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Click to edit Master title style<= /div>
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------=_NextPart_01C70A57.76D1C540 Content-Location: file:///C:/B133CA08/PNH_files/master07.xml Content-Transfer-Encoding: quoted-printable Content-Type: text/xml; charset="utf-8" ------=_NextPart_01C70A57.76D1C540 Content-Location: file:///C:/B133CA08/PNH_files/pres.xml Content-Transfer-Encoding: quoted-printable Content-Type: text/xml; charset="utf-8" ------=_NextPart_01C70A57.76D1C540 Content-Location: file:///C:/B133CA08/PNH_files/slide0001.htm Content-Transfer-Encoding: quoted-printable Content-Type: text/html; charset="us-ascii" Paroxysmal Nocturnal Hemoglobinuria (PNH)
Paroxysmal Nocturnal Hemoglobinuria (PNH)
PNH is an acquired chronic hemolytic anemia which arises from a somatic mutatio= n in a hematopoietic stem cell.  M= ost hematopoitic cell lines may be affected by the intrinsic membrane defect= . This defect renders the red cells highly susceptible to complement mediated <= /b>= lysis resulting in the characteristic hemolysis.
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Paro= xysmal Nocturnal Hemoglobinuria (PNH)
3D=History
3D=Epidemiology
3D=Clinical Features
&= #8211;Relationship to Aplastic Anemia
&= #8211;other diseases
3D=Pathogenesis
3D=Laboratory Diagnosis
3D=Therapy
3D"*"
Topics to be considered
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 H= istory
Investigator   Year        = ;            &n= bsp;       Contribution
<= b>
Gull    1866     Described noctu= rnal and paroxysmal nature of “intermittent haematinuria” in a young = tanner. Strubing         1882 Distinguished PNH from parox= ysmal cold haemoglobinuria and march haemoglobinuria. <= /span>= Attributed the problem to t= he 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 &= #13;
and Micheli= 1931 hemolysis. Their names became eponymous for PNH = in Europe. <= /span>Ham        = ;          1937- Identified the role of compl= ement in lysis of PNH red 1939 cells. Deve= loped the acidified serum test, also called the Ham test, which is stil= l 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<= /div>
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Epidemiology
3D=Rare disease -
&= #8211;frequency unknown
= 211;thought to be on the same order as aplastic anemia (2-6 per million)&= #13;
3D=Median age at diagnosis
&= #8211;~ 35 yrs
&= #8211;PNH reported at extremes of age
3D=Female:Male ratio =3D 1.2:1.0
3D=No increased risk of PNH in patient relatives
3D=Median Survival after diagnosis ~ 10-15 yrs
3D"*"
3D"*"
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Clinical Features
3D"=Major symptoms (Hemolysis, Cytopenia, and tendency to thrombosis)
= 211;chronic hemolysis with acute exacerbations (hallmark)
=3D"*"most patient at some stage
=3D"*"only 1/3 exhibit hemolysis at diagnosis
3D"*"Recurrent attacks of intravascular hemolys= is are usually associated with;
&= #8211;hemoglobinuria
&= #8211;abdominal pain
&= #8211;dysphagia
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&= #8211;cytopenia (varying severity)
=3D"*"isolated subclinical thrombocytopenia
=3D"*"classical severe aplastic anemia
&= #8211;tendency to thrombosis
3D"*"venous thrombosis (40%) of patients, main cause o= f morbidity
3D"=Variable expression of above often causes considerable delay in the diagnosis
3D=Major cause of death
&= #8211;venous thrombosis
&= #8211;complications from progressive pancytopenia
3D"*"
3D"*"
Clinical Features
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3D"=25% of PNH patients survive >25 years - one half of these go on to spontaneous remission
3D=Remission patients
&= #8211;hematological values revert to normal
&= #8211;no PHN rbcs or granulocytes detected
= 211;PNH lymphocytes - still detected but no clinical consequence <= /span>
3D=Higher incidence of acute leukemia (6%)
–“preleukemic condition” most likely bone marrow failure not PNH
Clinical Features - Long term
