Clinico-cytometric classification of PNH · 2019-03-28 · Clinico-cytometric classification of PNH Definition Clone size (by FCM) Hemolysis BMF Classic (or florid) Large + - PNH

Clinico-cytometric classification of PNH

Definition Clone size

(by FCM)

Hemolysis BMF

Classic (or florid) Large + -

PNH in the

setting of other

BM disorders

Small, unable to

counterbalance

BMF

+ +

Subclinical Minimal - +

Cytometric demonstration of PNH clone is a prerequisite

The cut-off point is 0.01%

(neutrophils are the preferred cells to assess clone size)

Parker, Blood 2005, modified

neutrophils

“RAP-CaRe”Rapid Approach to PNH identification

in Campania Region

1. Gating by CD15 (Lewis X) Typing by FLAER(Fluorescent Aerolysin)

FLAERr-Alexa 488

3

87

7 7

96

74

14

39

14

1 1

35

12,3

0,1

97

0,6

70

4

48

6

29

99

84

0,24

0,46

0

20

40

60

80

100

120

1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27

% o

f cl

on

alce

lls

PNH Testing Situation in Campania between 2011-2013 and 2013-2015

RAP-CaRe

PNH clones

6/1/2011-5/31/2013 6/1/2013-5/31/2015

CenterTests

performedClones Clones/Million

Ceinge / Univ Federico II -Napoli

115 5

3,57Ospedale Cardarelli - Napoli 29 5

Osp. San Gennaro - Napoli 26 1

Osp. Santobono - Napoli 12 0

Osp. S. Sebastiano - Caserta 40 12,23

Osp. SG Moscati - Aversa 59 1

Osp. SG Moscati - Avellino 89 3 6,83

Osp. Ruggi D'Aragona –Salerno

83 24,53

Ospedale Tortora - Pagani 106 3

Osp. Rummo - Benevento 13 0 0

TOTAL 572 21 3,61

Distribution of size classes of PNH3 clones in the national study ClonePNH

Skewness 0.2

0.0

1-0

.1

0.1

-11

-10

10

-20

20

-30

30

-40

40

-50

50

-60

60

-70

70

-80

80

-90

90

-10

0% of clonal cells

A

Skewness 1.1

0.0

1-0

.1

0.1

-1

1-1

0

10

-20

20

-30

30

-40

40

-50

50

-60

60

-70

70

-80

80

-90

90

-10

0

Distribution of size classes of PNH3 clones in the regional study RAP-CaRe

RAP-CARE studyrationale and reagent panel

A sample was sent to our lab for confirmation

Neutrophils

Confirmation test (Borowitz guidelines)

Confirmation test

Confirmation test

1.Screening test was always confirmed by «guideline panel» in RAP-CaRe study(21/21)

2.In retrospective cases, CD15/FLAER assaywas always consistent with «guidelineassay»

3.Screening test was always negative in normal donors and non-PNH patients

September 2006, women, 44y old

Budd Chiari Syndrome

PLT: 100.000/ml

Trilinear bone marrow

Therapy with low MW heparin, then Warfarin

April 2014, Hematological controlWBC 3890/ml, Neutrophils 2330/ml; Hb 13.6 gr/dl, Plt83.000/mlBudd Chiari confirmed.No LDH, no hemolysisSuspect of PNH

Case Report

PNH3 PNH2

Granulocytes

Confirmation test

Monocytes

Red Blood Cells

Eosinophils

PNH2

Lymphocytes

Platelets

RAP CARE-GranulocytesD, 29 anni

Patient suffered from two episodes of atypical thrombosis in abdominal veins

MonocytesD, 29 anni

Red cellsD, 29 anni

LymphocytesD, 29 anni

PlateletsD, 29 anni

No hemolysis, no PNH platelets: subclinical PNH?

A «neutro-less» PNH

Screening panel

Guidelinepanel

Extended investigation panel

CD15FLAER

FLAERCD24CD14CD45CD15CD64

FLAERCD24CD14CD45CD15CD64

GPACD59CD45

GPACD59CD45

CD45R0

CD3CD19CD56

CD61CD55CD59

N

N,M

Ery

N,M

Ery

Eos

L

P

A classification scheme, proposed by the International PNH

Interest Group, includes 3 main categories of PNH:

1. Classical PNH, which includes hemolytic and thrombotic

patients;

2. PNH in the context of other primary bone marrow disorders,

such as aplastic anemia or myelodysplastic syndrome;

3. Subclinical PNH, in which patients have small PNH clones butno clinical or laboratory evidence of hemolysis or thrombosis.

HLAS. Caruso

P. Bruno

G. Ciardiello

Programma di Citometria, Immunologia Cellulare e dei Trapianti

CitometriaG. Scalia

M. Raia

F. Visconte

L. Gentile

V. Cerbone

C. Ossato

V. Cosimato

Manipolazione

Stem CellsA Feliciello

F. Ferruzzi

P. Catapano

CD45-APC-Vio770, human (clone: 5B1)

Brand MILTENYI

Alternative Name LCA; T200

Vol. Per Test 10 µl

Isotype mouse IgG2a

Storage Buffer Aqueous buffered solution containing stabilizer and 0.05% sodium

azide.

