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Flow cytometric analysis of leukemia
and detection of minimal residual
disease.
Brent Wood MD, PhD
Professor, Laboratory Medicine and Pathology
University of Washington, Seattle
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Assess Data Quality
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Sample Exhaustion
Air in system gives rise to many spurious signals Event gate to exclude non-real events www.tc
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Laser Delay
Fluidic instability - Monitor events over time to detect www.tcs.r
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Laser Delay
Original
Gated
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Degeneration
Increase SS Decrease FS
08-03307
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Degeneration
Decrease in intensity for many antigens 08-03307
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Viability Gate
08-03307
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Viability Gate
All cells Viable cells 08-03307
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Doublet Discrimination
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Doublet Discrimination
• Doublets = > one cell in laser simultaneously
– High cell concentrations
– Cell aggregates, sample preparation
– High sample aspiration pressure
• Doublets have composite
properties
• Can exclude using
height, area, or width
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Informative Antibody
Combinations
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Abnormal population identification
• Normal
– Antigens expressed in consistent and reproducible patterns with maturation
• Neoplastic
– Increased or decreased normal antigens
– Asynchronous maturational expression
– Aberrant antigen expression
– Homogeneous expression www.tcs.r
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Normal B cell Maturation
Wood and Borowitz (2006) Henry’s Laboratory Medicine www.tc
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Antibody Combinations
PB FITC PE PE-TR PC55 PC7 A594 APC A700 APC7
B cells 20 k l - 5 19 38 10 - 45
T cells 8 2 5 34 56 3 4 7 30 45
Blasts DR 15 33 19 117 13 38 34 71 45
Myeloid DR 64 123 4 14 13 38 34 16 45 www.tcs.r
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Wood, Methods in Cell Biology, 2004 www.tc
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Precursor B cell Lymphoblastic
Leukemia
Wood and Borowitz (2006) Henry’s Laboratory Medicine www.tc
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Follicle Center B cells
08-01359
08-03324
Follicular Lymphoma
Follicular Hyperplasia
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Normal T cell Maturation
Wood and Borowitz (2010) Henry’s Laboratory Medicine www.tc
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Precursor T-cell Lymphoblastic Leukemia
Cherian and Wood (2012) Flow Cytometry in Evaluation of Hematopoietic Neoplasms: A Case-Based Approach
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Cell Type Identification
Borowitz et al (1993) AJCP 100:534-40.
Steltzer et al (1993) Ann NY Acad Sci 667:265-280
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Normal Blast Maturation
Wood (2004) Methods Cell Biology 75:559-576
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Blasts - Abnormal Antigen Intensity
Wood (2007) Clinics in Lab Medicine 27:551-575 www.tc
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Blasts - Aberrant Lymphoid Antigens
Wood (2007) Clinics in Lab Medicine 27:551-575 www.tc
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Blasts - Aberrant Maturation
Wood (2007) Clinics in Lab Medicine 27:551-575 www.tc
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Normal Granulocytic Maturation
Wood and Borowitz (2006) Henry’s Laboratory Methods www.tc
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Normal Granulocytic Maturation
Wood (2004) Methods Cell Biology 75:559-576
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Acute Promyelocytic Leukemia
Wood and Borowitz (2006) Henry’s Laboratory Medicine www.tc
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Wood (2007) Clinics in Lab Medicine 27:551-575 www.tc
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Normal Monocytic Maturation
Wood and Borowitz (2006) Henry’s Laboratory Methods www.tc
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Normal Monocytic Maturation
Wood (2004) Methods Cell Biology 75:559-576
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Acute Monocytic Leukemia
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Minimal Residual Disease
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34 J.J.M. van Dongen, FDA ALL MRD Workshop 4/2012
Response, MRD, and Relapse
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Informative Antibody
Combinations
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ALL Informative Antigens
From Krampera et al (2006) Haematologica 91:1109-1112
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0.1% abnormal immature B cells
ALL MRD
06-01469 www.tc
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Residual Acute Myeloid Leukemia
Chen & Wood (2016) Blood Reviews 31:63–75 www.tc
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COG MRD Panels
• B lineage ALL
• T lineage ALL
FITC PE PerCP-
Cy5.5
PE-Cy7 APC APC-H7
Tube 1 CD20 CD10 CD38 CD19 CD58 CD45
Tube 2 CD9 CD13/33 CD34 CD19 CD10 CD45
PB FITC PE PE-
TR
PE-
Cy5
PE-
Cy7
A594 APC APC-
H7
Tube 1 CD16 cCD3 CD7 CD56 CD5 CD38 CD3 CD45
Tube 2 CD8
BV421
CD48 CD5 CD34 CD16+
56
CD3 CD4 CD7 CD45 www.tcs.r
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AML MRD Panel
PB FITC PE PE-
TR
PE-
CyX
PE-
Cy7
A594 APC APC-
A700
APC-
H7
1 DR 15 33 19 117 13 38 34 71 45
2 DR 64 123 4 14 13 38 34 16 45
3 56 7 5 33 38 34 45
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Analytical Sensitivity
Wood BL (2016) Cytometry B Clin Cytom. 90(1):47-53 www.tc
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Immunophenotypic Stability
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MRD Identification
• Pre-treatment
– Identify abnormal immunophenotypic features
• Post-treatment
– Identify population different from normal
• “Difference from Normal”
– Discrete population defined by multiparametric gating
– Use pre-treatment immunophenotype as starting point
• “Leukemia-Associated Immunophenotype (LAIP)”
– Enumerate events in predefined gate determined pretreatment
– Assumes stability of leukemia and background populations
– Evaluate multiple LAIP when possible
– Harmonize by allowing flexibility in gating www.tcs.r
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Immunophenotypic Stability
• ALL
762510
Wood BL (2016) Cytometry B Clin Cytom. 90(1):47-53
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Immunophenotypic Stability
T-ALL
Roshal, et al (2010) Cytometry B 78:139-46 www.tcs.r
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MRD by Flow Cytometry
• Advantages
Fast
Cost effective
Large instrument base
Generally applicable
• Challenges
Subjective interpretation
Immunophenotypic change after therapy
Good to Moderate sensitivity
Poorly standardized www.tcs.r
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Acknowledgements
• Hematopathology Laboratory at UWMC
• Michael Borowitz MD PhD
• Entire COG ALL subcommittee
– Chairs, Steven Hunger, Mignon Loh
• Adaptive Biotechnologies
– Harlan Robins (FHCRC)
– Lanny Kirsch
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