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TRANSCRIPT
INTERNATIONAL SOCIETY OF LABORATORY
HEMATOLOGY
30TH INTERNATIONAL SYMPOSIUM
04 - 06 May 2017 HONOLULU HAWAII
bull Michael Keeney ART FCSMLS(D)
bull Coordinator Special Hematology London Health
Sciences Centre Canada
bull Associate Scientist Lawson Health Research
Institute
copy 2016 College of American Pathologists Materials are used with the permission of the faculty
Financial Disclosure
bull Consultant for Beckman Coulter
Objectives
bull Review B cell maturation
bull Highlight using case studies the
importance of level of marker expression
in addition to positivenegative
bull CD45 negative cells ndash a closer look
3
emission
intensity
wavelength
PE FITC
525nm
bandpass
filter
575nm
bandpass
filter
ldquoTruerdquoPE signal
Unwanted signal
appearing in PE
channel
Compensation settings determined at PMT
settings on population of interest
Unstained
Lymphocytes
Using single labeled
lymphocytes
Using multiple labeled
lymphocytes
UNCOMPENSATED
Using multiple labeled
lymphocytes
COMPENSATED
Unstained
lymphocytes
Considerations
bull Ideally compensation should be performed with the
same fluorochromes produced by the same
manufacturer as the antibodies under investigation
bull Instrument PMTrsquos must be set at appropriate level
(determined in single color staining) for each
fluorochrome)
bull The antibody used for compensation must be
equivalent to brightest signal likely to be encountered
in your assay
Alternative to real time compensation
bull Files collected in listmode can be analysed offline using several software packages
bull A control file can be run with mutually exclusive populations and then the settings from this file used to compensate any other data run with the same fluorochromes at the same PMT settings
bull Archival data can be reanalyzed ndash data is not ldquolostrdquo if incorrect compensation set at time of acquisition
Compensation must be done on the
actual fluorochrome used in the test
CD34 ANALYSIS WITH 7-AAD VIABILITY DYE
FL4-FL2 = 20
Appropriate compensation
Appropriate Compensation
CD34+
36uL
CD34 ANALYSIS WITH 7-AAD VIABILITY DYE
FL4-FL2 -10
Under compensation
Under compensation
CD34+
8uL
Recognizing bad flow data
Time Gating
Doublet Discrimination
DOUBLETS CAN CREATE ERRONEOUS RESULTS
False
CD19+CD5+
population
Current 8-10 color analysis on
bench top analyzers round bull Major advances in computing power optics
electronics and fluorochrome technology
bull uarr Colors = uarr Complexity
ndash what combinations
ndash what fluorochromes
London Health Custom Design Tube
Antigen Fluorochrome
CD8 FITC
Kappa FITC
CD4 PE
Lambda PE
CD19 ECD
CD20 PE-Cy55
CD5 PE-Cy7
CD10 APC
CD56 APC-Alexa700
Near-IR Viability Dye Excitation 633nm
Emission 780nm
CD3 Pacific Blue
CD45 Krome Orange
Result Reporting Bethesda Guidelines
bull Report percent abnormal population
bull Antibody Distribution
ndash Negative Positive Partial
bull Antibody Fluorescence
- Dim mdash Positive uniform cell population with a median fluorescence intensity abnormally less than that seen on an antigen positive normal leukocyte population Example CD20 in CLL
- Bright mdash Positive uniform cell population with a median fluorescence intensity abnormally greater than that seen on an antigen positive normal leukocyte population Example CD20 in HCL
- Heterogeneous mdash Variably intense staining not a tight uniform cluster of positive immunofluorescence staining
Example CD38 on maturing B cells
Bethesda Reporting
Normal
Dim
CLL
CD19+ CD20+(dim)
CD5+(dim)Kappa+(dim)
Discussion
bull The availability of new fluorochromes coupled with
multilaser instruments with simplified setup and
compensation makes routine ten color analysis possible for
the routine clinical laboratory
bull Knowledge of fluorochromes antibody and antigen
interactions required
bull Full understanding of normal versus abnormal population
phenotype required
bull Sophisticated data analysis programs coupled with
education essential for successful implementation ndash for all
staff involved in testing and analysis
Normal B Cell Maturation
Wood and Borowitz (2006) Henryrsquos Laboratory Medicine
Normal B cell Maturation Pattern
The three pearls
Case 1
bull Nine month old male seen by GP for nodules in his
scalp suggestive of lymphoid infiltrate incidental
finding of bilateral enlarged kidneys
bull Referred to hematology as WBC 16300uL increased
Plt
bull WBC 12300uL Hb 111gL Plt 536x109L
bull Normal differential
bull Scalp lymphoid infiltrate suspicious for leukemia
bull Bone marrow aspirate flow ordered
Case 1
34
BM 14 B cells
5 CD10dimneg
CD20 negCD38 bright
plasma cells
Case 1 BM
Case 1 Bone marrow morphology
bull Cellular marrow
bull Erythropoeisis 20-25
bull Granulopoesis 40-50 orderly maturation
bull Lymphocytes 30 10- 15 ldquoblast likerdquo
bull Increased megakaryocytes
Case 1 2 week follow up
77 B cells ndash 90
CD10 dimneg
CD20 neg bright CD38
pB-ALL
Case 1 BM 2 week follow up
Case 1 Cytogenetics
bull Complex karyotype
bull Structural abnormalities in chromosome
123491417 and 19
bull t(1719) rare in B cell acute lymphoblastic
leukemia
Case 1 Follow up
bull On high risk protocol AALL 0232 for infantile B-ALL
bull MRD 008 at Day 42
bull Cord blood transplant
bull Relapse 5 months post transplant
Pearl of Pathology
bull Differentiating precursor B cells (hematogones) from B-
ALL can be challenging Standardized analysis and
clear understanding of ldquonormalrdquo essential including
arrested maturation
Case 2
bull 4 year old girl referred to pediatric hematology with
pancytopenia
bull WBC 6800uL(Neuts 400uL) Hb 42gL Plt 28
bull Aplastic anemia
bull Bone marrow ordered aspirate biopsy flow and
cytogenetics
Case 2 BM flow cytometry
Polyclonal B cells
helper suppressor T cells
No increase in blasts
Maturing myeloid population
Case 2 CD45 neg B-ALL
Case 2 BM
Case 2
bull CD45neg
bull CD19+CD10+
bull CD20(partialdim)
bull CD34 neg
bull SigampcIg neg
bull Cytogenetics
bull Gain of X chromosome and
468911131415171821
DNA Index
122 Diploid
Case 2
bull Currently in remission after completion of
AALL0932 standard risk B-ALL therapy
Case 2
Pearls of Pathology
CD45 negative B-ALL is not uncommon
bull Always look at ungated set of plots for each marker
bull Eg SS versus each fluorescence parameter
Case 3
bull 58 year old male with pararenal mass
bull WBC 4000uL Hb 98gL Plt 229
bull ldquoblastsrdquo reported on blood film
bull Peripheral blood sent for flow cytometry
Case 3 PB flow cytometry
Dim CD45 CD19+CD10+
CD34- Sig neg
Case 3 PB flow cytometry
Dim CD45 CD19+CD10+
CD20(partial)CD5 neg
SIg cIg neg
Case 3 BM flow cytometry
Case 3 BM biopsy
Case 3 BM biopsy
Case 3 BM biopsy report
bull Diffuse infiltrate large lymphoid cells
bull Scant to moderate cytoplasm
bull Large irregular nuclei with prominent nucleoli
bull Increased mitotic activity
bull Positive for BCL-2 BCL6 CD10 and MUM1
bull Negative for TdT CD5
bull Cytogenetics FISH on para renal mass ndash C-MYC and
BCL2 rearrangements detected
Case 3 Doubletriple hit lymphoma
(DHLTHL)
bull Morphologically heterogeneous B cell neoplasms
characterized by rearrangement of the MYC proto-
oncogene with concurrent BCL2 andor BCL6 gene
rearrangement
bull WHO 2008 criteria - most cases of DHL and THL
classified DLBCL or B-cell lymphoma unclassifiable
with features intermediate between DLBCL and Burkitt
lymphoma (BCLU)
Case 3 DHLTHL
bull Retrospective study from one institution features of 13 cases (9 DHL4
THL)
bull median age was 59 years (range 30-74) eight females five males
bull enlarging lymphadenopathymasses (11 patients) abnormal peripheral
blood findings (2 patients)
bull TdT positivity (four cases two THLtwo DHL) dim CD45 expression (7
cases) lack of CD20 (2 cases) or lack of sIg (3 cases) and blastoid
morphology (2 cases)
bull Conclusion Expression of TdT in a B-cell lymphoma with mature
features or expression of surface light chain in a case otherwise
suggestive of B-lymphoblastic leukemialymphoma should prompt
an expedited evaluation for DHLTHL
Leuk Lymphoma 2016 Feb 181-10 [Epub ahead of print]
Double- and triple-hit lymphomas can present with features suggestive
of immaturity including TdT expression and create diagnostic challenges
Moench L Sachs Z Aasen G Dolan M Dayton V Courville EL
Case 4
bull 55 year old female
bull Previous history hyperparathyroidism
bull Parathyroidectomy in 1999
bull Mild persistent hypercalcemia
bull WBC 4800uL hemoglobin 121 gL Plt 296
bull Total protein 90gL IgG 34gL monoclonal peak
209gL
Case 4 Monoclonal IgG patient
Polyclonal B cells
With maturing hematogones
helper suppressor T cells
less than 3 blasts
Maturing myeloid population
Case 4 BM Ungated
Case 4 cytoplasmic immunoglobulin
Need KL
Case 4 BM Multiple Myeloma
Case 4 follow up
bull Smouldering myeloma
bull Recently developed CRAB (hyperCalcemia Renal
insufficiency Anemia Bone lesions
bull Serum free light chain ratio 123
bull Bone marrow aspirate gt60 plasma cells
bull Currently on treatment pending bone marrow
transplant
Case 5
bull 65 year old male
bull Slight lymphocytosis detected on routine CBC analysis
(lymph 4500uL)
bull Asymptomatic sent for flow cytometry
Case 5 PB
Case 5 flow cytometry KL ratio 0625
Case 5 Monoclonal B cell lymphocytosis
KL 18 KL 056
Case 5 Monoclonal B cell lymphocytosis
bull WHO defines CLL as abnormal lymphocyte count
gt5x10E9L (CD19+CD5+ light chain restricted) in the
absence of other clinical symptoms
bull Patients with small B cell clones are often detected as
an incidental finding
bull WHO 2016
bull CD19+CD5+ lt05x10E9L ldquoLow count MBLrdquo
raquo 05-5x10E9L ldquoHigh count MBLrdquo
raquo Non CLL type phenotype MBL also recognized
Monoclonal B cell Lymphocytosis
bull CLL immunophenotype - vast majority (75)
bull Atypical MBL
bull normal levels both CD5 and CD20 with variable
expression of CD23 (always exclude Mantle-cell
Lymphoma in CD23 negative cases)
bull Non-CLL-phenotype (CD5minus) MBL
bull Lacking CD5 expression and CD10 likely resembling B-
cell lymphomas of marginal-zone origin
Case 5 What is the normal KL Ratio
bull What is the normal or abnormal kappalambda ratio for
entire ldquopopulationrdquo of all B cells
bull Traditional view
ndash Kappa Lambda 21 or 31 ( 12 -13)
ndash Range 14 60
Case 5 What is the normal KL ratio
bull Wrong question
bull It doesnrsquot matter how many of the B cells are clonal but rather
that their exists a clonal population of B cells within the total
population
bull Need to Identify malignant B cells within a population of
normal B cells
bull In order to interpret a pathological immunophenotype it is
necessary to refer to quantitative and qualitative values of
normal B-cell subpopulations
bull Pearl Identify the abnormal phenotype first then look at KL
ratio
Case 6
bull 12 year old male presents to ER with fatigue bruising
Lkc 2200uL
bull Hb 84gL
bull Plt 32
bull Diff ndash neutropenia no blasts noted few ldquoatypicalrdquo cells
bull RBC ndash non specific changes
Leukemia
Case 6 PB
Case 6 BM
Case 6
bull BM aspirate ndash Hypocellular relative lymphocytosis
marked reduction in myeloid lineages
hypoplasticaplastic anemia
bull Flow Cytometry ndash less than 3 blasts maturing
myeloid 68 lymphocytes polyclonal B helper
suppressor T
bull Unusual pattern of CD13CD16 staining noted
Case 6 flow cytometry Aplastic Anemia
Normal Patient
Normal
Patient
Patient
Normal
Case 6 RBC Test for PNH CD235a v s CD59
Case 6 Neutrophil test for PNH clone
FLAER CD24 CD45CD15
18 PNH clone
Case 6
bull Follow up
bull Patient on steroids acyclovir and anti-thymocyte
globulin
bull PNH clone Neutrophils 96 RBC 1 Nov 2015
bull In remission as of April 2017
- Lkc 5500uL (Neuts 23)
- Hb 129gL
- Plt 208 X10e9
Summary
bull B cell malignancies are the most common
hematological disorders seen in flow cytometry
bull Many have complex overlapping phenotypes
bull Important to look at ungated plots as well as gating on
specific subpopulations
bull Level of expression of markers is as important as
positive or negative (think CD13 vs CD16)
68
References
bull Craig FE Foon KA Flow cytometric immunophenotyping for hematologic neoplasms Blood
2008 Apr 15111(8)3941-67
bull Wood BL Principles of minimal residual disease detection for hematopoietic neoplasms by flow
cytometry Cytometry B Clin Cytom 2015 Apr 23
bull Shanafelt TD Ghia P Lanasa MC Landgren O Rawstron AC Monoclonal B-cell
lymphocytosis (MBL) biology natural history and clinical management Leukemia 2010
Mar24(3)512-20
bull Swerdlow SH Campo E Pileri SA Harris NL Stein H Siebert R Advani RGhielmini M Salles
GA Zelenetz AD Jaffe ES The 2016 revision of the World Health Organization classification of
lymphoid neoplasms Blood 2016 May 19127(20)2375-90
bull 3 Moench L Sachs Z Aasen G Dolan M Dayton V Courville EL Double- and triple-hit
lymphomas can present with features suggestive of immaturity including TdT expression and
create diagnostic challenges Leuk Lymphoma 2016572626-35
bull Sutherland DR Keeney M Illingworth A Practical guidelines for the high-sensitivity detection
and monitoring of paroxysmal nocturnal hemoglobinuria clones by flow cytometry Cytometry B
Clin Cytom 2012 Jul82(4)195-208
69
copy 2016 College of American Pathologists Materials are used with the permission of the faculty
Financial Disclosure
bull Consultant for Beckman Coulter
Objectives
bull Review B cell maturation
bull Highlight using case studies the
importance of level of marker expression
in addition to positivenegative
bull CD45 negative cells ndash a closer look
3
emission
intensity
wavelength
PE FITC
525nm
bandpass
filter
575nm
bandpass
filter
ldquoTruerdquoPE signal
Unwanted signal
appearing in PE
channel
Compensation settings determined at PMT
settings on population of interest
Unstained
Lymphocytes
Using single labeled
lymphocytes
Using multiple labeled
lymphocytes
UNCOMPENSATED
Using multiple labeled
lymphocytes
COMPENSATED
Unstained
lymphocytes
Considerations
bull Ideally compensation should be performed with the
same fluorochromes produced by the same
manufacturer as the antibodies under investigation
bull Instrument PMTrsquos must be set at appropriate level
(determined in single color staining) for each
fluorochrome)
bull The antibody used for compensation must be
equivalent to brightest signal likely to be encountered
in your assay
Alternative to real time compensation
bull Files collected in listmode can be analysed offline using several software packages
bull A control file can be run with mutually exclusive populations and then the settings from this file used to compensate any other data run with the same fluorochromes at the same PMT settings
bull Archival data can be reanalyzed ndash data is not ldquolostrdquo if incorrect compensation set at time of acquisition
Compensation must be done on the
actual fluorochrome used in the test
CD34 ANALYSIS WITH 7-AAD VIABILITY DYE
FL4-FL2 = 20
Appropriate compensation
Appropriate Compensation
CD34+
36uL
CD34 ANALYSIS WITH 7-AAD VIABILITY DYE
FL4-FL2 -10
Under compensation
Under compensation
CD34+
8uL
Recognizing bad flow data
Time Gating
Doublet Discrimination
DOUBLETS CAN CREATE ERRONEOUS RESULTS
False
CD19+CD5+
population
Current 8-10 color analysis on
bench top analyzers round bull Major advances in computing power optics
electronics and fluorochrome technology
bull uarr Colors = uarr Complexity
ndash what combinations
ndash what fluorochromes
London Health Custom Design Tube
Antigen Fluorochrome
CD8 FITC
Kappa FITC
CD4 PE
Lambda PE
CD19 ECD
CD20 PE-Cy55
CD5 PE-Cy7
CD10 APC
CD56 APC-Alexa700
Near-IR Viability Dye Excitation 633nm
Emission 780nm
CD3 Pacific Blue
CD45 Krome Orange
Result Reporting Bethesda Guidelines
bull Report percent abnormal population
bull Antibody Distribution
ndash Negative Positive Partial
bull Antibody Fluorescence
- Dim mdash Positive uniform cell population with a median fluorescence intensity abnormally less than that seen on an antigen positive normal leukocyte population Example CD20 in CLL
- Bright mdash Positive uniform cell population with a median fluorescence intensity abnormally greater than that seen on an antigen positive normal leukocyte population Example CD20 in HCL
- Heterogeneous mdash Variably intense staining not a tight uniform cluster of positive immunofluorescence staining
Example CD38 on maturing B cells
Bethesda Reporting
Normal
Dim
CLL
CD19+ CD20+(dim)
CD5+(dim)Kappa+(dim)
Discussion
bull The availability of new fluorochromes coupled with
multilaser instruments with simplified setup and
compensation makes routine ten color analysis possible for
the routine clinical laboratory
bull Knowledge of fluorochromes antibody and antigen
interactions required
bull Full understanding of normal versus abnormal population
phenotype required
bull Sophisticated data analysis programs coupled with
education essential for successful implementation ndash for all
staff involved in testing and analysis
Normal B Cell Maturation
Wood and Borowitz (2006) Henryrsquos Laboratory Medicine
Normal B cell Maturation Pattern
The three pearls
Case 1
bull Nine month old male seen by GP for nodules in his
scalp suggestive of lymphoid infiltrate incidental
finding of bilateral enlarged kidneys
bull Referred to hematology as WBC 16300uL increased
Plt
bull WBC 12300uL Hb 111gL Plt 536x109L
bull Normal differential
bull Scalp lymphoid infiltrate suspicious for leukemia
bull Bone marrow aspirate flow ordered
Case 1
34
BM 14 B cells
5 CD10dimneg
CD20 negCD38 bright
plasma cells
Case 1 BM
Case 1 Bone marrow morphology
bull Cellular marrow
bull Erythropoeisis 20-25
bull Granulopoesis 40-50 orderly maturation
bull Lymphocytes 30 10- 15 ldquoblast likerdquo
bull Increased megakaryocytes
Case 1 2 week follow up
77 B cells ndash 90
CD10 dimneg
CD20 neg bright CD38
pB-ALL
Case 1 BM 2 week follow up
Case 1 Cytogenetics
bull Complex karyotype
bull Structural abnormalities in chromosome
123491417 and 19
bull t(1719) rare in B cell acute lymphoblastic
leukemia
Case 1 Follow up
bull On high risk protocol AALL 0232 for infantile B-ALL
bull MRD 008 at Day 42
bull Cord blood transplant
bull Relapse 5 months post transplant
Pearl of Pathology
bull Differentiating precursor B cells (hematogones) from B-
ALL can be challenging Standardized analysis and
clear understanding of ldquonormalrdquo essential including
arrested maturation
Case 2
bull 4 year old girl referred to pediatric hematology with
pancytopenia
bull WBC 6800uL(Neuts 400uL) Hb 42gL Plt 28
bull Aplastic anemia
bull Bone marrow ordered aspirate biopsy flow and
cytogenetics
Case 2 BM flow cytometry
Polyclonal B cells
helper suppressor T cells
No increase in blasts
Maturing myeloid population
Case 2 CD45 neg B-ALL
Case 2 BM
Case 2
bull CD45neg
bull CD19+CD10+
bull CD20(partialdim)
bull CD34 neg
bull SigampcIg neg
bull Cytogenetics
bull Gain of X chromosome and
468911131415171821
DNA Index
122 Diploid
Case 2
bull Currently in remission after completion of
AALL0932 standard risk B-ALL therapy
Case 2
Pearls of Pathology
CD45 negative B-ALL is not uncommon
bull Always look at ungated set of plots for each marker
bull Eg SS versus each fluorescence parameter
Case 3
bull 58 year old male with pararenal mass
bull WBC 4000uL Hb 98gL Plt 229
bull ldquoblastsrdquo reported on blood film
bull Peripheral blood sent for flow cytometry
Case 3 PB flow cytometry
Dim CD45 CD19+CD10+
CD34- Sig neg
Case 3 PB flow cytometry
Dim CD45 CD19+CD10+
CD20(partial)CD5 neg
SIg cIg neg
Case 3 BM flow cytometry
Case 3 BM biopsy
Case 3 BM biopsy
Case 3 BM biopsy report
bull Diffuse infiltrate large lymphoid cells
bull Scant to moderate cytoplasm
bull Large irregular nuclei with prominent nucleoli
bull Increased mitotic activity
bull Positive for BCL-2 BCL6 CD10 and MUM1
bull Negative for TdT CD5
bull Cytogenetics FISH on para renal mass ndash C-MYC and
BCL2 rearrangements detected
Case 3 Doubletriple hit lymphoma
(DHLTHL)
bull Morphologically heterogeneous B cell neoplasms
characterized by rearrangement of the MYC proto-
oncogene with concurrent BCL2 andor BCL6 gene
rearrangement
bull WHO 2008 criteria - most cases of DHL and THL
classified DLBCL or B-cell lymphoma unclassifiable
with features intermediate between DLBCL and Burkitt
lymphoma (BCLU)
Case 3 DHLTHL
bull Retrospective study from one institution features of 13 cases (9 DHL4
THL)
bull median age was 59 years (range 30-74) eight females five males
bull enlarging lymphadenopathymasses (11 patients) abnormal peripheral
blood findings (2 patients)
bull TdT positivity (four cases two THLtwo DHL) dim CD45 expression (7
cases) lack of CD20 (2 cases) or lack of sIg (3 cases) and blastoid
morphology (2 cases)
bull Conclusion Expression of TdT in a B-cell lymphoma with mature
features or expression of surface light chain in a case otherwise
suggestive of B-lymphoblastic leukemialymphoma should prompt
an expedited evaluation for DHLTHL
Leuk Lymphoma 2016 Feb 181-10 [Epub ahead of print]
Double- and triple-hit lymphomas can present with features suggestive
of immaturity including TdT expression and create diagnostic challenges
Moench L Sachs Z Aasen G Dolan M Dayton V Courville EL
Case 4
bull 55 year old female
bull Previous history hyperparathyroidism
bull Parathyroidectomy in 1999
bull Mild persistent hypercalcemia
bull WBC 4800uL hemoglobin 121 gL Plt 296
bull Total protein 90gL IgG 34gL monoclonal peak
209gL
Case 4 Monoclonal IgG patient
Polyclonal B cells
With maturing hematogones
helper suppressor T cells
less than 3 blasts
Maturing myeloid population
Case 4 BM Ungated
Case 4 cytoplasmic immunoglobulin
Need KL
Case 4 BM Multiple Myeloma
Case 4 follow up
bull Smouldering myeloma
bull Recently developed CRAB (hyperCalcemia Renal
insufficiency Anemia Bone lesions
bull Serum free light chain ratio 123
bull Bone marrow aspirate gt60 plasma cells
bull Currently on treatment pending bone marrow
transplant
Case 5
bull 65 year old male
bull Slight lymphocytosis detected on routine CBC analysis
(lymph 4500uL)
bull Asymptomatic sent for flow cytometry
Case 5 PB
Case 5 flow cytometry KL ratio 0625
Case 5 Monoclonal B cell lymphocytosis
KL 18 KL 056
Case 5 Monoclonal B cell lymphocytosis
bull WHO defines CLL as abnormal lymphocyte count
gt5x10E9L (CD19+CD5+ light chain restricted) in the
absence of other clinical symptoms
bull Patients with small B cell clones are often detected as
an incidental finding
bull WHO 2016
bull CD19+CD5+ lt05x10E9L ldquoLow count MBLrdquo
raquo 05-5x10E9L ldquoHigh count MBLrdquo
raquo Non CLL type phenotype MBL also recognized
Monoclonal B cell Lymphocytosis
bull CLL immunophenotype - vast majority (75)
bull Atypical MBL
bull normal levels both CD5 and CD20 with variable
expression of CD23 (always exclude Mantle-cell
Lymphoma in CD23 negative cases)
bull Non-CLL-phenotype (CD5minus) MBL
bull Lacking CD5 expression and CD10 likely resembling B-
cell lymphomas of marginal-zone origin
Case 5 What is the normal KL Ratio
bull What is the normal or abnormal kappalambda ratio for
entire ldquopopulationrdquo of all B cells
bull Traditional view
ndash Kappa Lambda 21 or 31 ( 12 -13)
ndash Range 14 60
Case 5 What is the normal KL ratio
bull Wrong question
bull It doesnrsquot matter how many of the B cells are clonal but rather
that their exists a clonal population of B cells within the total
population
bull Need to Identify malignant B cells within a population of
normal B cells
bull In order to interpret a pathological immunophenotype it is
necessary to refer to quantitative and qualitative values of
normal B-cell subpopulations
bull Pearl Identify the abnormal phenotype first then look at KL
ratio
Case 6
bull 12 year old male presents to ER with fatigue bruising
Lkc 2200uL
bull Hb 84gL
bull Plt 32
bull Diff ndash neutropenia no blasts noted few ldquoatypicalrdquo cells
bull RBC ndash non specific changes
Leukemia
Case 6 PB
Case 6 BM
Case 6
bull BM aspirate ndash Hypocellular relative lymphocytosis
marked reduction in myeloid lineages
hypoplasticaplastic anemia
bull Flow Cytometry ndash less than 3 blasts maturing
myeloid 68 lymphocytes polyclonal B helper
suppressor T
bull Unusual pattern of CD13CD16 staining noted
Case 6 flow cytometry Aplastic Anemia
Normal Patient
Normal
Patient
Patient
Normal
Case 6 RBC Test for PNH CD235a v s CD59
Case 6 Neutrophil test for PNH clone
FLAER CD24 CD45CD15
18 PNH clone
Case 6
bull Follow up
bull Patient on steroids acyclovir and anti-thymocyte
globulin
bull PNH clone Neutrophils 96 RBC 1 Nov 2015
bull In remission as of April 2017
- Lkc 5500uL (Neuts 23)
- Hb 129gL
- Plt 208 X10e9
Summary
bull B cell malignancies are the most common
hematological disorders seen in flow cytometry
bull Many have complex overlapping phenotypes
bull Important to look at ungated plots as well as gating on
specific subpopulations
bull Level of expression of markers is as important as
positive or negative (think CD13 vs CD16)
68
References
bull Craig FE Foon KA Flow cytometric immunophenotyping for hematologic neoplasms Blood
2008 Apr 15111(8)3941-67
bull Wood BL Principles of minimal residual disease detection for hematopoietic neoplasms by flow
cytometry Cytometry B Clin Cytom 2015 Apr 23
bull Shanafelt TD Ghia P Lanasa MC Landgren O Rawstron AC Monoclonal B-cell
lymphocytosis (MBL) biology natural history and clinical management Leukemia 2010
Mar24(3)512-20
bull Swerdlow SH Campo E Pileri SA Harris NL Stein H Siebert R Advani RGhielmini M Salles
GA Zelenetz AD Jaffe ES The 2016 revision of the World Health Organization classification of
lymphoid neoplasms Blood 2016 May 19127(20)2375-90
bull 3 Moench L Sachs Z Aasen G Dolan M Dayton V Courville EL Double- and triple-hit
lymphomas can present with features suggestive of immaturity including TdT expression and
create diagnostic challenges Leuk Lymphoma 2016572626-35
bull Sutherland DR Keeney M Illingworth A Practical guidelines for the high-sensitivity detection
and monitoring of paroxysmal nocturnal hemoglobinuria clones by flow cytometry Cytometry B
Clin Cytom 2012 Jul82(4)195-208
69
Objectives
bull Review B cell maturation
bull Highlight using case studies the
importance of level of marker expression
in addition to positivenegative
bull CD45 negative cells ndash a closer look
3
emission
intensity
wavelength
PE FITC
525nm
bandpass
filter
575nm
bandpass
filter
ldquoTruerdquoPE signal
Unwanted signal
appearing in PE
channel
Compensation settings determined at PMT
settings on population of interest
Unstained
Lymphocytes
Using single labeled
lymphocytes
Using multiple labeled
lymphocytes
UNCOMPENSATED
Using multiple labeled
lymphocytes
COMPENSATED
Unstained
lymphocytes
Considerations
bull Ideally compensation should be performed with the
same fluorochromes produced by the same
manufacturer as the antibodies under investigation
bull Instrument PMTrsquos must be set at appropriate level
(determined in single color staining) for each
fluorochrome)
bull The antibody used for compensation must be
equivalent to brightest signal likely to be encountered
in your assay
Alternative to real time compensation
bull Files collected in listmode can be analysed offline using several software packages
bull A control file can be run with mutually exclusive populations and then the settings from this file used to compensate any other data run with the same fluorochromes at the same PMT settings
bull Archival data can be reanalyzed ndash data is not ldquolostrdquo if incorrect compensation set at time of acquisition
Compensation must be done on the
actual fluorochrome used in the test
CD34 ANALYSIS WITH 7-AAD VIABILITY DYE
FL4-FL2 = 20
Appropriate compensation
Appropriate Compensation
CD34+
36uL
CD34 ANALYSIS WITH 7-AAD VIABILITY DYE
FL4-FL2 -10
Under compensation
Under compensation
CD34+
8uL
Recognizing bad flow data
Time Gating
Doublet Discrimination
DOUBLETS CAN CREATE ERRONEOUS RESULTS
False
CD19+CD5+
population
Current 8-10 color analysis on
bench top analyzers round bull Major advances in computing power optics
electronics and fluorochrome technology
bull uarr Colors = uarr Complexity
ndash what combinations
ndash what fluorochromes
London Health Custom Design Tube
Antigen Fluorochrome
CD8 FITC
Kappa FITC
CD4 PE
Lambda PE
CD19 ECD
CD20 PE-Cy55
CD5 PE-Cy7
CD10 APC
CD56 APC-Alexa700
Near-IR Viability Dye Excitation 633nm
Emission 780nm
CD3 Pacific Blue
CD45 Krome Orange
Result Reporting Bethesda Guidelines
bull Report percent abnormal population
bull Antibody Distribution
ndash Negative Positive Partial
bull Antibody Fluorescence
- Dim mdash Positive uniform cell population with a median fluorescence intensity abnormally less than that seen on an antigen positive normal leukocyte population Example CD20 in CLL
- Bright mdash Positive uniform cell population with a median fluorescence intensity abnormally greater than that seen on an antigen positive normal leukocyte population Example CD20 in HCL
- Heterogeneous mdash Variably intense staining not a tight uniform cluster of positive immunofluorescence staining
Example CD38 on maturing B cells
Bethesda Reporting
Normal
Dim
CLL
CD19+ CD20+(dim)
CD5+(dim)Kappa+(dim)
Discussion
bull The availability of new fluorochromes coupled with
multilaser instruments with simplified setup and
compensation makes routine ten color analysis possible for
the routine clinical laboratory
bull Knowledge of fluorochromes antibody and antigen
interactions required
bull Full understanding of normal versus abnormal population
phenotype required
bull Sophisticated data analysis programs coupled with
education essential for successful implementation ndash for all
staff involved in testing and analysis
Normal B Cell Maturation
Wood and Borowitz (2006) Henryrsquos Laboratory Medicine
Normal B cell Maturation Pattern
The three pearls
Case 1
bull Nine month old male seen by GP for nodules in his
scalp suggestive of lymphoid infiltrate incidental
finding of bilateral enlarged kidneys
bull Referred to hematology as WBC 16300uL increased
Plt
bull WBC 12300uL Hb 111gL Plt 536x109L
bull Normal differential
bull Scalp lymphoid infiltrate suspicious for leukemia
bull Bone marrow aspirate flow ordered
Case 1
34
BM 14 B cells
5 CD10dimneg
CD20 negCD38 bright
plasma cells
Case 1 BM
Case 1 Bone marrow morphology
bull Cellular marrow
bull Erythropoeisis 20-25
bull Granulopoesis 40-50 orderly maturation
bull Lymphocytes 30 10- 15 ldquoblast likerdquo
bull Increased megakaryocytes
Case 1 2 week follow up
77 B cells ndash 90
CD10 dimneg
CD20 neg bright CD38
pB-ALL
Case 1 BM 2 week follow up
Case 1 Cytogenetics
bull Complex karyotype
bull Structural abnormalities in chromosome
123491417 and 19
bull t(1719) rare in B cell acute lymphoblastic
leukemia
Case 1 Follow up
bull On high risk protocol AALL 0232 for infantile B-ALL
bull MRD 008 at Day 42
bull Cord blood transplant
bull Relapse 5 months post transplant
Pearl of Pathology
bull Differentiating precursor B cells (hematogones) from B-
ALL can be challenging Standardized analysis and
clear understanding of ldquonormalrdquo essential including
arrested maturation
Case 2
bull 4 year old girl referred to pediatric hematology with
pancytopenia
bull WBC 6800uL(Neuts 400uL) Hb 42gL Plt 28
bull Aplastic anemia
bull Bone marrow ordered aspirate biopsy flow and
cytogenetics
Case 2 BM flow cytometry
Polyclonal B cells
helper suppressor T cells
No increase in blasts
Maturing myeloid population
Case 2 CD45 neg B-ALL
Case 2 BM
Case 2
bull CD45neg
bull CD19+CD10+
bull CD20(partialdim)
bull CD34 neg
bull SigampcIg neg
bull Cytogenetics
bull Gain of X chromosome and
468911131415171821
DNA Index
122 Diploid
Case 2
bull Currently in remission after completion of
AALL0932 standard risk B-ALL therapy
Case 2
Pearls of Pathology
CD45 negative B-ALL is not uncommon
bull Always look at ungated set of plots for each marker
bull Eg SS versus each fluorescence parameter
Case 3
bull 58 year old male with pararenal mass
bull WBC 4000uL Hb 98gL Plt 229
bull ldquoblastsrdquo reported on blood film
bull Peripheral blood sent for flow cytometry
Case 3 PB flow cytometry
Dim CD45 CD19+CD10+
CD34- Sig neg
Case 3 PB flow cytometry
Dim CD45 CD19+CD10+
CD20(partial)CD5 neg
SIg cIg neg
Case 3 BM flow cytometry
Case 3 BM biopsy
Case 3 BM biopsy
Case 3 BM biopsy report
bull Diffuse infiltrate large lymphoid cells
bull Scant to moderate cytoplasm
bull Large irregular nuclei with prominent nucleoli
bull Increased mitotic activity
bull Positive for BCL-2 BCL6 CD10 and MUM1
bull Negative for TdT CD5
bull Cytogenetics FISH on para renal mass ndash C-MYC and
BCL2 rearrangements detected
Case 3 Doubletriple hit lymphoma
(DHLTHL)
bull Morphologically heterogeneous B cell neoplasms
characterized by rearrangement of the MYC proto-
oncogene with concurrent BCL2 andor BCL6 gene
rearrangement
bull WHO 2008 criteria - most cases of DHL and THL
classified DLBCL or B-cell lymphoma unclassifiable
with features intermediate between DLBCL and Burkitt
lymphoma (BCLU)
Case 3 DHLTHL
bull Retrospective study from one institution features of 13 cases (9 DHL4
THL)
bull median age was 59 years (range 30-74) eight females five males
bull enlarging lymphadenopathymasses (11 patients) abnormal peripheral
blood findings (2 patients)
bull TdT positivity (four cases two THLtwo DHL) dim CD45 expression (7
cases) lack of CD20 (2 cases) or lack of sIg (3 cases) and blastoid
morphology (2 cases)
bull Conclusion Expression of TdT in a B-cell lymphoma with mature
features or expression of surface light chain in a case otherwise
suggestive of B-lymphoblastic leukemialymphoma should prompt
an expedited evaluation for DHLTHL
Leuk Lymphoma 2016 Feb 181-10 [Epub ahead of print]
Double- and triple-hit lymphomas can present with features suggestive
of immaturity including TdT expression and create diagnostic challenges
Moench L Sachs Z Aasen G Dolan M Dayton V Courville EL
Case 4
bull 55 year old female
bull Previous history hyperparathyroidism
bull Parathyroidectomy in 1999
bull Mild persistent hypercalcemia
bull WBC 4800uL hemoglobin 121 gL Plt 296
bull Total protein 90gL IgG 34gL monoclonal peak
209gL
Case 4 Monoclonal IgG patient
Polyclonal B cells
With maturing hematogones
helper suppressor T cells
less than 3 blasts
Maturing myeloid population
Case 4 BM Ungated
Case 4 cytoplasmic immunoglobulin
Need KL
Case 4 BM Multiple Myeloma
Case 4 follow up
bull Smouldering myeloma
bull Recently developed CRAB (hyperCalcemia Renal
insufficiency Anemia Bone lesions
bull Serum free light chain ratio 123
bull Bone marrow aspirate gt60 plasma cells
bull Currently on treatment pending bone marrow
transplant
Case 5
bull 65 year old male
bull Slight lymphocytosis detected on routine CBC analysis
(lymph 4500uL)
bull Asymptomatic sent for flow cytometry
Case 5 PB
Case 5 flow cytometry KL ratio 0625
Case 5 Monoclonal B cell lymphocytosis
KL 18 KL 056
Case 5 Monoclonal B cell lymphocytosis
bull WHO defines CLL as abnormal lymphocyte count
gt5x10E9L (CD19+CD5+ light chain restricted) in the
absence of other clinical symptoms
bull Patients with small B cell clones are often detected as
an incidental finding
bull WHO 2016
bull CD19+CD5+ lt05x10E9L ldquoLow count MBLrdquo
raquo 05-5x10E9L ldquoHigh count MBLrdquo
raquo Non CLL type phenotype MBL also recognized
Monoclonal B cell Lymphocytosis
bull CLL immunophenotype - vast majority (75)
bull Atypical MBL
bull normal levels both CD5 and CD20 with variable
expression of CD23 (always exclude Mantle-cell
Lymphoma in CD23 negative cases)
bull Non-CLL-phenotype (CD5minus) MBL
bull Lacking CD5 expression and CD10 likely resembling B-
cell lymphomas of marginal-zone origin
Case 5 What is the normal KL Ratio
bull What is the normal or abnormal kappalambda ratio for
entire ldquopopulationrdquo of all B cells
bull Traditional view
ndash Kappa Lambda 21 or 31 ( 12 -13)
ndash Range 14 60
Case 5 What is the normal KL ratio
bull Wrong question
bull It doesnrsquot matter how many of the B cells are clonal but rather
that their exists a clonal population of B cells within the total
population
bull Need to Identify malignant B cells within a population of
normal B cells
bull In order to interpret a pathological immunophenotype it is
necessary to refer to quantitative and qualitative values of
normal B-cell subpopulations
bull Pearl Identify the abnormal phenotype first then look at KL
ratio
Case 6
bull 12 year old male presents to ER with fatigue bruising
Lkc 2200uL
bull Hb 84gL
bull Plt 32
bull Diff ndash neutropenia no blasts noted few ldquoatypicalrdquo cells
bull RBC ndash non specific changes
Leukemia
Case 6 PB
Case 6 BM
Case 6
bull BM aspirate ndash Hypocellular relative lymphocytosis
marked reduction in myeloid lineages
hypoplasticaplastic anemia
bull Flow Cytometry ndash less than 3 blasts maturing
myeloid 68 lymphocytes polyclonal B helper
suppressor T
bull Unusual pattern of CD13CD16 staining noted
Case 6 flow cytometry Aplastic Anemia
Normal Patient
Normal
Patient
Patient
Normal
Case 6 RBC Test for PNH CD235a v s CD59
Case 6 Neutrophil test for PNH clone
FLAER CD24 CD45CD15
18 PNH clone
Case 6
bull Follow up
bull Patient on steroids acyclovir and anti-thymocyte
globulin
bull PNH clone Neutrophils 96 RBC 1 Nov 2015
bull In remission as of April 2017
- Lkc 5500uL (Neuts 23)
- Hb 129gL
- Plt 208 X10e9
Summary
bull B cell malignancies are the most common
hematological disorders seen in flow cytometry
bull Many have complex overlapping phenotypes
bull Important to look at ungated plots as well as gating on
specific subpopulations
bull Level of expression of markers is as important as
positive or negative (think CD13 vs CD16)
68
References
bull Craig FE Foon KA Flow cytometric immunophenotyping for hematologic neoplasms Blood
2008 Apr 15111(8)3941-67
bull Wood BL Principles of minimal residual disease detection for hematopoietic neoplasms by flow
cytometry Cytometry B Clin Cytom 2015 Apr 23
bull Shanafelt TD Ghia P Lanasa MC Landgren O Rawstron AC Monoclonal B-cell
lymphocytosis (MBL) biology natural history and clinical management Leukemia 2010
Mar24(3)512-20
bull Swerdlow SH Campo E Pileri SA Harris NL Stein H Siebert R Advani RGhielmini M Salles
GA Zelenetz AD Jaffe ES The 2016 revision of the World Health Organization classification of
lymphoid neoplasms Blood 2016 May 19127(20)2375-90
bull 3 Moench L Sachs Z Aasen G Dolan M Dayton V Courville EL Double- and triple-hit
lymphomas can present with features suggestive of immaturity including TdT expression and
create diagnostic challenges Leuk Lymphoma 2016572626-35
bull Sutherland DR Keeney M Illingworth A Practical guidelines for the high-sensitivity detection
and monitoring of paroxysmal nocturnal hemoglobinuria clones by flow cytometry Cytometry B
Clin Cytom 2012 Jul82(4)195-208
69
emission
intensity
wavelength
PE FITC
525nm
bandpass
filter
575nm
bandpass
filter
ldquoTruerdquoPE signal
Unwanted signal
appearing in PE
channel
Compensation settings determined at PMT
settings on population of interest
Unstained
Lymphocytes
Using single labeled
lymphocytes
Using multiple labeled
lymphocytes
UNCOMPENSATED
Using multiple labeled
lymphocytes
COMPENSATED
Unstained
lymphocytes
Considerations
bull Ideally compensation should be performed with the
same fluorochromes produced by the same
manufacturer as the antibodies under investigation
bull Instrument PMTrsquos must be set at appropriate level
(determined in single color staining) for each
fluorochrome)
bull The antibody used for compensation must be
equivalent to brightest signal likely to be encountered
in your assay
Alternative to real time compensation
bull Files collected in listmode can be analysed offline using several software packages
bull A control file can be run with mutually exclusive populations and then the settings from this file used to compensate any other data run with the same fluorochromes at the same PMT settings
bull Archival data can be reanalyzed ndash data is not ldquolostrdquo if incorrect compensation set at time of acquisition
Compensation must be done on the
actual fluorochrome used in the test
CD34 ANALYSIS WITH 7-AAD VIABILITY DYE
FL4-FL2 = 20
Appropriate compensation
Appropriate Compensation
CD34+
36uL
CD34 ANALYSIS WITH 7-AAD VIABILITY DYE
FL4-FL2 -10
Under compensation
Under compensation
CD34+
8uL
Recognizing bad flow data
Time Gating
Doublet Discrimination
DOUBLETS CAN CREATE ERRONEOUS RESULTS
False
CD19+CD5+
population
Current 8-10 color analysis on
bench top analyzers round bull Major advances in computing power optics
electronics and fluorochrome technology
bull uarr Colors = uarr Complexity
ndash what combinations
ndash what fluorochromes
London Health Custom Design Tube
Antigen Fluorochrome
CD8 FITC
Kappa FITC
CD4 PE
Lambda PE
CD19 ECD
CD20 PE-Cy55
CD5 PE-Cy7
CD10 APC
CD56 APC-Alexa700
Near-IR Viability Dye Excitation 633nm
Emission 780nm
CD3 Pacific Blue
CD45 Krome Orange
Result Reporting Bethesda Guidelines
bull Report percent abnormal population
bull Antibody Distribution
ndash Negative Positive Partial
bull Antibody Fluorescence
- Dim mdash Positive uniform cell population with a median fluorescence intensity abnormally less than that seen on an antigen positive normal leukocyte population Example CD20 in CLL
- Bright mdash Positive uniform cell population with a median fluorescence intensity abnormally greater than that seen on an antigen positive normal leukocyte population Example CD20 in HCL
- Heterogeneous mdash Variably intense staining not a tight uniform cluster of positive immunofluorescence staining
Example CD38 on maturing B cells
Bethesda Reporting
Normal
Dim
CLL
CD19+ CD20+(dim)
CD5+(dim)Kappa+(dim)
Discussion
bull The availability of new fluorochromes coupled with
multilaser instruments with simplified setup and
compensation makes routine ten color analysis possible for
the routine clinical laboratory
bull Knowledge of fluorochromes antibody and antigen
interactions required
bull Full understanding of normal versus abnormal population
phenotype required
bull Sophisticated data analysis programs coupled with
education essential for successful implementation ndash for all
staff involved in testing and analysis
Normal B Cell Maturation
Wood and Borowitz (2006) Henryrsquos Laboratory Medicine
Normal B cell Maturation Pattern
The three pearls
Case 1
bull Nine month old male seen by GP for nodules in his
scalp suggestive of lymphoid infiltrate incidental
finding of bilateral enlarged kidneys
bull Referred to hematology as WBC 16300uL increased
Plt
bull WBC 12300uL Hb 111gL Plt 536x109L
bull Normal differential
bull Scalp lymphoid infiltrate suspicious for leukemia
bull Bone marrow aspirate flow ordered
Case 1
34
BM 14 B cells
5 CD10dimneg
CD20 negCD38 bright
plasma cells
Case 1 BM
Case 1 Bone marrow morphology
bull Cellular marrow
bull Erythropoeisis 20-25
bull Granulopoesis 40-50 orderly maturation
bull Lymphocytes 30 10- 15 ldquoblast likerdquo
bull Increased megakaryocytes
Case 1 2 week follow up
77 B cells ndash 90
CD10 dimneg
CD20 neg bright CD38
pB-ALL
Case 1 BM 2 week follow up
Case 1 Cytogenetics
bull Complex karyotype
bull Structural abnormalities in chromosome
123491417 and 19
bull t(1719) rare in B cell acute lymphoblastic
leukemia
Case 1 Follow up
bull On high risk protocol AALL 0232 for infantile B-ALL
bull MRD 008 at Day 42
bull Cord blood transplant
bull Relapse 5 months post transplant
Pearl of Pathology
bull Differentiating precursor B cells (hematogones) from B-
ALL can be challenging Standardized analysis and
clear understanding of ldquonormalrdquo essential including
arrested maturation
Case 2
bull 4 year old girl referred to pediatric hematology with
pancytopenia
bull WBC 6800uL(Neuts 400uL) Hb 42gL Plt 28
bull Aplastic anemia
bull Bone marrow ordered aspirate biopsy flow and
cytogenetics
Case 2 BM flow cytometry
Polyclonal B cells
helper suppressor T cells
No increase in blasts
Maturing myeloid population
Case 2 CD45 neg B-ALL
Case 2 BM
Case 2
bull CD45neg
bull CD19+CD10+
bull CD20(partialdim)
bull CD34 neg
bull SigampcIg neg
bull Cytogenetics
bull Gain of X chromosome and
468911131415171821
DNA Index
122 Diploid
Case 2
bull Currently in remission after completion of
AALL0932 standard risk B-ALL therapy
Case 2
Pearls of Pathology
CD45 negative B-ALL is not uncommon
bull Always look at ungated set of plots for each marker
bull Eg SS versus each fluorescence parameter
Case 3
bull 58 year old male with pararenal mass
bull WBC 4000uL Hb 98gL Plt 229
bull ldquoblastsrdquo reported on blood film
bull Peripheral blood sent for flow cytometry
Case 3 PB flow cytometry
Dim CD45 CD19+CD10+
CD34- Sig neg
Case 3 PB flow cytometry
Dim CD45 CD19+CD10+
CD20(partial)CD5 neg
SIg cIg neg
Case 3 BM flow cytometry
Case 3 BM biopsy
Case 3 BM biopsy
Case 3 BM biopsy report
bull Diffuse infiltrate large lymphoid cells
bull Scant to moderate cytoplasm
bull Large irregular nuclei with prominent nucleoli
bull Increased mitotic activity
bull Positive for BCL-2 BCL6 CD10 and MUM1
bull Negative for TdT CD5
bull Cytogenetics FISH on para renal mass ndash C-MYC and
BCL2 rearrangements detected
Case 3 Doubletriple hit lymphoma
(DHLTHL)
bull Morphologically heterogeneous B cell neoplasms
characterized by rearrangement of the MYC proto-
oncogene with concurrent BCL2 andor BCL6 gene
rearrangement
bull WHO 2008 criteria - most cases of DHL and THL
classified DLBCL or B-cell lymphoma unclassifiable
with features intermediate between DLBCL and Burkitt
lymphoma (BCLU)
Case 3 DHLTHL
bull Retrospective study from one institution features of 13 cases (9 DHL4
THL)
bull median age was 59 years (range 30-74) eight females five males
bull enlarging lymphadenopathymasses (11 patients) abnormal peripheral
blood findings (2 patients)
bull TdT positivity (four cases two THLtwo DHL) dim CD45 expression (7
cases) lack of CD20 (2 cases) or lack of sIg (3 cases) and blastoid
morphology (2 cases)
bull Conclusion Expression of TdT in a B-cell lymphoma with mature
features or expression of surface light chain in a case otherwise
suggestive of B-lymphoblastic leukemialymphoma should prompt
an expedited evaluation for DHLTHL
Leuk Lymphoma 2016 Feb 181-10 [Epub ahead of print]
Double- and triple-hit lymphomas can present with features suggestive
of immaturity including TdT expression and create diagnostic challenges
Moench L Sachs Z Aasen G Dolan M Dayton V Courville EL
Case 4
bull 55 year old female
bull Previous history hyperparathyroidism
bull Parathyroidectomy in 1999
bull Mild persistent hypercalcemia
bull WBC 4800uL hemoglobin 121 gL Plt 296
bull Total protein 90gL IgG 34gL monoclonal peak
209gL
Case 4 Monoclonal IgG patient
Polyclonal B cells
With maturing hematogones
helper suppressor T cells
less than 3 blasts
Maturing myeloid population
Case 4 BM Ungated
Case 4 cytoplasmic immunoglobulin
Need KL
Case 4 BM Multiple Myeloma
Case 4 follow up
bull Smouldering myeloma
bull Recently developed CRAB (hyperCalcemia Renal
insufficiency Anemia Bone lesions
bull Serum free light chain ratio 123
bull Bone marrow aspirate gt60 plasma cells
bull Currently on treatment pending bone marrow
transplant
Case 5
bull 65 year old male
bull Slight lymphocytosis detected on routine CBC analysis
(lymph 4500uL)
bull Asymptomatic sent for flow cytometry
Case 5 PB
Case 5 flow cytometry KL ratio 0625
Case 5 Monoclonal B cell lymphocytosis
KL 18 KL 056
Case 5 Monoclonal B cell lymphocytosis
bull WHO defines CLL as abnormal lymphocyte count
gt5x10E9L (CD19+CD5+ light chain restricted) in the
absence of other clinical symptoms
bull Patients with small B cell clones are often detected as
an incidental finding
bull WHO 2016
bull CD19+CD5+ lt05x10E9L ldquoLow count MBLrdquo
raquo 05-5x10E9L ldquoHigh count MBLrdquo
raquo Non CLL type phenotype MBL also recognized
Monoclonal B cell Lymphocytosis
bull CLL immunophenotype - vast majority (75)
bull Atypical MBL
bull normal levels both CD5 and CD20 with variable
expression of CD23 (always exclude Mantle-cell
Lymphoma in CD23 negative cases)
bull Non-CLL-phenotype (CD5minus) MBL
bull Lacking CD5 expression and CD10 likely resembling B-
cell lymphomas of marginal-zone origin
Case 5 What is the normal KL Ratio
bull What is the normal or abnormal kappalambda ratio for
entire ldquopopulationrdquo of all B cells
bull Traditional view
ndash Kappa Lambda 21 or 31 ( 12 -13)
ndash Range 14 60
Case 5 What is the normal KL ratio
bull Wrong question
bull It doesnrsquot matter how many of the B cells are clonal but rather
that their exists a clonal population of B cells within the total
population
bull Need to Identify malignant B cells within a population of
normal B cells
bull In order to interpret a pathological immunophenotype it is
necessary to refer to quantitative and qualitative values of
normal B-cell subpopulations
bull Pearl Identify the abnormal phenotype first then look at KL
ratio
Case 6
bull 12 year old male presents to ER with fatigue bruising
Lkc 2200uL
bull Hb 84gL
bull Plt 32
bull Diff ndash neutropenia no blasts noted few ldquoatypicalrdquo cells
bull RBC ndash non specific changes
Leukemia
Case 6 PB
Case 6 BM
Case 6
bull BM aspirate ndash Hypocellular relative lymphocytosis
marked reduction in myeloid lineages
hypoplasticaplastic anemia
bull Flow Cytometry ndash less than 3 blasts maturing
myeloid 68 lymphocytes polyclonal B helper
suppressor T
bull Unusual pattern of CD13CD16 staining noted
Case 6 flow cytometry Aplastic Anemia
Normal Patient
Normal
Patient
Patient
Normal
Case 6 RBC Test for PNH CD235a v s CD59
Case 6 Neutrophil test for PNH clone
FLAER CD24 CD45CD15
18 PNH clone
Case 6
bull Follow up
bull Patient on steroids acyclovir and anti-thymocyte
globulin
bull PNH clone Neutrophils 96 RBC 1 Nov 2015
bull In remission as of April 2017
- Lkc 5500uL (Neuts 23)
- Hb 129gL
- Plt 208 X10e9
Summary
bull B cell malignancies are the most common
hematological disorders seen in flow cytometry
bull Many have complex overlapping phenotypes
bull Important to look at ungated plots as well as gating on
specific subpopulations
bull Level of expression of markers is as important as
positive or negative (think CD13 vs CD16)
68
References
bull Craig FE Foon KA Flow cytometric immunophenotyping for hematologic neoplasms Blood
2008 Apr 15111(8)3941-67
bull Wood BL Principles of minimal residual disease detection for hematopoietic neoplasms by flow
cytometry Cytometry B Clin Cytom 2015 Apr 23
bull Shanafelt TD Ghia P Lanasa MC Landgren O Rawstron AC Monoclonal B-cell
lymphocytosis (MBL) biology natural history and clinical management Leukemia 2010
Mar24(3)512-20
bull Swerdlow SH Campo E Pileri SA Harris NL Stein H Siebert R Advani RGhielmini M Salles
GA Zelenetz AD Jaffe ES The 2016 revision of the World Health Organization classification of
lymphoid neoplasms Blood 2016 May 19127(20)2375-90
bull 3 Moench L Sachs Z Aasen G Dolan M Dayton V Courville EL Double- and triple-hit
lymphomas can present with features suggestive of immaturity including TdT expression and
create diagnostic challenges Leuk Lymphoma 2016572626-35
bull Sutherland DR Keeney M Illingworth A Practical guidelines for the high-sensitivity detection
and monitoring of paroxysmal nocturnal hemoglobinuria clones by flow cytometry Cytometry B
Clin Cytom 2012 Jul82(4)195-208
69
Compensation settings determined at PMT
settings on population of interest
Unstained
Lymphocytes
Using single labeled
lymphocytes
Using multiple labeled
lymphocytes
UNCOMPENSATED
Using multiple labeled
lymphocytes
COMPENSATED
Unstained
lymphocytes
Considerations
bull Ideally compensation should be performed with the
same fluorochromes produced by the same
manufacturer as the antibodies under investigation
bull Instrument PMTrsquos must be set at appropriate level
(determined in single color staining) for each
fluorochrome)
bull The antibody used for compensation must be
equivalent to brightest signal likely to be encountered
in your assay
Alternative to real time compensation
bull Files collected in listmode can be analysed offline using several software packages
bull A control file can be run with mutually exclusive populations and then the settings from this file used to compensate any other data run with the same fluorochromes at the same PMT settings
bull Archival data can be reanalyzed ndash data is not ldquolostrdquo if incorrect compensation set at time of acquisition
Compensation must be done on the
actual fluorochrome used in the test
CD34 ANALYSIS WITH 7-AAD VIABILITY DYE
FL4-FL2 = 20
Appropriate compensation
Appropriate Compensation
CD34+
36uL
CD34 ANALYSIS WITH 7-AAD VIABILITY DYE
FL4-FL2 -10
Under compensation
Under compensation
CD34+
8uL
Recognizing bad flow data
Time Gating
Doublet Discrimination
DOUBLETS CAN CREATE ERRONEOUS RESULTS
False
CD19+CD5+
population
Current 8-10 color analysis on
bench top analyzers round bull Major advances in computing power optics
electronics and fluorochrome technology
bull uarr Colors = uarr Complexity
ndash what combinations
ndash what fluorochromes
London Health Custom Design Tube
Antigen Fluorochrome
CD8 FITC
Kappa FITC
CD4 PE
Lambda PE
CD19 ECD
CD20 PE-Cy55
CD5 PE-Cy7
CD10 APC
CD56 APC-Alexa700
Near-IR Viability Dye Excitation 633nm
Emission 780nm
CD3 Pacific Blue
CD45 Krome Orange
Result Reporting Bethesda Guidelines
bull Report percent abnormal population
bull Antibody Distribution
ndash Negative Positive Partial
bull Antibody Fluorescence
- Dim mdash Positive uniform cell population with a median fluorescence intensity abnormally less than that seen on an antigen positive normal leukocyte population Example CD20 in CLL
- Bright mdash Positive uniform cell population with a median fluorescence intensity abnormally greater than that seen on an antigen positive normal leukocyte population Example CD20 in HCL
- Heterogeneous mdash Variably intense staining not a tight uniform cluster of positive immunofluorescence staining
Example CD38 on maturing B cells
Bethesda Reporting
Normal
Dim
CLL
CD19+ CD20+(dim)
CD5+(dim)Kappa+(dim)
Discussion
bull The availability of new fluorochromes coupled with
multilaser instruments with simplified setup and
compensation makes routine ten color analysis possible for
the routine clinical laboratory
bull Knowledge of fluorochromes antibody and antigen
interactions required
bull Full understanding of normal versus abnormal population
phenotype required
bull Sophisticated data analysis programs coupled with
education essential for successful implementation ndash for all
staff involved in testing and analysis
Normal B Cell Maturation
Wood and Borowitz (2006) Henryrsquos Laboratory Medicine
Normal B cell Maturation Pattern
The three pearls
Case 1
bull Nine month old male seen by GP for nodules in his
scalp suggestive of lymphoid infiltrate incidental
finding of bilateral enlarged kidneys
bull Referred to hematology as WBC 16300uL increased
Plt
bull WBC 12300uL Hb 111gL Plt 536x109L
bull Normal differential
bull Scalp lymphoid infiltrate suspicious for leukemia
bull Bone marrow aspirate flow ordered
Case 1
34
BM 14 B cells
5 CD10dimneg
CD20 negCD38 bright
plasma cells
Case 1 BM
Case 1 Bone marrow morphology
bull Cellular marrow
bull Erythropoeisis 20-25
bull Granulopoesis 40-50 orderly maturation
bull Lymphocytes 30 10- 15 ldquoblast likerdquo
bull Increased megakaryocytes
Case 1 2 week follow up
77 B cells ndash 90
CD10 dimneg
CD20 neg bright CD38
pB-ALL
Case 1 BM 2 week follow up
Case 1 Cytogenetics
bull Complex karyotype
bull Structural abnormalities in chromosome
123491417 and 19
bull t(1719) rare in B cell acute lymphoblastic
leukemia
Case 1 Follow up
bull On high risk protocol AALL 0232 for infantile B-ALL
bull MRD 008 at Day 42
bull Cord blood transplant
bull Relapse 5 months post transplant
Pearl of Pathology
bull Differentiating precursor B cells (hematogones) from B-
ALL can be challenging Standardized analysis and
clear understanding of ldquonormalrdquo essential including
arrested maturation
Case 2
bull 4 year old girl referred to pediatric hematology with
pancytopenia
bull WBC 6800uL(Neuts 400uL) Hb 42gL Plt 28
bull Aplastic anemia
bull Bone marrow ordered aspirate biopsy flow and
cytogenetics
Case 2 BM flow cytometry
Polyclonal B cells
helper suppressor T cells
No increase in blasts
Maturing myeloid population
Case 2 CD45 neg B-ALL
Case 2 BM
Case 2
bull CD45neg
bull CD19+CD10+
bull CD20(partialdim)
bull CD34 neg
bull SigampcIg neg
bull Cytogenetics
bull Gain of X chromosome and
468911131415171821
DNA Index
122 Diploid
Case 2
bull Currently in remission after completion of
AALL0932 standard risk B-ALL therapy
Case 2
Pearls of Pathology
CD45 negative B-ALL is not uncommon
bull Always look at ungated set of plots for each marker
bull Eg SS versus each fluorescence parameter
Case 3
bull 58 year old male with pararenal mass
bull WBC 4000uL Hb 98gL Plt 229
bull ldquoblastsrdquo reported on blood film
bull Peripheral blood sent for flow cytometry
Case 3 PB flow cytometry
Dim CD45 CD19+CD10+
CD34- Sig neg
Case 3 PB flow cytometry
Dim CD45 CD19+CD10+
CD20(partial)CD5 neg
SIg cIg neg
Case 3 BM flow cytometry
Case 3 BM biopsy
Case 3 BM biopsy
Case 3 BM biopsy report
bull Diffuse infiltrate large lymphoid cells
bull Scant to moderate cytoplasm
bull Large irregular nuclei with prominent nucleoli
bull Increased mitotic activity
bull Positive for BCL-2 BCL6 CD10 and MUM1
bull Negative for TdT CD5
bull Cytogenetics FISH on para renal mass ndash C-MYC and
BCL2 rearrangements detected
Case 3 Doubletriple hit lymphoma
(DHLTHL)
bull Morphologically heterogeneous B cell neoplasms
characterized by rearrangement of the MYC proto-
oncogene with concurrent BCL2 andor BCL6 gene
rearrangement
bull WHO 2008 criteria - most cases of DHL and THL
classified DLBCL or B-cell lymphoma unclassifiable
with features intermediate between DLBCL and Burkitt
lymphoma (BCLU)
Case 3 DHLTHL
bull Retrospective study from one institution features of 13 cases (9 DHL4
THL)
bull median age was 59 years (range 30-74) eight females five males
bull enlarging lymphadenopathymasses (11 patients) abnormal peripheral
blood findings (2 patients)
bull TdT positivity (four cases two THLtwo DHL) dim CD45 expression (7
cases) lack of CD20 (2 cases) or lack of sIg (3 cases) and blastoid
morphology (2 cases)
bull Conclusion Expression of TdT in a B-cell lymphoma with mature
features or expression of surface light chain in a case otherwise
suggestive of B-lymphoblastic leukemialymphoma should prompt
an expedited evaluation for DHLTHL
