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

2. 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

3. 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

4. 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

5. PMN MATURATION PATHWAY CD11a/CD11b/CD45/CD1410 1 2 3 4
CD11a FITC -> 10 1 2 3 4
CD11a FITC ->
last step increase

6. PMN MATURATION PATHWAY CD43/CD11b/CD45/CD1410 1 2 3 4
CD43 FITC -> 10 1 2 3 4
CD43 FITC ->
slow decrease

7. PMN MATURATION PATHWAY CD99/CD11b/CD45/CD1410 1 2 3 4
CD99 FITC -> 10 1 2 3 4
CD99 FITC ->
two phases

8. PMN MATURATION PATHWAY CD66b/CD66c/CD45/CD1410 1 2 3 4
CD66b FITC -> 10 1 2 3 4
CD66b FITC ->
the star model

9. PMN MATURATION PATHWAY CD24/CD11c/CD45/CD1410 1 2 3 4
CD24 FITC -> 10 1 2 3 4
CD24 FITC ->
the anvil model

10. PMN MATURATION PATHWAY CD35/CD11b/CD45/CD1410 1 2 3 4
CD35 FITC -> 10 1 2 3 4
CD35 FITC ->
last step increase

11. 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

12. CYTOMETRIC EVIDENCE OF IMMATURE (CD14-) and ATYPICAL (CD56+) MONOCYTES10 1 2 3 4
CD14 APC ->
grans 10 1 2 3 4
CD14 APC ->

13. ERYTHROID DEVELOPMENT CD71 - CD105 - LDS751 - CD452 3
proerythro
Intermediate
mature erythroblasts
FWSC 1 2 3 1 2 3
CD105

14. 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

15. 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

16. 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

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

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

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

20. Ogata, Blood 2006

21. 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

22. Flow vs No-Flow
ST 30’ 3-3

23. 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