Flow Cytometric Studies of MDS Increased CD 34

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Flow Cytometric Studies of MDS: • Increased CD 34+ cells (>3% of nonerythroid cells)

Flow Cytometric Studies of MDS: • Increased CD 34+ cells (>3% of nonerythroid cells) in MDS BM Kanter-Lewensohn et al. Eur. J. , Haematol. 56: 124 -129, 1996 • Decreased CD 71 in erythroblasts – Also in anemia of chronic disease Kuiper-Kramer et al 97: 127 -131, 1997

MOLECULES ANALYZED IN THIS STUDY CD 45 R 0 Tyr phosphatase CD 24 (CD

MOLECULES ANALYZED IN THIS STUDY CD 45 R 0 Tyr phosphatase CD 24 (CD 52/HSA) CD 11 a b 2 integrin CD 15 (x hapten, Lewis x) CD 11 b b 2 integrin CD 10 (NEP) CD 11 c b 2 integrin CD 13 (APN) CD 43 Sialomucin CD 35 (CR 1) CD 33 (Sialoadhesin) CD 203 (97 A 6) CD 64 (Fc receptor) CD 99 (E 2, MIC 2) CD 16 (Fc receptor) CD 14 (LPS receptor) CD 66 b (CEA family) CD 36 (Thrombosp. rec) CD 66 c (CEA family) CD 56 (NCAM) Del Vecchio, 2006

GRANULOID CELLS IN NORMAL BONE MARROW CD 13 CD 11 b the concave pattern

GRANULOID CELLS IN NORMAL BONE MARROW CD 13 CD 11 b the concave pattern the right angle CD 16 the mature population CD 10 CD 11 b the reverse right angle CD 64 CD 15

GRANULOID CELLS IN MDS BONE MARROW CD 13 CD 11 b the convex pattern

GRANULOID CELLS IN MDS BONE MARROW CD 13 CD 11 b the convex pattern the disrupted right angle CD 16 the disrupted reverse right angle CD 64 CD 10 CD 11 b the missing population CD 15

PMN MATURATION PATHWAY CD 11 a/CD 11 b/CD 45/CD 14 10 0 10 1

PMN MATURATION PATHWAY CD 11 a/CD 11 b/CD 45/CD 14 10 0 10 1 CD 11 a FITC -> 10 2 10 3 10 4 10 0 10 1 CD 11 a FITC -> last step increase 10 2 10 3 10 4

PMN MATURATION PATHWAY CD 43/CD 11 b/CD 45/CD 14 10 0 10 1 10

PMN MATURATION PATHWAY CD 43/CD 11 b/CD 45/CD 14 10 0 10 1 10 2 10 3 10 4 10 0 10 1 CD 43 FITC -> slow decrease 10 2 10 3 10 4

PMN MATURATION PATHWAY CD 99/CD 11 b/CD 45/CD 14 10 0 10 1 10

PMN MATURATION PATHWAY CD 99/CD 11 b/CD 45/CD 14 10 0 10 1 10 2 10 3 10 4 10 0 10 1 CD 99 FITC -> two phases 10 2 10 3 10 4

PMN MATURATION PATHWAY CD 66 b/CD 66 c/CD 45/CD 14 10 0 10 1

PMN MATURATION PATHWAY CD 66 b/CD 66 c/CD 45/CD 14 10 0 10 1 10 2 10 3 10 4 10 0 10 1 CD 66 b FITC -> the star model 10 2 10 3 10 4

PMN MATURATION PATHWAY CD 24/CD 11 c/CD 45/CD 14 10 0 10 1 10

PMN MATURATION PATHWAY CD 24/CD 11 c/CD 45/CD 14 10 0 10 1 10 2 10 3 10 4 10 0 10 1 CD 24 FITC -> the anvil model 10 2 10 3 10 4

PMN MATURATION PATHWAY CD 35/CD 11 b/CD 45/CD 14 10 0 10 1 CD

PMN MATURATION PATHWAY CD 35/CD 11 b/CD 45/CD 14 10 0 10 1 CD 35 FITC -> 10 2 10 3 10 4 10 0 10 1 CD 35 FITC -> last step increase 10 2 10 3 10 4