Leuk Lymphoma 2016 Feb 181-10 [Epub ahead of print]
Double- and triple-hit lymphomas can present with features suggestive
of immaturity including TdT expression and create diagnostic challenges
Moench L Sachs Z Aasen G Dolan M Dayton V Courville EL
Case 4
bull 55 year old female
bull Previous history hyperparathyroidism
bull Parathyroidectomy in 1999
bull Mild persistent hypercalcemia
bull WBC 4800uL hemoglobin 121 gL Plt 296
bull Total protein 90gL IgG 34gL monoclonal peak
209gL
Case 4 Monoclonal IgG patient
Polyclonal B cells
With maturing hematogones
helper suppressor T cells
less than 3 blasts
Maturing myeloid population
Case 4 BM Ungated
Case 4 cytoplasmic immunoglobulin
Need KL
Case 4 BM Multiple Myeloma
Case 4 follow up
bull Smouldering myeloma
bull Recently developed CRAB (hyperCalcemia Renal
insufficiency Anemia Bone lesions
bull Serum free light chain ratio 123
bull Bone marrow aspirate gt60 plasma cells
bull Currently on treatment pending bone marrow
transplant
Case 5
bull 65 year old male
bull Slight lymphocytosis detected on routine CBC analysis
(lymph 4500uL)
bull Asymptomatic sent for flow cytometry
Case 5 PB
Case 5 flow cytometry KL ratio 0625
Case 5 Monoclonal B cell lymphocytosis
KL 18 KL 056
Case 5 Monoclonal B cell lymphocytosis
bull WHO defines CLL as abnormal lymphocyte count
gt5x10E9L (CD19+CD5+ light chain restricted) in the
absence of other clinical symptoms
bull Patients with small B cell clones are often detected as
an incidental finding
bull WHO 2016
bull CD19+CD5+ lt05x10E9L ldquoLow count MBLrdquo
raquo 05-5x10E9L ldquoHigh count MBLrdquo
raquo Non CLL type phenotype MBL also recognized
Monoclonal B cell Lymphocytosis
bull CLL immunophenotype - vast majority (75)
bull Atypical MBL
bull normal levels both CD5 and CD20 with variable
expression of CD23 (always exclude Mantle-cell
Lymphoma in CD23 negative cases)
bull Non-CLL-phenotype (CD5minus) MBL
bull Lacking CD5 expression and CD10 likely resembling B-
cell lymphomas of marginal-zone origin
Case 5 What is the normal KL Ratio
bull What is the normal or abnormal kappalambda ratio for
entire ldquopopulationrdquo of all B cells
bull Traditional view
ndash Kappa Lambda 21 or 31 ( 12 -13)
ndash Range 14 60
Case 5 What is the normal KL ratio
bull Wrong question
bull It doesnrsquot matter how many of the B cells are clonal but rather
that their exists a clonal population of B cells within the total
population
bull Need to Identify malignant B cells within a population of
normal B cells
bull In order to interpret a pathological immunophenotype it is
necessary to refer to quantitative and qualitative values of
normal B-cell subpopulations
bull Pearl Identify the abnormal phenotype first then look at KL
ratio
Case 6
bull 12 year old male presents to ER with fatigue bruising
Lkc 2200uL
bull Hb 84gL
bull Plt 32
bull Diff ndash neutropenia no blasts noted few ldquoatypicalrdquo cells
bull RBC ndash non specific changes
Leukemia
Case 6 PB
Case 6 BM
Case 6
bull BM aspirate ndash Hypocellular relative lymphocytosis
marked reduction in myeloid lineages
hypoplasticaplastic anemia
bull Flow Cytometry ndash less than 3 blasts maturing
myeloid 68 lymphocytes polyclonal B helper
suppressor T
bull Unusual pattern of CD13CD16 staining noted
Case 6 flow cytometry Aplastic Anemia
Normal Patient
Normal
Patient
Patient
Normal
Case 6 RBC Test for PNH CD235a v s CD59
Case 6 Neutrophil test for PNH clone
FLAER CD24 CD45CD15
18 PNH clone
Case 6
bull Follow up
bull Patient on steroids acyclovir and anti-thymocyte
globulin
bull PNH clone Neutrophils 96 RBC 1 Nov 2015
bull In remission as of April 2017
- Lkc 5500uL (Neuts 23)
- Hb 129gL
- Plt 208 X10e9
Summary
bull B cell malignancies are the most common
hematological disorders seen in flow cytometry
bull Many have complex overlapping phenotypes
bull Important to look at ungated plots as well as gating on
specific subpopulations
bull Level of expression of markers is as important as
positive or negative (think CD13 vs CD16)
68
References
bull Craig FE Foon KA Flow cytometric immunophenotyping for hematologic neoplasms Blood
2008 Apr 15111(8)3941-67
bull Wood BL Principles of minimal residual disease detection for hematopoietic neoplasms by flow
cytometry Cytometry B Clin Cytom 2015 Apr 23
bull Shanafelt TD Ghia P Lanasa MC Landgren O Rawstron AC Monoclonal B-cell
lymphocytosis (MBL) biology natural history and clinical management Leukemia 2010
Mar24(3)512-20
bull Swerdlow SH Campo E Pileri SA Harris NL Stein H Siebert R Advani RGhielmini M Salles
GA Zelenetz AD Jaffe ES The 2016 revision of the World Health Organization classification of
lymphoid neoplasms Blood 2016 May 19127(20)2375-90
bull 3 Moench L Sachs Z Aasen G Dolan M Dayton V Courville EL Double- and triple-hit
lymphomas can present with features suggestive of immaturity including TdT expression and
create diagnostic challenges Leuk Lymphoma 2016572626-35
bull Sutherland DR Keeney M Illingworth A Practical guidelines for the high-sensitivity detection
and monitoring of paroxysmal nocturnal hemoglobinuria clones by flow cytometry Cytometry B
Clin Cytom 2012 Jul82(4)195-208
69
Considerations
bull Ideally compensation should be performed with the
same fluorochromes produced by the same
manufacturer as the antibodies under investigation
bull Instrument PMTrsquos must be set at appropriate level
(determined in single color staining) for each
fluorochrome)
bull The antibody used for compensation must be
equivalent to brightest signal likely to be encountered
in your assay
Alternative to real time compensation
bull Files collected in listmode can be analysed offline using several software packages
bull A control file can be run with mutually exclusive populations and then the settings from this file used to compensate any other data run with the same fluorochromes at the same PMT settings
bull Archival data can be reanalyzed ndash data is not ldquolostrdquo if incorrect compensation set at time of acquisition
Compensation must be done on the
actual fluorochrome used in the test
CD34 ANALYSIS WITH 7-AAD VIABILITY DYE
FL4-FL2 = 20
Appropriate compensation
Appropriate Compensation
CD34+
36uL
CD34 ANALYSIS WITH 7-AAD VIABILITY DYE
FL4-FL2 -10
Under compensation
Under compensation
CD34+
8uL
Recognizing bad flow data
Time Gating
Doublet Discrimination
DOUBLETS CAN CREATE ERRONEOUS RESULTS
False
CD19+CD5+
population
Current 8-10 color analysis on
bench top analyzers round bull Major advances in computing power optics
electronics and fluorochrome technology
bull uarr Colors = uarr Complexity
ndash what combinations
ndash what fluorochromes
London Health Custom Design Tube
Antigen Fluorochrome
CD8 FITC
Kappa FITC
CD4 PE
Lambda PE
CD19 ECD
CD20 PE-Cy55
CD5 PE-Cy7
CD10 APC
CD56 APC-Alexa700
Near-IR Viability Dye Excitation 633nm
Emission 780nm
CD3 Pacific Blue
CD45 Krome Orange
Result Reporting Bethesda Guidelines
bull Report percent abnormal population
bull Antibody Distribution
ndash Negative Positive Partial
bull Antibody Fluorescence
- Dim mdash Positive uniform cell population with a median fluorescence intensity abnormally less than that seen on an antigen positive normal leukocyte population Example CD20 in CLL
- Bright mdash Positive uniform cell population with a median fluorescence intensity abnormally greater than that seen on an antigen positive normal leukocyte population Example CD20 in HCL
- Heterogeneous mdash Variably intense staining not a tight uniform cluster of positive immunofluorescence staining
Example CD38 on maturing B cells
Bethesda Reporting
Normal
Dim
CLL
CD19+ CD20+(dim)
CD5+(dim)Kappa+(dim)
Discussion
bull The availability of new fluorochromes coupled with
multilaser instruments with simplified setup and
compensation makes routine ten color analysis possible for
the routine clinical laboratory
bull Knowledge of fluorochromes antibody and antigen
interactions required
bull Full understanding of normal versus abnormal population
phenotype required
bull Sophisticated data analysis programs coupled with
education essential for successful implementation ndash for all
staff involved in testing and analysis
Normal B Cell Maturation
Wood and Borowitz (2006) Henryrsquos Laboratory Medicine
Normal B cell Maturation Pattern
The three pearls
Case 1
bull Nine month old male seen by GP for nodules in his
scalp suggestive of lymphoid infiltrate incidental
finding of bilateral enlarged kidneys
bull Referred to hematology as WBC 16300uL increased
Plt
bull WBC 12300uL Hb 111gL Plt 536x109L
bull Normal differential
bull Scalp lymphoid infiltrate suspicious for leukemia
bull Bone marrow aspirate flow ordered
Case 1
34
BM 14 B cells
5 CD10dimneg
CD20 negCD38 bright
plasma cells
Case 1 BM
Case 1 Bone marrow morphology
bull Cellular marrow
bull Erythropoeisis 20-25
bull Granulopoesis 40-50 orderly maturation
bull Lymphocytes 30 10- 15 ldquoblast likerdquo
bull Increased megakaryocytes
Case 1 2 week follow up
77 B cells ndash 90
CD10 dimneg
CD20 neg bright CD38
pB-ALL
Case 1 BM 2 week follow up
Case 1 Cytogenetics
bull Complex karyotype
bull Structural abnormalities in chromosome
123491417 and 19
bull t(1719) rare in B cell acute lymphoblastic
leukemia
Case 1 Follow up
bull On high risk protocol AALL 0232 for infantile B-ALL
bull MRD 008 at Day 42
bull Cord blood transplant
bull Relapse 5 months post transplant
Pearl of Pathology
bull Differentiating precursor B cells (hematogones) from B-
ALL can be challenging Standardized analysis and
clear understanding of ldquonormalrdquo essential including
arrested maturation
Case 2
bull 4 year old girl referred to pediatric hematology with
pancytopenia
bull WBC 6800uL(Neuts 400uL) Hb 42gL Plt 28
bull Aplastic anemia
bull Bone marrow ordered aspirate biopsy flow and
cytogenetics
Case 2 BM flow cytometry
Polyclonal B cells
helper suppressor T cells
No increase in blasts
Maturing myeloid population
Case 2 CD45 neg B-ALL
Case 2 BM
Case 2
bull CD45neg
bull CD19+CD10+
bull CD20(partialdim)
bull CD34 neg
bull SigampcIg neg
bull Cytogenetics
bull Gain of X chromosome and
468911131415171821
DNA Index
122 Diploid
Case 2
bull Currently in remission after completion of
AALL0932 standard risk B-ALL therapy
Case 2
Pearls of Pathology
CD45 negative B-ALL is not uncommon
bull Always look at ungated set of plots for each marker
bull Eg SS versus each fluorescence parameter
Case 3
bull 58 year old male with pararenal mass
bull WBC 4000uL Hb 98gL Plt 229
bull ldquoblastsrdquo reported on blood film
bull Peripheral blood sent for flow cytometry
Case 3 PB flow cytometry
Dim CD45 CD19+CD10+
CD34- Sig neg
Case 3 PB flow cytometry
Dim CD45 CD19+CD10+
CD20(partial)CD5 neg
SIg cIg neg
Case 3 BM flow cytometry
Case 3 BM biopsy
Case 3 BM biopsy
Case 3 BM biopsy report
bull Diffuse infiltrate large lymphoid cells
bull Scant to moderate cytoplasm
bull Large irregular nuclei with prominent nucleoli
bull Increased mitotic activity
bull Positive for BCL-2 BCL6 CD10 and MUM1
bull Negative for TdT CD5
bull Cytogenetics FISH on para renal mass ndash C-MYC and
BCL2 rearrangements detected
Case 3 Doubletriple hit lymphoma
(DHLTHL)
bull Morphologically heterogeneous B cell neoplasms
characterized by rearrangement of the MYC proto-
oncogene with concurrent BCL2 andor BCL6 gene
rearrangement
bull WHO 2008 criteria - most cases of DHL and THL
classified DLBCL or B-cell lymphoma unclassifiable
with features intermediate between DLBCL and Burkitt
lymphoma (BCLU)
Case 3 DHLTHL
bull Retrospective study from one institution features of 13 cases (9 DHL4
THL)
bull median age was 59 years (range 30-74) eight females five males
bull enlarging lymphadenopathymasses (11 patients) abnormal peripheral
blood findings (2 patients)
bull TdT positivity (four cases two THLtwo DHL) dim CD45 expression (7
cases) lack of CD20 (2 cases) or lack of sIg (3 cases) and blastoid
morphology (2 cases)
bull Conclusion Expression of TdT in a B-cell lymphoma with mature
features or expression of surface light chain in a case otherwise
suggestive of B-lymphoblastic leukemialymphoma should prompt
an expedited evaluation for DHLTHL
Leuk Lymphoma 2016 Feb 181-10 [Epub ahead of print]
Double- and triple-hit lymphomas can present with features suggestive
of immaturity including TdT expression and create diagnostic challenges
Moench L Sachs Z Aasen G Dolan M Dayton V Courville EL
Case 4
bull 55 year old female
bull Previous history hyperparathyroidism
bull Parathyroidectomy in 1999
bull Mild persistent hypercalcemia
bull WBC 4800uL hemoglobin 121 gL Plt 296
bull Total protein 90gL IgG 34gL monoclonal peak
209gL
Case 4 Monoclonal IgG patient
Polyclonal B cells
With maturing hematogones
helper suppressor T cells
less than 3 blasts
Maturing myeloid population
Case 4 BM Ungated
Case 4 cytoplasmic immunoglobulin
Need KL
Case 4 BM Multiple Myeloma
Case 4 follow up
bull Smouldering myeloma
bull Recently developed CRAB (hyperCalcemia Renal
insufficiency Anemia Bone lesions
bull Serum free light chain ratio 123
bull Bone marrow aspirate gt60 plasma cells
bull Currently on treatment pending bone marrow
transplant
Case 5
bull 65 year old male
bull Slight lymphocytosis detected on routine CBC analysis
(lymph 4500uL)
bull Asymptomatic sent for flow cytometry
Case 5 PB
Case 5 flow cytometry KL ratio 0625
Case 5 Monoclonal B cell lymphocytosis
KL 18 KL 056
Case 5 Monoclonal B cell lymphocytosis
bull WHO defines CLL as abnormal lymphocyte count
gt5x10E9L (CD19+CD5+ light chain restricted) in the
absence of other clinical symptoms
bull Patients with small B cell clones are often detected as
an incidental finding
bull WHO 2016
bull CD19+CD5+ lt05x10E9L ldquoLow count MBLrdquo
raquo 05-5x10E9L ldquoHigh count MBLrdquo
raquo Non CLL type phenotype MBL also recognized
Monoclonal B cell Lymphocytosis
bull CLL immunophenotype - vast majority (75)
bull Atypical MBL
bull normal levels both CD5 and CD20 with variable
expression of CD23 (always exclude Mantle-cell
Lymphoma in CD23 negative cases)
bull Non-CLL-phenotype (CD5minus) MBL
bull Lacking CD5 expression and CD10 likely resembling B-
cell lymphomas of marginal-zone origin
Case 5 What is the normal KL Ratio
bull What is the normal or abnormal kappalambda ratio for
entire ldquopopulationrdquo of all B cells
bull Traditional view
ndash Kappa Lambda 21 or 31 ( 12 -13)
ndash Range 14 60
Case 5 What is the normal KL ratio
bull Wrong question
bull It doesnrsquot matter how many of the B cells are clonal but rather
that their exists a clonal population of B cells within the total
population
bull Need to Identify malignant B cells within a population of
normal B cells
bull In order to interpret a pathological immunophenotype it is
necessary to refer to quantitative and qualitative values of
normal B-cell subpopulations
bull Pearl Identify the abnormal phenotype first then look at KL
ratio
Case 6
bull 12 year old male presents to ER with fatigue bruising
Lkc 2200uL
bull Hb 84gL
bull Plt 32
bull Diff ndash neutropenia no blasts noted few ldquoatypicalrdquo cells
bull RBC ndash non specific changes
Leukemia
Case 6 PB
Case 6 BM
Case 6
bull BM aspirate ndash Hypocellular relative lymphocytosis
marked reduction in myeloid lineages
hypoplasticaplastic anemia
bull Flow Cytometry ndash less than 3 blasts maturing
myeloid 68 lymphocytes polyclonal B helper
suppressor T
bull Unusual pattern of CD13CD16 staining noted
Case 6 flow cytometry Aplastic Anemia
Normal Patient
Normal
Patient
Patient
Normal
Case 6 RBC Test for PNH CD235a v s CD59
Case 6 Neutrophil test for PNH clone
FLAER CD24 CD45CD15
18 PNH clone
Case 6
bull Follow up
bull Patient on steroids acyclovir and anti-thymocyte
globulin
bull PNH clone Neutrophils 96 RBC 1 Nov 2015
bull In remission as of April 2017
- Lkc 5500uL (Neuts 23)
- Hb 129gL
- Plt 208 X10e9
Summary
bull B cell malignancies are the most common
hematological disorders seen in flow cytometry
bull Many have complex overlapping phenotypes
bull Important to look at ungated plots as well as gating on
specific subpopulations
bull Level of expression of markers is as important as
positive or negative (think CD13 vs CD16)
68
References
bull Craig FE Foon KA Flow cytometric immunophenotyping for hematologic neoplasms Blood
2008 Apr 15111(8)3941-67
bull Wood BL Principles of minimal residual disease detection for hematopoietic neoplasms by flow
cytometry Cytometry B Clin Cytom 2015 Apr 23
bull Shanafelt TD Ghia P Lanasa MC Landgren O Rawstron AC Monoclonal B-cell
lymphocytosis (MBL) biology natural history and clinical management Leukemia 2010
Mar24(3)512-20
bull Swerdlow SH Campo E Pileri SA Harris NL Stein H Siebert R Advani RGhielmini M Salles
GA Zelenetz AD Jaffe ES The 2016 revision of the World Health Organization classification of
lymphoid neoplasms Blood 2016 May 19127(20)2375-90
bull 3 Moench L Sachs Z Aasen G Dolan M Dayton V Courville EL Double- and triple-hit
lymphomas can present with features suggestive of immaturity including TdT expression and
create diagnostic challenges Leuk Lymphoma 2016572626-35
bull Sutherland DR Keeney M Illingworth A Practical guidelines for the high-sensitivity detection
and monitoring of paroxysmal nocturnal hemoglobinuria clones by flow cytometry Cytometry B
Clin Cytom 2012 Jul82(4)195-208
69
Alternative to real time compensation
bull Files collected in listmode can be analysed offline using several software packages
bull A control file can be run with mutually exclusive populations and then the settings from this file used to compensate any other data run with the same fluorochromes at the same PMT settings
bull Archival data can be reanalyzed ndash data is not ldquolostrdquo if incorrect compensation set at time of acquisition
Compensation must be done on the
actual fluorochrome used in the test
CD34 ANALYSIS WITH 7-AAD VIABILITY DYE
FL4-FL2 = 20
Appropriate compensation
Appropriate Compensation
CD34+
36uL
CD34 ANALYSIS WITH 7-AAD VIABILITY DYE
FL4-FL2 -10
Under compensation
Under compensation
CD34+
8uL
Recognizing bad flow data
Time Gating
Doublet Discrimination
DOUBLETS CAN CREATE ERRONEOUS RESULTS
False
CD19+CD5+
population
Current 8-10 color analysis on
bench top analyzers round bull Major advances in computing power optics
electronics and fluorochrome technology
bull uarr Colors = uarr Complexity
ndash what combinations
ndash what fluorochromes
London Health Custom Design Tube
Antigen Fluorochrome
CD8 FITC
Kappa FITC
CD4 PE
Lambda PE
CD19 ECD
CD20 PE-Cy55
CD5 PE-Cy7
CD10 APC
CD56 APC-Alexa700
Near-IR Viability Dye Excitation 633nm
Emission 780nm
CD3 Pacific Blue
CD45 Krome Orange
Result Reporting Bethesda Guidelines
bull Report percent abnormal population
bull Antibody Distribution
ndash Negative Positive Partial
bull Antibody Fluorescence
- Dim mdash Positive uniform cell population with a median fluorescence intensity abnormally less than that seen on an antigen positive normal leukocyte population Example CD20 in CLL
- Bright mdash Positive uniform cell population with a median fluorescence intensity abnormally greater than that seen on an antigen positive normal leukocyte population Example CD20 in HCL
- Heterogeneous mdash Variably intense staining not a tight uniform cluster of positive immunofluorescence staining
Example CD38 on maturing B cells
Bethesda Reporting
Normal
Dim
CLL
CD19+ CD20+(dim)
CD5+(dim)Kappa+(dim)
Discussion
bull The availability of new fluorochromes coupled with
multilaser instruments with simplified setup and
compensation makes routine ten color analysis possible for
the routine clinical laboratory
bull Knowledge of fluorochromes antibody and antigen
interactions required
bull Full understanding of normal versus abnormal population
phenotype required
bull Sophisticated data analysis programs coupled with
education essential for successful implementation ndash for all
staff involved in testing and analysis
Normal B Cell Maturation
Wood and Borowitz (2006) Henryrsquos Laboratory Medicine
Normal B cell Maturation Pattern
The three pearls
Case 1
bull Nine month old male seen by GP for nodules in his
scalp suggestive of lymphoid infiltrate incidental
finding of bilateral enlarged kidneys
bull Referred to hematology as WBC 16300uL increased
Plt
bull WBC 12300uL Hb 111gL Plt 536x109L
bull Normal differential
bull Scalp lymphoid infiltrate suspicious for leukemia
bull Bone marrow aspirate flow ordered
Case 1
34
BM 14 B cells
5 CD10dimneg
CD20 negCD38 bright
plasma cells
Case 1 BM
Case 1 Bone marrow morphology
bull Cellular marrow
bull Erythropoeisis 20-25
bull Granulopoesis 40-50 orderly maturation
bull Lymphocytes 30 10- 15 ldquoblast likerdquo
bull Increased megakaryocytes
Case 1 2 week follow up
77 B cells ndash 90
CD10 dimneg
CD20 neg bright CD38
pB-ALL
Case 1 BM 2 week follow up
Case 1 Cytogenetics
bull Complex karyotype
bull Structural abnormalities in chromosome
123491417 and 19
bull t(1719) rare in B cell acute lymphoblastic
leukemia
Case 1 Follow up
bull On high risk protocol AALL 0232 for infantile B-ALL
bull MRD 008 at Day 42
bull Cord blood transplant
bull Relapse 5 months post transplant
Pearl of Pathology
bull Differentiating precursor B cells (hematogones) from B-
ALL can be challenging Standardized analysis and
clear understanding of ldquonormalrdquo essential including
arrested maturation
Case 2
bull 4 year old girl referred to pediatric hematology with
pancytopenia
bull WBC 6800uL(Neuts 400uL) Hb 42gL Plt 28
bull Aplastic anemia
bull Bone marrow ordered aspirate biopsy flow and
cytogenetics
Case 2 BM flow cytometry
Polyclonal B cells
helper suppressor T cells
No increase in blasts
Maturing myeloid population
Case 2 CD45 neg B-ALL
Case 2 BM
Case 2
bull CD45neg
bull CD19+CD10+
bull CD20(partialdim)
bull CD34 neg
bull SigampcIg neg
bull Cytogenetics
bull Gain of X chromosome and
468911131415171821
DNA Index
122 Diploid
Case 2
bull Currently in remission after completion of
AALL0932 standard risk B-ALL therapy
Case 2
Pearls of Pathology
CD45 negative B-ALL is not uncommon
bull Always look at ungated set of plots for each marker
bull Eg SS versus each fluorescence parameter
Case 3
bull 58 year old male with pararenal mass
bull WBC 4000uL Hb 98gL Plt 229
bull ldquoblastsrdquo reported on blood film
bull Peripheral blood sent for flow cytometry
Case 3 PB flow cytometry
Dim CD45 CD19+CD10+
CD34- Sig neg
Case 3 PB flow cytometry
Dim CD45 CD19+CD10+
CD20(partial)CD5 neg
SIg cIg neg
Case 3 BM flow cytometry
Case 3 BM biopsy
Case 3 BM biopsy
Case 3 BM biopsy report
bull Diffuse infiltrate large lymphoid cells
bull Scant to moderate cytoplasm
bull Large irregular nuclei with prominent nucleoli
bull Increased mitotic activity
bull Positive for BCL-2 BCL6 CD10 and MUM1
bull Negative for TdT CD5
bull Cytogenetics FISH on para renal mass ndash C-MYC and
BCL2 rearrangements detected
Case 3 Doubletriple hit lymphoma
(DHLTHL)
bull Morphologically heterogeneous B cell neoplasms
characterized by rearrangement of the MYC proto-
oncogene with concurrent BCL2 andor BCL6 gene
rearrangement
bull WHO 2008 criteria - most cases of DHL and THL
classified DLBCL or B-cell lymphoma unclassifiable
with features intermediate between DLBCL and Burkitt
lymphoma (BCLU)
Case 3 DHLTHL
bull Retrospective study from one institution features of 13 cases (9 DHL4
THL)
bull median age was 59 years (range 30-74) eight females five males
bull enlarging lymphadenopathymasses (11 patients) abnormal peripheral
blood findings (2 patients)
bull TdT positivity (four cases two THLtwo DHL) dim CD45 expression (7
cases) lack of CD20 (2 cases) or lack of sIg (3 cases) and blastoid
morphology (2 cases)
bull Conclusion Expression of TdT in a B-cell lymphoma with mature
features or expression of surface light chain in a case otherwise
suggestive of B-lymphoblastic leukemialymphoma should prompt
an expedited evaluation for DHLTHL
Leuk Lymphoma 2016 Feb 181-10 [Epub ahead of print]
Double- and triple-hit lymphomas can present with features suggestive
of immaturity including TdT expression and create diagnostic challenges
Moench L Sachs Z Aasen G Dolan M Dayton V Courville EL
Case 4
bull 55 year old female
bull Previous history hyperparathyroidism
bull Parathyroidectomy in 1999
bull Mild persistent hypercalcemia
bull WBC 4800uL hemoglobin 121 gL Plt 296
bull Total protein 90gL IgG 34gL monoclonal peak
209gL
Case 4 Monoclonal IgG patient
Polyclonal B cells
With maturing hematogones
helper suppressor T cells
less than 3 blasts
Maturing myeloid population
Case 4 BM Ungated
Case 4 cytoplasmic immunoglobulin
Need KL
Case 4 BM Multiple Myeloma
Case 4 follow up
bull Smouldering myeloma
bull Recently developed CRAB (hyperCalcemia Renal
insufficiency Anemia Bone lesions
bull Serum free light chain ratio 123
bull Bone marrow aspirate gt60 plasma cells
bull Currently on treatment pending bone marrow
transplant
Case 5
bull 65 year old male
bull Slight lymphocytosis detected on routine CBC analysis
(lymph 4500uL)
bull Asymptomatic sent for flow cytometry
Case 5 PB
Case 5 flow cytometry KL ratio 0625
Case 5 Monoclonal B cell lymphocytosis
KL 18 KL 056
Case 5 Monoclonal B cell lymphocytosis
bull WHO defines CLL as abnormal lymphocyte count
gt5x10E9L (CD19+CD5+ light chain restricted) in the
absence of other clinical symptoms
bull Patients with small B cell clones are often detected as
an incidental finding
bull WHO 2016
bull CD19+CD5+ lt05x10E9L ldquoLow count MBLrdquo
raquo 05-5x10E9L ldquoHigh count MBLrdquo
raquo Non CLL type phenotype MBL also recognized
Monoclonal B cell Lymphocytosis
bull CLL immunophenotype - vast majority (75)
bull Atypical MBL
bull normal levels both CD5 and CD20 with variable
expression of CD23 (always exclude Mantle-cell
Lymphoma in CD23 negative cases)
bull Non-CLL-phenotype (CD5minus) MBL
bull Lacking CD5 expression and CD10 likely resembling B-
cell lymphomas of marginal-zone origin
Case 5 What is the normal KL Ratio
bull What is the normal or abnormal kappalambda ratio for
entire ldquopopulationrdquo of all B cells
bull Traditional view
ndash Kappa Lambda 21 or 31 ( 12 -13)
ndash Range 14 60
Case 5 What is the normal KL ratio
bull Wrong question
bull It doesnrsquot matter how many of the B cells are clonal but rather
that their exists a clonal population of B cells within the total
population
bull Need to Identify malignant B cells within a population of
normal B cells
bull In order to interpret a pathological immunophenotype it is
necessary to refer to quantitative and qualitative values of
normal B-cell subpopulations
bull Pearl Identify the abnormal phenotype first then look at KL
ratio
Case 6
bull 12 year old male presents to ER with fatigue bruising
Lkc 2200uL
bull Hb 84gL
bull Plt 32
bull Diff ndash neutropenia no blasts noted few ldquoatypicalrdquo cells
bull RBC ndash non specific changes
Leukemia
Case 6 PB
Case 6 BM
Case 6
bull BM aspirate ndash Hypocellular relative lymphocytosis
marked reduction in myeloid lineages
hypoplasticaplastic anemia
bull Flow Cytometry ndash less than 3 blasts maturing
myeloid 68 lymphocytes polyclonal B helper
suppressor T
bull Unusual pattern of CD13CD16 staining noted
Case 6 flow cytometry Aplastic Anemia
Normal Patient
Normal
Patient
Patient
Normal
Case 6 RBC Test for PNH CD235a v s CD59
Case 6 Neutrophil test for PNH clone
FLAER CD24 CD45CD15
18 PNH clone
Case 6
bull Follow up
bull Patient on steroids acyclovir and anti-thymocyte
globulin
bull PNH clone Neutrophils 96 RBC 1 Nov 2015
bull In remission as of April 2017
- Lkc 5500uL (Neuts 23)
- Hb 129gL
- Plt 208 X10e9
Summary
bull B cell malignancies are the most common
hematological disorders seen in flow cytometry
bull Many have complex overlapping phenotypes
bull Important to look at ungated plots as well as gating on
specific subpopulations
bull Level of expression of markers is as important as
positive or negative (think CD13 vs CD16)
68
References
bull Craig FE Foon KA Flow cytometric immunophenotyping for hematologic neoplasms Blood
2008 Apr 15111(8)3941-67
bull Wood BL Principles of minimal residual disease detection for hematopoietic neoplasms by flow
cytometry Cytometry B Clin Cytom 2015 Apr 23
bull Shanafelt TD Ghia P Lanasa MC Landgren O Rawstron AC Monoclonal B-cell
lymphocytosis (MBL) biology natural history and clinical management Leukemia 2010
Mar24(3)512-20
bull Swerdlow SH Campo E Pileri SA Harris NL Stein H Siebert R Advani RGhielmini M Salles
GA Zelenetz AD Jaffe ES The 2016 revision of the World Health Organization classification of
lymphoid neoplasms Blood 2016 May 19127(20)2375-90
bull 3 Moench L Sachs Z Aasen G Dolan M Dayton V Courville EL Double- and triple-hit
lymphomas can present with features suggestive of immaturity including TdT expression and
create diagnostic challenges Leuk Lymphoma 2016572626-35
bull Sutherland DR Keeney M Illingworth A Practical guidelines for the high-sensitivity detection
and monitoring of paroxysmal nocturnal hemoglobinuria clones by flow cytometry Cytometry B
Clin Cytom 2012 Jul82(4)195-208
69
Compensation must be done on the
actual fluorochrome used in the test
CD34 ANALYSIS WITH 7-AAD VIABILITY DYE
FL4-FL2 = 20
Appropriate compensation
Appropriate Compensation
CD34+
36uL
CD34 ANALYSIS WITH 7-AAD VIABILITY DYE
FL4-FL2 -10
Under compensation
Under compensation
CD34+
8uL
Recognizing bad flow data
Time Gating
Doublet Discrimination
DOUBLETS CAN CREATE ERRONEOUS RESULTS
False
CD19+CD5+
population
Current 8-10 color analysis on
bench top analyzers round bull Major advances in computing power optics
electronics and fluorochrome technology
bull uarr Colors = uarr Complexity
ndash what combinations
ndash what fluorochromes
London Health Custom Design Tube
Antigen Fluorochrome
CD8 FITC
Kappa FITC
CD4 PE
Lambda PE
CD19 ECD
CD20 PE-Cy55
CD5 PE-Cy7
CD10 APC
CD56 APC-Alexa700
Near-IR Viability Dye Excitation 633nm
Emission 780nm
CD3 Pacific Blue
CD45 Krome Orange
Result Reporting Bethesda Guidelines
bull Report percent abnormal population
bull Antibody Distribution
ndash Negative Positive Partial
bull Antibody Fluorescence
- Dim mdash Positive uniform cell population with a median fluorescence intensity abnormally less than that seen on an antigen positive normal leukocyte population Example CD20 in CLL
- Bright mdash Positive uniform cell population with a median fluorescence intensity abnormally greater than that seen on an antigen positive normal leukocyte population Example CD20 in HCL
- Heterogeneous mdash Variably intense staining not a tight uniform cluster of positive immunofluorescence staining
Example CD38 on maturing B cells
Bethesda Reporting
Normal
Dim
CLL
CD19+ CD20+(dim)
CD5+(dim)Kappa+(dim)
Discussion
bull The availability of new fluorochromes coupled with
multilaser instruments with simplified setup and
compensation makes routine ten color analysis possible for
the routine clinical laboratory
bull Knowledge of fluorochromes antibody and antigen
interactions required
bull Full understanding of normal versus abnormal population
phenotype required
bull Sophisticated data analysis programs coupled with
education essential for successful implementation ndash for all
staff involved in testing and analysis
Normal B Cell Maturation
Wood and Borowitz (2006) Henryrsquos Laboratory Medicine
Normal B cell Maturation Pattern
The three pearls
Case 1
bull Nine month old male seen by GP for nodules in his
scalp suggestive of lymphoid infiltrate incidental
finding of bilateral enlarged kidneys
bull Referred to hematology as WBC 16300uL increased
Plt
bull WBC 12300uL Hb 111gL Plt 536x109L
bull Normal differential
bull Scalp lymphoid infiltrate suspicious for leukemia
bull Bone marrow aspirate flow ordered
Case 1
34
BM 14 B cells
5 CD10dimneg
CD20 negCD38 bright
plasma cells
Case 1 BM
Case 1 Bone marrow morphology
bull Cellular marrow
bull Erythropoeisis 20-25
bull Granulopoesis 40-50 orderly maturation
bull Lymphocytes 30 10- 15 ldquoblast likerdquo
bull Increased megakaryocytes
Case 1 2 week follow up
77 B cells ndash 90
CD10 dimneg
CD20 neg bright CD38
pB-ALL
Case 1 BM 2 week follow up
Case 1 Cytogenetics
bull Complex karyotype
bull Structural abnormalities in chromosome
123491417 and 19
bull t(1719) rare in B cell acute lymphoblastic
leukemia
Case 1 Follow up
bull On high risk protocol AALL 0232 for infantile B-ALL
bull MRD 008 at Day 42
bull Cord blood transplant
bull Relapse 5 months post transplant
Pearl of Pathology
bull Differentiating precursor B cells (hematogones) from B-
ALL can be challenging Standardized analysis and
clear understanding of ldquonormalrdquo essential including
arrested maturation
Case 2
bull 4 year old girl referred to pediatric hematology with
pancytopenia
bull WBC 6800uL(Neuts 400uL) Hb 42gL Plt 28
bull Aplastic anemia
bull Bone marrow ordered aspirate biopsy flow and
cytogenetics
Case 2 BM flow cytometry
Polyclonal B cells
helper suppressor T cells
No increase in blasts
Maturing myeloid population
Case 2 CD45 neg B-ALL
Case 2 BM
Case 2
bull CD45neg
bull CD19+CD10+
bull CD20(partialdim)
bull CD34 neg
bull SigampcIg neg
bull Cytogenetics
bull Gain of X chromosome and
468911131415171821
DNA Index
122 Diploid
Case 2
bull Currently in remission after completion of
AALL0932 standard risk B-ALL therapy
Case 2
Pearls of Pathology
CD45 negative B-ALL is not uncommon
bull Always look at ungated set of plots for each marker
bull Eg SS versus each fluorescence parameter
Case 3
bull 58 year old male with pararenal mass
bull WBC 4000uL Hb 98gL Plt 229
bull ldquoblastsrdquo reported on blood film
bull Peripheral blood sent for flow cytometry
Case 3 PB flow cytometry
Dim CD45 CD19+CD10+
CD34- Sig neg
Case 3 PB flow cytometry
Dim CD45 CD19+CD10+
CD20(partial)CD5 neg
SIg cIg neg
Case 3 BM flow cytometry
Case 3 BM biopsy
Case 3 BM biopsy
Case 3 BM biopsy report
bull Diffuse infiltrate large lymphoid cells
bull Scant to moderate cytoplasm
bull Large irregular nuclei with prominent nucleoli
bull Increased mitotic activity
bull Positive for BCL-2 BCL6 CD10 and MUM1
bull Negative for TdT CD5
bull Cytogenetics FISH on para renal mass ndash C-MYC and
BCL2 rearrangements detected
Case 3 Doubletriple hit lymphoma
(DHLTHL)
bull Morphologically heterogeneous B cell neoplasms
characterized by rearrangement of the MYC proto-
oncogene with concurrent BCL2 andor BCL6 gene
rearrangement
bull WHO 2008 criteria - most cases of DHL and THL
classified DLBCL or B-cell lymphoma unclassifiable
with features intermediate between DLBCL and Burkitt
lymphoma (BCLU)
Case 3 DHLTHL
bull Retrospective study from one institution features of 13 cases (9 DHL4
THL)
bull median age was 59 years (range 30-74) eight females five males
bull enlarging lymphadenopathymasses (11 patients) abnormal peripheral
blood findings (2 patients)
bull TdT positivity (four cases two THLtwo DHL) dim CD45 expression (7
cases) lack of CD20 (2 cases) or lack of sIg (3 cases) and blastoid
morphology (2 cases)
bull Conclusion Expression of TdT in a B-cell lymphoma with mature
features or expression of surface light chain in a case otherwise
suggestive of B-lymphoblastic leukemialymphoma should prompt
an expedited evaluation for DHLTHL
Leuk Lymphoma 2016 Feb 181-10 [Epub ahead of print]
Double- and triple-hit lymphomas can present with features suggestive
of immaturity including TdT expression and create diagnostic challenges
Moench L Sachs Z Aasen G Dolan M Dayton V Courville EL
Case 4
bull 55 year old female
bull Previous history hyperparathyroidism
bull Parathyroidectomy in 1999
bull Mild persistent hypercalcemia
bull WBC 4800uL hemoglobin 121 gL Plt 296
bull Total protein 90gL IgG 34gL monoclonal peak
209gL
Case 4 Monoclonal IgG patient
Polyclonal B cells
With maturing hematogones
helper suppressor T cells
less than 3 blasts
Maturing myeloid population
Case 4 BM Ungated
Case 4 cytoplasmic immunoglobulin
Need KL
Case 4 BM Multiple Myeloma
Case 4 follow up
bull Smouldering myeloma
bull Recently developed CRAB (hyperCalcemia Renal
insufficiency Anemia Bone lesions
bull Serum free light chain ratio 123
bull Bone marrow aspirate gt60 plasma cells
bull Currently on treatment pending bone marrow
transplant
Case 5
bull 65 year old male
bull Slight lymphocytosis detected on routine CBC analysis
(lymph 4500uL)
bull Asymptomatic sent for flow cytometry
Case 5 PB
Case 5 flow cytometry KL ratio 0625
Case 5 Monoclonal B cell lymphocytosis
KL 18 KL 056
Case 5 Monoclonal B cell lymphocytosis
bull WHO defines CLL as abnormal lymphocyte count
gt5x10E9L (CD19+CD5+ light chain restricted) in the
absence of other clinical symptoms
bull Patients with small B cell clones are often detected as
an incidental finding
bull WHO 2016
bull CD19+CD5+ lt05x10E9L ldquoLow count MBLrdquo
raquo 05-5x10E9L ldquoHigh count MBLrdquo
raquo Non CLL type phenotype MBL also recognized
Monoclonal B cell Lymphocytosis
bull CLL immunophenotype - vast majority (75)
bull Atypical MBL
bull normal levels both CD5 and CD20 with variable
expression of CD23 (always exclude Mantle-cell
Lymphoma in CD23 negative cases)
bull Non-CLL-phenotype (CD5minus) MBL
bull Lacking CD5 expression and CD10 likely resembling B-
cell lymphomas of marginal-zone origin
Case 5 What is the normal KL Ratio
bull What is the normal or abnormal kappalambda ratio for
entire ldquopopulationrdquo of all B cells
bull Traditional view
ndash Kappa Lambda 21 or 31 ( 12 -13)
ndash Range 14 60
Case 5 What is the normal KL ratio
bull Wrong question
bull It doesnrsquot matter how many of the B cells are clonal but rather
that their exists a clonal population of B cells within the total
population
bull Need to Identify malignant B cells within a population of
normal B cells
bull In order to interpret a pathological immunophenotype it is
necessary to refer to quantitative and qualitative values of
normal B-cell subpopulations
bull Pearl Identify the abnormal phenotype first then look at KL
ratio
Case 6
bull 12 year old male presents to ER with fatigue bruising
Lkc 2200uL
bull Hb 84gL
bull Plt 32
bull Diff ndash neutropenia no blasts noted few ldquoatypicalrdquo cells
bull RBC ndash non specific changes
Leukemia
Case 6 PB
Case 6 BM
Case 6
bull BM aspirate ndash Hypocellular relative lymphocytosis
marked reduction in myeloid lineages
hypoplasticaplastic anemia
bull Flow Cytometry ndash less than 3 blasts maturing
myeloid 68 lymphocytes polyclonal B helper
suppressor T
bull Unusual pattern of CD13CD16 staining noted
Case 6 flow cytometry Aplastic Anemia
Normal Patient
Normal
Patient
Patient
Normal
Case 6 RBC Test for PNH CD235a v s CD59
Case 6 Neutrophil test for PNH clone
FLAER CD24 CD45CD15
18 PNH clone
Case 6
bull Follow up
bull Patient on steroids acyclovir and anti-thymocyte
globulin
bull PNH clone Neutrophils 96 RBC 1 Nov 2015
bull In remission as of April 2017
- Lkc 5500uL (Neuts 23)
- Hb 129gL
- Plt 208 X10e9
Summary
bull B cell malignancies are the most common
hematological disorders seen in flow cytometry
bull Many have complex overlapping phenotypes
bull Important to look at ungated plots as well as gating on
specific subpopulations
bull Level of expression of markers is as important as
positive or negative (think CD13 vs CD16)
68
References
bull Craig FE Foon KA Flow cytometric immunophenotyping for hematologic neoplasms Blood
2008 Apr 15111(8)3941-67
bull Wood BL Principles of minimal residual disease detection for hematopoietic neoplasms by flow
cytometry Cytometry B Clin Cytom 2015 Apr 23
bull Shanafelt TD Ghia P Lanasa MC Landgren O Rawstron AC Monoclonal B-cell
lymphocytosis (MBL) biology natural history and clinical management Leukemia 2010
Mar24(3)512-20
bull Swerdlow SH Campo E Pileri SA Harris NL Stein H Siebert R Advani RGhielmini M Salles
GA Zelenetz AD Jaffe ES The 2016 revision of the World Health Organization classification of
lymphoid neoplasms Blood 2016 May 19127(20)2375-90
bull 3 Moench L Sachs Z Aasen G Dolan M Dayton V Courville EL Double- and triple-hit
lymphomas can present with features suggestive of immaturity including TdT expression and
create diagnostic challenges Leuk Lymphoma 2016572626-35
bull Sutherland DR Keeney M Illingworth A Practical guidelines for the high-sensitivity detection
and monitoring of paroxysmal nocturnal hemoglobinuria clones by flow cytometry Cytometry B
Clin Cytom 2012 Jul82(4)195-208
69
CD34 ANALYSIS WITH 7-AAD VIABILITY DYE
FL4-FL2 = 20
Appropriate compensation
Appropriate Compensation
CD34+
36uL
CD34 ANALYSIS WITH 7-AAD VIABILITY DYE
FL4-FL2 -10
Under compensation
Under compensation
CD34+
8uL
Recognizing bad flow data
Time Gating
Doublet Discrimination
DOUBLETS CAN CREATE ERRONEOUS RESULTS
False
CD19+CD5+
population
Current 8-10 color analysis on
bench top analyzers round bull Major advances in computing power optics
electronics and fluorochrome technology
bull uarr Colors = uarr Complexity
ndash what combinations
ndash what fluorochromes
London Health Custom Design Tube
Antigen Fluorochrome
CD8 FITC
Kappa FITC
CD4 PE
Lambda PE
CD19 ECD
CD20 PE-Cy55
CD5 PE-Cy7
CD10 APC
CD56 APC-Alexa700
Near-IR Viability Dye Excitation 633nm
Emission 780nm
CD3 Pacific Blue
CD45 Krome Orange
Result Reporting Bethesda Guidelines
bull Report percent abnormal population
bull Antibody Distribution
ndash Negative Positive Partial
bull Antibody Fluorescence
- Dim mdash Positive uniform cell population with a median fluorescence intensity abnormally less than that seen on an antigen positive normal leukocyte population Example CD20 in CLL
- Bright mdash Positive uniform cell population with a median fluorescence intensity abnormally greater than that seen on an antigen positive normal leukocyte population Example CD20 in HCL
- Heterogeneous mdash Variably intense staining not a tight uniform cluster of positive immunofluorescence staining
Example CD38 on maturing B cells
Bethesda Reporting
Normal
Dim
CLL
CD19+ CD20+(dim)
CD5+(dim)Kappa+(dim)
Discussion
bull The availability of new fluorochromes coupled with
multilaser instruments with simplified setup and
compensation makes routine ten color analysis possible for
the routine clinical laboratory
bull Knowledge of fluorochromes antibody and antigen
interactions required
bull Full understanding of normal versus abnormal population
phenotype required
bull Sophisticated data analysis programs coupled with
education essential for successful implementation ndash for all
staff involved in testing and analysis
Normal B Cell Maturation
Wood and Borowitz (2006) Henryrsquos Laboratory Medicine
Normal B cell Maturation Pattern
The three pearls
Case 1
bull Nine month old male seen by GP for nodules in his
scalp suggestive of lymphoid infiltrate incidental
finding of bilateral enlarged kidneys
bull Referred to hematology as WBC 16300uL increased
Plt
bull WBC 12300uL Hb 111gL Plt 536x109L
bull Normal differential
bull Scalp lymphoid infiltrate suspicious for leukemia
bull Bone marrow aspirate flow ordered
Case 1
34
BM 14 B cells
5 CD10dimneg
CD20 negCD38 bright
plasma cells
Case 1 BM
Case 1 Bone marrow morphology
bull Cellular marrow
bull Erythropoeisis 20-25
bull Granulopoesis 40-50 orderly maturation
bull Lymphocytes 30 10- 15 ldquoblast likerdquo
bull Increased megakaryocytes
Case 1 2 week follow up
77 B cells ndash 90
CD10 dimneg
CD20 neg bright CD38
pB-ALL
Case 1 BM 2 week follow up
Case 1 Cytogenetics
bull Complex karyotype
bull Structural abnormalities in chromosome
123491417 and 19
bull t(1719) rare in B cell acute lymphoblastic
leukemia
Case 1 Follow up
bull On high risk protocol AALL 0232 for infantile B-ALL
bull MRD 008 at Day 42
bull Cord blood transplant
bull Relapse 5 months post transplant
Pearl of Pathology
bull Differentiating precursor B cells (hematogones) from B-
ALL can be challenging Standardized analysis and
clear understanding of ldquonormalrdquo essential including
arrested maturation
Case 2
bull 4 year old girl referred to pediatric hematology with
pancytopenia
bull WBC 6800uL(Neuts 400uL) Hb 42gL Plt 28
bull Aplastic anemia
bull Bone marrow ordered aspirate biopsy flow and
cytogenetics
Case 2 BM flow cytometry
Polyclonal B cells
helper suppressor T cells
No increase in blasts
Maturing myeloid population
Case 2 CD45 neg B-ALL
Case 2 BM
Case 2
bull CD45neg
bull CD19+CD10+
bull CD20(partialdim)
bull CD34 neg
bull SigampcIg neg
bull Cytogenetics
bull Gain of X chromosome and
468911131415171821
DNA Index
122 Diploid
Case 2
bull Currently in remission after completion of
AALL0932 standard risk B-ALL therapy
Case 2
Pearls of Pathology
CD45 negative B-ALL is not uncommon
bull Always look at ungated set of plots for each marker
bull Eg SS versus each fluorescence parameter
Case 3
bull 58 year old male with pararenal mass
bull WBC 4000uL Hb 98gL Plt 229
bull ldquoblastsrdquo reported on blood film
bull Peripheral blood sent for flow cytometry
Case 3 PB flow cytometry
Dim CD45 CD19+CD10+
CD34- Sig neg
Case 3 PB flow cytometry
Dim CD45 CD19+CD10+
CD20(partial)CD5 neg
SIg cIg neg
Case 3 BM flow cytometry
Case 3 BM biopsy
Case 3 BM biopsy
Case 3 BM biopsy report
bull Diffuse infiltrate large lymphoid cells
bull Scant to moderate cytoplasm
bull Large irregular nuclei with prominent nucleoli
bull Increased mitotic activity
bull Positive for BCL-2 BCL6 CD10 and MUM1
bull Negative for TdT CD5
bull Cytogenetics FISH on para renal mass ndash C-MYC and
BCL2 rearrangements detected
Case 3 Doubletriple hit lymphoma
(DHLTHL)
bull Morphologically heterogeneous B cell neoplasms
characterized by rearrangement of the MYC proto-
oncogene with concurrent BCL2 andor BCL6 gene
rearrangement
bull WHO 2008 criteria - most cases of DHL and THL
classified DLBCL or B-cell lymphoma unclassifiable
with features intermediate between DLBCL and Burkitt
lymphoma (BCLU)
Case 3 DHLTHL
bull Retrospective study from one institution features of 13 cases (9 DHL4
THL)
bull median age was 59 years (range 30-74) eight females five males
bull enlarging lymphadenopathymasses (11 patients) abnormal peripheral
blood findings (2 patients)
bull TdT positivity (four cases two THLtwo DHL) dim CD45 expression (7
cases) lack of CD20 (2 cases) or lack of sIg (3 cases) and blastoid
morphology (2 cases)
bull Conclusion Expression of TdT in a B-cell lymphoma with mature
features or expression of surface light chain in a case otherwise
suggestive of B-lymphoblastic leukemialymphoma should prompt
an expedited evaluation for DHLTHL
Leuk Lymphoma 2016 Feb 181-10 [Epub ahead of print]
Double- and triple-hit lymphomas can present with features suggestive
of immaturity including TdT expression and create diagnostic challenges
Moench L Sachs Z Aasen G Dolan M Dayton V Courville EL
Case 4
bull 55 year old female
bull Previous history hyperparathyroidism
bull Parathyroidectomy in 1999
bull Mild persistent hypercalcemia
bull WBC 4800uL hemoglobin 121 gL Plt 296
bull Total protein 90gL IgG 34gL monoclonal peak
209gL
Case 4 Monoclonal IgG patient
Polyclonal B cells
With maturing hematogones
helper suppressor T cells
less than 3 blasts
Maturing myeloid population
Case 4 BM Ungated
Case 4 cytoplasmic immunoglobulin
Need KL
Case 4 BM Multiple Myeloma
Case 4 follow up
bull Smouldering myeloma
bull Recently developed CRAB (hyperCalcemia Renal
insufficiency Anemia Bone lesions
bull Serum free light chain ratio 123
bull Bone marrow aspirate gt60 plasma cells
bull Currently on treatment pending bone marrow
transplant
Case 5
bull 65 year old male
bull Slight lymphocytosis detected on routine CBC analysis
(lymph 4500uL)
bull Asymptomatic sent for flow cytometry
Case 5 PB
Case 5 flow cytometry KL ratio 0625
Case 5 Monoclonal B cell lymphocytosis
KL 18 KL 056
Case 5 Monoclonal B cell lymphocytosis
bull WHO defines CLL as abnormal lymphocyte count
gt5x10E9L (CD19+CD5+ light chain restricted) in the
absence of other clinical symptoms
bull Patients with small B cell clones are often detected as
an incidental finding
bull WHO 2016
bull CD19+CD5+ lt05x10E9L ldquoLow count MBLrdquo
raquo 05-5x10E9L ldquoHigh count MBLrdquo
raquo Non CLL type phenotype MBL also recognized
Monoclonal B cell Lymphocytosis
bull CLL immunophenotype - vast majority (75)
bull Atypical MBL
bull normal levels both CD5 and CD20 with variable
expression of CD23 (always exclude Mantle-cell
Lymphoma in CD23 negative cases)
bull Non-CLL-phenotype (CD5minus) MBL
bull Lacking CD5 expression and CD10 likely resembling B-
cell lymphomas of marginal-zone origin
Case 5 What is the normal KL Ratio
bull What is the normal or abnormal kappalambda ratio for
entire ldquopopulationrdquo of all B cells
bull Traditional view
ndash Kappa Lambda 21 or 31 ( 12 -13)
ndash Range 14 60
Case 5 What is the normal KL ratio
bull Wrong question
bull It doesnrsquot matter how many of the B cells are clonal but rather
that their exists a clonal population of B cells within the total
population
bull Need to Identify malignant B cells within a population of
normal B cells
bull In order to interpret a pathological immunophenotype it is
necessary to refer to quantitative and qualitative values of
normal B-cell subpopulations
bull Pearl Identify the abnormal phenotype first then look at KL
ratio
Case 6
bull 12 year old male presents to ER with fatigue bruising
Lkc 2200uL
bull Hb 84gL
bull Plt 32
bull Diff ndash neutropenia no blasts noted few ldquoatypicalrdquo cells
bull RBC ndash non specific changes
Leukemia
Case 6 PB
Case 6 BM
Case 6
bull BM aspirate ndash Hypocellular relative lymphocytosis
marked reduction in myeloid lineages
hypoplasticaplastic anemia
bull Flow Cytometry ndash less than 3 blasts maturing
myeloid 68 lymphocytes polyclonal B helper
suppressor T
bull Unusual pattern of CD13CD16 staining noted
Case 6 flow cytometry Aplastic Anemia
Normal Patient
Normal
Patient
Patient
Normal
Case 6 RBC Test for PNH CD235a v s CD59
Case 6 Neutrophil test for PNH clone
FLAER CD24 CD45CD15
18 PNH clone
Case 6
bull Follow up
bull Patient on steroids acyclovir and anti-thymocyte
globulin
bull PNH clone Neutrophils 96 RBC 1 Nov 2015
bull In remission as of April 2017
- Lkc 5500uL (Neuts 23)
- Hb 129gL
- Plt 208 X10e9
Summary
bull B cell malignancies are the most common
hematological disorders seen in flow cytometry
bull Many have complex overlapping phenotypes
bull Important to look at ungated plots as well as gating on
specific subpopulations
bull Level of expression of markers is as important as
positive or negative (think CD13 vs CD16)
68
References
bull Craig FE Foon KA Flow cytometric immunophenotyping for hematologic neoplasms Blood
2008 Apr 15111(8)3941-67
bull Wood BL Principles of minimal residual disease detection for hematopoietic neoplasms by flow
cytometry Cytometry B Clin Cytom 2015 Apr 23
bull Shanafelt TD Ghia P Lanasa MC Landgren O Rawstron AC Monoclonal B-cell
lymphocytosis (MBL) biology natural history and clinical management Leukemia 2010
Mar24(3)512-20
bull Swerdlow SH Campo E Pileri SA Harris NL Stein H Siebert R Advani RGhielmini M Salles
GA Zelenetz AD Jaffe ES The 2016 revision of the World Health Organization classification of
lymphoid neoplasms Blood 2016 May 19127(20)2375-90
bull 3 Moench L Sachs Z Aasen G Dolan M Dayton V Courville EL Double- and triple-hit
lymphomas can present with features suggestive of immaturity including TdT expression and
create diagnostic challenges Leuk Lymphoma 2016572626-35
bull Sutherland DR Keeney M Illingworth A Practical guidelines for the high-sensitivity detection
and monitoring of paroxysmal nocturnal hemoglobinuria clones by flow cytometry Cytometry B
Clin Cytom 2012 Jul82(4)195-208
69
CD34 ANALYSIS WITH 7-AAD VIABILITY DYE
FL4-FL2 -10
Under compensation
Under compensation
CD34+
8uL
Recognizing bad flow data
Time Gating
Doublet Discrimination
DOUBLETS CAN CREATE ERRONEOUS RESULTS
False
CD19+CD5+
population
Current 8-10 color analysis on
bench top analyzers round bull Major advances in computing power optics
electronics and fluorochrome technology
bull uarr Colors = uarr Complexity
ndash what combinations
ndash what fluorochromes
London Health Custom Design Tube
Antigen Fluorochrome
CD8 FITC
Kappa FITC
CD4 PE
Lambda PE
CD19 ECD
CD20 PE-Cy55
CD5 PE-Cy7
CD10 APC
CD56 APC-Alexa700
Near-IR Viability Dye Excitation 633nm
Emission 780nm
CD3 Pacific Blue
CD45 Krome Orange
Result Reporting Bethesda Guidelines
bull Report percent abnormal population
bull Antibody Distribution
ndash Negative Positive Partial
bull Antibody Fluorescence
- Dim mdash Positive uniform cell population with a median fluorescence intensity abnormally less than that seen on an antigen positive normal leukocyte population Example CD20 in CLL
- Bright mdash Positive uniform cell population with a median fluorescence intensity abnormally greater than that seen on an antigen positive normal leukocyte population Example CD20 in HCL
- Heterogeneous mdash Variably intense staining not a tight uniform cluster of positive immunofluorescence staining
Example CD38 on maturing B cells
Bethesda Reporting
Normal
Dim
CLL
CD19+ CD20+(dim)
CD5+(dim)Kappa+(dim)
Discussion
bull The availability of new fluorochromes coupled with
multilaser instruments with simplified setup and
compensation makes routine ten color analysis possible for
the routine clinical laboratory
bull Knowledge of fluorochromes antibody and antigen
interactions required
bull Full understanding of normal versus abnormal population
phenotype required
bull Sophisticated data analysis programs coupled with
education essential for successful implementation ndash for all
staff involved in testing and analysis
Normal B Cell Maturation
Wood and Borowitz (2006) Henryrsquos Laboratory Medicine
Normal B cell Maturation Pattern
The three pearls
Case 1
bull Nine month old male seen by GP for nodules in his
scalp suggestive of lymphoid infiltrate incidental
finding of bilateral enlarged kidneys
bull Referred to hematology as WBC 16300uL increased
Plt
bull WBC 12300uL Hb 111gL Plt 536x109L
bull Normal differential
bull Scalp lymphoid infiltrate suspicious for leukemia
bull Bone marrow aspirate flow ordered
Case 1
34
BM 14 B cells
5 CD10dimneg
CD20 negCD38 bright
plasma cells
Case 1 BM
Case 1 Bone marrow morphology
bull Cellular marrow
bull Erythropoeisis 20-25
bull Granulopoesis 40-50 orderly maturation
bull Lymphocytes 30 10- 15 ldquoblast likerdquo
bull Increased megakaryocytes
Case 1 2 week follow up
77 B cells ndash 90
CD10 dimneg
CD20 neg bright CD38
pB-ALL
Case 1 BM 2 week follow up
Case 1 Cytogenetics
bull Complex karyotype
bull Structural abnormalities in chromosome
123491417 and 19
bull t(1719) rare in B cell acute lymphoblastic
leukemia
Case 1 Follow up
bull On high risk protocol AALL 0232 for infantile B-ALL
bull MRD 008 at Day 42
bull Cord blood transplant
bull Relapse 5 months post transplant
Pearl of Pathology
bull Differentiating precursor B cells (hematogones) from B-
ALL can be challenging Standardized analysis and
clear understanding of ldquonormalrdquo essential including
arrested maturation
Case 2
bull 4 year old girl referred to pediatric hematology with
pancytopenia
bull WBC 6800uL(Neuts 400uL) Hb 42gL Plt 28
bull Aplastic anemia
bull Bone marrow ordered aspirate biopsy flow and
cytogenetics
Case 2 BM flow cytometry
Polyclonal B cells
helper suppressor T cells
No increase in blasts
Maturing myeloid population
Case 2 CD45 neg B-ALL
Case 2 BM
Case 2
bull CD45neg
bull CD19+CD10+
bull CD20(partialdim)
bull CD34 neg
bull SigampcIg neg
bull Cytogenetics
bull Gain of X chromosome and
468911131415171821
DNA Index
122 Diploid
Case 2
bull Currently in remission after completion of
AALL0932 standard risk B-ALL therapy
Case 2
Pearls of Pathology
CD45 negative B-ALL is not uncommon
bull Always look at ungated set of plots for each marker
bull Eg SS versus each fluorescence parameter
Case 3
bull 58 year old male with pararenal mass
bull WBC 4000uL Hb 98gL Plt 229
bull ldquoblastsrdquo reported on blood film
bull Peripheral blood sent for flow cytometry
Case 3 PB flow cytometry
Dim CD45 CD19+CD10+
CD34- Sig neg
Case 3 PB flow cytometry
Dim CD45 CD19+CD10+
CD20(partial)CD5 neg
SIg cIg neg
Case 3 BM flow cytometry
Case 3 BM biopsy
Case 3 BM biopsy
Case 3 BM biopsy report
bull Diffuse infiltrate large lymphoid cells
bull Scant to moderate cytoplasm
bull Large irregular nuclei with prominent nucleoli
bull Increased mitotic activity
bull Positive for BCL-2 BCL6 CD10 and MUM1
bull Negative for TdT CD5
bull Cytogenetics FISH on para renal mass ndash C-MYC and
BCL2 rearrangements detected
Case 3 Doubletriple hit lymphoma
(DHLTHL)
bull Morphologically heterogeneous B cell neoplasms
characterized by rearrangement of the MYC proto-
oncogene with concurrent BCL2 andor BCL6 gene
rearrangement
bull WHO 2008 criteria - most cases of DHL and THL
classified DLBCL or B-cell lymphoma unclassifiable
with features intermediate between DLBCL and Burkitt
lymphoma (BCLU)
Case 3 DHLTHL
bull Retrospective study from one institution features of 13 cases (9 DHL4
THL)
bull median age was 59 years (range 30-74) eight females five males
bull enlarging lymphadenopathymasses (11 patients) abnormal peripheral
blood findings (2 patients)
bull TdT positivity (four cases two THLtwo DHL) dim CD45 expression (7
cases) lack of CD20 (2 cases) or lack of sIg (3 cases) and blastoid
morphology (2 cases)
bull Conclusion Expression of TdT in a B-cell lymphoma with mature
features or expression of surface light chain in a case otherwise
suggestive of B-lymphoblastic leukemialymphoma should prompt
an expedited evaluation for DHLTHL
Leuk Lymphoma 2016 Feb 181-10 [Epub ahead of print]
Double- and triple-hit lymphomas can present with features suggestive
of immaturity including TdT expression and create diagnostic challenges
Moench L Sachs Z Aasen G Dolan M Dayton V Courville EL
Case 4
bull 55 year old female
bull Previous history hyperparathyroidism
bull Parathyroidectomy in 1999
bull Mild persistent hypercalcemia
bull WBC 4800uL hemoglobin 121 gL Plt 296
bull Total protein 90gL IgG 34gL monoclonal peak
209gL
Case 4 Monoclonal IgG patient
Polyclonal B cells
With maturing hematogones
helper suppressor T cells
less than 3 blasts
Maturing myeloid population
Case 4 BM Ungated
Case 4 cytoplasmic immunoglobulin
Need KL
Case 4 BM Multiple Myeloma
Case 4 follow up
bull Smouldering myeloma
bull Recently developed CRAB (hyperCalcemia Renal
insufficiency Anemia Bone lesions
bull Serum free light chain ratio 123
bull Bone marrow aspirate gt60 plasma cells
bull Currently on treatment pending bone marrow
transplant
Case 5
bull 65 year old male
bull Slight lymphocytosis detected on routine CBC analysis
(lymph 4500uL)
bull Asymptomatic sent for flow cytometry
Case 5 PB
