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Anaemia PNH
1. Paroxysmal NocturnalParoxysmal Nocturnal
Hemoglobinuria (PNH)Hemoglobinuria (PNH)
PNH is an acquired chronic hemolytic anemia whichPNH is an acquired chronic hemolytic anemia which
arises from a somatic mutation in a hematopoietic stemarises from a somatic mutation in a hematopoietic stem
cell. Most hematopoitic cell lines may be affected bycell. Most hematopoitic cell lines may be affected by
the intrinsic membrane defect. This defect renders thethe intrinsic membrane defect. This defect renders the
red cells highly susceptible to complement mediatedred cells highly susceptible to complement mediated
lysis resulting in the characteristic hemolysis.lysis resulting in the characteristic hemolysis.
2. Paroxysmal Nocturnal HemoglobinuriaParoxysmal Nocturnal Hemoglobinuria
(PNH)(PNH)
HistoryHistory
EpidemiologyEpidemiology
Clinical FeaturesClinical Features
– Relationship to Aplastic AnemiaRelationship to Aplastic Anemia
– other diseasesother diseases
PathogenesisPathogenesis
Laboratory DiagnosisLaboratory Diagnosis
TherapyTherapy
Topics to be consideredTopics to be considered
3. HistoryHistory
Investigator Year ContributionInvestigator Year Contribution
GullGull 1866 Described nocturnal and paroxysmal nature of1866 Described nocturnal and paroxysmal nature of
“intermittent haematinuria” in a young“intermittent haematinuria” in a young
tanner.tanner.
StrubingStrubing 18821882 Distinguished PNH from paroxysmal coldDistinguished PNH from paroxysmal cold
haemoglobinuria and march haemoglobinuria.haemoglobinuria and march haemoglobinuria.
Attributed the problem to the red cells.Attributed the problem to the red cells.
van den Burghvan den Burgh 19111911 Red cells lysed in acidified serum.Red cells lysed in acidified serum.
Suggested a roleSuggested a role for complement.for complement.
EnnekingEnneking 19281928 Coined the name “paroxysmalCoined the name “paroxysmal
nocturnalnocturnal haemoglobinuria”.haemoglobinuria”.
MarchiafavaMarchiafava 1928-1928- Described perpetual hemosiderinemia in absenceDescribed perpetual hemosiderinemia in absence
ofof
and Micheliand Micheli 19311931 hemolysis. Their names became eponymous forhemolysis. Their names became eponymous for
PNHPNH in Europe.in Europe.
HamHam 1937-1937- Identified the role of complement in lysis ofIdentified the role of complement in lysis of
PNH redPNH red 19391939 cells. Developed the acidified serum test,cells. Developed the acidified serum test,
also calledalso called the Ham test, which is still used tothe Ham test, which is still used to
diagnose PNH.diagnose PNH. Demonstrated that only a portion ofDemonstrated that only a portion of
PNH red cells arePNH red cells are abnormally sensitive to complement.abnormally sensitive to complement.
DavitzDavitz 19861986 Suggests defect in membrane protein anchoringSuggests defect in membrane protein anchoring
4. EpidemiologyEpidemiology
Rare disease -Rare disease -
– frequency unknownfrequency unknown
– thought to be on the same order as aplasticthought to be on the same order as aplastic
anemia (2-6 per million)anemia (2-6 per million)
Median age at diagnosisMedian age at diagnosis
– ~ 35 yrs~ 35 yrs
– PNH reported at extremes of agePNH reported at extremes of age
Female:Male ratio = 1.2:1.0Female:Male ratio = 1.2:1.0
No increased risk of PNH in patient relativesNo increased risk of PNH in patient relatives
Median Survival after diagnosis ~ 10-15 yrsMedian Survival after diagnosis ~ 10-15 yrs
5. Clinical FeaturesClinical Features
Major symptoms (Hemolysis, Cytopenia,Major symptoms (Hemolysis, Cytopenia,
and tendency to thrombosis)and tendency to thrombosis)
– chronic hemolysis with acute exacerbationschronic hemolysis with acute exacerbations
(hallmark)(hallmark)
most patient at some stagemost patient at some stage
only 1/3 exhibit hemolysis at diagnosisonly 1/3 exhibit hemolysis at diagnosis
Recurrent attacks of intravascular hemolysis areRecurrent attacks of intravascular hemolysis are
usually associated with;usually associated with;
– hemoglobinuriahemoglobinuria
– abdominal painabdominal pain
– dysphagiadysphagia
6. – cytopenia (varying severity)cytopenia (varying severity)
isolated subclinical thrombocytopeniaisolated subclinical thrombocytopenia
classical severe aplastic anemiaclassical severe aplastic anemia
– tendency to thrombosistendency to thrombosis
