Paroxysmal Nocturnal Paroxysmal Nocturnal Hemoglobinuria (PNH)Hemoglobinuria (PNH)

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

Paroxysmal Nocturnal Hemoglobinuria Paroxysmal 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

HistoryHistoryInvestigator Year ContributionInvestigator Year Contribution

Gull Gull 1866 Described nocturnal and paroxysmal nature of 1866 Described nocturnal and paroxysmal nature of “intermittent haematinuria” in a young “intermittent haematinuria” in a young

tanner.tanner.Strubing Strubing 18821882 Distinguished PNH from paroxysmal cold Distinguished PNH from paroxysmal cold

haemoglobinuria and march haemoglobinuria and march haemoglobinuria. haemoglobinuria. Attributed the problem to Attributed the problem to the red cells. the red cells. van den Burgh van den Burgh 1911 1911 Red cells lysed in acidified serum. Red cells lysed in acidified serum. Suggested a role Suggested a role for complement.for complement.Enneking Enneking 1928 1928 Coined the name “paroxysmal Coined the name “paroxysmal nocturnal nocturnal haemoglobinuria”.haemoglobinuria”.Marchiafava Marchiafava 1928- 1928- Described perpetual hemosiderinemia in Described perpetual hemosiderinemia in absence of absence of and Micheli and Micheli 19311931 hemolysis. Their names became eponymous for hemolysis. Their names became eponymous for PNH PNH in Europe.in Europe.Ham Ham 1937-1937- Identified the role of complement in Identified the role of complement in lysis of PNH red lysis of PNH red 19391939 cells. Developed the acidified cells. Developed the acidified serum test, also called serum test, also called the Ham test, which is still the Ham test, which is still used to diagnose PNH. used to diagnose PNH. Demonstrated that only a Demonstrated that only a portion of PNH red cells are portion of PNH red cells are abnormally abnormally sensitive to complement.sensitive to complement.DavitzDavitz 19861986 Suggests defect in membrane protein Suggests defect in membrane protein anchoring anchoring system responsiblesystem responsibleHall & RosseHall & Rosse 19961996 Flow cytometry for the diagnosis of PNHFlow cytometry for the diagnosis of PNH

EpidemiologyEpidemiology Rare disease - Rare disease -

– frequency unknownfrequency unknown– thought to be on the same order as aplastic thought to be on the same order as aplastic

anemia (2-6 per million)anemia (2-6 per million) Median age at diagnosis Median 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 No increased risk of PNH in patient

relativesrelatives Median Survival after diagnosis ~ 10-15 Median Survival after diagnosis ~ 10-15

yrsyrs

Clinical FeaturesClinical Features

Major symptoms (Hemolysis, Major symptoms (Hemolysis, Cytopenia, and tendency to Cytopenia, and tendency to thrombosis)thrombosis)– chronic hemolysis with acute chronic hemolysis with acute

exacerbations (hallmark)exacerbations (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 Recurrent attacks of intravascular

hemolysis are usually associated with;hemolysis are usually associated with;– hemoglobinuriahemoglobinuria– abdominal painabdominal pain– dysphagiadysphagia

– 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 venous thrombosis (40%) of patients, main cause

of morbidityof morbidity

Variable expression of above often Variable expression of above often causes considerable delay in the causes considerable delay in the diagnosisdiagnosis

Major cause of deathMajor cause of death– venous thrombosisvenous thrombosis– complications from progressive pancytopeniacomplications from progressive pancytopenia

Clinical FeaturesClinical Features

25% of PNH patients survive >25 years - 25% of PNH patients survive >25 years - one half of these go on to spontaneous one half of these go on to spontaneous remissionremission

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 clinical PNH lymphocytes - still detected but no clinical

consequenceconsequence Higher incidence of acute leukemia (6%)Higher incidence of acute leukemia (6%)

– ““preleukemic condition” most likely bone preleukemic condition” most likely bone marrow failure not PNH marrow failure not PNH

Clinical Features - Long termClinical Features - Long term

AA described as pancytopenia with AA described as pancytopenia with nonfunctioning bone marrow. Cytopenia nonfunctioning bone marrow. Cytopenia in one or all cell lineages also common to in one or all cell lineages also common to PNHPNH

