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FLOWCYTOMETRY

FCA
Flow cytometry menjadi metode yang
lebih disukai dalam menentukan
lineage dan maturasi dari sel leukemia.
Advantage :
fast
objective
quantitative method

FCA
The scope of FCA :
Menentukan lineage dari sel leukemia
Analisa klonal, biasanya kombinasi dengan
pemeriksaan molekuler dan sitogenetika.
Analisa maturasi seluler dan heterogenitas diantara
populasi sel ganas/leukemia
Aplikasi observasi pada kontrol, monitoring terapi
dan deteksi residual minimal dari penyakit.

Myeloid cell maturation

Lymphoid cell maturation

Penggunaan FCA
Diagnostik
Leukemia akut
Krisus blast pada CML atau transformasi leukemia pada MDS
Kronik lymphoproliferative disorder (T,B,NK)

Flow cytometer

Limitations With Light Scattering


Some Information Can Be Obtained
FSC Correlates With Cell Size
SSC Correlates With Internal Complexity
To Distinguish Between 2 Cell types
A. Size Has To Be Different OR
B. Internal Complexity i.e amount of granules

If These Two Parameters Are The Same,


Then No Distinction Can Be Made

Fluorescence And Antibodies


To The Rescue

Direct versus indirect labeling of


antigens

B or T Cell
marker

B or T cell
specific Ab

B or T cell
specific Ab

B or T Cell
marker

Flow Cytometry Is A Powerful Technique


For Characterizing Immune Cells
Allows
Allows
Allows
Allows

For Detection Of Surface Markers Of Cells


For Detection Of Intracellular Factors
Detection Of Secreted Factors By Cells
For Detection Of DNA Content

Antigen dideteksi dengan monoklonal antibodi


yang dilabel flourochrom secara direct
immunofloresence.

Fluorescent Dyes And Antibodies


Fluorochromes Are Molecules That Emit
Fluorescence Upon Excitation With Light
Ex. FITC (Fluorescein Isothiocyanate)
PE (Phycoerythrin)
PerCP (Peridinin Chlorophyll Protein)
APC (Allophycocyanin)

Architecture Of A FacsCalibur
Instrument

FAKULTAS KEDOKTERAN UGM


BAGIAN PATOLOGI KLINIK

File: DIMAS SURBAKTI.003


Acquisition Date: 28-Aug-14
Gate: Blastgate
Quad Location: 44, 113

Gedung Radioputro LT 5
Jln Kesehatan Sekip Bulaksumur
Yogyakarta telp. 0274-710 3748

Quad Events % Gated % Total


UL
31
0.38
0.31
UR
0
0.00
0.00
LL
8204
99.53
82.04
LR
8
0.10
0.08

Atas Permintaan dokter : dr.USI SUKORINI, Sp.PK-K


Nama Pasien
: TN. DIMAS SURBAKTI
No RM
: -/ RS DR. SARDJITO
Jenis Sampel
: BMP

File: DIMAS SURBAKTI.004


Acquisition Date: 28-Aug-14
Gate: Blastgate
Quad Location: 46, 27
Quad Events % Gated % Total
UL
69
1.06
0.69
UR
5
0.08
0.05
LL
6403
98.58
64.03
LR
18
0.28
0.18

R2

File: DIMAS SURBAKTI.005


Acquisition Date: 28-Aug-14
Gate: Blastgate
Quad Location: 87, 18

Pada
Pada populasi
populasi Blast
Blast ditemukan
ditemukan CD
CD
CD10
CD10
Quad Events % Gated % Total
UL
2237
34.95
22.37
UR
1
0.02
0.01
LL
4142
64.71
41.42
LR
21
0.33
0.21

File: DIMAS SURBAKTI.001


Sample ID: DIMAS SURBAKTI
Patient ID: 0828.14
Acquisition Date: 28-Aug-14
Gate: Blastgate
Quad Location: 39, 22

File: DIMAS SURBAKTI.006


Sample ID: DIMAS SURBAKTI
Patient ID: 0828.14
Acquisition Date: 28-Aug-14
Gate: Blastgate
Quad Location: 44, 15

Quad Events % Gated % Total


UL
7
0.10
0.07
UR
2
0.03
0.02
LL
6722
99.66
67.22
LR
14
0.21
0.14

Quad Events % Gated % Total


UL
1290
7.80
6.45
UR
265
1.60
1.32
LL
14988
90.59
74.94
LR
1
0.01
0.01

File: DIMAS SURBAKTI.002


Acquisition Date: 28-Aug-14
Gate: Blastgate
Quad Location: 39, 22
Quad Events % Gated % Total
UL
5305
68.05
53.05
UR
241
3.09
2.41
LL
2234
28.66
22.34
LR
16
0.21
0.16

KESAN :
Pada POPULASI SEL BLAST DITEMUKAN CD34 dan CD10

jogjakarta, 28 AGUSTUS 20
Laboratory doctor

dr. Teguh Triyono/dr. Umi

Principle of the test


primary immunological gating i.e. to focus on cells
specifically stained by one of the antibodies first (e.g.
precursor cell associated, T or B or myeloid specific) and
then to analyze the other features of the immunologically
identified populations at a second step of the analysis.
The abnormal population identified based on scatter or
immunological characteristics is then further characterized
regarding cellular lineage, degree of maturation,
abnormalities of antigen coexpression and heterogeneity in
comparison to normal hematopoietic cells.
A diagnosis is assigned in conjunction with clinical
findings, morphology and cytochemistry based on the
typical patterns of antigen expression in different clinically
characterized disorders

Selection of antigens
A "minimal" primary panel may be used for lineage
assignment of the predominant blast population,
followed by a secondary panel of mAbs characterizing
the definite phenotype and maturational stage of the
blast population depending on the results of the primary
panel.
This sequential immunophenotyping of blasts is
associated with some savings in reagent costs but
requires more time and planning.

Selection of antigens
Lineage derivation can be precisely determined by the
analysis of functionally important cytoplasmic antigens
such as
myeloperoxidase for myeloid cells
CD79 and CD22 for B-cell lineage
CD3 for T-cell lineage
CD33 and CD13 for myeloid
CD19 for B-cell and CD7 for T-cell lineage are also
diagnostically helpful, but they are not always as precise as
the detection by cytoplasmic markers.
The additional analysis of CD65 and CD117 may increase the
sensitivity for the detection of myeloid cells.

Selection of antigens
Taken together:
myeloperoxidase plus CD13/CD33 (myeloid)
CD79 plus CD19 (B-cells)
CD3 plus CD7 (T-cells)

respectively identifies >98% of acute leukemias as


myeloid, B, or T and allocates 1.5-2% into the acute
undifferentiated category

AML subtype
.

B-cell ALL
The further goal in B-lineage ALL is the maturational
analysis of B-cell precursor ALL subtypes, i.e. pre-pre-B
ALL (or pro-B-ALL), common B-ALL, pre-B ALL and surface
Ig-positive B-ALL.
The secondary panel should include the surface
membrane markers CD22 and CD24 as additional B-cell
associated markers, as well as CD5 for the identification
of subtypes of mature B-lymphatic neoplasias

T-ALL subtypes
The goal is the discrimination of T-ALL and mature T-cell
malignancies.
The secondary panel should include CD1a, CD2, CD5 as
well as mAbs to T-cell receptor (TcR) a/b and g/d chains.
Furthermore the determination of CD4 and CD8 and their
coexpression may be helpful in the analysis of T cell
(im)maturity