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Practical Application of Multi-dimensional Flow Cytometry in Hematological Disorders
李啟誠 , 朱崧肇 , 蔡喜修 , 許淑敏 , 謝馨慧 Michael R. Loken
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Introduction
• Immunophenotyping has become a powerful
tool in the characterization of different
hematological disorders
• The key is to realize normal cellular
differentiation pathways on the flow
cytometric histographs and detect any
“deviation” from the normal patterns
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Different in strategy of flow cytometric development
• rather than to design novel markers and apply more and more fluorescences, which are high-priced, time consuming, and only suitable for study of MRD but not the other hematological disorder such as MDS.
• For any suspicious cell population found on flow, the best way is to sort them and send for molecular confirmation.
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Types of antigeneic abnormalities
• Lineage infidelity
• Maturational asynchrony
• Antigeneic absence
• Quantational abnormality
Wells DA, Benesch M, Loken MR et al, Blood, 2003, 102: 394-403
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Sensitivity of MRD by flow cytometry
• Two variables: degree of phenotypic difference between target cells and remaining cells; number of cells can be analyzed
• 1 in 107 or more similar to PCR • Maximal sensitivity of 1 in 105 cells during analysis in
clinical sample• Consistent sensitivity: 1 in 104
Campana D, Acta haematologica, 2004, 112:8-15
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Principle of flow cytometry
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Forward Light Scatter
Laser
Red Orange Green Fluorescence Detectors
Right Angle Light
Structure of flow cytometry
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Normal bone marrow with orderly myeloid maturation
Promye
Mye
Meta
Neu
Promye
Mye/Meta
Neu
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I II III IV
B Lymphoid Maturation
CD 10
FMC 7
CD 34
CD 45
CD 19
CD 20
CD 22 TdT
CD 5
CD 23 Antigen E
xpre
ssio
n 103
102
101
Normal B lymphoid maturation
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Normal B lymphoid maturation
CD19+
6 7
8 9
10 30 50 70 90 120
FSC -->
10
11
02
10
31
04
SS
C -
->
ML
101 102 103 104
SSC -->1
01
10
21
03
10
4C
D45 P
erC
P -
->
LymphMonoMyeloid
101 102 103 104
SSC -->
10
11
02
10
31
04
CD
19-A
PC
-->
CD19+
101 102 103 104
CD20 FITC -->
10
11
02
10
31
04
CD
45 P
erC
P -
->
101 102 103 104
CD10 PE -->
10
11
02
10
31
04
CD
45 P
erC
P -
->
101 102 103 104
CD20 FITC -->1
01
10
21
03
10
4C
D10 P
E -
->
III
III
IV
III
IIIIV
T cell
III
III, IVT
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Normal B lymphoid maturation
CD19+
6 7
8 9
10 30 50 70 90 120
FSC -->
10
11
02
10
31
04
SS
C -
->
ML
101 102 103 104
SSC -->1
01
10
21
03
10
4C
D45 P
erC
P -
->
LymphMonoMyeloid
101 102 103 104
CD19-APC -->
10
11
02
10
31
04
CD
45 P
erC
P -
->101 102 103 104
CD22 FITC -->
10
11
02
10
31
04
CD
45 P
erC
P -
->
101 102 103 104
CD34 PE -->
10
11
02
10
31
04
CD
45 P
erC
P -
->
101 102 103 104
CD22 FITC -->1
01
10
21
03
10
4C
D34 P
E -
->
I
I
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Antibodies panels used for myeloid assessment
Tube FITC PE PerCP
# 1 isotope isotope CD45
# 2 HLA-DR CD11b CD45
# 3 CD5 CD19 CD45
# 4 CD56 CD38 CD45
# 5 CD16 CD13 CD45
# 6 CD15 CD34 CD45
# 7 CD14 CD33 CD45
# 8 CD7 CD56 CD45
# 9 HLA-DR CD34 CD45
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Antibodies panels used for B-lymphoid assessment
Tube FITC PE PerCP
