practical application of multi- dimensional flow cytometry in hematological disorders 李啟誠,...
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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
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
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.
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
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
Principle of flow cytometry
Forward Light Scatter
Laser
Red Orange Green Fluorescence Detectors
Right Angle Light
Structure of flow cytometry
Normal bone marrow with orderly myeloid maturation
Promye
Mye
Meta
Neu
Promye
Mye/Meta
Neu
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
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
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
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
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
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
Minimal Residual Detection for AML
Diagnostic sample MRD 4.0%
Over-expression of CD34 & HLA-DR
Diagnostic sample MRD 0.1%
Aberrant expression of CD56 & CD7
Diagnostic sample MRD 0.6%
Aberrant expression of CD19
Minimal Residual Detection for ALL
Diagnostic sample MRD 0.03%
Precursor B-ALL, D15 s/p induction C/T
CALLA (-) precursor B-ALL
Relapse s/p BMT, MRD 0.3%
Flow Cytometric Diagnosis of MDS
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-
53 y/o female, MDS, CD56+ on blast, monocyte & neutrophil
Flow Cytometric Diagnosis of Lymphoma
66 y/o man, Maltoma, lacrimal gland, for staging
HE staining of BM
7.0% clonal marginal zone lymphoma, lower CD45 as compared to lymphocyte, CD19+, CD20+, FMC7+, kappa+, lambda-
L26 staining
Flow Cytometric Diagnosis of Multiple myeloma
68 y/o female, multiple myeloma with cytoplasmic lambda restriction, (30.0%)
AML, M4eo, in remission; MGUS, 0.2% cyto-kappa+
Flow Cytometric Analysis of Tissue Sample
55 y/o man, left axillary LN: CD10+, CD20+, CD19/lambda+, diagnostic of follicular center B-cell lymphoma
Flow Cytometric Diagnosis of Non-malignant Disorder
27 y/o female, macrocytic anemia, Acid-Ham test (-) PB flow: PNH, heterozygous clone
Neutrophil: dim CD16
Monocyte: lose CD14
BM flow
Application of Cell Sorting for MRD Confirmation
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
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+
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
A)
B)
C)
Patient specific Tumor Clonality Profile
Monitoring Specimen (0.3 % tumor)
Tumor Cell Sort (Monitoring Specimen)
Cell sorting for precursor B-ALL
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
Diagnostic Techniques
Morphology; IHC
Flow Cytometry Cytogenetics
FISH
Molecular Analysis;
DNA/RNA
Strategy of development at SCTC
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)
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
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
Thank You!