Case 5 flow cytometry KL ratio 0625
Case 5 Monoclonal B cell lymphocytosis
KL 18 KL 056
Case 5 Monoclonal B cell lymphocytosis
bull WHO defines CLL as abnormal lymphocyte count
gt5x10E9L (CD19+CD5+ light chain restricted) in the
absence of other clinical symptoms
bull Patients with small B cell clones are often detected as
an incidental finding
bull WHO 2016
bull CD19+CD5+ lt05x10E9L ldquoLow count MBLrdquo
raquo 05-5x10E9L ldquoHigh count MBLrdquo
raquo Non CLL type phenotype MBL also recognized
Monoclonal B cell Lymphocytosis
bull CLL immunophenotype - vast majority (75)
bull Atypical MBL
bull normal levels both CD5 and CD20 with variable
expression of CD23 (always exclude Mantle-cell
Lymphoma in CD23 negative cases)
bull Non-CLL-phenotype (CD5minus) MBL
bull Lacking CD5 expression and CD10 likely resembling B-
cell lymphomas of marginal-zone origin
Case 5 What is the normal KL Ratio
bull What is the normal or abnormal kappalambda ratio for
entire ldquopopulationrdquo of all B cells
bull Traditional view
ndash Kappa Lambda 21 or 31 ( 12 -13)
ndash Range 14 60
Case 5 What is the normal KL ratio
bull Wrong question
bull It doesnrsquot matter how many of the B cells are clonal but rather
that their exists a clonal population of B cells within the total
population
bull Need to Identify malignant B cells within a population of
normal B cells
bull In order to interpret a pathological immunophenotype it is
necessary to refer to quantitative and qualitative values of
normal B-cell subpopulations
bull Pearl Identify the abnormal phenotype first then look at KL
ratio
Case 6
bull 12 year old male presents to ER with fatigue bruising
Lkc 2200uL
bull Hb 84gL
bull Plt 32
bull Diff ndash neutropenia no blasts noted few ldquoatypicalrdquo cells
bull RBC ndash non specific changes
Leukemia
Case 6 PB
Case 6 BM
Case 6
bull BM aspirate ndash Hypocellular relative lymphocytosis
marked reduction in myeloid lineages
hypoplasticaplastic anemia
bull Flow Cytometry ndash less than 3 blasts maturing
myeloid 68 lymphocytes polyclonal B helper
suppressor T
bull Unusual pattern of CD13CD16 staining noted
Case 6 flow cytometry Aplastic Anemia
Normal Patient
Normal
Patient
Patient
Normal
Case 6 RBC Test for PNH CD235a v s CD59
Case 6 Neutrophil test for PNH clone
FLAER CD24 CD45CD15
18 PNH clone
Case 6
bull Follow up
bull Patient on steroids acyclovir and anti-thymocyte
globulin
bull PNH clone Neutrophils 96 RBC 1 Nov 2015
bull In remission as of April 2017
- Lkc 5500uL (Neuts 23)
- Hb 129gL
- Plt 208 X10e9
Summary
bull B cell malignancies are the most common
hematological disorders seen in flow cytometry
bull Many have complex overlapping phenotypes
bull Important to look at ungated plots as well as gating on
specific subpopulations
bull Level of expression of markers is as important as
positive or negative (think CD13 vs CD16)
68
References
bull Craig FE Foon KA Flow cytometric immunophenotyping for hematologic neoplasms Blood
2008 Apr 15111(8)3941-67
bull Wood BL Principles of minimal residual disease detection for hematopoietic neoplasms by flow
cytometry Cytometry B Clin Cytom 2015 Apr 23
bull Shanafelt TD Ghia P Lanasa MC Landgren O Rawstron AC Monoclonal B-cell
lymphocytosis (MBL) biology natural history and clinical management Leukemia 2010
Mar24(3)512-20
bull Swerdlow SH Campo E Pileri SA Harris NL Stein H Siebert R Advani RGhielmini M Salles
GA Zelenetz AD Jaffe ES The 2016 revision of the World Health Organization classification of
lymphoid neoplasms Blood 2016 May 19127(20)2375-90
bull 3 Moench L Sachs Z Aasen G Dolan M Dayton V Courville EL Double- and triple-hit
lymphomas can present with features suggestive of immaturity including TdT expression and
create diagnostic challenges Leuk Lymphoma 2016572626-35
bull Sutherland DR Keeney M Illingworth A Practical guidelines for the high-sensitivity detection
and monitoring of paroxysmal nocturnal hemoglobinuria clones by flow cytometry Cytometry B
Clin Cytom 2012 Jul82(4)195-208
69
Recognizing bad flow data
Time Gating
Doublet Discrimination
DOUBLETS CAN CREATE ERRONEOUS RESULTS
False
CD19+CD5+
population
Current 8-10 color analysis on
bench top analyzers round bull Major advances in computing power optics
electronics and fluorochrome technology
bull uarr Colors = uarr Complexity
ndash what combinations
ndash what fluorochromes
London Health Custom Design Tube
Antigen Fluorochrome
CD8 FITC
Kappa FITC
CD4 PE
Lambda PE
CD19 ECD
CD20 PE-Cy55
CD5 PE-Cy7
CD10 APC
CD56 APC-Alexa700
Near-IR Viability Dye Excitation 633nm
Emission 780nm
CD3 Pacific Blue
CD45 Krome Orange
Result Reporting Bethesda Guidelines
bull Report percent abnormal population
bull Antibody Distribution
ndash Negative Positive Partial
bull Antibody Fluorescence
- Dim mdash Positive uniform cell population with a median fluorescence intensity abnormally less than that seen on an antigen positive normal leukocyte population Example CD20 in CLL
- Bright mdash Positive uniform cell population with a median fluorescence intensity abnormally greater than that seen on an antigen positive normal leukocyte population Example CD20 in HCL
- Heterogeneous mdash Variably intense staining not a tight uniform cluster of positive immunofluorescence staining
Example CD38 on maturing B cells
Bethesda Reporting
Normal
Dim
CLL
CD19+ CD20+(dim)
CD5+(dim)Kappa+(dim)
Discussion
bull The availability of new fluorochromes coupled with
multilaser instruments with simplified setup and
compensation makes routine ten color analysis possible for
the routine clinical laboratory
bull Knowledge of fluorochromes antibody and antigen
interactions required
bull Full understanding of normal versus abnormal population
phenotype required
bull Sophisticated data analysis programs coupled with
education essential for successful implementation ndash for all
staff involved in testing and analysis
Normal B Cell Maturation
Wood and Borowitz (2006) Henryrsquos Laboratory Medicine
Normal B cell Maturation Pattern
The three pearls
Case 1
bull Nine month old male seen by GP for nodules in his
scalp suggestive of lymphoid infiltrate incidental
finding of bilateral enlarged kidneys
bull Referred to hematology as WBC 16300uL increased
Plt
bull WBC 12300uL Hb 111gL Plt 536x109L
bull Normal differential
bull Scalp lymphoid infiltrate suspicious for leukemia
bull Bone marrow aspirate flow ordered
Case 1
34
BM 14 B cells
5 CD10dimneg
CD20 negCD38 bright
plasma cells
Case 1 BM
Case 1 Bone marrow morphology
bull Cellular marrow
bull Erythropoeisis 20-25
bull Granulopoesis 40-50 orderly maturation
bull Lymphocytes 30 10- 15 ldquoblast likerdquo
bull Increased megakaryocytes
Case 1 2 week follow up
77 B cells ndash 90
CD10 dimneg
CD20 neg bright CD38
pB-ALL
Case 1 BM 2 week follow up
Case 1 Cytogenetics
bull Complex karyotype
bull Structural abnormalities in chromosome
123491417 and 19
bull t(1719) rare in B cell acute lymphoblastic
leukemia
Case 1 Follow up
bull On high risk protocol AALL 0232 for infantile B-ALL
bull MRD 008 at Day 42
bull Cord blood transplant
bull Relapse 5 months post transplant
Pearl of Pathology
bull Differentiating precursor B cells (hematogones) from B-
ALL can be challenging Standardized analysis and
clear understanding of ldquonormalrdquo essential including
arrested maturation
Case 2
bull 4 year old girl referred to pediatric hematology with
pancytopenia
bull WBC 6800uL(Neuts 400uL) Hb 42gL Plt 28
bull Aplastic anemia
bull Bone marrow ordered aspirate biopsy flow and
cytogenetics
Case 2 BM flow cytometry
Polyclonal B cells
helper suppressor T cells
No increase in blasts
Maturing myeloid population
Case 2 CD45 neg B-ALL
Case 2 BM
Case 2
bull CD45neg
bull CD19+CD10+
bull CD20(partialdim)
bull CD34 neg
bull SigampcIg neg
bull Cytogenetics
bull Gain of X chromosome and
468911131415171821
DNA Index
122 Diploid
Case 2
bull Currently in remission after completion of
AALL0932 standard risk B-ALL therapy
Case 2
Pearls of Pathology
CD45 negative B-ALL is not uncommon
bull Always look at ungated set of plots for each marker
bull Eg SS versus each fluorescence parameter
Case 3
bull 58 year old male with pararenal mass
bull WBC 4000uL Hb 98gL Plt 229
bull ldquoblastsrdquo reported on blood film
bull Peripheral blood sent for flow cytometry
Case 3 PB flow cytometry
Dim CD45 CD19+CD10+
CD34- Sig neg
Case 3 PB flow cytometry
Dim CD45 CD19+CD10+
CD20(partial)CD5 neg
SIg cIg neg
Case 3 BM flow cytometry
Case 3 BM biopsy
Case 3 BM biopsy
Case 3 BM biopsy report
bull Diffuse infiltrate large lymphoid cells
bull Scant to moderate cytoplasm
bull Large irregular nuclei with prominent nucleoli
bull Increased mitotic activity
bull Positive for BCL-2 BCL6 CD10 and MUM1
bull Negative for TdT CD5
bull Cytogenetics FISH on para renal mass ndash C-MYC and
BCL2 rearrangements detected
Case 3 Doubletriple hit lymphoma
(DHLTHL)
bull Morphologically heterogeneous B cell neoplasms
characterized by rearrangement of the MYC proto-
oncogene with concurrent BCL2 andor BCL6 gene
rearrangement
bull WHO 2008 criteria - most cases of DHL and THL
classified DLBCL or B-cell lymphoma unclassifiable
with features intermediate between DLBCL and Burkitt
lymphoma (BCLU)
Case 3 DHLTHL
bull Retrospective study from one institution features of 13 cases (9 DHL4
THL)
bull median age was 59 years (range 30-74) eight females five males
bull enlarging lymphadenopathymasses (11 patients) abnormal peripheral
blood findings (2 patients)
bull TdT positivity (four cases two THLtwo DHL) dim CD45 expression (7
cases) lack of CD20 (2 cases) or lack of sIg (3 cases) and blastoid
morphology (2 cases)
bull Conclusion Expression of TdT in a B-cell lymphoma with mature
features or expression of surface light chain in a case otherwise
suggestive of B-lymphoblastic leukemialymphoma should prompt
an expedited evaluation for DHLTHL
Leuk Lymphoma 2016 Feb 181-10 [Epub ahead of print]
Double- and triple-hit lymphomas can present with features suggestive
of immaturity including TdT expression and create diagnostic challenges
Moench L Sachs Z Aasen G Dolan M Dayton V Courville EL
Case 4
bull 55 year old female
bull Previous history hyperparathyroidism
bull Parathyroidectomy in 1999
bull Mild persistent hypercalcemia
bull WBC 4800uL hemoglobin 121 gL Plt 296
bull Total protein 90gL IgG 34gL monoclonal peak
209gL
Case 4 Monoclonal IgG patient
Polyclonal B cells
With maturing hematogones
helper suppressor T cells
less than 3 blasts
Maturing myeloid population
Case 4 BM Ungated
Case 4 cytoplasmic immunoglobulin
Need KL
Case 4 BM Multiple Myeloma
Case 4 follow up
bull Smouldering myeloma
bull Recently developed CRAB (hyperCalcemia Renal
insufficiency Anemia Bone lesions
bull Serum free light chain ratio 123
bull Bone marrow aspirate gt60 plasma cells
bull Currently on treatment pending bone marrow
transplant
Case 5
bull 65 year old male
bull Slight lymphocytosis detected on routine CBC analysis
(lymph 4500uL)
bull Asymptomatic sent for flow cytometry
Case 5 PB
Case 5 flow cytometry KL ratio 0625
Case 5 Monoclonal B cell lymphocytosis
KL 18 KL 056
Case 5 Monoclonal B cell lymphocytosis
bull WHO defines CLL as abnormal lymphocyte count
gt5x10E9L (CD19+CD5+ light chain restricted) in the
absence of other clinical symptoms
bull Patients with small B cell clones are often detected as
an incidental finding
bull WHO 2016
bull CD19+CD5+ lt05x10E9L ldquoLow count MBLrdquo
raquo 05-5x10E9L ldquoHigh count MBLrdquo
raquo Non CLL type phenotype MBL also recognized
Monoclonal B cell Lymphocytosis
bull CLL immunophenotype - vast majority (75)
bull Atypical MBL
bull normal levels both CD5 and CD20 with variable
expression of CD23 (always exclude Mantle-cell
Lymphoma in CD23 negative cases)
bull Non-CLL-phenotype (CD5minus) MBL
bull Lacking CD5 expression and CD10 likely resembling B-
cell lymphomas of marginal-zone origin
Case 5 What is the normal KL Ratio
bull What is the normal or abnormal kappalambda ratio for
entire ldquopopulationrdquo of all B cells
bull Traditional view
ndash Kappa Lambda 21 or 31 ( 12 -13)
ndash Range 14 60
Case 5 What is the normal KL ratio
bull Wrong question
bull It doesnrsquot matter how many of the B cells are clonal but rather
that their exists a clonal population of B cells within the total
population
bull Need to Identify malignant B cells within a population of
normal B cells
bull In order to interpret a pathological immunophenotype it is
necessary to refer to quantitative and qualitative values of
normal B-cell subpopulations
bull Pearl Identify the abnormal phenotype first then look at KL
ratio
Case 6
bull 12 year old male presents to ER with fatigue bruising
Lkc 2200uL
bull Hb 84gL
bull Plt 32
bull Diff ndash neutropenia no blasts noted few ldquoatypicalrdquo cells
bull RBC ndash non specific changes
Leukemia
Case 6 PB
Case 6 BM
Case 6
bull BM aspirate ndash Hypocellular relative lymphocytosis
marked reduction in myeloid lineages
hypoplasticaplastic anemia
bull Flow Cytometry ndash less than 3 blasts maturing
myeloid 68 lymphocytes polyclonal B helper
suppressor T
bull Unusual pattern of CD13CD16 staining noted
Case 6 flow cytometry Aplastic Anemia
Normal Patient
Normal
Patient
Patient
Normal
Case 6 RBC Test for PNH CD235a v s CD59
Case 6 Neutrophil test for PNH clone
FLAER CD24 CD45CD15
18 PNH clone
Case 6
bull Follow up
bull Patient on steroids acyclovir and anti-thymocyte
globulin
bull PNH clone Neutrophils 96 RBC 1 Nov 2015
bull In remission as of April 2017
- Lkc 5500uL (Neuts 23)
- Hb 129gL
- Plt 208 X10e9
Summary
bull B cell malignancies are the most common
hematological disorders seen in flow cytometry
bull Many have complex overlapping phenotypes
bull Important to look at ungated plots as well as gating on
specific subpopulations
bull Level of expression of markers is as important as
positive or negative (think CD13 vs CD16)
68
References
bull Craig FE Foon KA Flow cytometric immunophenotyping for hematologic neoplasms Blood
2008 Apr 15111(8)3941-67
bull Wood BL Principles of minimal residual disease detection for hematopoietic neoplasms by flow
cytometry Cytometry B Clin Cytom 2015 Apr 23
bull Shanafelt TD Ghia P Lanasa MC Landgren O Rawstron AC Monoclonal B-cell
lymphocytosis (MBL) biology natural history and clinical management Leukemia 2010
Mar24(3)512-20
bull Swerdlow SH Campo E Pileri SA Harris NL Stein H Siebert R Advani RGhielmini M Salles
GA Zelenetz AD Jaffe ES The 2016 revision of the World Health Organization classification of
lymphoid neoplasms Blood 2016 May 19127(20)2375-90
bull 3 Moench L Sachs Z Aasen G Dolan M Dayton V Courville EL Double- and triple-hit
lymphomas can present with features suggestive of immaturity including TdT expression and
create diagnostic challenges Leuk Lymphoma 2016572626-35
bull Sutherland DR Keeney M Illingworth A Practical guidelines for the high-sensitivity detection
and monitoring of paroxysmal nocturnal hemoglobinuria clones by flow cytometry Cytometry B
Clin Cytom 2012 Jul82(4)195-208
69
Time Gating
Doublet Discrimination
DOUBLETS CAN CREATE ERRONEOUS RESULTS
False
CD19+CD5+
population
Current 8-10 color analysis on
bench top analyzers round bull Major advances in computing power optics
electronics and fluorochrome technology
bull uarr Colors = uarr Complexity
ndash what combinations
ndash what fluorochromes
London Health Custom Design Tube
Antigen Fluorochrome
CD8 FITC
Kappa FITC
CD4 PE
Lambda PE
CD19 ECD
CD20 PE-Cy55
CD5 PE-Cy7
CD10 APC
CD56 APC-Alexa700
Near-IR Viability Dye Excitation 633nm
Emission 780nm
CD3 Pacific Blue
CD45 Krome Orange
Result Reporting Bethesda Guidelines
bull Report percent abnormal population
bull Antibody Distribution
ndash Negative Positive Partial
bull Antibody Fluorescence
- Dim mdash Positive uniform cell population with a median fluorescence intensity abnormally less than that seen on an antigen positive normal leukocyte population Example CD20 in CLL
- Bright mdash Positive uniform cell population with a median fluorescence intensity abnormally greater than that seen on an antigen positive normal leukocyte population Example CD20 in HCL
- Heterogeneous mdash Variably intense staining not a tight uniform cluster of positive immunofluorescence staining
Example CD38 on maturing B cells
Bethesda Reporting
Normal
Dim
CLL
CD19+ CD20+(dim)
CD5+(dim)Kappa+(dim)
Discussion
bull The availability of new fluorochromes coupled with
multilaser instruments with simplified setup and
compensation makes routine ten color analysis possible for
the routine clinical laboratory
bull Knowledge of fluorochromes antibody and antigen
interactions required
bull Full understanding of normal versus abnormal population
phenotype required
bull Sophisticated data analysis programs coupled with
education essential for successful implementation ndash for all
staff involved in testing and analysis
Normal B Cell Maturation
Wood and Borowitz (2006) Henryrsquos Laboratory Medicine
Normal B cell Maturation Pattern
The three pearls
Case 1
bull Nine month old male seen by GP for nodules in his
scalp suggestive of lymphoid infiltrate incidental
finding of bilateral enlarged kidneys
bull Referred to hematology as WBC 16300uL increased
Plt
bull WBC 12300uL Hb 111gL Plt 536x109L
bull Normal differential
bull Scalp lymphoid infiltrate suspicious for leukemia
bull Bone marrow aspirate flow ordered
Case 1
34
BM 14 B cells
5 CD10dimneg
CD20 negCD38 bright
plasma cells
Case 1 BM
Case 1 Bone marrow morphology
bull Cellular marrow
bull Erythropoeisis 20-25
bull Granulopoesis 40-50 orderly maturation
bull Lymphocytes 30 10- 15 ldquoblast likerdquo
bull Increased megakaryocytes
Case 1 2 week follow up
77 B cells ndash 90
CD10 dimneg
CD20 neg bright CD38
pB-ALL
Case 1 BM 2 week follow up
Case 1 Cytogenetics
bull Complex karyotype
bull Structural abnormalities in chromosome
123491417 and 19
bull t(1719) rare in B cell acute lymphoblastic
leukemia
Case 1 Follow up
bull On high risk protocol AALL 0232 for infantile B-ALL
bull MRD 008 at Day 42
bull Cord blood transplant
bull Relapse 5 months post transplant
Pearl of Pathology
bull Differentiating precursor B cells (hematogones) from B-
ALL can be challenging Standardized analysis and
clear understanding of ldquonormalrdquo essential including
arrested maturation
Case 2
bull 4 year old girl referred to pediatric hematology with
pancytopenia
bull WBC 6800uL(Neuts 400uL) Hb 42gL Plt 28
bull Aplastic anemia
bull Bone marrow ordered aspirate biopsy flow and
cytogenetics
Case 2 BM flow cytometry
Polyclonal B cells
helper suppressor T cells
No increase in blasts
Maturing myeloid population
Case 2 CD45 neg B-ALL
Case 2 BM
Case 2
bull CD45neg
bull CD19+CD10+
bull CD20(partialdim)
bull CD34 neg
bull SigampcIg neg
bull Cytogenetics
bull Gain of X chromosome and
468911131415171821
DNA Index
122 Diploid
Case 2
bull Currently in remission after completion of
AALL0932 standard risk B-ALL therapy
Case 2
Pearls of Pathology
CD45 negative B-ALL is not uncommon
bull Always look at ungated set of plots for each marker
bull Eg SS versus each fluorescence parameter
Case 3
bull 58 year old male with pararenal mass
bull WBC 4000uL Hb 98gL Plt 229
bull ldquoblastsrdquo reported on blood film
bull Peripheral blood sent for flow cytometry
Case 3 PB flow cytometry
Dim CD45 CD19+CD10+
CD34- Sig neg
Case 3 PB flow cytometry
Dim CD45 CD19+CD10+
CD20(partial)CD5 neg
SIg cIg neg
Case 3 BM flow cytometry
Case 3 BM biopsy
Case 3 BM biopsy
Case 3 BM biopsy report
bull Diffuse infiltrate large lymphoid cells
bull Scant to moderate cytoplasm
bull Large irregular nuclei with prominent nucleoli
bull Increased mitotic activity
bull Positive for BCL-2 BCL6 CD10 and MUM1
bull Negative for TdT CD5
bull Cytogenetics FISH on para renal mass ndash C-MYC and
BCL2 rearrangements detected
Case 3 Doubletriple hit lymphoma
(DHLTHL)
bull Morphologically heterogeneous B cell neoplasms
characterized by rearrangement of the MYC proto-
oncogene with concurrent BCL2 andor BCL6 gene
rearrangement
bull WHO 2008 criteria - most cases of DHL and THL
classified DLBCL or B-cell lymphoma unclassifiable
with features intermediate between DLBCL and Burkitt
lymphoma (BCLU)
Case 3 DHLTHL
bull Retrospective study from one institution features of 13 cases (9 DHL4
THL)
bull median age was 59 years (range 30-74) eight females five males
bull enlarging lymphadenopathymasses (11 patients) abnormal peripheral
blood findings (2 patients)
bull TdT positivity (four cases two THLtwo DHL) dim CD45 expression (7
cases) lack of CD20 (2 cases) or lack of sIg (3 cases) and blastoid
morphology (2 cases)
bull Conclusion Expression of TdT in a B-cell lymphoma with mature
features or expression of surface light chain in a case otherwise
suggestive of B-lymphoblastic leukemialymphoma should prompt
an expedited evaluation for DHLTHL
Leuk Lymphoma 2016 Feb 181-10 [Epub ahead of print]
Double- and triple-hit lymphomas can present with features suggestive
of immaturity including TdT expression and create diagnostic challenges
Moench L Sachs Z Aasen G Dolan M Dayton V Courville EL
Case 4
bull 55 year old female
bull Previous history hyperparathyroidism
bull Parathyroidectomy in 1999
bull Mild persistent hypercalcemia
bull WBC 4800uL hemoglobin 121 gL Plt 296
bull Total protein 90gL IgG 34gL monoclonal peak
209gL
Case 4 Monoclonal IgG patient
Polyclonal B cells
With maturing hematogones
helper suppressor T cells
less than 3 blasts
Maturing myeloid population
Case 4 BM Ungated
Case 4 cytoplasmic immunoglobulin
Need KL
Case 4 BM Multiple Myeloma
Case 4 follow up
bull Smouldering myeloma
bull Recently developed CRAB (hyperCalcemia Renal
insufficiency Anemia Bone lesions
bull Serum free light chain ratio 123
bull Bone marrow aspirate gt60 plasma cells
bull Currently on treatment pending bone marrow
transplant
Case 5
bull 65 year old male
bull Slight lymphocytosis detected on routine CBC analysis
(lymph 4500uL)
bull Asymptomatic sent for flow cytometry
Case 5 PB
Case 5 flow cytometry KL ratio 0625
Case 5 Monoclonal B cell lymphocytosis
KL 18 KL 056
Case 5 Monoclonal B cell lymphocytosis
bull WHO defines CLL as abnormal lymphocyte count
gt5x10E9L (CD19+CD5+ light chain restricted) in the
absence of other clinical symptoms
bull Patients with small B cell clones are often detected as
an incidental finding
bull WHO 2016
bull CD19+CD5+ lt05x10E9L ldquoLow count MBLrdquo
raquo 05-5x10E9L ldquoHigh count MBLrdquo
raquo Non CLL type phenotype MBL also recognized
Monoclonal B cell Lymphocytosis
bull CLL immunophenotype - vast majority (75)
bull Atypical MBL
bull normal levels both CD5 and CD20 with variable
expression of CD23 (always exclude Mantle-cell
Lymphoma in CD23 negative cases)
bull Non-CLL-phenotype (CD5minus) MBL
bull Lacking CD5 expression and CD10 likely resembling B-
cell lymphomas of marginal-zone origin
Case 5 What is the normal KL Ratio
bull What is the normal or abnormal kappalambda ratio for
entire ldquopopulationrdquo of all B cells
bull Traditional view
ndash Kappa Lambda 21 or 31 ( 12 -13)
ndash Range 14 60
Case 5 What is the normal KL ratio
bull Wrong question
bull It doesnrsquot matter how many of the B cells are clonal but rather
that their exists a clonal population of B cells within the total
population
bull Need to Identify malignant B cells within a population of
normal B cells
bull In order to interpret a pathological immunophenotype it is
necessary to refer to quantitative and qualitative values of
normal B-cell subpopulations
bull Pearl Identify the abnormal phenotype first then look at KL
ratio
Case 6
bull 12 year old male presents to ER with fatigue bruising
Lkc 2200uL
bull Hb 84gL
bull Plt 32
bull Diff ndash neutropenia no blasts noted few ldquoatypicalrdquo cells
bull RBC ndash non specific changes
Leukemia
Case 6 PB
Case 6 BM
Case 6
bull BM aspirate ndash Hypocellular relative lymphocytosis
marked reduction in myeloid lineages
hypoplasticaplastic anemia
bull Flow Cytometry ndash less than 3 blasts maturing
myeloid 68 lymphocytes polyclonal B helper
suppressor T
bull Unusual pattern of CD13CD16 staining noted
Case 6 flow cytometry Aplastic Anemia
Normal Patient
Normal
Patient
Patient
Normal
Case 6 RBC Test for PNH CD235a v s CD59
Case 6 Neutrophil test for PNH clone
FLAER CD24 CD45CD15
18 PNH clone
Case 6
bull Follow up
bull Patient on steroids acyclovir and anti-thymocyte
globulin
bull PNH clone Neutrophils 96 RBC 1 Nov 2015
bull In remission as of April 2017
- Lkc 5500uL (Neuts 23)
- Hb 129gL
- Plt 208 X10e9
Summary
bull B cell malignancies are the most common
hematological disorders seen in flow cytometry
bull Many have complex overlapping phenotypes
bull Important to look at ungated plots as well as gating on
specific subpopulations
bull Level of expression of markers is as important as
positive or negative (think CD13 vs CD16)
68
References
bull Craig FE Foon KA Flow cytometric immunophenotyping for hematologic neoplasms Blood
2008 Apr 15111(8)3941-67
bull Wood BL Principles of minimal residual disease detection for hematopoietic neoplasms by flow
cytometry Cytometry B Clin Cytom 2015 Apr 23
bull Shanafelt TD Ghia P Lanasa MC Landgren O Rawstron AC Monoclonal B-cell
lymphocytosis (MBL) biology natural history and clinical management Leukemia 2010
Mar24(3)512-20
bull Swerdlow SH Campo E Pileri SA Harris NL Stein H Siebert R Advani RGhielmini M Salles
GA Zelenetz AD Jaffe ES The 2016 revision of the World Health Organization classification of
lymphoid neoplasms Blood 2016 May 19127(20)2375-90
bull 3 Moench L Sachs Z Aasen G Dolan M Dayton V Courville EL Double- and triple-hit
lymphomas can present with features suggestive of immaturity including TdT expression and
create diagnostic challenges Leuk Lymphoma 2016572626-35
bull Sutherland DR Keeney M Illingworth A Practical guidelines for the high-sensitivity detection
and monitoring of paroxysmal nocturnal hemoglobinuria clones by flow cytometry Cytometry B
Clin Cytom 2012 Jul82(4)195-208
69
Doublet Discrimination
DOUBLETS CAN CREATE ERRONEOUS RESULTS
False
CD19+CD5+
population
Current 8-10 color analysis on
bench top analyzers round bull Major advances in computing power optics
electronics and fluorochrome technology
bull uarr Colors = uarr Complexity
ndash what combinations
ndash what fluorochromes
London Health Custom Design Tube
Antigen Fluorochrome
CD8 FITC
Kappa FITC
CD4 PE
Lambda PE
CD19 ECD
CD20 PE-Cy55
CD5 PE-Cy7
CD10 APC
CD56 APC-Alexa700
Near-IR Viability Dye Excitation 633nm
Emission 780nm
CD3 Pacific Blue
CD45 Krome Orange
Result Reporting Bethesda Guidelines
bull Report percent abnormal population
bull Antibody Distribution
ndash Negative Positive Partial
bull Antibody Fluorescence
- Dim mdash Positive uniform cell population with a median fluorescence intensity abnormally less than that seen on an antigen positive normal leukocyte population Example CD20 in CLL
- Bright mdash Positive uniform cell population with a median fluorescence intensity abnormally greater than that seen on an antigen positive normal leukocyte population Example CD20 in HCL
- Heterogeneous mdash Variably intense staining not a tight uniform cluster of positive immunofluorescence staining
Example CD38 on maturing B cells
Bethesda Reporting
Normal
Dim
CLL
CD19+ CD20+(dim)
CD5+(dim)Kappa+(dim)
Discussion
bull The availability of new fluorochromes coupled with
multilaser instruments with simplified setup and
compensation makes routine ten color analysis possible for
the routine clinical laboratory
bull Knowledge of fluorochromes antibody and antigen
interactions required
bull Full understanding of normal versus abnormal population
phenotype required
bull Sophisticated data analysis programs coupled with
education essential for successful implementation ndash for all
staff involved in testing and analysis
Normal B Cell Maturation
Wood and Borowitz (2006) Henryrsquos Laboratory Medicine
Normal B cell Maturation Pattern
The three pearls
Case 1
bull Nine month old male seen by GP for nodules in his
scalp suggestive of lymphoid infiltrate incidental
finding of bilateral enlarged kidneys
bull Referred to hematology as WBC 16300uL increased
Plt
bull WBC 12300uL Hb 111gL Plt 536x109L
bull Normal differential
bull Scalp lymphoid infiltrate suspicious for leukemia
bull Bone marrow aspirate flow ordered
Case 1
34
BM 14 B cells
5 CD10dimneg
CD20 negCD38 bright
plasma cells
Case 1 BM
Case 1 Bone marrow morphology
bull Cellular marrow
bull Erythropoeisis 20-25
bull Granulopoesis 40-50 orderly maturation
bull Lymphocytes 30 10- 15 ldquoblast likerdquo
bull Increased megakaryocytes
Case 1 2 week follow up
77 B cells ndash 90
CD10 dimneg
CD20 neg bright CD38
pB-ALL
Case 1 BM 2 week follow up
Case 1 Cytogenetics
bull Complex karyotype
bull Structural abnormalities in chromosome
123491417 and 19
bull t(1719) rare in B cell acute lymphoblastic
leukemia
Case 1 Follow up
bull On high risk protocol AALL 0232 for infantile B-ALL
bull MRD 008 at Day 42
bull Cord blood transplant
bull Relapse 5 months post transplant
Pearl of Pathology
bull Differentiating precursor B cells (hematogones) from B-
ALL can be challenging Standardized analysis and
clear understanding of ldquonormalrdquo essential including
arrested maturation
Case 2
bull 4 year old girl referred to pediatric hematology with
pancytopenia
bull WBC 6800uL(Neuts 400uL) Hb 42gL Plt 28
bull Aplastic anemia
bull Bone marrow ordered aspirate biopsy flow and
cytogenetics
Case 2 BM flow cytometry
Polyclonal B cells
helper suppressor T cells
No increase in blasts
Maturing myeloid population
Case 2 CD45 neg B-ALL
Case 2 BM
Case 2
bull CD45neg
bull CD19+CD10+
bull CD20(partialdim)
bull CD34 neg
bull SigampcIg neg
bull Cytogenetics
bull Gain of X chromosome and
468911131415171821
DNA Index
122 Diploid
Case 2
bull Currently in remission after completion of
AALL0932 standard risk B-ALL therapy
Case 2
Pearls of Pathology
CD45 negative B-ALL is not uncommon
bull Always look at ungated set of plots for each marker
bull Eg SS versus each fluorescence parameter
Case 3
bull 58 year old male with pararenal mass
bull WBC 4000uL Hb 98gL Plt 229
bull ldquoblastsrdquo reported on blood film
bull Peripheral blood sent for flow cytometry
Case 3 PB flow cytometry
Dim CD45 CD19+CD10+
CD34- Sig neg
Case 3 PB flow cytometry
Dim CD45 CD19+CD10+
CD20(partial)CD5 neg
SIg cIg neg
Case 3 BM flow cytometry
Case 3 BM biopsy
Case 3 BM biopsy
Case 3 BM biopsy report
bull Diffuse infiltrate large lymphoid cells
bull Scant to moderate cytoplasm
bull Large irregular nuclei with prominent nucleoli
bull Increased mitotic activity
bull Positive for BCL-2 BCL6 CD10 and MUM1
bull Negative for TdT CD5
bull Cytogenetics FISH on para renal mass ndash C-MYC and
BCL2 rearrangements detected
Case 3 Doubletriple hit lymphoma
(DHLTHL)
bull Morphologically heterogeneous B cell neoplasms
characterized by rearrangement of the MYC proto-
oncogene with concurrent BCL2 andor BCL6 gene
rearrangement
bull WHO 2008 criteria - most cases of DHL and THL
classified DLBCL or B-cell lymphoma unclassifiable
with features intermediate between DLBCL and Burkitt
lymphoma (BCLU)
Case 3 DHLTHL
bull Retrospective study from one institution features of 13 cases (9 DHL4
THL)
bull median age was 59 years (range 30-74) eight females five males
bull enlarging lymphadenopathymasses (11 patients) abnormal peripheral
blood findings (2 patients)
bull TdT positivity (four cases two THLtwo DHL) dim CD45 expression (7
cases) lack of CD20 (2 cases) or lack of sIg (3 cases) and blastoid
morphology (2 cases)
bull Conclusion Expression of TdT in a B-cell lymphoma with mature
features or expression of surface light chain in a case otherwise
suggestive of B-lymphoblastic leukemialymphoma should prompt
an expedited evaluation for DHLTHL
Leuk Lymphoma 2016 Feb 181-10 [Epub ahead of print]
Double- and triple-hit lymphomas can present with features suggestive
of immaturity including TdT expression and create diagnostic challenges
Moench L Sachs Z Aasen G Dolan M Dayton V Courville EL
Case 4
bull 55 year old female
bull Previous history hyperparathyroidism
bull Parathyroidectomy in 1999
bull Mild persistent hypercalcemia
bull WBC 4800uL hemoglobin 121 gL Plt 296
bull Total protein 90gL IgG 34gL monoclonal peak
209gL
Case 4 Monoclonal IgG patient
Polyclonal B cells
With maturing hematogones
helper suppressor T cells
less than 3 blasts
Maturing myeloid population
Case 4 BM Ungated
Case 4 cytoplasmic immunoglobulin
Need KL
Case 4 BM Multiple Myeloma
Case 4 follow up
bull Smouldering myeloma
bull Recently developed CRAB (hyperCalcemia Renal
insufficiency Anemia Bone lesions
bull Serum free light chain ratio 123
bull Bone marrow aspirate gt60 plasma cells
bull Currently on treatment pending bone marrow
transplant
Case 5
bull 65 year old male
bull Slight lymphocytosis detected on routine CBC analysis
(lymph 4500uL)
bull Asymptomatic sent for flow cytometry
Case 5 PB
Case 5 flow cytometry KL ratio 0625
Case 5 Monoclonal B cell lymphocytosis
KL 18 KL 056
Case 5 Monoclonal B cell lymphocytosis
bull WHO defines CLL as abnormal lymphocyte count
gt5x10E9L (CD19+CD5+ light chain restricted) in the
absence of other clinical symptoms
bull Patients with small B cell clones are often detected as
an incidental finding
bull WHO 2016
bull CD19+CD5+ lt05x10E9L ldquoLow count MBLrdquo
raquo 05-5x10E9L ldquoHigh count MBLrdquo
raquo Non CLL type phenotype MBL also recognized
Monoclonal B cell Lymphocytosis
bull CLL immunophenotype - vast majority (75)
bull Atypical MBL
bull normal levels both CD5 and CD20 with variable
expression of CD23 (always exclude Mantle-cell
Lymphoma in CD23 negative cases)
bull Non-CLL-phenotype (CD5minus) MBL
bull Lacking CD5 expression and CD10 likely resembling B-
cell lymphomas of marginal-zone origin
Case 5 What is the normal KL Ratio
bull What is the normal or abnormal kappalambda ratio for
entire ldquopopulationrdquo of all B cells
bull Traditional view
ndash Kappa Lambda 21 or 31 ( 12 -13)
ndash Range 14 60
Case 5 What is the normal KL ratio
bull Wrong question
bull It doesnrsquot matter how many of the B cells are clonal but rather
that their exists a clonal population of B cells within the total
population
bull Need to Identify malignant B cells within a population of
normal B cells
bull In order to interpret a pathological immunophenotype it is
necessary to refer to quantitative and qualitative values of
normal B-cell subpopulations
bull Pearl Identify the abnormal phenotype first then look at KL
ratio
Case 6
bull 12 year old male presents to ER with fatigue bruising
Lkc 2200uL
bull Hb 84gL
bull Plt 32
bull Diff ndash neutropenia no blasts noted few ldquoatypicalrdquo cells
bull RBC ndash non specific changes
Leukemia
Case 6 PB
Case 6 BM
Case 6
bull BM aspirate ndash Hypocellular relative lymphocytosis
marked reduction in myeloid lineages
hypoplasticaplastic anemia
bull Flow Cytometry ndash less than 3 blasts maturing
myeloid 68 lymphocytes polyclonal B helper
suppressor T
bull Unusual pattern of CD13CD16 staining noted
Case 6 flow cytometry Aplastic Anemia
Normal Patient
Normal
Patient
Patient
Normal
Case 6 RBC Test for PNH CD235a v s CD59
Case 6 Neutrophil test for PNH clone
FLAER CD24 CD45CD15
18 PNH clone
Case 6
bull Follow up
bull Patient on steroids acyclovir and anti-thymocyte
globulin
bull PNH clone Neutrophils 96 RBC 1 Nov 2015
bull In remission as of April 2017
- Lkc 5500uL (Neuts 23)
- Hb 129gL
- Plt 208 X10e9
Summary
bull B cell malignancies are the most common
hematological disorders seen in flow cytometry
bull Many have complex overlapping phenotypes
bull Important to look at ungated plots as well as gating on
specific subpopulations
bull Level of expression of markers is as important as
positive or negative (think CD13 vs CD16)
68
References
bull Craig FE Foon KA Flow cytometric immunophenotyping for hematologic neoplasms Blood
2008 Apr 15111(8)3941-67
bull Wood BL Principles of minimal residual disease detection for hematopoietic neoplasms by flow
cytometry Cytometry B Clin Cytom 2015 Apr 23
bull Shanafelt TD Ghia P Lanasa MC Landgren O Rawstron AC Monoclonal B-cell
lymphocytosis (MBL) biology natural history and clinical management Leukemia 2010
Mar24(3)512-20
bull Swerdlow SH Campo E Pileri SA Harris NL Stein H Siebert R Advani RGhielmini M Salles
GA Zelenetz AD Jaffe ES The 2016 revision of the World Health Organization classification of
lymphoid neoplasms Blood 2016 May 19127(20)2375-90
bull 3 Moench L Sachs Z Aasen G Dolan M Dayton V Courville EL Double- and triple-hit
lymphomas can present with features suggestive of immaturity including TdT expression and
create diagnostic challenges Leuk Lymphoma 2016572626-35
bull Sutherland DR Keeney M Illingworth A Practical guidelines for the high-sensitivity detection
and monitoring of paroxysmal nocturnal hemoglobinuria clones by flow cytometry Cytometry B
Clin Cytom 2012 Jul82(4)195-208
69
Current 8-10 color analysis on
bench top analyzers round bull Major advances in computing power optics
electronics and fluorochrome technology
bull uarr Colors = uarr Complexity
ndash what combinations
ndash what fluorochromes
London Health Custom Design Tube
Antigen Fluorochrome
CD8 FITC
Kappa FITC
CD4 PE
Lambda PE
CD19 ECD
CD20 PE-Cy55
CD5 PE-Cy7
CD10 APC
CD56 APC-Alexa700
Near-IR Viability Dye Excitation 633nm
Emission 780nm
CD3 Pacific Blue
CD45 Krome Orange
Result Reporting Bethesda Guidelines
bull Report percent abnormal population
bull Antibody Distribution
ndash Negative Positive Partial
bull Antibody Fluorescence
- Dim mdash Positive uniform cell population with a median fluorescence intensity abnormally less than that seen on an antigen positive normal leukocyte population Example CD20 in CLL
- Bright mdash Positive uniform cell population with a median fluorescence intensity abnormally greater than that seen on an antigen positive normal leukocyte population Example CD20 in HCL
- Heterogeneous mdash Variably intense staining not a tight uniform cluster of positive immunofluorescence staining
Example CD38 on maturing B cells
Bethesda Reporting
Normal
Dim
CLL
CD19+ CD20+(dim)
CD5+(dim)Kappa+(dim)
Discussion
bull The availability of new fluorochromes coupled with
multilaser instruments with simplified setup and
compensation makes routine ten color analysis possible for
the routine clinical laboratory
bull Knowledge of fluorochromes antibody and antigen
interactions required
bull Full understanding of normal versus abnormal population
phenotype required
bull Sophisticated data analysis programs coupled with
education essential for successful implementation ndash for all
staff involved in testing and analysis
Normal B Cell Maturation
Wood and Borowitz (2006) Henryrsquos Laboratory Medicine
Normal B cell Maturation Pattern
The three pearls
Case 1
bull Nine month old male seen by GP for nodules in his
scalp suggestive of lymphoid infiltrate incidental
finding of bilateral enlarged kidneys
bull Referred to hematology as WBC 16300uL increased
Plt
bull WBC 12300uL Hb 111gL Plt 536x109L
bull Normal differential
bull Scalp lymphoid infiltrate suspicious for leukemia
bull Bone marrow aspirate flow ordered
Case 1
34
BM 14 B cells
5 CD10dimneg
CD20 negCD38 bright
plasma cells
Case 1 BM
Case 1 Bone marrow morphology
bull Cellular marrow
bull Erythropoeisis 20-25
bull Granulopoesis 40-50 orderly maturation
bull Lymphocytes 30 10- 15 ldquoblast likerdquo
bull Increased megakaryocytes
Case 1 2 week follow up
77 B cells ndash 90
CD10 dimneg
CD20 neg bright CD38
pB-ALL
Case 1 BM 2 week follow up
Case 1 Cytogenetics
bull Complex karyotype
bull Structural abnormalities in chromosome
123491417 and 19
bull t(1719) rare in B cell acute lymphoblastic
leukemia
Case 1 Follow up
bull On high risk protocol AALL 0232 for infantile B-ALL
bull MRD 008 at Day 42
bull Cord blood transplant
bull Relapse 5 months post transplant
Pearl of Pathology
bull Differentiating precursor B cells (hematogones) from B-
ALL can be challenging Standardized analysis and
clear understanding of ldquonormalrdquo essential including
arrested maturation
Case 2
bull 4 year old girl referred to pediatric hematology with
pancytopenia
bull WBC 6800uL(Neuts 400uL) Hb 42gL Plt 28
bull Aplastic anemia
bull Bone marrow ordered aspirate biopsy flow and
cytogenetics
Case 2 BM flow cytometry
Polyclonal B cells
helper suppressor T cells
No increase in blasts
Maturing myeloid population
Case 2 CD45 neg B-ALL
Case 2 BM
Case 2
bull CD45neg
bull CD19+CD10+
bull CD20(partialdim)
bull CD34 neg
bull SigampcIg neg
bull Cytogenetics
bull Gain of X chromosome and
468911131415171821
DNA Index
122 Diploid
Case 2
bull Currently in remission after completion of
AALL0932 standard risk B-ALL therapy
Case 2
Pearls of Pathology
CD45 negative B-ALL is not uncommon
bull Always look at ungated set of plots for each marker
bull Eg SS versus each fluorescence parameter
Case 3
bull 58 year old male with pararenal mass
bull WBC 4000uL Hb 98gL Plt 229
bull ldquoblastsrdquo reported on blood film
bull Peripheral blood sent for flow cytometry
Case 3 PB flow cytometry
Dim CD45 CD19+CD10+
CD34- Sig neg
Case 3 PB flow cytometry
Dim CD45 CD19+CD10+
CD20(partial)CD5 neg
SIg cIg neg
Case 3 BM flow cytometry
Case 3 BM biopsy
Case 3 BM biopsy
Case 3 BM biopsy report
bull Diffuse infiltrate large lymphoid cells
bull Scant to moderate cytoplasm
bull Large irregular nuclei with prominent nucleoli
bull Increased mitotic activity
bull Positive for BCL-2 BCL6 CD10 and MUM1
bull Negative for TdT CD5
bull Cytogenetics FISH on para renal mass ndash C-MYC and
BCL2 rearrangements detected
Case 3 Doubletriple hit lymphoma
(DHLTHL)
bull Morphologically heterogeneous B cell neoplasms
characterized by rearrangement of the MYC proto-
oncogene with concurrent BCL2 andor BCL6 gene
rearrangement
bull WHO 2008 criteria - most cases of DHL and THL
classified DLBCL or B-cell lymphoma unclassifiable
with features intermediate between DLBCL and Burkitt
lymphoma (BCLU)
Case 3 DHLTHL
bull Retrospective study from one institution features of 13 cases (9 DHL4
THL)
bull median age was 59 years (range 30-74) eight females five males
bull enlarging lymphadenopathymasses (11 patients) abnormal peripheral
blood findings (2 patients)
bull TdT positivity (four cases two THLtwo DHL) dim CD45 expression (7
cases) lack of CD20 (2 cases) or lack of sIg (3 cases) and blastoid
morphology (2 cases)
bull Conclusion Expression of TdT in a B-cell lymphoma with mature
features or expression of surface light chain in a case otherwise
suggestive of B-lymphoblastic leukemialymphoma should prompt
an expedited evaluation for DHLTHL
Leuk Lymphoma 2016 Feb 181-10 [Epub ahead of print]
Double- and triple-hit lymphomas can present with features suggestive
of immaturity including TdT expression and create diagnostic challenges
Moench L Sachs Z Aasen G Dolan M Dayton V Courville EL
Case 4
bull 55 year old female
bull Previous history hyperparathyroidism
bull Parathyroidectomy in 1999
bull Mild persistent hypercalcemia
bull WBC 4800uL hemoglobin 121 gL Plt 296
bull Total protein 90gL IgG 34gL monoclonal peak
209gL
Case 4 Monoclonal IgG patient
Polyclonal B cells
With maturing hematogones
helper suppressor T cells
less than 3 blasts
Maturing myeloid population
Case 4 BM Ungated
Case 4 cytoplasmic immunoglobulin
Need KL
Case 4 BM Multiple Myeloma
Case 4 follow up
bull Smouldering myeloma
bull Recently developed CRAB (hyperCalcemia Renal
insufficiency Anemia Bone lesions
bull Serum free light chain ratio 123
bull Bone marrow aspirate gt60 plasma cells
bull Currently on treatment pending bone marrow
transplant
Case 5
bull 65 year old male
bull Slight lymphocytosis detected on routine CBC analysis
(lymph 4500uL)
bull Asymptomatic sent for flow cytometry
Case 5 PB
Case 5 flow cytometry KL ratio 0625
Case 5 Monoclonal B cell lymphocytosis
KL 18 KL 056
Case 5 Monoclonal B cell lymphocytosis
bull WHO defines CLL as abnormal lymphocyte count
gt5x10E9L (CD19+CD5+ light chain restricted) in the
absence of other clinical symptoms
bull Patients with small B cell clones are often detected as
an incidental finding
bull WHO 2016
bull CD19+CD5+ lt05x10E9L ldquoLow count MBLrdquo
raquo 05-5x10E9L ldquoHigh count MBLrdquo
raquo Non CLL type phenotype MBL also recognized
Monoclonal B cell Lymphocytosis
bull CLL immunophenotype - vast majority (75)
bull Atypical MBL
bull normal levels both CD5 and CD20 with variable
expression of CD23 (always exclude Mantle-cell
Lymphoma in CD23 negative cases)
bull Non-CLL-phenotype (CD5minus) MBL
bull Lacking CD5 expression and CD10 likely resembling B-
cell lymphomas of marginal-zone origin
Case 5 What is the normal KL Ratio
bull What is the normal or abnormal kappalambda ratio for
entire ldquopopulationrdquo of all B cells
bull Traditional view
ndash Kappa Lambda 21 or 31 ( 12 -13)
ndash Range 14 60
Case 5 What is the normal KL ratio
bull Wrong question
bull It doesnrsquot matter how many of the B cells are clonal but rather
that their exists a clonal population of B cells within the total
population
bull Need to Identify malignant B cells within a population of
normal B cells
bull In order to interpret a pathological immunophenotype it is
necessary to refer to quantitative and qualitative values of
normal B-cell subpopulations
bull Pearl Identify the abnormal phenotype first then look at KL
ratio
Case 6
bull 12 year old male presents to ER with fatigue bruising
Lkc 2200uL
bull Hb 84gL
bull Plt 32
bull Diff ndash neutropenia no blasts noted few ldquoatypicalrdquo cells
bull RBC ndash non specific changes
Leukemia
Case 6 PB
Case 6 BM
Case 6
bull BM aspirate ndash Hypocellular relative lymphocytosis
marked reduction in myeloid lineages
hypoplasticaplastic anemia
bull Flow Cytometry ndash less than 3 blasts maturing
myeloid 68 lymphocytes polyclonal B helper
suppressor T
bull Unusual pattern of CD13CD16 staining noted
Case 6 flow cytometry Aplastic Anemia
Normal Patient
Normal
Patient
Patient
Normal
Case 6 RBC Test for PNH CD235a v s CD59
Case 6 Neutrophil test for PNH clone
FLAER CD24 CD45CD15
18 PNH clone
Case 6
bull Follow up
bull Patient on steroids acyclovir and anti-thymocyte
globulin
bull PNH clone Neutrophils 96 RBC 1 Nov 2015
bull In remission as of April 2017
- Lkc 5500uL (Neuts 23)
- Hb 129gL
- Plt 208 X10e9
Summary
bull B cell malignancies are the most common
hematological disorders seen in flow cytometry
bull Many have complex overlapping phenotypes
bull Important to look at ungated plots as well as gating on
specific subpopulations
bull Level of expression of markers is as important as
positive or negative (think CD13 vs CD16)
68
References
bull Craig FE Foon KA Flow cytometric immunophenotyping for hematologic neoplasms Blood
2008 Apr 15111(8)3941-67
bull Wood BL Principles of minimal residual disease detection for hematopoietic neoplasms by flow
cytometry Cytometry B Clin Cytom 2015 Apr 23
bull Shanafelt TD Ghia P Lanasa MC Landgren O Rawstron AC Monoclonal B-cell
lymphocytosis (MBL) biology natural history and clinical management Leukemia 2010
Mar24(3)512-20
bull Swerdlow SH Campo E Pileri SA Harris NL Stein H Siebert R Advani RGhielmini M Salles
GA Zelenetz AD Jaffe ES The 2016 revision of the World Health Organization classification of
lymphoid neoplasms Blood 2016 May 19127(20)2375-90
bull 3 Moench L Sachs Z Aasen G Dolan M Dayton V Courville EL Double- and triple-hit
lymphomas can present with features suggestive of immaturity including TdT expression and
create diagnostic challenges Leuk Lymphoma 2016572626-35
bull Sutherland DR Keeney M Illingworth A Practical guidelines for the high-sensitivity detection
and monitoring of paroxysmal nocturnal hemoglobinuria clones by flow cytometry Cytometry B
Clin Cytom 2012 Jul82(4)195-208
69
London Health Custom Design Tube
Antigen Fluorochrome
CD8 FITC
Kappa FITC
CD4 PE
Lambda PE
CD19 ECD
CD20 PE-Cy55
CD5 PE-Cy7
CD10 APC
CD56 APC-Alexa700
Near-IR Viability Dye Excitation 633nm
Emission 780nm
CD3 Pacific Blue
CD45 Krome Orange
Result Reporting Bethesda Guidelines
bull Report percent abnormal population
bull Antibody Distribution
ndash Negative Positive Partial
bull Antibody Fluorescence
- Dim mdash Positive uniform cell population with a median fluorescence intensity abnormally less than that seen on an antigen positive normal leukocyte population Example CD20 in CLL
- Bright mdash Positive uniform cell population with a median fluorescence intensity abnormally greater than that seen on an antigen positive normal leukocyte population Example CD20 in HCL
- Heterogeneous mdash Variably intense staining not a tight uniform cluster of positive immunofluorescence staining
Example CD38 on maturing B cells
Bethesda Reporting
Normal
Dim
CLL
CD19+ CD20+(dim)
CD5+(dim)Kappa+(dim)
Discussion
bull The availability of new fluorochromes coupled with
multilaser instruments with simplified setup and
compensation makes routine ten color analysis possible for
the routine clinical laboratory
bull Knowledge of fluorochromes antibody and antigen
interactions required
bull Full understanding of normal versus abnormal population
phenotype required
bull Sophisticated data analysis programs coupled with
education essential for successful implementation ndash for all
staff involved in testing and analysis
Normal B Cell Maturation
Wood and Borowitz (2006) Henryrsquos Laboratory Medicine
Normal B cell Maturation Pattern
The three pearls
Case 1
bull Nine month old male seen by GP for nodules in his
scalp suggestive of lymphoid infiltrate incidental
finding of bilateral enlarged kidneys
bull Referred to hematology as WBC 16300uL increased
Plt
bull WBC 12300uL Hb 111gL Plt 536x109L
bull Normal differential
bull Scalp lymphoid infiltrate suspicious for leukemia
bull Bone marrow aspirate flow ordered
Case 1
34
BM 14 B cells
5 CD10dimneg
CD20 negCD38 bright
plasma cells
Case 1 BM
Case 1 Bone marrow morphology
bull Cellular marrow
bull Erythropoeisis 20-25
bull Granulopoesis 40-50 orderly maturation
bull Lymphocytes 30 10- 15 ldquoblast likerdquo
bull Increased megakaryocytes
Case 1 2 week follow up
77 B cells ndash 90
CD10 dimneg
CD20 neg bright CD38
pB-ALL
Case 1 BM 2 week follow up
Case 1 Cytogenetics
bull Complex karyotype
bull Structural abnormalities in chromosome
123491417 and 19
bull t(1719) rare in B cell acute lymphoblastic
leukemia
Case 1 Follow up
bull On high risk protocol AALL 0232 for infantile B-ALL
bull MRD 008 at Day 42
bull Cord blood transplant
bull Relapse 5 months post transplant
Pearl of Pathology
bull Differentiating precursor B cells (hematogones) from B-
ALL can be challenging Standardized analysis and
clear understanding of ldquonormalrdquo essential including
arrested maturation
Case 2
bull 4 year old girl referred to pediatric hematology with
pancytopenia
bull WBC 6800uL(Neuts 400uL) Hb 42gL Plt 28
bull Aplastic anemia
bull Bone marrow ordered aspirate biopsy flow and
cytogenetics
Case 2 BM flow cytometry
Polyclonal B cells
helper suppressor T cells
No increase in blasts
Maturing myeloid population
Case 2 CD45 neg B-ALL
Case 2 BM
Case 2
bull CD45neg
bull CD19+CD10+
bull CD20(partialdim)
bull CD34 neg
bull SigampcIg neg
bull Cytogenetics
bull Gain of X chromosome and
468911131415171821
DNA Index
122 Diploid
Case 2
bull Currently in remission after completion of
AALL0932 standard risk B-ALL therapy
Case 2
Pearls of Pathology
CD45 negative B-ALL is not uncommon
bull Always look at ungated set of plots for each marker
bull Eg SS versus each fluorescence parameter
Case 3
bull 58 year old male with pararenal mass
bull WBC 4000uL Hb 98gL Plt 229
bull ldquoblastsrdquo reported on blood film
bull Peripheral blood sent for flow cytometry
Case 3 PB flow cytometry
Dim CD45 CD19+CD10+
CD34- Sig neg
Case 3 PB flow cytometry
Dim CD45 CD19+CD10+
CD20(partial)CD5 neg
SIg cIg neg
Case 3 BM flow cytometry
Case 3 BM biopsy
Case 3 BM biopsy
Case 3 BM biopsy report
bull Diffuse infiltrate large lymphoid cells
bull Scant to moderate cytoplasm
bull Large irregular nuclei with prominent nucleoli
bull Increased mitotic activity
bull Positive for BCL-2 BCL6 CD10 and MUM1
bull Negative for TdT CD5
bull Cytogenetics FISH on para renal mass ndash C-MYC and
BCL2 rearrangements detected
Case 3 Doubletriple hit lymphoma
(DHLTHL)
bull Morphologically heterogeneous B cell neoplasms
characterized by rearrangement of the MYC proto-
oncogene with concurrent BCL2 andor BCL6 gene
rearrangement
bull WHO 2008 criteria - most cases of DHL and THL
classified DLBCL or B-cell lymphoma unclassifiable
with features intermediate between DLBCL and Burkitt
lymphoma (BCLU)
Case 3 DHLTHL
bull Retrospective study from one institution features of 13 cases (9 DHL4
THL)
bull median age was 59 years (range 30-74) eight females five males
bull enlarging lymphadenopathymasses (11 patients) abnormal peripheral
blood findings (2 patients)
bull TdT positivity (four cases two THLtwo DHL) dim CD45 expression (7
cases) lack of CD20 (2 cases) or lack of sIg (3 cases) and blastoid
morphology (2 cases)
bull Conclusion Expression of TdT in a B-cell lymphoma with mature
features or expression of surface light chain in a case otherwise
suggestive of B-lymphoblastic leukemialymphoma should prompt
an expedited evaluation for DHLTHL
Leuk Lymphoma 2016 Feb 181-10 [Epub ahead of print]
Double- and triple-hit lymphomas can present with features suggestive
of immaturity including TdT expression and create diagnostic challenges
Moench L Sachs Z Aasen G Dolan M Dayton V Courville EL
Case 4
bull 55 year old female
bull Previous history hyperparathyroidism
bull Parathyroidectomy in 1999
bull Mild persistent hypercalcemia
bull WBC 4800uL hemoglobin 121 gL Plt 296
bull Total protein 90gL IgG 34gL monoclonal peak
209gL
Case 4 Monoclonal IgG patient
Polyclonal B cells
With maturing hematogones
helper suppressor T cells
less than 3 blasts
Maturing myeloid population
Case 4 BM Ungated
Case 4 cytoplasmic immunoglobulin
Need KL
Case 4 BM Multiple Myeloma
Case 4 follow up
bull Smouldering myeloma
bull Recently developed CRAB (hyperCalcemia Renal
insufficiency Anemia Bone lesions
bull Serum free light chain ratio 123
bull Bone marrow aspirate gt60 plasma cells
bull Currently on treatment pending bone marrow
transplant
Case 5
bull 65 year old male
bull Slight lymphocytosis detected on routine CBC analysis
(lymph 4500uL)
bull Asymptomatic sent for flow cytometry
Case 5 PB
Case 5 flow cytometry KL ratio 0625
Case 5 Monoclonal B cell lymphocytosis
KL 18 KL 056
Case 5 Monoclonal B cell lymphocytosis
bull WHO defines CLL as abnormal lymphocyte count
gt5x10E9L (CD19+CD5+ light chain restricted) in the
absence of other clinical symptoms
bull Patients with small B cell clones are often detected as
an incidental finding
bull WHO 2016
bull CD19+CD5+ lt05x10E9L ldquoLow count MBLrdquo
raquo 05-5x10E9L ldquoHigh count MBLrdquo
raquo Non CLL type phenotype MBL also recognized
Monoclonal B cell Lymphocytosis
bull CLL immunophenotype - vast majority (75)
bull Atypical MBL
bull normal levels both CD5 and CD20 with variable
expression of CD23 (always exclude Mantle-cell
Lymphoma in CD23 negative cases)
bull Non-CLL-phenotype (CD5minus) MBL
bull Lacking CD5 expression and CD10 likely resembling B-
cell lymphomas of marginal-zone origin
Case 5 What is the normal KL Ratio
bull What is the normal or abnormal kappalambda ratio for
entire ldquopopulationrdquo of all B cells
bull Traditional view
ndash Kappa Lambda 21 or 31 ( 12 -13)
ndash Range 14 60
Case 5 What is the normal KL ratio
bull Wrong question
bull It doesnrsquot matter how many of the B cells are clonal but rather
that their exists a clonal population of B cells within the total
population
bull Need to Identify malignant B cells within a population of
normal B cells
bull In order to interpret a pathological immunophenotype it is
necessary to refer to quantitative and qualitative values of
normal B-cell subpopulations
bull Pearl Identify the abnormal phenotype first then look at KL
ratio
Case 6
bull 12 year old male presents to ER with fatigue bruising
Lkc 2200uL
bull Hb 84gL
bull Plt 32
bull Diff ndash neutropenia no blasts noted few ldquoatypicalrdquo cells
bull RBC ndash non specific changes
Leukemia
Case 6 PB
Case 6 BM
Case 6
bull BM aspirate ndash Hypocellular relative lymphocytosis
marked reduction in myeloid lineages
hypoplasticaplastic anemia
bull Flow Cytometry ndash less than 3 blasts maturing
myeloid 68 lymphocytes polyclonal B helper
suppressor T
bull Unusual pattern of CD13CD16 staining noted
Case 6 flow cytometry Aplastic Anemia
Normal Patient
Normal
Patient
Patient
Normal
Case 6 RBC Test for PNH CD235a v s CD59
Case 6 Neutrophil test for PNH clone
FLAER CD24 CD45CD15
18 PNH clone
Case 6
bull Follow up
bull Patient on steroids acyclovir and anti-thymocyte
globulin
bull PNH clone Neutrophils 96 RBC 1 Nov 2015
bull In remission as of April 2017
- Lkc 5500uL (Neuts 23)
- Hb 129gL
- Plt 208 X10e9
Summary
bull B cell malignancies are the most common
hematological disorders seen in flow cytometry
bull Many have complex overlapping phenotypes
bull Important to look at ungated plots as well as gating on
specific subpopulations
bull Level of expression of markers is as important as
positive or negative (think CD13 vs CD16)
68
References
bull Craig FE Foon KA Flow cytometric immunophenotyping for hematologic neoplasms Blood
2008 Apr 15111(8)3941-67
bull Wood BL Principles of minimal residual disease detection for hematopoietic neoplasms by flow
cytometry Cytometry B Clin Cytom 2015 Apr 23
bull Shanafelt TD Ghia P Lanasa MC Landgren O Rawstron AC Monoclonal B-cell
lymphocytosis (MBL) biology natural history and clinical management Leukemia 2010
Mar24(3)512-20
bull Swerdlow SH Campo E Pileri SA Harris NL Stein H Siebert R Advani RGhielmini M Salles
GA Zelenetz AD Jaffe ES The 2016 revision of the World Health Organization classification of
lymphoid neoplasms Blood 2016 May 19127(20)2375-90
bull 3 Moench L Sachs Z Aasen G Dolan M Dayton V Courville EL Double- and triple-hit
lymphomas can present with features suggestive of immaturity including TdT expression and
create diagnostic challenges Leuk Lymphoma 2016572626-35
bull Sutherland DR Keeney M Illingworth A Practical guidelines for the high-sensitivity detection
and monitoring of paroxysmal nocturnal hemoglobinuria clones by flow cytometry Cytometry B
Clin Cytom 2012 Jul82(4)195-208
69
Result Reporting Bethesda Guidelines
bull Report percent abnormal population
bull Antibody Distribution
ndash Negative Positive Partial
bull Antibody Fluorescence
