombosis
Retinal v. thrombosis
Deep vein thrombosis
Arterial thrombosis:
Stroke
MI
Pancytopenia:
fatigue, infections,
bleeding
MDS
AML (rare)

18. Presenting Features in 80 Patients with Paroxysmal Nocturnal Hemoglobinuria
Signs and Symptoms Number of Patients
Symptoms of anemia 28
Hemoglobinuria 21
Hemorrhagic signs and symptoms 14
Aplastic anemia 10
Gastrointestinal symptoms 8
Hemolytic anemia and jaundice 7
Iron deficiency anemia 5
Thrombosis or embolism 5
Infections 4
Neurologic signs and symptoms 3
From Dacie JV, Lewis SM. Paroxysmal nocturnal haemoglobinuria: clinical manifestations, haematology, and
nature of the disease. Ser Haematol 1972;5:3–23.

19. Classification of PNH
Subcategory Rate of
intravascular
hemolysis
Bone marrow % GPI-AP-
deficient
Neutrophils
Classic PNH Marked; visible
hemoglobinuria
frequent
Normocellular to
hypercellular with
erythroid
hyperplasia and
normal or near-
normal
morphology
>50%
PNH in the setting
of another
specified bone
marrow disorder
Mild to moderate;
visible
hemoglobinuria is
intermittent or
absent
Evidence of a
concomitant BM
failure syndrome
Variable;
usually<30%
Subclinical PNH No clinical or
biochemical e/o
intravascular
hemolysis
Evidence of a
concomitant BM
failure syndrome
<1%

20. Laboratory Findings
• Increase urobilinogen
• Hemoglobinuria
• Classic hallmark of PNH
• Occurs in only about 25-50% of patients
• Begins insidiously
• Infrequent brown urine passed upon awakening
• urine is usually darkly discolored in the morning and clears during
the day.
• Hemosiderinuria
• Albuminuria
• Decrease S. Haptoglobin
• Increase S. Indirect bilirubin
• Increase LDH

21. • Blood-
• Anemia
• RBCs normocytic, may appear hypochromic and
microcytic
• Occasional red cell fragments
• Polychromatophilia
• Nucleated red blood cells may be found
• Reticulocyte counts may increased
• Leukopenia
• Thrombocytopenia of moderate to severe degree

22. • BONE MARROW-
• BM aspirate/biopsy specimens are examined.
• May be normocellular to hypercellular with erythroid
hyperplasia.
• 50% of the nucleated cells may be normoblasts.
• May be hypocellular in concomitant BM failure.
• Finding of dysplasia or certain chromosomal
abnormalities helpful in diagnosis of MDS.
• Number of megakaryocytes may be decreased.

23. • Cytogenetic study-
• Non random chromosomal aberrations specific for PNH
involves rearrangement of the HMGA2 locus on 12q13-
15.

24. LABORATORY TESTS FOR THE DIAGNOSIS OF PNH
•HAM Test
•Sucrose Lysis Test
•Sephacryl Gel Card Test
•Flow cytometry

25. Acidified Serum Test (Ham Test)
• First described in 1937.
• The patient’s red cells are exposed at 37 deg C to the
action of normal or patient’s own serum suitably
acidified to the optimum Ph for lysis (pH 6.5-7.0)
• Patient’s red cells can be
obtained from defibrinated,
heparinized, oxalated, citrated
or EDTA blood.
Thomas Hale Ham (1905-1987)

28. Significance of Acidified Serum Lysis Test
• PNH
Positive HAM
Test
• CDA Type II or HEMPASFalse Positive
• Markedly spherocytic
red cells
Positive test in
inactivated
serum

29. Sucrose Lysis Test (sugar water test)
• Red cells absorb complement components from serum at
low ionic concentrations.
• PNH cells , because of their great sensitivity , undergo
lysis but normal red cells do not.
• An iso-osmotic solution of sucrose (92.4 g/l) is required.
• Sucrose provides a medium of low ionic strength that
promotes the binding of complement to the red cells.
• In PNH, lysis usually varies from 10% to 80%.

30. 1st test tube (0.05 ml of normal
AB- or ABO- compatible serum
diluted in 0.85 ml of sucrose soln. )
2nd test tube (0.05 ml of serum
diluted in 0.85 ml saline)
0.1 ml 50%
washed red
cells
Incubate 37 deg C for
30 mn, centrifuge
Incubate 37 deg C for 30
mn, centrifuge
Check for
lysis in
spectrometer
Check for lysis
in
spectrometer
0.1 ml 50%
washed red
cells
Tube used as blankNo
hemolysis
Excludes PNH
Hemolysis
(10-80%) PNH

31. • Less specific for PNH; detects haemolysis associated
with other blood-cell disorders
• False negative in patients who have recently received
a transfusion.

