Paroxysmal Nocturnal Hemoglobinuria PNH

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Paroxysmal Nocturnal Hemoglobinuria (PNH)
PNH is an acquired chronic hemolytic anemia which
arises from a somatic mutation in a hematopoietic
stem cell. Most hematopoitic cell lines may be
affected by the intrinsic membrane defect. This
defect renders the red cells highly susceptible
to complement mediated lysis resulting in the
characteristic hemolysis.
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Paroxysmal Nocturnal Hemoglobinuria (PNH)
Topics to be considered

• History
• Epidemiology
• Clinical Features
• Relationship to Aplastic Anemia
• other diseases
• Pathogenesis
• Laboratory Diagnosis
• Therapy

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History
Investigator Year
Contribution Gull 1866 Described
nocturnal and paroxysmal nature of
intermittent haematinuria in a young
tanner. Strubing 1882 Distinguished PNH
from paroxysmal cold haemoglobinuria and
march haemoglobinuria. Attributed the problem
to the red cells. van den Burgh 1911 Red
cells lysed in acidified serum. Suggested a role
for complement. Enneking 1928
Coined the name paroxysmal nocturnal
haemoglobinuria. Marchiafava 1928-
Described perpetual hemosiderinemia in absence
of and Micheli 1931 hemolysis. Their names
became eponymous for PNH in Europe. Ham
1937- Identified the role of
complement in lysis of PNH red 1939 cells.
Developed the acidified serum test, also called
the Ham test, which is still used to diagnose
PNH. Demonstrated that only a portion of PNH
red cells are abnormally sensitive to
complement. Davitz 1986 Suggests defect in
membrane protein anchoring system
responsible Hall Rosse 1996 Flow cytometry for
the diagnosis of PNH
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Epidemiology

• Rare disease -
• frequency unknown
• thought to be on the same order as aplastic
  anemia (2-6 per million)
• Median age at diagnosis
• 35 yrs
• PNH reported at extremes of age
• FemaleMale ratio 1.21.0
• No increased risk of PNH in patient relatives
• Median Survival after diagnosis 10-15 yrs

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Clinical Features

• Major symptoms (Hemolysis, Cytopenia, and
  tendency to thrombosis)
• chronic hemolysis with acute exacerbations
  (hallmark)
• most patient at some stage
• only 1/3 exhibit hemolysis at diagnosis
• Recurrent attacks of intravascular hemolysis are
  usually associated with
• hemoglobinuria
• abdominal pain
• dysphagia

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Clinical Features

• cytopenia (varying severity)
• isolated subclinical thrombocytopenia
• classical severe aplastic anemia
• tendency to thrombosis
• venous thrombosis (40) of patients, main cause
  of morbidity
• Variable expression of above often causes
  considerable delay in the diagnosis
• Major cause of death
• venous thrombosis
• complications from progressive pancytopenia

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Clinical Features - Long term

• 25 of PNH patients survive gt25 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 most likely bone marrow
  failure not PNH

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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

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Pathogenesis - The Defect

• Defect - Somatic mutation of PIG-A gene
  (phosphatidylinositol glycan complementation
  group A) located on the X chromosome in a clone
  of a hematopoietic stem cell
• gt100 mutations in PIG - A gene known in PNH
• The mutations (mostly deletions or insertions)
  generally result in stop codons - yielding
  truncated proteins which may be non or partially
  functional - explains heterogeneity seen in PNH

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Pathogenesis - The Defect
GPI Anchor

• PIG - A gene codes for 60 kDa protein
  glycosyltransferase which effects the first step
  in the synthesis of the glycolipid GPI anchor
  (glycosylphosphatidylinositol). Results in clones
  lacking GPI anchor - in turn, attached proteins

PIG - A protein
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Pathogenesis - The DefectGPI Anchor deficiency

• PNH blood cells deficient in GPI anchor lack
  membrane proteins linked via the anchor
• Membrane proteins w/o anchor degraded in ER
• Severity size of deficiency - variable -
  clinical/diagnostic implications
• GPI anchor highly conserved in all eukaryotic
  cells
• Variant surface proteins of Trypanosomes - GPI
  linked
• Shed by cleavage of GPI anchor - immune system
  avoid
• Swapping GPI linked proteins - CD55 complement
  resistance - Schistosoma mansoni
• In Humans
• signal transduction, co-receptors
• advantage to this type of anchor?

