Flow Cytometric Studies of MDS: Increased CD34+ cells (>3% of nonerythroid cells) in MDS BM Kanter-Lewensohn et al. Eur.J., Haematol.56:124-129, 1996 Decreased CD71 in erythroblasts Also in anemia of chronic disease Kuiper-Kramer et al 97:127-131, 1997

MOLECULES ANALYZED IN THIS STUDY CD45R0 Tyr phosphatase CD11a b2 integrin CD11b b2 integrin CD11c b2 integrin CD43 Sialomucin CD33 (Sialoadhesin) CD64 (Fc receptor) CD16 (Fc receptor) CD66b (CEA family) CD66c (CEA family) CD24 (CD52/HSA) CD15 (x hapten, Lewis x) CD10 (NEP) CD13 (APN) CD35 (CR1) CD203 (97A6) CD99 (E2, MIC2) CD14 (LPS receptor) CD36 (Thrombosp. rec) CD56 (NCAM) Del Vecchio, 2006

GRANULOID CELLS IN NORMAL BONE MARROW the concave pattern CD11b CD13 the right angle CD16 CD16 the reverse right angle the mature population CD11b CD10 CD64 CD15

the missing population GRANULOID CELLS IN MDS BONE MARROW the convex pattern CD11b CD13 the disrupted right angle CD16 CD16 the missing population CD11b CD10 the disrupted reverse right angle CD64 CD15

PMN MATURATION PATHWAY CD11a/CD11b/CD45/CD14 10 1 2 3 4 CD11a FITC -> 10 1 2 3 4 CD11a FITC -> last step increase

PMN MATURATION PATHWAY CD43/CD11b/CD45/CD14 10 1 2 3 4 CD43 FITC -> 10 1 2 3 4 CD43 FITC -> slow decrease

PMN MATURATION PATHWAY CD99/CD11b/CD45/CD14 10 1 2 3 4 CD99 FITC -> 10 1 2 3 4 CD99 FITC -> two phases

PMN MATURATION PATHWAY CD66b/CD66c/CD45/CD14 10 1 2 3 4 CD66b FITC -> 10 1 2 3 4 CD66b FITC -> the star model

PMN MATURATION PATHWAY CD24/CD11c/CD45/CD14 10 1 2 3 4 CD24 FITC -> 10 1 2 3 4 CD24 FITC -> the anvil model

PMN MATURATION PATHWAY CD35/CD11b/CD45/CD14 10 1 2 3 4 CD35 FITC -> 10 1 2 3 4 CD35 FITC -> last step increase

CD36/CD11b/CD45/CD14 Monocytes SSC-H -> 256 512 768 1024 256 512 768 1024 SSC-H -> lymphocytes CD14+ monocytes CD14- monoblasts CD11b+ granulocytes CD11b-immature myeloid cells CD36/CD11b/CD45/CD14

CYTOMETRIC EVIDENCE OF IMMATURE (CD14-) and ATYPICAL (CD56+) MONOCYTES 10 1 2 3 4 CD14 APC -> grans 10 1 2 3 4 CD14 APC ->

ERYTHROID DEVELOPMENT CD71 - CD105 - LDS751 - CD45 2 3 proerythro Intermediate mature erythroblasts FWSC 1 2 3 1 2 3 CD105

CASES INCLUDED IN THE STUDY DISEASE N RA 57 RCMD 25 RAEB 12 CMML 23 PV 11 ET 5 CML MF 9 AML 59 APL ALL 13 MA 24 IPT 30 NHL A TOT 350 117 CASES INCLUDED IN THE STUDY Hemolytic Iron deficiency Hypoplastic

Analytical tools to diagnose MDS by FCM Abnormality Lineage CD16/CD11b Myeloid CD16/CD13 CD64/CD11b % of My1 gate cells % of area 2 cells Monocytes % of CD14+CD56+ % of CD34 cells Blasts CD105/FSC pattern Erythroid

MULTIMARKER SET ANALYSIS CD16-CD11b abn or CD16-CD13 abn CD64-CD11b abn >% of My1 gate >% of area2 or >% of CD14+CD56+ >CD105/FSC abn >% of CD34+ AND

MULTIMARKER SET ANALYSIS OF MDS P<0.001 100 A RA RCMD RAEB CMML

MULTIMARKER SET ANALYSIS OF OTHER CLONAL HEMATOLOGICAL DISEASES P<0.001 A APL AML ALL MF PV ET

Ag MDS RS MDS wo RS P MtF 4.9 1.1 <0.01 Della Porta 2006

Ogata, Blood 2006

IPSS score N Flow score Myeloblasts by flow 0 (low) 13 1 (0-4) 2.5 0.5-1(int-1) 52 2 (0-6) 2.9 1.5-2 (int-2) 31 4 (0-7) 6 2.5 or more (high) 17 6 (3-9) 11 Denise Wells, 2003

Flow vs No-Flow ST 30’ 3-3

Cytometric typing of MDS XY HOSPITAL Immunology Unit Cytometric typing of MDS CD19 -> CD105 PE -> First name:Xxxxxxxxx Last Name:Yyyyyyyyy Date of birth: dd.mm.yyyy Case number: 2412 Division: Hematology Specimen: Bone Marrow First obs:_X_Follow up:___ Diagnostic suspect: MDS. SSC-H -> Antigen studied: CD45, CD16, CD11b, CD13, CD64, CD56, CD19, CD71, CD105, CD34, CD33, CD14. Technique: multicolor immunofluorescence Bone marrow aspirate characterized by abundant cellularity (50000 cells/ml). Granulocytopoiesis shows dysmaturation features, with marked alteration of CD16/CDC11b, CD16/CD13 and CD64/CD11b patterns. Presence of atypical CD56+ monocytes. Normal expression of CD71 on erythroblasts. Normal erythroid maturation pattern assessed by FWSC-CD105. Taken together, the BM characterization suggests the condition of dysgranulo-dysmonopoiesis. Date of typing Signature