Specimen Peripheral Blood in EDTA

Method Summary Stain first, then lyse with ammonium chloride, resuspend in FACS

Flow

Instrument BD FACS CANTO II

Viable cells

Lymphocytes

Monocytes

Platelets and debris

Granulocytes

CD

45-A

PC

-Vio

770-A

SSC-A

CD24 PE Mouse Anti-Human (Clone: ML5)

Brand BD Pharmingen

Alternative Name Heat Stable Antigen Homologue (HSA); Ba-1; CD24A

Vol. Per Test 5 µl

Isotype Mouse IgG2a, κ

Storage Buffer Aqueous buffered solution containing BSA and ≤0.09% sodium

azide.



Flow


Granulocytes CD15+

CD64 PeCy5 Mouse anti-Human (Clone: 22)

Brand Beckman Coulter

Alternative Name receptor for IgG (FcγRI)

Vol. Per Test 10µl

Isotype IgG1, Mouse

Storage Buffer Aqueous buffered solution containing 2 mg/mL BSA and 0.1%

sodium azide.



Flow


Monocytes CD64+

CD33 PeCy7 Mouse anti-Human (Clone: D3HL60.251)


Alternative Name gp67

Vol. Per Test 10µl

Isotype IgG1, Mouse


sodium azide.



Flow


Monocytes CD33++

Brand eBioscience

Vol. Per Test 5µl

Isotype Mouse IgG1, Kappa

Storage Buffer Aqueous buffered solution containing 0.09% sodium azide.


Method Summary Stain first, then lyse with ammonium chloride resuspend in FACS

Flow


CD14 PE-Cyanine7 Mouse Anti-Human (Clone: 61D3)

Monocytes CD64+

Brand BioLegend

Alternative Name Lewis X, 3-FAL, 3-FL, LNFP III, Lex , SSEA-1, X-hapten

Vol. Per Test (5-20)µl

Isotype Mouse IgM, κ

Storage Buffer Aqueous buffered solution

containing 0.2% BSA and 0.09% sodium azide.



Flow


CD15 APC Mouse anti-Human (Clone: HI98)

Granulocytes CD15+

CD235a FITC Mouse anti-Human (Clone: 11E4B-7-6)


Alternative Name Glycophorin A

Vol. Per Test 20µl

Isotype IgG1, Mouse


sodium azide.


Method Summary PB diluted 1:500, then stain and resuspend in FACS Flow


Viable cells

Singletons

CD45-APC-Vio770-AC

D235a F

ITC

-A

Red blood cells

Brand eBIOSCIENCE

Alternative Name Protectin

Vol. Per Test 5µl

Isotype IgG1, kappa

Storage Buffer Aqueous buffered solution containing 0.09% sodium azide, may

contain carrier protein/stabilizer. .


Method Summary PB diluted 1:500, then stain and resuspend in FACS Flow


CD59 PE Mouse Anti-Human (Clone: OV9A2)

Red blood cells

CD

235a F

ITC

-A

CD45-APC-Vio770-A

CD157 PE Mouse Anti-Human (Clone: SY11B5)

Brand eBIOSCIENCE

Alternative Name BST-1, BP-3/IF-7

Vol. Per Test 5µl

Isotype Mouse, IgG1, kappa

Storage Buffer Aqueous buffered solution containing 0.09% sodium azide, may

contain carrier protein/stabilizer .



Flow


Monocytes CD64+

Granulocytes CD15+

Monocytes Granulocytes

Brand LAGITRE

Vol. Per Test 5µl

Storage Buffer Aqueous buffered solution containing sodium azide.



Flow


FLAER Alexa Fluor 488 proaerolysin

Monocytes CD33++

Granulocytes CD15+

FLAER Alexa Fluor 488-A

Count

Granulocytes

FLAER Alexa Fluor 488-A

Count

Monocytes

Target cells Markers Application Source

RBC CD59-PE GPI-RBC MEM43 Invitrogen

RBC GPA-FITC Gating on RBC

KC16 - BC

RBC Typing in PNH

Target cells Markers Application Source

WBC FLAER-Alexa488 GPI Cedarlane

CD24-PE GPI-Neutrophils ALB9-BC

CD14-PE GPI-Monocytes RMO52-BC

CD14-APC700 GPI-Monocytes RMO52-BC

CD157-PE GPI-Monocytesand Neutrophils

SY11B5eBiosciences

CD64-PC5 CD64-ECD CD64-PC7

GatingMonocytes

22-BC22-BC22-BC

CD15-PC5 GatingNeutrophils

80H5-BC

CD45-PC7 Pattern J33-BC

CD45KrO Pattern J33-BC

WBC Typing in PNH

Clinico-cytometric classification of PNH · 2019-03-28 · Clinico-cytometric classification of PNH Definition Clone size (by FCM) Hemolysis BMF Classic (or florid) Large + - PNH

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