Clinico-cytometric classification of PNH

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2 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

3 RAP-CaRe Rapid Approach to PNH identification in Campania Region neutrophils FLAERr-Alexa Gating by CD15 (Lewis X) Typing by FLAER (Fluorescent Aerolysin)

4 % of clonal cells PNH Testing Situation in Campania between and /1/2011-5/31/2013 6/1/2013-5/31/ RAP-CaRe , ,1 0,6 0,2 0, PNH clones

5 Center Ceinge / Univ Federico II - Napoli Tests performed Clones Ospedale Cardarelli - Napoli 29 5 Osp. San Gennaro - Napoli 26 1 Osp. Santobono - Napoli 12 0 Osp. S. Sebastiano - Caserta 40 1 Osp. SG Moscati - Aversa 59 1 Clones/Million 3,57 2,23 Osp. SG Moscati - Avellino ,83 Osp. Ruggi D'Aragona Salerno 83 2 Ospedale Tortora - Pagani Osp. Rummo - Benevento ,53 TOTAL ,61

6 Distribution of size classes of PNH3 clones in the national study ClonePNH Skewness 0.2 A % of clonal cells

7 Distribution of size classes of PNH3 clones in the regional study RAP-CaRe Skewness 1.1

8 RAP-CARE study rationale and reagent panel A sample was sent to our lab for confirmation

9 Confirmation test (Borowitz guidelines) Neutrophils

10 Confirmation test

11 Confirmation test 1.Screening test was always confirmed by «guideline panel» in RAP-CaRe study (21/21) 2.In retrospective cases, CD15/FLAER assay was always consistent with «guideline assay» 3.Screening test was always negative in normal donors and non-pnh patients

12 Case Report September 2006, women, 44y old Budd Chiari Syndrome PLT: /ml Trilinear bone marrow Therapy with low MW heparin, then Warfarin April 2014, Hematological control WBC 3890/ml, Neutrophils 2330/ml; Hb 13.6 gr/dl, Plt /ml Budd Chiari confirmed. No LDH, no hemolysis Suspect of PNH

13 Granulocytes PNH3 PNH2 Confirmation test

14 Monocytes

15 Red Blood Cells

16 Eosinophils PNH2

17 Lymphocytes

18 Platelets

19 D, 29 anni RAP CARE-Granulocytes Patient suffered from two episodes of atypical thrombosis in abdominal veins

20 D, 29 anni Monocytes

21 D, 29 anni Red cells

22 D, 29 anni Lymphocytes

23 D, 29 anni Platelets No hemolysis, no PNH platelets: subclinical PNH?

24 A «neutro-less» PNH

25 Screening panel Guideline panel Extended investigation panel CD15 FLAER N FLAER CD24 CD14 CD45 CD15 CD64 GPA CD59 CD45 N,M Ery FLAER CD24 CD14 CD45 CD15 CD64 CD45R0 CD3 CD19 CD56 GPA CD59 CD45 N,M Eos L Ery CD61 CD55 CD59 P

26 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 but no clinical or laboratory evidence of hemolysis or thrombosis.

27 Programma di Citometria, Immunologia Cellulare e dei Trapianti Citometria G. Scalia M. Raia F. Visconte L. Gentile V. Cerbone C. Ossato V. Cosimato HLA S. Caruso P. Bruno G. Ciardiello Manipolazione Stem Cells A Feliciello F. Ferruzzi P. Catapano

28 CD45-APC-Vio770-A CD45-APC-Vio770, human (clone: 5B1) Brand Alternative Name LCA; T200 MILTENYI Vol. Per Test 10 µl Isotype Storage Buffer Specimen Method Summary Instrument mouse IgG2a Aqueous buffered solution containing stabilizer and 0.05% sodium azide. Peripheral Blood in EDTA Stain first, then lyse with ammonium chloride, resuspend in FACS Flow BD FACS CANTO II Lymphocytes Monocytes Granulocytes Viable cells Platelets and debris SSC-A

29 CD24 PE Mouse Anti-Human (Clone: ML5) Brand Alternative Name BD Pharmingen Heat Stable Antigen Homologue (HSA); Ba-1; CD24A Vol. Per Test 5 µl Isotype Storage Buffer Specimen Method Summary Instrument Mouse IgG 2a, κ Aqueous buffered solution containing BSA and 0.09% sodium azide. Peripheral Blood in EDTA Stain first, then lyse with ammonium chloride, resuspend in FACS Flow BD FACS CANTO II Granulocytes CD15+

