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CEINGE – Biotecnologie AvanzateDipartimento di Biochimica e Biotecnologie Mediche
Università Federico II, Napoli
Ruolo della citometrianello studio del clone
EPN
Luigi Del VecchioFirenze, 25 novembre 2010
PNH PATHOGENESISPIG-Agene63 552 41
Cytoplasm
Transmembraneprotein
COOH
GPI-AnchoredProtein
NH2
COOH
NH2
Extracellularspace
PHOSPHO-ETHANOLAMINE
C=OC=O
O=P-O
O
O-
O
CH2
INOS
GLUMAN
C=O
N
CH2
CH2
NH
O-P-O-
O
O
MAN
PHOSPHATIDYL-INOSITOL
GLYCAN CORE
PROTEIN
MAN
H2CHC
O
O
O O
(α 1-2)(α 1-6)
(α 1-4)
GPI-anchor
GPI Linked Proteins in Blood CellsCD59, CD90, CD109
CD48CD55CD58CD59CD52
CD48CD24CD73CD55CD58CD59CD108
CD55CD58CD59CD109
CD59CD55CD58
CD24CD66bCD16CD66cCD87CD55CD58CD59CD48CD109CD157
CD14 CD55 CD58 CD59CD48 CD52 CD87 CD109
CD157 CD16
CD48CD52CD73CD55CD58CD59CD87CD108CD109
Stem Cell
NK cells
PMN
PlateletsMonocytes
B cells
T cells
RBC
PATHOPHYSIOLOGY OF PNHThe dual hypothesis (Rotoli and Luzzatto, Baillieres Clin Haematol 1989)
Mutazioni fisiologiche Danno autoimmune
Prevalenza del clone Espansione del clone
1. Chronic hemolytic anemia with paroxistic crises
Intravascular hemolysis, complement mediated
2. Propensity to thromboembolisms
Often at unusual site, especially veins (cerebralveins, hepatic veins, splenic vein)
THE CLINICAL TRIAD OF PNH
3. Variable cytopenia
Deficiency of CD55/CD59
As a consequence of hemolysis. Deficiency of CD87on neutrophils and of CD55/CD59 on PLT.
Condition allowing the growth ofPNH clone
L.Luzzatto 1996
Fate of PNH clone
What patients to test for PNH?
Usually 3 types of patients: Cytopenias
• Genuine aplastic anaemia (established on BM trephine biopsy)• Pancytopenia/neutropenia/isolated cytopenia
Thrombosis• Budd-Chiari, hepatic/mesenteric vein thrombosis• Other more likely causes of thrombosis must be investigated
Haemolysis/anaemia• Intravascular haemolysis and haemoglobinuria (paroxysmal &
nocturnal!)
PNH screening by flow cytometryMain cell lineages
Red blood cellsGranulocytes and monocytes
a
b
RBC analysis
Log amplification for FSC/SSC MoAbs to CD59 (MEM43)
granulocytes
Monocytes
ColorsLab Cells First Second Third Fourth Fifth Sixth Gating strategy
3 color G FLAER CD24 CD15 CD15/SSChi
3 color M FLAER CD14 CD33 CD33hi/SSCint
4 color G FLAER CD24 CD15 CD45 CD15hi/SSChi
4 color M FLAER CD14 CD33 CD45 CD33hi/SSClo
4 color G+M FLAER CD24 CD14 CD33 CD33hi/SSClo
5 color G+M FLAER CD24 CD14 CD15 CD33 CD15hi/CD33low/SSChi
(grans) andCD33hi/SSCint (monos)
6 color G+M FLAER CD24 CD14 CD15 CD33 CD45 CD15hi/CD45int/SSChi/CD33lo (grans) and
CD45hi/SSCint/CD33hi
(monos)
L. Del Vecchio, 2010
Assessment of PNH clone size
Red cell clone sizea) Type III and/or Type II red cellsb) Proportion of PNH red cells is related to
recent transfusion and hemolysis1. PNH granulocytes
a) More accurate reflection of PNH clone size2. PNH monocytes
a) Validates the size of clone
Normal cases –report normal expression ofGPI-linked antigens – no PNH clonesdetected.
New cases – report GPI deficiency, celllineages tested & PNH clone sizes.
Monitoring – report PNH clone sizes and anychanges
Proposal of reporting format in ClonePNHMagazine (www.clonePnh.com)
Cut-off point?
Reporting results
Classification of PNHDefinition Haemolysis BMF
Classic PNH + -PNH in the setting ofother BM disorders
+ +Subclinical PNH - +
Detection of PNH clone is cytometric,but final diagnosis is not only cytometric
www.clonePnh.com 26 ottobre 2010