1. Aggiornamento in Ematologia Clinica
Diagnosi differenziale dei processi linfoproliferativi leucemizzati
Catania, 6-7 novembre 2008

2. FCM studies -> diagnosis of lymphoid neoplasms through the identification of phenotypically abnormal cells belonging to the lymphoid lineage and recognition of phenotypes characteristic of separate disease entities.
FCM can be used to identify expression of targets for potential antibody-directed therapy.
FCM can provide prognostic information such as CD38, ZAP-70 and CD49d expression in chronic lymphocytic leukemia/small lymphocytic lymphoma (CLL/SLL).
Following therapy, flow cytometry is becoming an established method for the evaluation of minimal residual disease.

3. FCM LYMPHOMA TYPING
BLOOD
BONE MARROW
LYMPH NODES

4. Survival in homing tissues
Natural Abs and IgA
B1 cell
Germinal centre
Marginal zone B cell
IgM
Proliferation and mutation
memory B cell
apoptotic cell
IgG IgA IgE
Follicular B cell
Selection and CSR
Survival in homing tissues
IgM
week1
week2
week3

5. The wrong approach to FCM characterization of NHL
CD5+ CD23+ CD10-
CLL ?
CD5+ CD23- CD10-
MCL
CD5- CD10+ FL
CD5- CD10-
MZL
This simplistic (and misleading) view of NHL typing is one of the main reasons for the unsufficient scientific communication between histo-pathologists and FCMetrists

6. CD5+CD10- CD5-CD10+ CLL MCL PLL MZL LPL FL BL HCL CD5-CD10- CD5+CD10+
DLBCL
LPL FL
DLBCL BL
HCL
CD11c+ CD103+ CD25+ CD22++
CD11c+ CD103± CD25+ CD138+
cIg+ sIg- CD138+
CD5-CD10-
CD5+CD10+ FL
DLBCL
HCL
MCL
MZL FL
DLBCL BL
MCL
CD23- CD43+ Bcl-1
CD23- CD43+ Bcl-1

7. CD5 CD22 CD79a/b CD10 CD23 CD103 CD19 CD25 FMC-7 CD20 CD13 CD138 CD45
Reagents of clinical utility in mature B-lymphoid neoplasms (from Craig & Foon, 2008, modified)
CD5
CD22
CD79a/b
CD10
CD23
CD103
CD19
CD25
FMC-7
CD20
CD13
CD138
CD45
CD33
bcl-2
k and l
CD34
k and l (cy)
CD9
CD38
Zap-70
CD11c
CD43
TdT
CD15
CD58
cIgM    

8. NOVEL RISK STRATIFICATION: NON-MUTATED VERSUS MUTATED CLL
LDT<12
LDT>12
CD38high
CD38low
Zap-70 high
Zap-70 low NM M
CD49dhigh
CD49dlow
CD69
CD71
p53 defects
normal p53
poor risk FISH (17p-;11q-)
good risk FISH (13q-)

9. A type of phenotypic aberrancy is abnormal expression of antigens (eg, bcl-2 expression on CD10 B cells).
Normal germinal center B cells and hematogones are both CD10+ and bcl-2-, whereas bcl-2 is expressed by most other B-cell subsets.
Abnormally increased bcl-2 expression can be found in most FL, some diffuse large B-cell lymphoma (DLBCL), and some B-lineage ALL.
In contrast, Burkitt lymphoma (BL) is usually CD10+ and bcl-2-.

10. FL (bone marrow) bcl-2/CD3/CD45/CD19 BCL2 -> B-CELLS T-CELLS PMN 101 2 3 4
B-CELLS
T-CELLS
PMN

11. Differential diagnosis between FL and regenerative BM

12. Identification of composite lymphomas

13. Structure and expression of CD200
Rosenblum M, Yancey K, Olasz E, Truitt R. J Dermatol Sci 2006;41:

14. Function of anti-CD200
Treg
TH2
CD200+ Tumor Cell
TH1

15. CD200 expression may help in differential diagnosis between mantle cell lymphoma and B-cell chronic lymphocytic leukemia
CD200 is OX2
Membrane gp, Ig superfamily, T and B
Immunosuppressive/ tolerogenic
Myeloma and B-CLL
Cancer stem cells + -
CLL 84
MCL 3 5
Palumbo GA, Catania

16. Expression of CD200 in LPD and MM
L. Brunetti, ASH 2008

17. Consistently expressed with high fluorescence intensity
Expression of CD200 in HCL
hairy
Consistently expressed with high fluorescence intensity

18. Significance of small populations of phenotypically abnormal B cells.
In the staging of patients with NHL, FCM identification of a small population of abnormal cells can be used to determine the presence of involvement by the neoplasm (particularly if the phenotype matches that of the original diagnostic specimen).
In patients without a previous diagnosis of NHL, the significance of a small population of abnormal B cells (less than 5% of the total cells analyzed) is less clear.
Small populations of B cells with abnormal phenotypes have been reported in peripheral blood and bone marrow specimens, and are not necessarily associated with a diagnosable neoplasm.
Therefore, if a small population of phenotypically abnormal B cells is identified in a patient with no previous diagnosis of a lymphoid neoplasm, it should not be used to establish a new diagnosis of malignancy, but correlated with the morphology, clinical information, and other findings.

19. A case of MZL

20. Clinical role of flow cytometry in redefining bone marrow involvement in diffuse large B-cell lymphoma – a new perspective
p=0.173
p=0.026
Talaulikar D, Histopathology 2008

21. % pos cells HyperCVAD M Jan 9 Apr 28 Jun 1 Sep 1 ABMT Apr 3 22 18 0.20
0.10
0.20 18 22

22. Flow cytometry and morphology on the same cell
Abnormal localization of selected Antigens
David Basiji

23. Centro di Citometria Clinica e Sperimentale
M.Fiammetta Romano
Giulia Scalia
Rita Bisogni
Giovanna Abate
Marisa Gorrese
Maddalena Raia
Ceterina Pascariello
Marica Gemei
Angela Gravetti
Annalisa Di Santi
Lorenzo Brunetti