30 CD64 PeCy5 Mouse anti-human (Clone: 22) Brand Alternative Name Beckman Coulter 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. Specimen Peripheral Blood in EDTA Method Summary Instrument Stain first, then lyse with ammonium chloride, resuspend in FACS Flow BD FACS CANTO II Monocytes CD64+

31 Brand Alternative Name CD33 PeCy7 Mouse anti-human (Clone: D3HL60.251) gp67 Vol. Per Test 10µl Isotype Beckman Coulter IgG1, Mouse Storage Buffer Aqueous buffered solution containing 2 mg/ml BSA and 0.1% sodium azide. Specimen Peripheral Blood in EDTA Method Summary Instrument Stain first, then lyse with ammonium chloride, resuspend in FACS Flow BD FACS CANTO II Monocytes CD33++

32 CD14 PE-Cyanine7 Mouse Anti-Human (Clone: 61D3) Brand Vol. Per Test 5µl ebioscience Isotype Storage Buffer Specimen Method Summary Instrument Mouse IgG1, Kappa Aqueous buffered solution containing 0.09% sodium azide. Peripheral Blood in EDTA Stain first, then lyse with ammonium chloride resuspend in FACS Flow BD FACS CANTO II Monocytes CD64+

33 CD15 APC Mouse anti-human (Clone: HI98) Brand Alternative Name Vol. Per Test Isotype Storage Buffer Specimen Method Summary Instrument BioLegend Lewis X, 3-FAL, 3-FL, LNFP III, Lex, SSEA-1, X-hapten (5-20)µl Mouse IgM, κ Aqueous buffered solution containing 0.2% BSA and 0.09% sodium azide. Peripheral Blood in EDTA Stain first, then lyse with ammonium chloride resuspend in FACS Flow BD FACS CANTO II Granulocytes CD15+

34 CD235a FITC-A CD235a FITC Mouse anti-human (Clone: 11E4B-7-6) Brand Alternative Name Beckman Coulter Glycophorin A Vol. Per Test 20µl Isotype IgG1, Mouse Storage Buffer Aqueous buffered solution containing 2 mg/ml BSA and 0.1% sodium azide. Specimen Peripheral Blood in EDTA Method Summary Instrument PB diluted 1:500, then stain and resuspend in FACS Flow BD FACS CANTO II Viable cells Singletons Red blood cells CD45-APC-Vio770-A

35 CD235a FITC-A CD59 PE Mouse Anti-Human (Clone: OV9A2) Brand Alternative Name Protectin ebioscience Vol. Per Test 5µl Isotype Storage Buffer Specimen Method Summary Instrument IgG1, kappa Aqueous buffered solution containing 0.09% sodium azide, may contain carrier protein/stabilizer.. Peripheral Blood in EDTA PB diluted 1:500, then stain and resuspend in FACS Flow BD FACS CANTO II Red blood cells CD45-APC-Vio770-A

36 CD157 PE Mouse Anti-Human (Clone: SY11B5) Brand Alternative Name ebioscience BST-1, BP-3/IF-7 Vol. Per Test 5µl Isotype Storage Buffer Specimen Method Summary Instrument Mouse, IgG1, kappa Aqueous buffered solution containing 0.09% sodium azide, may contain carrier protein/stabilizer. Peripheral Blood in EDTA Stain first, then lyse with ammonium chloride resuspend in FACS Flow BD FACS CANTO II Granulocytes CD15+ Monocytes Granulocytes Monocytes CD64+

37 Count Count FLAER Alexa Fluor 488 proaerolysin Brand Vol. Per Test 5µl LAGITRE Storage Buffer Specimen Method Summary Instrument Aqueous buffered solution containing sodium azide. Peripheral Blood in EDTA Stain first, then lyse with ammonium chloride resuspend in FACS Flow BD FACS CANTO II Granulocytes Monocytes Monocytes CD33++ Granulocytes CD15+ FLAER Alexa Fluor 488-A FLAER Alexa Fluor 488-A

38 RBC Typing in PNH Target cells Markers Application Source RBC CD59-PE GPI-RBC MEM43 Invitrogen RBC GPA-FITC Gating on RBC KC16 - BC

39 WBC 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 CD64-PC5 CD64-ECD CD64-PC7 CD15-PC5 GPI-Monocytes and Neutrophils Gating Monocytes Gating Neutrophils SY11B5 ebiosciences 22-BC 22-BC 22-BC 80H5-BC CD45-PC7 Pattern J33-BC CD45KrO Pattern J33-BC