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3D"*"AA described as pancytopenia w= ith nonfunctioning bone marrow. Cytopenia in one or all cell lineages also common to PNH 3D"*"High percentage of patients with AA develo= p clinical PNH or have lab evidence of PNH abnormality at some point (52%) 3D"*"Supports the theory that bone marrow failu= re supports the abnormal PNH cells - more later
Clinical Features - Relationship to aplastic anemia (AA)
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Pat= hogenesis - The Defect
3D"=Defect - Somatic mutation of PIG-A gene (phosphatidylinositol glyc= an complementation group A)  located on the X chromosome in a clone of a hematopoietic stem cell
&#= 8211;>100 mutations in PIG - A gene known in PNH
= 211;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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3D"*"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 <= /span>turn, attached proteins
<= span style=3D'font-size:88%;visibility:hidden'>3D"*"
Pat= hogenesis - The Defect
GPI Anchor
PIG - A protein<= span style=3D'mso-special-format:lastCR;display:none'>
Protein
Phosphoethanolamine
Mannose
Mannose
Glucosamine
Mannose
Phosphatidylinositol
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PNH blood cells deficient in GPI anchor lack <= span style=3D'font-size:88%'>membrane proteins linked via the anchor
–M= embrane proteins w/o anchor degraded in ER
Severity & size of deficiency - variable - <= span style=3D'font-size:88%'>clinical/diagnostic implications
= 3D"*"GPI anchor highly conserved in all eukaryotic cells
–Va= riant surface proteins of Trypanosomes - GPI linked
–S= hed by cleavage of GPI anchor - immune system avoid
–S= wapping GPI linked proteins - CD55 complement resistance - = Schistosoma mansoni
–In Humans
3D"*"signal transduction, co-receptors
3D"*"advantage to this type of anchor?
3D"*"
Pathogenesis - The Defect
GPI Anchor deficiency
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Proteins anchored by GPI Anchor
and=
Surface Proteins Missing on PNH Blood Cells
<= b>
Antigen<= span style=3D'mso-spacerun:yes'>        = ;            &n= bsp;            = ;            &n= bsp;            = ;       Expression Pattern
<= b>
Enzymes<= /u><= b>
Acetylcholi= nesterase (AchE)        = ;       Red blood cell= s
Ecto-5'-nuc= leotidase (CD73)       Some B- and T-lymphocytes
Neutrophil alkaline phosphatase(NAP) Neutrophils
ADP-rybosyl transferase     Some T-lymphs, Neutrophils&= #13;
<= b>
Adhesion molecules
Blast-I/CD4= 8        = ;            &n= bsp;         Lymphocytes= 3;
Lymphocyte function-
associated antigen-3(LFA-3 or CD58) All blood cells
CD66b Neutrophils =
<= b>
<= b>
Compleme= nt regulating surface proteins
Decay accelerating factor (DAF or CD55)        = ;    All blood cells&= #13;
Homologous restriction factor, &#= 13;
Membrance inhibitor of reactive lysis = All blood cells
(MIRL or CD= 59)
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Surface Proteins Missing on PNH Blood Cells
<= b>
Antigen<= span style=3D'mso-spacerun:yes'>        = ;            &n= bsp;            = ;            &n= bsp;            = ;       Expression Pattern
<= b>
Receptor= s<= b>
Fc-g receptor III (Fc g Rlll or CD16)        Neutrophils, NK-cells, macrophages,
some T-lymphocytes =
Monocyte differentiation antigen Monocytes, macrophages
(CD14) =
Urokinase-t= ype Plasminogen Monocytes, granulocytes
Activator Receptor (u-PAR, CD87)
<= b>
Blood gr= oup antigens
Comer antig= ens (DAF)        = ;            &n= bsp;       Red blood cells=
Yt antigens (AchE)        = ;            &n= bsp;      Red blood cells=
Holley Greg= ory antigen        = ;            Red blood cells=
John Milton= Hagen antigen (JMH)    Red blood cells, lymphocytes&= #13;
Dombrock reside        = ;            &n= bsp;   Red blood cell= s
<= b>
Neutroph= il antigens
NB1/NB2        = ;            &n= bsp;            = ;          Neutrophils=