# 1 isotope isotope CD45
# 2 HLA-DR CD11b CD45
# 3 CD5 CD19 CD45
# 4 CD56 CD38 CD45
# 5 CD20 CD10 CD45
# 6 CD22 CD34 CD45
# 7 kappa CD19 CD45
# 8 lambda CD19 CD45
# 9 FMC7 CD19 CD45
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Examples of flow cytometric application in hematological disorders
• Minimal residual detection for AML
• Minimal residual detection for ALL
• Flow cytometric diagnosis of MDS
• Flow cytometric diagnosis of Lymphoma
• Flow cytometric diagnosis of multiple myeloma
• Flow cytometric analysis of tissue sample
• Flow cytometric diagnosis of non-malignant disorder
• Application of cell sorting for MRD confirmation
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Minimal Residual Detection for AML
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Diagnostic sample MRD 4.0%
Over-expression of CD34 & HLA-DR
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Diagnostic sample MRD 0.1%
Aberrant expression of CD56 & CD7
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Diagnostic sample MRD 0.6%
Aberrant expression of CD19
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Minimal Residual Detection for ALL
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Diagnostic sample MRD 0.03%
Precursor B-ALL, D15 s/p induction C/T
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CALLA (-) precursor B-ALL
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Relapse s/p BMT, MRD 0.3%
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Flow Cytometric Diagnosis of MDS
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56 y/o female, pancytopenia, blast 5.0%, CD13+, CD33+, aberrant CD15+, aberrant CD34-, asynchronous myeloid maturation on CD13 vs CD16 panel
Convex shape
CD15+ CD34-
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53 y/o female, MDS, CD56+ on blast, monocyte & neutrophil
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Flow Cytometric Diagnosis of Lymphoma
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66 y/o man, Maltoma, lacrimal gland, for staging
HE staining of BM
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7.0% clonal marginal zone lymphoma, lower CD45 as compared to lymphocyte, CD19+, CD20+, FMC7+, kappa+, lambda-
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L26 staining
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Flow Cytometric Diagnosis of Multiple myeloma
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68 y/o female, multiple myeloma with cytoplasmic lambda restriction, (30.0%)
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AML, M4eo, in remission; MGUS, 0.2% cyto-kappa+
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Flow Cytometric Analysis of Tissue Sample
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55 y/o man, left axillary LN: CD10+, CD20+, CD19/lambda+, diagnostic of follicular center B-cell lymphoma
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Flow Cytometric Diagnosis of Non-malignant Disorder
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27 y/o female, macrocytic anemia, Acid-Ham test (-) PB flow: PNH, heterozygous clone
Neutrophil: dim CD16
Monocyte: lose CD14
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BM flow
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Application of Cell Sorting for MRD Confirmation
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Ab-Lymph
Lymph
10 11
12 13
E-7282
10 30 50 70 90 120
FSC -->
10
11
02
10
31
04
SS
C -
-> MLe-7282
101 102 103 104
SSC -->1
01
10
21
03
10
4C
D45 P
erC
P -
->
LymphMono
Myeloid
Blast
e-7282
101 102 103 104
CD5 FITC -->
10
11
02
10
31
04
CD
19 P
E -
->
Ab-Lymph
Lymph
10 11
12 13
e-7282
101 102 103 104
CD20 FITC -->
10
11
02
10
31
04
CD
10 P
E -
->
Ab-Lymph
Lymph
10 11
12 13
e-7282
101 102 103 104
CD20 FITC -->
10
11
02
10
31
04
CD
5 P
E -
->
Ab-LymphLymph
10 11
12 13
e-7282
101 102 103 104
CD23 FITC -->