- Dim mdash Positive uniform cell population with a median fluorescence intensity abnormally less than that seen on an antigen positive normal leukocyte population Example CD20 in CLL
- Bright mdash Positive uniform cell population with a median fluorescence intensity abnormally greater than that seen on an antigen positive normal leukocyte population Example CD20 in HCL
- Heterogeneous mdash Variably intense staining not a tight uniform cluster of positive immunofluorescence staining
Example CD38 on maturing B cells
Bethesda Reporting
Normal
Dim
CLL
CD19+ CD20+(dim)
CD5+(dim)Kappa+(dim)
Discussion
bull The availability of new fluorochromes coupled with
multilaser instruments with simplified setup and
compensation makes routine ten color analysis possible for
the routine clinical laboratory
bull Knowledge of fluorochromes antibody and antigen
interactions required
bull Full understanding of normal versus abnormal population
phenotype required
bull Sophisticated data analysis programs coupled with
education essential for successful implementation ndash for all
staff involved in testing and analysis
Normal B Cell Maturation
Wood and Borowitz (2006) Henryrsquos Laboratory Medicine
Normal B cell Maturation Pattern
The three pearls
Case 1
bull Nine month old male seen by GP for nodules in his
scalp suggestive of lymphoid infiltrate incidental
finding of bilateral enlarged kidneys
bull Referred to hematology as WBC 16300uL increased
Plt
bull WBC 12300uL Hb 111gL Plt 536x109L
bull Normal differential
bull Scalp lymphoid infiltrate suspicious for leukemia
bull Bone marrow aspirate flow ordered
Case 1
34
BM 14 B cells
5 CD10dimneg
CD20 negCD38 bright
plasma cells
Case 1 BM
Case 1 Bone marrow morphology
bull Cellular marrow
bull Erythropoeisis 20-25
bull Granulopoesis 40-50 orderly maturation
bull Lymphocytes 30 10- 15 ldquoblast likerdquo
bull Increased megakaryocytes
Case 1 2 week follow up
77 B cells ndash 90
CD10 dimneg
CD20 neg bright CD38
pB-ALL
Case 1 BM 2 week follow up
Case 1 Cytogenetics
bull Complex karyotype
bull Structural abnormalities in chromosome
123491417 and 19
bull t(1719) rare in B cell acute lymphoblastic
leukemia
Case 1 Follow up
bull On high risk protocol AALL 0232 for infantile B-ALL
bull MRD 008 at Day 42
bull Cord blood transplant
bull Relapse 5 months post transplant
Pearl of Pathology
bull Differentiating precursor B cells (hematogones) from B-
ALL can be challenging Standardized analysis and
clear understanding of ldquonormalrdquo essential including
arrested maturation
Case 2
bull 4 year old girl referred to pediatric hematology with
pancytopenia
bull WBC 6800uL(Neuts 400uL) Hb 42gL Plt 28
bull Aplastic anemia
bull Bone marrow ordered aspirate biopsy flow and
cytogenetics
Case 2 BM flow cytometry
Polyclonal B cells
helper suppressor T cells
No increase in blasts
Maturing myeloid population
Case 2 CD45 neg B-ALL
Case 2 BM
Case 2
bull CD45neg
bull CD19+CD10+
bull CD20(partialdim)
bull CD34 neg
bull SigampcIg neg
bull Cytogenetics
bull Gain of X chromosome and
468911131415171821
DNA Index
122 Diploid
Case 2
bull Currently in remission after completion of
AALL0932 standard risk B-ALL therapy
Case 2
Pearls of Pathology
CD45 negative B-ALL is not uncommon
bull Always look at ungated set of plots for each marker
bull Eg SS versus each fluorescence parameter
Case 3
bull 58 year old male with pararenal mass
bull WBC 4000uL Hb 98gL Plt 229
bull ldquoblastsrdquo reported on blood film
bull Peripheral blood sent for flow cytometry
Case 3 PB flow cytometry
Dim CD45 CD19+CD10+
CD34- Sig neg
Case 3 PB flow cytometry
Dim CD45 CD19+CD10+
CD20(partial)CD5 neg
SIg cIg neg
Case 3 BM flow cytometry
Case 3 BM biopsy
Case 3 BM biopsy
Case 3 BM biopsy report
bull Diffuse infiltrate large lymphoid cells
bull Scant to moderate cytoplasm
bull Large irregular nuclei with prominent nucleoli
bull Increased mitotic activity
bull Positive for BCL-2 BCL6 CD10 and MUM1
bull Negative for TdT CD5
bull Cytogenetics FISH on para renal mass ndash C-MYC and
BCL2 rearrangements detected
Case 3 Doubletriple hit lymphoma
(DHLTHL)
bull Morphologically heterogeneous B cell neoplasms
characterized by rearrangement of the MYC proto-
oncogene with concurrent BCL2 andor BCL6 gene
rearrangement
bull WHO 2008 criteria - most cases of DHL and THL
classified DLBCL or B-cell lymphoma unclassifiable
with features intermediate between DLBCL and Burkitt
lymphoma (BCLU)
Case 3 DHLTHL
bull Retrospective study from one institution features of 13 cases (9 DHL4
THL)
bull median age was 59 years (range 30-74) eight females five males
bull enlarging lymphadenopathymasses (11 patients) abnormal peripheral
blood findings (2 patients)
bull TdT positivity (four cases two THLtwo DHL) dim CD45 expression (7
cases) lack of CD20 (2 cases) or lack of sIg (3 cases) and blastoid
morphology (2 cases)
bull Conclusion Expression of TdT in a B-cell lymphoma with mature
features or expression of surface light chain in a case otherwise
suggestive of B-lymphoblastic leukemialymphoma should prompt
an expedited evaluation for DHLTHL
Leuk Lymphoma 2016 Feb 181-10 [Epub ahead of print]
Double- and triple-hit lymphomas can present with features suggestive
of immaturity including TdT expression and create diagnostic challenges
Moench L Sachs Z Aasen G Dolan M Dayton V Courville EL
Case 4
bull 55 year old female
bull Previous history hyperparathyroidism
bull Parathyroidectomy in 1999
bull Mild persistent hypercalcemia
bull WBC 4800uL hemoglobin 121 gL Plt 296
bull Total protein 90gL IgG 34gL monoclonal peak
209gL
Case 4 Monoclonal IgG patient
Polyclonal B cells
With maturing hematogones
helper suppressor T cells
less than 3 blasts
Maturing myeloid population
Case 4 BM Ungated
Case 4 cytoplasmic immunoglobulin
Need KL
Case 4 BM Multiple Myeloma
Case 4 follow up
bull Smouldering myeloma
bull Recently developed CRAB (hyperCalcemia Renal
insufficiency Anemia Bone lesions
bull Serum free light chain ratio 123
bull Bone marrow aspirate gt60 plasma cells
bull Currently on treatment pending bone marrow
transplant
Case 5
bull 65 year old male
bull Slight lymphocytosis detected on routine CBC analysis
(lymph 4500uL)
bull Asymptomatic sent for flow cytometry
Case 5 PB
Case 5 flow cytometry KL ratio 0625
Case 5 Monoclonal B cell lymphocytosis
KL 18 KL 056
Case 5 Monoclonal B cell lymphocytosis
bull WHO defines CLL as abnormal lymphocyte count
gt5x10E9L (CD19+CD5+ light chain restricted) in the
absence of other clinical symptoms
bull Patients with small B cell clones are often detected as
an incidental finding
bull WHO 2016
bull CD19+CD5+ lt05x10E9L ldquoLow count MBLrdquo
raquo 05-5x10E9L ldquoHigh count MBLrdquo
raquo Non CLL type phenotype MBL also recognized
Monoclonal B cell Lymphocytosis
bull CLL immunophenotype - vast majority (75)
bull Atypical MBL
bull normal levels both CD5 and CD20 with variable
expression of CD23 (always exclude Mantle-cell
Lymphoma in CD23 negative cases)
bull Non-CLL-phenotype (CD5minus) MBL
bull Lacking CD5 expression and CD10 likely resembling B-
cell lymphomas of marginal-zone origin
Case 5 What is the normal KL Ratio
bull What is the normal or abnormal kappalambda ratio for
entire ldquopopulationrdquo of all B cells
bull Traditional view
ndash Kappa Lambda 21 or 31 ( 12 -13)
ndash Range 14 60
Case 5 What is the normal KL ratio
bull Wrong question
bull It doesnrsquot matter how many of the B cells are clonal but rather
that their exists a clonal population of B cells within the total
population
bull Need to Identify malignant B cells within a population of
normal B cells
bull In order to interpret a pathological immunophenotype it is
necessary to refer to quantitative and qualitative values of
normal B-cell subpopulations
bull Pearl Identify the abnormal phenotype first then look at KL
ratio
Case 6
bull 12 year old male presents to ER with fatigue bruising
Lkc 2200uL
bull Hb 84gL
bull Plt 32
bull Diff ndash neutropenia no blasts noted few ldquoatypicalrdquo cells
bull RBC ndash non specific changes
Leukemia
Case 6 PB
Case 6 BM
Case 6
bull BM aspirate ndash Hypocellular relative lymphocytosis
marked reduction in myeloid lineages
hypoplasticaplastic anemia
bull Flow Cytometry ndash less than 3 blasts maturing
myeloid 68 lymphocytes polyclonal B helper
suppressor T
bull Unusual pattern of CD13CD16 staining noted
Case 6 flow cytometry Aplastic Anemia
Normal Patient
Normal
Patient
Patient
Normal
Case 6 RBC Test for PNH CD235a v s CD59
Case 6 Neutrophil test for PNH clone
FLAER CD24 CD45CD15
18 PNH clone
Case 6
bull Follow up
bull Patient on steroids acyclovir and anti-thymocyte
globulin
bull PNH clone Neutrophils 96 RBC 1 Nov 2015
bull In remission as of April 2017
- Lkc 5500uL (Neuts 23)
- Hb 129gL
- Plt 208 X10e9
Summary
bull B cell malignancies are the most common
hematological disorders seen in flow cytometry
bull Many have complex overlapping phenotypes
bull Important to look at ungated plots as well as gating on
specific subpopulations
bull Level of expression of markers is as important as
positive or negative (think CD13 vs CD16)
68
References
bull Craig FE Foon KA Flow cytometric immunophenotyping for hematologic neoplasms Blood
2008 Apr 15111(8)3941-67
bull Wood BL Principles of minimal residual disease detection for hematopoietic neoplasms by flow
cytometry Cytometry B Clin Cytom 2015 Apr 23
bull Shanafelt TD Ghia P Lanasa MC Landgren O Rawstron AC Monoclonal B-cell
lymphocytosis (MBL) biology natural history and clinical management Leukemia 2010
Mar24(3)512-20
bull Swerdlow SH Campo E Pileri SA Harris NL Stein H Siebert R Advani RGhielmini M Salles
GA Zelenetz AD Jaffe ES The 2016 revision of the World Health Organization classification of
lymphoid neoplasms Blood 2016 May 19127(20)2375-90
bull 3 Moench L Sachs Z Aasen G Dolan M Dayton V Courville EL Double- and triple-hit
lymphomas can present with features suggestive of immaturity including TdT expression and
create diagnostic challenges Leuk Lymphoma 2016572626-35
bull Sutherland DR Keeney M Illingworth A Practical guidelines for the high-sensitivity detection
and monitoring of paroxysmal nocturnal hemoglobinuria clones by flow cytometry Cytometry B
Clin Cytom 2012 Jul82(4)195-208
69
Bethesda Reporting
Normal
Dim
CLL
CD19+ CD20+(dim)
CD5+(dim)Kappa+(dim)
Discussion
bull The availability of new fluorochromes coupled with
multilaser instruments with simplified setup and
compensation makes routine ten color analysis possible for
the routine clinical laboratory
bull Knowledge of fluorochromes antibody and antigen
interactions required
bull Full understanding of normal versus abnormal population
phenotype required
bull Sophisticated data analysis programs coupled with
education essential for successful implementation ndash for all
staff involved in testing and analysis
Normal B Cell Maturation
Wood and Borowitz (2006) Henryrsquos Laboratory Medicine
Normal B cell Maturation Pattern
The three pearls
Case 1
bull Nine month old male seen by GP for nodules in his
scalp suggestive of lymphoid infiltrate incidental
finding of bilateral enlarged kidneys
bull Referred to hematology as WBC 16300uL increased
Plt
bull WBC 12300uL Hb 111gL Plt 536x109L
bull Normal differential
bull Scalp lymphoid infiltrate suspicious for leukemia
bull Bone marrow aspirate flow ordered
Case 1
34
BM 14 B cells
5 CD10dimneg
CD20 negCD38 bright
plasma cells
Case 1 BM
Case 1 Bone marrow morphology
bull Cellular marrow
bull Erythropoeisis 20-25
bull Granulopoesis 40-50 orderly maturation
bull Lymphocytes 30 10- 15 ldquoblast likerdquo
bull Increased megakaryocytes
Case 1 2 week follow up
77 B cells ndash 90
CD10 dimneg
CD20 neg bright CD38
pB-ALL
Case 1 BM 2 week follow up
Case 1 Cytogenetics
bull Complex karyotype
bull Structural abnormalities in chromosome
123491417 and 19
bull t(1719) rare in B cell acute lymphoblastic
leukemia
Case 1 Follow up
bull On high risk protocol AALL 0232 for infantile B-ALL
bull MRD 008 at Day 42
bull Cord blood transplant
bull Relapse 5 months post transplant
Pearl of Pathology
bull Differentiating precursor B cells (hematogones) from B-
ALL can be challenging Standardized analysis and
clear understanding of ldquonormalrdquo essential including
arrested maturation
Case 2
bull 4 year old girl referred to pediatric hematology with
pancytopenia
bull WBC 6800uL(Neuts 400uL) Hb 42gL Plt 28
bull Aplastic anemia
bull Bone marrow ordered aspirate biopsy flow and
cytogenetics
Case 2 BM flow cytometry
Polyclonal B cells
helper suppressor T cells
No increase in blasts
Maturing myeloid population
Case 2 CD45 neg B-ALL
Case 2 BM
Case 2
bull CD45neg
bull CD19+CD10+
bull CD20(partialdim)
bull CD34 neg
bull SigampcIg neg
bull Cytogenetics
bull Gain of X chromosome and
468911131415171821
DNA Index
122 Diploid
Case 2
bull Currently in remission after completion of
AALL0932 standard risk B-ALL therapy
Case 2
Pearls of Pathology
CD45 negative B-ALL is not uncommon
bull Always look at ungated set of plots for each marker
bull Eg SS versus each fluorescence parameter
Case 3
bull 58 year old male with pararenal mass
bull WBC 4000uL Hb 98gL Plt 229
bull ldquoblastsrdquo reported on blood film
bull Peripheral blood sent for flow cytometry
Case 3 PB flow cytometry
Dim CD45 CD19+CD10+
CD34- Sig neg
Case 3 PB flow cytometry
Dim CD45 CD19+CD10+
CD20(partial)CD5 neg
SIg cIg neg
Case 3 BM flow cytometry
Case 3 BM biopsy
Case 3 BM biopsy
Case 3 BM biopsy report
bull Diffuse infiltrate large lymphoid cells
bull Scant to moderate cytoplasm
bull Large irregular nuclei with prominent nucleoli
bull Increased mitotic activity
bull Positive for BCL-2 BCL6 CD10 and MUM1
bull Negative for TdT CD5
bull Cytogenetics FISH on para renal mass ndash C-MYC and
BCL2 rearrangements detected
Case 3 Doubletriple hit lymphoma
(DHLTHL)
bull Morphologically heterogeneous B cell neoplasms
characterized by rearrangement of the MYC proto-
oncogene with concurrent BCL2 andor BCL6 gene
rearrangement
bull WHO 2008 criteria - most cases of DHL and THL
classified DLBCL or B-cell lymphoma unclassifiable
with features intermediate between DLBCL and Burkitt
lymphoma (BCLU)
Case 3 DHLTHL
bull Retrospective study from one institution features of 13 cases (9 DHL4
THL)
bull median age was 59 years (range 30-74) eight females five males
bull enlarging lymphadenopathymasses (11 patients) abnormal peripheral
blood findings (2 patients)
bull TdT positivity (four cases two THLtwo DHL) dim CD45 expression (7
cases) lack of CD20 (2 cases) or lack of sIg (3 cases) and blastoid
morphology (2 cases)
bull Conclusion Expression of TdT in a B-cell lymphoma with mature
features or expression of surface light chain in a case otherwise
suggestive of B-lymphoblastic leukemialymphoma should prompt
an expedited evaluation for DHLTHL
Leuk Lymphoma 2016 Feb 181-10 [Epub ahead of print]
Double- and triple-hit lymphomas can present with features suggestive
of immaturity including TdT expression and create diagnostic challenges
Moench L Sachs Z Aasen G Dolan M Dayton V Courville EL
Case 4
bull 55 year old female
bull Previous history hyperparathyroidism
bull Parathyroidectomy in 1999
bull Mild persistent hypercalcemia
bull WBC 4800uL hemoglobin 121 gL Plt 296
bull Total protein 90gL IgG 34gL monoclonal peak
209gL
Case 4 Monoclonal IgG patient
Polyclonal B cells
With maturing hematogones
helper suppressor T cells
less than 3 blasts
Maturing myeloid population
Case 4 BM Ungated
Case 4 cytoplasmic immunoglobulin
Need KL
Case 4 BM Multiple Myeloma
Case 4 follow up
bull Smouldering myeloma
bull Recently developed CRAB (hyperCalcemia Renal
insufficiency Anemia Bone lesions
bull Serum free light chain ratio 123
bull Bone marrow aspirate gt60 plasma cells
bull Currently on treatment pending bone marrow
transplant
Case 5
bull 65 year old male
bull Slight lymphocytosis detected on routine CBC analysis
(lymph 4500uL)
bull Asymptomatic sent for flow cytometry
Case 5 PB
Case 5 flow cytometry KL ratio 0625
Case 5 Monoclonal B cell lymphocytosis
KL 18 KL 056
Case 5 Monoclonal B cell lymphocytosis
bull WHO defines CLL as abnormal lymphocyte count
gt5x10E9L (CD19+CD5+ light chain restricted) in the
absence of other clinical symptoms
bull Patients with small B cell clones are often detected as
an incidental finding
bull WHO 2016
bull CD19+CD5+ lt05x10E9L ldquoLow count MBLrdquo
raquo 05-5x10E9L ldquoHigh count MBLrdquo
raquo Non CLL type phenotype MBL also recognized
Monoclonal B cell Lymphocytosis
bull CLL immunophenotype - vast majority (75)
bull Atypical MBL
bull normal levels both CD5 and CD20 with variable
expression of CD23 (always exclude Mantle-cell
Lymphoma in CD23 negative cases)
bull Non-CLL-phenotype (CD5minus) MBL
bull Lacking CD5 expression and CD10 likely resembling B-
cell lymphomas of marginal-zone origin
Case 5 What is the normal KL Ratio
bull What is the normal or abnormal kappalambda ratio for
entire ldquopopulationrdquo of all B cells
bull Traditional view
ndash Kappa Lambda 21 or 31 ( 12 -13)
ndash Range 14 60
Case 5 What is the normal KL ratio
bull Wrong question
bull It doesnrsquot matter how many of the B cells are clonal but rather
that their exists a clonal population of B cells within the total
population
bull Need to Identify malignant B cells within a population of
normal B cells
bull In order to interpret a pathological immunophenotype it is
necessary to refer to quantitative and qualitative values of
normal B-cell subpopulations
bull Pearl Identify the abnormal phenotype first then look at KL
ratio
Case 6
bull 12 year old male presents to ER with fatigue bruising
Lkc 2200uL
bull Hb 84gL
bull Plt 32
bull Diff ndash neutropenia no blasts noted few ldquoatypicalrdquo cells
bull RBC ndash non specific changes
Leukemia
Case 6 PB
Case 6 BM
Case 6
bull BM aspirate ndash Hypocellular relative lymphocytosis
marked reduction in myeloid lineages
hypoplasticaplastic anemia
bull Flow Cytometry ndash less than 3 blasts maturing
myeloid 68 lymphocytes polyclonal B helper
suppressor T
bull Unusual pattern of CD13CD16 staining noted
Case 6 flow cytometry Aplastic Anemia
Normal Patient
Normal
Patient
Patient
Normal
Case 6 RBC Test for PNH CD235a v s CD59
Case 6 Neutrophil test for PNH clone
FLAER CD24 CD45CD15
18 PNH clone
Case 6
bull Follow up
bull Patient on steroids acyclovir and anti-thymocyte
globulin
bull PNH clone Neutrophils 96 RBC 1 Nov 2015
bull In remission as of April 2017
- Lkc 5500uL (Neuts 23)
- Hb 129gL
- Plt 208 X10e9
Summary
bull B cell malignancies are the most common
hematological disorders seen in flow cytometry
bull Many have complex overlapping phenotypes
bull Important to look at ungated plots as well as gating on
specific subpopulations
bull Level of expression of markers is as important as
positive or negative (think CD13 vs CD16)
68
References
bull Craig FE Foon KA Flow cytometric immunophenotyping for hematologic neoplasms Blood
2008 Apr 15111(8)3941-67
bull Wood BL Principles of minimal residual disease detection for hematopoietic neoplasms by flow
cytometry Cytometry B Clin Cytom 2015 Apr 23
bull Shanafelt TD Ghia P Lanasa MC Landgren O Rawstron AC Monoclonal B-cell
lymphocytosis (MBL) biology natural history and clinical management Leukemia 2010
Mar24(3)512-20
bull Swerdlow SH Campo E Pileri SA Harris NL Stein H Siebert R Advani RGhielmini M Salles
GA Zelenetz AD Jaffe ES The 2016 revision of the World Health Organization classification of
lymphoid neoplasms Blood 2016 May 19127(20)2375-90
bull 3 Moench L Sachs Z Aasen G Dolan M Dayton V Courville EL Double- and triple-hit
lymphomas can present with features suggestive of immaturity including TdT expression and
create diagnostic challenges Leuk Lymphoma 2016572626-35
bull Sutherland DR Keeney M Illingworth A Practical guidelines for the high-sensitivity detection
and monitoring of paroxysmal nocturnal hemoglobinuria clones by flow cytometry Cytometry B
Clin Cytom 2012 Jul82(4)195-208
69
Discussion
bull The availability of new fluorochromes coupled with
multilaser instruments with simplified setup and
compensation makes routine ten color analysis possible for
the routine clinical laboratory
bull Knowledge of fluorochromes antibody and antigen
interactions required
bull Full understanding of normal versus abnormal population
phenotype required
bull Sophisticated data analysis programs coupled with
education essential for successful implementation ndash for all
staff involved in testing and analysis
Normal B Cell Maturation
Wood and Borowitz (2006) Henryrsquos Laboratory Medicine
Normal B cell Maturation Pattern
The three pearls
Case 1
bull Nine month old male seen by GP for nodules in his
scalp suggestive of lymphoid infiltrate incidental
finding of bilateral enlarged kidneys
bull Referred to hematology as WBC 16300uL increased
Plt
bull WBC 12300uL Hb 111gL Plt 536x109L
bull Normal differential
bull Scalp lymphoid infiltrate suspicious for leukemia
bull Bone marrow aspirate flow ordered
Case 1
34
BM 14 B cells
5 CD10dimneg
CD20 negCD38 bright
plasma cells
Case 1 BM
Case 1 Bone marrow morphology
bull Cellular marrow
bull Erythropoeisis 20-25
bull Granulopoesis 40-50 orderly maturation
bull Lymphocytes 30 10- 15 ldquoblast likerdquo
bull Increased megakaryocytes
Case 1 2 week follow up
77 B cells ndash 90
CD10 dimneg
CD20 neg bright CD38
pB-ALL
Case 1 BM 2 week follow up
Case 1 Cytogenetics
bull Complex karyotype
bull Structural abnormalities in chromosome
123491417 and 19
bull t(1719) rare in B cell acute lymphoblastic
leukemia
Case 1 Follow up
bull On high risk protocol AALL 0232 for infantile B-ALL
bull MRD 008 at Day 42
bull Cord blood transplant
bull Relapse 5 months post transplant
Pearl of Pathology
bull Differentiating precursor B cells (hematogones) from B-
ALL can be challenging Standardized analysis and
clear understanding of ldquonormalrdquo essential including
arrested maturation
Case 2
bull 4 year old girl referred to pediatric hematology with
pancytopenia
bull WBC 6800uL(Neuts 400uL) Hb 42gL Plt 28
bull Aplastic anemia
bull Bone marrow ordered aspirate biopsy flow and
cytogenetics
Case 2 BM flow cytometry
Polyclonal B cells
helper suppressor T cells
No increase in blasts
Maturing myeloid population
Case 2 CD45 neg B-ALL
Case 2 BM
Case 2
bull CD45neg
bull CD19+CD10+
bull CD20(partialdim)
bull CD34 neg
bull SigampcIg neg
bull Cytogenetics
bull Gain of X chromosome and
468911131415171821
DNA Index
122 Diploid
Case 2
bull Currently in remission after completion of
AALL0932 standard risk B-ALL therapy
Case 2
Pearls of Pathology
CD45 negative B-ALL is not uncommon
bull Always look at ungated set of plots for each marker
bull Eg SS versus each fluorescence parameter
Case 3
bull 58 year old male with pararenal mass
bull WBC 4000uL Hb 98gL Plt 229
bull ldquoblastsrdquo reported on blood film
bull Peripheral blood sent for flow cytometry
Case 3 PB flow cytometry
Dim CD45 CD19+CD10+
CD34- Sig neg
Case 3 PB flow cytometry
Dim CD45 CD19+CD10+
CD20(partial)CD5 neg
SIg cIg neg
Case 3 BM flow cytometry
Case 3 BM biopsy
Case 3 BM biopsy
Case 3 BM biopsy report
bull Diffuse infiltrate large lymphoid cells
bull Scant to moderate cytoplasm
bull Large irregular nuclei with prominent nucleoli
bull Increased mitotic activity
bull Positive for BCL-2 BCL6 CD10 and MUM1
bull Negative for TdT CD5
bull Cytogenetics FISH on para renal mass ndash C-MYC and
BCL2 rearrangements detected
Case 3 Doubletriple hit lymphoma
(DHLTHL)
bull Morphologically heterogeneous B cell neoplasms
characterized by rearrangement of the MYC proto-
oncogene with concurrent BCL2 andor BCL6 gene
rearrangement
bull WHO 2008 criteria - most cases of DHL and THL
classified DLBCL or B-cell lymphoma unclassifiable
with features intermediate between DLBCL and Burkitt
lymphoma (BCLU)
Case 3 DHLTHL
bull Retrospective study from one institution features of 13 cases (9 DHL4
THL)
bull median age was 59 years (range 30-74) eight females five males
bull enlarging lymphadenopathymasses (11 patients) abnormal peripheral
blood findings (2 patients)
bull TdT positivity (four cases two THLtwo DHL) dim CD45 expression (7
cases) lack of CD20 (2 cases) or lack of sIg (3 cases) and blastoid
morphology (2 cases)
bull Conclusion Expression of TdT in a B-cell lymphoma with mature
features or expression of surface light chain in a case otherwise
suggestive of B-lymphoblastic leukemialymphoma should prompt
an expedited evaluation for DHLTHL
Leuk Lymphoma 2016 Feb 181-10 [Epub ahead of print]
Double- and triple-hit lymphomas can present with features suggestive
of immaturity including TdT expression and create diagnostic challenges
Moench L Sachs Z Aasen G Dolan M Dayton V Courville EL
Case 4
bull 55 year old female
bull Previous history hyperparathyroidism
bull Parathyroidectomy in 1999
bull Mild persistent hypercalcemia
bull WBC 4800uL hemoglobin 121 gL Plt 296
bull Total protein 90gL IgG 34gL monoclonal peak
209gL
Case 4 Monoclonal IgG patient
Polyclonal B cells
With maturing hematogones
helper suppressor T cells
less than 3 blasts
Maturing myeloid population
Case 4 BM Ungated
Case 4 cytoplasmic immunoglobulin
Need KL
Case 4 BM Multiple Myeloma
Case 4 follow up
bull Smouldering myeloma
bull Recently developed CRAB (hyperCalcemia Renal
insufficiency Anemia Bone lesions
bull Serum free light chain ratio 123
bull Bone marrow aspirate gt60 plasma cells
bull Currently on treatment pending bone marrow
transplant
Case 5
bull 65 year old male
bull Slight lymphocytosis detected on routine CBC analysis
(lymph 4500uL)
bull Asymptomatic sent for flow cytometry
Case 5 PB
Case 5 flow cytometry KL ratio 0625
Case 5 Monoclonal B cell lymphocytosis
KL 18 KL 056
Case 5 Monoclonal B cell lymphocytosis
bull WHO defines CLL as abnormal lymphocyte count
gt5x10E9L (CD19+CD5+ light chain restricted) in the
absence of other clinical symptoms
bull Patients with small B cell clones are often detected as
an incidental finding
bull WHO 2016
bull CD19+CD5+ lt05x10E9L ldquoLow count MBLrdquo
raquo 05-5x10E9L ldquoHigh count MBLrdquo
raquo Non CLL type phenotype MBL also recognized
Monoclonal B cell Lymphocytosis
bull CLL immunophenotype - vast majority (75)
bull Atypical MBL
bull normal levels both CD5 and CD20 with variable
expression of CD23 (always exclude Mantle-cell
Lymphoma in CD23 negative cases)
bull Non-CLL-phenotype (CD5minus) MBL
bull Lacking CD5 expression and CD10 likely resembling B-
cell lymphomas of marginal-zone origin
Case 5 What is the normal KL Ratio
bull What is the normal or abnormal kappalambda ratio for
entire ldquopopulationrdquo of all B cells
bull Traditional view
ndash Kappa Lambda 21 or 31 ( 12 -13)
ndash Range 14 60
Case 5 What is the normal KL ratio
bull Wrong question
bull It doesnrsquot matter how many of the B cells are clonal but rather
that their exists a clonal population of B cells within the total
population
bull Need to Identify malignant B cells within a population of
normal B cells
bull In order to interpret a pathological immunophenotype it is
necessary to refer to quantitative and qualitative values of
normal B-cell subpopulations
bull Pearl Identify the abnormal phenotype first then look at KL
ratio
Case 6
bull 12 year old male presents to ER with fatigue bruising
Lkc 2200uL
bull Hb 84gL
bull Plt 32
bull Diff ndash neutropenia no blasts noted few ldquoatypicalrdquo cells
bull RBC ndash non specific changes
Leukemia
Case 6 PB
Case 6 BM
Case 6
bull BM aspirate ndash Hypocellular relative lymphocytosis
marked reduction in myeloid lineages
hypoplasticaplastic anemia
bull Flow Cytometry ndash less than 3 blasts maturing
myeloid 68 lymphocytes polyclonal B helper
suppressor T
bull Unusual pattern of CD13CD16 staining noted
Case 6 flow cytometry Aplastic Anemia
Normal Patient
Normal
Patient
Patient
Normal
Case 6 RBC Test for PNH CD235a v s CD59
Case 6 Neutrophil test for PNH clone
FLAER CD24 CD45CD15
18 PNH clone
Case 6
bull Follow up
bull Patient on steroids acyclovir and anti-thymocyte
globulin
bull PNH clone Neutrophils 96 RBC 1 Nov 2015
bull In remission as of April 2017
- Lkc 5500uL (Neuts 23)
- Hb 129gL
- Plt 208 X10e9
Summary
bull B cell malignancies are the most common
hematological disorders seen in flow cytometry
bull Many have complex overlapping phenotypes
bull Important to look at ungated plots as well as gating on
specific subpopulations
bull Level of expression of markers is as important as
positive or negative (think CD13 vs CD16)
68
References
bull Craig FE Foon KA Flow cytometric immunophenotyping for hematologic neoplasms Blood
2008 Apr 15111(8)3941-67
bull Wood BL Principles of minimal residual disease detection for hematopoietic neoplasms by flow
cytometry Cytometry B Clin Cytom 2015 Apr 23
bull Shanafelt TD Ghia P Lanasa MC Landgren O Rawstron AC Monoclonal B-cell
lymphocytosis (MBL) biology natural history and clinical management Leukemia 2010
Mar24(3)512-20
bull Swerdlow SH Campo E Pileri SA Harris NL Stein H Siebert R Advani RGhielmini M Salles
GA Zelenetz AD Jaffe ES The 2016 revision of the World Health Organization classification of
lymphoid neoplasms Blood 2016 May 19127(20)2375-90
bull 3 Moench L Sachs Z Aasen G Dolan M Dayton V Courville EL Double- and triple-hit
lymphomas can present with features suggestive of immaturity including TdT expression and
create diagnostic challenges Leuk Lymphoma 2016572626-35
bull Sutherland DR Keeney M Illingworth A Practical guidelines for the high-sensitivity detection
and monitoring of paroxysmal nocturnal hemoglobinuria clones by flow cytometry Cytometry B
Clin Cytom 2012 Jul82(4)195-208
69
Normal B Cell Maturation
Wood and Borowitz (2006) Henryrsquos Laboratory Medicine
Normal B cell Maturation Pattern
The three pearls
Case 1
bull Nine month old male seen by GP for nodules in his
scalp suggestive of lymphoid infiltrate incidental
finding of bilateral enlarged kidneys
bull Referred to hematology as WBC 16300uL increased
Plt
bull WBC 12300uL Hb 111gL Plt 536x109L
bull Normal differential
bull Scalp lymphoid infiltrate suspicious for leukemia
bull Bone marrow aspirate flow ordered
Case 1
34
BM 14 B cells
5 CD10dimneg
CD20 negCD38 bright
plasma cells
Case 1 BM
Case 1 Bone marrow morphology
bull Cellular marrow
bull Erythropoeisis 20-25
bull Granulopoesis 40-50 orderly maturation
bull Lymphocytes 30 10- 15 ldquoblast likerdquo
bull Increased megakaryocytes
Case 1 2 week follow up
77 B cells ndash 90
CD10 dimneg
CD20 neg bright CD38
pB-ALL
Case 1 BM 2 week follow up
Case 1 Cytogenetics
bull Complex karyotype
bull Structural abnormalities in chromosome
123491417 and 19
bull t(1719) rare in B cell acute lymphoblastic
leukemia
Case 1 Follow up
bull On high risk protocol AALL 0232 for infantile B-ALL
bull MRD 008 at Day 42
bull Cord blood transplant
bull Relapse 5 months post transplant
Pearl of Pathology
bull Differentiating precursor B cells (hematogones) from B-
ALL can be challenging Standardized analysis and
clear understanding of ldquonormalrdquo essential including
arrested maturation
Case 2
bull 4 year old girl referred to pediatric hematology with
pancytopenia
bull WBC 6800uL(Neuts 400uL) Hb 42gL Plt 28
bull Aplastic anemia
bull Bone marrow ordered aspirate biopsy flow and
cytogenetics
Case 2 BM flow cytometry
Polyclonal B cells
helper suppressor T cells
No increase in blasts
Maturing myeloid population
Case 2 CD45 neg B-ALL
Case 2 BM
Case 2
bull CD45neg
bull CD19+CD10+
bull CD20(partialdim)
bull CD34 neg
bull SigampcIg neg
bull Cytogenetics
bull Gain of X chromosome and
468911131415171821
DNA Index
122 Diploid
Case 2
bull Currently in remission after completion of
AALL0932 standard risk B-ALL therapy
Case 2
Pearls of Pathology
CD45 negative B-ALL is not uncommon
bull Always look at ungated set of plots for each marker
bull Eg SS versus each fluorescence parameter
Case 3
bull 58 year old male with pararenal mass
bull WBC 4000uL Hb 98gL Plt 229
bull ldquoblastsrdquo reported on blood film
bull Peripheral blood sent for flow cytometry
Case 3 PB flow cytometry
Dim CD45 CD19+CD10+
CD34- Sig neg
Case 3 PB flow cytometry
Dim CD45 CD19+CD10+
CD20(partial)CD5 neg
SIg cIg neg
Case 3 BM flow cytometry
Case 3 BM biopsy
Case 3 BM biopsy
Case 3 BM biopsy report
bull Diffuse infiltrate large lymphoid cells
bull Scant to moderate cytoplasm
bull Large irregular nuclei with prominent nucleoli
bull Increased mitotic activity
bull Positive for BCL-2 BCL6 CD10 and MUM1
bull Negative for TdT CD5
bull Cytogenetics FISH on para renal mass ndash C-MYC and
BCL2 rearrangements detected
Case 3 Doubletriple hit lymphoma
(DHLTHL)
bull Morphologically heterogeneous B cell neoplasms
characterized by rearrangement of the MYC proto-
oncogene with concurrent BCL2 andor BCL6 gene
rearrangement
bull WHO 2008 criteria - most cases of DHL and THL
classified DLBCL or B-cell lymphoma unclassifiable
with features intermediate between DLBCL and Burkitt
lymphoma (BCLU)
Case 3 DHLTHL
bull Retrospective study from one institution features of 13 cases (9 DHL4
THL)
bull median age was 59 years (range 30-74) eight females five males
bull enlarging lymphadenopathymasses (11 patients) abnormal peripheral
blood findings (2 patients)
bull TdT positivity (four cases two THLtwo DHL) dim CD45 expression (7
cases) lack of CD20 (2 cases) or lack of sIg (3 cases) and blastoid
morphology (2 cases)
bull Conclusion Expression of TdT in a B-cell lymphoma with mature
features or expression of surface light chain in a case otherwise
suggestive of B-lymphoblastic leukemialymphoma should prompt
an expedited evaluation for DHLTHL
Leuk Lymphoma 2016 Feb 181-10 [Epub ahead of print]
Double- and triple-hit lymphomas can present with features suggestive
of immaturity including TdT expression and create diagnostic challenges
Moench L Sachs Z Aasen G Dolan M Dayton V Courville EL
Case 4
bull 55 year old female
bull Previous history hyperparathyroidism
bull Parathyroidectomy in 1999
bull Mild persistent hypercalcemia
bull WBC 4800uL hemoglobin 121 gL Plt 296
bull Total protein 90gL IgG 34gL monoclonal peak
209gL
Case 4 Monoclonal IgG patient
Polyclonal B cells
With maturing hematogones
helper suppressor T cells
less than 3 blasts
Maturing myeloid population
Case 4 BM Ungated
Case 4 cytoplasmic immunoglobulin
Need KL
Case 4 BM Multiple Myeloma
Case 4 follow up
bull Smouldering myeloma
bull Recently developed CRAB (hyperCalcemia Renal
insufficiency Anemia Bone lesions
bull Serum free light chain ratio 123
bull Bone marrow aspirate gt60 plasma cells
bull Currently on treatment pending bone marrow
transplant
Case 5
bull 65 year old male
bull Slight lymphocytosis detected on routine CBC analysis
(lymph 4500uL)
bull Asymptomatic sent for flow cytometry
Case 5 PB
Case 5 flow cytometry KL ratio 0625
Case 5 Monoclonal B cell lymphocytosis
KL 18 KL 056
Case 5 Monoclonal B cell lymphocytosis
bull WHO defines CLL as abnormal lymphocyte count
gt5x10E9L (CD19+CD5+ light chain restricted) in the
absence of other clinical symptoms
bull Patients with small B cell clones are often detected as
an incidental finding
bull WHO 2016
bull CD19+CD5+ lt05x10E9L ldquoLow count MBLrdquo
raquo 05-5x10E9L ldquoHigh count MBLrdquo
raquo Non CLL type phenotype MBL also recognized
Monoclonal B cell Lymphocytosis
bull CLL immunophenotype - vast majority (75)
bull Atypical MBL
bull normal levels both CD5 and CD20 with variable
expression of CD23 (always exclude Mantle-cell
Lymphoma in CD23 negative cases)
bull Non-CLL-phenotype (CD5minus) MBL
bull Lacking CD5 expression and CD10 likely resembling B-
cell lymphomas of marginal-zone origin
Case 5 What is the normal KL Ratio
bull What is the normal or abnormal kappalambda ratio for
entire ldquopopulationrdquo of all B cells
bull Traditional view
ndash Kappa Lambda 21 or 31 ( 12 -13)
ndash Range 14 60
Case 5 What is the normal KL ratio
bull Wrong question
bull It doesnrsquot matter how many of the B cells are clonal but rather
that their exists a clonal population of B cells within the total
population
bull Need to Identify malignant B cells within a population of
normal B cells
bull In order to interpret a pathological immunophenotype it is
necessary to refer to quantitative and qualitative values of
normal B-cell subpopulations
bull Pearl Identify the abnormal phenotype first then look at KL
ratio
Case 6
bull 12 year old male presents to ER with fatigue bruising
Lkc 2200uL
bull Hb 84gL
bull Plt 32
bull Diff ndash neutropenia no blasts noted few ldquoatypicalrdquo cells
bull RBC ndash non specific changes
Leukemia
Case 6 PB
Case 6 BM
Case 6
bull BM aspirate ndash Hypocellular relative lymphocytosis
marked reduction in myeloid lineages
hypoplasticaplastic anemia
bull Flow Cytometry ndash less than 3 blasts maturing
myeloid 68 lymphocytes polyclonal B helper
suppressor T
bull Unusual pattern of CD13CD16 staining noted
Case 6 flow cytometry Aplastic Anemia
Normal Patient
Normal
Patient
Patient
Normal
Case 6 RBC Test for PNH CD235a v s CD59
Case 6 Neutrophil test for PNH clone
FLAER CD24 CD45CD15
18 PNH clone
Case 6
bull Follow up
bull Patient on steroids acyclovir and anti-thymocyte
globulin
bull PNH clone Neutrophils 96 RBC 1 Nov 2015
bull In remission as of April 2017
- Lkc 5500uL (Neuts 23)
- Hb 129gL
- Plt 208 X10e9
Summary
bull B cell malignancies are the most common
hematological disorders seen in flow cytometry
bull Many have complex overlapping phenotypes
bull Important to look at ungated plots as well as gating on
specific subpopulations
bull Level of expression of markers is as important as
positive or negative (think CD13 vs CD16)
68
References
bull Craig FE Foon KA Flow cytometric immunophenotyping for hematologic neoplasms Blood
2008 Apr 15111(8)3941-67
bull Wood BL Principles of minimal residual disease detection for hematopoietic neoplasms by flow
cytometry Cytometry B Clin Cytom 2015 Apr 23
bull Shanafelt TD Ghia P Lanasa MC Landgren O Rawstron AC Monoclonal B-cell
lymphocytosis (MBL) biology natural history and clinical management Leukemia 2010
Mar24(3)512-20
bull Swerdlow SH Campo E Pileri SA Harris NL Stein H Siebert R Advani RGhielmini M Salles
GA Zelenetz AD Jaffe ES The 2016 revision of the World Health Organization classification of
lymphoid neoplasms Blood 2016 May 19127(20)2375-90
bull 3 Moench L Sachs Z Aasen G Dolan M Dayton V Courville EL Double- and triple-hit
lymphomas can present with features suggestive of immaturity including TdT expression and
create diagnostic challenges Leuk Lymphoma 2016572626-35
bull Sutherland DR Keeney M Illingworth A Practical guidelines for the high-sensitivity detection
and monitoring of paroxysmal nocturnal hemoglobinuria clones by flow cytometry Cytometry B
Clin Cytom 2012 Jul82(4)195-208
69
Normal B cell Maturation Pattern
The three pearls
Case 1
bull Nine month old male seen by GP for nodules in his
scalp suggestive of lymphoid infiltrate incidental
finding of bilateral enlarged kidneys
bull Referred to hematology as WBC 16300uL increased
Plt
bull WBC 12300uL Hb 111gL Plt 536x109L
bull Normal differential
bull Scalp lymphoid infiltrate suspicious for leukemia
bull Bone marrow aspirate flow ordered
Case 1
34
BM 14 B cells
5 CD10dimneg
CD20 negCD38 bright
plasma cells
Case 1 BM
Case 1 Bone marrow morphology
bull Cellular marrow
bull Erythropoeisis 20-25
bull Granulopoesis 40-50 orderly maturation
bull Lymphocytes 30 10- 15 ldquoblast likerdquo
bull Increased megakaryocytes
Case 1 2 week follow up
77 B cells ndash 90
CD10 dimneg
CD20 neg bright CD38
pB-ALL
Case 1 BM 2 week follow up
Case 1 Cytogenetics
bull Complex karyotype
bull Structural abnormalities in chromosome
123491417 and 19
bull t(1719) rare in B cell acute lymphoblastic
leukemia
Case 1 Follow up
bull On high risk protocol AALL 0232 for infantile B-ALL
bull MRD 008 at Day 42
bull Cord blood transplant
bull Relapse 5 months post transplant
Pearl of Pathology
bull Differentiating precursor B cells (hematogones) from B-
ALL can be challenging Standardized analysis and
clear understanding of ldquonormalrdquo essential including
arrested maturation
Case 2
bull 4 year old girl referred to pediatric hematology with
pancytopenia
bull WBC 6800uL(Neuts 400uL) Hb 42gL Plt 28
bull Aplastic anemia
bull Bone marrow ordered aspirate biopsy flow and
cytogenetics
Case 2 BM flow cytometry
Polyclonal B cells
helper suppressor T cells
No increase in blasts
Maturing myeloid population
Case 2 CD45 neg B-ALL
Case 2 BM
Case 2
bull CD45neg
bull CD19+CD10+
bull CD20(partialdim)
bull CD34 neg
bull SigampcIg neg
bull Cytogenetics
bull Gain of X chromosome and
468911131415171821
DNA Index
122 Diploid
Case 2
bull Currently in remission after completion of
AALL0932 standard risk B-ALL therapy
Case 2
Pearls of Pathology
CD45 negative B-ALL is not uncommon
bull Always look at ungated set of plots for each marker
bull Eg SS versus each fluorescence parameter
Case 3
bull 58 year old male with pararenal mass
bull WBC 4000uL Hb 98gL Plt 229
bull ldquoblastsrdquo reported on blood film
bull Peripheral blood sent for flow cytometry
Case 3 PB flow cytometry
Dim CD45 CD19+CD10+
CD34- Sig neg
Case 3 PB flow cytometry
Dim CD45 CD19+CD10+
CD20(partial)CD5 neg
SIg cIg neg
Case 3 BM flow cytometry
Case 3 BM biopsy
Case 3 BM biopsy
Case 3 BM biopsy report
bull Diffuse infiltrate large lymphoid cells
bull Scant to moderate cytoplasm
bull Large irregular nuclei with prominent nucleoli
bull Increased mitotic activity
bull Positive for BCL-2 BCL6 CD10 and MUM1
bull Negative for TdT CD5
bull Cytogenetics FISH on para renal mass ndash C-MYC and
BCL2 rearrangements detected
Case 3 Doubletriple hit lymphoma
(DHLTHL)
bull Morphologically heterogeneous B cell neoplasms
characterized by rearrangement of the MYC proto-
oncogene with concurrent BCL2 andor BCL6 gene
rearrangement
bull WHO 2008 criteria - most cases of DHL and THL
classified DLBCL or B-cell lymphoma unclassifiable
with features intermediate between DLBCL and Burkitt
lymphoma (BCLU)
Case 3 DHLTHL
bull Retrospective study from one institution features of 13 cases (9 DHL4
THL)
bull median age was 59 years (range 30-74) eight females five males
bull enlarging lymphadenopathymasses (11 patients) abnormal peripheral
blood findings (2 patients)
bull TdT positivity (four cases two THLtwo DHL) dim CD45 expression (7
cases) lack of CD20 (2 cases) or lack of sIg (3 cases) and blastoid
morphology (2 cases)
bull Conclusion Expression of TdT in a B-cell lymphoma with mature
features or expression of surface light chain in a case otherwise
suggestive of B-lymphoblastic leukemialymphoma should prompt
an expedited evaluation for DHLTHL
Leuk Lymphoma 2016 Feb 181-10 [Epub ahead of print]
Double- and triple-hit lymphomas can present with features suggestive
of immaturity including TdT expression and create diagnostic challenges
Moench L Sachs Z Aasen G Dolan M Dayton V Courville EL
Case 4
bull 55 year old female
bull Previous history hyperparathyroidism
bull Parathyroidectomy in 1999
bull Mild persistent hypercalcemia
bull WBC 4800uL hemoglobin 121 gL Plt 296
bull Total protein 90gL IgG 34gL monoclonal peak
209gL
Case 4 Monoclonal IgG patient
Polyclonal B cells
With maturing hematogones
helper suppressor T cells
less than 3 blasts
Maturing myeloid population
Case 4 BM Ungated
Case 4 cytoplasmic immunoglobulin
Need KL
Case 4 BM Multiple Myeloma
Case 4 follow up
bull Smouldering myeloma
bull Recently developed CRAB (hyperCalcemia Renal
insufficiency Anemia Bone lesions
bull Serum free light chain ratio 123
bull Bone marrow aspirate gt60 plasma cells
bull Currently on treatment pending bone marrow
transplant
Case 5
bull 65 year old male
bull Slight lymphocytosis detected on routine CBC analysis
(lymph 4500uL)
bull Asymptomatic sent for flow cytometry
Case 5 PB
Case 5 flow cytometry KL ratio 0625
Case 5 Monoclonal B cell lymphocytosis
KL 18 KL 056
Case 5 Monoclonal B cell lymphocytosis
bull WHO defines CLL as abnormal lymphocyte count
gt5x10E9L (CD19+CD5+ light chain restricted) in the
absence of other clinical symptoms
bull Patients with small B cell clones are often detected as
an incidental finding
bull WHO 2016
bull CD19+CD5+ lt05x10E9L ldquoLow count MBLrdquo
raquo 05-5x10E9L ldquoHigh count MBLrdquo
raquo Non CLL type phenotype MBL also recognized
Monoclonal B cell Lymphocytosis
bull CLL immunophenotype - vast majority (75)
bull Atypical MBL
bull normal levels both CD5 and CD20 with variable
expression of CD23 (always exclude Mantle-cell
Lymphoma in CD23 negative cases)
bull Non-CLL-phenotype (CD5minus) MBL
bull Lacking CD5 expression and CD10 likely resembling B-
cell lymphomas of marginal-zone origin
Case 5 What is the normal KL Ratio
bull What is the normal or abnormal kappalambda ratio for
entire ldquopopulationrdquo of all B cells
bull Traditional view
ndash Kappa Lambda 21 or 31 ( 12 -13)
ndash Range 14 60
Case 5 What is the normal KL ratio
bull Wrong question
bull It doesnrsquot matter how many of the B cells are clonal but rather
that their exists a clonal population of B cells within the total
population
bull Need to Identify malignant B cells within a population of
normal B cells
bull In order to interpret a pathological immunophenotype it is
necessary to refer to quantitative and qualitative values of
normal B-cell subpopulations
bull Pearl Identify the abnormal phenotype first then look at KL
ratio
Case 6
bull 12 year old male presents to ER with fatigue bruising
Lkc 2200uL
bull Hb 84gL
bull Plt 32
bull Diff ndash neutropenia no blasts noted few ldquoatypicalrdquo cells
bull RBC ndash non specific changes
Leukemia
Case 6 PB
Case 6 BM
Case 6
bull BM aspirate ndash Hypocellular relative lymphocytosis
marked reduction in myeloid lineages
hypoplasticaplastic anemia
bull Flow Cytometry ndash less than 3 blasts maturing
myeloid 68 lymphocytes polyclonal B helper
suppressor T
bull Unusual pattern of CD13CD16 staining noted
Case 6 flow cytometry Aplastic Anemia
Normal Patient
Normal
Patient
Patient
Normal
Case 6 RBC Test for PNH CD235a v s CD59
Case 6 Neutrophil test for PNH clone
FLAER CD24 CD45CD15
18 PNH clone
Case 6
bull Follow up
bull Patient on steroids acyclovir and anti-thymocyte
globulin
bull PNH clone Neutrophils 96 RBC 1 Nov 2015
bull In remission as of April 2017
- Lkc 5500uL (Neuts 23)
- Hb 129gL
- Plt 208 X10e9
Summary
bull B cell malignancies are the most common
hematological disorders seen in flow cytometry
bull Many have complex overlapping phenotypes
bull Important to look at ungated plots as well as gating on
specific subpopulations
bull Level of expression of markers is as important as
positive or negative (think CD13 vs CD16)
68
References
bull Craig FE Foon KA Flow cytometric immunophenotyping for hematologic neoplasms Blood
2008 Apr 15111(8)3941-67
bull Wood BL Principles of minimal residual disease detection for hematopoietic neoplasms by flow
cytometry Cytometry B Clin Cytom 2015 Apr 23
bull Shanafelt TD Ghia P Lanasa MC Landgren O Rawstron AC Monoclonal B-cell
lymphocytosis (MBL) biology natural history and clinical management Leukemia 2010
Mar24(3)512-20
bull Swerdlow SH Campo E Pileri SA Harris NL Stein H Siebert R Advani RGhielmini M Salles
GA Zelenetz AD Jaffe ES The 2016 revision of the World Health Organization classification of
lymphoid neoplasms Blood 2016 May 19127(20)2375-90
bull 3 Moench L Sachs Z Aasen G Dolan M Dayton V Courville EL Double- and triple-hit
lymphomas can present with features suggestive of immaturity including TdT expression and
create diagnostic challenges Leuk Lymphoma 2016572626-35
bull Sutherland DR Keeney M Illingworth A Practical guidelines for the high-sensitivity detection
and monitoring of paroxysmal nocturnal hemoglobinuria clones by flow cytometry Cytometry B
Clin Cytom 2012 Jul82(4)195-208
69
The three pearls
Case 1
bull Nine month old male seen by GP for nodules in his
scalp suggestive of lymphoid infiltrate incidental
finding of bilateral enlarged kidneys
bull Referred to hematology as WBC 16300uL increased
Plt
bull WBC 12300uL Hb 111gL Plt 536x109L
bull Normal differential
bull Scalp lymphoid infiltrate suspicious for leukemia
bull Bone marrow aspirate flow ordered
Case 1
34
BM 14 B cells
5 CD10dimneg
CD20 negCD38 bright
plasma cells
Case 1 BM
Case 1 Bone marrow morphology
bull Cellular marrow
bull Erythropoeisis 20-25
bull Granulopoesis 40-50 orderly maturation
bull Lymphocytes 30 10- 15 ldquoblast likerdquo
bull Increased megakaryocytes
Case 1 2 week follow up
77 B cells ndash 90
CD10 dimneg
CD20 neg bright CD38
pB-ALL
Case 1 BM 2 week follow up
Case 1 Cytogenetics
bull Complex karyotype
bull Structural abnormalities in chromosome
123491417 and 19
bull t(1719) rare in B cell acute lymphoblastic
leukemia
Case 1 Follow up
bull On high risk protocol AALL 0232 for infantile B-ALL
bull MRD 008 at Day 42
bull Cord blood transplant
bull Relapse 5 months post transplant
Pearl of Pathology
bull Differentiating precursor B cells (hematogones) from B-
ALL can be challenging Standardized analysis and
clear understanding of ldquonormalrdquo essential including
arrested maturation
Case 2
bull 4 year old girl referred to pediatric hematology with
pancytopenia
bull WBC 6800uL(Neuts 400uL) Hb 42gL Plt 28
bull Aplastic anemia
bull Bone marrow ordered aspirate biopsy flow and
cytogenetics
Case 2 BM flow cytometry
Polyclonal B cells
helper suppressor T cells
No increase in blasts
Maturing myeloid population
Case 2 CD45 neg B-ALL
Case 2 BM
Case 2
bull CD45neg
bull CD19+CD10+
bull CD20(partialdim)
bull CD34 neg
bull SigampcIg neg
bull Cytogenetics
bull Gain of X chromosome and
468911131415171821
DNA Index
122 Diploid
Case 2
bull Currently in remission after completion of
AALL0932 standard risk B-ALL therapy
Case 2
Pearls of Pathology
CD45 negative B-ALL is not uncommon
bull Always look at ungated set of plots for each marker
bull Eg SS versus each fluorescence parameter
Case 3
bull 58 year old male with pararenal mass
bull WBC 4000uL Hb 98gL Plt 229
bull ldquoblastsrdquo reported on blood film
bull Peripheral blood sent for flow cytometry
Case 3 PB flow cytometry
Dim CD45 CD19+CD10+
CD34- Sig neg
Case 3 PB flow cytometry
Dim CD45 CD19+CD10+
CD20(partial)CD5 neg
SIg cIg neg
Case 3 BM flow cytometry
Case 3 BM biopsy
Case 3 BM biopsy
Case 3 BM biopsy report
bull Diffuse infiltrate large lymphoid cells
bull Scant to moderate cytoplasm
bull Large irregular nuclei with prominent nucleoli
bull Increased mitotic activity
bull Positive for BCL-2 BCL6 CD10 and MUM1
bull Negative for TdT CD5
bull Cytogenetics FISH on para renal mass ndash C-MYC and
BCL2 rearrangements detected
Case 3 Doubletriple hit lymphoma
(DHLTHL)
bull Morphologically heterogeneous B cell neoplasms
characterized by rearrangement of the MYC proto-
oncogene with concurrent BCL2 andor BCL6 gene
rearrangement
bull WHO 2008 criteria - most cases of DHL and THL
classified DLBCL or B-cell lymphoma unclassifiable
with features intermediate between DLBCL and Burkitt
lymphoma (BCLU)
Case 3 DHLTHL
bull Retrospective study from one institution features of 13 cases (9 DHL4
THL)
bull median age was 59 years (range 30-74) eight females five males
bull enlarging lymphadenopathymasses (11 patients) abnormal peripheral
blood findings (2 patients)
bull TdT positivity (four cases two THLtwo DHL) dim CD45 expression (7
cases) lack of CD20 (2 cases) or lack of sIg (3 cases) and blastoid
morphology (2 cases)
bull Conclusion Expression of TdT in a B-cell lymphoma with mature
features or expression of surface light chain in a case otherwise
suggestive of B-lymphoblastic leukemialymphoma should prompt
an expedited evaluation for DHLTHL
Leuk Lymphoma 2016 Feb 181-10 [Epub ahead of print]
Double- and triple-hit lymphomas can present with features suggestive
of immaturity including TdT expression and create diagnostic challenges
Moench L Sachs Z Aasen G Dolan M Dayton V Courville EL
Case 4
bull 55 year old female
bull Previous history hyperparathyroidism
bull Parathyroidectomy in 1999
bull Mild persistent hypercalcemia
bull WBC 4800uL hemoglobin 121 gL Plt 296
bull Total protein 90gL IgG 34gL monoclonal peak
209gL
Case 4 Monoclonal IgG patient
Polyclonal B cells
With maturing hematogones
helper suppressor T cells
less than 3 blasts
Maturing myeloid population
Case 4 BM Ungated
Case 4 cytoplasmic immunoglobulin
Need KL
Case 4 BM Multiple Myeloma
Case 4 follow up
bull Smouldering myeloma
bull Recently developed CRAB (hyperCalcemia Renal
insufficiency Anemia Bone lesions
bull Serum free light chain ratio 123
bull Bone marrow aspirate gt60 plasma cells
bull Currently on treatment pending bone marrow
transplant
Case 5
bull 65 year old male
bull Slight lymphocytosis detected on routine CBC analysis
(lymph 4500uL)
bull Asymptomatic sent for flow cytometry
Case 5 PB
Case 5 flow cytometry KL ratio 0625
Case 5 Monoclonal B cell lymphocytosis
KL 18 KL 056
Case 5 Monoclonal B cell lymphocytosis
bull WHO defines CLL as abnormal lymphocyte count
gt5x10E9L (CD19+CD5+ light chain restricted) in the
absence of other clinical symptoms
bull Patients with small B cell clones are often detected as
an incidental finding
bull WHO 2016
bull CD19+CD5+ lt05x10E9L ldquoLow count MBLrdquo
raquo 05-5x10E9L ldquoHigh count MBLrdquo
raquo Non CLL type phenotype MBL also recognized
Monoclonal B cell Lymphocytosis
bull CLL immunophenotype - vast majority (75)
bull Atypical MBL
bull normal levels both CD5 and CD20 with variable
expression of CD23 (always exclude Mantle-cell
Lymphoma in CD23 negative cases)
bull Non-CLL-phenotype (CD5minus) MBL
bull Lacking CD5 expression and CD10 likely resembling B-
cell lymphomas of marginal-zone origin
Case 5 What is the normal KL Ratio
bull What is the normal or abnormal kappalambda ratio for
entire ldquopopulationrdquo of all B cells
bull Traditional view
ndash Kappa Lambda 21 or 31 ( 12 -13)
ndash Range 14 60
Case 5 What is the normal KL ratio
bull Wrong question
bull It doesnrsquot matter how many of the B cells are clonal but rather
that their exists a clonal population of B cells within the total
population
bull Need to Identify malignant B cells within a population of
normal B cells
bull In order to interpret a pathological immunophenotype it is
necessary to refer to quantitative and qualitative values of
normal B-cell subpopulations
bull Pearl Identify the abnormal phenotype first then look at KL
ratio
Case 6
bull 12 year old male presents to ER with fatigue bruising
Lkc 2200uL
bull Hb 84gL
bull Plt 32
bull Diff ndash neutropenia no blasts noted few ldquoatypicalrdquo cells
bull RBC ndash non specific changes
Leukemia
Case 6 PB
Case 6 BM
Case 6
bull BM aspirate ndash Hypocellular relative lymphocytosis
marked reduction in myeloid lineages
hypoplasticaplastic anemia
bull Flow Cytometry ndash less than 3 blasts maturing
myeloid 68 lymphocytes polyclonal B helper
suppressor T
bull Unusual pattern of CD13CD16 staining noted
Case 6 flow cytometry Aplastic Anemia
Normal Patient
Normal
Patient
Patient
Normal
Case 6 RBC Test for PNH CD235a v s CD59
Case 6 Neutrophil test for PNH clone
FLAER CD24 CD45CD15
18 PNH clone
Case 6
bull Follow up
bull Patient on steroids acyclovir and anti-thymocyte
globulin
bull PNH clone Neutrophils 96 RBC 1 Nov 2015
bull In remission as of April 2017
- Lkc 5500uL (Neuts 23)
- Hb 129gL
- Plt 208 X10e9
Summary
bull B cell malignancies are the most common
hematological disorders seen in flow cytometry
bull Many have complex overlapping phenotypes
bull Important to look at ungated plots as well as gating on
specific subpopulations
bull Level of expression of markers is as important as
positive or negative (think CD13 vs CD16)
68
References
bull Craig FE Foon KA Flow cytometric immunophenotyping for hematologic neoplasms Blood
2008 Apr 15111(8)3941-67
bull Wood BL Principles of minimal residual disease detection for hematopoietic neoplasms by flow
cytometry Cytometry B Clin Cytom 2015 Apr 23
bull Shanafelt TD Ghia P Lanasa MC Landgren O Rawstron AC Monoclonal B-cell
lymphocytosis (MBL) biology natural history and clinical management Leukemia 2010
Mar24(3)512-20
bull Swerdlow SH Campo E Pileri SA Harris NL Stein H Siebert R Advani RGhielmini M Salles
GA Zelenetz AD Jaffe ES The 2016 revision of the World Health Organization classification of
lymphoid neoplasms Blood 2016 May 19127(20)2375-90
bull 3 Moench L Sachs Z Aasen G Dolan M Dayton V Courville EL Double- and triple-hit
lymphomas can present with features suggestive of immaturity including TdT expression and
create diagnostic challenges Leuk Lymphoma 2016572626-35
bull Sutherland DR Keeney M Illingworth A Practical guidelines for the high-sensitivity detection
and monitoring of paroxysmal nocturnal hemoglobinuria clones by flow cytometry Cytometry B
Clin Cytom 2012 Jul82(4)195-208
69
Case 1
bull Nine month old male seen by GP for nodules in his
scalp suggestive of lymphoid infiltrate incidental
finding of bilateral enlarged kidneys
bull Referred to hematology as WBC 16300uL increased
Plt
bull WBC 12300uL Hb 111gL Plt 536x109L
bull Normal differential
bull Scalp lymphoid infiltrate suspicious for leukemia
bull Bone marrow aspirate flow ordered
Case 1
34
BM 14 B cells
5 CD10dimneg
CD20 negCD38 bright
plasma cells
Case 1 BM
Case 1 Bone marrow morphology
bull Cellular marrow
bull Erythropoeisis 20-25
bull Granulopoesis 40-50 orderly maturation
bull Lymphocytes 30 10- 15 ldquoblast likerdquo
bull Increased megakaryocytes
Case 1 2 week follow up
77 B cells ndash 90
CD10 dimneg
CD20 neg bright CD38
pB-ALL
Case 1 BM 2 week follow up
Case 1 Cytogenetics
bull Complex karyotype
bull Structural abnormalities in chromosome
123491417 and 19
bull t(1719) rare in B cell acute lymphoblastic
leukemia
Case 1 Follow up
bull On high risk protocol AALL 0232 for infantile B-ALL
bull MRD 008 at Day 42
bull Cord blood transplant
bull Relapse 5 months post transplant
Pearl of Pathology
bull Differentiating precursor B cells (hematogones) from B-
ALL can be challenging Standardized analysis and
clear understanding of ldquonormalrdquo essential including
arrested maturation
Case 2
bull 4 year old girl referred to pediatric hematology with
pancytopenia
bull WBC 6800uL(Neuts 400uL) Hb 42gL Plt 28
bull Aplastic anemia
bull Bone marrow ordered aspirate biopsy flow and
cytogenetics
Case 2 BM flow cytometry
Polyclonal B cells
helper suppressor T cells
No increase in blasts
Maturing myeloid population
Case 2 CD45 neg B-ALL
Case 2 BM
Case 2
bull CD45neg
bull CD19+CD10+
bull CD20(partialdim)
bull CD34 neg
bull SigampcIg neg
bull Cytogenetics
bull Gain of X chromosome and
468911131415171821
DNA Index
122 Diploid