32. Sephacryl Gel Card Test
• Diluents and microtyping cards containing sephacryl and
rabbit anti-mouse antibody within the gel matrix, from Dia
Med-ID (St. Morat, Switzerland) were used.
• Ten healthy donors served as negative control.

33. C C
T T
PNH: demonstrating button at bottom indicating lack of DAF and MIRL antigens on red cell surface
In tubes 4 and 5 confirming PNH

34. Cells lacked
CD55 or CD59
Do not
agglutinate
Pellet at the
bottom
Cells that
express CD55
and CD59
Agglutinate Clump on top
## Presence of a cell trail just beneath the surface of gel was disregarded.
PNH
NO PNH

35. FLOWCYTOMETRY
• Flow cytometer has replaced HAM test as primary method
for diagnosis of PNH ( gold standard).
• Patient’s red cells are stained with a fluorescein antibody
that is specific for one of several GPI linked proteins (CD55,
CD58, CD59), which are deficient in PNH red cells.
• Stained cells are then analysed with flow cytometer.
• The simplest method is to analyze expression of MIRL (CD59)
on erythrocytes because it is normally present in relatively
high density.
• Concern that recent red cell transfusion might result in a
false-negative result

36. Paroxysmal Nocturnal Hemoglobinuria (PNH)
Phenotypes

38. Example of flow cytometric analysis. Granulocytes that
express neither CD59 nor CD55 make up the PNH clone,
and are found in the lower left quadrant
(26 % of cells). Normal granulocytes that express both
markers are in the right upper quadrant (74 % of cells)

39. Subclinical paroxysmal nocturnal hemoglobinuria (PNH).
A- normal volunteer-
No GPI-AP deficient erythrocytes were among ∼100,000 cells
counted in this analysis
B- patient with aplastic anemia but with no clinical
evidence of PNH.
0.077% of the erythrocytes failed to express CD55 and CD59

40. • Fluorescent Aerolysin (FLAER)-
• Also flow cytometry based assay that utilizes a
fluorescent labelled inactive variant of the protein
aerolysin (toxin of the bacterium Aeromonas hydrophila).
• Used for serial analysis for predicting both disease course
and clinical course especially in assessing for thrombotic
events.
• Can detect the clone in granulocytic and monocytic
lineages in one assay.
• Lack of FLAER binding to granulocytes as measured by
flow cytometry is sufficient for dx of PNH.

41. • It is specific for PNH,
because it detects all GPI-
APs,
Advantage
• FLAER reagent does not
bind well to RBCsDisadvantage

42. Treatment
• With the exception of marrow transplantation, no definitive
therapy is available.
• Management of the Anemia
• Androgenic Steroids, Cortical Steroids, and
Immunosuppressive therapy
• Transfusions
• Iron
• Splenectomy
• Folate
• Complement Inhibitors (eculizumab)
• Management of Non-hemolytic Anemia
• Bone Marrow Transplantation
• Prevention and Treatment of Thrombosis

43. Disease Course and Prognosis
• Median survival of 10 to 15 years.
• Major causes of morbidity and mortality are thrombosis,
bleeding, and infections.
• Development of other clonal myelopathies including MDS
and acute leukemia adversely affect prognosis.
• Patients with aplastic anemia and the refractory anemia
variant of MDS should undergo screening for PNH-sc at
diagnosis and yearly thereafter.
• Other clonal myelopathies that have been reported in
association with PNH include myelofibrosis, chronic
lymphocytic leukemia, chronic myelocytic leukemia,
polycythemia vera, and erythroleukemia.

44. Future Directions for Clinical and Basic Research
• Guidelines for management of the thrombophilia of PNH.
• Guidelines for bone marrow and stem cell
transplantation.
• Guidelines for how best to use eculizumab.
• Guidelines for management of pregnancy and PNH.
• More clearly defining the effects of ethnic/geographic
differences on the natural history of PNH.