venous thrombosis (40%) of patients, main cause ofvenous thrombosis (40%) of patients, main cause of
morbiditymorbidity
Variable expression of above often causesVariable expression of above often causes
considerable delay in the diagnosisconsiderable delay in the diagnosis
Major cause of deathMajor cause of death
– venous thrombosisvenous thrombosis
– complications from progressive pancytopeniacomplications from progressive pancytopenia
Clinical FeaturesClinical Features
7. 25% of PNH patients survive >25 years - one25% of PNH patients survive >25 years - one
half of these go on to spontaneous remissionhalf of these go on to spontaneous remission
Remission patientsRemission patients
– hematological values revert to normalhematological values revert to normal
– no PHN rbcs or granulocytes detectedno PHN rbcs or granulocytes detected
– PNH lymphocytes - still detected but no clinicalPNH lymphocytes - still detected but no clinical
consequenceconsequence
Higher incidence of acute leukemia (6%)Higher incidence of acute leukemia (6%)
– ““preleukemic condition” most likely bone marrowpreleukemic condition” most likely bone marrow
failure not PNHfailure not PNH
Clinical Features - Long termClinical Features - Long term
8. AA described as pancytopenia withAA described as pancytopenia with
nonfunctioning bone marrow. Cytopenia innonfunctioning bone marrow. Cytopenia in
one or all cell lineages also common to PNHone or all cell lineages also common to PNH
High percentage of patients with AA developHigh percentage of patients with AA develop
clinical PNH or have lab evidence of PNHclinical PNH or have lab evidence of PNH
abnormality at some point (52%)abnormality at some point (52%)
Supports the theory that bone marrow failureSupports the theory that bone marrow failure
supports the abnormal PNH cells - more latersupports the abnormal PNH cells - more later
Clinical Features - Relationship toClinical Features - Relationship to
aplastic anemia (AA)aplastic anemia (AA)
9. Pathogenesis - The DefectPathogenesis - The Defect
Defect - Somatic mutation ofDefect - Somatic mutation of PIG-APIG-A genegene
(phosphatidylinositol glycan(phosphatidylinositol glycan
complementation group A) located on the Xcomplementation group A) located on the X
chromosome in a clone of a hematopoieticchromosome in a clone of a hematopoietic
stem cellstem cell
– >100 mutations in>100 mutations in PIG - APIG - A gene known in PNHgene known in PNH
– The mutations (mostly deletions or insertions)The mutations (mostly deletions or insertions)
generally result in stop codons - yieldinggenerally result in stop codons - yielding
truncated proteins which may be non or partiallytruncated proteins which may be non or partially
functional - explains heterogeneity seen in PNHfunctional - explains heterogeneity seen in PNH
10. PIG - APIG - A gene codes for 60 kDa proteingene codes for 60 kDa protein
glycosyltransferase which effects the firstglycosyltransferase which effects the first
step in the synthesis of the glycolipid GPIstep in the synthesis of the glycolipid GPI
anchor (glycosylphosphatidylinositol).anchor (glycosylphosphatidylinositol).
Results in clones lacking GPI anchor - inResults in clones lacking GPI anchor - in
turn, attached proteinsturn, attached proteins
Pathogenesis - The DefectPathogenesis - The Defect
GPI AnchorGPI Anchor
PIG - APIG - A proteinprotein
11. PNH blood cells deficient in GPI anchor lackPNH blood cells deficient in GPI anchor lack
membrane proteins linked via the anchormembrane proteins linked via the anchor
– Membrane proteins w/o anchor degraded in ERMembrane proteins w/o anchor degraded in ER
Severity & size of deficiency - variable -Severity & size of deficiency - variable -
clinical/diagnostic implicationsclinical/diagnostic implications
GPI anchor highly conserved in all eukaryotic cellsGPI anchor highly conserved in all eukaryotic cells
– Variant surface proteins of Trypanosomes - GPI linkedVariant surface proteins of Trypanosomes - GPI linked
– Shed by cleavage of GPI anchor - immune system avoidShed by cleavage of GPI anchor - immune system avoid
– Swapping GPI linked proteins - CD55 complementSwapping GPI linked proteins - CD55 complement
resistance -resistance - Schistosoma mansoniSchistosoma mansoni
– In HumansIn Humans
signal transduction, co-receptorssignal transduction, co-receptors
advantage to this type of anchor?advantage to this type of anchor?