Case 2
bull Currently in remission after completion of
AALL0932 standard risk B-ALL therapy
Case 2
Pearls of Pathology
CD45 negative B-ALL is not uncommon
bull Always look at ungated set of plots for each marker
bull Eg SS versus each fluorescence parameter
Case 3
bull 58 year old male with pararenal mass
bull WBC 4000uL Hb 98gL Plt 229
bull ldquoblastsrdquo reported on blood film
bull Peripheral blood sent for flow cytometry
Case 3 PB flow cytometry
Dim CD45 CD19+CD10+
CD34- Sig neg
Case 3 PB flow cytometry
Dim CD45 CD19+CD10+
CD20(partial)CD5 neg
SIg cIg neg
Case 3 BM flow cytometry
Case 3 BM biopsy
Case 3 BM biopsy
Case 3 BM biopsy report
bull Diffuse infiltrate large lymphoid cells
bull Scant to moderate cytoplasm
bull Large irregular nuclei with prominent nucleoli
bull Increased mitotic activity
bull Positive for BCL-2 BCL6 CD10 and MUM1
bull Negative for TdT CD5
bull Cytogenetics FISH on para renal mass ndash C-MYC and
BCL2 rearrangements detected
Case 3 Doubletriple hit lymphoma
(DHLTHL)
bull Morphologically heterogeneous B cell neoplasms
characterized by rearrangement of the MYC proto-
oncogene with concurrent BCL2 andor BCL6 gene
rearrangement
bull WHO 2008 criteria - most cases of DHL and THL
classified DLBCL or B-cell lymphoma unclassifiable
with features intermediate between DLBCL and Burkitt
lymphoma (BCLU)
Case 3 DHLTHL
bull Retrospective study from one institution features of 13 cases (9 DHL4
THL)
bull median age was 59 years (range 30-74) eight females five males
bull enlarging lymphadenopathymasses (11 patients) abnormal peripheral
blood findings (2 patients)
bull TdT positivity (four cases two THLtwo DHL) dim CD45 expression (7
cases) lack of CD20 (2 cases) or lack of sIg (3 cases) and blastoid
morphology (2 cases)
bull Conclusion Expression of TdT in a B-cell lymphoma with mature
features or expression of surface light chain in a case otherwise
suggestive of B-lymphoblastic leukemialymphoma should prompt
an expedited evaluation for DHLTHL
Leuk Lymphoma 2016 Feb 181-10 [Epub ahead of print]
Double- and triple-hit lymphomas can present with features suggestive
of immaturity including TdT expression and create diagnostic challenges
Moench L Sachs Z Aasen G Dolan M Dayton V Courville EL
Case 4
bull 55 year old female
bull Previous history hyperparathyroidism
bull Parathyroidectomy in 1999
bull Mild persistent hypercalcemia
bull WBC 4800uL hemoglobin 121 gL Plt 296
bull Total protein 90gL IgG 34gL monoclonal peak
209gL
Case 4 Monoclonal IgG patient
Polyclonal B cells
With maturing hematogones
helper suppressor T cells
less than 3 blasts
Maturing myeloid population
Case 4 BM Ungated
Case 4 cytoplasmic immunoglobulin
Need KL
Case 4 BM Multiple Myeloma
Case 4 follow up
bull Smouldering myeloma
bull Recently developed CRAB (hyperCalcemia Renal
insufficiency Anemia Bone lesions
bull Serum free light chain ratio 123
bull Bone marrow aspirate gt60 plasma cells
bull Currently on treatment pending bone marrow
transplant
Case 5
bull 65 year old male
bull Slight lymphocytosis detected on routine CBC analysis
(lymph 4500uL)
bull Asymptomatic sent for flow cytometry
Case 5 PB
Case 5 flow cytometry KL ratio 0625
Case 5 Monoclonal B cell lymphocytosis
KL 18 KL 056
Case 5 Monoclonal B cell lymphocytosis
bull WHO defines CLL as abnormal lymphocyte count
gt5x10E9L (CD19+CD5+ light chain restricted) in the
absence of other clinical symptoms
bull Patients with small B cell clones are often detected as
an incidental finding
bull WHO 2016
bull CD19+CD5+ lt05x10E9L ldquoLow count MBLrdquo
raquo 05-5x10E9L ldquoHigh count MBLrdquo
raquo Non CLL type phenotype MBL also recognized
Monoclonal B cell Lymphocytosis
bull CLL immunophenotype - vast majority (75)
bull Atypical MBL
bull normal levels both CD5 and CD20 with variable
expression of CD23 (always exclude Mantle-cell
Lymphoma in CD23 negative cases)
bull Non-CLL-phenotype (CD5minus) MBL
bull Lacking CD5 expression and CD10 likely resembling B-
cell lymphomas of marginal-zone origin
Case 5 What is the normal KL Ratio
bull What is the normal or abnormal kappalambda ratio for
entire ldquopopulationrdquo of all B cells
bull Traditional view
ndash Kappa Lambda 21 or 31 ( 12 -13)
ndash Range 14 60
Case 5 What is the normal KL ratio
bull Wrong question
bull It doesnrsquot matter how many of the B cells are clonal but rather
that their exists a clonal population of B cells within the total
population
bull Need to Identify malignant B cells within a population of
normal B cells
bull In order to interpret a pathological immunophenotype it is
necessary to refer to quantitative and qualitative values of
normal B-cell subpopulations
bull Pearl Identify the abnormal phenotype first then look at KL
ratio
Case 6
bull 12 year old male presents to ER with fatigue bruising
Lkc 2200uL
bull Hb 84gL
bull Plt 32
bull Diff ndash neutropenia no blasts noted few ldquoatypicalrdquo cells
bull RBC ndash non specific changes
Leukemia
Case 6 PB
Case 6 BM
Case 6
bull BM aspirate ndash Hypocellular relative lymphocytosis
marked reduction in myeloid lineages
hypoplasticaplastic anemia
bull Flow Cytometry ndash less than 3 blasts maturing
myeloid 68 lymphocytes polyclonal B helper
suppressor T
bull Unusual pattern of CD13CD16 staining noted
Case 6 flow cytometry Aplastic Anemia
Normal Patient
Normal
Patient
Patient
Normal
Case 6 RBC Test for PNH CD235a v s CD59
Case 6 Neutrophil test for PNH clone
FLAER CD24 CD45CD15
18 PNH clone
Case 6
bull Follow up
bull Patient on steroids acyclovir and anti-thymocyte
globulin
bull PNH clone Neutrophils 96 RBC 1 Nov 2015
bull In remission as of April 2017
- Lkc 5500uL (Neuts 23)
- Hb 129gL
- Plt 208 X10e9
Summary
bull B cell malignancies are the most common
hematological disorders seen in flow cytometry
bull Many have complex overlapping phenotypes
bull Important to look at ungated plots as well as gating on
specific subpopulations
bull Level of expression of markers is as important as
positive or negative (think CD13 vs CD16)
68
References
bull Craig FE Foon KA Flow cytometric immunophenotyping for hematologic neoplasms Blood
2008 Apr 15111(8)3941-67
bull Wood BL Principles of minimal residual disease detection for hematopoietic neoplasms by flow
cytometry Cytometry B Clin Cytom 2015 Apr 23
bull Shanafelt TD Ghia P Lanasa MC Landgren O Rawstron AC Monoclonal B-cell
lymphocytosis (MBL) biology natural history and clinical management Leukemia 2010
Mar24(3)512-20
bull Swerdlow SH Campo E Pileri SA Harris NL Stein H Siebert R Advani RGhielmini M Salles
GA Zelenetz AD Jaffe ES The 2016 revision of the World Health Organization classification of
lymphoid neoplasms Blood 2016 May 19127(20)2375-90
bull 3 Moench L Sachs Z Aasen G Dolan M Dayton V Courville EL Double- and triple-hit
lymphomas can present with features suggestive of immaturity including TdT expression and
create diagnostic challenges Leuk Lymphoma 2016572626-35
bull Sutherland DR Keeney M Illingworth A Practical guidelines for the high-sensitivity detection
and monitoring of paroxysmal nocturnal hemoglobinuria clones by flow cytometry Cytometry B
Clin Cytom 2012 Jul82(4)195-208
69
Case 1
34
BM 14 B cells
5 CD10dimneg
CD20 negCD38 bright
plasma cells
Case 1 BM
Case 1 Bone marrow morphology
bull Cellular marrow
bull Erythropoeisis 20-25
bull Granulopoesis 40-50 orderly maturation
bull Lymphocytes 30 10- 15 ldquoblast likerdquo
bull Increased megakaryocytes
Case 1 2 week follow up
77 B cells ndash 90
CD10 dimneg
CD20 neg bright CD38
pB-ALL
Case 1 BM 2 week follow up
Case 1 Cytogenetics
bull Complex karyotype
bull Structural abnormalities in chromosome
123491417 and 19
bull t(1719) rare in B cell acute lymphoblastic
leukemia
Case 1 Follow up
bull On high risk protocol AALL 0232 for infantile B-ALL
bull MRD 008 at Day 42
bull Cord blood transplant
bull Relapse 5 months post transplant
Pearl of Pathology
bull Differentiating precursor B cells (hematogones) from B-
ALL can be challenging Standardized analysis and
clear understanding of ldquonormalrdquo essential including
arrested maturation
Case 2
bull 4 year old girl referred to pediatric hematology with
pancytopenia
bull WBC 6800uL(Neuts 400uL) Hb 42gL Plt 28
bull Aplastic anemia
bull Bone marrow ordered aspirate biopsy flow and
cytogenetics
Case 2 BM flow cytometry
Polyclonal B cells
helper suppressor T cells
No increase in blasts
Maturing myeloid population
Case 2 CD45 neg B-ALL
Case 2 BM
Case 2
bull CD45neg
bull CD19+CD10+
bull CD20(partialdim)
bull CD34 neg
bull SigampcIg neg
bull Cytogenetics
bull Gain of X chromosome and
468911131415171821
DNA Index
122 Diploid
Case 2
bull Currently in remission after completion of
AALL0932 standard risk B-ALL therapy
Case 2
Pearls of Pathology
CD45 negative B-ALL is not uncommon
bull Always look at ungated set of plots for each marker
bull Eg SS versus each fluorescence parameter
Case 3
bull 58 year old male with pararenal mass
bull WBC 4000uL Hb 98gL Plt 229
bull ldquoblastsrdquo reported on blood film
bull Peripheral blood sent for flow cytometry
Case 3 PB flow cytometry
Dim CD45 CD19+CD10+
CD34- Sig neg
Case 3 PB flow cytometry
Dim CD45 CD19+CD10+
CD20(partial)CD5 neg
SIg cIg neg
Case 3 BM flow cytometry
Case 3 BM biopsy
Case 3 BM biopsy
Case 3 BM biopsy report
bull Diffuse infiltrate large lymphoid cells
bull Scant to moderate cytoplasm
bull Large irregular nuclei with prominent nucleoli
bull Increased mitotic activity
bull Positive for BCL-2 BCL6 CD10 and MUM1
bull Negative for TdT CD5
bull Cytogenetics FISH on para renal mass ndash C-MYC and
BCL2 rearrangements detected
Case 3 Doubletriple hit lymphoma
(DHLTHL)
bull Morphologically heterogeneous B cell neoplasms
characterized by rearrangement of the MYC proto-
oncogene with concurrent BCL2 andor BCL6 gene
rearrangement
bull WHO 2008 criteria - most cases of DHL and THL
classified DLBCL or B-cell lymphoma unclassifiable
with features intermediate between DLBCL and Burkitt
lymphoma (BCLU)
Case 3 DHLTHL
bull Retrospective study from one institution features of 13 cases (9 DHL4
THL)
bull median age was 59 years (range 30-74) eight females five males
bull enlarging lymphadenopathymasses (11 patients) abnormal peripheral
blood findings (2 patients)
bull TdT positivity (four cases two THLtwo DHL) dim CD45 expression (7
cases) lack of CD20 (2 cases) or lack of sIg (3 cases) and blastoid
morphology (2 cases)
bull Conclusion Expression of TdT in a B-cell lymphoma with mature
features or expression of surface light chain in a case otherwise
suggestive of B-lymphoblastic leukemialymphoma should prompt
an expedited evaluation for DHLTHL
Leuk Lymphoma 2016 Feb 181-10 [Epub ahead of print]
Double- and triple-hit lymphomas can present with features suggestive
of immaturity including TdT expression and create diagnostic challenges
Moench L Sachs Z Aasen G Dolan M Dayton V Courville EL
Case 4
bull 55 year old female
bull Previous history hyperparathyroidism
bull Parathyroidectomy in 1999
bull Mild persistent hypercalcemia
bull WBC 4800uL hemoglobin 121 gL Plt 296
bull Total protein 90gL IgG 34gL monoclonal peak
209gL
Case 4 Monoclonal IgG patient
Polyclonal B cells
With maturing hematogones
helper suppressor T cells
less than 3 blasts
Maturing myeloid population
Case 4 BM Ungated
Case 4 cytoplasmic immunoglobulin
Need KL
Case 4 BM Multiple Myeloma
Case 4 follow up
bull Smouldering myeloma
bull Recently developed CRAB (hyperCalcemia Renal
insufficiency Anemia Bone lesions
bull Serum free light chain ratio 123
bull Bone marrow aspirate gt60 plasma cells
bull Currently on treatment pending bone marrow
transplant
Case 5
bull 65 year old male
bull Slight lymphocytosis detected on routine CBC analysis
(lymph 4500uL)
bull Asymptomatic sent for flow cytometry
Case 5 PB
Case 5 flow cytometry KL ratio 0625
Case 5 Monoclonal B cell lymphocytosis
KL 18 KL 056
Case 5 Monoclonal B cell lymphocytosis
bull WHO defines CLL as abnormal lymphocyte count
gt5x10E9L (CD19+CD5+ light chain restricted) in the
absence of other clinical symptoms
bull Patients with small B cell clones are often detected as
an incidental finding
bull WHO 2016
bull CD19+CD5+ lt05x10E9L ldquoLow count MBLrdquo
raquo 05-5x10E9L ldquoHigh count MBLrdquo
raquo Non CLL type phenotype MBL also recognized
Monoclonal B cell Lymphocytosis
bull CLL immunophenotype - vast majority (75)
bull Atypical MBL
bull normal levels both CD5 and CD20 with variable
expression of CD23 (always exclude Mantle-cell
Lymphoma in CD23 negative cases)
bull Non-CLL-phenotype (CD5minus) MBL
bull Lacking CD5 expression and CD10 likely resembling B-
cell lymphomas of marginal-zone origin
Case 5 What is the normal KL Ratio
bull What is the normal or abnormal kappalambda ratio for
entire ldquopopulationrdquo of all B cells
bull Traditional view
ndash Kappa Lambda 21 or 31 ( 12 -13)
ndash Range 14 60
Case 5 What is the normal KL ratio
bull Wrong question
bull It doesnrsquot matter how many of the B cells are clonal but rather
that their exists a clonal population of B cells within the total
population
bull Need to Identify malignant B cells within a population of
normal B cells
bull In order to interpret a pathological immunophenotype it is
necessary to refer to quantitative and qualitative values of
normal B-cell subpopulations
bull Pearl Identify the abnormal phenotype first then look at KL
ratio
Case 6
bull 12 year old male presents to ER with fatigue bruising
Lkc 2200uL
bull Hb 84gL
bull Plt 32
bull Diff ndash neutropenia no blasts noted few ldquoatypicalrdquo cells
bull RBC ndash non specific changes
Leukemia
Case 6 PB
Case 6 BM
Case 6
bull BM aspirate ndash Hypocellular relative lymphocytosis
marked reduction in myeloid lineages
hypoplasticaplastic anemia
bull Flow Cytometry ndash less than 3 blasts maturing
myeloid 68 lymphocytes polyclonal B helper
suppressor T
bull Unusual pattern of CD13CD16 staining noted
Case 6 flow cytometry Aplastic Anemia
Normal Patient
Normal
Patient
Patient
Normal
Case 6 RBC Test for PNH CD235a v s CD59
Case 6 Neutrophil test for PNH clone
FLAER CD24 CD45CD15
18 PNH clone
Case 6
bull Follow up
bull Patient on steroids acyclovir and anti-thymocyte
globulin
bull PNH clone Neutrophils 96 RBC 1 Nov 2015
bull In remission as of April 2017
- Lkc 5500uL (Neuts 23)
- Hb 129gL
- Plt 208 X10e9
Summary
bull B cell malignancies are the most common
hematological disorders seen in flow cytometry
bull Many have complex overlapping phenotypes
bull Important to look at ungated plots as well as gating on
specific subpopulations
bull Level of expression of markers is as important as
positive or negative (think CD13 vs CD16)
68
References
bull Craig FE Foon KA Flow cytometric immunophenotyping for hematologic neoplasms Blood
2008 Apr 15111(8)3941-67
bull Wood BL Principles of minimal residual disease detection for hematopoietic neoplasms by flow
cytometry Cytometry B Clin Cytom 2015 Apr 23
bull Shanafelt TD Ghia P Lanasa MC Landgren O Rawstron AC Monoclonal B-cell
lymphocytosis (MBL) biology natural history and clinical management Leukemia 2010
Mar24(3)512-20
bull Swerdlow SH Campo E Pileri SA Harris NL Stein H Siebert R Advani RGhielmini M Salles
GA Zelenetz AD Jaffe ES The 2016 revision of the World Health Organization classification of
lymphoid neoplasms Blood 2016 May 19127(20)2375-90
bull 3 Moench L Sachs Z Aasen G Dolan M Dayton V Courville EL Double- and triple-hit
lymphomas can present with features suggestive of immaturity including TdT expression and
create diagnostic challenges Leuk Lymphoma 2016572626-35
bull Sutherland DR Keeney M Illingworth A Practical guidelines for the high-sensitivity detection
and monitoring of paroxysmal nocturnal hemoglobinuria clones by flow cytometry Cytometry B
Clin Cytom 2012 Jul82(4)195-208
69
Case 1 BM
Case 1 Bone marrow morphology
bull Cellular marrow
bull Erythropoeisis 20-25
bull Granulopoesis 40-50 orderly maturation
bull Lymphocytes 30 10- 15 ldquoblast likerdquo
bull Increased megakaryocytes
Case 1 2 week follow up
77 B cells ndash 90
CD10 dimneg
CD20 neg bright CD38
pB-ALL
Case 1 BM 2 week follow up
Case 1 Cytogenetics
bull Complex karyotype
bull Structural abnormalities in chromosome
123491417 and 19
bull t(1719) rare in B cell acute lymphoblastic
leukemia
Case 1 Follow up
bull On high risk protocol AALL 0232 for infantile B-ALL
bull MRD 008 at Day 42
bull Cord blood transplant
bull Relapse 5 months post transplant
Pearl of Pathology
bull Differentiating precursor B cells (hematogones) from B-
ALL can be challenging Standardized analysis and
clear understanding of ldquonormalrdquo essential including
arrested maturation
Case 2
bull 4 year old girl referred to pediatric hematology with
pancytopenia
bull WBC 6800uL(Neuts 400uL) Hb 42gL Plt 28
bull Aplastic anemia
bull Bone marrow ordered aspirate biopsy flow and
cytogenetics
Case 2 BM flow cytometry
Polyclonal B cells
helper suppressor T cells
No increase in blasts
Maturing myeloid population
Case 2 CD45 neg B-ALL
Case 2 BM
Case 2
bull CD45neg
bull CD19+CD10+
bull CD20(partialdim)
bull CD34 neg
bull SigampcIg neg
bull Cytogenetics
bull Gain of X chromosome and
468911131415171821
DNA Index
122 Diploid
Case 2
bull Currently in remission after completion of
AALL0932 standard risk B-ALL therapy
Case 2
Pearls of Pathology
CD45 negative B-ALL is not uncommon
bull Always look at ungated set of plots for each marker
bull Eg SS versus each fluorescence parameter
Case 3
bull 58 year old male with pararenal mass
bull WBC 4000uL Hb 98gL Plt 229
bull ldquoblastsrdquo reported on blood film
bull Peripheral blood sent for flow cytometry
Case 3 PB flow cytometry
Dim CD45 CD19+CD10+
CD34- Sig neg
Case 3 PB flow cytometry
Dim CD45 CD19+CD10+
CD20(partial)CD5 neg
SIg cIg neg
Case 3 BM flow cytometry
Case 3 BM biopsy
Case 3 BM biopsy
Case 3 BM biopsy report
bull Diffuse infiltrate large lymphoid cells
bull Scant to moderate cytoplasm
bull Large irregular nuclei with prominent nucleoli
bull Increased mitotic activity
bull Positive for BCL-2 BCL6 CD10 and MUM1
bull Negative for TdT CD5
bull Cytogenetics FISH on para renal mass ndash C-MYC and
BCL2 rearrangements detected
Case 3 Doubletriple hit lymphoma
(DHLTHL)
bull Morphologically heterogeneous B cell neoplasms
characterized by rearrangement of the MYC proto-
oncogene with concurrent BCL2 andor BCL6 gene
rearrangement
bull WHO 2008 criteria - most cases of DHL and THL
classified DLBCL or B-cell lymphoma unclassifiable
with features intermediate between DLBCL and Burkitt
lymphoma (BCLU)
Case 3 DHLTHL
bull Retrospective study from one institution features of 13 cases (9 DHL4
THL)
bull median age was 59 years (range 30-74) eight females five males
bull enlarging lymphadenopathymasses (11 patients) abnormal peripheral
blood findings (2 patients)
bull TdT positivity (four cases two THLtwo DHL) dim CD45 expression (7
cases) lack of CD20 (2 cases) or lack of sIg (3 cases) and blastoid
morphology (2 cases)
bull Conclusion Expression of TdT in a B-cell lymphoma with mature
features or expression of surface light chain in a case otherwise
suggestive of B-lymphoblastic leukemialymphoma should prompt
an expedited evaluation for DHLTHL
Leuk Lymphoma 2016 Feb 181-10 [Epub ahead of print]
Double- and triple-hit lymphomas can present with features suggestive
of immaturity including TdT expression and create diagnostic challenges
Moench L Sachs Z Aasen G Dolan M Dayton V Courville EL
Case 4
bull 55 year old female
bull Previous history hyperparathyroidism
bull Parathyroidectomy in 1999
bull Mild persistent hypercalcemia
bull WBC 4800uL hemoglobin 121 gL Plt 296
bull Total protein 90gL IgG 34gL monoclonal peak
209gL
Case 4 Monoclonal IgG patient
Polyclonal B cells
With maturing hematogones
helper suppressor T cells
less than 3 blasts
Maturing myeloid population
Case 4 BM Ungated
Case 4 cytoplasmic immunoglobulin
Need KL
Case 4 BM Multiple Myeloma
Case 4 follow up
bull Smouldering myeloma
bull Recently developed CRAB (hyperCalcemia Renal
insufficiency Anemia Bone lesions
bull Serum free light chain ratio 123
bull Bone marrow aspirate gt60 plasma cells
bull Currently on treatment pending bone marrow
transplant
Case 5
bull 65 year old male
bull Slight lymphocytosis detected on routine CBC analysis
(lymph 4500uL)
bull Asymptomatic sent for flow cytometry
Case 5 PB
Case 5 flow cytometry KL ratio 0625
Case 5 Monoclonal B cell lymphocytosis
KL 18 KL 056
Case 5 Monoclonal B cell lymphocytosis
bull WHO defines CLL as abnormal lymphocyte count
gt5x10E9L (CD19+CD5+ light chain restricted) in the
absence of other clinical symptoms
bull Patients with small B cell clones are often detected as
an incidental finding
bull WHO 2016
bull CD19+CD5+ lt05x10E9L ldquoLow count MBLrdquo
raquo 05-5x10E9L ldquoHigh count MBLrdquo
raquo Non CLL type phenotype MBL also recognized
Monoclonal B cell Lymphocytosis
bull CLL immunophenotype - vast majority (75)
bull Atypical MBL
bull normal levels both CD5 and CD20 with variable
expression of CD23 (always exclude Mantle-cell
Lymphoma in CD23 negative cases)
bull Non-CLL-phenotype (CD5minus) MBL
bull Lacking CD5 expression and CD10 likely resembling B-
cell lymphomas of marginal-zone origin
Case 5 What is the normal KL Ratio
bull What is the normal or abnormal kappalambda ratio for
entire ldquopopulationrdquo of all B cells
bull Traditional view
ndash Kappa Lambda 21 or 31 ( 12 -13)
ndash Range 14 60
Case 5 What is the normal KL ratio
bull Wrong question
bull It doesnrsquot matter how many of the B cells are clonal but rather
that their exists a clonal population of B cells within the total
population
bull Need to Identify malignant B cells within a population of
normal B cells
bull In order to interpret a pathological immunophenotype it is
necessary to refer to quantitative and qualitative values of
normal B-cell subpopulations
bull Pearl Identify the abnormal phenotype first then look at KL
ratio
Case 6
bull 12 year old male presents to ER with fatigue bruising
Lkc 2200uL
bull Hb 84gL
bull Plt 32
bull Diff ndash neutropenia no blasts noted few ldquoatypicalrdquo cells
bull RBC ndash non specific changes
Leukemia
Case 6 PB
Case 6 BM
Case 6
bull BM aspirate ndash Hypocellular relative lymphocytosis
marked reduction in myeloid lineages
hypoplasticaplastic anemia
bull Flow Cytometry ndash less than 3 blasts maturing
myeloid 68 lymphocytes polyclonal B helper
suppressor T
bull Unusual pattern of CD13CD16 staining noted
Case 6 flow cytometry Aplastic Anemia
Normal Patient
Normal
Patient
Patient
Normal
Case 6 RBC Test for PNH CD235a v s CD59
Case 6 Neutrophil test for PNH clone
FLAER CD24 CD45CD15
18 PNH clone
Case 6
bull Follow up
bull Patient on steroids acyclovir and anti-thymocyte
globulin
bull PNH clone Neutrophils 96 RBC 1 Nov 2015
bull In remission as of April 2017
- Lkc 5500uL (Neuts 23)
- Hb 129gL
- Plt 208 X10e9
Summary
bull B cell malignancies are the most common
hematological disorders seen in flow cytometry
bull Many have complex overlapping phenotypes
bull Important to look at ungated plots as well as gating on
specific subpopulations
bull Level of expression of markers is as important as
positive or negative (think CD13 vs CD16)
68
References
bull Craig FE Foon KA Flow cytometric immunophenotyping for hematologic neoplasms Blood
2008 Apr 15111(8)3941-67
bull Wood BL Principles of minimal residual disease detection for hematopoietic neoplasms by flow
cytometry Cytometry B Clin Cytom 2015 Apr 23
bull Shanafelt TD Ghia P Lanasa MC Landgren O Rawstron AC Monoclonal B-cell
lymphocytosis (MBL) biology natural history and clinical management Leukemia 2010
Mar24(3)512-20
bull Swerdlow SH Campo E Pileri SA Harris NL Stein H Siebert R Advani RGhielmini M Salles
GA Zelenetz AD Jaffe ES The 2016 revision of the World Health Organization classification of
lymphoid neoplasms Blood 2016 May 19127(20)2375-90
bull 3 Moench L Sachs Z Aasen G Dolan M Dayton V Courville EL Double- and triple-hit
lymphomas can present with features suggestive of immaturity including TdT expression and
create diagnostic challenges Leuk Lymphoma 2016572626-35
bull Sutherland DR Keeney M Illingworth A Practical guidelines for the high-sensitivity detection
and monitoring of paroxysmal nocturnal hemoglobinuria clones by flow cytometry Cytometry B
Clin Cytom 2012 Jul82(4)195-208
69
Case 1 Bone marrow morphology
bull Cellular marrow
bull Erythropoeisis 20-25
bull Granulopoesis 40-50 orderly maturation
bull Lymphocytes 30 10- 15 ldquoblast likerdquo
bull Increased megakaryocytes
Case 1 2 week follow up
77 B cells ndash 90
CD10 dimneg
CD20 neg bright CD38
pB-ALL
Case 1 BM 2 week follow up
Case 1 Cytogenetics
bull Complex karyotype
bull Structural abnormalities in chromosome
123491417 and 19
bull t(1719) rare in B cell acute lymphoblastic
leukemia
Case 1 Follow up
bull On high risk protocol AALL 0232 for infantile B-ALL
bull MRD 008 at Day 42
bull Cord blood transplant
bull Relapse 5 months post transplant
Pearl of Pathology
bull Differentiating precursor B cells (hematogones) from B-
ALL can be challenging Standardized analysis and
clear understanding of ldquonormalrdquo essential including
arrested maturation
Case 2
bull 4 year old girl referred to pediatric hematology with
pancytopenia
bull WBC 6800uL(Neuts 400uL) Hb 42gL Plt 28
bull Aplastic anemia
bull Bone marrow ordered aspirate biopsy flow and
cytogenetics
Case 2 BM flow cytometry
Polyclonal B cells
helper suppressor T cells
No increase in blasts
Maturing myeloid population
Case 2 CD45 neg B-ALL
Case 2 BM
Case 2
bull CD45neg
bull CD19+CD10+
bull CD20(partialdim)
bull CD34 neg
bull SigampcIg neg
bull Cytogenetics
bull Gain of X chromosome and
468911131415171821
DNA Index
122 Diploid
Case 2
bull Currently in remission after completion of
AALL0932 standard risk B-ALL therapy
Case 2
Pearls of Pathology
CD45 negative B-ALL is not uncommon
bull Always look at ungated set of plots for each marker
bull Eg SS versus each fluorescence parameter
Case 3
bull 58 year old male with pararenal mass
bull WBC 4000uL Hb 98gL Plt 229
bull ldquoblastsrdquo reported on blood film
bull Peripheral blood sent for flow cytometry
Case 3 PB flow cytometry
Dim CD45 CD19+CD10+
CD34- Sig neg
Case 3 PB flow cytometry
Dim CD45 CD19+CD10+
CD20(partial)CD5 neg
SIg cIg neg
Case 3 BM flow cytometry
Case 3 BM biopsy
Case 3 BM biopsy
Case 3 BM biopsy report
bull Diffuse infiltrate large lymphoid cells
bull Scant to moderate cytoplasm
bull Large irregular nuclei with prominent nucleoli
bull Increased mitotic activity
bull Positive for BCL-2 BCL6 CD10 and MUM1
bull Negative for TdT CD5
bull Cytogenetics FISH on para renal mass ndash C-MYC and
BCL2 rearrangements detected
Case 3 Doubletriple hit lymphoma
(DHLTHL)
bull Morphologically heterogeneous B cell neoplasms
characterized by rearrangement of the MYC proto-
oncogene with concurrent BCL2 andor BCL6 gene
rearrangement
bull WHO 2008 criteria - most cases of DHL and THL
classified DLBCL or B-cell lymphoma unclassifiable
with features intermediate between DLBCL and Burkitt
lymphoma (BCLU)
Case 3 DHLTHL
bull Retrospective study from one institution features of 13 cases (9 DHL4
THL)
bull median age was 59 years (range 30-74) eight females five males
bull enlarging lymphadenopathymasses (11 patients) abnormal peripheral
blood findings (2 patients)
bull TdT positivity (four cases two THLtwo DHL) dim CD45 expression (7
cases) lack of CD20 (2 cases) or lack of sIg (3 cases) and blastoid
morphology (2 cases)
bull Conclusion Expression of TdT in a B-cell lymphoma with mature
features or expression of surface light chain in a case otherwise
suggestive of B-lymphoblastic leukemialymphoma should prompt
an expedited evaluation for DHLTHL
Leuk Lymphoma 2016 Feb 181-10 [Epub ahead of print]
Double- and triple-hit lymphomas can present with features suggestive
of immaturity including TdT expression and create diagnostic challenges
Moench L Sachs Z Aasen G Dolan M Dayton V Courville EL
Case 4
bull 55 year old female
bull Previous history hyperparathyroidism
bull Parathyroidectomy in 1999
bull Mild persistent hypercalcemia
bull WBC 4800uL hemoglobin 121 gL Plt 296
bull Total protein 90gL IgG 34gL monoclonal peak
209gL
Case 4 Monoclonal IgG patient
Polyclonal B cells
With maturing hematogones
helper suppressor T cells
less than 3 blasts
Maturing myeloid population
Case 4 BM Ungated
Case 4 cytoplasmic immunoglobulin
Need KL
Case 4 BM Multiple Myeloma
Case 4 follow up
bull Smouldering myeloma
bull Recently developed CRAB (hyperCalcemia Renal
insufficiency Anemia Bone lesions
bull Serum free light chain ratio 123
bull Bone marrow aspirate gt60 plasma cells
bull Currently on treatment pending bone marrow
transplant
Case 5
bull 65 year old male
bull Slight lymphocytosis detected on routine CBC analysis
(lymph 4500uL)
bull Asymptomatic sent for flow cytometry
Case 5 PB
Case 5 flow cytometry KL ratio 0625
Case 5 Monoclonal B cell lymphocytosis
KL 18 KL 056
Case 5 Monoclonal B cell lymphocytosis
bull WHO defines CLL as abnormal lymphocyte count
gt5x10E9L (CD19+CD5+ light chain restricted) in the
absence of other clinical symptoms
bull Patients with small B cell clones are often detected as
an incidental finding
bull WHO 2016
bull CD19+CD5+ lt05x10E9L ldquoLow count MBLrdquo
raquo 05-5x10E9L ldquoHigh count MBLrdquo
raquo Non CLL type phenotype MBL also recognized
Monoclonal B cell Lymphocytosis
bull CLL immunophenotype - vast majority (75)
bull Atypical MBL
bull normal levels both CD5 and CD20 with variable
expression of CD23 (always exclude Mantle-cell
Lymphoma in CD23 negative cases)
bull Non-CLL-phenotype (CD5minus) MBL
bull Lacking CD5 expression and CD10 likely resembling B-
cell lymphomas of marginal-zone origin
Case 5 What is the normal KL Ratio
bull What is the normal or abnormal kappalambda ratio for
entire ldquopopulationrdquo of all B cells
bull Traditional view
ndash Kappa Lambda 21 or 31 ( 12 -13)
ndash Range 14 60
Case 5 What is the normal KL ratio
bull Wrong question
bull It doesnrsquot matter how many of the B cells are clonal but rather
that their exists a clonal population of B cells within the total
population
bull Need to Identify malignant B cells within a population of
normal B cells
bull In order to interpret a pathological immunophenotype it is
necessary to refer to quantitative and qualitative values of
normal B-cell subpopulations
bull Pearl Identify the abnormal phenotype first then look at KL
ratio
Case 6
bull 12 year old male presents to ER with fatigue bruising
Lkc 2200uL
bull Hb 84gL
bull Plt 32
bull Diff ndash neutropenia no blasts noted few ldquoatypicalrdquo cells
bull RBC ndash non specific changes
Leukemia
Case 6 PB
Case 6 BM
Case 6
bull BM aspirate ndash Hypocellular relative lymphocytosis
marked reduction in myeloid lineages
hypoplasticaplastic anemia
bull Flow Cytometry ndash less than 3 blasts maturing
myeloid 68 lymphocytes polyclonal B helper
suppressor T
bull Unusual pattern of CD13CD16 staining noted
Case 6 flow cytometry Aplastic Anemia
Normal Patient
Normal
Patient
Patient
Normal
Case 6 RBC Test for PNH CD235a v s CD59
Case 6 Neutrophil test for PNH clone
FLAER CD24 CD45CD15
18 PNH clone
Case 6
bull Follow up
bull Patient on steroids acyclovir and anti-thymocyte
globulin
bull PNH clone Neutrophils 96 RBC 1 Nov 2015
bull In remission as of April 2017
- Lkc 5500uL (Neuts 23)
- Hb 129gL
- Plt 208 X10e9
Summary
bull B cell malignancies are the most common
hematological disorders seen in flow cytometry
bull Many have complex overlapping phenotypes
bull Important to look at ungated plots as well as gating on
specific subpopulations
bull Level of expression of markers is as important as
positive or negative (think CD13 vs CD16)
68
References
bull Craig FE Foon KA Flow cytometric immunophenotyping for hematologic neoplasms Blood
2008 Apr 15111(8)3941-67
bull Wood BL Principles of minimal residual disease detection for hematopoietic neoplasms by flow
cytometry Cytometry B Clin Cytom 2015 Apr 23
bull Shanafelt TD Ghia P Lanasa MC Landgren O Rawstron AC Monoclonal B-cell
lymphocytosis (MBL) biology natural history and clinical management Leukemia 2010
Mar24(3)512-20
bull Swerdlow SH Campo E Pileri SA Harris NL Stein H Siebert R Advani RGhielmini M Salles
GA Zelenetz AD Jaffe ES The 2016 revision of the World Health Organization classification of
lymphoid neoplasms Blood 2016 May 19127(20)2375-90
bull 3 Moench L Sachs Z Aasen G Dolan M Dayton V Courville EL Double- and triple-hit
lymphomas can present with features suggestive of immaturity including TdT expression and
create diagnostic challenges Leuk Lymphoma 2016572626-35
bull Sutherland DR Keeney M Illingworth A Practical guidelines for the high-sensitivity detection
and monitoring of paroxysmal nocturnal hemoglobinuria clones by flow cytometry Cytometry B
Clin Cytom 2012 Jul82(4)195-208
69
Case 1 2 week follow up
77 B cells ndash 90
CD10 dimneg
CD20 neg bright CD38
pB-ALL
Case 1 BM 2 week follow up
Case 1 Cytogenetics
bull Complex karyotype
bull Structural abnormalities in chromosome
123491417 and 19
bull t(1719) rare in B cell acute lymphoblastic
leukemia
Case 1 Follow up
bull On high risk protocol AALL 0232 for infantile B-ALL
bull MRD 008 at Day 42
bull Cord blood transplant
bull Relapse 5 months post transplant
Pearl of Pathology
bull Differentiating precursor B cells (hematogones) from B-
ALL can be challenging Standardized analysis and
clear understanding of ldquonormalrdquo essential including
arrested maturation
Case 2
bull 4 year old girl referred to pediatric hematology with
pancytopenia
bull WBC 6800uL(Neuts 400uL) Hb 42gL Plt 28
bull Aplastic anemia
bull Bone marrow ordered aspirate biopsy flow and
cytogenetics
Case 2 BM flow cytometry
Polyclonal B cells
helper suppressor T cells
No increase in blasts
Maturing myeloid population
Case 2 CD45 neg B-ALL
Case 2 BM
Case 2
bull CD45neg
bull CD19+CD10+
bull CD20(partialdim)
bull CD34 neg
bull SigampcIg neg
bull Cytogenetics
bull Gain of X chromosome and
468911131415171821
DNA Index
122 Diploid
Case 2
bull Currently in remission after completion of
AALL0932 standard risk B-ALL therapy
Case 2
Pearls of Pathology
CD45 negative B-ALL is not uncommon
bull Always look at ungated set of plots for each marker
bull Eg SS versus each fluorescence parameter
Case 3
bull 58 year old male with pararenal mass
bull WBC 4000uL Hb 98gL Plt 229
bull ldquoblastsrdquo reported on blood film
bull Peripheral blood sent for flow cytometry
Case 3 PB flow cytometry
Dim CD45 CD19+CD10+
CD34- Sig neg
Case 3 PB flow cytometry
Dim CD45 CD19+CD10+
CD20(partial)CD5 neg
SIg cIg neg
Case 3 BM flow cytometry
Case 3 BM biopsy
Case 3 BM biopsy
Case 3 BM biopsy report
bull Diffuse infiltrate large lymphoid cells
bull Scant to moderate cytoplasm
bull Large irregular nuclei with prominent nucleoli
bull Increased mitotic activity
bull Positive for BCL-2 BCL6 CD10 and MUM1
bull Negative for TdT CD5
bull Cytogenetics FISH on para renal mass ndash C-MYC and
BCL2 rearrangements detected
Case 3 Doubletriple hit lymphoma
(DHLTHL)
bull Morphologically heterogeneous B cell neoplasms
characterized by rearrangement of the MYC proto-
oncogene with concurrent BCL2 andor BCL6 gene
rearrangement
bull WHO 2008 criteria - most cases of DHL and THL
classified DLBCL or B-cell lymphoma unclassifiable
with features intermediate between DLBCL and Burkitt
lymphoma (BCLU)
Case 3 DHLTHL
bull Retrospective study from one institution features of 13 cases (9 DHL4
THL)
bull median age was 59 years (range 30-74) eight females five males
bull enlarging lymphadenopathymasses (11 patients) abnormal peripheral
blood findings (2 patients)
bull TdT positivity (four cases two THLtwo DHL) dim CD45 expression (7
cases) lack of CD20 (2 cases) or lack of sIg (3 cases) and blastoid
morphology (2 cases)
bull Conclusion Expression of TdT in a B-cell lymphoma with mature
features or expression of surface light chain in a case otherwise
suggestive of B-lymphoblastic leukemialymphoma should prompt
an expedited evaluation for DHLTHL
Leuk Lymphoma 2016 Feb 181-10 [Epub ahead of print]
Double- and triple-hit lymphomas can present with features suggestive
of immaturity including TdT expression and create diagnostic challenges
Moench L Sachs Z Aasen G Dolan M Dayton V Courville EL
Case 4
bull 55 year old female
bull Previous history hyperparathyroidism
bull Parathyroidectomy in 1999
bull Mild persistent hypercalcemia
bull WBC 4800uL hemoglobin 121 gL Plt 296
bull Total protein 90gL IgG 34gL monoclonal peak
209gL
Case 4 Monoclonal IgG patient
Polyclonal B cells
With maturing hematogones
helper suppressor T cells
less than 3 blasts
Maturing myeloid population
Case 4 BM Ungated
Case 4 cytoplasmic immunoglobulin
Need KL
Case 4 BM Multiple Myeloma
Case 4 follow up
bull Smouldering myeloma
bull Recently developed CRAB (hyperCalcemia Renal
insufficiency Anemia Bone lesions
bull Serum free light chain ratio 123
bull Bone marrow aspirate gt60 plasma cells
bull Currently on treatment pending bone marrow
transplant
Case 5
bull 65 year old male
bull Slight lymphocytosis detected on routine CBC analysis
(lymph 4500uL)
bull Asymptomatic sent for flow cytometry
Case 5 PB
Case 5 flow cytometry KL ratio 0625
Case 5 Monoclonal B cell lymphocytosis
KL 18 KL 056
Case 5 Monoclonal B cell lymphocytosis
bull WHO defines CLL as abnormal lymphocyte count
gt5x10E9L (CD19+CD5+ light chain restricted) in the
absence of other clinical symptoms
bull Patients with small B cell clones are often detected as
an incidental finding
bull WHO 2016
bull CD19+CD5+ lt05x10E9L ldquoLow count MBLrdquo
raquo 05-5x10E9L ldquoHigh count MBLrdquo
raquo Non CLL type phenotype MBL also recognized
Monoclonal B cell Lymphocytosis
bull CLL immunophenotype - vast majority (75)
bull Atypical MBL
bull normal levels both CD5 and CD20 with variable
expression of CD23 (always exclude Mantle-cell
Lymphoma in CD23 negative cases)
bull Non-CLL-phenotype (CD5minus) MBL
bull Lacking CD5 expression and CD10 likely resembling B-
cell lymphomas of marginal-zone origin
Case 5 What is the normal KL Ratio
bull What is the normal or abnormal kappalambda ratio for
entire ldquopopulationrdquo of all B cells
bull Traditional view
ndash Kappa Lambda 21 or 31 ( 12 -13)
ndash Range 14 60
Case 5 What is the normal KL ratio
bull Wrong question
bull It doesnrsquot matter how many of the B cells are clonal but rather
that their exists a clonal population of B cells within the total
population
bull Need to Identify malignant B cells within a population of
normal B cells
bull In order to interpret a pathological immunophenotype it is
necessary to refer to quantitative and qualitative values of
normal B-cell subpopulations
bull Pearl Identify the abnormal phenotype first then look at KL
ratio
Case 6
bull 12 year old male presents to ER with fatigue bruising
Lkc 2200uL
bull Hb 84gL
bull Plt 32
bull Diff ndash neutropenia no blasts noted few ldquoatypicalrdquo cells
bull RBC ndash non specific changes
Leukemia
Case 6 PB
Case 6 BM
Case 6
bull BM aspirate ndash Hypocellular relative lymphocytosis
marked reduction in myeloid lineages
hypoplasticaplastic anemia
bull Flow Cytometry ndash less than 3 blasts maturing
myeloid 68 lymphocytes polyclonal B helper
suppressor T
bull Unusual pattern of CD13CD16 staining noted
Case 6 flow cytometry Aplastic Anemia
Normal Patient
Normal
Patient
Patient
Normal
Case 6 RBC Test for PNH CD235a v s CD59
Case 6 Neutrophil test for PNH clone
FLAER CD24 CD45CD15
18 PNH clone
Case 6
bull Follow up
bull Patient on steroids acyclovir and anti-thymocyte
globulin
bull PNH clone Neutrophils 96 RBC 1 Nov 2015
bull In remission as of April 2017
- Lkc 5500uL (Neuts 23)
- Hb 129gL
- Plt 208 X10e9
Summary
bull B cell malignancies are the most common
hematological disorders seen in flow cytometry
bull Many have complex overlapping phenotypes
bull Important to look at ungated plots as well as gating on
specific subpopulations
bull Level of expression of markers is as important as
positive or negative (think CD13 vs CD16)
68
References
bull Craig FE Foon KA Flow cytometric immunophenotyping for hematologic neoplasms Blood
2008 Apr 15111(8)3941-67
bull Wood BL Principles of minimal residual disease detection for hematopoietic neoplasms by flow
cytometry Cytometry B Clin Cytom 2015 Apr 23
bull Shanafelt TD Ghia P Lanasa MC Landgren O Rawstron AC Monoclonal B-cell
lymphocytosis (MBL) biology natural history and clinical management Leukemia 2010
Mar24(3)512-20
bull Swerdlow SH Campo E Pileri SA Harris NL Stein H Siebert R Advani RGhielmini M Salles
GA Zelenetz AD Jaffe ES The 2016 revision of the World Health Organization classification of
lymphoid neoplasms Blood 2016 May 19127(20)2375-90
bull 3 Moench L Sachs Z Aasen G Dolan M Dayton V Courville EL Double- and triple-hit
lymphomas can present with features suggestive of immaturity including TdT expression and
create diagnostic challenges Leuk Lymphoma 2016572626-35
bull Sutherland DR Keeney M Illingworth A Practical guidelines for the high-sensitivity detection
and monitoring of paroxysmal nocturnal hemoglobinuria clones by flow cytometry Cytometry B
Clin Cytom 2012 Jul82(4)195-208
69
Case 1 BM 2 week follow up
Case 1 Cytogenetics
bull Complex karyotype
bull Structural abnormalities in chromosome
123491417 and 19
bull t(1719) rare in B cell acute lymphoblastic
leukemia
Case 1 Follow up
bull On high risk protocol AALL 0232 for infantile B-ALL
bull MRD 008 at Day 42
bull Cord blood transplant
bull Relapse 5 months post transplant
Pearl of Pathology
bull Differentiating precursor B cells (hematogones) from B-
ALL can be challenging Standardized analysis and
clear understanding of ldquonormalrdquo essential including
arrested maturation
Case 2
bull 4 year old girl referred to pediatric hematology with
pancytopenia
bull WBC 6800uL(Neuts 400uL) Hb 42gL Plt 28
bull Aplastic anemia
bull Bone marrow ordered aspirate biopsy flow and
cytogenetics
Case 2 BM flow cytometry
Polyclonal B cells
helper suppressor T cells
No increase in blasts
Maturing myeloid population
Case 2 CD45 neg B-ALL
Case 2 BM
Case 2
bull CD45neg
bull CD19+CD10+
bull CD20(partialdim)
bull CD34 neg
bull SigampcIg neg
bull Cytogenetics
bull Gain of X chromosome and
468911131415171821
DNA Index
122 Diploid
Case 2
bull Currently in remission after completion of
AALL0932 standard risk B-ALL therapy
Case 2
Pearls of Pathology
CD45 negative B-ALL is not uncommon
bull Always look at ungated set of plots for each marker
bull Eg SS versus each fluorescence parameter
Case 3
bull 58 year old male with pararenal mass
bull WBC 4000uL Hb 98gL Plt 229
bull ldquoblastsrdquo reported on blood film
bull Peripheral blood sent for flow cytometry
Case 3 PB flow cytometry
Dim CD45 CD19+CD10+
CD34- Sig neg
Case 3 PB flow cytometry
Dim CD45 CD19+CD10+
CD20(partial)CD5 neg
SIg cIg neg
Case 3 BM flow cytometry
Case 3 BM biopsy
Case 3 BM biopsy
Case 3 BM biopsy report
bull Diffuse infiltrate large lymphoid cells
bull Scant to moderate cytoplasm
bull Large irregular nuclei with prominent nucleoli
bull Increased mitotic activity
bull Positive for BCL-2 BCL6 CD10 and MUM1
bull Negative for TdT CD5
bull Cytogenetics FISH on para renal mass ndash C-MYC and
BCL2 rearrangements detected
Case 3 Doubletriple hit lymphoma
(DHLTHL)
bull Morphologically heterogeneous B cell neoplasms
characterized by rearrangement of the MYC proto-
oncogene with concurrent BCL2 andor BCL6 gene
rearrangement
bull WHO 2008 criteria - most cases of DHL and THL
classified DLBCL or B-cell lymphoma unclassifiable
with features intermediate between DLBCL and Burkitt
lymphoma (BCLU)
Case 3 DHLTHL
bull Retrospective study from one institution features of 13 cases (9 DHL4
THL)
bull median age was 59 years (range 30-74) eight females five males
bull enlarging lymphadenopathymasses (11 patients) abnormal peripheral
blood findings (2 patients)
bull TdT positivity (four cases two THLtwo DHL) dim CD45 expression (7
cases) lack of CD20 (2 cases) or lack of sIg (3 cases) and blastoid
morphology (2 cases)
bull Conclusion Expression of TdT in a B-cell lymphoma with mature
features or expression of surface light chain in a case otherwise
suggestive of B-lymphoblastic leukemialymphoma should prompt
an expedited evaluation for DHLTHL
Leuk Lymphoma 2016 Feb 181-10 [Epub ahead of print]
Double- and triple-hit lymphomas can present with features suggestive
of immaturity including TdT expression and create diagnostic challenges
Moench L Sachs Z Aasen G Dolan M Dayton V Courville EL
Case 4
bull 55 year old female
bull Previous history hyperparathyroidism
bull Parathyroidectomy in 1999
bull Mild persistent hypercalcemia
bull WBC 4800uL hemoglobin 121 gL Plt 296
bull Total protein 90gL IgG 34gL monoclonal peak
209gL
Case 4 Monoclonal IgG patient
Polyclonal B cells
With maturing hematogones
helper suppressor T cells
less than 3 blasts
Maturing myeloid population
Case 4 BM Ungated
Case 4 cytoplasmic immunoglobulin
Need KL
Case 4 BM Multiple Myeloma
Case 4 follow up
bull Smouldering myeloma
bull Recently developed CRAB (hyperCalcemia Renal
insufficiency Anemia Bone lesions
bull Serum free light chain ratio 123
bull Bone marrow aspirate gt60 plasma cells
bull Currently on treatment pending bone marrow
transplant
Case 5
bull 65 year old male
bull Slight lymphocytosis detected on routine CBC analysis
(lymph 4500uL)
bull Asymptomatic sent for flow cytometry
Case 5 PB
Case 5 flow cytometry KL ratio 0625
Case 5 Monoclonal B cell lymphocytosis
KL 18 KL 056
Case 5 Monoclonal B cell lymphocytosis
bull WHO defines CLL as abnormal lymphocyte count
gt5x10E9L (CD19+CD5+ light chain restricted) in the
absence of other clinical symptoms
bull Patients with small B cell clones are often detected as
an incidental finding
bull WHO 2016
bull CD19+CD5+ lt05x10E9L ldquoLow count MBLrdquo
raquo 05-5x10E9L ldquoHigh count MBLrdquo
raquo Non CLL type phenotype MBL also recognized
Monoclonal B cell Lymphocytosis
bull CLL immunophenotype - vast majority (75)
bull Atypical MBL
bull normal levels both CD5 and CD20 with variable
expression of CD23 (always exclude Mantle-cell
Lymphoma in CD23 negative cases)
bull Non-CLL-phenotype (CD5minus) MBL
bull Lacking CD5 expression and CD10 likely resembling B-
cell lymphomas of marginal-zone origin
Case 5 What is the normal KL Ratio
bull What is the normal or abnormal kappalambda ratio for
entire ldquopopulationrdquo of all B cells
bull Traditional view
ndash Kappa Lambda 21 or 31 ( 12 -13)
ndash Range 14 60
Case 5 What is the normal KL ratio
bull Wrong question
bull It doesnrsquot matter how many of the B cells are clonal but rather
that their exists a clonal population of B cells within the total
population
bull Need to Identify malignant B cells within a population of
normal B cells
bull In order to interpret a pathological immunophenotype it is
necessary to refer to quantitative and qualitative values of
normal B-cell subpopulations
bull Pearl Identify the abnormal phenotype first then look at KL
ratio
Case 6
bull 12 year old male presents to ER with fatigue bruising
Lkc 2200uL
bull Hb 84gL
bull Plt 32
bull Diff ndash neutropenia no blasts noted few ldquoatypicalrdquo cells
bull RBC ndash non specific changes
Leukemia
Case 6 PB
Case 6 BM
Case 6
bull BM aspirate ndash Hypocellular relative lymphocytosis
marked reduction in myeloid lineages
hypoplasticaplastic anemia
bull Flow Cytometry ndash less than 3 blasts maturing
myeloid 68 lymphocytes polyclonal B helper
suppressor T
bull Unusual pattern of CD13CD16 staining noted
Case 6 flow cytometry Aplastic Anemia
Normal Patient
Normal
Patient
Patient
Normal
Case 6 RBC Test for PNH CD235a v s CD59
Case 6 Neutrophil test for PNH clone
FLAER CD24 CD45CD15
18 PNH clone
Case 6
bull Follow up
bull Patient on steroids acyclovir and anti-thymocyte
globulin
bull PNH clone Neutrophils 96 RBC 1 Nov 2015
bull In remission as of April 2017
- Lkc 5500uL (Neuts 23)
- Hb 129gL
- Plt 208 X10e9
Summary
bull B cell malignancies are the most common
hematological disorders seen in flow cytometry
bull Many have complex overlapping phenotypes
bull Important to look at ungated plots as well as gating on
specific subpopulations
bull Level of expression of markers is as important as
positive or negative (think CD13 vs CD16)
68
References
bull Craig FE Foon KA Flow cytometric immunophenotyping for hematologic neoplasms Blood
2008 Apr 15111(8)3941-67
bull Wood BL Principles of minimal residual disease detection for hematopoietic neoplasms by flow
cytometry Cytometry B Clin Cytom 2015 Apr 23
bull Shanafelt TD Ghia P Lanasa MC Landgren O Rawstron AC Monoclonal B-cell
lymphocytosis (MBL) biology natural history and clinical management Leukemia 2010
Mar24(3)512-20
bull Swerdlow SH Campo E Pileri SA Harris NL Stein H Siebert R Advani RGhielmini M Salles
GA Zelenetz AD Jaffe ES The 2016 revision of the World Health Organization classification of
lymphoid neoplasms Blood 2016 May 19127(20)2375-90
bull 3 Moench L Sachs Z Aasen G Dolan M Dayton V Courville EL Double- and triple-hit
lymphomas can present with features suggestive of immaturity including TdT expression and
create diagnostic challenges Leuk Lymphoma 2016572626-35
bull Sutherland DR Keeney M Illingworth A Practical guidelines for the high-sensitivity detection
and monitoring of paroxysmal nocturnal hemoglobinuria clones by flow cytometry Cytometry B
Clin Cytom 2012 Jul82(4)195-208
69
Case 1 Cytogenetics
bull Complex karyotype
bull Structural abnormalities in chromosome
123491417 and 19
bull t(1719) rare in B cell acute lymphoblastic
leukemia
Case 1 Follow up
bull On high risk protocol AALL 0232 for infantile B-ALL
bull MRD 008 at Day 42
bull Cord blood transplant
bull Relapse 5 months post transplant
Pearl of Pathology
bull Differentiating precursor B cells (hematogones) from B-
ALL can be challenging Standardized analysis and
clear understanding of ldquonormalrdquo essential including
arrested maturation
Case 2
bull 4 year old girl referred to pediatric hematology with
pancytopenia
bull WBC 6800uL(Neuts 400uL) Hb 42gL Plt 28
bull Aplastic anemia
bull Bone marrow ordered aspirate biopsy flow and
cytogenetics
Case 2 BM flow cytometry
Polyclonal B cells
helper suppressor T cells
No increase in blasts
Maturing myeloid population
Case 2 CD45 neg B-ALL
Case 2 BM
Case 2
bull CD45neg
bull CD19+CD10+
bull CD20(partialdim)
bull CD34 neg
bull SigampcIg neg
bull Cytogenetics
bull Gain of X chromosome and
468911131415171821
DNA Index
122 Diploid
Case 2
bull Currently in remission after completion of
AALL0932 standard risk B-ALL therapy
Case 2
Pearls of Pathology
CD45 negative B-ALL is not uncommon
bull Always look at ungated set of plots for each marker
bull Eg SS versus each fluorescence parameter
Case 3
bull 58 year old male with pararenal mass
bull WBC 4000uL Hb 98gL Plt 229
bull ldquoblastsrdquo reported on blood film
bull Peripheral blood sent for flow cytometry
Case 3 PB flow cytometry
Dim CD45 CD19+CD10+
CD34- Sig neg
Case 3 PB flow cytometry
Dim CD45 CD19+CD10+
CD20(partial)CD5 neg
SIg cIg neg
Case 3 BM flow cytometry
Case 3 BM biopsy
Case 3 BM biopsy
Case 3 BM biopsy report
bull Diffuse infiltrate large lymphoid cells
bull Scant to moderate cytoplasm
bull Large irregular nuclei with prominent nucleoli
bull Increased mitotic activity
bull Positive for BCL-2 BCL6 CD10 and MUM1
bull Negative for TdT CD5
bull Cytogenetics FISH on para renal mass ndash C-MYC and
BCL2 rearrangements detected
Case 3 Doubletriple hit lymphoma
(DHLTHL)
bull Morphologically heterogeneous B cell neoplasms
characterized by rearrangement of the MYC proto-
oncogene with concurrent BCL2 andor BCL6 gene
rearrangement
bull WHO 2008 criteria - most cases of DHL and THL
classified DLBCL or B-cell lymphoma unclassifiable
with features intermediate between DLBCL and Burkitt
lymphoma (BCLU)
Case 3 DHLTHL
bull Retrospective study from one institution features of 13 cases (9 DHL4
THL)
bull median age was 59 years (range 30-74) eight females five males
bull enlarging lymphadenopathymasses (11 patients) abnormal peripheral
blood findings (2 patients)
bull TdT positivity (four cases two THLtwo DHL) dim CD45 expression (7
cases) lack of CD20 (2 cases) or lack of sIg (3 cases) and blastoid
morphology (2 cases)
bull Conclusion Expression of TdT in a B-cell lymphoma with mature
features or expression of surface light chain in a case otherwise
suggestive of B-lymphoblastic leukemialymphoma should prompt
an expedited evaluation for DHLTHL
Leuk Lymphoma 2016 Feb 181-10 [Epub ahead of print]
Double- and triple-hit lymphomas can present with features suggestive
of immaturity including TdT expression and create diagnostic challenges
Moench L Sachs Z Aasen G Dolan M Dayton V Courville EL
Case 4
bull 55 year old female
bull Previous history hyperparathyroidism
bull Parathyroidectomy in 1999
bull Mild persistent hypercalcemia
bull WBC 4800uL hemoglobin 121 gL Plt 296
bull Total protein 90gL IgG 34gL monoclonal peak
209gL
Case 4 Monoclonal IgG patient
Polyclonal B cells
With maturing hematogones
helper suppressor T cells
less than 3 blasts
Maturing myeloid population
Case 4 BM Ungated
Case 4 cytoplasmic immunoglobulin
Need KL
Case 4 BM Multiple Myeloma
Case 4 follow up
bull Smouldering myeloma
bull Recently developed CRAB (hyperCalcemia Renal
insufficiency Anemia Bone lesions
bull Serum free light chain ratio 123
bull Bone marrow aspirate gt60 plasma cells
bull Currently on treatment pending bone marrow
transplant
Case 5
bull 65 year old male
bull Slight lymphocytosis detected on routine CBC analysis
(lymph 4500uL)
bull Asymptomatic sent for flow cytometry
Case 5 PB
Case 5 flow cytometry KL ratio 0625
Case 5 Monoclonal B cell lymphocytosis
KL 18 KL 056
Case 5 Monoclonal B cell lymphocytosis
bull WHO defines CLL as abnormal lymphocyte count
gt5x10E9L (CD19+CD5+ light chain restricted) in the
absence of other clinical symptoms
bull Patients with small B cell clones are often detected as
an incidental finding
bull WHO 2016
bull CD19+CD5+ lt05x10E9L ldquoLow count MBLrdquo
raquo 05-5x10E9L ldquoHigh count MBLrdquo
raquo Non CLL type phenotype MBL also recognized
Monoclonal B cell Lymphocytosis
bull CLL immunophenotype - vast majority (75)
bull Atypical MBL
bull normal levels both CD5 and CD20 with variable
expression of CD23 (always exclude Mantle-cell
Lymphoma in CD23 negative cases)
bull Non-CLL-phenotype (CD5minus) MBL
bull Lacking CD5 expression and CD10 likely resembling B-
cell lymphomas of marginal-zone origin
Case 5 What is the normal KL Ratio
bull What is the normal or abnormal kappalambda ratio for
entire ldquopopulationrdquo of all B cells
bull Traditional view
ndash Kappa Lambda 21 or 31 ( 12 -13)
ndash Range 14 60
Case 5 What is the normal KL ratio
bull Wrong question
bull It doesnrsquot matter how many of the B cells are clonal but rather
that their exists a clonal population of B cells within the total
population
bull Need to Identify malignant B cells within a population of
normal B cells
bull In order to interpret a pathological immunophenotype it is
necessary to refer to quantitative and qualitative values of
normal B-cell subpopulations
bull Pearl Identify the abnormal phenotype first then look at KL
ratio
Case 6
bull 12 year old male presents to ER with fatigue bruising
Lkc 2200uL
bull Hb 84gL
bull Plt 32
bull Diff ndash neutropenia no blasts noted few ldquoatypicalrdquo cells
bull RBC ndash non specific changes
Leukemia
Case 6 PB
Case 6 BM
Case 6
bull BM aspirate ndash Hypocellular relative lymphocytosis
marked reduction in myeloid lineages
hypoplasticaplastic anemia
bull Flow Cytometry ndash less than 3 blasts maturing
myeloid 68 lymphocytes polyclonal B helper
suppressor T
bull Unusual pattern of CD13CD16 staining noted
Case 6 flow cytometry Aplastic Anemia
Normal Patient
Normal
Patient
Patient
Normal
Case 6 RBC Test for PNH CD235a v s CD59
Case 6 Neutrophil test for PNH clone
FLAER CD24 CD45CD15
18 PNH clone
Case 6
bull Follow up
bull Patient on steroids acyclovir and anti-thymocyte
globulin
bull PNH clone Neutrophils 96 RBC 1 Nov 2015
bull In remission as of April 2017
- Lkc 5500uL (Neuts 23)
- Hb 129gL
- Plt 208 X10e9
Summary
bull B cell malignancies are the most common
hematological disorders seen in flow cytometry
bull Many have complex overlapping phenotypes
bull Important to look at ungated plots as well as gating on
specific subpopulations
bull Level of expression of markers is as important as
positive or negative (think CD13 vs CD16)
68
References
bull Craig FE Foon KA Flow cytometric immunophenotyping for hematologic neoplasms Blood
2008 Apr 15111(8)3941-67
bull Wood BL Principles of minimal residual disease detection for hematopoietic neoplasms by flow
cytometry Cytometry B Clin Cytom 2015 Apr 23
bull Shanafelt TD Ghia P Lanasa MC Landgren O Rawstron AC Monoclonal B-cell
lymphocytosis (MBL) biology natural history and clinical management Leukemia 2010
Mar24(3)512-20
bull Swerdlow SH Campo E Pileri SA Harris NL Stein H Siebert R Advani RGhielmini M Salles
GA Zelenetz AD Jaffe ES The 2016 revision of the World Health Organization classification of
lymphoid neoplasms Blood 2016 May 19127(20)2375-90
bull 3 Moench L Sachs Z Aasen G Dolan M Dayton V Courville EL Double- and triple-hit
lymphomas can present with features suggestive of immaturity including TdT expression and
create diagnostic challenges Leuk Lymphoma 2016572626-35
bull Sutherland DR Keeney M Illingworth A Practical guidelines for the high-sensitivity detection
and monitoring of paroxysmal nocturnal hemoglobinuria clones by flow cytometry Cytometry B
Clin Cytom 2012 Jul82(4)195-208
69
Case 1 Follow up
bull On high risk protocol AALL 0232 for infantile B-ALL
bull MRD 008 at Day 42
bull Cord blood transplant
bull Relapse 5 months post transplant
Pearl of Pathology
bull Differentiating precursor B cells (hematogones) from B-
ALL can be challenging Standardized analysis and
clear understanding of ldquonormalrdquo essential including
arrested maturation
Case 2
bull 4 year old girl referred to pediatric hematology with
pancytopenia
bull WBC 6800uL(Neuts 400uL) Hb 42gL Plt 28
bull Aplastic anemia
bull Bone marrow ordered aspirate biopsy flow and
cytogenetics
Case 2 BM flow cytometry
Polyclonal B cells
helper suppressor T cells
No increase in blasts
Maturing myeloid population
Case 2 CD45 neg B-ALL
Case 2 BM
Case 2
bull CD45neg
bull CD19+CD10+
bull CD20(partialdim)
bull CD34 neg
bull SigampcIg neg
bull Cytogenetics
bull Gain of X chromosome and
468911131415171821
DNA Index
122 Diploid