10
11
02
10
31
04
CD
19 P
E -
->Ab-Lymph
Lymph
10 11
12 13
e-7282
101 102 103 104
CD25 FITC -->
10
11
02
10
31
04
CD
22 P
E -
->
Ab-Lymph
Lymph
10 11
12 13
e-7282
101 102 103 104
FMC7 FITC -->
10
11
02
10
31
04
CD
19 P
E -
->
Ab-Lymph
Lymph
10 11
12 13
e-7282
101 102 103 104
KAPPA FITC -->
10
11
02
10
31
04
CD
19 P
E -
->
Ab-Lymph
Lymph
10 11
12 13
e-7282
101 102 103 104
LAMBDA -->
10
11
02
10
31
04
CD
19 P
E -
->
Ab-Lymph
Lymph
10 11
12 13
e-7282
101 102 103 104
KAPPA FITC -->
10
11
02
10
31
04
CD
10 P
E -
->Ab-Lymph
Lymph
10 11
12 13
E-7282
101 102 103 104
LAMBDA FITC -->
10
11
02
10
31
04
CD
10 P
E -
->
53 y/o man, follicular center B-cell lymphoma
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Monoclonal Peaks 345.7 bp for FR1 and 280.7 bp for FR2 in unsorted and sorted cell fraction (CD10+/CD45+) not in sorted control cell fraction (CD10-/CD45+) [blue=FR1; black=FR2; green=FR3; red=size standard]
Cell sorting for follicular B-cell lymphoma
Unsorted cells
Sorted cell: CD10+/CD45+ Sorted cell: CD10-/CD45+
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Abn_Blasts
6 7
8 9
E-8409
1030507090 120
FSC -->
101
102
103
104
SS
C -
-> MLE-8409
101 102 103 104
SSC -->
101
102
103
104
CD
45 P
erC
P -
->
LymphMono
Myeloid
Blasts
Abn_Blasts
E-8409
101 102 103 104
CD19 PE -->
101
102
103
104
CD
45 P
erC
P -
->
Abn_Blasts_
E-8409
101 102 103 104
CD5 FITC -->
101
102
103
104
CD
19 P
E -
->
Abn_Blasts
6 7
8 9
E-8409
101 102 103 104
CD10 PE -->
101
102
103
104
CD
45 P
erC
P -
->
Abn_Blasts_
E-8409
101 102 103 104
CD20 FITC -->
101
102
103
104
CD
10 P
E -
->
Abn_Blasts
6 7
8 9
E-8409
101 102 103 104
CD34 PE -->
101
102
103
104
CD
45 P
erC
P -
->Abn_Blasts_
E-8409
101 102 103 104
CD22 FITC -->
101
102
103
104
CD
34 P
E -
->
19 y/o female, precursor B-ALL s/p BMT, 0.3% relapse
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A)
B)
C)
Patient specific Tumor Clonality Profile
Monitoring Specimen (0.3 % tumor)
Tumor Cell Sort (Monitoring Specimen)
Cell sorting for precursor B-ALL
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Conclusion
• Hierarchical approach to diagnostics• Integration of multiple techniques• Establish diagnosis and prognosis with
minimum of tests• Develop strategy for monitoring treatment• Requires close interaction between
hematologist/pathologist/hematological core Lab
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Diagnostic Techniques
Morphology; IHC
Flow Cytometry Cytogenetics
FISH
Molecular Analysis;
DNA/RNA
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Strategy of development at SCTC
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Standard processing & instant report
• BM or PB or tissue sample come in
• Immediate morphology & flow screening (within 24 hours)
• Molecular study (sorted or unsorted) if needed (within 48 hours)
• Cytogenetics or FISH (sorted or unsorted) if needed (within 72 hours)
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Member Recruitment & Training
• Technician for morphology & flow cytometry
• Technician for molecular analysis (include all hematological malignancies)
• Flow & sorting training: Hematologics, Seattle
• Honor Dr. Michael Loken by:
Inviting Hematologics affiliate to SCTC
Honor Dr. Loken to be distinguished professor at SCTC
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Compassionate support to hematological society in Taiwan
• Cooperate with non-medical centers that can not perform standard hematological study
• Free charge for half a year since 2009
• Establish potential strategical alliances in the long run, including publication and patients referral
• Charge as government controlled insurance fee after solid relationship created
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Thank You!