Pathogenesis - The DefectPathogenesis - The Defect
GPI Anchor deficiencyGPI Anchor deficiency
12. Proteins anchored by GPI AnchorProteins anchored by GPI Anchor
andand
Surface Proteins Missing on PNH Blood CellsSurface Proteins Missing on PNH Blood Cells
Antigen Expression PatternAntigen Expression Pattern
EnzymesEnzymes
Acetylcholinesterase (AchE)Acetylcholinesterase (AchE) Red blood cellsRed blood cells
Ecto-5'-nucleotidase (CD73)Ecto-5'-nucleotidase (CD73) Some B- and T-lymphocytesSome B- and T-lymphocytes
Neutrophil alkaline phosphatase(NAP)Neutrophil alkaline phosphatase(NAP) NeutrophilsNeutrophils
ADP-rybosyl transferaseADP-rybosyl transferase Some T-lymphs, NeutrophilsSome T-lymphs, Neutrophils
Adhesion moleculesAdhesion molecules
Blast-I/CD48Blast-I/CD48 LymphocytesLymphocytes
Lymphocyte function-Lymphocyte function-
associated antigen-3(LFA-3 or CD58)associated antigen-3(LFA-3 or CD58) All blood cellsAll blood cells
CD66bCD66b NeutrophilsNeutrophils
Complement regulating surface proteinsComplement regulating surface proteins
Decay accelerating factor (DAF or CD55)Decay accelerating factor (DAF or CD55) All blood cellsAll blood cells
Homologous restriction factor,Homologous restriction factor,
Membrance inhibitor of reactive lysisMembrance inhibitor of reactive lysis All blood cellsAll blood cells
(MIRL or CD59)(MIRL or CD59)
13. Surface Proteins Missing on PNH Blood CellsSurface Proteins Missing on PNH Blood Cells
Antigen Expression PatternAntigen Expression Pattern
ReceptorsReceptors
Fc-Fc-γγ receptor III (Fcreceptor III (Fc γγ Rlll or CD16)Rlll or CD16) Neutrophils, NK-cells,Neutrophils, NK-cells,
macrophages,macrophages,
some T-lymphocytessome T-lymphocytes
Monocyte differentiation antigenMonocyte differentiation antigen Monocytes, macrophagesMonocytes, macrophages
(CD14)(CD14)
Urokinase-type PlasminogenUrokinase-type Plasminogen Monocytes, granulocytesMonocytes, granulocytes
Activator Receptor (u-PAR, CD87)Activator Receptor (u-PAR, CD87)
Blood group antigensBlood group antigens
Comer antigens (DAF)Comer antigens (DAF) Red blood cellsRed blood cells
Yt antigens (AchE)Yt antigens (AchE) Red blood cellsRed blood cells
Holley Gregory antigenHolley Gregory antigen Red blood cellsRed blood cells
John Milton Hagen antigen (JMH)John Milton Hagen antigen (JMH) Red blood cells, lymphocytesRed blood cells, lymphocytes
Dombrock resideDombrock reside Red blood cellsRed blood cells
Neutrophil antigensNeutrophil antigens
NB1/NB2NB1/NB2 NeutrophilsNeutrophils
15. Pathogenesis - Functional consequences ofPathogenesis - Functional consequences of
lack of GPI linked proteinslack of GPI linked proteins
In vivoIn vivo function of many of these membrane proteinsfunction of many of these membrane proteins
not fully understoodnot fully understood
However, CD55 and CD59 functions are well knownHowever, CD55 and CD59 functions are well known
– CD55 (decay accelerating factor) inhibits the formation orCD55 (decay accelerating factor) inhibits the formation or