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Surface Proteins Missing on PNH Blood Cells
<= b>
Antigen<= span style=3D'mso-spacerun:yes'>        = ;            &n= bsp;            = ;            &n= bsp;            = ;       Expression Pattern
<= b>
Other su= rface proteins
of unkno= wn functions
CAMPATH-1 a= ntigen (CDw52)        = ;            &n= bsp; Lymphocytes, monocytes
CD24 B-lymphocytes, Neutrophils,=
eosinophils =
p5O-80 Neutrophils =
GP500        = ;            &n= bsp;            = ;       Platelets =
GPI75        = ;            &n= bsp;    Platelets <= /b>
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Pathogenesis - Functional consequences of lack of GPI linked proteins
<= img src=3Dimage003.gif alt=3D"*" style=3D'position:absolute;top:23.8%;left:-4.= 02%; width:2.41%;height:57.14%'>In vivo function of many = of these membrane proteins not fully understood
= 3D"*"However, CD55 and CD59 functions are well known
–CD55 (decay accelerating factor) inhibits the form= ation or destabilizes compl= ement C3 convertase (C4bC2a)
–CD59 (memb= rane inhibitor of reactive lysis, protectin, homologous restriction factor) Protects the membra= ne from attack by the C5-C9 complex
–Inherited absences of both proteins in humans have= been described
<= img src=3Dimage004.gif alt=3D"*" style=3D'position:absolute;top:18.75%;left:-3= .31%; width:2.29%;height:56.25%'>Most inherited deficiencies of CD55 - no distinct clinical hemolytic syndrome
= 3D"*"Inherited absence of CD59 - produces a clinical disease similar to PNH with hemolysis and recurrent thrombotic eve= nts
------=_NextPart_01C70A57.76D1C540 Content-Location: file:///C:/B133CA08/PNH_files/slide0016.htm Content-Transfer-Encoding: quoted-printable Content-Type: text/html; charset="us-ascii" Paroxysmal Nocturnal Hemoglobinuria (PNH)
C5b-8
RBC Membrane
Protectin (CD59)
<= /span>
= <= /span>
<= /span>
= <= /span>
C9 unable to
insert in catalytically
active form
Mechanism for hemolysis in PNH via lack of CD59
3D"Oval:
C5b-8
RBC Membrane
C9 able to
insert in catalytically
active form and polymerize
<= /span>
= <= /span>
= <= /span>
= <= /span>
<= span style=3D'color:black;mso-special-format:lastCR;display:none'> <= /span>
<= /span>
<= /span>
<= span style=3D'color:black;mso-special-format:lastCR;display:none'> <= /span>
= <= /span>
= <= /span>
= NORMAL
P= NH
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R0lGODlhGgIEAHcAMSH+GlNvZnR3YXJlOiBNaWNyb3NvZnQgT2ZmaWNlACH5BAEAAAAALAIAAQAW AgMAgAAAAP///wInjI+py+0Po5y02ouz3rz7D4biSJbmiabqyrbuC8fyTNf2jef6zisFADs= ------=_NextPart_01C70A57.76D1C540 Content-Location: file:///C:/B133CA08/PNH_files/slide0017.htm Content-Transfer-Encoding: quoted-printable Content-Type: text/html; charset="us-ascii" Paroxysmal Nocturnal Hemoglobinuria (PNH)
Pathogenesis - Clonal evolution and cellular selection
<= img src=3Dimage003.gif alt=3D"*" style=3D'position:absolute;top:23.8%;left:-3.= 74%; width:2.24%;height:57.14%'>Expansion of abnormal hematopoietic stem cell required = for PNH disease expression
–T= heories for expansion
<= img src=3Dimage004.gif alt=3D"*" style=3D'position:absolute;top:18.75%;left:-2= .74%; width:1.89%;height:56.25%'>Blood cells lacking GPI-linked proteins have intrinsic ability to gro= w abnormally fast
–In vitro  growth studies demonstrate that t= here 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.
<= img src=3Dimage004.gif alt=3D"*" style=3D'position:absolute;top:25.0%;left:-2.= 72%; width:1.88%;height:56.25%'>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 phenot= ypically hematopoitic cells
–Close association with AA - PNH hematopoitic cells= cells may be more resist= ant to the IS than normal hematopoitic cells.
<= img src=3Dimage013.gif alt=3D"*" style=3D'position:absolute;top:21.42%;left:-3= .12%; width:1.92%;height:57.14%'>Evidence in AA is that the decrease in hematopoitic cells is due to <= /span>increased apoptosis via cytotoxic T cells by direc= t cell contact or cytokines (escape = via deficiency in GPI linked protein???) 