Monocytes CD 14+ monocytes lymphocytes CD 14 monoblasts CD 11 b+ granulocytes CD 11

Monocytes CD 14+ monocytes lymphocytes CD 14 monoblasts CD 11 b+ granulocytes CD 11 b-immature myeloid cells 0 256 512 768 1024 SSC-H -> CD 36/CD 11 b/CD 45/CD 14

CYTOMETRIC EVIDENCE OF IMMATURE (CD 14 -) and ATYPICAL (CD 56+) MONOCYTES grans 10

CYTOMETRIC EVIDENCE OF IMMATURE (CD 14 -) and ATYPICAL (CD 56+) MONOCYTES grans 10 0 10 1 10 2 CD 14 APC -> 10 3 10 4

ERYTHROID DEVELOPMENT CD 71 - CD 105 - LDS 751 - CD 45 FWSC

ERYTHROID DEVELOPMENT CD 71 - CD 105 - LDS 751 - CD 45 FWSC proerythro Intermediate mature erythroblasts 1 2 3 1 1 2 3 CD 105

CASES INCLUDED IN THE STUDY Hemolytic Iron deficiency Hypoplastic DISEASE RA RCMD RAEB CMML

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

Analytical tools to diagnose MDS by FCM Abnormality CD 16/CD 11 b CD 16/CD

Analytical tools to diagnose MDS by FCM Abnormality CD 16/CD 11 b CD 16/CD 13 CD 64/CD 11 b % of My 1 gate cells % of area 2 cells % of CD 14+CD 56+ % of CD 34 cells CD 105/FSC pattern Lineage Myeloid Monocytes Blasts Erythroid

MULTIMARKER SET ANALYSIS CD 16 -CD 11 b abn >% of area 2 or

MULTIMARKER SET ANALYSIS CD 16 -CD 11 b abn >% of area 2 or or CD 16 -CD 13 abn or CD 64 -CD 11 b abn or >% of My 1 gate >% of CD 14+CD 56+ AND or >CD 105/FSC abn or >% of CD 34+

MULTIMARKER SET ANALYSIS OF MDS P<0. 001 100 A RA RCMD RAEB CMML

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

MULTIMARKER SET ANALYSIS OF OTHER CLONAL HEMATOLOGICAL DISEASES P<0. 001 A APL AML ALL MF PV ET

Ag Mt. F MDS RS wo RS 4. 9 1. 1 P <0. 01

Ag Mt. F MDS RS wo RS 4. 9 1. 1 P <0. 01 Della Porta 2006

Ogata, Blood 2006

Ogata, Blood 2006

IPSS score N Flow score Myeloblasts by flow 0 (low) 13 1 (0 -4)

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

Flow vs No-Flow ST 30’ 3 -3

XY HOSPITAL Immunology Unit Cytometric typing of MDS SSC-H -> CD 19 -> CD

XY HOSPITAL Immunology Unit Cytometric typing of MDS SSC-H -> CD 19 -> CD 105 PE -> Antigen studied: CD 45, CD 16, CD 11 b, CD 13, CD 64, CD 56, CD 19, CD 71, CD 105, CD 34, CD 33, CD 14. Technique: multicolor immunofluorescence First name: Xxxxx Last Name: Yyyyy Date of birth: dd. mm. yyyy Case number: 2412 Division: Hematology Specimen: Bone Marrow First obs: _X_Follow up: ___ Diagnostic suspect: MDS. Bone marrow aspirate characterized by abundant cellularity (50000 cells/ml). Granulocytopoiesis shows dysmaturation features, with marked alteration of CD 16/CDC 11 b, CD 16/CD 13 and CD 64/CD 11 b patterns. Presence of atypical CD 56+ monocytes. Normal expression of CD 71 on erythroblasts. Normal erythroid maturation pattern assessed by FWSCCD 105. Taken together, the BM characterization suggests the condition of dysgranulodysmonopoiesis. Date of typing Signature

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