destabilizes complement C3 convertase (C4bC2a)destabilizes complement C3 convertase (C4bC2a)
– CD59 (membrane inhibitor of reactive lysis, protectin,CD59 (membrane inhibitor of reactive lysis, protectin,
homologous restriction factor) Protects the membrane fromhomologous restriction factor) Protects the membrane from
attack by the C5-C9 complexattack by the C5-C9 complex
– Inherited absences of both proteins in humans have beenInherited absences of both proteins in humans have been
describeddescribed
Most inherited deficiencies of CD55 - no distinct clinicalMost inherited deficiencies of CD55 - no distinct clinical
hemolytic syndromehemolytic syndrome
Inherited absence of CD59 - produces a clinical disease similarInherited absence of CD59 - produces a clinical disease similar
to PNH with hemolysis and recurrent thrombotic eventsto PNH with hemolysis and recurrent thrombotic events
16. Mechanism for hemolysis in PNH via lack of CD59Mechanism for hemolysis in PNH via lack of CD59
(CD59)
(CD59)
17. Pathogenesis - Clonal evolution and cellularPathogenesis - Clonal evolution and cellular
selectionselection
Expansion of abnormal hematopoietic stem cell requiredExpansion of abnormal hematopoietic stem cell required
for PNH disease expressionfor PNH disease expression
– Theories for expansionTheories for expansion
Blood cells lacking GPI-linked proteins have intrinsic ability to growBlood cells lacking GPI-linked proteins have intrinsic ability to grow
abnormally fastabnormally fast
– In vitroIn vitro growth studies demonstrate that there are no differences in growthgrowth studies demonstrate that there are no differences in growth
between normal progenitors and PNH phenotype progenitorsbetween normal progenitors and PNH phenotype progenitors
– In vivoIn vivo - mice deficient for- mice deficient for PIG -APIG -A gene also demonstrates no growthgene also demonstrates no growth
advantage to repopulation of BM.advantage to repopulation of BM.
Additional environmental factors exert selective pressure in favor ofAdditional environmental factors exert selective pressure in favor of
expansion of GPI anchor deficient blood cellsexpansion of GPI anchor deficient blood cells
– PNH hematopoitic cells perferentially engraft SCID mice compared toPNH hematopoitic cells perferentially engraft SCID mice compared to
phenotypically hematopoitic cellsphenotypically hematopoitic cells
– Close association with AA - PNH hematopoitic cells cells may be moreClose association with AA - PNH hematopoitic cells cells may be more
resistant to the IS than normal hematopoitic cells.resistant to the IS than normal hematopoitic cells.
Evidence in AA is that the decrease in hematopoitic cells is due toEvidence in AA is that the decrease in hematopoitic cells is due to
increased apoptosis via cytotoxic T cells by direct cell contact orincreased apoptosis via cytotoxic T cells by direct cell contact or
cytokines (escape via deficiency in GPI linked protein???)cytokines (escape via deficiency in GPI linked protein???)
