FACS - applications

1
FACS - applications
2
RBC (PNH, Fetal Hgb)
Platelets (function)
neutrophil
HSC (CD34)
(AML, CML)
monocyte
Lymphocyte (CLL, ALL, MM, immunology)
3
Leukemia - diagnosis
2. Changes in normal populations pattern
?
Side scatter (SSC)
?
CD45
1. Defined population in the blast hole (most
cases)
4
Leukemia ?
Normal BM
SSC
CD45
5
M0 myeloblastic without differentiation

• Facs MPO7 HLA Dr
• CD34CD13CD33CD7

1000
800
1000
600
Side scatter
800
400
600
Side scatter
200
400
1
3
2
4
200
10
10
10
10
CD45
1
3
2
4
10
10
10
10
MPO7
6
M1/M2 myeloblastic with differentiation

• Most common in adults
• FACS HLA DrCD34CD13
• CD33CD15

1000
4
800
10
Side scatter
600
3
10
CD34
400
2
10
200
1
10
1
3
2
4
10
10
10
10
CD45
1
3
2
4
CD33
10
10
10
10
7
M3 promyelocytic leukemia

• T(1517) gt PML-RARa fusion gene
• FACS HLA DR-CD34-/CD13
• CD33CD15

1000
800
4
10
Side scatter
600
3
10
CD34
400
2
10
200
1
10
1
3
2
4
10
10
10
10
1
3
2
4
10
10
10
10
CD33
CD45
8
M4 myelo/monocytic M5 monocytic

• FACS HLA DrCD34-/
• CD13CD33CD15CD14

1000
800
4
10
Blasts monocytes
600
Side scatter
3
10
CD33
400
2
10
200
1
10
1
3
2
4
10
10
10
10
1
3
2
4
10
10
10
10
CD45
CD14
9
M6 erythroleukemia M7
megakaryoblastic

• FACS CD45-/CD71CD34

1000
800
600
Side scatter
400
200
1000
CD45
1
3
2
4
10
10
10
10
800
600
Side scatter
400
200

• FACS CD41a CD34CD62

1
3
2
4
10
10
10
10
CD45
10
CML blast crisis ?
1000
800

• FACS Not diagnostic for
• CML unless excess of
• blasts present (CD34)

Side scatter
600
400
200
1
3
2
4
CD45
10
10
10
10
4
10
3
10
CD34
2
10
1
10
1
3
2
4
10
10
10
10
CD13
11
B cell malignancies (precursor)
Diseases (leukemia/ lymphoma)
Pro-B
Pre-B
m
CD38 CD34 CD10 CD19
CD38 CD79a CD34 CD10 CD19
12
B cell malignancies (peripheral)
(clonal LC) CD5
ZAP-70
B-CLL / SLL CD19, 20(dim), 22 (dim), 23 B-PLL
(trnsf) CD19, 20, 22, FMC7 Mantle cell Ly
CD19, 20, 22
13
B cell malignancies (peripheral)
(clonal LC) CD5 -
Follicular lymphoma CD19, 20, 22, 10(),
23() B PLL de novo CD19, 20, 22,
FMC7 Marginal cell, MALT, splenic CD19, 20, 22,
11c Hairy cell leukemia CD19, 20, 22, 79a, 25,
103 Burkitt ly CD19, 20, 10, sIgM Burkitt like
ly 10 neg
14
ZAP70 analysis (CLL)
Orchard JA, Ibbotson RE, Davis Z, et al. ZAP-70
expression and prognosis in chronic lymphocytic
leukaemia. Lancet. 2004363 105-111. Rassenti
LZ, Huynh L, Toy TL, et al. ZAP-70 compared with
immunoglobulin heavy-chain gene mutation status
as a predictor of disease progression in chronic
lymphocytic leukemia. N Engl J Med. 2004351
893-901.
15
ZAP70 analysis (CLL)
Wiestner A., Blood, 1 March 2005, Vol. 105, 1839-1840.
Antigen stimulation of the leukemic clone is
increasingly implicated in the pathogenesis of
CLL. that expression of ZAP-70 in CLL B cells
renders IgM signaling more effective and thereby
could contribute to the more rapidly progressive
clinical course of ZAP-70positive CLL.


16
ZAP70 analysis (CLL)
17
ZAP70 analysis (CLL)
T-cells
Negative
NK-cells
CLL
Positive
18
Regulatory T-cells
CD4/CD25 T lymphocytes Autoimmunity, BMT,
Lymphoproliferative diseases FoxP3 expression
FoxP3