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Proteins anchored by GPI Anchorand
Surface Proteins Missing on PNH Blood
Cells Antigen
Expression
Pattern Enzymes Acetylcholinesterase (AchE)
Red blood cells Ecto-5'-nucleotidase
(CD73) Some B- and T-lymphocytes Neutrophi
l alkaline phosphatase(NAP) Neutrophils ADP-rybo
syl transferase Some T-lymphs,
Neutrophils Adhesion molecules Blast-I/CD48
Lymphocytes Lymphocyte
function- associated antigen-3(LFA-3 or CD58)
All blood cells CD66b Neutrophils Complem
ent regulating surface proteins Decay
accelerating factor (DAF or CD55) All
blood cells Homologous restriction
factor, Membrance inhibitor of reactive lysis
All blood cells (MIRL or CD59)
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Surface Proteins Missing on PNH Blood
Cells Antigen
Expression
Pattern Receptors Fc-? receptor III (Fc ? Rlll
or CD16) Neutrophils, NK-cells,
macrophages, some T-lymphocytes Monocyte
differentiation antigen Monocytes,
macrophages (CD14) Urokinase-type Plasminogen
Monocytes, granulocytes Activator Receptor
(u-PAR, CD87) Blood group antigens Comer
antigens (DAF) Red
blood cells Yt antigens (AchE)
Red blood cells Holley Gregory antigen
Red blood cells John Milton
Hagen antigen (JMH) Red blood cells,
lymphocytes Dombrock reside
Red blood cells Neutrophil antigens NB1/NB2

Neutrophils
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Surface Proteins Missing on PNH Blood
Cells Antigen
Expression
Pattern Other surface proteins of unknown
functions CAMPATH-1 antigen (CDw52)
Lymphocytes, monocytes CD24
B-lymphocytes, Neutrophils,
eosinophils p5O-80 Neutrophils GP500

Platelets GPI75
Platelets
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Pathogenesis - Functional consequences of lack of
GPI linked proteins

• In vivo function of many of these membrane
  proteins not fully understood
• However, 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,
  protectin, homologous restriction factor)
  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

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Mechanism for hemolysis in PNH via lack of CD59
(CD59)
(CD59)
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Pathogenesis - Clonal evolution and cellular
selection

• Expansion of abnormal hematopoietic stem cell
  required for PNH disease expression
• Theories for expansion
• Blood cells lacking GPI-linked proteins have
  intrinsic ability to grow abnormally fast
• In vitro growth studies demonstrate that there
  are no differences in growth between normal
  progenitors and PNH phenotype progenitors
• In vivo - mice deficient for PIG -A gene also
  demonstrates no growth advantage to repopulation
  of BM.
• Additional environmental factors exert selective
  pressure in favor of expansion of GPI anchor
  deficient blood cells
• PNH hematopoitic cells perferentially engraft
  SCID mice compared to phenotypically hematopoitic
  cells
• Close association with AA - PNH hematopoitic
  cells cells may be more resistant to the IS than
  normal hematopoitic cells.
• Evidence in AA is that the decrease in
  hematopoitic cells is due to increased apoptosis
  via cytotoxic T cells by direct cell contact or
  cytokines (escape via deficiency in GPI linked
  protein???)

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Laboratory Evaluation of PNH

• Acidified Serum Test (Ham Test 1939)
• Acidified serum activates alternative complement
  pathway resulting in lysis of patients rbcs
• May be positive in congenitial dyserythropoietic
  anemia
• Still in use today
• Sucrose Hemolysis Test (1970)
• 10 sucrose provides low ionic strength which
  promotes complement binding resulting in lysis of
  patients rbcs
• May be positive in megaloblastic anemia,
  autoimmune hemolytic anemia, others
• Less specific than Ham test

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Laboratory Evaluation of PNH

• PNH Diagnosis by Flow Cytometry (1986)
• Considered method of choice for diagnosis of PNH
  (1996)
• Detects actual PNH clones lacking GPI anchored
  proteins
• More sensitive and specific than Ham and sucrose
  hemolysis test

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PNH Diagnosis by Flow Cytometry
Of the long list of GPI anchored protein,
monoclonal antibodies to the following antigens
have been used in the diagnosis of PNH The most
useful Abs are to CD14, 16, 55, 59, and 66. Are
all required? Probably not - more studies needed
Antigen Cell Lineage Function CD14 monocytes LP
S receptor, MDF CD16 neutrophils Fc?III
receptor CD24 neutrophils B-cell
differentiation marker CD55 all
lineages DAF CD58 all lineages possible
adhesion CD59 all lineages MIRL, HRF,
protectin CD66b neutrophils CEA-related
glycoprotein
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PNH Diagnosis by Flow Cytometry

• Antigen expression is generally categorized into
  three antigen density groups
• 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 granulocytes
  monocytes)

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PNH Diagnosis by Flow Cytometry
Examples of variable GPI linked CD59 expression
on granulocytes on four PNH patients
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PNH Diagnosis by Flow Cytometry
Example of variable expression of several GPI
linked Ags on several lineages
From Purdue Cytometry CD-ROM vol3 97
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PNH Diagnosis by Flow Cytometry

• Flow Cytometry is method of choice but only
  supportive for/against diagnosis
• More studies are needed to better define whether
  the type (I, II, or III), cell lineage, and size
  of thecirculating clone can provide additional
  prognosticinformation.
• Theoretically - should be very valuable

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Therapy

• Bone Marrow Transplantation
• Only curative treatment
• chronic condition (possiblity of spontaneous
  remission) - BMT should be avoided
• Immunosuppressive therapy
• Antilymphocyte globulin /or cyclosporine A
• Does not alter proportion of PNH hemopoiesis
• Steroids - experimental - controlled studies ??
• Growth Factors
• Some improvement
• no evidence that normal clones respond better
  than PNH clones