High percentage of patients with AA High percentage of patients with AA develop clinical PNH or have lab evidence develop clinical PNH or have lab evidence of PNH abnormality at some point (52%)of PNH abnormality at some point (52%)

Supports the theory that bone marrow Supports the theory that bone marrow failure supports the abnormal PNH cells - failure supports the abnormal PNH cells - more latermore later

Clinical Features - Relationship Clinical Features - Relationship to aplastic anemia (AA)to aplastic anemia (AA)

Pathogenesis - The DefectPathogenesis - The Defect

Defect - Somatic mutation of Defect - Somatic mutation of PIG-APIG-A gene gene (phosphatidylinositol glycan (phosphatidylinositol glycan complementation group A) located on complementation group A) located on the X chromosome in a clone of a the X chromosome in a clone of a hematopoietic stem cell hematopoietic stem cell – >100 mutations in >100 mutations in PIG - APIG - A gene known in gene known in

PNHPNH– The mutations (mostly deletions or The mutations (mostly deletions or

insertions) generally result in stop codons - insertions) generally result in stop codons - yielding truncated proteins which may be yielding truncated proteins which may be non or partially functional - explains non or partially functional - explains heterogeneity seen in PNHheterogeneity seen in PNH

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

Pathogenesis - The DefectPathogenesis - The Defect

GPI GPI AnchorAnchor

PIG - APIG - A protein protein

PNH blood cells deficient in GPI anchor lack PNH 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 ER Membrane 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 complement Swapping 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 DefectGPI Anchor deficiencyGPI Anchor deficiency

Proteins anchored by GPI AnchorProteins anchored by GPI Anchorandand

Surface Proteins Missing on PNH Blood CellsSurface Proteins Missing on PNH Blood Cells

Antigen Expression PatternAntigen Expression Pattern

EnzymesEnzymesAcetylcholinesterase (AchE) Acetylcholinesterase (AchE) Red blood cellsRed blood cellsEcto-5'-nucleotidase (CD73) Ecto-5'-nucleotidase (CD73) Some B- and T-Some B- and T-lymphocyteslymphocytesNeutrophil alkaline phosphatase(NAP) Neutrophil alkaline phosphatase(NAP) NeutrophilsNeutrophilsADP-rybosyl transferase ADP-rybosyl transferase Some T-lymphs, Some T-lymphs, NeutrophilsNeutrophils

Adhesion moleculesAdhesion moleculesBlast-I/CD48 Blast-I/CD48 LymphocytesLymphocytesLymphocyte function- Lymphocyte function- associated antigen-3(LFA-3 or CD58) associated antigen-3(LFA-3 or CD58) All blood cellsAll blood cellsCD66bCD66b NeutrophilsNeutrophils

Complement regulating surface proteinsComplement regulating surface proteinsDecay accelerating factor (DAF or CD55) Decay accelerating factor (DAF or CD55) All blood cellsAll blood cellsHomologous restriction factor,Homologous restriction factor,Membrance inhibitor of reactive lysis Membrance inhibitor of reactive lysis All blood cellsAll blood cells(MIRL or CD59)(MIRL or CD59)

Surface Proteins Missing on PNH Blood CellsSurface Proteins Missing on PNH Blood Cells

Antigen Expression PatternAntigen Expression Pattern

ReceptorsReceptorsFc-Fc- receptor III (Fc receptor III (Fc Rlll or CD16) Rlll or CD16) Neutrophils, NK-cells, Neutrophils, NK-cells, macrophages,macrophages,

some T-lymphocytessome T-lymphocytesMonocyte differentiation antigen Monocyte differentiation antigen Monocytes, macrophagesMonocytes, macrophages(CD14)(CD14)Urokinase-type Plasminogen Urokinase-type Plasminogen Monocytes, granulocytesMonocytes, granulocytesActivator Receptor (u-PAR, CD87)Activator Receptor (u-PAR, CD87)

Blood group antigensBlood group antigensComer antigens (DAF) Comer antigens (DAF) Red blood cellsRed blood cellsYt antigens (AchE) Yt antigens (AchE) Red blood cellsRed blood cellsHolley Gregory antigen Holley Gregory antigen Red blood cellsRed blood cellsJohn Milton Hagen antigen (JMH) John Milton Hagen antigen (JMH) Red blood cells, lymphocytesRed blood cells, lymphocytesDombrock reside Dombrock reside Red blood cellsRed blood cells

