Download Imaging Cytometry and the Diagnosis of Haematological Malignancies

Imaging Cytometry and the
Diagnosis of Haematological
Malignancies
Dr Kathy Fuller
Translational Cancer Pathology Laboratory
Don’t we already use flow?
Flow analysis of an AML case
What is imaging cytometry?
A technique that combines the high-throughput power of flow cytometry with the
cellular localisation information provided by immunofluorescence microscopy.
AMNIS ImageStreamX
Why use imaging cytometry?
Advantages of flow cytometry
Advantages of microscopy
•
•
•
•
•
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Multi-parameter analysis (1-8)
Rapid analysis (2-10,000 cells/s)
Significant number of cells sampled
Sensitive identification of rare events
(eg MRD)
•
Multi-parameter analysis (1-4)
Cell location in tissue sections
preserved (eg BMT)
Detect location of fluor in cell (eg
nuclear or cytoplasmic)
Disadvantages of flow cytometry
Disadvantages of microscopy
• No tissue context
• Unable to detect intracellular location
• Analysis slower
• Smaller number of cells samples
• Rare event detection less sensitive
Why use imaging cytometry?
Advantages of Imaging cytometry
•
•
•
•
Multi-parameter analysis (1-8)
Rapid analysis (2-10,000 cells/s)
Significant number of cells sampled
Sensitive identification of rare events
(eg MRD)
Multi-parameter analysis (1-4)
Cell location in tissue sections
preserved (eg BMT)
• Detect location of fluor in cell (eg
nuclear or cytoplasmic)
Disadvantages of imaging cytometry
• Instrument not currently available in routine diagnostic laboratories
Acute myeloid leukaemia: diagnosis
Acute promyelocytic leukaemia (APML) is a subtype of acute myeloid leukaemia (AML)
with disrupted PML bodies. These have an abnormal diffuse staining pattern in APML cells
using immunofluorescence microscopy.
 results in a fusion protein, which disrupts the normal function of the RARa gene,
causing a block of the normal differentiation of granulocytes at the promyelocyte stage
 associated with a complex coagulopathy, which can lead to fatal haemorrhage
 rapid diagnosis is essential, as prompt initiation of specific therapy greatly improves
survival by reducing the incidence of bleeding complications
Acute myeloid leukaemia: diagnosis
Acute promyelocytic leukaemia (APML) is a subtype of acute myeloid leukaemia (AML)
with disrupted PML bodies. These have an abnormal diffuse staining pattern in APML cells
using immunofluorescence microscopy.
 results in a fusion protein, which disrupts the normal function of the RARa gene,
causing a block of the normal differentiation of granulocytes at the promyelocyte stage.
 associated with a complex coagulopathy, which can lead to fatal haemorrhage
 rapid diagnosis is essential, as prompt initiation of specific therapy greatly improves
survival by reducing the incidence of bleeding complications
“normal” AML pattern
Disrupted PML bodies in APML
[t(15;17); PML-RARA]
Acute myeloid leukaemia: diagnosis
High modulation/pixel variation
PML-FITC
Low modulation/pixel variation
PML-FITC
Lizz Grimwade and Wendy Erber
Acute myeloid leukaemia: diagnosis
PML
BF
Nuc
Grimwade et al, J.Clin.Pathol. 2010
Acute myeloid leukaemia: prognosis
 Nucleophosmin (NPM) is a protein that functions as a molecular chaperone, preventing
protein aggregation in the nucleolus and regulating p53 levels
 Mutations in the C-terminal region (exon 12) of NPM1 gene occur in 30% de novo AML
 These mutations lead to increased nuclear export and aberrant cytoplasmic accumulation
of the mutant NPM protein
 AML with NPM mutations have a good prognosis
 Current methods of assessment require visualisation of cytoplasmic NPM protein on
sections of bone marrow or direct DNA analysis.
Lizz Grimwade and Wendy Erber
Acute myeloid leukaemia: prognosis
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NPM-FITC (green) and DRAQ5 (pink) IS100 images
(E and F) wild-type AML similar staining patterns
(G) mutated NPM1 AML ‘‘dissimilar’’ staining pattern
(H) nuclear & cytoplasmic NPM staining visible in mutated case (I) DRAQ5 signal removed
(J, K…) NPM1 mutated cases show cytoplasmic NPM, a good prognosis subtype
Grimwade et al. CPA 2012;81A:896-900
FISH v FICTION
Fluorescence in situ hybridisation (FISH)
• technique which uses a fluorescent probe to detect DNA sequences (eg
BCR/ABL1 in Chronic Myeloid Leukaemia)
• poor sensitivity where the morphology of the abnormal cells is not distinctly
different from normal cells
Fluorescence Immunophenotyping and Interphase Cytogenetics as a
Tool for the Investigation of Neoplasms (FICTION)
• technique that combines fluorescently labelled antibodies to detect cell
surface antigens with FISH
• enables chromosomal defects to be identified in phenotypically identified
cells on interphase preparations, blood/bone marrow smears, cytospins
and tissue sections
Limitations
• Acid denaturation not compatible with all immunophenotyping antibodies
or fluorophores
• Low number of cells counted, low sensitivity for MRD detection
immunoFISH by imaging cytometry
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High-throughput FISH analysis of cells in solution
10 fluorescent parameters
Acquire up to 6000 events per second
Automated analysis or “spot counting”
immunoFISH
Optimise an imaging flow cytometry protocol for FISH analysis in combination
with cell immunophenotyping.