Case 2
bull Currently in remission after completion of
AALL0932 standard risk B-ALL therapy
Case 2
Pearls of Pathology
CD45 negative B-ALL is not uncommon
bull Always look at ungated set of plots for each marker
bull Eg SS versus each fluorescence parameter
Case 3
bull 58 year old male with pararenal mass
bull WBC 4000uL Hb 98gL Plt 229
bull ldquoblastsrdquo reported on blood film
bull Peripheral blood sent for flow cytometry
Case 3 PB flow cytometry
Dim CD45 CD19+CD10+
CD34- Sig neg
Case 3 PB flow cytometry
Dim CD45 CD19+CD10+
CD20(partial)CD5 neg
SIg cIg neg
Case 3 BM flow cytometry
Case 3 BM biopsy
Case 3 BM biopsy
Case 3 BM biopsy report
bull Diffuse infiltrate large lymphoid cells
bull Scant to moderate cytoplasm
bull Large irregular nuclei with prominent nucleoli
bull Increased mitotic activity
bull Positive for BCL-2 BCL6 CD10 and MUM1
bull Negative for TdT CD5
bull Cytogenetics FISH on para renal mass ndash C-MYC and
BCL2 rearrangements detected
Case 3 Doubletriple hit lymphoma
(DHLTHL)
bull Morphologically heterogeneous B cell neoplasms
characterized by rearrangement of the MYC proto-
oncogene with concurrent BCL2 andor BCL6 gene
rearrangement
bull WHO 2008 criteria - most cases of DHL and THL
classified DLBCL or B-cell lymphoma unclassifiable
with features intermediate between DLBCL and Burkitt
lymphoma (BCLU)
Case 3 DHLTHL
bull Retrospective study from one institution features of 13 cases (9 DHL4
THL)
bull median age was 59 years (range 30-74) eight females five males
bull enlarging lymphadenopathymasses (11 patients) abnormal peripheral
blood findings (2 patients)
bull TdT positivity (four cases two THLtwo DHL) dim CD45 expression (7
cases) lack of CD20 (2 cases) or lack of sIg (3 cases) and blastoid
morphology (2 cases)
bull Conclusion Expression of TdT in a B-cell lymphoma with mature
features or expression of surface light chain in a case otherwise
suggestive of B-lymphoblastic leukemialymphoma should prompt
an expedited evaluation for DHLTHL
Leuk Lymphoma 2016 Feb 181-10 [Epub ahead of print]
Double- and triple-hit lymphomas can present with features suggestive
of immaturity including TdT expression and create diagnostic challenges
Moench L Sachs Z Aasen G Dolan M Dayton V Courville EL
Case 4
bull 55 year old female
bull Previous history hyperparathyroidism
bull Parathyroidectomy in 1999
bull Mild persistent hypercalcemia
bull WBC 4800uL hemoglobin 121 gL Plt 296
bull Total protein 90gL IgG 34gL monoclonal peak
209gL
Case 4 Monoclonal IgG patient
Polyclonal B cells
With maturing hematogones
helper suppressor T cells
less than 3 blasts
Maturing myeloid population
Case 4 BM Ungated
Case 4 cytoplasmic immunoglobulin
Need KL
Case 4 BM Multiple Myeloma
Case 4 follow up
bull Smouldering myeloma
bull Recently developed CRAB (hyperCalcemia Renal
insufficiency Anemia Bone lesions
bull Serum free light chain ratio 123
bull Bone marrow aspirate gt60 plasma cells
bull Currently on treatment pending bone marrow
transplant
Case 5
bull 65 year old male
bull Slight lymphocytosis detected on routine CBC analysis
(lymph 4500uL)
bull Asymptomatic sent for flow cytometry
Case 5 PB
Case 5 flow cytometry KL ratio 0625
Case 5 Monoclonal B cell lymphocytosis
KL 18 KL 056
Case 5 Monoclonal B cell lymphocytosis
bull WHO defines CLL as abnormal lymphocyte count
gt5x10E9L (CD19+CD5+ light chain restricted) in the
absence of other clinical symptoms
bull Patients with small B cell clones are often detected as
an incidental finding
bull WHO 2016
bull CD19+CD5+ lt05x10E9L ldquoLow count MBLrdquo
raquo 05-5x10E9L ldquoHigh count MBLrdquo
raquo Non CLL type phenotype MBL also recognized
Monoclonal B cell Lymphocytosis
bull CLL immunophenotype - vast majority (75)
bull Atypical MBL
bull normal levels both CD5 and CD20 with variable
expression of CD23 (always exclude Mantle-cell
Lymphoma in CD23 negative cases)
bull Non-CLL-phenotype (CD5minus) MBL
bull Lacking CD5 expression and CD10 likely resembling B-
cell lymphomas of marginal-zone origin
Case 5 What is the normal KL Ratio
bull What is the normal or abnormal kappalambda ratio for
entire ldquopopulationrdquo of all B cells
bull Traditional view
ndash Kappa Lambda 21 or 31 ( 12 -13)
ndash Range 14 60
Case 5 What is the normal KL ratio
bull Wrong question
bull It doesnrsquot matter how many of the B cells are clonal but rather
that their exists a clonal population of B cells within the total
population
bull Need to Identify malignant B cells within a population of
normal B cells
bull In order to interpret a pathological immunophenotype it is
necessary to refer to quantitative and qualitative values of
normal B-cell subpopulations
bull Pearl Identify the abnormal phenotype first then look at KL
ratio
Case 6
bull 12 year old male presents to ER with fatigue bruising
Lkc 2200uL
bull Hb 84gL
bull Plt 32
bull Diff ndash neutropenia no blasts noted few ldquoatypicalrdquo cells
bull RBC ndash non specific changes
Leukemia
Case 6 PB
Case 6 BM
Case 6
bull BM aspirate ndash Hypocellular relative lymphocytosis
marked reduction in myeloid lineages
hypoplasticaplastic anemia
bull Flow Cytometry ndash less than 3 blasts maturing
myeloid 68 lymphocytes polyclonal B helper
suppressor T
bull Unusual pattern of CD13CD16 staining noted
Case 6 flow cytometry Aplastic Anemia
Normal Patient
Normal
Patient
Patient
Normal
Case 6 RBC Test for PNH CD235a v s CD59
Case 6 Neutrophil test for PNH clone
FLAER CD24 CD45CD15
18 PNH clone
Case 6
bull Follow up
bull Patient on steroids acyclovir and anti-thymocyte
globulin
bull PNH clone Neutrophils 96 RBC 1 Nov 2015
bull In remission as of April 2017
- Lkc 5500uL (Neuts 23)
- Hb 129gL
- Plt 208 X10e9
Summary
bull B cell malignancies are the most common
hematological disorders seen in flow cytometry
bull Many have complex overlapping phenotypes
bull Important to look at ungated plots as well as gating on
specific subpopulations
bull Level of expression of markers is as important as
positive or negative (think CD13 vs CD16)
68
References
bull Craig FE Foon KA Flow cytometric immunophenotyping for hematologic neoplasms Blood
2008 Apr 15111(8)3941-67
bull Wood BL Principles of minimal residual disease detection for hematopoietic neoplasms by flow
cytometry Cytometry B Clin Cytom 2015 Apr 23
bull Shanafelt TD Ghia P Lanasa MC Landgren O Rawstron AC Monoclonal B-cell
lymphocytosis (MBL) biology natural history and clinical management Leukemia 2010
Mar24(3)512-20
bull Swerdlow SH Campo E Pileri SA Harris NL Stein H Siebert R Advani RGhielmini M Salles
GA Zelenetz AD Jaffe ES The 2016 revision of the World Health Organization classification of
lymphoid neoplasms Blood 2016 May 19127(20)2375-90
bull 3 Moench L Sachs Z Aasen G Dolan M Dayton V Courville EL Double- and triple-hit
lymphomas can present with features suggestive of immaturity including TdT expression and
create diagnostic challenges Leuk Lymphoma 2016572626-35
bull Sutherland DR Keeney M Illingworth A Practical guidelines for the high-sensitivity detection
and monitoring of paroxysmal nocturnal hemoglobinuria clones by flow cytometry Cytometry B
Clin Cytom 2012 Jul82(4)195-208
69
Case 2
bull 4 year old girl referred to pediatric hematology with
pancytopenia
bull WBC 6800uL(Neuts 400uL) Hb 42gL Plt 28
bull Aplastic anemia
bull Bone marrow ordered aspirate biopsy flow and
cytogenetics
Case 2 BM flow cytometry
Polyclonal B cells
helper suppressor T cells
No increase in blasts
Maturing myeloid population
Case 2 CD45 neg B-ALL
Case 2 BM
Case 2
bull CD45neg
bull CD19+CD10+
bull CD20(partialdim)
bull CD34 neg
bull SigampcIg neg
bull Cytogenetics
bull Gain of X chromosome and
468911131415171821
DNA Index
122 Diploid
Case 2
bull Currently in remission after completion of
AALL0932 standard risk B-ALL therapy
Case 2
Pearls of Pathology
CD45 negative B-ALL is not uncommon
bull Always look at ungated set of plots for each marker
bull Eg SS versus each fluorescence parameter
Case 3
bull 58 year old male with pararenal mass
bull WBC 4000uL Hb 98gL Plt 229
bull ldquoblastsrdquo reported on blood film
bull Peripheral blood sent for flow cytometry
Case 3 PB flow cytometry
Dim CD45 CD19+CD10+
CD34- Sig neg
Case 3 PB flow cytometry
Dim CD45 CD19+CD10+
CD20(partial)CD5 neg
SIg cIg neg
Case 3 BM flow cytometry
Case 3 BM biopsy
Case 3 BM biopsy
Case 3 BM biopsy report
bull Diffuse infiltrate large lymphoid cells
bull Scant to moderate cytoplasm
bull Large irregular nuclei with prominent nucleoli
bull Increased mitotic activity
bull Positive for BCL-2 BCL6 CD10 and MUM1
bull Negative for TdT CD5
bull Cytogenetics FISH on para renal mass ndash C-MYC and
BCL2 rearrangements detected
Case 3 Doubletriple hit lymphoma
(DHLTHL)
bull Morphologically heterogeneous B cell neoplasms
characterized by rearrangement of the MYC proto-
oncogene with concurrent BCL2 andor BCL6 gene
rearrangement
bull WHO 2008 criteria - most cases of DHL and THL
classified DLBCL or B-cell lymphoma unclassifiable
with features intermediate between DLBCL and Burkitt
lymphoma (BCLU)
Case 3 DHLTHL
bull Retrospective study from one institution features of 13 cases (9 DHL4
THL)
bull median age was 59 years (range 30-74) eight females five males
bull enlarging lymphadenopathymasses (11 patients) abnormal peripheral
blood findings (2 patients)
bull TdT positivity (four cases two THLtwo DHL) dim CD45 expression (7
cases) lack of CD20 (2 cases) or lack of sIg (3 cases) and blastoid
morphology (2 cases)
bull Conclusion Expression of TdT in a B-cell lymphoma with mature
features or expression of surface light chain in a case otherwise
suggestive of B-lymphoblastic leukemialymphoma should prompt
an expedited evaluation for DHLTHL
Leuk Lymphoma 2016 Feb 181-10 [Epub ahead of print]
Double- and triple-hit lymphomas can present with features suggestive
of immaturity including TdT expression and create diagnostic challenges
Moench L Sachs Z Aasen G Dolan M Dayton V Courville EL
Case 4
bull 55 year old female
bull Previous history hyperparathyroidism
bull Parathyroidectomy in 1999
bull Mild persistent hypercalcemia
bull WBC 4800uL hemoglobin 121 gL Plt 296
bull Total protein 90gL IgG 34gL monoclonal peak
209gL
Case 4 Monoclonal IgG patient
Polyclonal B cells
With maturing hematogones
helper suppressor T cells
less than 3 blasts
Maturing myeloid population
Case 4 BM Ungated
Case 4 cytoplasmic immunoglobulin
Need KL
Case 4 BM Multiple Myeloma
Case 4 follow up
bull Smouldering myeloma
bull Recently developed CRAB (hyperCalcemia Renal
insufficiency Anemia Bone lesions
bull Serum free light chain ratio 123
bull Bone marrow aspirate gt60 plasma cells
bull Currently on treatment pending bone marrow
transplant
Case 5
bull 65 year old male
bull Slight lymphocytosis detected on routine CBC analysis
(lymph 4500uL)
bull Asymptomatic sent for flow cytometry
Case 5 PB
Case 5 flow cytometry KL ratio 0625
Case 5 Monoclonal B cell lymphocytosis
KL 18 KL 056
Case 5 Monoclonal B cell lymphocytosis
bull WHO defines CLL as abnormal lymphocyte count
gt5x10E9L (CD19+CD5+ light chain restricted) in the
absence of other clinical symptoms
bull Patients with small B cell clones are often detected as
an incidental finding
bull WHO 2016
bull CD19+CD5+ lt05x10E9L ldquoLow count MBLrdquo
raquo 05-5x10E9L ldquoHigh count MBLrdquo
raquo Non CLL type phenotype MBL also recognized
Monoclonal B cell Lymphocytosis
bull CLL immunophenotype - vast majority (75)
bull Atypical MBL
bull normal levels both CD5 and CD20 with variable
expression of CD23 (always exclude Mantle-cell
Lymphoma in CD23 negative cases)
bull Non-CLL-phenotype (CD5minus) MBL
bull Lacking CD5 expression and CD10 likely resembling B-
cell lymphomas of marginal-zone origin
Case 5 What is the normal KL Ratio
bull What is the normal or abnormal kappalambda ratio for
entire ldquopopulationrdquo of all B cells
bull Traditional view
ndash Kappa Lambda 21 or 31 ( 12 -13)
ndash Range 14 60
Case 5 What is the normal KL ratio
bull Wrong question
bull It doesnrsquot matter how many of the B cells are clonal but rather
that their exists a clonal population of B cells within the total
population
bull Need to Identify malignant B cells within a population of
normal B cells
bull In order to interpret a pathological immunophenotype it is
necessary to refer to quantitative and qualitative values of
normal B-cell subpopulations
bull Pearl Identify the abnormal phenotype first then look at KL
ratio
Case 6
bull 12 year old male presents to ER with fatigue bruising
Lkc 2200uL
bull Hb 84gL
bull Plt 32
bull Diff ndash neutropenia no blasts noted few ldquoatypicalrdquo cells
bull RBC ndash non specific changes
Leukemia
Case 6 PB
Case 6 BM
Case 6
bull BM aspirate ndash Hypocellular relative lymphocytosis
marked reduction in myeloid lineages
hypoplasticaplastic anemia
bull Flow Cytometry ndash less than 3 blasts maturing
myeloid 68 lymphocytes polyclonal B helper
suppressor T
bull Unusual pattern of CD13CD16 staining noted
Case 6 flow cytometry Aplastic Anemia
Normal Patient
Normal
Patient
Patient
Normal
Case 6 RBC Test for PNH CD235a v s CD59
Case 6 Neutrophil test for PNH clone
FLAER CD24 CD45CD15
18 PNH clone
Case 6
bull Follow up
bull Patient on steroids acyclovir and anti-thymocyte
globulin
bull PNH clone Neutrophils 96 RBC 1 Nov 2015
bull In remission as of April 2017
- Lkc 5500uL (Neuts 23)
- Hb 129gL
- Plt 208 X10e9
Summary
bull B cell malignancies are the most common
hematological disorders seen in flow cytometry
bull Many have complex overlapping phenotypes
bull Important to look at ungated plots as well as gating on
specific subpopulations
bull Level of expression of markers is as important as
positive or negative (think CD13 vs CD16)
68
References
bull Craig FE Foon KA Flow cytometric immunophenotyping for hematologic neoplasms Blood
2008 Apr 15111(8)3941-67
bull Wood BL Principles of minimal residual disease detection for hematopoietic neoplasms by flow
cytometry Cytometry B Clin Cytom 2015 Apr 23
bull Shanafelt TD Ghia P Lanasa MC Landgren O Rawstron AC Monoclonal B-cell
lymphocytosis (MBL) biology natural history and clinical management Leukemia 2010
Mar24(3)512-20
bull Swerdlow SH Campo E Pileri SA Harris NL Stein H Siebert R Advani RGhielmini M Salles
GA Zelenetz AD Jaffe ES The 2016 revision of the World Health Organization classification of
lymphoid neoplasms Blood 2016 May 19127(20)2375-90
bull 3 Moench L Sachs Z Aasen G Dolan M Dayton V Courville EL Double- and triple-hit
lymphomas can present with features suggestive of immaturity including TdT expression and
create diagnostic challenges Leuk Lymphoma 2016572626-35
bull Sutherland DR Keeney M Illingworth A Practical guidelines for the high-sensitivity detection
and monitoring of paroxysmal nocturnal hemoglobinuria clones by flow cytometry Cytometry B
Clin Cytom 2012 Jul82(4)195-208
69
Case 2 BM flow cytometry
Polyclonal B cells
helper suppressor T cells
No increase in blasts
Maturing myeloid population
Case 2 CD45 neg B-ALL
Case 2 BM
Case 2
bull CD45neg
bull CD19+CD10+
bull CD20(partialdim)
bull CD34 neg
bull SigampcIg neg
bull Cytogenetics
bull Gain of X chromosome and
468911131415171821
DNA Index
122 Diploid
Case 2
bull Currently in remission after completion of
AALL0932 standard risk B-ALL therapy
Case 2
Pearls of Pathology
CD45 negative B-ALL is not uncommon
bull Always look at ungated set of plots for each marker
bull Eg SS versus each fluorescence parameter
Case 3
bull 58 year old male with pararenal mass
bull WBC 4000uL Hb 98gL Plt 229
bull ldquoblastsrdquo reported on blood film
bull Peripheral blood sent for flow cytometry
Case 3 PB flow cytometry
Dim CD45 CD19+CD10+
CD34- Sig neg
Case 3 PB flow cytometry
Dim CD45 CD19+CD10+
CD20(partial)CD5 neg
SIg cIg neg
Case 3 BM flow cytometry
Case 3 BM biopsy
Case 3 BM biopsy
Case 3 BM biopsy report
bull Diffuse infiltrate large lymphoid cells
bull Scant to moderate cytoplasm
bull Large irregular nuclei with prominent nucleoli
bull Increased mitotic activity
bull Positive for BCL-2 BCL6 CD10 and MUM1
bull Negative for TdT CD5
bull Cytogenetics FISH on para renal mass ndash C-MYC and
BCL2 rearrangements detected
Case 3 Doubletriple hit lymphoma
(DHLTHL)
bull Morphologically heterogeneous B cell neoplasms
characterized by rearrangement of the MYC proto-
oncogene with concurrent BCL2 andor BCL6 gene
rearrangement
bull WHO 2008 criteria - most cases of DHL and THL
classified DLBCL or B-cell lymphoma unclassifiable
with features intermediate between DLBCL and Burkitt
lymphoma (BCLU)
Case 3 DHLTHL
bull Retrospective study from one institution features of 13 cases (9 DHL4
THL)
bull median age was 59 years (range 30-74) eight females five males
bull enlarging lymphadenopathymasses (11 patients) abnormal peripheral
blood findings (2 patients)
bull TdT positivity (four cases two THLtwo DHL) dim CD45 expression (7
cases) lack of CD20 (2 cases) or lack of sIg (3 cases) and blastoid
morphology (2 cases)
bull Conclusion Expression of TdT in a B-cell lymphoma with mature
features or expression of surface light chain in a case otherwise
suggestive of B-lymphoblastic leukemialymphoma should prompt
an expedited evaluation for DHLTHL
Leuk Lymphoma 2016 Feb 181-10 [Epub ahead of print]
Double- and triple-hit lymphomas can present with features suggestive
of immaturity including TdT expression and create diagnostic challenges
Moench L Sachs Z Aasen G Dolan M Dayton V Courville EL
Case 4
bull 55 year old female
bull Previous history hyperparathyroidism
bull Parathyroidectomy in 1999
bull Mild persistent hypercalcemia
bull WBC 4800uL hemoglobin 121 gL Plt 296
bull Total protein 90gL IgG 34gL monoclonal peak
209gL
Case 4 Monoclonal IgG patient
Polyclonal B cells
With maturing hematogones
helper suppressor T cells
less than 3 blasts
Maturing myeloid population
Case 4 BM Ungated
Case 4 cytoplasmic immunoglobulin
Need KL
Case 4 BM Multiple Myeloma
Case 4 follow up
bull Smouldering myeloma
bull Recently developed CRAB (hyperCalcemia Renal
insufficiency Anemia Bone lesions
bull Serum free light chain ratio 123
bull Bone marrow aspirate gt60 plasma cells
bull Currently on treatment pending bone marrow
transplant
Case 5
bull 65 year old male
bull Slight lymphocytosis detected on routine CBC analysis
(lymph 4500uL)
bull Asymptomatic sent for flow cytometry
Case 5 PB
Case 5 flow cytometry KL ratio 0625
Case 5 Monoclonal B cell lymphocytosis
KL 18 KL 056
Case 5 Monoclonal B cell lymphocytosis
bull WHO defines CLL as abnormal lymphocyte count
gt5x10E9L (CD19+CD5+ light chain restricted) in the
absence of other clinical symptoms
bull Patients with small B cell clones are often detected as
an incidental finding
bull WHO 2016
bull CD19+CD5+ lt05x10E9L ldquoLow count MBLrdquo
raquo 05-5x10E9L ldquoHigh count MBLrdquo
raquo Non CLL type phenotype MBL also recognized
Monoclonal B cell Lymphocytosis
bull CLL immunophenotype - vast majority (75)
bull Atypical MBL
bull normal levels both CD5 and CD20 with variable
expression of CD23 (always exclude Mantle-cell
Lymphoma in CD23 negative cases)
bull Non-CLL-phenotype (CD5minus) MBL
bull Lacking CD5 expression and CD10 likely resembling B-
cell lymphomas of marginal-zone origin
Case 5 What is the normal KL Ratio
bull What is the normal or abnormal kappalambda ratio for
entire ldquopopulationrdquo of all B cells
bull Traditional view
ndash Kappa Lambda 21 or 31 ( 12 -13)
ndash Range 14 60
Case 5 What is the normal KL ratio
bull Wrong question
bull It doesnrsquot matter how many of the B cells are clonal but rather
that their exists a clonal population of B cells within the total
population
bull Need to Identify malignant B cells within a population of
normal B cells
bull In order to interpret a pathological immunophenotype it is
necessary to refer to quantitative and qualitative values of
normal B-cell subpopulations
bull Pearl Identify the abnormal phenotype first then look at KL
ratio
Case 6
bull 12 year old male presents to ER with fatigue bruising
Lkc 2200uL
bull Hb 84gL
bull Plt 32
bull Diff ndash neutropenia no blasts noted few ldquoatypicalrdquo cells
bull RBC ndash non specific changes
Leukemia
Case 6 PB
Case 6 BM
Case 6
bull BM aspirate ndash Hypocellular relative lymphocytosis
marked reduction in myeloid lineages
hypoplasticaplastic anemia
bull Flow Cytometry ndash less than 3 blasts maturing
myeloid 68 lymphocytes polyclonal B helper
suppressor T
bull Unusual pattern of CD13CD16 staining noted
Case 6 flow cytometry Aplastic Anemia
Normal Patient
Normal
Patient
Patient
Normal
Case 6 RBC Test for PNH CD235a v s CD59
Case 6 Neutrophil test for PNH clone
FLAER CD24 CD45CD15
18 PNH clone
Case 6
bull Follow up
bull Patient on steroids acyclovir and anti-thymocyte
globulin
bull PNH clone Neutrophils 96 RBC 1 Nov 2015
bull In remission as of April 2017
- Lkc 5500uL (Neuts 23)
- Hb 129gL
- Plt 208 X10e9
Summary
bull B cell malignancies are the most common
hematological disorders seen in flow cytometry
bull Many have complex overlapping phenotypes
bull Important to look at ungated plots as well as gating on
specific subpopulations
bull Level of expression of markers is as important as
positive or negative (think CD13 vs CD16)
68
References
bull Craig FE Foon KA Flow cytometric immunophenotyping for hematologic neoplasms Blood
2008 Apr 15111(8)3941-67
bull Wood BL Principles of minimal residual disease detection for hematopoietic neoplasms by flow
cytometry Cytometry B Clin Cytom 2015 Apr 23
bull Shanafelt TD Ghia P Lanasa MC Landgren O Rawstron AC Monoclonal B-cell
lymphocytosis (MBL) biology natural history and clinical management Leukemia 2010
Mar24(3)512-20
bull Swerdlow SH Campo E Pileri SA Harris NL Stein H Siebert R Advani RGhielmini M Salles
GA Zelenetz AD Jaffe ES The 2016 revision of the World Health Organization classification of
lymphoid neoplasms Blood 2016 May 19127(20)2375-90
bull 3 Moench L Sachs Z Aasen G Dolan M Dayton V Courville EL Double- and triple-hit
lymphomas can present with features suggestive of immaturity including TdT expression and
create diagnostic challenges Leuk Lymphoma 2016572626-35
bull Sutherland DR Keeney M Illingworth A Practical guidelines for the high-sensitivity detection
and monitoring of paroxysmal nocturnal hemoglobinuria clones by flow cytometry Cytometry B
Clin Cytom 2012 Jul82(4)195-208
69
Case 2 CD45 neg B-ALL
Case 2 BM
Case 2
bull CD45neg
bull CD19+CD10+
bull CD20(partialdim)
bull CD34 neg
bull SigampcIg neg
bull Cytogenetics
bull Gain of X chromosome and
468911131415171821
DNA Index
122 Diploid
Case 2
bull Currently in remission after completion of
AALL0932 standard risk B-ALL therapy
Case 2
Pearls of Pathology
CD45 negative B-ALL is not uncommon
bull Always look at ungated set of plots for each marker
bull Eg SS versus each fluorescence parameter
Case 3
bull 58 year old male with pararenal mass
bull WBC 4000uL Hb 98gL Plt 229
bull ldquoblastsrdquo reported on blood film
bull Peripheral blood sent for flow cytometry
Case 3 PB flow cytometry
Dim CD45 CD19+CD10+
CD34- Sig neg
Case 3 PB flow cytometry
Dim CD45 CD19+CD10+
CD20(partial)CD5 neg
SIg cIg neg
Case 3 BM flow cytometry
Case 3 BM biopsy
Case 3 BM biopsy
Case 3 BM biopsy report
bull Diffuse infiltrate large lymphoid cells
bull Scant to moderate cytoplasm
bull Large irregular nuclei with prominent nucleoli
bull Increased mitotic activity
bull Positive for BCL-2 BCL6 CD10 and MUM1
bull Negative for TdT CD5
bull Cytogenetics FISH on para renal mass ndash C-MYC and
BCL2 rearrangements detected
Case 3 Doubletriple hit lymphoma
(DHLTHL)
bull Morphologically heterogeneous B cell neoplasms
characterized by rearrangement of the MYC proto-
oncogene with concurrent BCL2 andor BCL6 gene
rearrangement
bull WHO 2008 criteria - most cases of DHL and THL
classified DLBCL or B-cell lymphoma unclassifiable
with features intermediate between DLBCL and Burkitt
lymphoma (BCLU)
Case 3 DHLTHL
bull Retrospective study from one institution features of 13 cases (9 DHL4
THL)
bull median age was 59 years (range 30-74) eight females five males
bull enlarging lymphadenopathymasses (11 patients) abnormal peripheral
blood findings (2 patients)
bull TdT positivity (four cases two THLtwo DHL) dim CD45 expression (7
cases) lack of CD20 (2 cases) or lack of sIg (3 cases) and blastoid
morphology (2 cases)
bull Conclusion Expression of TdT in a B-cell lymphoma with mature
features or expression of surface light chain in a case otherwise
suggestive of B-lymphoblastic leukemialymphoma should prompt
an expedited evaluation for DHLTHL
Leuk Lymphoma 2016 Feb 181-10 [Epub ahead of print]
Double- and triple-hit lymphomas can present with features suggestive
of immaturity including TdT expression and create diagnostic challenges
Moench L Sachs Z Aasen G Dolan M Dayton V Courville EL
Case 4
bull 55 year old female
bull Previous history hyperparathyroidism
bull Parathyroidectomy in 1999
bull Mild persistent hypercalcemia
bull WBC 4800uL hemoglobin 121 gL Plt 296
bull Total protein 90gL IgG 34gL monoclonal peak
209gL
Case 4 Monoclonal IgG patient
Polyclonal B cells
With maturing hematogones
helper suppressor T cells
less than 3 blasts
Maturing myeloid population
Case 4 BM Ungated
Case 4 cytoplasmic immunoglobulin
Need KL
Case 4 BM Multiple Myeloma
Case 4 follow up
bull Smouldering myeloma
bull Recently developed CRAB (hyperCalcemia Renal
insufficiency Anemia Bone lesions
bull Serum free light chain ratio 123
bull Bone marrow aspirate gt60 plasma cells
bull Currently on treatment pending bone marrow
transplant
Case 5
bull 65 year old male
bull Slight lymphocytosis detected on routine CBC analysis
(lymph 4500uL)
bull Asymptomatic sent for flow cytometry
Case 5 PB
Case 5 flow cytometry KL ratio 0625
Case 5 Monoclonal B cell lymphocytosis
KL 18 KL 056
Case 5 Monoclonal B cell lymphocytosis
bull WHO defines CLL as abnormal lymphocyte count
gt5x10E9L (CD19+CD5+ light chain restricted) in the
absence of other clinical symptoms
bull Patients with small B cell clones are often detected as
an incidental finding
bull WHO 2016
bull CD19+CD5+ lt05x10E9L ldquoLow count MBLrdquo
raquo 05-5x10E9L ldquoHigh count MBLrdquo
raquo Non CLL type phenotype MBL also recognized
Monoclonal B cell Lymphocytosis
bull CLL immunophenotype - vast majority (75)
bull Atypical MBL
bull normal levels both CD5 and CD20 with variable
expression of CD23 (always exclude Mantle-cell
Lymphoma in CD23 negative cases)
bull Non-CLL-phenotype (CD5minus) MBL
bull Lacking CD5 expression and CD10 likely resembling B-
cell lymphomas of marginal-zone origin
Case 5 What is the normal KL Ratio
bull What is the normal or abnormal kappalambda ratio for
entire ldquopopulationrdquo of all B cells
bull Traditional view
ndash Kappa Lambda 21 or 31 ( 12 -13)
ndash Range 14 60
Case 5 What is the normal KL ratio
bull Wrong question
bull It doesnrsquot matter how many of the B cells are clonal but rather
that their exists a clonal population of B cells within the total
population
bull Need to Identify malignant B cells within a population of
normal B cells
bull In order to interpret a pathological immunophenotype it is
necessary to refer to quantitative and qualitative values of
normal B-cell subpopulations
bull Pearl Identify the abnormal phenotype first then look at KL
ratio
Case 6
bull 12 year old male presents to ER with fatigue bruising
Lkc 2200uL
bull Hb 84gL
bull Plt 32
bull Diff ndash neutropenia no blasts noted few ldquoatypicalrdquo cells
bull RBC ndash non specific changes
Leukemia
Case 6 PB
Case 6 BM
Case 6
bull BM aspirate ndash Hypocellular relative lymphocytosis
marked reduction in myeloid lineages
hypoplasticaplastic anemia
bull Flow Cytometry ndash less than 3 blasts maturing
myeloid 68 lymphocytes polyclonal B helper
suppressor T
bull Unusual pattern of CD13CD16 staining noted
Case 6 flow cytometry Aplastic Anemia
Normal Patient
Normal
Patient
Patient
Normal
Case 6 RBC Test for PNH CD235a v s CD59
Case 6 Neutrophil test for PNH clone
FLAER CD24 CD45CD15
18 PNH clone
Case 6
bull Follow up
bull Patient on steroids acyclovir and anti-thymocyte
globulin
bull PNH clone Neutrophils 96 RBC 1 Nov 2015
bull In remission as of April 2017
- Lkc 5500uL (Neuts 23)
- Hb 129gL
- Plt 208 X10e9
Summary
bull B cell malignancies are the most common
hematological disorders seen in flow cytometry
bull Many have complex overlapping phenotypes
bull Important to look at ungated plots as well as gating on
specific subpopulations
bull Level of expression of markers is as important as
positive or negative (think CD13 vs CD16)
68
References
bull Craig FE Foon KA Flow cytometric immunophenotyping for hematologic neoplasms Blood
2008 Apr 15111(8)3941-67
bull Wood BL Principles of minimal residual disease detection for hematopoietic neoplasms by flow
cytometry Cytometry B Clin Cytom 2015 Apr 23
bull Shanafelt TD Ghia P Lanasa MC Landgren O Rawstron AC Monoclonal B-cell
lymphocytosis (MBL) biology natural history and clinical management Leukemia 2010
Mar24(3)512-20
bull Swerdlow SH Campo E Pileri SA Harris NL Stein H Siebert R Advani RGhielmini M Salles
GA Zelenetz AD Jaffe ES The 2016 revision of the World Health Organization classification of
lymphoid neoplasms Blood 2016 May 19127(20)2375-90
bull 3 Moench L Sachs Z Aasen G Dolan M Dayton V Courville EL Double- and triple-hit
lymphomas can present with features suggestive of immaturity including TdT expression and
create diagnostic challenges Leuk Lymphoma 2016572626-35
bull Sutherland DR Keeney M Illingworth A Practical guidelines for the high-sensitivity detection
and monitoring of paroxysmal nocturnal hemoglobinuria clones by flow cytometry Cytometry B
Clin Cytom 2012 Jul82(4)195-208
69
Case 2 BM
Case 2
bull CD45neg
bull CD19+CD10+
bull CD20(partialdim)
bull CD34 neg
bull SigampcIg neg
bull Cytogenetics
bull Gain of X chromosome and
468911131415171821
DNA Index
122 Diploid
Case 2
bull Currently in remission after completion of
AALL0932 standard risk B-ALL therapy
Case 2
Pearls of Pathology
CD45 negative B-ALL is not uncommon
bull Always look at ungated set of plots for each marker
bull Eg SS versus each fluorescence parameter
Case 3
bull 58 year old male with pararenal mass
bull WBC 4000uL Hb 98gL Plt 229
bull ldquoblastsrdquo reported on blood film
bull Peripheral blood sent for flow cytometry
Case 3 PB flow cytometry
Dim CD45 CD19+CD10+
CD34- Sig neg
Case 3 PB flow cytometry
Dim CD45 CD19+CD10+
CD20(partial)CD5 neg
SIg cIg neg
Case 3 BM flow cytometry
Case 3 BM biopsy
Case 3 BM biopsy
Case 3 BM biopsy report
bull Diffuse infiltrate large lymphoid cells
bull Scant to moderate cytoplasm
bull Large irregular nuclei with prominent nucleoli
bull Increased mitotic activity
bull Positive for BCL-2 BCL6 CD10 and MUM1
bull Negative for TdT CD5
bull Cytogenetics FISH on para renal mass ndash C-MYC and
BCL2 rearrangements detected
Case 3 Doubletriple hit lymphoma
(DHLTHL)
bull Morphologically heterogeneous B cell neoplasms
characterized by rearrangement of the MYC proto-
oncogene with concurrent BCL2 andor BCL6 gene
rearrangement
bull WHO 2008 criteria - most cases of DHL and THL
classified DLBCL or B-cell lymphoma unclassifiable
with features intermediate between DLBCL and Burkitt
lymphoma (BCLU)
Case 3 DHLTHL
bull Retrospective study from one institution features of 13 cases (9 DHL4
THL)
bull median age was 59 years (range 30-74) eight females five males
bull enlarging lymphadenopathymasses (11 patients) abnormal peripheral
blood findings (2 patients)
bull TdT positivity (four cases two THLtwo DHL) dim CD45 expression (7
cases) lack of CD20 (2 cases) or lack of sIg (3 cases) and blastoid
morphology (2 cases)
bull Conclusion Expression of TdT in a B-cell lymphoma with mature
features or expression of surface light chain in a case otherwise
suggestive of B-lymphoblastic leukemialymphoma should prompt
an expedited evaluation for DHLTHL
Leuk Lymphoma 2016 Feb 181-10 [Epub ahead of print]
Double- and triple-hit lymphomas can present with features suggestive
of immaturity including TdT expression and create diagnostic challenges
Moench L Sachs Z Aasen G Dolan M Dayton V Courville EL
Case 4
bull 55 year old female
bull Previous history hyperparathyroidism
bull Parathyroidectomy in 1999
bull Mild persistent hypercalcemia
bull WBC 4800uL hemoglobin 121 gL Plt 296
bull Total protein 90gL IgG 34gL monoclonal peak
209gL
Case 4 Monoclonal IgG patient
Polyclonal B cells
With maturing hematogones
helper suppressor T cells
less than 3 blasts
Maturing myeloid population
Case 4 BM Ungated
Case 4 cytoplasmic immunoglobulin
Need KL
Case 4 BM Multiple Myeloma
Case 4 follow up
bull Smouldering myeloma
bull Recently developed CRAB (hyperCalcemia Renal
insufficiency Anemia Bone lesions
bull Serum free light chain ratio 123
bull Bone marrow aspirate gt60 plasma cells
bull Currently on treatment pending bone marrow
transplant
Case 5
bull 65 year old male
bull Slight lymphocytosis detected on routine CBC analysis
(lymph 4500uL)
bull Asymptomatic sent for flow cytometry
Case 5 PB
Case 5 flow cytometry KL ratio 0625
Case 5 Monoclonal B cell lymphocytosis
KL 18 KL 056
Case 5 Monoclonal B cell lymphocytosis
bull WHO defines CLL as abnormal lymphocyte count
gt5x10E9L (CD19+CD5+ light chain restricted) in the
absence of other clinical symptoms
bull Patients with small B cell clones are often detected as
an incidental finding
bull WHO 2016
bull CD19+CD5+ lt05x10E9L ldquoLow count MBLrdquo
raquo 05-5x10E9L ldquoHigh count MBLrdquo
raquo Non CLL type phenotype MBL also recognized
Monoclonal B cell Lymphocytosis
bull CLL immunophenotype - vast majority (75)
bull Atypical MBL
bull normal levels both CD5 and CD20 with variable
expression of CD23 (always exclude Mantle-cell
Lymphoma in CD23 negative cases)
bull Non-CLL-phenotype (CD5minus) MBL
bull Lacking CD5 expression and CD10 likely resembling B-
cell lymphomas of marginal-zone origin
Case 5 What is the normal KL Ratio
bull What is the normal or abnormal kappalambda ratio for
entire ldquopopulationrdquo of all B cells
bull Traditional view
ndash Kappa Lambda 21 or 31 ( 12 -13)
ndash Range 14 60
Case 5 What is the normal KL ratio
bull Wrong question
bull It doesnrsquot matter how many of the B cells are clonal but rather
that their exists a clonal population of B cells within the total
population
bull Need to Identify malignant B cells within a population of
normal B cells
bull In order to interpret a pathological immunophenotype it is
necessary to refer to quantitative and qualitative values of
normal B-cell subpopulations
bull Pearl Identify the abnormal phenotype first then look at KL
ratio
Case 6
bull 12 year old male presents to ER with fatigue bruising
Lkc 2200uL
bull Hb 84gL
bull Plt 32
bull Diff ndash neutropenia no blasts noted few ldquoatypicalrdquo cells
bull RBC ndash non specific changes
Leukemia
Case 6 PB
Case 6 BM
Case 6
bull BM aspirate ndash Hypocellular relative lymphocytosis
marked reduction in myeloid lineages
hypoplasticaplastic anemia
bull Flow Cytometry ndash less than 3 blasts maturing
myeloid 68 lymphocytes polyclonal B helper
suppressor T
bull Unusual pattern of CD13CD16 staining noted
Case 6 flow cytometry Aplastic Anemia
Normal Patient
Normal
Patient
Patient
Normal
Case 6 RBC Test for PNH CD235a v s CD59
Case 6 Neutrophil test for PNH clone
FLAER CD24 CD45CD15
18 PNH clone
Case 6
bull Follow up
bull Patient on steroids acyclovir and anti-thymocyte
globulin
bull PNH clone Neutrophils 96 RBC 1 Nov 2015
bull In remission as of April 2017
- Lkc 5500uL (Neuts 23)
- Hb 129gL
- Plt 208 X10e9
Summary
bull B cell malignancies are the most common
hematological disorders seen in flow cytometry
bull Many have complex overlapping phenotypes
bull Important to look at ungated plots as well as gating on
specific subpopulations
bull Level of expression of markers is as important as
positive or negative (think CD13 vs CD16)
68
References
bull Craig FE Foon KA Flow cytometric immunophenotyping for hematologic neoplasms Blood
2008 Apr 15111(8)3941-67
bull Wood BL Principles of minimal residual disease detection for hematopoietic neoplasms by flow
cytometry Cytometry B Clin Cytom 2015 Apr 23
bull Shanafelt TD Ghia P Lanasa MC Landgren O Rawstron AC Monoclonal B-cell
lymphocytosis (MBL) biology natural history and clinical management Leukemia 2010
Mar24(3)512-20
bull Swerdlow SH Campo E Pileri SA Harris NL Stein H Siebert R Advani RGhielmini M Salles
GA Zelenetz AD Jaffe ES The 2016 revision of the World Health Organization classification of
lymphoid neoplasms Blood 2016 May 19127(20)2375-90
bull 3 Moench L Sachs Z Aasen G Dolan M Dayton V Courville EL Double- and triple-hit
lymphomas can present with features suggestive of immaturity including TdT expression and
create diagnostic challenges Leuk Lymphoma 2016572626-35
bull Sutherland DR Keeney M Illingworth A Practical guidelines for the high-sensitivity detection
and monitoring of paroxysmal nocturnal hemoglobinuria clones by flow cytometry Cytometry B
Clin Cytom 2012 Jul82(4)195-208
69
Case 2
bull CD45neg
bull CD19+CD10+
bull CD20(partialdim)
bull CD34 neg
bull SigampcIg neg
bull Cytogenetics
bull Gain of X chromosome and
468911131415171821
DNA Index
122 Diploid
Case 2
bull Currently in remission after completion of
AALL0932 standard risk B-ALL therapy
Case 2
Pearls of Pathology
CD45 negative B-ALL is not uncommon
bull Always look at ungated set of plots for each marker
bull Eg SS versus each fluorescence parameter
Case 3
bull 58 year old male with pararenal mass
bull WBC 4000uL Hb 98gL Plt 229
bull ldquoblastsrdquo reported on blood film
bull Peripheral blood sent for flow cytometry
Case 3 PB flow cytometry
Dim CD45 CD19+CD10+
CD34- Sig neg
Case 3 PB flow cytometry
Dim CD45 CD19+CD10+
CD20(partial)CD5 neg
SIg cIg neg
Case 3 BM flow cytometry
Case 3 BM biopsy
Case 3 BM biopsy
Case 3 BM biopsy report
bull Diffuse infiltrate large lymphoid cells
bull Scant to moderate cytoplasm
bull Large irregular nuclei with prominent nucleoli
bull Increased mitotic activity
bull Positive for BCL-2 BCL6 CD10 and MUM1
bull Negative for TdT CD5
bull Cytogenetics FISH on para renal mass ndash C-MYC and
BCL2 rearrangements detected
Case 3 Doubletriple hit lymphoma
(DHLTHL)
bull Morphologically heterogeneous B cell neoplasms
characterized by rearrangement of the MYC proto-
oncogene with concurrent BCL2 andor BCL6 gene
rearrangement
bull WHO 2008 criteria - most cases of DHL and THL
classified DLBCL or B-cell lymphoma unclassifiable
with features intermediate between DLBCL and Burkitt
lymphoma (BCLU)
Case 3 DHLTHL
bull Retrospective study from one institution features of 13 cases (9 DHL4
THL)
bull median age was 59 years (range 30-74) eight females five males
bull enlarging lymphadenopathymasses (11 patients) abnormal peripheral
blood findings (2 patients)
bull TdT positivity (four cases two THLtwo DHL) dim CD45 expression (7
cases) lack of CD20 (2 cases) or lack of sIg (3 cases) and blastoid
morphology (2 cases)
bull Conclusion Expression of TdT in a B-cell lymphoma with mature
features or expression of surface light chain in a case otherwise
suggestive of B-lymphoblastic leukemialymphoma should prompt
an expedited evaluation for DHLTHL
Leuk Lymphoma 2016 Feb 181-10 [Epub ahead of print]
Double- and triple-hit lymphomas can present with features suggestive
of immaturity including TdT expression and create diagnostic challenges
Moench L Sachs Z Aasen G Dolan M Dayton V Courville EL
Case 4
bull 55 year old female
bull Previous history hyperparathyroidism
bull Parathyroidectomy in 1999
bull Mild persistent hypercalcemia
bull WBC 4800uL hemoglobin 121 gL Plt 296
bull Total protein 90gL IgG 34gL monoclonal peak
209gL
Case 4 Monoclonal IgG patient
Polyclonal B cells
With maturing hematogones
helper suppressor T cells
less than 3 blasts
Maturing myeloid population
Case 4 BM Ungated
Case 4 cytoplasmic immunoglobulin
Need KL
Case 4 BM Multiple Myeloma
Case 4 follow up
bull Smouldering myeloma
bull Recently developed CRAB (hyperCalcemia Renal
insufficiency Anemia Bone lesions
bull Serum free light chain ratio 123
bull Bone marrow aspirate gt60 plasma cells
bull Currently on treatment pending bone marrow
transplant
Case 5
bull 65 year old male
bull Slight lymphocytosis detected on routine CBC analysis
(lymph 4500uL)
bull Asymptomatic sent for flow cytometry
Case 5 PB
Case 5 flow cytometry KL ratio 0625
Case 5 Monoclonal B cell lymphocytosis
KL 18 KL 056
Case 5 Monoclonal B cell lymphocytosis
bull WHO defines CLL as abnormal lymphocyte count
gt5x10E9L (CD19+CD5+ light chain restricted) in the
absence of other clinical symptoms
bull Patients with small B cell clones are often detected as
an incidental finding
bull WHO 2016
bull CD19+CD5+ lt05x10E9L ldquoLow count MBLrdquo
raquo 05-5x10E9L ldquoHigh count MBLrdquo
raquo Non CLL type phenotype MBL also recognized
Monoclonal B cell Lymphocytosis
bull CLL immunophenotype - vast majority (75)
bull Atypical MBL
bull normal levels both CD5 and CD20 with variable
expression of CD23 (always exclude Mantle-cell
Lymphoma in CD23 negative cases)
bull Non-CLL-phenotype (CD5minus) MBL
bull Lacking CD5 expression and CD10 likely resembling B-
cell lymphomas of marginal-zone origin
Case 5 What is the normal KL Ratio
bull What is the normal or abnormal kappalambda ratio for
entire ldquopopulationrdquo of all B cells
bull Traditional view
ndash Kappa Lambda 21 or 31 ( 12 -13)
ndash Range 14 60
Case 5 What is the normal KL ratio
bull Wrong question
bull It doesnrsquot matter how many of the B cells are clonal but rather
that their exists a clonal population of B cells within the total
population
bull Need to Identify malignant B cells within a population of
normal B cells
bull In order to interpret a pathological immunophenotype it is
necessary to refer to quantitative and qualitative values of
normal B-cell subpopulations
bull Pearl Identify the abnormal phenotype first then look at KL
ratio
Case 6
bull 12 year old male presents to ER with fatigue bruising
Lkc 2200uL
bull Hb 84gL
bull Plt 32
bull Diff ndash neutropenia no blasts noted few ldquoatypicalrdquo cells
bull RBC ndash non specific changes
Leukemia
Case 6 PB
Case 6 BM
Case 6
bull BM aspirate ndash Hypocellular relative lymphocytosis
marked reduction in myeloid lineages
hypoplasticaplastic anemia
bull Flow Cytometry ndash less than 3 blasts maturing
myeloid 68 lymphocytes polyclonal B helper
suppressor T
bull Unusual pattern of CD13CD16 staining noted
Case 6 flow cytometry Aplastic Anemia
Normal Patient
Normal
Patient
Patient
Normal
Case 6 RBC Test for PNH CD235a v s CD59
Case 6 Neutrophil test for PNH clone
FLAER CD24 CD45CD15
18 PNH clone
Case 6
bull Follow up
bull Patient on steroids acyclovir and anti-thymocyte
globulin
bull PNH clone Neutrophils 96 RBC 1 Nov 2015
bull In remission as of April 2017
- Lkc 5500uL (Neuts 23)
- Hb 129gL
- Plt 208 X10e9
Summary
bull B cell malignancies are the most common
hematological disorders seen in flow cytometry
bull Many have complex overlapping phenotypes
bull Important to look at ungated plots as well as gating on
specific subpopulations
bull Level of expression of markers is as important as
positive or negative (think CD13 vs CD16)
68
References
bull Craig FE Foon KA Flow cytometric immunophenotyping for hematologic neoplasms Blood
2008 Apr 15111(8)3941-67
bull Wood BL Principles of minimal residual disease detection for hematopoietic neoplasms by flow
cytometry Cytometry B Clin Cytom 2015 Apr 23
bull Shanafelt TD Ghia P Lanasa MC Landgren O Rawstron AC Monoclonal B-cell
lymphocytosis (MBL) biology natural history and clinical management Leukemia 2010
Mar24(3)512-20
bull Swerdlow SH Campo E Pileri SA Harris NL Stein H Siebert R Advani RGhielmini M Salles
GA Zelenetz AD Jaffe ES The 2016 revision of the World Health Organization classification of
lymphoid neoplasms Blood 2016 May 19127(20)2375-90
bull 3 Moench L Sachs Z Aasen G Dolan M Dayton V Courville EL Double- and triple-hit
lymphomas can present with features suggestive of immaturity including TdT expression and
create diagnostic challenges Leuk Lymphoma 2016572626-35
bull Sutherland DR Keeney M Illingworth A Practical guidelines for the high-sensitivity detection
and monitoring of paroxysmal nocturnal hemoglobinuria clones by flow cytometry Cytometry B
Clin Cytom 2012 Jul82(4)195-208
69
Case 2
bull Currently in remission after completion of
AALL0932 standard risk B-ALL therapy
Case 2
Pearls of Pathology
CD45 negative B-ALL is not uncommon
bull Always look at ungated set of plots for each marker
bull Eg SS versus each fluorescence parameter
Case 3
bull 58 year old male with pararenal mass
bull WBC 4000uL Hb 98gL Plt 229
bull ldquoblastsrdquo reported on blood film
bull Peripheral blood sent for flow cytometry
Case 3 PB flow cytometry
Dim CD45 CD19+CD10+
CD34- Sig neg
Case 3 PB flow cytometry
Dim CD45 CD19+CD10+
CD20(partial)CD5 neg
SIg cIg neg
Case 3 BM flow cytometry
Case 3 BM biopsy
Case 3 BM biopsy
Case 3 BM biopsy report
bull Diffuse infiltrate large lymphoid cells
bull Scant to moderate cytoplasm
bull Large irregular nuclei with prominent nucleoli
bull Increased mitotic activity
bull Positive for BCL-2 BCL6 CD10 and MUM1
bull Negative for TdT CD5
bull Cytogenetics FISH on para renal mass ndash C-MYC and
BCL2 rearrangements detected
Case 3 Doubletriple hit lymphoma
(DHLTHL)
bull Morphologically heterogeneous B cell neoplasms
characterized by rearrangement of the MYC proto-
oncogene with concurrent BCL2 andor BCL6 gene
rearrangement
bull WHO 2008 criteria - most cases of DHL and THL
classified DLBCL or B-cell lymphoma unclassifiable
with features intermediate between DLBCL and Burkitt
lymphoma (BCLU)
Case 3 DHLTHL
bull Retrospective study from one institution features of 13 cases (9 DHL4
THL)
bull median age was 59 years (range 30-74) eight females five males
bull enlarging lymphadenopathymasses (11 patients) abnormal peripheral
blood findings (2 patients)
bull TdT positivity (four cases two THLtwo DHL) dim CD45 expression (7
cases) lack of CD20 (2 cases) or lack of sIg (3 cases) and blastoid
morphology (2 cases)
bull Conclusion Expression of TdT in a B-cell lymphoma with mature
features or expression of surface light chain in a case otherwise
suggestive of B-lymphoblastic leukemialymphoma should prompt
an expedited evaluation for DHLTHL
Leuk Lymphoma 2016 Feb 181-10 [Epub ahead of print]
Double- and triple-hit lymphomas can present with features suggestive
of immaturity including TdT expression and create diagnostic challenges
Moench L Sachs Z Aasen G Dolan M Dayton V Courville EL
Case 4
bull 55 year old female
bull Previous history hyperparathyroidism
bull Parathyroidectomy in 1999
bull Mild persistent hypercalcemia
bull WBC 4800uL hemoglobin 121 gL Plt 296
bull Total protein 90gL IgG 34gL monoclonal peak
209gL
Case 4 Monoclonal IgG patient
Polyclonal B cells
With maturing hematogones
helper suppressor T cells
less than 3 blasts
Maturing myeloid population
Case 4 BM Ungated
Case 4 cytoplasmic immunoglobulin
Need KL
Case 4 BM Multiple Myeloma
Case 4 follow up
bull Smouldering myeloma
bull Recently developed CRAB (hyperCalcemia Renal
insufficiency Anemia Bone lesions
bull Serum free light chain ratio 123
bull Bone marrow aspirate gt60 plasma cells
bull Currently on treatment pending bone marrow
transplant
Case 5
bull 65 year old male
bull Slight lymphocytosis detected on routine CBC analysis
(lymph 4500uL)
bull Asymptomatic sent for flow cytometry
Case 5 PB
Case 5 flow cytometry KL ratio 0625
Case 5 Monoclonal B cell lymphocytosis
KL 18 KL 056
Case 5 Monoclonal B cell lymphocytosis
bull WHO defines CLL as abnormal lymphocyte count
gt5x10E9L (CD19+CD5+ light chain restricted) in the
absence of other clinical symptoms
bull Patients with small B cell clones are often detected as
an incidental finding
bull WHO 2016
bull CD19+CD5+ lt05x10E9L ldquoLow count MBLrdquo
raquo 05-5x10E9L ldquoHigh count MBLrdquo
raquo Non CLL type phenotype MBL also recognized
Monoclonal B cell Lymphocytosis
bull CLL immunophenotype - vast majority (75)
bull Atypical MBL
bull normal levels both CD5 and CD20 with variable
expression of CD23 (always exclude Mantle-cell
Lymphoma in CD23 negative cases)
bull Non-CLL-phenotype (CD5minus) MBL
bull Lacking CD5 expression and CD10 likely resembling B-
cell lymphomas of marginal-zone origin
Case 5 What is the normal KL Ratio
bull What is the normal or abnormal kappalambda ratio for
entire ldquopopulationrdquo of all B cells
bull Traditional view
ndash Kappa Lambda 21 or 31 ( 12 -13)
ndash Range 14 60
Case 5 What is the normal KL ratio
bull Wrong question
bull It doesnrsquot matter how many of the B cells are clonal but rather
that their exists a clonal population of B cells within the total
population
bull Need to Identify malignant B cells within a population of
normal B cells
bull In order to interpret a pathological immunophenotype it is
necessary to refer to quantitative and qualitative values of
normal B-cell subpopulations
bull Pearl Identify the abnormal phenotype first then look at KL
ratio
Case 6
bull 12 year old male presents to ER with fatigue bruising
Lkc 2200uL
bull Hb 84gL
bull Plt 32
bull Diff ndash neutropenia no blasts noted few ldquoatypicalrdquo cells
bull RBC ndash non specific changes
Leukemia
Case 6 PB
Case 6 BM
Case 6
bull BM aspirate ndash Hypocellular relative lymphocytosis
marked reduction in myeloid lineages
hypoplasticaplastic anemia
bull Flow Cytometry ndash less than 3 blasts maturing
myeloid 68 lymphocytes polyclonal B helper
suppressor T
bull Unusual pattern of CD13CD16 staining noted
Case 6 flow cytometry Aplastic Anemia
Normal Patient
Normal
Patient
Patient
Normal
Case 6 RBC Test for PNH CD235a v s CD59
Case 6 Neutrophil test for PNH clone
FLAER CD24 CD45CD15
18 PNH clone
Case 6
bull Follow up
bull Patient on steroids acyclovir and anti-thymocyte
globulin
bull PNH clone Neutrophils 96 RBC 1 Nov 2015
bull In remission as of April 2017
- Lkc 5500uL (Neuts 23)
- Hb 129gL
- Plt 208 X10e9
Summary
bull B cell malignancies are the most common
hematological disorders seen in flow cytometry
bull Many have complex overlapping phenotypes
bull Important to look at ungated plots as well as gating on
specific subpopulations
bull Level of expression of markers is as important as
positive or negative (think CD13 vs CD16)
68
References
bull Craig FE Foon KA Flow cytometric immunophenotyping for hematologic neoplasms Blood
2008 Apr 15111(8)3941-67
bull Wood BL Principles of minimal residual disease detection for hematopoietic neoplasms by flow
cytometry Cytometry B Clin Cytom 2015 Apr 23
bull Shanafelt TD Ghia P Lanasa MC Landgren O Rawstron AC Monoclonal B-cell
lymphocytosis (MBL) biology natural history and clinical management Leukemia 2010
Mar24(3)512-20
bull Swerdlow SH Campo E Pileri SA Harris NL Stein H Siebert R Advani RGhielmini M Salles
GA Zelenetz AD Jaffe ES The 2016 revision of the World Health Organization classification of
lymphoid neoplasms Blood 2016 May 19127(20)2375-90
bull 3 Moench L Sachs Z Aasen G Dolan M Dayton V Courville EL Double- and triple-hit
lymphomas can present with features suggestive of immaturity including TdT expression and
create diagnostic challenges Leuk Lymphoma 2016572626-35
bull Sutherland DR Keeney M Illingworth A Practical guidelines for the high-sensitivity detection
and monitoring of paroxysmal nocturnal hemoglobinuria clones by flow cytometry Cytometry B
Clin Cytom 2012 Jul82(4)195-208
69
Case 2
Pearls of Pathology
CD45 negative B-ALL is not uncommon
bull Always look at ungated set of plots for each marker
bull Eg SS versus each fluorescence parameter
Case 3
bull 58 year old male with pararenal mass
bull WBC 4000uL Hb 98gL Plt 229
bull ldquoblastsrdquo reported on blood film
bull Peripheral blood sent for flow cytometry
Case 3 PB flow cytometry
Dim CD45 CD19+CD10+
CD34- Sig neg
Case 3 PB flow cytometry
Dim CD45 CD19+CD10+
CD20(partial)CD5 neg
SIg cIg neg
Case 3 BM flow cytometry
Case 3 BM biopsy
Case 3 BM biopsy
Case 3 BM biopsy report
bull Diffuse infiltrate large lymphoid cells
bull Scant to moderate cytoplasm
bull Large irregular nuclei with prominent nucleoli
bull Increased mitotic activity
bull Positive for BCL-2 BCL6 CD10 and MUM1
bull Negative for TdT CD5
bull Cytogenetics FISH on para renal mass ndash C-MYC and
BCL2 rearrangements detected
Case 3 Doubletriple hit lymphoma
(DHLTHL)
bull Morphologically heterogeneous B cell neoplasms
characterized by rearrangement of the MYC proto-
oncogene with concurrent BCL2 andor BCL6 gene
rearrangement
bull WHO 2008 criteria - most cases of DHL and THL
classified DLBCL or B-cell lymphoma unclassifiable
with features intermediate between DLBCL and Burkitt
lymphoma (BCLU)
Case 3 DHLTHL
bull Retrospective study from one institution features of 13 cases (9 DHL4
THL)
bull median age was 59 years (range 30-74) eight females five males
bull enlarging lymphadenopathymasses (11 patients) abnormal peripheral
blood findings (2 patients)
bull TdT positivity (four cases two THLtwo DHL) dim CD45 expression (7
cases) lack of CD20 (2 cases) or lack of sIg (3 cases) and blastoid
morphology (2 cases)
bull Conclusion Expression of TdT in a B-cell lymphoma with mature
features or expression of surface light chain in a case otherwise
suggestive of B-lymphoblastic leukemialymphoma should prompt
an expedited evaluation for DHLTHL
Leuk Lymphoma 2016 Feb 181-10 [Epub ahead of print]
Double- and triple-hit lymphomas can present with features suggestive
of immaturity including TdT expression and create diagnostic challenges
Moench L Sachs Z Aasen G Dolan M Dayton V Courville EL
Case 4
bull 55 year old female
bull Previous history hyperparathyroidism
bull Parathyroidectomy in 1999
bull Mild persistent hypercalcemia
bull WBC 4800uL hemoglobin 121 gL Plt 296
bull Total protein 90gL IgG 34gL monoclonal peak
209gL
Case 4 Monoclonal IgG patient
Polyclonal B cells
With maturing hematogones
helper suppressor T cells
less than 3 blasts
Maturing myeloid population
Case 4 BM Ungated
Case 4 cytoplasmic immunoglobulin
Need KL
Case 4 BM Multiple Myeloma
Case 4 follow up
bull Smouldering myeloma
bull Recently developed CRAB (hyperCalcemia Renal
insufficiency Anemia Bone lesions
bull Serum free light chain ratio 123
bull Bone marrow aspirate gt60 plasma cells
bull Currently on treatment pending bone marrow
transplant
Case 5
bull 65 year old male
bull Slight lymphocytosis detected on routine CBC analysis
(lymph 4500uL)
bull Asymptomatic sent for flow cytometry
Case 5 PB
Case 5 flow cytometry KL ratio 0625
Case 5 Monoclonal B cell lymphocytosis
KL 18 KL 056
Case 5 Monoclonal B cell lymphocytosis
bull WHO defines CLL as abnormal lymphocyte count
gt5x10E9L (CD19+CD5+ light chain restricted) in the
absence of other clinical symptoms
bull Patients with small B cell clones are often detected as
an incidental finding
bull WHO 2016
bull CD19+CD5+ lt05x10E9L ldquoLow count MBLrdquo
raquo 05-5x10E9L ldquoHigh count MBLrdquo
raquo Non CLL type phenotype MBL also recognized
Monoclonal B cell Lymphocytosis
bull CLL immunophenotype - vast majority (75)
bull Atypical MBL
bull normal levels both CD5 and CD20 with variable
expression of CD23 (always exclude Mantle-cell
Lymphoma in CD23 negative cases)
bull Non-CLL-phenotype (CD5minus) MBL
bull Lacking CD5 expression and CD10 likely resembling B-
cell lymphomas of marginal-zone origin
Case 5 What is the normal KL Ratio
bull What is the normal or abnormal kappalambda ratio for
entire ldquopopulationrdquo of all B cells
bull Traditional view
ndash Kappa Lambda 21 or 31 ( 12 -13)
ndash Range 14 60
Case 5 What is the normal KL ratio
bull Wrong question
bull It doesnrsquot matter how many of the B cells are clonal but rather
that their exists a clonal population of B cells within the total
population
bull Need to Identify malignant B cells within a population of
normal B cells
bull In order to interpret a pathological immunophenotype it is
necessary to refer to quantitative and qualitative values of
normal B-cell subpopulations
bull Pearl Identify the abnormal phenotype first then look at KL
ratio
Case 6
bull 12 year old male presents to ER with fatigue bruising
Lkc 2200uL
bull Hb 84gL
bull Plt 32
bull Diff ndash neutropenia no blasts noted few ldquoatypicalrdquo cells
bull RBC ndash non specific changes
Leukemia
Case 6 PB
Case 6 BM
Case 6
bull BM aspirate ndash Hypocellular relative lymphocytosis
marked reduction in myeloid lineages
hypoplasticaplastic anemia
bull Flow Cytometry ndash less than 3 blasts maturing
myeloid 68 lymphocytes polyclonal B helper
suppressor T
bull Unusual pattern of CD13CD16 staining noted
Case 6 flow cytometry Aplastic Anemia
Normal Patient
Normal
Patient
Patient
Normal
Case 6 RBC Test for PNH CD235a v s CD59
Case 6 Neutrophil test for PNH clone
FLAER CD24 CD45CD15
18 PNH clone
Case 6
bull Follow up
bull Patient on steroids acyclovir and anti-thymocyte
globulin
bull PNH clone Neutrophils 96 RBC 1 Nov 2015
bull In remission as of April 2017
- Lkc 5500uL (Neuts 23)
- Hb 129gL
- Plt 208 X10e9
Summary
bull B cell malignancies are the most common
hematological disorders seen in flow cytometry
bull Many have complex overlapping phenotypes
bull Important to look at ungated plots as well as gating on
specific subpopulations
bull Level of expression of markers is as important as
positive or negative (think CD13 vs CD16)
68
References
bull Craig FE Foon KA Flow cytometric immunophenotyping for hematologic neoplasms Blood
2008 Apr 15111(8)3941-67
bull Wood BL Principles of minimal residual disease detection for hematopoietic neoplasms by flow
cytometry Cytometry B Clin Cytom 2015 Apr 23
bull Shanafelt TD Ghia P Lanasa MC Landgren O Rawstron AC Monoclonal B-cell
lymphocytosis (MBL) biology natural history and clinical management Leukemia 2010
Mar24(3)512-20
bull Swerdlow SH Campo E Pileri SA Harris NL Stein H Siebert R Advani RGhielmini M Salles
GA Zelenetz AD Jaffe ES The 2016 revision of the World Health Organization classification of
lymphoid neoplasms Blood 2016 May 19127(20)2375-90
bull 3 Moench L Sachs Z Aasen G Dolan M Dayton V Courville EL Double- and triple-hit
lymphomas can present with features suggestive of immaturity including TdT expression and
create diagnostic challenges Leuk Lymphoma 2016572626-35
bull Sutherland DR Keeney M Illingworth A Practical guidelines for the high-sensitivity detection
and monitoring of paroxysmal nocturnal hemoglobinuria clones by flow cytometry Cytometry B
Clin Cytom 2012 Jul82(4)195-208
69
Case 3