Neutrophil antigensNeutrophil antigensNB1/NB2 NB1/NB2 NeutrophilsNeutrophils

Surface Proteins Missing on PNH Blood CellsSurface Proteins Missing on PNH Blood Cells

Antigen Expression PatternAntigen Expression Pattern

Other surface proteins Other surface proteins of unknown functionsof unknown functionsCAMPATH-1 antigen (CDw52) CAMPATH-1 antigen (CDw52) Lymphocytes, Lymphocytes, monocytesmonocytesCD24 CD24 B-lymphocytes, B-lymphocytes, Neutrophils, Neutrophils,

eosinophilseosinophilsp5O-80p5O-80 NeutrophilsNeutrophilsGP500 GP500 PlateletsPlateletsGPI75 GPI75 PlateletsPlatelets

Pathogenesis - Functional consequences of Pathogenesis - Functional consequences of lack of GPI linked proteinslack of GPI linked proteins

In vivoIn vivo function of many of these membrane function of many of these membrane proteins not fully understoodproteins not fully understood

However, CD55 and CD59 functions are well However, CD55 and CD59 functions are well knownknown– CD55 (decay accelerating factor) inhibits the CD55 (decay accelerating factor) inhibits the

formation or destabilizes complement C3 convertase formation or destabilizes complement C3 convertase (C4bC2a)(C4bC2a)

– CD59 (membrane inhibitor of reactive lysis, CD59 (membrane inhibitor of reactive lysis, protectin, homologous restriction factor) Protects the protectin, homologous restriction factor) Protects the membrane from attack by the C5-C9 complexmembrane from attack by the C5-C9 complex

– Inherited absences of both proteins in humans have Inherited absences of both proteins in humans have been describedbeen described

Most inherited deficiencies of CD55 - no distinct clinical Most inherited deficiencies of CD55 - no distinct clinical hemolytic syndromehemolytic syndrome

Inherited absence of CD59 - produces a clinical disease Inherited absence of CD59 - produces a clinical disease similar to PNH with hemolysis and recurrent thrombotic similar to PNH with hemolysis and recurrent thrombotic eventsevents

Mechanism for hemolysis in PNH via lack of CD59Mechanism for hemolysis in PNH via lack of CD59

(CD59)

(CD59)

Pathogenesis - Clonal evolution and Pathogenesis - Clonal evolution and cellular selectioncellular selection

Expansion of abnormal hematopoietic stem cell Expansion of abnormal hematopoietic stem cell required for PNH disease expression required for PNH disease expression – Theories for expansionTheories for expansion

Blood cells lacking GPI-linked proteins have intrinsic ability to Blood cells lacking GPI-linked proteins have intrinsic ability to grow abnormally fastgrow abnormally fast

– In vitroIn vitro growth studies demonstrate that there are no differences in growth studies demonstrate that there are no differences in growth between normal progenitors and PNH phenotype growth between normal progenitors and PNH phenotype progenitorsprogenitors

– In vivoIn vivo - mice deficient for - mice deficient for PIG -A PIG -A gene also demonstrates no gene also demonstrates no growth advantage to repopulation of BM.growth advantage to repopulation of BM.

Additional environmental factors exert selective pressure in Additional environmental factors exert selective pressure in favor of expansion of GPI anchor deficient blood cellsfavor of expansion of GPI anchor deficient blood cells

– PNH hematopoitic cells perferentially engraft SCID mice compared PNH hematopoitic cells perferentially engraft SCID mice compared to phenotypically hematopoitic cellsto phenotypically hematopoitic cells

– Close association with AA - PNH hematopoitic cells cells may be Close association with AA - PNH hematopoitic cells cells may be more resistant to the IS than normal hematopoitic cells. more resistant to the IS than normal hematopoitic cells.

Evidence in AA is that the decrease in hematopoitic cells is due Evidence in AA is that the decrease in hematopoitic cells is due to increased apoptosis via cytotoxic T cells by direct cell to increased apoptosis via cytotoxic T cells by direct cell contact or cytokines (escape via deficiency in GPI linked contact or cytokines (escape via deficiency in GPI linked protein???) protein???)