------=_NextPart_01C70A57.76D1C540 Content-Location: file:///C:/B133CA08/PNH_files/image013.gif Content-Transfer-Encoding: base64 Content-Type: image/gif R0lGODlhDwAPAHcAACH/C01TT0ZGSUNFOS4wDQAAAAFzUkdCAK7OHOkAIf8LTVNPRkZJQ0U5LjAY AAAADG1zT1BNU09GRklDRTkuMAlmS20eACH/C01TT0ZGSUNFOS4wGAAAAAxjbVBQSkNtcDA3MTIC AAAFC2oeAgAh+QQBAAACACwAAAAADwAPAIOZZplmZmbAwMCZmcxmmZmZmWZmmWaZzJnMmZkBAgMB AgMBAgMBAgMBAgMBAgMBAgMESlDISau9OE8wCigEYABBOR1FFxplOyEDEhYB6UrDQSAgzZoSmOzT uglyu54NKBASVoFfYNMBEJdTCarTixqd1+IEySP4vrFZrRgBADs= ------=_NextPart_01C70A57.76D1C540 Content-Location: file:///C:/B133CA08/PNH_files/slide0018.htm Content-Transfer-Encoding: quoted-printable Content-Type: text/html; charset="us-ascii" Paroxysmal Nocturnal Hemoglobinuria (PNH)
Lab= oratory Evaluation of PNH
= 3D"*"Acidified Serum Test (Ham Test 1939)
–A= cidified serum activates alternative complement pathway resulting in lysis of patient’s rbcs=
–May be positive in congenitial dyserythropoietic <= /span>anemia
–S= till in use today
= 3D"*"Sucrose Hemolysis Test (1970)
–10% sucrose provides low ionic strength which promotes complement binding resulting in lysis of = patient’s rbcs
–M= ay be positive in megaloblastic anemia, autoimmune hemolytic anemia, others
–L= ess specific than Ham test
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= 3D"*"PNH Diagnosis by Flow Cytometry (1986)
–Considered method of choice for diagnosis of PNH (1996) =
= 211;Detects actual PNH clones lacking GPI anchored proteins
–More sensitive and specific than Ham and sucrose hemolysis test = ;
–
Lab= oratory Evaluation of PNH
------=_NextPart_01C70A57.76D1C540 Content-Location: file:///C:/B133CA08/PNH_files/slide0020.htm Content-Transfer-Encoding: quoted-printable Content-Type: text/html; charset="us-ascii" Paroxysmal Nocturnal Hemoglobinuria (PNH)
PNH Diagnosis by Flow Cytometry
Antigen <= /u>Cell Lineage Function
CD14 monocytes LPS receptor, MDF
CD16 neutrophils FcgIII receptor
CD24 neutrophils B-cell differentiation marker
CD55 all lineages DAF
CD58 all lineages possible adhe= sion
CD59 all lineages MIRL, HRF, protectin <= /div>
CD66b neutrophils CEA-related glycoprotein
Of the long list of GPI anchored protein, monoclonal antibodies to the following antigens have been used in the diagnosis of PNH <= /span>
The most useful Abs are to C= D14, 16, 55, 59, and 66.  Are all = required?  Probably not - more studies neede= d <= /span>
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Antigen expression is generally categorized into three antigen density groups
–t= ype I Normal Ag expression
–t= ype II Intermediate Ag expression
–t= ype III No Ag expression
3D"*"= Patient samples that demonstrate cell populations with diminished or absent GPI-linked <= /span>proteins (Type II or III cells) with multiple antibodies are considered to be consistent with = PNH.
3D"*"= Should examine multiple lineages (ie = granulocytes & monocytes)
3D"*"=
3D"*"=
PNH Diagnosis by Flow Cytometry
------=_NextPart_01C70A57.76D1C540 Content-Location: file:///C:/B133CA08/PNH_files/slide0022.htm Content-Transfer-Encoding: quoted-printable Content-Type: text/html; charset="us-ascii" Paroxysmal Nocturnal Hemoglobinuria (PNH)
PNH Diagnosis by Flow Cytometry
Examples of variable GPI linked CD59 and CD55 expression= on granulocytes on PNH patient = and normal control
Healthy
Control
PNH
Patient
Type I
Type II
Normal
expression
Normal
expression
Type II
Type I
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