46. MCQs

47. 1
• Pancytopenia with cellular marrow is seen in:
A. PNH
B. G6PD deficiency
C. Acquired aplastic anemia
D. Thalassemia

48. 1
• Pancytopenia with cellular marrow is seen in:
A. PNH
B. G6PD deficiency
C. Acquired aplastic anemia
D. Thalassemia

49. 2
• Commonest cause of Budd Chiari syndrome is
a. Valve in the IVC
b. hepatocellular carcinoma
c. Paroxysmal nocturnal hemoglobinuria
d. Renal cell carcinoma

50. 2
• Commonest cause of Budd Chiari syndrome is
a. Valve in the IVC
b. hepatocellular carcinoma
c. Paroxysmal nocturnal hemoglobinuria
d. Renal cell carcinoma

51. 3
• False regarding PNH
A-intracorpuscular defect acquired at stem cell
level
B-hemolytic anemia
C-More common in children
D-bone marrow failure

52. 3
• False regarding PNH
A-intracorpuscular defect acquired at stem cell
level
B-hemolytic anemia
C-More common in children
D-bone marrow failure

53. 4
• HAM test (Acidified serum test) is done for
a) G.P.I. anchor protein
b) Complement defect
c) Spectrin defect
d) Mannose binding protein

54. 4
• HAM test (Acidified serum test) is done for
a) G.P.I. anchor protein
b) Complement defect
c) Spectrin defect
d) Mannose binding protein

55. 5
• Which of the following is a disadvantage of
FLAER:
a)Low specificity for PNH
b)Inadequate binding to WBC’s.
c)Inadequate binding to RBC’s due to presence
of large amounts of glycophorin in red cells.
d)Inadequate binding to RBC’s due to presence
of large amounts of hemoglobin in red cells.

56. 5
• Which of the following is a disadvantage of
FLAER:
a)Low specificity for PNH
b)Inadequate binding to WBC’s.
c)Inadequate binding to RBC’s due to presence
of large amounts of glycophorin in red cells.
d)Inadequate binding to RBC’s due to presence
of large amounts of hemoglobin in red cells.

57. 6
• PIGA gene is located on:
a)Chromosome 6
b) Chromosome 1
c)X chromosome
d)Y chromosome

58. 6
• PIGA gene is located on:
a)Chromosome 6
b) Chromosome 1
c)X chromosome
d)Y chromosome

59. 7
• Recommended screening protocol for PNH-sc in
patients with Aplastic Anemia and Refractory Anemia
variant of MDS are:
a)Screening for PNH-sc at diagnosis and yearly thereafter.
b)Screening for PNH-sc at diagnosis and six monthly
thereafter.
c)Screening for PNH-sc at diagnosis and three yearly
thereafter.
d)Screening will depend upon manifestation of signs and
symptoms of intravascular hemolysis and may be
carried out as and when required.

60. 7
• Recommended screening protocol for PNH-sc in
patients with Aplastic Anemia and Refractory Anemia
variant of MDS are:
a)Screening for PNH-sc at diagnosis and yearly thereafter.
b)Screening for PNH-sc at diagnosis and six monthly
thereafter.
c)Screening for PNH-sc at diagnosis and three yearly
thereafter.
d)Screening will depend upon manifestation of signs and
symptoms of intravascular hemolysis and may be
carried out as and when required.

61. 8
• All of the following are true regarding lab findings
in PNH except:
a)The RBC’s are usually macrocytic but may
occasionally be hypochromic and microcytic
because of iron deficiency from acute or chronic
hemoglobinuria.
b)Normoblasts may be found in the peripheral
blood smear.
c)NAP expression is low or absent.
d)Direct antiglobulin test is positive prior to
treatment.

62. 8
• All of the following are true regarding lab findings
in PNH except:
a)The RBC’s are usually macrocytic but may
occasionally be hypochromic and microcytic
because of iron deficiency from acute or chronic
hemoglobinuria.
b)Normoblasts may be found in the peripheral
blood smear.
c)NAP expression is low or absent.
d)Direct antiglobulin test is positive prior to
treatment.

63. 9
• PNH is:
a) A Hereditary disorder
b)An acquired disorder
c)Both
d)None

64. 9
• PNH is:
a) A Hereditary disorder
b)An acquired disorder
c)Both
d)None

65. 10
• All of the following statements are true
regarding HAM test except:
A- test is considered positive when all of the
patient’s RBCs undergo lysis
B- the optimum pH for lysis is 6.5-7.0
C- in unacidified normal serum, less lysis or even
no lysis occurs
D- the serum should be used within few hours of
collection

66. 10
• All of the following statements are true
regarding HAM test except:
A- test is considered positive when all of the
patient’s RBCs undergo lysis
B- the optimum pH for lysis is 6.5-7.0
C- in unacidified normal serum, less lysis or even
no lysis occurs
D- the serum should be used within few hours of
collection