Laboratory Evaluation of PNHLaboratory Evaluation of PNH

Acidified Serum Test (Ham Test 1939)Acidified Serum Test (Ham Test 1939)– Acidified serum activates alternative Acidified serum activates alternative

complement pathway resulting in lysis of complement pathway resulting in lysis of patient’s rbcspatient’s rbcs

– May be positive in congenitial dyserythropoietic May 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 which 10% sucrose provides low ionic strength which promotes complement binding resulting in lysis promotes complement binding resulting in lysis of patient’s rbcsof patient’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

PNH Diagnosis by Flow Cytometry PNH Diagnosis by Flow Cytometry (1986)(1986)– Considered method of choice for Considered method of choice for

diagnosis of PNH (1996) diagnosis of PNH (1996) – Detects actual PNH clones lacking GPI Detects actual PNH clones lacking GPI

anchored proteinsanchored proteins– More sensitive and specific than Ham More sensitive and specific than Ham

and sucrose hemolysis testand sucrose hemolysis test

Laboratory Evaluation of PNHLaboratory Evaluation of PNH

PNH Diagnosis by Flow CytometryPNH Diagnosis by Flow Cytometry

AntigenAntigen Cell LineageCell Lineage FunctionFunction

CD14CD14 monocytesmonocytes LPS receptor, MDFLPS receptor, MDF

CD16CD16 neutrophilsneutrophils FcFcIII receptorIII receptor

CD24CD24 neutrophilsneutrophils B-cell differentiation B-cell differentiation markermarker

CD55CD55 all lineagesall lineages DAFDAF

CD58CD58 all lineagesall lineages possible adhesionpossible adhesion

CD59CD59 all lineagesall lineages MIRL, HRF, protectinMIRL, HRF, protectin

CD66bCD66b neutrophilsneutrophils CEA-related CEA-related glycoproteinglycoprotein

Of the long list of GPI anchored protein, monoclonal Of the long list of GPI anchored protein, monoclonal antibodies to the following antigens have been used in antibodies to the following antigens have been used in the diagnosis of PNHthe diagnosis of PNH

The most useful Abs are to CD14, 16, 55, 59, and 66. The most useful Abs are to CD14, 16, 55, 59, and 66. Are all required? Probably not - more studies needed Are all required? Probably not - more studies needed

Antigen expression is generally categorized Antigen expression is generally categorized into three antigen density groupsinto three 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 cell Patient samples that demonstrate cell populations with diminished or absent GPI-populations with diminished or absent GPI-linked proteins (Type II or III cells) with linked proteins (Type II or III cells) with multiple antibodies are considered to be multiple antibodies are considered to be consistent with PNH.consistent with PNH.

Should examine multiple lineages (ie Should examine multiple lineages (ie granulocytes & monocytes)granulocytes & monocytes)

PNH Diagnosis by Flow CytometryPNH Diagnosis by Flow Cytometry

PNH Diagnosis by Flow CytometryPNH Diagnosis by Flow CytometryExamples of variable GPI linked CD59 expression on Examples of variable GPI linked CD59 expression on granulocytes on four PNH patientsgranulocytes on four PNH patients

PNH Diagnosis by Flow CytometryPNH Diagnosis by Flow Cytometry

Example of variable Example of variable expression of expression of several GPI linked several GPI linked Ags on several Ags on several lineageslineages

From Purdue Cytometry CD-ROM vol3 97

Flow Cytometry is method of choice but only Flow Cytometry is method of choice but only supportive for/against diagnosissupportive for/against diagnosis

More studies are needed to better define More studies are needed to better define whether the type (I, II, or III), cell lineage, whether the type (I, II, or III), cell lineage, and size of theand size of thecirculating clone can provide additional circulating clone can provide additional prognosticprognosticinformation.information.

Theoretically - should be very valuableTheoretically - should be very valuable

PNH Diagnosis by Flow CytometryPNH Diagnosis by Flow Cytometry

TherapyTherapy

Bone Marrow TransplantationBone Marrow Transplantation– Only curative treatmentOnly curative treatment– chronic condition (possiblity of spontaneous chronic condition (possiblity of spontaneous

remission) - BMT should be avoidedremission) - BMT 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 no evidence that normal clones respond better

than PNH clonesthan PNH clones