Method
Immunophenotyping
Fixation
Permeabilisation
Acid denaturation
Hybridisation
Nuclear staining
Hoechst 33342
Acquisition
AMNIS ISX
60x lens EDF
immunoFISH by imaging cytometry
Data analysis - select single cells
All (Brightfield)
Single cells
Aspect Ratio
1
0.8
Multicellular events
0.6
0.4
0.2
0
300
600
Area
900
1.2e3
Area, Aspect Ratio
Population
All
Single cells
Multicellular events
Count %Gated
25021 100
20692 82.7
3972
15.9
Data analysis - select cells in G1
Using Hoechst - DNA fluorescence
R2
Normalized Frequency
5
4
3
R3
2
1
0
0
1.5e5
1e5
5e4
Intensity_MC_Ch07
G1
G2
2e5
Select subpopulations
In focus
200
200
150
150
100
Frequency
Frequency
CD45-V500 (+)
In focus
CD45+
50
100
50
0
0
-1e3 0 1e3
1e4
1e5
CD45-V500 intensity
1e6
-1e3 0 1e3
1e4
1e5
CD45-V500 intensity
CD45+
CD3-BB515 (+)
1e6
CD3-BB515 intensity
CD3-BB515 intensity
T-cells
1e5
1e4
1e3
1e6
CD45+
1e6
T-cells
CD19-BV605 (+)
CD45+
B-cells
0
1e5
1e4
B-cells
1e3
0
-1e3
-1e3
-1e3 0 1e3
1e4
1e5
CD19-BV605 intensity
1e6
-1e3 0 1e3
1e4
1e5
CD19-BV605 intensity
1e6
Henry Hui
Select subpopulations
Calculate “spot count”
Cells (Hoechst+)
Normalized Frequency
70
60
2- spots
50
40
30
' 3- spots '
20
10
1- spot
3+ spots
0
0
3
6
9
Spot Count (Channel 3 560 - 595nm)
Spot Count (Channel 3 560 - 595nm)
Population
Cells (Hoechst+) & R2 & R1
2- spots & Cells (Hoechst+) & R2 & R1
' 3- spots ' & Cells (Hoechst+) & R2 & R1
1- spot & Cells (Hoechst+) & R2 & R1
3+ spots & Cells (Hoechst+) & R2 & R1
Count %Gated
6382
100
4694
73.6
300
4.7
1280
20.1
87
1.36
Henry Hui
One spot or two?
1- spot
All
5
Normalized Frequency
Normalized Frequency
4
3
2
2+ spots; cell clumps
1
2-spots single cells
0
-1e3 0 1e3
1e4
1e5
Channel 3 560 - 595nm
1e6
Count
10000
6945
2827
%Gated
100
69.4
28.3
3
2
1
0
1e6
1e5
1e4
-1e3 0 1e3
Channel 3 (560 - 595nm)
Channel 3 560 - 595nm
Population
All
2-spots single cells
2+ spots; cell clumps
4
Geo. Mean
28505.23
22266.28
61511.81
1e7
Channel 3 (560 - 595nm)
Population
1- spot & Cells (Hoechst+) & R2 & R1
Count %Gated
100
1280
Geo. Mean
22343.57
Calculation from Minderman et al. CPA 2012;81(9):776-84
Conclusion
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ImmunoFISH requires careful consideration of cellular fixation, antibodyfluorophore selection and inclusion of a nuclear marker.
•
enables the automated FISH analysis of large numbers of cells identified
by their cell phenotype, even when they only make up a subset of cells in
the sample, providing a more accurate analysis of chromosomal
abnormalities.
•
has potential to be applied to the detection of aneuploidy, deletions,
translocations or fusions in phenotypically identified cells.
•
could be used for staging of neoplastic diseases and detection of minimal
residual disease detection following therapy.
 Imaging cytometry can enhance current flow cytometric diagnostic and
prognostic analyses
Acknowledgements
Translational Cancer Pathology Laboratory, UWA
Prof Wendy Erber
Mr Henry Hui
Staff & student blood donors 
Department of Haematology, Addenbrooke’s Hospital, Cambridge
Dr Lizz Grimwade
Translational Renal Research Group, Harry Perkins Institute of Medical Research
Dr Aron Chakera
Ms Sophia Bennet
PathWest Laboratory Medicine, SCGH
Dr Kym Mina
Dr Ashleigh Murch