18. Laboratory Evaluation of PNHLaboratory Evaluation of PNH
Acidified Serum Test (Ham Test 1939)Acidified Serum Test (Ham Test 1939)
– Acidified serum activates alternative complementAcidified serum activates alternative complement
pathway resulting in lysis of patient’s rbcspathway resulting in lysis of patient’s rbcs
– May be positive in congenitial dyserythropoieticMay be positive in congenitial dyserythropoietic
anemiaanemia
– Still in use todayStill in use today
Sucrose Hemolysis Test (1970)Sucrose Hemolysis Test (1970)
– 10% sucrose provides low ionic strength which10% sucrose provides low ionic strength which
promotes complement binding resulting in lysis ofpromotes complement binding resulting in lysis of
patient’s rbcspatient’s rbcs
– May be positive in megaloblastic anemia,May be positive in megaloblastic anemia,
autoimmune hemolytic anemia, othersautoimmune hemolytic anemia, others
– Less specific than Ham testLess specific than Ham test
19. PNH Diagnosis by Flow Cytometry (1986)PNH Diagnosis by Flow Cytometry (1986)
– Considered method of choice for diagnosisConsidered method of choice for diagnosis
of PNH (1996)of PNH (1996)
– Detects actual PNH clones lacking GPIDetects actual PNH clones lacking GPI
anchored proteinsanchored proteins
– More sensitive and specific than Ham andMore sensitive and specific than Ham and
sucrose hemolysis testsucrose hemolysis test
Laboratory Evaluation of PNHLaboratory Evaluation of PNH
20. PNH Diagnosis by Flow CytometryPNH Diagnosis by Flow Cytometry
AntigenAntigen Cell LineageCell Lineage FunctionFunction
CD14CD14 monocytesmonocytes LPS receptor, MDFLPS receptor, MDF
CD16CD16 neutrophilsneutrophils FcFcγγIII receptorIII receptor
CD24CD24 neutrophilsneutrophils B-cell differentiation markerB-cell differentiation marker
CD55CD55 all lineagesall lineages DAFDAF
CD58CD58 all lineagesall lineages possible adhesionpossible adhesion
CD59CD59 all lineagesall lineages MIRL, HRF, protectinMIRL, HRF, protectin
CD66bCD66b neutrophilsneutrophils CEA-relatedCEA-related
Of the long list of GPI anchored protein, monoclonal antibodies toOf the long list of GPI anchored protein, monoclonal antibodies to
the following antigens have been used in the diagnosis of PNHthe following antigens have been used in the diagnosis of PNH
The most useful Abs are to CD14, 16, 55, 59, and 66. Are allThe most useful Abs are to CD14, 16, 55, 59, and 66. Are all
required? Probably not - more studies neededrequired? Probably not - more studies needed
21. Antigen expression is generally categorized intoAntigen expression is generally categorized into
three antigen density groupsthree antigen density groups
– type I Normal Ag expressiontype I Normal Ag expression
– type II Intermediate Ag expressiontype II Intermediate Ag expression
– type III No Ag expressiontype III No Ag expression
Patient samples that demonstrate cellPatient samples that demonstrate cell
populations with diminished or absent GPI-linkedpopulations with diminished or absent GPI-linked
proteins (Type II or III cells) with multipleproteins (Type II or III cells) with multiple
antibodies are considered to be consistent withantibodies are considered to be consistent with
PNH.PNH.
Should examine multiple lineages (ieShould examine multiple lineages (ie
granulocytes & monocytes)granulocytes & monocytes)
PNH Diagnosis by Flow CytometryPNH Diagnosis by Flow Cytometry
22. PNH Diagnosis by Flow CytometryPNH Diagnosis by Flow Cytometry
Examples of variable GPI linked CD59 expression onExamples of variable GPI linked CD59 expression on
granulocytes on four PNH patientsgranulocytes on four PNH patients
23. PNH Diagnosis by Flow CytometryPNH Diagnosis by Flow Cytometry
Example of variableExample of variable
expression of severalexpression of several
GPI linked Ags onGPI linked Ags on
several lineagesseveral lineages
From Purdue Cytometry CD-ROM vol3 97
24. Flow Cytometry is method of choice but onlyFlow Cytometry is method of choice but only
supportive for/against diagnosissupportive for/against diagnosis
More studies are needed to better define whetherMore studies are needed to better define whether
the type (I, II, or III), cell lineage, and size of thethe type (I, II, or III), cell lineage, and size of the
circulating clone can provide additional prognosticcirculating clone can provide additional prognostic
information.information.
Theoretically - should be very valuableTheoretically - should be very valuable
PNH Diagnosis by Flow CytometryPNH Diagnosis by Flow Cytometry
25. TherapyTherapy
Bone Marrow TransplantationBone Marrow Transplantation
– Only curative treatmentOnly curative treatment
– chronic condition (possiblity of spontaneous remission) -chronic condition (possiblity of spontaneous remission) -
BMT should be avoidedBMT should be avoided
Immunosuppressive therapyImmunosuppressive therapy
– Antilymphocyte globulin &/or cyclosporine AAntilymphocyte globulin &/or cyclosporine A
Does not alter proportion of PNH hemopoiesisDoes not alter proportion of PNH hemopoiesis
– Steroids - experimental - controlled studies ??Steroids - experimental - controlled studies ??
Growth FactorsGrowth Factors
– Some improvementSome improvement
– no evidence that normal clones respond better thanno evidence that normal clones respond better than
PNH clonesPNH clones