bull 58 year old male with pararenal mass
bull WBC 4000uL Hb 98gL Plt 229
bull ldquoblastsrdquo reported on blood film
bull Peripheral blood sent for flow cytometry
Case 3 PB flow cytometry
Dim CD45 CD19+CD10+
CD34- Sig neg
Case 3 PB flow cytometry
Dim CD45 CD19+CD10+
CD20(partial)CD5 neg
SIg cIg neg
Case 3 BM flow cytometry
Case 3 BM biopsy
Case 3 BM biopsy
Case 3 BM biopsy report
bull Diffuse infiltrate large lymphoid cells
bull Scant to moderate cytoplasm
bull Large irregular nuclei with prominent nucleoli
bull Increased mitotic activity
bull Positive for BCL-2 BCL6 CD10 and MUM1
bull Negative for TdT CD5
bull Cytogenetics FISH on para renal mass ndash C-MYC and
BCL2 rearrangements detected
Case 3 Doubletriple hit lymphoma
(DHLTHL)
bull Morphologically heterogeneous B cell neoplasms
characterized by rearrangement of the MYC proto-
oncogene with concurrent BCL2 andor BCL6 gene
rearrangement
bull WHO 2008 criteria - most cases of DHL and THL
classified DLBCL or B-cell lymphoma unclassifiable
with features intermediate between DLBCL and Burkitt
lymphoma (BCLU)
Case 3 DHLTHL
bull Retrospective study from one institution features of 13 cases (9 DHL4
THL)
bull median age was 59 years (range 30-74) eight females five males
bull enlarging lymphadenopathymasses (11 patients) abnormal peripheral
blood findings (2 patients)
bull TdT positivity (four cases two THLtwo DHL) dim CD45 expression (7
cases) lack of CD20 (2 cases) or lack of sIg (3 cases) and blastoid
morphology (2 cases)
bull Conclusion Expression of TdT in a B-cell lymphoma with mature
features or expression of surface light chain in a case otherwise
suggestive of B-lymphoblastic leukemialymphoma should prompt
an expedited evaluation for DHLTHL
Leuk Lymphoma 2016 Feb 181-10 [Epub ahead of print]
Double- and triple-hit lymphomas can present with features suggestive
of immaturity including TdT expression and create diagnostic challenges
Moench L Sachs Z Aasen G Dolan M Dayton V Courville EL
Case 4
bull 55 year old female
bull Previous history hyperparathyroidism
bull Parathyroidectomy in 1999
bull Mild persistent hypercalcemia
bull WBC 4800uL hemoglobin 121 gL Plt 296
bull Total protein 90gL IgG 34gL monoclonal peak
209gL
Case 4 Monoclonal IgG patient
Polyclonal B cells
With maturing hematogones
helper suppressor T cells
less than 3 blasts
Maturing myeloid population
Case 4 BM Ungated
Case 4 cytoplasmic immunoglobulin
Need KL
Case 4 BM Multiple Myeloma
Case 4 follow up
bull Smouldering myeloma
bull Recently developed CRAB (hyperCalcemia Renal
insufficiency Anemia Bone lesions
bull Serum free light chain ratio 123
bull Bone marrow aspirate gt60 plasma cells
bull Currently on treatment pending bone marrow
transplant
Case 5
bull 65 year old male
bull Slight lymphocytosis detected on routine CBC analysis
(lymph 4500uL)
bull Asymptomatic sent for flow cytometry
Case 5 PB
Case 5 flow cytometry KL ratio 0625
Case 5 Monoclonal B cell lymphocytosis
KL 18 KL 056
Case 5 Monoclonal B cell lymphocytosis
bull WHO defines CLL as abnormal lymphocyte count
gt5x10E9L (CD19+CD5+ light chain restricted) in the
absence of other clinical symptoms
bull Patients with small B cell clones are often detected as
an incidental finding
bull WHO 2016
bull CD19+CD5+ lt05x10E9L ldquoLow count MBLrdquo
raquo 05-5x10E9L ldquoHigh count MBLrdquo
raquo Non CLL type phenotype MBL also recognized
Monoclonal B cell Lymphocytosis
bull CLL immunophenotype - vast majority (75)
bull Atypical MBL
bull normal levels both CD5 and CD20 with variable
expression of CD23 (always exclude Mantle-cell
Lymphoma in CD23 negative cases)
bull Non-CLL-phenotype (CD5minus) MBL
bull Lacking CD5 expression and CD10 likely resembling B-
cell lymphomas of marginal-zone origin
Case 5 What is the normal KL Ratio
bull What is the normal or abnormal kappalambda ratio for
entire ldquopopulationrdquo of all B cells
bull Traditional view
ndash Kappa Lambda 21 or 31 ( 12 -13)
ndash Range 14 60
Case 5 What is the normal KL ratio
bull Wrong question
bull It doesnrsquot matter how many of the B cells are clonal but rather
that their exists a clonal population of B cells within the total
population
bull Need to Identify malignant B cells within a population of
normal B cells
bull In order to interpret a pathological immunophenotype it is
necessary to refer to quantitative and qualitative values of
normal B-cell subpopulations
bull Pearl Identify the abnormal phenotype first then look at KL
ratio
Case 6
bull 12 year old male presents to ER with fatigue bruising
Lkc 2200uL
bull Hb 84gL
bull Plt 32
bull Diff ndash neutropenia no blasts noted few ldquoatypicalrdquo cells
bull RBC ndash non specific changes
Leukemia
Case 6 PB
Case 6 BM
Case 6
bull BM aspirate ndash Hypocellular relative lymphocytosis
marked reduction in myeloid lineages
hypoplasticaplastic anemia
bull Flow Cytometry ndash less than 3 blasts maturing
myeloid 68 lymphocytes polyclonal B helper
suppressor T
bull Unusual pattern of CD13CD16 staining noted
Case 6 flow cytometry Aplastic Anemia
Normal Patient
Normal
Patient
Patient
Normal
Case 6 RBC Test for PNH CD235a v s CD59
Case 6 Neutrophil test for PNH clone
FLAER CD24 CD45CD15
18 PNH clone
Case 6
bull Follow up
bull Patient on steroids acyclovir and anti-thymocyte
globulin
bull PNH clone Neutrophils 96 RBC 1 Nov 2015
bull In remission as of April 2017
- Lkc 5500uL (Neuts 23)
- Hb 129gL
- Plt 208 X10e9
Summary
bull B cell malignancies are the most common
hematological disorders seen in flow cytometry
bull Many have complex overlapping phenotypes
bull Important to look at ungated plots as well as gating on
specific subpopulations
bull Level of expression of markers is as important as
positive or negative (think CD13 vs CD16)
68
References
bull Craig FE Foon KA Flow cytometric immunophenotyping for hematologic neoplasms Blood
2008 Apr 15111(8)3941-67
bull Wood BL Principles of minimal residual disease detection for hematopoietic neoplasms by flow
cytometry Cytometry B Clin Cytom 2015 Apr 23
bull Shanafelt TD Ghia P Lanasa MC Landgren O Rawstron AC Monoclonal B-cell
lymphocytosis (MBL) biology natural history and clinical management Leukemia 2010
Mar24(3)512-20
bull Swerdlow SH Campo E Pileri SA Harris NL Stein H Siebert R Advani RGhielmini M Salles
GA Zelenetz AD Jaffe ES The 2016 revision of the World Health Organization classification of
lymphoid neoplasms Blood 2016 May 19127(20)2375-90
bull 3 Moench L Sachs Z Aasen G Dolan M Dayton V Courville EL Double- and triple-hit
lymphomas can present with features suggestive of immaturity including TdT expression and
create diagnostic challenges Leuk Lymphoma 2016572626-35
bull Sutherland DR Keeney M Illingworth A Practical guidelines for the high-sensitivity detection
and monitoring of paroxysmal nocturnal hemoglobinuria clones by flow cytometry Cytometry B
Clin Cytom 2012 Jul82(4)195-208
69
Case 3 PB flow cytometry
Dim CD45 CD19+CD10+
CD34- Sig neg
Case 3 PB flow cytometry
Dim CD45 CD19+CD10+
CD20(partial)CD5 neg
SIg cIg neg
Case 3 BM flow cytometry
Case 3 BM biopsy
Case 3 BM biopsy
Case 3 BM biopsy report
bull Diffuse infiltrate large lymphoid cells
bull Scant to moderate cytoplasm
bull Large irregular nuclei with prominent nucleoli
bull Increased mitotic activity
bull Positive for BCL-2 BCL6 CD10 and MUM1
bull Negative for TdT CD5
bull Cytogenetics FISH on para renal mass ndash C-MYC and
BCL2 rearrangements detected
Case 3 Doubletriple hit lymphoma
(DHLTHL)
bull Morphologically heterogeneous B cell neoplasms
characterized by rearrangement of the MYC proto-
oncogene with concurrent BCL2 andor BCL6 gene
rearrangement
bull WHO 2008 criteria - most cases of DHL and THL
classified DLBCL or B-cell lymphoma unclassifiable
with features intermediate between DLBCL and Burkitt
lymphoma (BCLU)
Case 3 DHLTHL
bull Retrospective study from one institution features of 13 cases (9 DHL4
THL)
bull median age was 59 years (range 30-74) eight females five males
bull enlarging lymphadenopathymasses (11 patients) abnormal peripheral
blood findings (2 patients)
bull TdT positivity (four cases two THLtwo DHL) dim CD45 expression (7
cases) lack of CD20 (2 cases) or lack of sIg (3 cases) and blastoid
morphology (2 cases)
bull Conclusion Expression of TdT in a B-cell lymphoma with mature
features or expression of surface light chain in a case otherwise
suggestive of B-lymphoblastic leukemialymphoma should prompt
an expedited evaluation for DHLTHL
Leuk Lymphoma 2016 Feb 181-10 [Epub ahead of print]
Double- and triple-hit lymphomas can present with features suggestive
of immaturity including TdT expression and create diagnostic challenges
Moench L Sachs Z Aasen G Dolan M Dayton V Courville EL
Case 4
bull 55 year old female
bull Previous history hyperparathyroidism
bull Parathyroidectomy in 1999
bull Mild persistent hypercalcemia
bull WBC 4800uL hemoglobin 121 gL Plt 296
bull Total protein 90gL IgG 34gL monoclonal peak
209gL
Case 4 Monoclonal IgG patient
Polyclonal B cells
With maturing hematogones
helper suppressor T cells
less than 3 blasts
Maturing myeloid population
Case 4 BM Ungated
Case 4 cytoplasmic immunoglobulin
Need KL
Case 4 BM Multiple Myeloma
Case 4 follow up
bull Smouldering myeloma
bull Recently developed CRAB (hyperCalcemia Renal
insufficiency Anemia Bone lesions
bull Serum free light chain ratio 123
bull Bone marrow aspirate gt60 plasma cells
bull Currently on treatment pending bone marrow
transplant
Case 5
bull 65 year old male
bull Slight lymphocytosis detected on routine CBC analysis
(lymph 4500uL)
bull Asymptomatic sent for flow cytometry
Case 5 PB
Case 5 flow cytometry KL ratio 0625
Case 5 Monoclonal B cell lymphocytosis
KL 18 KL 056
Case 5 Monoclonal B cell lymphocytosis
bull WHO defines CLL as abnormal lymphocyte count
gt5x10E9L (CD19+CD5+ light chain restricted) in the
absence of other clinical symptoms
bull Patients with small B cell clones are often detected as
an incidental finding
bull WHO 2016
bull CD19+CD5+ lt05x10E9L ldquoLow count MBLrdquo
raquo 05-5x10E9L ldquoHigh count MBLrdquo
raquo Non CLL type phenotype MBL also recognized
Monoclonal B cell Lymphocytosis
bull CLL immunophenotype - vast majority (75)
bull Atypical MBL
bull normal levels both CD5 and CD20 with variable
expression of CD23 (always exclude Mantle-cell
Lymphoma in CD23 negative cases)
bull Non-CLL-phenotype (CD5minus) MBL
bull Lacking CD5 expression and CD10 likely resembling B-
cell lymphomas of marginal-zone origin
Case 5 What is the normal KL Ratio
bull What is the normal or abnormal kappalambda ratio for
entire ldquopopulationrdquo of all B cells
bull Traditional view
ndash Kappa Lambda 21 or 31 ( 12 -13)
ndash Range 14 60
Case 5 What is the normal KL ratio
bull Wrong question
bull It doesnrsquot matter how many of the B cells are clonal but rather
that their exists a clonal population of B cells within the total
population
bull Need to Identify malignant B cells within a population of
normal B cells
bull In order to interpret a pathological immunophenotype it is
necessary to refer to quantitative and qualitative values of
normal B-cell subpopulations
bull Pearl Identify the abnormal phenotype first then look at KL
ratio
Case 6
bull 12 year old male presents to ER with fatigue bruising
Lkc 2200uL
bull Hb 84gL
bull Plt 32
bull Diff ndash neutropenia no blasts noted few ldquoatypicalrdquo cells
bull RBC ndash non specific changes
Leukemia
Case 6 PB
Case 6 BM
Case 6
bull BM aspirate ndash Hypocellular relative lymphocytosis
marked reduction in myeloid lineages
hypoplasticaplastic anemia
bull Flow Cytometry ndash less than 3 blasts maturing
myeloid 68 lymphocytes polyclonal B helper
suppressor T
bull Unusual pattern of CD13CD16 staining noted
Case 6 flow cytometry Aplastic Anemia
Normal Patient
Normal
Patient
Patient
Normal
Case 6 RBC Test for PNH CD235a v s CD59
Case 6 Neutrophil test for PNH clone
FLAER CD24 CD45CD15
18 PNH clone
Case 6
bull Follow up
bull Patient on steroids acyclovir and anti-thymocyte
globulin
bull PNH clone Neutrophils 96 RBC 1 Nov 2015
bull In remission as of April 2017
- Lkc 5500uL (Neuts 23)
- Hb 129gL
- Plt 208 X10e9
Summary
bull B cell malignancies are the most common
hematological disorders seen in flow cytometry
bull Many have complex overlapping phenotypes
bull Important to look at ungated plots as well as gating on
specific subpopulations
bull Level of expression of markers is as important as
positive or negative (think CD13 vs CD16)
68
References
bull Craig FE Foon KA Flow cytometric immunophenotyping for hematologic neoplasms Blood
2008 Apr 15111(8)3941-67
bull Wood BL Principles of minimal residual disease detection for hematopoietic neoplasms by flow
cytometry Cytometry B Clin Cytom 2015 Apr 23
bull Shanafelt TD Ghia P Lanasa MC Landgren O Rawstron AC Monoclonal B-cell
lymphocytosis (MBL) biology natural history and clinical management Leukemia 2010
Mar24(3)512-20
bull Swerdlow SH Campo E Pileri SA Harris NL Stein H Siebert R Advani RGhielmini M Salles
GA Zelenetz AD Jaffe ES The 2016 revision of the World Health Organization classification of
lymphoid neoplasms Blood 2016 May 19127(20)2375-90
bull 3 Moench L Sachs Z Aasen G Dolan M Dayton V Courville EL Double- and triple-hit
lymphomas can present with features suggestive of immaturity including TdT expression and
create diagnostic challenges Leuk Lymphoma 2016572626-35
bull Sutherland DR Keeney M Illingworth A Practical guidelines for the high-sensitivity detection
and monitoring of paroxysmal nocturnal hemoglobinuria clones by flow cytometry Cytometry B
Clin Cytom 2012 Jul82(4)195-208
69
Case 3 PB flow cytometry
Dim CD45 CD19+CD10+
CD20(partial)CD5 neg
SIg cIg neg
Case 3 BM flow cytometry
Case 3 BM biopsy
Case 3 BM biopsy
Case 3 BM biopsy report
bull Diffuse infiltrate large lymphoid cells
bull Scant to moderate cytoplasm
bull Large irregular nuclei with prominent nucleoli
bull Increased mitotic activity
bull Positive for BCL-2 BCL6 CD10 and MUM1
bull Negative for TdT CD5
bull Cytogenetics FISH on para renal mass ndash C-MYC and
BCL2 rearrangements detected
Case 3 Doubletriple hit lymphoma
(DHLTHL)
bull Morphologically heterogeneous B cell neoplasms
characterized by rearrangement of the MYC proto-
oncogene with concurrent BCL2 andor BCL6 gene
rearrangement
bull WHO 2008 criteria - most cases of DHL and THL
classified DLBCL or B-cell lymphoma unclassifiable
with features intermediate between DLBCL and Burkitt
lymphoma (BCLU)
Case 3 DHLTHL
bull Retrospective study from one institution features of 13 cases (9 DHL4
THL)
bull median age was 59 years (range 30-74) eight females five males
bull enlarging lymphadenopathymasses (11 patients) abnormal peripheral
blood findings (2 patients)
bull TdT positivity (four cases two THLtwo DHL) dim CD45 expression (7
cases) lack of CD20 (2 cases) or lack of sIg (3 cases) and blastoid
morphology (2 cases)
bull Conclusion Expression of TdT in a B-cell lymphoma with mature
features or expression of surface light chain in a case otherwise
suggestive of B-lymphoblastic leukemialymphoma should prompt
an expedited evaluation for DHLTHL
Leuk Lymphoma 2016 Feb 181-10 [Epub ahead of print]
Double- and triple-hit lymphomas can present with features suggestive
of immaturity including TdT expression and create diagnostic challenges
Moench L Sachs Z Aasen G Dolan M Dayton V Courville EL
Case 4
bull 55 year old female
bull Previous history hyperparathyroidism
bull Parathyroidectomy in 1999
bull Mild persistent hypercalcemia
bull WBC 4800uL hemoglobin 121 gL Plt 296
bull Total protein 90gL IgG 34gL monoclonal peak
209gL
Case 4 Monoclonal IgG patient
Polyclonal B cells
With maturing hematogones
helper suppressor T cells
less than 3 blasts
Maturing myeloid population
Case 4 BM Ungated
Case 4 cytoplasmic immunoglobulin
Need KL
Case 4 BM Multiple Myeloma
Case 4 follow up
bull Smouldering myeloma
bull Recently developed CRAB (hyperCalcemia Renal
insufficiency Anemia Bone lesions
bull Serum free light chain ratio 123
bull Bone marrow aspirate gt60 plasma cells
bull Currently on treatment pending bone marrow
transplant
Case 5
bull 65 year old male
bull Slight lymphocytosis detected on routine CBC analysis
(lymph 4500uL)
bull Asymptomatic sent for flow cytometry
Case 5 PB
Case 5 flow cytometry KL ratio 0625
Case 5 Monoclonal B cell lymphocytosis
KL 18 KL 056
Case 5 Monoclonal B cell lymphocytosis
bull WHO defines CLL as abnormal lymphocyte count
gt5x10E9L (CD19+CD5+ light chain restricted) in the
absence of other clinical symptoms
bull Patients with small B cell clones are often detected as
an incidental finding
bull WHO 2016
bull CD19+CD5+ lt05x10E9L ldquoLow count MBLrdquo
raquo 05-5x10E9L ldquoHigh count MBLrdquo
raquo Non CLL type phenotype MBL also recognized
Monoclonal B cell Lymphocytosis
bull CLL immunophenotype - vast majority (75)
bull Atypical MBL
bull normal levels both CD5 and CD20 with variable
expression of CD23 (always exclude Mantle-cell
Lymphoma in CD23 negative cases)
bull Non-CLL-phenotype (CD5minus) MBL
bull Lacking CD5 expression and CD10 likely resembling B-
cell lymphomas of marginal-zone origin
Case 5 What is the normal KL Ratio
bull What is the normal or abnormal kappalambda ratio for
entire ldquopopulationrdquo of all B cells
bull Traditional view
ndash Kappa Lambda 21 or 31 ( 12 -13)
ndash Range 14 60
Case 5 What is the normal KL ratio
bull Wrong question
bull It doesnrsquot matter how many of the B cells are clonal but rather
that their exists a clonal population of B cells within the total
population
bull Need to Identify malignant B cells within a population of
normal B cells
bull In order to interpret a pathological immunophenotype it is
necessary to refer to quantitative and qualitative values of
normal B-cell subpopulations
bull Pearl Identify the abnormal phenotype first then look at KL
ratio
Case 6
bull 12 year old male presents to ER with fatigue bruising
Lkc 2200uL
bull Hb 84gL
bull Plt 32
bull Diff ndash neutropenia no blasts noted few ldquoatypicalrdquo cells
bull RBC ndash non specific changes
Leukemia
Case 6 PB
Case 6 BM
Case 6
bull BM aspirate ndash Hypocellular relative lymphocytosis
marked reduction in myeloid lineages
hypoplasticaplastic anemia
bull Flow Cytometry ndash less than 3 blasts maturing
myeloid 68 lymphocytes polyclonal B helper
suppressor T
bull Unusual pattern of CD13CD16 staining noted
Case 6 flow cytometry Aplastic Anemia
Normal Patient
Normal
Patient
Patient
Normal
Case 6 RBC Test for PNH CD235a v s CD59
Case 6 Neutrophil test for PNH clone
FLAER CD24 CD45CD15
18 PNH clone
Case 6
bull Follow up
bull Patient on steroids acyclovir and anti-thymocyte
globulin
bull PNH clone Neutrophils 96 RBC 1 Nov 2015
bull In remission as of April 2017
- Lkc 5500uL (Neuts 23)
- Hb 129gL
- Plt 208 X10e9
Summary
bull B cell malignancies are the most common
hematological disorders seen in flow cytometry
bull Many have complex overlapping phenotypes
bull Important to look at ungated plots as well as gating on
specific subpopulations
bull Level of expression of markers is as important as
positive or negative (think CD13 vs CD16)
68
References
bull Craig FE Foon KA Flow cytometric immunophenotyping for hematologic neoplasms Blood
2008 Apr 15111(8)3941-67
bull Wood BL Principles of minimal residual disease detection for hematopoietic neoplasms by flow
cytometry Cytometry B Clin Cytom 2015 Apr 23
bull Shanafelt TD Ghia P Lanasa MC Landgren O Rawstron AC Monoclonal B-cell
lymphocytosis (MBL) biology natural history and clinical management Leukemia 2010
Mar24(3)512-20
bull Swerdlow SH Campo E Pileri SA Harris NL Stein H Siebert R Advani RGhielmini M Salles
GA Zelenetz AD Jaffe ES The 2016 revision of the World Health Organization classification of
lymphoid neoplasms Blood 2016 May 19127(20)2375-90
bull 3 Moench L Sachs Z Aasen G Dolan M Dayton V Courville EL Double- and triple-hit
lymphomas can present with features suggestive of immaturity including TdT expression and
create diagnostic challenges Leuk Lymphoma 2016572626-35
bull Sutherland DR Keeney M Illingworth A Practical guidelines for the high-sensitivity detection
and monitoring of paroxysmal nocturnal hemoglobinuria clones by flow cytometry Cytometry B
Clin Cytom 2012 Jul82(4)195-208
69
Case 3 BM flow cytometry
Case 3 BM biopsy
Case 3 BM biopsy
Case 3 BM biopsy report
bull Diffuse infiltrate large lymphoid cells
bull Scant to moderate cytoplasm
bull Large irregular nuclei with prominent nucleoli
bull Increased mitotic activity
bull Positive for BCL-2 BCL6 CD10 and MUM1
bull Negative for TdT CD5
bull Cytogenetics FISH on para renal mass ndash C-MYC and
BCL2 rearrangements detected
Case 3 Doubletriple hit lymphoma
(DHLTHL)
bull Morphologically heterogeneous B cell neoplasms
characterized by rearrangement of the MYC proto-
oncogene with concurrent BCL2 andor BCL6 gene
rearrangement
bull WHO 2008 criteria - most cases of DHL and THL
classified DLBCL or B-cell lymphoma unclassifiable
with features intermediate between DLBCL and Burkitt
lymphoma (BCLU)
Case 3 DHLTHL
bull Retrospective study from one institution features of 13 cases (9 DHL4
THL)
bull median age was 59 years (range 30-74) eight females five males
bull enlarging lymphadenopathymasses (11 patients) abnormal peripheral
blood findings (2 patients)
bull TdT positivity (four cases two THLtwo DHL) dim CD45 expression (7
cases) lack of CD20 (2 cases) or lack of sIg (3 cases) and blastoid
morphology (2 cases)
bull Conclusion Expression of TdT in a B-cell lymphoma with mature
features or expression of surface light chain in a case otherwise
suggestive of B-lymphoblastic leukemialymphoma should prompt
an expedited evaluation for DHLTHL
Leuk Lymphoma 2016 Feb 181-10 [Epub ahead of print]
Double- and triple-hit lymphomas can present with features suggestive
of immaturity including TdT expression and create diagnostic challenges
Moench L Sachs Z Aasen G Dolan M Dayton V Courville EL
Case 4
bull 55 year old female
bull Previous history hyperparathyroidism
bull Parathyroidectomy in 1999
bull Mild persistent hypercalcemia
bull WBC 4800uL hemoglobin 121 gL Plt 296
bull Total protein 90gL IgG 34gL monoclonal peak
209gL
Case 4 Monoclonal IgG patient
Polyclonal B cells
With maturing hematogones
helper suppressor T cells
less than 3 blasts
Maturing myeloid population
Case 4 BM Ungated
Case 4 cytoplasmic immunoglobulin
Need KL
Case 4 BM Multiple Myeloma
Case 4 follow up
bull Smouldering myeloma
bull Recently developed CRAB (hyperCalcemia Renal
insufficiency Anemia Bone lesions
bull Serum free light chain ratio 123
bull Bone marrow aspirate gt60 plasma cells
bull Currently on treatment pending bone marrow
transplant
Case 5
bull 65 year old male
bull Slight lymphocytosis detected on routine CBC analysis
(lymph 4500uL)
bull Asymptomatic sent for flow cytometry
Case 5 PB
Case 5 flow cytometry KL ratio 0625
Case 5 Monoclonal B cell lymphocytosis
KL 18 KL 056
Case 5 Monoclonal B cell lymphocytosis
bull WHO defines CLL as abnormal lymphocyte count
gt5x10E9L (CD19+CD5+ light chain restricted) in the
absence of other clinical symptoms
bull Patients with small B cell clones are often detected as
an incidental finding
bull WHO 2016
bull CD19+CD5+ lt05x10E9L ldquoLow count MBLrdquo
raquo 05-5x10E9L ldquoHigh count MBLrdquo
raquo Non CLL type phenotype MBL also recognized
Monoclonal B cell Lymphocytosis
bull CLL immunophenotype - vast majority (75)
bull Atypical MBL
bull normal levels both CD5 and CD20 with variable
expression of CD23 (always exclude Mantle-cell
Lymphoma in CD23 negative cases)
bull Non-CLL-phenotype (CD5minus) MBL
bull Lacking CD5 expression and CD10 likely resembling B-
cell lymphomas of marginal-zone origin
Case 5 What is the normal KL Ratio
bull What is the normal or abnormal kappalambda ratio for
entire ldquopopulationrdquo of all B cells
bull Traditional view
ndash Kappa Lambda 21 or 31 ( 12 -13)
ndash Range 14 60
Case 5 What is the normal KL ratio
bull Wrong question
bull It doesnrsquot matter how many of the B cells are clonal but rather
that their exists a clonal population of B cells within the total
population
bull Need to Identify malignant B cells within a population of
normal B cells
bull In order to interpret a pathological immunophenotype it is
necessary to refer to quantitative and qualitative values of
normal B-cell subpopulations
bull Pearl Identify the abnormal phenotype first then look at KL
ratio
Case 6
bull 12 year old male presents to ER with fatigue bruising
Lkc 2200uL
bull Hb 84gL
bull Plt 32
bull Diff ndash neutropenia no blasts noted few ldquoatypicalrdquo cells
bull RBC ndash non specific changes
Leukemia
Case 6 PB
Case 6 BM
Case 6
bull BM aspirate ndash Hypocellular relative lymphocytosis
marked reduction in myeloid lineages
hypoplasticaplastic anemia
bull Flow Cytometry ndash less than 3 blasts maturing
myeloid 68 lymphocytes polyclonal B helper
suppressor T
bull Unusual pattern of CD13CD16 staining noted
Case 6 flow cytometry Aplastic Anemia
Normal Patient
Normal
Patient
Patient
Normal
Case 6 RBC Test for PNH CD235a v s CD59
Case 6 Neutrophil test for PNH clone
FLAER CD24 CD45CD15
18 PNH clone
Case 6
bull Follow up
bull Patient on steroids acyclovir and anti-thymocyte
globulin
bull PNH clone Neutrophils 96 RBC 1 Nov 2015
bull In remission as of April 2017
- Lkc 5500uL (Neuts 23)
- Hb 129gL
- Plt 208 X10e9
Summary
bull B cell malignancies are the most common
hematological disorders seen in flow cytometry
bull Many have complex overlapping phenotypes
bull Important to look at ungated plots as well as gating on
specific subpopulations
bull Level of expression of markers is as important as
positive or negative (think CD13 vs CD16)
68
References
bull Craig FE Foon KA Flow cytometric immunophenotyping for hematologic neoplasms Blood
2008 Apr 15111(8)3941-67
bull Wood BL Principles of minimal residual disease detection for hematopoietic neoplasms by flow
cytometry Cytometry B Clin Cytom 2015 Apr 23
bull Shanafelt TD Ghia P Lanasa MC Landgren O Rawstron AC Monoclonal B-cell
lymphocytosis (MBL) biology natural history and clinical management Leukemia 2010
Mar24(3)512-20
bull Swerdlow SH Campo E Pileri SA Harris NL Stein H Siebert R Advani RGhielmini M Salles
GA Zelenetz AD Jaffe ES The 2016 revision of the World Health Organization classification of
lymphoid neoplasms Blood 2016 May 19127(20)2375-90
bull 3 Moench L Sachs Z Aasen G Dolan M Dayton V Courville EL Double- and triple-hit
lymphomas can present with features suggestive of immaturity including TdT expression and
create diagnostic challenges Leuk Lymphoma 2016572626-35
bull Sutherland DR Keeney M Illingworth A Practical guidelines for the high-sensitivity detection
and monitoring of paroxysmal nocturnal hemoglobinuria clones by flow cytometry Cytometry B
Clin Cytom 2012 Jul82(4)195-208
69
Case 3 BM biopsy
Case 3 BM biopsy
Case 3 BM biopsy report
bull Diffuse infiltrate large lymphoid cells
bull Scant to moderate cytoplasm
bull Large irregular nuclei with prominent nucleoli
bull Increased mitotic activity
bull Positive for BCL-2 BCL6 CD10 and MUM1
bull Negative for TdT CD5
bull Cytogenetics FISH on para renal mass ndash C-MYC and
BCL2 rearrangements detected
Case 3 Doubletriple hit lymphoma
(DHLTHL)
bull Morphologically heterogeneous B cell neoplasms
characterized by rearrangement of the MYC proto-
oncogene with concurrent BCL2 andor BCL6 gene
rearrangement
bull WHO 2008 criteria - most cases of DHL and THL
classified DLBCL or B-cell lymphoma unclassifiable
with features intermediate between DLBCL and Burkitt
lymphoma (BCLU)
Case 3 DHLTHL
bull Retrospective study from one institution features of 13 cases (9 DHL4
THL)
bull median age was 59 years (range 30-74) eight females five males
bull enlarging lymphadenopathymasses (11 patients) abnormal peripheral
blood findings (2 patients)
bull TdT positivity (four cases two THLtwo DHL) dim CD45 expression (7
cases) lack of CD20 (2 cases) or lack of sIg (3 cases) and blastoid
morphology (2 cases)
bull Conclusion Expression of TdT in a B-cell lymphoma with mature
features or expression of surface light chain in a case otherwise
suggestive of B-lymphoblastic leukemialymphoma should prompt
an expedited evaluation for DHLTHL
Leuk Lymphoma 2016 Feb 181-10 [Epub ahead of print]
Double- and triple-hit lymphomas can present with features suggestive
of immaturity including TdT expression and create diagnostic challenges
Moench L Sachs Z Aasen G Dolan M Dayton V Courville EL
Case 4
bull 55 year old female
bull Previous history hyperparathyroidism
bull Parathyroidectomy in 1999
bull Mild persistent hypercalcemia
bull WBC 4800uL hemoglobin 121 gL Plt 296
bull Total protein 90gL IgG 34gL monoclonal peak
209gL
Case 4 Monoclonal IgG patient
Polyclonal B cells
With maturing hematogones
helper suppressor T cells
less than 3 blasts
Maturing myeloid population
Case 4 BM Ungated
Case 4 cytoplasmic immunoglobulin
Need KL
Case 4 BM Multiple Myeloma
Case 4 follow up
bull Smouldering myeloma
bull Recently developed CRAB (hyperCalcemia Renal
insufficiency Anemia Bone lesions
bull Serum free light chain ratio 123
bull Bone marrow aspirate gt60 plasma cells
bull Currently on treatment pending bone marrow
transplant
Case 5
bull 65 year old male
bull Slight lymphocytosis detected on routine CBC analysis
(lymph 4500uL)
bull Asymptomatic sent for flow cytometry
Case 5 PB
Case 5 flow cytometry KL ratio 0625
Case 5 Monoclonal B cell lymphocytosis
KL 18 KL 056
Case 5 Monoclonal B cell lymphocytosis
bull WHO defines CLL as abnormal lymphocyte count
gt5x10E9L (CD19+CD5+ light chain restricted) in the
absence of other clinical symptoms
bull Patients with small B cell clones are often detected as
an incidental finding
bull WHO 2016
bull CD19+CD5+ lt05x10E9L ldquoLow count MBLrdquo
raquo 05-5x10E9L ldquoHigh count MBLrdquo
raquo Non CLL type phenotype MBL also recognized
Monoclonal B cell Lymphocytosis
bull CLL immunophenotype - vast majority (75)
bull Atypical MBL
bull normal levels both CD5 and CD20 with variable
expression of CD23 (always exclude Mantle-cell
Lymphoma in CD23 negative cases)
bull Non-CLL-phenotype (CD5minus) MBL
bull Lacking CD5 expression and CD10 likely resembling B-
cell lymphomas of marginal-zone origin
Case 5 What is the normal KL Ratio
bull What is the normal or abnormal kappalambda ratio for
entire ldquopopulationrdquo of all B cells
bull Traditional view
ndash Kappa Lambda 21 or 31 ( 12 -13)
ndash Range 14 60
Case 5 What is the normal KL ratio
bull Wrong question
bull It doesnrsquot matter how many of the B cells are clonal but rather
that their exists a clonal population of B cells within the total
population
bull Need to Identify malignant B cells within a population of
normal B cells
bull In order to interpret a pathological immunophenotype it is
necessary to refer to quantitative and qualitative values of
normal B-cell subpopulations
bull Pearl Identify the abnormal phenotype first then look at KL
ratio
Case 6
bull 12 year old male presents to ER with fatigue bruising
Lkc 2200uL
bull Hb 84gL
bull Plt 32
bull Diff ndash neutropenia no blasts noted few ldquoatypicalrdquo cells
bull RBC ndash non specific changes
Leukemia
Case 6 PB
Case 6 BM
Case 6
bull BM aspirate ndash Hypocellular relative lymphocytosis
marked reduction in myeloid lineages
hypoplasticaplastic anemia
bull Flow Cytometry ndash less than 3 blasts maturing
myeloid 68 lymphocytes polyclonal B helper
suppressor T
bull Unusual pattern of CD13CD16 staining noted
Case 6 flow cytometry Aplastic Anemia
Normal Patient
Normal
Patient
Patient
Normal
Case 6 RBC Test for PNH CD235a v s CD59
Case 6 Neutrophil test for PNH clone
FLAER CD24 CD45CD15
18 PNH clone
Case 6
bull Follow up
bull Patient on steroids acyclovir and anti-thymocyte
globulin
bull PNH clone Neutrophils 96 RBC 1 Nov 2015
bull In remission as of April 2017
- Lkc 5500uL (Neuts 23)
- Hb 129gL
- Plt 208 X10e9
Summary
bull B cell malignancies are the most common
hematological disorders seen in flow cytometry
bull Many have complex overlapping phenotypes
bull Important to look at ungated plots as well as gating on
specific subpopulations
bull Level of expression of markers is as important as
positive or negative (think CD13 vs CD16)
68
References
bull Craig FE Foon KA Flow cytometric immunophenotyping for hematologic neoplasms Blood
2008 Apr 15111(8)3941-67
bull Wood BL Principles of minimal residual disease detection for hematopoietic neoplasms by flow
cytometry Cytometry B Clin Cytom 2015 Apr 23
bull Shanafelt TD Ghia P Lanasa MC Landgren O Rawstron AC Monoclonal B-cell
lymphocytosis (MBL) biology natural history and clinical management Leukemia 2010
Mar24(3)512-20
bull Swerdlow SH Campo E Pileri SA Harris NL Stein H Siebert R Advani RGhielmini M Salles
GA Zelenetz AD Jaffe ES The 2016 revision of the World Health Organization classification of
lymphoid neoplasms Blood 2016 May 19127(20)2375-90
bull 3 Moench L Sachs Z Aasen G Dolan M Dayton V Courville EL Double- and triple-hit
lymphomas can present with features suggestive of immaturity including TdT expression and
create diagnostic challenges Leuk Lymphoma 2016572626-35
bull Sutherland DR Keeney M Illingworth A Practical guidelines for the high-sensitivity detection
and monitoring of paroxysmal nocturnal hemoglobinuria clones by flow cytometry Cytometry B
Clin Cytom 2012 Jul82(4)195-208
69
Case 3 BM biopsy
Case 3 BM biopsy report
bull Diffuse infiltrate large lymphoid cells
bull Scant to moderate cytoplasm
bull Large irregular nuclei with prominent nucleoli
bull Increased mitotic activity
bull Positive for BCL-2 BCL6 CD10 and MUM1
bull Negative for TdT CD5
bull Cytogenetics FISH on para renal mass ndash C-MYC and
BCL2 rearrangements detected
Case 3 Doubletriple hit lymphoma
(DHLTHL)
bull Morphologically heterogeneous B cell neoplasms
characterized by rearrangement of the MYC proto-
oncogene with concurrent BCL2 andor BCL6 gene
rearrangement
bull WHO 2008 criteria - most cases of DHL and THL
classified DLBCL or B-cell lymphoma unclassifiable
with features intermediate between DLBCL and Burkitt
lymphoma (BCLU)
Case 3 DHLTHL
bull Retrospective study from one institution features of 13 cases (9 DHL4
THL)
bull median age was 59 years (range 30-74) eight females five males
bull enlarging lymphadenopathymasses (11 patients) abnormal peripheral
blood findings (2 patients)
bull TdT positivity (four cases two THLtwo DHL) dim CD45 expression (7
cases) lack of CD20 (2 cases) or lack of sIg (3 cases) and blastoid
morphology (2 cases)
bull Conclusion Expression of TdT in a B-cell lymphoma with mature
features or expression of surface light chain in a case otherwise
suggestive of B-lymphoblastic leukemialymphoma should prompt
an expedited evaluation for DHLTHL
Leuk Lymphoma 2016 Feb 181-10 [Epub ahead of print]
Double- and triple-hit lymphomas can present with features suggestive
of immaturity including TdT expression and create diagnostic challenges
Moench L Sachs Z Aasen G Dolan M Dayton V Courville EL
Case 4
bull 55 year old female
bull Previous history hyperparathyroidism
bull Parathyroidectomy in 1999
bull Mild persistent hypercalcemia
bull WBC 4800uL hemoglobin 121 gL Plt 296
bull Total protein 90gL IgG 34gL monoclonal peak
209gL
Case 4 Monoclonal IgG patient
Polyclonal B cells
With maturing hematogones
helper suppressor T cells
less than 3 blasts
Maturing myeloid population
Case 4 BM Ungated
Case 4 cytoplasmic immunoglobulin
Need KL
Case 4 BM Multiple Myeloma
Case 4 follow up
bull Smouldering myeloma
bull Recently developed CRAB (hyperCalcemia Renal
insufficiency Anemia Bone lesions
bull Serum free light chain ratio 123
bull Bone marrow aspirate gt60 plasma cells
bull Currently on treatment pending bone marrow
transplant
Case 5
bull 65 year old male
bull Slight lymphocytosis detected on routine CBC analysis
(lymph 4500uL)
bull Asymptomatic sent for flow cytometry
Case 5 PB
Case 5 flow cytometry KL ratio 0625
Case 5 Monoclonal B cell lymphocytosis
KL 18 KL 056
Case 5 Monoclonal B cell lymphocytosis
bull WHO defines CLL as abnormal lymphocyte count
gt5x10E9L (CD19+CD5+ light chain restricted) in the
absence of other clinical symptoms
bull Patients with small B cell clones are often detected as
an incidental finding
bull WHO 2016
bull CD19+CD5+ lt05x10E9L ldquoLow count MBLrdquo
raquo 05-5x10E9L ldquoHigh count MBLrdquo
raquo Non CLL type phenotype MBL also recognized
Monoclonal B cell Lymphocytosis
bull CLL immunophenotype - vast majority (75)
bull Atypical MBL
bull normal levels both CD5 and CD20 with variable
expression of CD23 (always exclude Mantle-cell
Lymphoma in CD23 negative cases)
bull Non-CLL-phenotype (CD5minus) MBL
bull Lacking CD5 expression and CD10 likely resembling B-
cell lymphomas of marginal-zone origin
Case 5 What is the normal KL Ratio
bull What is the normal or abnormal kappalambda ratio for
entire ldquopopulationrdquo of all B cells
bull Traditional view
ndash Kappa Lambda 21 or 31 ( 12 -13)
ndash Range 14 60
Case 5 What is the normal KL ratio
bull Wrong question
bull It doesnrsquot matter how many of the B cells are clonal but rather
that their exists a clonal population of B cells within the total
population
bull Need to Identify malignant B cells within a population of
normal B cells
bull In order to interpret a pathological immunophenotype it is
necessary to refer to quantitative and qualitative values of
normal B-cell subpopulations
bull Pearl Identify the abnormal phenotype first then look at KL
ratio
Case 6
bull 12 year old male presents to ER with fatigue bruising
Lkc 2200uL
bull Hb 84gL
bull Plt 32
bull Diff ndash neutropenia no blasts noted few ldquoatypicalrdquo cells
bull RBC ndash non specific changes
Leukemia
Case 6 PB
Case 6 BM
Case 6
bull BM aspirate ndash Hypocellular relative lymphocytosis
marked reduction in myeloid lineages
hypoplasticaplastic anemia
bull Flow Cytometry ndash less than 3 blasts maturing
myeloid 68 lymphocytes polyclonal B helper
suppressor T
bull Unusual pattern of CD13CD16 staining noted
Case 6 flow cytometry Aplastic Anemia
Normal Patient
Normal
Patient
Patient
Normal
Case 6 RBC Test for PNH CD235a v s CD59
Case 6 Neutrophil test for PNH clone
FLAER CD24 CD45CD15
18 PNH clone
Case 6
bull Follow up
bull Patient on steroids acyclovir and anti-thymocyte
globulin
bull PNH clone Neutrophils 96 RBC 1 Nov 2015
bull In remission as of April 2017
- Lkc 5500uL (Neuts 23)
- Hb 129gL
- Plt 208 X10e9
Summary
bull B cell malignancies are the most common
hematological disorders seen in flow cytometry
bull Many have complex overlapping phenotypes
bull Important to look at ungated plots as well as gating on
specific subpopulations
bull Level of expression of markers is as important as
positive or negative (think CD13 vs CD16)
68
References
bull Craig FE Foon KA Flow cytometric immunophenotyping for hematologic neoplasms Blood
2008 Apr 15111(8)3941-67
bull Wood BL Principles of minimal residual disease detection for hematopoietic neoplasms by flow
cytometry Cytometry B Clin Cytom 2015 Apr 23
bull Shanafelt TD Ghia P Lanasa MC Landgren O Rawstron AC Monoclonal B-cell
lymphocytosis (MBL) biology natural history and clinical management Leukemia 2010
Mar24(3)512-20
bull Swerdlow SH Campo E Pileri SA Harris NL Stein H Siebert R Advani RGhielmini M Salles
GA Zelenetz AD Jaffe ES The 2016 revision of the World Health Organization classification of
lymphoid neoplasms Blood 2016 May 19127(20)2375-90
bull 3 Moench L Sachs Z Aasen G Dolan M Dayton V Courville EL Double- and triple-hit
lymphomas can present with features suggestive of immaturity including TdT expression and
create diagnostic challenges Leuk Lymphoma 2016572626-35
bull Sutherland DR Keeney M Illingworth A Practical guidelines for the high-sensitivity detection
and monitoring of paroxysmal nocturnal hemoglobinuria clones by flow cytometry Cytometry B
Clin Cytom 2012 Jul82(4)195-208
69
Case 3 BM biopsy report
bull Diffuse infiltrate large lymphoid cells
bull Scant to moderate cytoplasm
bull Large irregular nuclei with prominent nucleoli
bull Increased mitotic activity
bull Positive for BCL-2 BCL6 CD10 and MUM1
bull Negative for TdT CD5
bull Cytogenetics FISH on para renal mass ndash C-MYC and
BCL2 rearrangements detected
Case 3 Doubletriple hit lymphoma
(DHLTHL)
bull Morphologically heterogeneous B cell neoplasms
characterized by rearrangement of the MYC proto-
oncogene with concurrent BCL2 andor BCL6 gene
rearrangement
bull WHO 2008 criteria - most cases of DHL and THL
classified DLBCL or B-cell lymphoma unclassifiable
with features intermediate between DLBCL and Burkitt
lymphoma (BCLU)
Case 3 DHLTHL
bull Retrospective study from one institution features of 13 cases (9 DHL4
THL)
bull median age was 59 years (range 30-74) eight females five males
bull enlarging lymphadenopathymasses (11 patients) abnormal peripheral
blood findings (2 patients)
bull TdT positivity (four cases two THLtwo DHL) dim CD45 expression (7
cases) lack of CD20 (2 cases) or lack of sIg (3 cases) and blastoid
morphology (2 cases)
bull Conclusion Expression of TdT in a B-cell lymphoma with mature
features or expression of surface light chain in a case otherwise
suggestive of B-lymphoblastic leukemialymphoma should prompt
an expedited evaluation for DHLTHL
Leuk Lymphoma 2016 Feb 181-10 [Epub ahead of print]
Double- and triple-hit lymphomas can present with features suggestive
of immaturity including TdT expression and create diagnostic challenges
Moench L Sachs Z Aasen G Dolan M Dayton V Courville EL
Case 4
bull 55 year old female
bull Previous history hyperparathyroidism
bull Parathyroidectomy in 1999
bull Mild persistent hypercalcemia
bull WBC 4800uL hemoglobin 121 gL Plt 296
bull Total protein 90gL IgG 34gL monoclonal peak
209gL
Case 4 Monoclonal IgG patient
Polyclonal B cells
With maturing hematogones
helper suppressor T cells
less than 3 blasts
Maturing myeloid population
Case 4 BM Ungated
Case 4 cytoplasmic immunoglobulin
Need KL
Case 4 BM Multiple Myeloma
Case 4 follow up
bull Smouldering myeloma
bull Recently developed CRAB (hyperCalcemia Renal
insufficiency Anemia Bone lesions
bull Serum free light chain ratio 123
bull Bone marrow aspirate gt60 plasma cells
bull Currently on treatment pending bone marrow
transplant
Case 5
bull 65 year old male
bull Slight lymphocytosis detected on routine CBC analysis
(lymph 4500uL)
bull Asymptomatic sent for flow cytometry
Case 5 PB
Case 5 flow cytometry KL ratio 0625
Case 5 Monoclonal B cell lymphocytosis
KL 18 KL 056
Case 5 Monoclonal B cell lymphocytosis
bull WHO defines CLL as abnormal lymphocyte count
gt5x10E9L (CD19+CD5+ light chain restricted) in the
absence of other clinical symptoms
bull Patients with small B cell clones are often detected as
an incidental finding
bull WHO 2016
bull CD19+CD5+ lt05x10E9L ldquoLow count MBLrdquo
raquo 05-5x10E9L ldquoHigh count MBLrdquo
raquo Non CLL type phenotype MBL also recognized
Monoclonal B cell Lymphocytosis
bull CLL immunophenotype - vast majority (75)
bull Atypical MBL
bull normal levels both CD5 and CD20 with variable
expression of CD23 (always exclude Mantle-cell
Lymphoma in CD23 negative cases)
bull Non-CLL-phenotype (CD5minus) MBL
bull Lacking CD5 expression and CD10 likely resembling B-
cell lymphomas of marginal-zone origin
Case 5 What is the normal KL Ratio
bull What is the normal or abnormal kappalambda ratio for
entire ldquopopulationrdquo of all B cells
bull Traditional view
ndash Kappa Lambda 21 or 31 ( 12 -13)
ndash Range 14 60
Case 5 What is the normal KL ratio
bull Wrong question
bull It doesnrsquot matter how many of the B cells are clonal but rather
that their exists a clonal population of B cells within the total
population
bull Need to Identify malignant B cells within a population of
normal B cells
bull In order to interpret a pathological immunophenotype it is
necessary to refer to quantitative and qualitative values of
normal B-cell subpopulations
bull Pearl Identify the abnormal phenotype first then look at KL
ratio
Case 6
bull 12 year old male presents to ER with fatigue bruising
Lkc 2200uL
bull Hb 84gL
bull Plt 32
bull Diff ndash neutropenia no blasts noted few ldquoatypicalrdquo cells
bull RBC ndash non specific changes
Leukemia
Case 6 PB
Case 6 BM
Case 6
bull BM aspirate ndash Hypocellular relative lymphocytosis
marked reduction in myeloid lineages
hypoplasticaplastic anemia
bull Flow Cytometry ndash less than 3 blasts maturing
myeloid 68 lymphocytes polyclonal B helper
suppressor T
bull Unusual pattern of CD13CD16 staining noted
Case 6 flow cytometry Aplastic Anemia
Normal Patient
Normal
Patient
Patient
Normal
Case 6 RBC Test for PNH CD235a v s CD59
Case 6 Neutrophil test for PNH clone
FLAER CD24 CD45CD15
18 PNH clone
Case 6
bull Follow up
bull Patient on steroids acyclovir and anti-thymocyte
globulin
bull PNH clone Neutrophils 96 RBC 1 Nov 2015
bull In remission as of April 2017
- Lkc 5500uL (Neuts 23)
- Hb 129gL
- Plt 208 X10e9
Summary
bull B cell malignancies are the most common
hematological disorders seen in flow cytometry
bull Many have complex overlapping phenotypes
bull Important to look at ungated plots as well as gating on
specific subpopulations
bull Level of expression of markers is as important as
positive or negative (think CD13 vs CD16)
68
References
bull Craig FE Foon KA Flow cytometric immunophenotyping for hematologic neoplasms Blood
2008 Apr 15111(8)3941-67
bull Wood BL Principles of minimal residual disease detection for hematopoietic neoplasms by flow
cytometry Cytometry B Clin Cytom 2015 Apr 23
bull Shanafelt TD Ghia P Lanasa MC Landgren O Rawstron AC Monoclonal B-cell
lymphocytosis (MBL) biology natural history and clinical management Leukemia 2010
Mar24(3)512-20
bull Swerdlow SH Campo E Pileri SA Harris NL Stein H Siebert R Advani RGhielmini M Salles
GA Zelenetz AD Jaffe ES The 2016 revision of the World Health Organization classification of
lymphoid neoplasms Blood 2016 May 19127(20)2375-90
bull 3 Moench L Sachs Z Aasen G Dolan M Dayton V Courville EL Double- and triple-hit
lymphomas can present with features suggestive of immaturity including TdT expression and
create diagnostic challenges Leuk Lymphoma 2016572626-35
bull Sutherland DR Keeney M Illingworth A Practical guidelines for the high-sensitivity detection
and monitoring of paroxysmal nocturnal hemoglobinuria clones by flow cytometry Cytometry B
Clin Cytom 2012 Jul82(4)195-208
69
Case 3 Doubletriple hit lymphoma
(DHLTHL)
bull Morphologically heterogeneous B cell neoplasms
characterized by rearrangement of the MYC proto-
oncogene with concurrent BCL2 andor BCL6 gene
rearrangement
bull WHO 2008 criteria - most cases of DHL and THL
classified DLBCL or B-cell lymphoma unclassifiable
with features intermediate between DLBCL and Burkitt
lymphoma (BCLU)
Case 3 DHLTHL
bull Retrospective study from one institution features of 13 cases (9 DHL4
THL)
bull median age was 59 years (range 30-74) eight females five males
bull enlarging lymphadenopathymasses (11 patients) abnormal peripheral
blood findings (2 patients)
bull TdT positivity (four cases two THLtwo DHL) dim CD45 expression (7
cases) lack of CD20 (2 cases) or lack of sIg (3 cases) and blastoid
morphology (2 cases)
bull Conclusion Expression of TdT in a B-cell lymphoma with mature
features or expression of surface light chain in a case otherwise
suggestive of B-lymphoblastic leukemialymphoma should prompt
an expedited evaluation for DHLTHL
Leuk Lymphoma 2016 Feb 181-10 [Epub ahead of print]
Double- and triple-hit lymphomas can present with features suggestive
of immaturity including TdT expression and create diagnostic challenges
Moench L Sachs Z Aasen G Dolan M Dayton V Courville EL
Case 4
bull 55 year old female
bull Previous history hyperparathyroidism
bull Parathyroidectomy in 1999
bull Mild persistent hypercalcemia
bull WBC 4800uL hemoglobin 121 gL Plt 296
bull Total protein 90gL IgG 34gL monoclonal peak
209gL
Case 4 Monoclonal IgG patient
Polyclonal B cells
With maturing hematogones
helper suppressor T cells
less than 3 blasts
Maturing myeloid population
Case 4 BM Ungated
Case 4 cytoplasmic immunoglobulin
Need KL
Case 4 BM Multiple Myeloma
Case 4 follow up
bull Smouldering myeloma
bull Recently developed CRAB (hyperCalcemia Renal
insufficiency Anemia Bone lesions
bull Serum free light chain ratio 123
bull Bone marrow aspirate gt60 plasma cells
bull Currently on treatment pending bone marrow
transplant
Case 5
bull 65 year old male
bull Slight lymphocytosis detected on routine CBC analysis
(lymph 4500uL)
bull Asymptomatic sent for flow cytometry
Case 5 PB
Case 5 flow cytometry KL ratio 0625
Case 5 Monoclonal B cell lymphocytosis
KL 18 KL 056
Case 5 Monoclonal B cell lymphocytosis
bull WHO defines CLL as abnormal lymphocyte count
gt5x10E9L (CD19+CD5+ light chain restricted) in the
absence of other clinical symptoms
bull Patients with small B cell clones are often detected as
an incidental finding
bull WHO 2016
bull CD19+CD5+ lt05x10E9L ldquoLow count MBLrdquo
raquo 05-5x10E9L ldquoHigh count MBLrdquo
raquo Non CLL type phenotype MBL also recognized
Monoclonal B cell Lymphocytosis
bull CLL immunophenotype - vast majority (75)
bull Atypical MBL
bull normal levels both CD5 and CD20 with variable
expression of CD23 (always exclude Mantle-cell
Lymphoma in CD23 negative cases)
bull Non-CLL-phenotype (CD5minus) MBL
bull Lacking CD5 expression and CD10 likely resembling B-
cell lymphomas of marginal-zone origin
Case 5 What is the normal KL Ratio
bull What is the normal or abnormal kappalambda ratio for
entire ldquopopulationrdquo of all B cells
bull Traditional view
ndash Kappa Lambda 21 or 31 ( 12 -13)
ndash Range 14 60
Case 5 What is the normal KL ratio
bull Wrong question
bull It doesnrsquot matter how many of the B cells are clonal but rather
that their exists a clonal population of B cells within the total
population
bull Need to Identify malignant B cells within a population of
normal B cells
bull In order to interpret a pathological immunophenotype it is
necessary to refer to quantitative and qualitative values of
normal B-cell subpopulations
bull Pearl Identify the abnormal phenotype first then look at KL
ratio
Case 6
bull 12 year old male presents to ER with fatigue bruising
Lkc 2200uL
bull Hb 84gL
bull Plt 32
bull Diff ndash neutropenia no blasts noted few ldquoatypicalrdquo cells
bull RBC ndash non specific changes
Leukemia
Case 6 PB
Case 6 BM
Case 6
bull BM aspirate ndash Hypocellular relative lymphocytosis
marked reduction in myeloid lineages
hypoplasticaplastic anemia
bull Flow Cytometry ndash less than 3 blasts maturing
myeloid 68 lymphocytes polyclonal B helper
suppressor T
bull Unusual pattern of CD13CD16 staining noted
Case 6 flow cytometry Aplastic Anemia
Normal Patient
Normal
Patient
Patient
Normal
Case 6 RBC Test for PNH CD235a v s CD59
Case 6 Neutrophil test for PNH clone
FLAER CD24 CD45CD15
18 PNH clone
Case 6
bull Follow up
bull Patient on steroids acyclovir and anti-thymocyte
globulin
bull PNH clone Neutrophils 96 RBC 1 Nov 2015
bull In remission as of April 2017
- Lkc 5500uL (Neuts 23)
- Hb 129gL
- Plt 208 X10e9
Summary
bull B cell malignancies are the most common
hematological disorders seen in flow cytometry
bull Many have complex overlapping phenotypes
bull Important to look at ungated plots as well as gating on
specific subpopulations
bull Level of expression of markers is as important as
positive or negative (think CD13 vs CD16)
68
References
bull Craig FE Foon KA Flow cytometric immunophenotyping for hematologic neoplasms Blood
2008 Apr 15111(8)3941-67
bull Wood BL Principles of minimal residual disease detection for hematopoietic neoplasms by flow
cytometry Cytometry B Clin Cytom 2015 Apr 23
bull Shanafelt TD Ghia P Lanasa MC Landgren O Rawstron AC Monoclonal B-cell
lymphocytosis (MBL) biology natural history and clinical management Leukemia 2010
Mar24(3)512-20
bull Swerdlow SH Campo E Pileri SA Harris NL Stein H Siebert R Advani RGhielmini M Salles
GA Zelenetz AD Jaffe ES The 2016 revision of the World Health Organization classification of
lymphoid neoplasms Blood 2016 May 19127(20)2375-90
bull 3 Moench L Sachs Z Aasen G Dolan M Dayton V Courville EL Double- and triple-hit
lymphomas can present with features suggestive of immaturity including TdT expression and
create diagnostic challenges Leuk Lymphoma 2016572626-35
bull Sutherland DR Keeney M Illingworth A Practical guidelines for the high-sensitivity detection
and monitoring of paroxysmal nocturnal hemoglobinuria clones by flow cytometry Cytometry B
Clin Cytom 2012 Jul82(4)195-208
69
Case 3 DHLTHL
bull Retrospective study from one institution features of 13 cases (9 DHL4
THL)
bull median age was 59 years (range 30-74) eight females five males
bull enlarging lymphadenopathymasses (11 patients) abnormal peripheral
blood findings (2 patients)
bull TdT positivity (four cases two THLtwo DHL) dim CD45 expression (7
cases) lack of CD20 (2 cases) or lack of sIg (3 cases) and blastoid
morphology (2 cases)
bull Conclusion Expression of TdT in a B-cell lymphoma with mature
features or expression of surface light chain in a case otherwise
suggestive of B-lymphoblastic leukemialymphoma should prompt
an expedited evaluation for DHLTHL
Leuk Lymphoma 2016 Feb 181-10 [Epub ahead of print]
Double- and triple-hit lymphomas can present with features suggestive
of immaturity including TdT expression and create diagnostic challenges
Moench L Sachs Z Aasen G Dolan M Dayton V Courville EL
Case 4
bull 55 year old female
bull Previous history hyperparathyroidism
bull Parathyroidectomy in 1999
bull Mild persistent hypercalcemia
bull WBC 4800uL hemoglobin 121 gL Plt 296
bull Total protein 90gL IgG 34gL monoclonal peak
209gL
Case 4 Monoclonal IgG patient
Polyclonal B cells
With maturing hematogones
helper suppressor T cells
less than 3 blasts
Maturing myeloid population
Case 4 BM Ungated
Case 4 cytoplasmic immunoglobulin
Need KL
Case 4 BM Multiple Myeloma
Case 4 follow up
bull Smouldering myeloma
bull Recently developed CRAB (hyperCalcemia Renal
insufficiency Anemia Bone lesions
bull Serum free light chain ratio 123
bull Bone marrow aspirate gt60 plasma cells
bull Currently on treatment pending bone marrow
transplant
Case 5
bull 65 year old male
bull Slight lymphocytosis detected on routine CBC analysis
(lymph 4500uL)
bull Asymptomatic sent for flow cytometry
Case 5 PB
Case 5 flow cytometry KL ratio 0625
Case 5 Monoclonal B cell lymphocytosis
KL 18 KL 056
Case 5 Monoclonal B cell lymphocytosis
bull WHO defines CLL as abnormal lymphocyte count
gt5x10E9L (CD19+CD5+ light chain restricted) in the
absence of other clinical symptoms
bull Patients with small B cell clones are often detected as
an incidental finding
bull WHO 2016
bull CD19+CD5+ lt05x10E9L ldquoLow count MBLrdquo
raquo 05-5x10E9L ldquoHigh count MBLrdquo
raquo Non CLL type phenotype MBL also recognized
Monoclonal B cell Lymphocytosis
bull CLL immunophenotype - vast majority (75)
bull Atypical MBL
bull normal levels both CD5 and CD20 with variable
expression of CD23 (always exclude Mantle-cell
Lymphoma in CD23 negative cases)
bull Non-CLL-phenotype (CD5minus) MBL
bull Lacking CD5 expression and CD10 likely resembling B-
cell lymphomas of marginal-zone origin
Case 5 What is the normal KL Ratio
bull What is the normal or abnormal kappalambda ratio for
entire ldquopopulationrdquo of all B cells
bull Traditional view
ndash Kappa Lambda 21 or 31 ( 12 -13)
ndash Range 14 60
Case 5 What is the normal KL ratio
bull Wrong question
bull It doesnrsquot matter how many of the B cells are clonal but rather
that their exists a clonal population of B cells within the total
population
bull Need to Identify malignant B cells within a population of
normal B cells
bull In order to interpret a pathological immunophenotype it is
necessary to refer to quantitative and qualitative values of
normal B-cell subpopulations
bull Pearl Identify the abnormal phenotype first then look at KL
ratio
Case 6
bull 12 year old male presents to ER with fatigue bruising
Lkc 2200uL
bull Hb 84gL
bull Plt 32
bull Diff ndash neutropenia no blasts noted few ldquoatypicalrdquo cells
bull RBC ndash non specific changes
Leukemia
Case 6 PB
Case 6 BM
Case 6
bull BM aspirate ndash Hypocellular relative lymphocytosis
marked reduction in myeloid lineages
hypoplasticaplastic anemia
bull Flow Cytometry ndash less than 3 blasts maturing
myeloid 68 lymphocytes polyclonal B helper
suppressor T
bull Unusual pattern of CD13CD16 staining noted
Case 6 flow cytometry Aplastic Anemia
Normal Patient
Normal
Patient
Patient
Normal
Case 6 RBC Test for PNH CD235a v s CD59
Case 6 Neutrophil test for PNH clone
FLAER CD24 CD45CD15
18 PNH clone
Case 6
bull Follow up
bull Patient on steroids acyclovir and anti-thymocyte
globulin
bull PNH clone Neutrophils 96 RBC 1 Nov 2015
bull In remission as of April 2017
- Lkc 5500uL (Neuts 23)
- Hb 129gL
- Plt 208 X10e9
Summary
bull B cell malignancies are the most common
hematological disorders seen in flow cytometry
bull Many have complex overlapping phenotypes
bull Important to look at ungated plots as well as gating on
specific subpopulations
bull Level of expression of markers is as important as
positive or negative (think CD13 vs CD16)
68
References
bull Craig FE Foon KA Flow cytometric immunophenotyping for hematologic neoplasms Blood
2008 Apr 15111(8)3941-67
bull Wood BL Principles of minimal residual disease detection for hematopoietic neoplasms by flow
cytometry Cytometry B Clin Cytom 2015 Apr 23
bull Shanafelt TD Ghia P Lanasa MC Landgren O Rawstron AC Monoclonal B-cell
lymphocytosis (MBL) biology natural history and clinical management Leukemia 2010
Mar24(3)512-20
bull Swerdlow SH Campo E Pileri SA Harris NL Stein H Siebert R Advani RGhielmini M Salles
GA Zelenetz AD Jaffe ES The 2016 revision of the World Health Organization classification of
lymphoid neoplasms Blood 2016 May 19127(20)2375-90
bull 3 Moench L Sachs Z Aasen G Dolan M Dayton V Courville EL Double- and triple-hit
lymphomas can present with features suggestive of immaturity including TdT expression and
create diagnostic challenges Leuk Lymphoma 2016572626-35
bull Sutherland DR Keeney M Illingworth A Practical guidelines for the high-sensitivity detection
and monitoring of paroxysmal nocturnal hemoglobinuria clones by flow cytometry Cytometry B
Clin Cytom 2012 Jul82(4)195-208
69
Case 4
bull 55 year old female
bull Previous history hyperparathyroidism
bull Parathyroidectomy in 1999
bull Mild persistent hypercalcemia
bull WBC 4800uL hemoglobin 121 gL Plt 296
bull Total protein 90gL IgG 34gL monoclonal peak
209gL
Case 4 Monoclonal IgG patient
Polyclonal B cells
With maturing hematogones
helper suppressor T cells
less than 3 blasts
Maturing myeloid population
Case 4 BM Ungated
Case 4 cytoplasmic immunoglobulin
Need KL
Case 4 BM Multiple Myeloma
Case 4 follow up
bull Smouldering myeloma
bull Recently developed CRAB (hyperCalcemia Renal
insufficiency Anemia Bone lesions
bull Serum free light chain ratio 123
bull Bone marrow aspirate gt60 plasma cells
bull Currently on treatment pending bone marrow
transplant
Case 5
bull 65 year old male
bull Slight lymphocytosis detected on routine CBC analysis
(lymph 4500uL)
bull Asymptomatic sent for flow cytometry
Case 5 PB
Case 5 flow cytometry KL ratio 0625
Case 5 Monoclonal B cell lymphocytosis
KL 18 KL 056
Case 5 Monoclonal B cell lymphocytosis
bull WHO defines CLL as abnormal lymphocyte count
gt5x10E9L (CD19+CD5+ light chain restricted) in the
absence of other clinical symptoms
bull Patients with small B cell clones are often detected as
an incidental finding
bull WHO 2016
bull CD19+CD5+ lt05x10E9L ldquoLow count MBLrdquo
raquo 05-5x10E9L ldquoHigh count MBLrdquo
raquo Non CLL type phenotype MBL also recognized
Monoclonal B cell Lymphocytosis
bull CLL immunophenotype - vast majority (75)
bull Atypical MBL
bull normal levels both CD5 and CD20 with variable
expression of CD23 (always exclude Mantle-cell
Lymphoma in CD23 negative cases)
bull Non-CLL-phenotype (CD5minus) MBL
bull Lacking CD5 expression and CD10 likely resembling B-
cell lymphomas of marginal-zone origin
Case 5 What is the normal KL Ratio
bull What is the normal or abnormal kappalambda ratio for
entire ldquopopulationrdquo of all B cells
bull Traditional view
ndash Kappa Lambda 21 or 31 ( 12 -13)
ndash Range 14 60
Case 5 What is the normal KL ratio
bull Wrong question
bull It doesnrsquot matter how many of the B cells are clonal but rather
that their exists a clonal population of B cells within the total
population
bull Need to Identify malignant B cells within a population of
normal B cells
bull In order to interpret a pathological immunophenotype it is
necessary to refer to quantitative and qualitative values of
normal B-cell subpopulations
bull Pearl Identify the abnormal phenotype first then look at KL
ratio
Case 6
bull 12 year old male presents to ER with fatigue bruising
Lkc 2200uL
bull Hb 84gL
bull Plt 32
bull Diff ndash neutropenia no blasts noted few ldquoatypicalrdquo cells
bull RBC ndash non specific changes
Leukemia
Case 6 PB
Case 6 BM
Case 6
bull BM aspirate ndash Hypocellular relative lymphocytosis
marked reduction in myeloid lineages
hypoplasticaplastic anemia
bull Flow Cytometry ndash less than 3 blasts maturing
myeloid 68 lymphocytes polyclonal B helper
suppressor T
bull Unusual pattern of CD13CD16 staining noted
Case 6 flow cytometry Aplastic Anemia
Normal Patient
Normal
Patient
Patient
Normal
Case 6 RBC Test for PNH CD235a v s CD59
Case 6 Neutrophil test for PNH clone
FLAER CD24 CD45CD15
18 PNH clone
Case 6
bull Follow up
bull Patient on steroids acyclovir and anti-thymocyte
globulin
bull PNH clone Neutrophils 96 RBC 1 Nov 2015
bull In remission as of April 2017
- Lkc 5500uL (Neuts 23)
- Hb 129gL
- Plt 208 X10e9
Summary
bull B cell malignancies are the most common
hematological disorders seen in flow cytometry
bull Many have complex overlapping phenotypes
bull Important to look at ungated plots as well as gating on
specific subpopulations
bull Level of expression of markers is as important as
positive or negative (think CD13 vs CD16)
68
References
bull Craig FE Foon KA Flow cytometric immunophenotyping for hematologic neoplasms Blood
2008 Apr 15111(8)3941-67
bull Wood BL Principles of minimal residual disease detection for hematopoietic neoplasms by flow
cytometry Cytometry B Clin Cytom 2015 Apr 23
bull Shanafelt TD Ghia P Lanasa MC Landgren O Rawstron AC Monoclonal B-cell
lymphocytosis (MBL) biology natural history and clinical management Leukemia 2010
Mar24(3)512-20
bull Swerdlow SH Campo E Pileri SA Harris NL Stein H Siebert R Advani RGhielmini M Salles
GA Zelenetz AD Jaffe ES The 2016 revision of the World Health Organization classification of
lymphoid neoplasms Blood 2016 May 19127(20)2375-90
bull 3 Moench L Sachs Z Aasen G Dolan M Dayton V Courville EL Double- and triple-hit
lymphomas can present with features suggestive of immaturity including TdT expression and
create diagnostic challenges Leuk Lymphoma 2016572626-35
bull Sutherland DR Keeney M Illingworth A Practical guidelines for the high-sensitivity detection
and monitoring of paroxysmal nocturnal hemoglobinuria clones by flow cytometry Cytometry B
Clin Cytom 2012 Jul82(4)195-208
69
Case 4 Monoclonal IgG patient
Polyclonal B cells
With maturing hematogones
helper suppressor T cells
less than 3 blasts
Maturing myeloid population
Case 4 BM Ungated
Case 4 cytoplasmic immunoglobulin
Need KL
Case 4 BM Multiple Myeloma
Case 4 follow up
bull Smouldering myeloma
bull Recently developed CRAB (hyperCalcemia Renal
insufficiency Anemia Bone lesions
bull Serum free light chain ratio 123
bull Bone marrow aspirate gt60 plasma cells
bull Currently on treatment pending bone marrow
transplant
Case 5
bull 65 year old male
bull Slight lymphocytosis detected on routine CBC analysis
(lymph 4500uL)
bull Asymptomatic sent for flow cytometry
Case 5 PB
Case 5 flow cytometry KL ratio 0625
Case 5 Monoclonal B cell lymphocytosis
KL 18 KL 056
Case 5 Monoclonal B cell lymphocytosis
bull WHO defines CLL as abnormal lymphocyte count
gt5x10E9L (CD19+CD5+ light chain restricted) in the
absence of other clinical symptoms
bull Patients with small B cell clones are often detected as
an incidental finding
bull WHO 2016
bull CD19+CD5+ lt05x10E9L ldquoLow count MBLrdquo
raquo 05-5x10E9L ldquoHigh count MBLrdquo
raquo Non CLL type phenotype MBL also recognized
Monoclonal B cell Lymphocytosis
bull CLL immunophenotype - vast majority (75)
bull Atypical MBL
bull normal levels both CD5 and CD20 with variable
expression of CD23 (always exclude Mantle-cell
Lymphoma in CD23 negative cases)
bull Non-CLL-phenotype (CD5minus) MBL
bull Lacking CD5 expression and CD10 likely resembling B-
cell lymphomas of marginal-zone origin
Case 5 What is the normal KL Ratio
bull What is the normal or abnormal kappalambda ratio for
entire ldquopopulationrdquo of all B cells
bull Traditional view
ndash Kappa Lambda 21 or 31 ( 12 -13)
ndash Range 14 60
Case 5 What is the normal KL ratio
bull Wrong question
bull It doesnrsquot matter how many of the B cells are clonal but rather
that their exists a clonal population of B cells within the total
population
bull Need to Identify malignant B cells within a population of
normal B cells
bull In order to interpret a pathological immunophenotype it is
necessary to refer to quantitative and qualitative values of
normal B-cell subpopulations
bull Pearl Identify the abnormal phenotype first then look at KL
ratio
Case 6
bull 12 year old male presents to ER with fatigue bruising
Lkc 2200uL
bull Hb 84gL
bull Plt 32
bull Diff ndash neutropenia no blasts noted few ldquoatypicalrdquo cells
bull RBC ndash non specific changes
Leukemia
Case 6 PB
Case 6 BM
Case 6
bull BM aspirate ndash Hypocellular relative lymphocytosis
marked reduction in myeloid lineages
hypoplasticaplastic anemia
bull Flow Cytometry ndash less than 3 blasts maturing
myeloid 68 lymphocytes polyclonal B helper
suppressor T
bull Unusual pattern of CD13CD16 staining noted
Case 6 flow cytometry Aplastic Anemia
Normal Patient
Normal
Patient
Patient
Normal
Case 6 RBC Test for PNH CD235a v s CD59
Case 6 Neutrophil test for PNH clone
FLAER CD24 CD45CD15
18 PNH clone
Case 6
bull Follow up
bull Patient on steroids acyclovir and anti-thymocyte
globulin
bull PNH clone Neutrophils 96 RBC 1 Nov 2015
bull In remission as of April 2017
- Lkc 5500uL (Neuts 23)
- Hb 129gL
- Plt 208 X10e9
Summary
bull B cell malignancies are the most common
hematological disorders seen in flow cytometry
bull Many have complex overlapping phenotypes
bull Important to look at ungated plots as well as gating on
specific subpopulations
bull Level of expression of markers is as important as
positive or negative (think CD13 vs CD16)
68
References
bull Craig FE Foon KA Flow cytometric immunophenotyping for hematologic neoplasms Blood
2008 Apr 15111(8)3941-67
bull Wood BL Principles of minimal residual disease detection for hematopoietic neoplasms by flow
cytometry Cytometry B Clin Cytom 2015 Apr 23
bull Shanafelt TD Ghia P Lanasa MC Landgren O Rawstron AC Monoclonal B-cell
lymphocytosis (MBL) biology natural history and clinical management Leukemia 2010
Mar24(3)512-20
bull Swerdlow SH Campo E Pileri SA Harris NL Stein H Siebert R Advani RGhielmini M Salles
GA Zelenetz AD Jaffe ES The 2016 revision of the World Health Organization classification of
lymphoid neoplasms Blood 2016 May 19127(20)2375-90
bull 3 Moench L Sachs Z Aasen G Dolan M Dayton V Courville EL Double- and triple-hit
lymphomas can present with features suggestive of immaturity including TdT expression and
create diagnostic challenges Leuk Lymphoma 2016572626-35
bull Sutherland DR Keeney M Illingworth A Practical guidelines for the high-sensitivity detection
and monitoring of paroxysmal nocturnal hemoglobinuria clones by flow cytometry Cytometry B
Clin Cytom 2012 Jul82(4)195-208
69
Case 4 BM Ungated
Case 4 cytoplasmic immunoglobulin
Need KL
Case 4 BM Multiple Myeloma
Case 4 follow up
bull Smouldering myeloma
bull Recently developed CRAB (hyperCalcemia Renal
insufficiency Anemia Bone lesions
bull Serum free light chain ratio 123
bull Bone marrow aspirate gt60 plasma cells
bull Currently on treatment pending bone marrow
transplant
Case 5
bull 65 year old male
bull Slight lymphocytosis detected on routine CBC analysis
(lymph 4500uL)
bull Asymptomatic sent for flow cytometry
Case 5 PB
Case 5 flow cytometry KL ratio 0625
Case 5 Monoclonal B cell lymphocytosis
KL 18 KL 056
Case 5 Monoclonal B cell lymphocytosis
bull WHO defines CLL as abnormal lymphocyte count
gt5x10E9L (CD19+CD5+ light chain restricted) in the
absence of other clinical symptoms
bull Patients with small B cell clones are often detected as
an incidental finding
bull WHO 2016
bull CD19+CD5+ lt05x10E9L ldquoLow count MBLrdquo
raquo 05-5x10E9L ldquoHigh count MBLrdquo
raquo Non CLL type phenotype MBL also recognized
Monoclonal B cell Lymphocytosis
bull CLL immunophenotype - vast majority (75)
bull Atypical MBL
bull normal levels both CD5 and CD20 with variable
expression of CD23 (always exclude Mantle-cell
Lymphoma in CD23 negative cases)
bull Non-CLL-phenotype (CD5minus) MBL
bull Lacking CD5 expression and CD10 likely resembling B-
cell lymphomas of marginal-zone origin
Case 5 What is the normal KL Ratio
bull What is the normal or abnormal kappalambda ratio for
entire ldquopopulationrdquo of all B cells
bull Traditional view
ndash Kappa Lambda 21 or 31 ( 12 -13)
ndash Range 14 60
Case 5 What is the normal KL ratio
bull Wrong question
bull It doesnrsquot matter how many of the B cells are clonal but rather
that their exists a clonal population of B cells within the total
population
bull Need to Identify malignant B cells within a population of
normal B cells
bull In order to interpret a pathological immunophenotype it is
necessary to refer to quantitative and qualitative values of
normal B-cell subpopulations
bull Pearl Identify the abnormal phenotype first then look at KL
ratio
Case 6
bull 12 year old male presents to ER with fatigue bruising
Lkc 2200uL
bull Hb 84gL
bull Plt 32
bull Diff ndash neutropenia no blasts noted few ldquoatypicalrdquo cells
bull RBC ndash non specific changes
Leukemia
Case 6 PB
Case 6 BM
Case 6
bull BM aspirate ndash Hypocellular relative lymphocytosis
marked reduction in myeloid lineages
hypoplasticaplastic anemia
bull Flow Cytometry ndash less than 3 blasts maturing
myeloid 68 lymphocytes polyclonal B helper
suppressor T
bull Unusual pattern of CD13CD16 staining noted
Case 6 flow cytometry Aplastic Anemia
Normal Patient
Normal
Patient
Patient
Normal
Case 6 RBC Test for PNH CD235a v s CD59
Case 6 Neutrophil test for PNH clone
FLAER CD24 CD45CD15
18 PNH clone
Case 6
bull Follow up
bull Patient on steroids acyclovir and anti-thymocyte
globulin
bull PNH clone Neutrophils 96 RBC 1 Nov 2015
bull In remission as of April 2017
- Lkc 5500uL (Neuts 23)
- Hb 129gL
- Plt 208 X10e9
Summary
bull B cell malignancies are the most common
hematological disorders seen in flow cytometry
bull Many have complex overlapping phenotypes
bull Important to look at ungated plots as well as gating on
specific subpopulations
bull Level of expression of markers is as important as
positive or negative (think CD13 vs CD16)
68
References
bull Craig FE Foon KA Flow cytometric immunophenotyping for hematologic neoplasms Blood
2008 Apr 15111(8)3941-67
bull Wood BL Principles of minimal residual disease detection for hematopoietic neoplasms by flow
cytometry Cytometry B Clin Cytom 2015 Apr 23
bull Shanafelt TD Ghia P Lanasa MC Landgren O Rawstron AC Monoclonal B-cell
lymphocytosis (MBL) biology natural history and clinical management Leukemia 2010
Mar24(3)512-20
bull Swerdlow SH Campo E Pileri SA Harris NL Stein H Siebert R Advani RGhielmini M Salles
GA Zelenetz AD Jaffe ES The 2016 revision of the World Health Organization classification of
lymphoid neoplasms Blood 2016 May 19127(20)2375-90
bull 3 Moench L Sachs Z Aasen G Dolan M Dayton V Courville EL Double- and triple-hit
lymphomas can present with features suggestive of immaturity including TdT expression and
create diagnostic challenges Leuk Lymphoma 2016572626-35
bull Sutherland DR Keeney M Illingworth A Practical guidelines for the high-sensitivity detection
and monitoring of paroxysmal nocturnal hemoglobinuria clones by flow cytometry Cytometry B
Clin Cytom 2012 Jul82(4)195-208
69
Case 4 cytoplasmic immunoglobulin
Need KL
Case 4 BM Multiple Myeloma
Case 4 follow up
bull Smouldering myeloma
bull Recently developed CRAB (hyperCalcemia Renal
insufficiency Anemia Bone lesions
bull Serum free light chain ratio 123
bull Bone marrow aspirate gt60 plasma cells
bull Currently on treatment pending bone marrow
transplant
Case 5
bull 65 year old male
bull Slight lymphocytosis detected on routine CBC analysis
(lymph 4500uL)
bull Asymptomatic sent for flow cytometry
Case 5 PB
Case 5 flow cytometry KL ratio 0625
Case 5 Monoclonal B cell lymphocytosis
KL 18 KL 056
Case 5 Monoclonal B cell lymphocytosis
bull WHO defines CLL as abnormal lymphocyte count
gt5x10E9L (CD19+CD5+ light chain restricted) in the
absence of other clinical symptoms
bull Patients with small B cell clones are often detected as
an incidental finding
bull WHO 2016
bull CD19+CD5+ lt05x10E9L ldquoLow count MBLrdquo
raquo 05-5x10E9L ldquoHigh count MBLrdquo
raquo Non CLL type phenotype MBL also recognized
Monoclonal B cell Lymphocytosis
bull CLL immunophenotype - vast majority (75)
bull Atypical MBL
bull normal levels both CD5 and CD20 with variable
expression of CD23 (always exclude Mantle-cell
Lymphoma in CD23 negative cases)
bull Non-CLL-phenotype (CD5minus) MBL
bull Lacking CD5 expression and CD10 likely resembling B-
cell lymphomas of marginal-zone origin
Case 5 What is the normal KL Ratio
bull What is the normal or abnormal kappalambda ratio for
entire ldquopopulationrdquo of all B cells
bull Traditional view
ndash Kappa Lambda 21 or 31 ( 12 -13)
ndash Range 14 60
Case 5 What is the normal KL ratio
bull Wrong question
bull It doesnrsquot matter how many of the B cells are clonal but rather
that their exists a clonal population of B cells within the total
population
bull Need to Identify malignant B cells within a population of
normal B cells
bull In order to interpret a pathological immunophenotype it is
necessary to refer to quantitative and qualitative values of
normal B-cell subpopulations
bull Pearl Identify the abnormal phenotype first then look at KL
ratio
Case 6
bull 12 year old male presents to ER with fatigue bruising
Lkc 2200uL
bull Hb 84gL
bull Plt 32
bull Diff ndash neutropenia no blasts noted few ldquoatypicalrdquo cells
bull RBC ndash non specific changes
Leukemia
Case 6 PB
Case 6 BM
Case 6
bull BM aspirate ndash Hypocellular relative lymphocytosis
marked reduction in myeloid lineages
hypoplasticaplastic anemia
bull Flow Cytometry ndash less than 3 blasts maturing
myeloid 68 lymphocytes polyclonal B helper
suppressor T
bull Unusual pattern of CD13CD16 staining noted
Case 6 flow cytometry Aplastic Anemia
Normal Patient
Normal
Patient
Patient
Normal
Case 6 RBC Test for PNH CD235a v s CD59
Case 6 Neutrophil test for PNH clone
FLAER CD24 CD45CD15
18 PNH clone
Case 6
bull Follow up
bull Patient on steroids acyclovir and anti-thymocyte
globulin
bull PNH clone Neutrophils 96 RBC 1 Nov 2015
bull In remission as of April 2017
- Lkc 5500uL (Neuts 23)
- Hb 129gL
- Plt 208 X10e9
Summary
bull B cell malignancies are the most common
hematological disorders seen in flow cytometry
bull Many have complex overlapping phenotypes
bull Important to look at ungated plots as well as gating on
specific subpopulations
bull Level of expression of markers is as important as
positive or negative (think CD13 vs CD16)
68
References
bull Craig FE Foon KA Flow cytometric immunophenotyping for hematologic neoplasms Blood
2008 Apr 15111(8)3941-67
bull Wood BL Principles of minimal residual disease detection for hematopoietic neoplasms by flow
cytometry Cytometry B Clin Cytom 2015 Apr 23
bull Shanafelt TD Ghia P Lanasa MC Landgren O Rawstron AC Monoclonal B-cell
lymphocytosis (MBL) biology natural history and clinical management Leukemia 2010
Mar24(3)512-20
bull Swerdlow SH Campo E Pileri SA Harris NL Stein H Siebert R Advani RGhielmini M Salles
GA Zelenetz AD Jaffe ES The 2016 revision of the World Health Organization classification of
lymphoid neoplasms Blood 2016 May 19127(20)2375-90
bull 3 Moench L Sachs Z Aasen G Dolan M Dayton V Courville EL Double- and triple-hit
lymphomas can present with features suggestive of immaturity including TdT expression and
create diagnostic challenges Leuk Lymphoma 2016572626-35
bull Sutherland DR Keeney M Illingworth A Practical guidelines for the high-sensitivity detection
and monitoring of paroxysmal nocturnal hemoglobinuria clones by flow cytometry Cytometry B
Clin Cytom 2012 Jul82(4)195-208
69
Case 4 BM Multiple Myeloma
Case 4 follow up
bull Smouldering myeloma
bull Recently developed CRAB (hyperCalcemia Renal
insufficiency Anemia Bone lesions
bull Serum free light chain ratio 123
bull Bone marrow aspirate gt60 plasma cells
bull Currently on treatment pending bone marrow
transplant
Case 5
bull 65 year old male
bull Slight lymphocytosis detected on routine CBC analysis
(lymph 4500uL)
bull Asymptomatic sent for flow cytometry
Case 5 PB
Case 5 flow cytometry KL ratio 0625
Case 5 Monoclonal B cell lymphocytosis
KL 18 KL 056
Case 5 Monoclonal B cell lymphocytosis
bull WHO defines CLL as abnormal lymphocyte count
gt5x10E9L (CD19+CD5+ light chain restricted) in the
absence of other clinical symptoms
bull Patients with small B cell clones are often detected as
an incidental finding
bull WHO 2016
bull CD19+CD5+ lt05x10E9L ldquoLow count MBLrdquo
raquo 05-5x10E9L ldquoHigh count MBLrdquo
raquo Non CLL type phenotype MBL also recognized
Monoclonal B cell Lymphocytosis
bull CLL immunophenotype - vast majority (75)
bull Atypical MBL
bull normal levels both CD5 and CD20 with variable
expression of CD23 (always exclude Mantle-cell
Lymphoma in CD23 negative cases)
bull Non-CLL-phenotype (CD5minus) MBL
bull Lacking CD5 expression and CD10 likely resembling B-
cell lymphomas of marginal-zone origin
Case 5 What is the normal KL Ratio
bull What is the normal or abnormal kappalambda ratio for
entire ldquopopulationrdquo of all B cells
bull Traditional view
ndash Kappa Lambda 21 or 31 ( 12 -13)
ndash Range 14 60
Case 5 What is the normal KL ratio
bull Wrong question
bull It doesnrsquot matter how many of the B cells are clonal but rather
that their exists a clonal population of B cells within the total
population
bull Need to Identify malignant B cells within a population of
normal B cells
bull In order to interpret a pathological immunophenotype it is
necessary to refer to quantitative and qualitative values of
normal B-cell subpopulations
bull Pearl Identify the abnormal phenotype first then look at KL
ratio
Case 6
bull 12 year old male presents to ER with fatigue bruising
Lkc 2200uL
bull Hb 84gL
bull Plt 32
bull Diff ndash neutropenia no blasts noted few ldquoatypicalrdquo cells
bull RBC ndash non specific changes
Leukemia
Case 6 PB
Case 6 BM
Case 6
bull BM aspirate ndash Hypocellular relative lymphocytosis
marked reduction in myeloid lineages
hypoplasticaplastic anemia
bull Flow Cytometry ndash less than 3 blasts maturing
myeloid 68 lymphocytes polyclonal B helper
suppressor T
bull Unusual pattern of CD13CD16 staining noted
Case 6 flow cytometry Aplastic Anemia
Normal Patient
Normal
Patient
Patient
Normal
Case 6 RBC Test for PNH CD235a v s CD59
Case 6 Neutrophil test for PNH clone
FLAER CD24 CD45CD15
18 PNH clone
Case 6
bull Follow up
bull Patient on steroids acyclovir and anti-thymocyte
globulin
bull PNH clone Neutrophils 96 RBC 1 Nov 2015
bull In remission as of April 2017
- Lkc 5500uL (Neuts 23)
- Hb 129gL
- Plt 208 X10e9
Summary
bull B cell malignancies are the most common
hematological disorders seen in flow cytometry
bull Many have complex overlapping phenotypes
bull Important to look at ungated plots as well as gating on
specific subpopulations
bull Level of expression of markers is as important as
positive or negative (think CD13 vs CD16)
68
References
bull Craig FE Foon KA Flow cytometric immunophenotyping for hematologic neoplasms Blood
2008 Apr 15111(8)3941-67
bull Wood BL Principles of minimal residual disease detection for hematopoietic neoplasms by flow
cytometry Cytometry B Clin Cytom 2015 Apr 23
bull Shanafelt TD Ghia P Lanasa MC Landgren O Rawstron AC Monoclonal B-cell
lymphocytosis (MBL) biology natural history and clinical management Leukemia 2010
Mar24(3)512-20
bull Swerdlow SH Campo E Pileri SA Harris NL Stein H Siebert R Advani RGhielmini M Salles
GA Zelenetz AD Jaffe ES The 2016 revision of the World Health Organization classification of
lymphoid neoplasms Blood 2016 May 19127(20)2375-90
bull 3 Moench L Sachs Z Aasen G Dolan M Dayton V Courville EL Double- and triple-hit
lymphomas can present with features suggestive of immaturity including TdT expression and
create diagnostic challenges Leuk Lymphoma 2016572626-35
bull Sutherland DR Keeney M Illingworth A Practical guidelines for the high-sensitivity detection
and monitoring of paroxysmal nocturnal hemoglobinuria clones by flow cytometry Cytometry B
Clin Cytom 2012 Jul82(4)195-208
69
Case 4 follow up
bull Smouldering myeloma
bull Recently developed CRAB (hyperCalcemia Renal
insufficiency Anemia Bone lesions
bull Serum free light chain ratio 123
bull Bone marrow aspirate gt60 plasma cells
bull Currently on treatment pending bone marrow
transplant
Case 5
bull 65 year old male
bull Slight lymphocytosis detected on routine CBC analysis
(lymph 4500uL)
bull Asymptomatic sent for flow cytometry
Case 5 PB
Case 5 flow cytometry KL ratio 0625
Case 5 Monoclonal B cell lymphocytosis
KL 18 KL 056
Case 5 Monoclonal B cell lymphocytosis
bull WHO defines CLL as abnormal lymphocyte count
gt5x10E9L (CD19+CD5+ light chain restricted) in the
absence of other clinical symptoms
bull Patients with small B cell clones are often detected as
an incidental finding
bull WHO 2016
bull CD19+CD5+ lt05x10E9L ldquoLow count MBLrdquo
raquo 05-5x10E9L ldquoHigh count MBLrdquo
raquo Non CLL type phenotype MBL also recognized
Monoclonal B cell Lymphocytosis
bull CLL immunophenotype - vast majority (75)
bull Atypical MBL
bull normal levels both CD5 and CD20 with variable
expression of CD23 (always exclude Mantle-cell
Lymphoma in CD23 negative cases)
bull Non-CLL-phenotype (CD5minus) MBL
bull Lacking CD5 expression and CD10 likely resembling B-
cell lymphomas of marginal-zone origin
Case 5 What is the normal KL Ratio
bull What is the normal or abnormal kappalambda ratio for
entire ldquopopulationrdquo of all B cells
bull Traditional view
ndash Kappa Lambda 21 or 31 ( 12 -13)
ndash Range 14 60
Case 5 What is the normal KL ratio
bull Wrong question
bull It doesnrsquot matter how many of the B cells are clonal but rather
that their exists a clonal population of B cells within the total
population
bull Need to Identify malignant B cells within a population of
normal B cells
bull In order to interpret a pathological immunophenotype it is
necessary to refer to quantitative and qualitative values of
normal B-cell subpopulations
bull Pearl Identify the abnormal phenotype first then look at KL
ratio
Case 6
bull 12 year old male presents to ER with fatigue bruising
Lkc 2200uL
bull Hb 84gL
bull Plt 32
bull Diff ndash neutropenia no blasts noted few ldquoatypicalrdquo cells
bull RBC ndash non specific changes
Leukemia
Case 6 PB
Case 6 BM
Case 6
bull BM aspirate ndash Hypocellular relative lymphocytosis
marked reduction in myeloid lineages
hypoplasticaplastic anemia
bull Flow Cytometry ndash less than 3 blasts maturing
myeloid 68 lymphocytes polyclonal B helper
suppressor T
bull Unusual pattern of CD13CD16 staining noted
Case 6 flow cytometry Aplastic Anemia
Normal Patient
Normal
Patient
Patient
Normal
Case 6 RBC Test for PNH CD235a v s CD59
Case 6 Neutrophil test for PNH clone
FLAER CD24 CD45CD15
18 PNH clone
Case 6
bull Follow up
bull Patient on steroids acyclovir and anti-thymocyte
globulin
bull PNH clone Neutrophils 96 RBC 1 Nov 2015
bull In remission as of April 2017
- Lkc 5500uL (Neuts 23)
- Hb 129gL
- Plt 208 X10e9
Summary
bull B cell malignancies are the most common
hematological disorders seen in flow cytometry
bull Many have complex overlapping phenotypes
bull Important to look at ungated plots as well as gating on
specific subpopulations
bull Level of expression of markers is as important as
positive or negative (think CD13 vs CD16)
68
References
bull Craig FE Foon KA Flow cytometric immunophenotyping for hematologic neoplasms Blood
2008 Apr 15111(8)3941-67
bull Wood BL Principles of minimal residual disease detection for hematopoietic neoplasms by flow
cytometry Cytometry B Clin Cytom 2015 Apr 23
bull Shanafelt TD Ghia P Lanasa MC Landgren O Rawstron AC Monoclonal B-cell
lymphocytosis (MBL) biology natural history and clinical management Leukemia 2010
Mar24(3)512-20
bull Swerdlow SH Campo E Pileri SA Harris NL Stein H Siebert R Advani RGhielmini M Salles
GA Zelenetz AD Jaffe ES The 2016 revision of the World Health Organization classification of
lymphoid neoplasms Blood 2016 May 19127(20)2375-90
bull 3 Moench L Sachs Z Aasen G Dolan M Dayton V Courville EL Double- and triple-hit
lymphomas can present with features suggestive of immaturity including TdT expression and
create diagnostic challenges Leuk Lymphoma 2016572626-35
bull Sutherland DR Keeney M Illingworth A Practical guidelines for the high-sensitivity detection
and monitoring of paroxysmal nocturnal hemoglobinuria clones by flow cytometry Cytometry B
Clin Cytom 2012 Jul82(4)195-208
69
Case 5
bull 65 year old male
bull Slight lymphocytosis detected on routine CBC analysis
(lymph 4500uL)
bull Asymptomatic sent for flow cytometry
Case 5 PB
Case 5 flow cytometry KL ratio 0625
Case 5 Monoclonal B cell lymphocytosis
KL 18 KL 056
Case 5 Monoclonal B cell lymphocytosis
bull WHO defines CLL as abnormal lymphocyte count
gt5x10E9L (CD19+CD5+ light chain restricted) in the
absence of other clinical symptoms
bull Patients with small B cell clones are often detected as
an incidental finding
bull WHO 2016
bull CD19+CD5+ lt05x10E9L ldquoLow count MBLrdquo
raquo 05-5x10E9L ldquoHigh count MBLrdquo
raquo Non CLL type phenotype MBL also recognized
Monoclonal B cell Lymphocytosis
bull CLL immunophenotype - vast majority (75)
bull Atypical MBL
bull normal levels both CD5 and CD20 with variable
expression of CD23 (always exclude Mantle-cell
Lymphoma in CD23 negative cases)
bull Non-CLL-phenotype (CD5minus) MBL
bull Lacking CD5 expression and CD10 likely resembling B-
cell lymphomas of marginal-zone origin
Case 5 What is the normal KL Ratio
bull What is the normal or abnormal kappalambda ratio for
entire ldquopopulationrdquo of all B cells
bull Traditional view
ndash Kappa Lambda 21 or 31 ( 12 -13)
ndash Range 14 60
Case 5 What is the normal KL ratio
bull Wrong question
bull It doesnrsquot matter how many of the B cells are clonal but rather
that their exists a clonal population of B cells within the total
population
bull Need to Identify malignant B cells within a population of
normal B cells
bull In order to interpret a pathological immunophenotype it is
necessary to refer to quantitative and qualitative values of
normal B-cell subpopulations
bull Pearl Identify the abnormal phenotype first then look at KL
ratio
Case 6
bull 12 year old male presents to ER with fatigue bruising
Lkc 2200uL
bull Hb 84gL
bull Plt 32
bull Diff ndash neutropenia no blasts noted few ldquoatypicalrdquo cells
bull RBC ndash non specific changes
Leukemia
Case 6 PB
Case 6 BM
Case 6
bull BM aspirate ndash Hypocellular relative lymphocytosis
marked reduction in myeloid lineages
hypoplasticaplastic anemia
bull Flow Cytometry ndash less than 3 blasts maturing
myeloid 68 lymphocytes polyclonal B helper
suppressor T
bull Unusual pattern of CD13CD16 staining noted
Case 6 flow cytometry Aplastic Anemia
Normal Patient
Normal
Patient
Patient
Normal
Case 6 RBC Test for PNH CD235a v s CD59
Case 6 Neutrophil test for PNH clone
FLAER CD24 CD45CD15
18 PNH clone
Case 6
bull Follow up
bull Patient on steroids acyclovir and anti-thymocyte
globulin
bull PNH clone Neutrophils 96 RBC 1 Nov 2015
bull In remission as of April 2017
- Lkc 5500uL (Neuts 23)
- Hb 129gL
- Plt 208 X10e9
Summary
bull B cell malignancies are the most common
hematological disorders seen in flow cytometry
bull Many have complex overlapping phenotypes
bull Important to look at ungated plots as well as gating on
specific subpopulations
bull Level of expression of markers is as important as
positive or negative (think CD13 vs CD16)
68
References
bull Craig FE Foon KA Flow cytometric immunophenotyping for hematologic neoplasms Blood
2008 Apr 15111(8)3941-67
bull Wood BL Principles of minimal residual disease detection for hematopoietic neoplasms by flow
cytometry Cytometry B Clin Cytom 2015 Apr 23
bull Shanafelt TD Ghia P Lanasa MC Landgren O Rawstron AC Monoclonal B-cell
lymphocytosis (MBL) biology natural history and clinical management Leukemia 2010
Mar24(3)512-20
bull Swerdlow SH Campo E Pileri SA Harris NL Stein H Siebert R Advani RGhielmini M Salles
GA Zelenetz AD Jaffe ES The 2016 revision of the World Health Organization classification of
lymphoid neoplasms Blood 2016 May 19127(20)2375-90
bull 3 Moench L Sachs Z Aasen G Dolan M Dayton V Courville EL Double- and triple-hit
lymphomas can present with features suggestive of immaturity including TdT expression and
create diagnostic challenges Leuk Lymphoma 2016572626-35
bull Sutherland DR Keeney M Illingworth A Practical guidelines for the high-sensitivity detection
and monitoring of paroxysmal nocturnal hemoglobinuria clones by flow cytometry Cytometry B
Clin Cytom 2012 Jul82(4)195-208
69
Case 5 PB
Case 5 flow cytometry KL ratio 0625
Case 5 Monoclonal B cell lymphocytosis
KL 18 KL 056
Case 5 Monoclonal B cell lymphocytosis
bull WHO defines CLL as abnormal lymphocyte count
gt5x10E9L (CD19+CD5+ light chain restricted) in the
absence of other clinical symptoms
bull Patients with small B cell clones are often detected as
an incidental finding
bull WHO 2016
bull CD19+CD5+ lt05x10E9L ldquoLow count MBLrdquo
raquo 05-5x10E9L ldquoHigh count MBLrdquo
raquo Non CLL type phenotype MBL also recognized
Monoclonal B cell Lymphocytosis
bull CLL immunophenotype - vast majority (75)
bull Atypical MBL
bull normal levels both CD5 and CD20 with variable
expression of CD23 (always exclude Mantle-cell
Lymphoma in CD23 negative cases)
bull Non-CLL-phenotype (CD5minus) MBL
bull Lacking CD5 expression and CD10 likely resembling B-
cell lymphomas of marginal-zone origin
Case 5 What is the normal KL Ratio
bull What is the normal or abnormal kappalambda ratio for
entire ldquopopulationrdquo of all B cells
bull Traditional view
ndash Kappa Lambda 21 or 31 ( 12 -13)
ndash Range 14 60
Case 5 What is the normal KL ratio
bull Wrong question
bull It doesnrsquot matter how many of the B cells are clonal but rather
that their exists a clonal population of B cells within the total
population
bull Need to Identify malignant B cells within a population of
normal B cells
bull In order to interpret a pathological immunophenotype it is
necessary to refer to quantitative and qualitative values of
normal B-cell subpopulations
bull Pearl Identify the abnormal phenotype first then look at KL
ratio
Case 6
bull 12 year old male presents to ER with fatigue bruising
Lkc 2200uL
bull Hb 84gL
bull Plt 32
bull Diff ndash neutropenia no blasts noted few ldquoatypicalrdquo cells
bull RBC ndash non specific changes
Leukemia
Case 6 PB
Case 6 BM
Case 6
bull BM aspirate ndash Hypocellular relative lymphocytosis
marked reduction in myeloid lineages
hypoplasticaplastic anemia
bull Flow Cytometry ndash less than 3 blasts maturing
myeloid 68 lymphocytes polyclonal B helper
suppressor T
bull Unusual pattern of CD13CD16 staining noted
Case 6 flow cytometry Aplastic Anemia
Normal Patient
Normal
Patient
Patient
Normal
Case 6 RBC Test for PNH CD235a v s CD59
Case 6 Neutrophil test for PNH clone
FLAER CD24 CD45CD15
18 PNH clone
Case 6
bull Follow up
bull Patient on steroids acyclovir and anti-thymocyte
globulin
bull PNH clone Neutrophils 96 RBC 1 Nov 2015
bull In remission as of April 2017
- Lkc 5500uL (Neuts 23)
- Hb 129gL
- Plt 208 X10e9
Summary
bull B cell malignancies are the most common
hematological disorders seen in flow cytometry
bull Many have complex overlapping phenotypes
bull Important to look at ungated plots as well as gating on
specific subpopulations
bull Level of expression of markers is as important as
positive or negative (think CD13 vs CD16)
68
References
bull Craig FE Foon KA Flow cytometric immunophenotyping for hematologic neoplasms Blood
2008 Apr 15111(8)3941-67
bull Wood BL Principles of minimal residual disease detection for hematopoietic neoplasms by flow
cytometry Cytometry B Clin Cytom 2015 Apr 23
bull Shanafelt TD Ghia P Lanasa MC Landgren O Rawstron AC Monoclonal B-cell
lymphocytosis (MBL) biology natural history and clinical management Leukemia 2010
Mar24(3)512-20
bull Swerdlow SH Campo E Pileri SA Harris NL Stein H Siebert R Advani RGhielmini M Salles
GA Zelenetz AD Jaffe ES The 2016 revision of the World Health Organization classification of
lymphoid neoplasms Blood 2016 May 19127(20)2375-90
bull 3 Moench L Sachs Z Aasen G Dolan M Dayton V Courville EL Double- and triple-hit
lymphomas can present with features suggestive of immaturity including TdT expression and
create diagnostic challenges Leuk Lymphoma 2016572626-35
bull Sutherland DR Keeney M Illingworth A Practical guidelines for the high-sensitivity detection
and monitoring of paroxysmal nocturnal hemoglobinuria clones by flow cytometry Cytometry B
Clin Cytom 2012 Jul82(4)195-208
69
Case 5 flow cytometry KL ratio 0625
Case 5 Monoclonal B cell lymphocytosis
KL 18 KL 056
Case 5 Monoclonal B cell lymphocytosis
bull WHO defines CLL as abnormal lymphocyte count
gt5x10E9L (CD19+CD5+ light chain restricted) in the
absence of other clinical symptoms
bull Patients with small B cell clones are often detected as
an incidental finding
bull WHO 2016
bull CD19+CD5+ lt05x10E9L ldquoLow count MBLrdquo
raquo 05-5x10E9L ldquoHigh count MBLrdquo
raquo Non CLL type phenotype MBL also recognized
Monoclonal B cell Lymphocytosis
bull CLL immunophenotype - vast majority (75)
bull Atypical MBL
bull normal levels both CD5 and CD20 with variable
expression of CD23 (always exclude Mantle-cell
Lymphoma in CD23 negative cases)
bull Non-CLL-phenotype (CD5minus) MBL
bull Lacking CD5 expression and CD10 likely resembling B-
cell lymphomas of marginal-zone origin
Case 5 What is the normal KL Ratio
bull What is the normal or abnormal kappalambda ratio for
entire ldquopopulationrdquo of all B cells
bull Traditional view
ndash Kappa Lambda 21 or 31 ( 12 -13)
ndash Range 14 60
Case 5 What is the normal KL ratio
bull Wrong question
bull It doesnrsquot matter how many of the B cells are clonal but rather
that their exists a clonal population of B cells within the total
population
bull Need to Identify malignant B cells within a population of
normal B cells
bull In order to interpret a pathological immunophenotype it is
necessary to refer to quantitative and qualitative values of
normal B-cell subpopulations
bull Pearl Identify the abnormal phenotype first then look at KL
ratio
Case 6
bull 12 year old male presents to ER with fatigue bruising
Lkc 2200uL
bull Hb 84gL
bull Plt 32
bull Diff ndash neutropenia no blasts noted few ldquoatypicalrdquo cells
bull RBC ndash non specific changes
Leukemia
Case 6 PB
Case 6 BM
Case 6
bull BM aspirate ndash Hypocellular relative lymphocytosis
marked reduction in myeloid lineages
hypoplasticaplastic anemia
bull Flow Cytometry ndash less than 3 blasts maturing
myeloid 68 lymphocytes polyclonal B helper
suppressor T
bull Unusual pattern of CD13CD16 staining noted
Case 6 flow cytometry Aplastic Anemia
Normal Patient
Normal
Patient
Patient
Normal
Case 6 RBC Test for PNH CD235a v s CD59
Case 6 Neutrophil test for PNH clone
FLAER CD24 CD45CD15
18 PNH clone
Case 6
bull Follow up
bull Patient on steroids acyclovir and anti-thymocyte
globulin
bull PNH clone Neutrophils 96 RBC 1 Nov 2015
bull In remission as of April 2017
- Lkc 5500uL (Neuts 23)
- Hb 129gL
- Plt 208 X10e9
Summary
bull B cell malignancies are the most common
hematological disorders seen in flow cytometry
bull Many have complex overlapping phenotypes
bull Important to look at ungated plots as well as gating on
specific subpopulations
bull Level of expression of markers is as important as
positive or negative (think CD13 vs CD16)
68
References
bull Craig FE Foon KA Flow cytometric immunophenotyping for hematologic neoplasms Blood
2008 Apr 15111(8)3941-67
bull Wood BL Principles of minimal residual disease detection for hematopoietic neoplasms by flow
cytometry Cytometry B Clin Cytom 2015 Apr 23
bull Shanafelt TD Ghia P Lanasa MC Landgren O Rawstron AC Monoclonal B-cell
lymphocytosis (MBL) biology natural history and clinical management Leukemia 2010
Mar24(3)512-20
bull Swerdlow SH Campo E Pileri SA Harris NL Stein H Siebert R Advani RGhielmini M Salles
GA Zelenetz AD Jaffe ES The 2016 revision of the World Health Organization classification of
lymphoid neoplasms Blood 2016 May 19127(20)2375-90
bull 3 Moench L Sachs Z Aasen G Dolan M Dayton V Courville EL Double- and triple-hit
lymphomas can present with features suggestive of immaturity including TdT expression and
create diagnostic challenges Leuk Lymphoma 2016572626-35
bull Sutherland DR Keeney M Illingworth A Practical guidelines for the high-sensitivity detection
and monitoring of paroxysmal nocturnal hemoglobinuria clones by flow cytometry Cytometry B
Clin Cytom 2012 Jul82(4)195-208
69
Case 5 Monoclonal B cell lymphocytosis
KL 18 KL 056
Case 5 Monoclonal B cell lymphocytosis
bull WHO defines CLL as abnormal lymphocyte count
gt5x10E9L (CD19+CD5+ light chain restricted) in the
absence of other clinical symptoms
bull Patients with small B cell clones are often detected as
an incidental finding
bull WHO 2016
bull CD19+CD5+ lt05x10E9L ldquoLow count MBLrdquo
raquo 05-5x10E9L ldquoHigh count MBLrdquo
raquo Non CLL type phenotype MBL also recognized
Monoclonal B cell Lymphocytosis
bull CLL immunophenotype - vast majority (75)
bull Atypical MBL
bull normal levels both CD5 and CD20 with variable
expression of CD23 (always exclude Mantle-cell
Lymphoma in CD23 negative cases)
bull Non-CLL-phenotype (CD5minus) MBL
bull Lacking CD5 expression and CD10 likely resembling B-
cell lymphomas of marginal-zone origin
Case 5 What is the normal KL Ratio
bull What is the normal or abnormal kappalambda ratio for
entire ldquopopulationrdquo of all B cells
bull Traditional view
ndash Kappa Lambda 21 or 31 ( 12 -13)
ndash Range 14 60
Case 5 What is the normal KL ratio
bull Wrong question
bull It doesnrsquot matter how many of the B cells are clonal but rather
that their exists a clonal population of B cells within the total
population
bull Need to Identify malignant B cells within a population of
normal B cells
bull In order to interpret a pathological immunophenotype it is
necessary to refer to quantitative and qualitative values of
normal B-cell subpopulations
bull Pearl Identify the abnormal phenotype first then look at KL
ratio
Case 6
bull 12 year old male presents to ER with fatigue bruising
Lkc 2200uL
bull Hb 84gL
bull Plt 32
bull Diff ndash neutropenia no blasts noted few ldquoatypicalrdquo cells
bull RBC ndash non specific changes
Leukemia
Case 6 PB
Case 6 BM
Case 6
bull BM aspirate ndash Hypocellular relative lymphocytosis
marked reduction in myeloid lineages
hypoplasticaplastic anemia
bull Flow Cytometry ndash less than 3 blasts maturing
myeloid 68 lymphocytes polyclonal B helper
suppressor T
bull Unusual pattern of CD13CD16 staining noted
Case 6 flow cytometry Aplastic Anemia
Normal Patient
Normal
Patient
Patient
Normal
Case 6 RBC Test for PNH CD235a v s CD59
Case 6 Neutrophil test for PNH clone
FLAER CD24 CD45CD15
18 PNH clone
Case 6
bull Follow up
bull Patient on steroids acyclovir and anti-thymocyte
globulin
bull PNH clone Neutrophils 96 RBC 1 Nov 2015
bull In remission as of April 2017
- Lkc 5500uL (Neuts 23)
- Hb 129gL
- Plt 208 X10e9
Summary
bull B cell malignancies are the most common
hematological disorders seen in flow cytometry
bull Many have complex overlapping phenotypes
bull Important to look at ungated plots as well as gating on
specific subpopulations
bull Level of expression of markers is as important as
positive or negative (think CD13 vs CD16)
68
References
bull Craig FE Foon KA Flow cytometric immunophenotyping for hematologic neoplasms Blood
2008 Apr 15111(8)3941-67
bull Wood BL Principles of minimal residual disease detection for hematopoietic neoplasms by flow
cytometry Cytometry B Clin Cytom 2015 Apr 23
bull Shanafelt TD Ghia P Lanasa MC Landgren O Rawstron AC Monoclonal B-cell
lymphocytosis (MBL) biology natural history and clinical management Leukemia 2010
Mar24(3)512-20
bull Swerdlow SH Campo E Pileri SA Harris NL Stein H Siebert R Advani RGhielmini M Salles
GA Zelenetz AD Jaffe ES The 2016 revision of the World Health Organization classification of
lymphoid neoplasms Blood 2016 May 19127(20)2375-90
bull 3 Moench L Sachs Z Aasen G Dolan M Dayton V Courville EL Double- and triple-hit
lymphomas can present with features suggestive of immaturity including TdT expression and
create diagnostic challenges Leuk Lymphoma 2016572626-35
bull Sutherland DR Keeney M Illingworth A Practical guidelines for the high-sensitivity detection
and monitoring of paroxysmal nocturnal hemoglobinuria clones by flow cytometry Cytometry B
Clin Cytom 2012 Jul82(4)195-208
69
Case 5 Monoclonal B cell lymphocytosis
bull WHO defines CLL as abnormal lymphocyte count
gt5x10E9L (CD19+CD5+ light chain restricted) in the
absence of other clinical symptoms
bull Patients with small B cell clones are often detected as
an incidental finding
bull WHO 2016
bull CD19+CD5+ lt05x10E9L ldquoLow count MBLrdquo
raquo 05-5x10E9L ldquoHigh count MBLrdquo
raquo Non CLL type phenotype MBL also recognized
Monoclonal B cell Lymphocytosis
bull CLL immunophenotype - vast majority (75)
bull Atypical MBL
bull normal levels both CD5 and CD20 with variable
expression of CD23 (always exclude Mantle-cell
Lymphoma in CD23 negative cases)
bull Non-CLL-phenotype (CD5minus) MBL
bull Lacking CD5 expression and CD10 likely resembling B-
cell lymphomas of marginal-zone origin
Case 5 What is the normal KL Ratio
bull What is the normal or abnormal kappalambda ratio for
entire ldquopopulationrdquo of all B cells
bull Traditional view
ndash Kappa Lambda 21 or 31 ( 12 -13)
ndash Range 14 60
Case 5 What is the normal KL ratio
bull Wrong question
bull It doesnrsquot matter how many of the B cells are clonal but rather
that their exists a clonal population of B cells within the total
population
bull Need to Identify malignant B cells within a population of
normal B cells
bull In order to interpret a pathological immunophenotype it is
necessary to refer to quantitative and qualitative values of
normal B-cell subpopulations
bull Pearl Identify the abnormal phenotype first then look at KL
ratio
Case 6
bull 12 year old male presents to ER with fatigue bruising
Lkc 2200uL
bull Hb 84gL
bull Plt 32
bull Diff ndash neutropenia no blasts noted few ldquoatypicalrdquo cells
bull RBC ndash non specific changes
Leukemia
Case 6 PB
Case 6 BM
Case 6
bull BM aspirate ndash Hypocellular relative lymphocytosis
marked reduction in myeloid lineages
hypoplasticaplastic anemia
bull Flow Cytometry ndash less than 3 blasts maturing
myeloid 68 lymphocytes polyclonal B helper
suppressor T
bull Unusual pattern of CD13CD16 staining noted
Case 6 flow cytometry Aplastic Anemia
Normal Patient
Normal
Patient
Patient
Normal
Case 6 RBC Test for PNH CD235a v s CD59
Case 6 Neutrophil test for PNH clone
FLAER CD24 CD45CD15
18 PNH clone
Case 6
bull Follow up
bull Patient on steroids acyclovir and anti-thymocyte
globulin
bull PNH clone Neutrophils 96 RBC 1 Nov 2015
bull In remission as of April 2017
- Lkc 5500uL (Neuts 23)
- Hb 129gL
- Plt 208 X10e9
Summary
bull B cell malignancies are the most common
hematological disorders seen in flow cytometry
bull Many have complex overlapping phenotypes
bull Important to look at ungated plots as well as gating on
specific subpopulations
bull Level of expression of markers is as important as
positive or negative (think CD13 vs CD16)
68
References
bull Craig FE Foon KA Flow cytometric immunophenotyping for hematologic neoplasms Blood
2008 Apr 15111(8)3941-67
bull Wood BL Principles of minimal residual disease detection for hematopoietic neoplasms by flow
cytometry Cytometry B Clin Cytom 2015 Apr 23
bull Shanafelt TD Ghia P Lanasa MC Landgren O Rawstron AC Monoclonal B-cell
lymphocytosis (MBL) biology natural history and clinical management Leukemia 2010
Mar24(3)512-20
bull Swerdlow SH Campo E Pileri SA Harris NL Stein H Siebert R Advani RGhielmini M Salles
GA Zelenetz AD Jaffe ES The 2016 revision of the World Health Organization classification of
lymphoid neoplasms Blood 2016 May 19127(20)2375-90
bull 3 Moench L Sachs Z Aasen G Dolan M Dayton V Courville EL Double- and triple-hit
lymphomas can present with features suggestive of immaturity including TdT expression and
create diagnostic challenges Leuk Lymphoma 2016572626-35
bull Sutherland DR Keeney M Illingworth A Practical guidelines for the high-sensitivity detection
and monitoring of paroxysmal nocturnal hemoglobinuria clones by flow cytometry Cytometry B
Clin Cytom 2012 Jul82(4)195-208
69
Monoclonal B cell Lymphocytosis
bull CLL immunophenotype - vast majority (75)
bull Atypical MBL
bull normal levels both CD5 and CD20 with variable
expression of CD23 (always exclude Mantle-cell
Lymphoma in CD23 negative cases)
bull Non-CLL-phenotype (CD5minus) MBL
bull Lacking CD5 expression and CD10 likely resembling B-
cell lymphomas of marginal-zone origin
Case 5 What is the normal KL Ratio
bull What is the normal or abnormal kappalambda ratio for
entire ldquopopulationrdquo of all B cells
bull Traditional view
ndash Kappa Lambda 21 or 31 ( 12 -13)
ndash Range 14 60
Case 5 What is the normal KL ratio
bull Wrong question
bull It doesnrsquot matter how many of the B cells are clonal but rather
that their exists a clonal population of B cells within the total
population
bull Need to Identify malignant B cells within a population of
normal B cells
bull In order to interpret a pathological immunophenotype it is
necessary to refer to quantitative and qualitative values of
normal B-cell subpopulations
bull Pearl Identify the abnormal phenotype first then look at KL
ratio
Case 6
bull 12 year old male presents to ER with fatigue bruising
Lkc 2200uL
bull Hb 84gL
bull Plt 32
bull Diff ndash neutropenia no blasts noted few ldquoatypicalrdquo cells
bull RBC ndash non specific changes
Leukemia
Case 6 PB
Case 6 BM
Case 6
bull BM aspirate ndash Hypocellular relative lymphocytosis
marked reduction in myeloid lineages
hypoplasticaplastic anemia
bull Flow Cytometry ndash less than 3 blasts maturing
myeloid 68 lymphocytes polyclonal B helper
suppressor T
bull Unusual pattern of CD13CD16 staining noted
Case 6 flow cytometry Aplastic Anemia
Normal Patient
Normal
Patient
Patient
Normal
Case 6 RBC Test for PNH CD235a v s CD59
Case 6 Neutrophil test for PNH clone
FLAER CD24 CD45CD15
18 PNH clone
Case 6
bull Follow up
bull Patient on steroids acyclovir and anti-thymocyte
globulin
bull PNH clone Neutrophils 96 RBC 1 Nov 2015
bull In remission as of April 2017
- Lkc 5500uL (Neuts 23)
- Hb 129gL
- Plt 208 X10e9
Summary
bull B cell malignancies are the most common
hematological disorders seen in flow cytometry
bull Many have complex overlapping phenotypes
bull Important to look at ungated plots as well as gating on
specific subpopulations
bull Level of expression of markers is as important as
positive or negative (think CD13 vs CD16)
68
References
bull Craig FE Foon KA Flow cytometric immunophenotyping for hematologic neoplasms Blood
2008 Apr 15111(8)3941-67
bull Wood BL Principles of minimal residual disease detection for hematopoietic neoplasms by flow
cytometry Cytometry B Clin Cytom 2015 Apr 23
bull Shanafelt TD Ghia P Lanasa MC Landgren O Rawstron AC Monoclonal B-cell
lymphocytosis (MBL) biology natural history and clinical management Leukemia 2010
Mar24(3)512-20
bull Swerdlow SH Campo E Pileri SA Harris NL Stein H Siebert R Advani RGhielmini M Salles
GA Zelenetz AD Jaffe ES The 2016 revision of the World Health Organization classification of
lymphoid neoplasms Blood 2016 May 19127(20)2375-90
bull 3 Moench L Sachs Z Aasen G Dolan M Dayton V Courville EL Double- and triple-hit
lymphomas can present with features suggestive of immaturity including TdT expression and
create diagnostic challenges Leuk Lymphoma 2016572626-35
bull Sutherland DR Keeney M Illingworth A Practical guidelines for the high-sensitivity detection
and monitoring of paroxysmal nocturnal hemoglobinuria clones by flow cytometry Cytometry B
Clin Cytom 2012 Jul82(4)195-208
69
Case 5 What is the normal KL Ratio
bull What is the normal or abnormal kappalambda ratio for
entire ldquopopulationrdquo of all B cells
bull Traditional view
ndash Kappa Lambda 21 or 31 ( 12 -13)
ndash Range 14 60
Case 5 What is the normal KL ratio
bull Wrong question
bull It doesnrsquot matter how many of the B cells are clonal but rather
that their exists a clonal population of B cells within the total
population
bull Need to Identify malignant B cells within a population of
normal B cells
bull In order to interpret a pathological immunophenotype it is
necessary to refer to quantitative and qualitative values of
normal B-cell subpopulations
bull Pearl Identify the abnormal phenotype first then look at KL
ratio
Case 6
bull 12 year old male presents to ER with fatigue bruising
Lkc 2200uL
bull Hb 84gL
bull Plt 32
bull Diff ndash neutropenia no blasts noted few ldquoatypicalrdquo cells
bull RBC ndash non specific changes
Leukemia
Case 6 PB
Case 6 BM
Case 6
bull BM aspirate ndash Hypocellular relative lymphocytosis
marked reduction in myeloid lineages
hypoplasticaplastic anemia
bull Flow Cytometry ndash less than 3 blasts maturing
myeloid 68 lymphocytes polyclonal B helper
suppressor T
bull Unusual pattern of CD13CD16 staining noted
Case 6 flow cytometry Aplastic Anemia
Normal Patient
Normal
Patient
Patient
Normal
Case 6 RBC Test for PNH CD235a v s CD59
Case 6 Neutrophil test for PNH clone
FLAER CD24 CD45CD15
18 PNH clone
Case 6
bull Follow up
bull Patient on steroids acyclovir and anti-thymocyte
globulin
bull PNH clone Neutrophils 96 RBC 1 Nov 2015
bull In remission as of April 2017
- Lkc 5500uL (Neuts 23)
- Hb 129gL
- Plt 208 X10e9
Summary
bull B cell malignancies are the most common
hematological disorders seen in flow cytometry
bull Many have complex overlapping phenotypes
bull Important to look at ungated plots as well as gating on
specific subpopulations
bull Level of expression of markers is as important as
positive or negative (think CD13 vs CD16)
68
References
bull Craig FE Foon KA Flow cytometric immunophenotyping for hematologic neoplasms Blood
2008 Apr 15111(8)3941-67
bull Wood BL Principles of minimal residual disease detection for hematopoietic neoplasms by flow
cytometry Cytometry B Clin Cytom 2015 Apr 23
bull Shanafelt TD Ghia P Lanasa MC Landgren O Rawstron AC Monoclonal B-cell
lymphocytosis (MBL) biology natural history and clinical management Leukemia 2010
Mar24(3)512-20
bull Swerdlow SH Campo E Pileri SA Harris NL Stein H Siebert R Advani RGhielmini M Salles
GA Zelenetz AD Jaffe ES The 2016 revision of the World Health Organization classification of
lymphoid neoplasms Blood 2016 May 19127(20)2375-90
bull 3 Moench L Sachs Z Aasen G Dolan M Dayton V Courville EL Double- and triple-hit
lymphomas can present with features suggestive of immaturity including TdT expression and
create diagnostic challenges Leuk Lymphoma 2016572626-35
bull Sutherland DR Keeney M Illingworth A Practical guidelines for the high-sensitivity detection
and monitoring of paroxysmal nocturnal hemoglobinuria clones by flow cytometry Cytometry B
Clin Cytom 2012 Jul82(4)195-208
69
Case 5 What is the normal KL ratio
bull Wrong question
bull It doesnrsquot matter how many of the B cells are clonal but rather
that their exists a clonal population of B cells within the total
population
bull Need to Identify malignant B cells within a population of
normal B cells
bull In order to interpret a pathological immunophenotype it is
necessary to refer to quantitative and qualitative values of
normal B-cell subpopulations
bull Pearl Identify the abnormal phenotype first then look at KL
ratio
Case 6
bull 12 year old male presents to ER with fatigue bruising
Lkc 2200uL
bull Hb 84gL
bull Plt 32
bull Diff ndash neutropenia no blasts noted few ldquoatypicalrdquo cells
bull RBC ndash non specific changes
Leukemia
Case 6 PB
Case 6 BM
Case 6
bull BM aspirate ndash Hypocellular relative lymphocytosis
marked reduction in myeloid lineages
hypoplasticaplastic anemia
bull Flow Cytometry ndash less than 3 blasts maturing
myeloid 68 lymphocytes polyclonal B helper
suppressor T
bull Unusual pattern of CD13CD16 staining noted
Case 6 flow cytometry Aplastic Anemia
Normal Patient
Normal
Patient
Patient
Normal
Case 6 RBC Test for PNH CD235a v s CD59
Case 6 Neutrophil test for PNH clone
FLAER CD24 CD45CD15
18 PNH clone
Case 6
bull Follow up
bull Patient on steroids acyclovir and anti-thymocyte
globulin
bull PNH clone Neutrophils 96 RBC 1 Nov 2015
bull In remission as of April 2017
- Lkc 5500uL (Neuts 23)
- Hb 129gL
- Plt 208 X10e9
Summary
bull B cell malignancies are the most common
hematological disorders seen in flow cytometry
bull Many have complex overlapping phenotypes
bull Important to look at ungated plots as well as gating on
specific subpopulations
bull Level of expression of markers is as important as
positive or negative (think CD13 vs CD16)
68
References
bull Craig FE Foon KA Flow cytometric immunophenotyping for hematologic neoplasms Blood
2008 Apr 15111(8)3941-67
bull Wood BL Principles of minimal residual disease detection for hematopoietic neoplasms by flow
cytometry Cytometry B Clin Cytom 2015 Apr 23
bull Shanafelt TD Ghia P Lanasa MC Landgren O Rawstron AC Monoclonal B-cell
lymphocytosis (MBL) biology natural history and clinical management Leukemia 2010
Mar24(3)512-20
bull Swerdlow SH Campo E Pileri SA Harris NL Stein H Siebert R Advani RGhielmini M Salles
GA Zelenetz AD Jaffe ES The 2016 revision of the World Health Organization classification of
lymphoid neoplasms Blood 2016 May 19127(20)2375-90
bull 3 Moench L Sachs Z Aasen G Dolan M Dayton V Courville EL Double- and triple-hit
lymphomas can present with features suggestive of immaturity including TdT expression and
create diagnostic challenges Leuk Lymphoma 2016572626-35
bull Sutherland DR Keeney M Illingworth A Practical guidelines for the high-sensitivity detection
and monitoring of paroxysmal nocturnal hemoglobinuria clones by flow cytometry Cytometry B
Clin Cytom 2012 Jul82(4)195-208
69
Case 6
bull 12 year old male presents to ER with fatigue bruising
Lkc 2200uL
bull Hb 84gL
bull Plt 32
bull Diff ndash neutropenia no blasts noted few ldquoatypicalrdquo cells
bull RBC ndash non specific changes
Leukemia
Case 6 PB
Case 6 BM
Case 6
bull BM aspirate ndash Hypocellular relative lymphocytosis
marked reduction in myeloid lineages
hypoplasticaplastic anemia
bull Flow Cytometry ndash less than 3 blasts maturing
myeloid 68 lymphocytes polyclonal B helper
suppressor T
bull Unusual pattern of CD13CD16 staining noted
Case 6 flow cytometry Aplastic Anemia
Normal Patient
Normal
Patient
Patient
Normal
Case 6 RBC Test for PNH CD235a v s CD59
Case 6 Neutrophil test for PNH clone
FLAER CD24 CD45CD15
18 PNH clone
Case 6
bull Follow up
bull Patient on steroids acyclovir and anti-thymocyte
globulin
bull PNH clone Neutrophils 96 RBC 1 Nov 2015
bull In remission as of April 2017
- Lkc 5500uL (Neuts 23)
- Hb 129gL
- Plt 208 X10e9
Summary
bull B cell malignancies are the most common
hematological disorders seen in flow cytometry
bull Many have complex overlapping phenotypes
bull Important to look at ungated plots as well as gating on
specific subpopulations
bull Level of expression of markers is as important as
positive or negative (think CD13 vs CD16)
68
References
bull Craig FE Foon KA Flow cytometric immunophenotyping for hematologic neoplasms Blood
2008 Apr 15111(8)3941-67
bull Wood BL Principles of minimal residual disease detection for hematopoietic neoplasms by flow
cytometry Cytometry B Clin Cytom 2015 Apr 23
bull Shanafelt TD Ghia P Lanasa MC Landgren O Rawstron AC Monoclonal B-cell
lymphocytosis (MBL) biology natural history and clinical management Leukemia 2010
Mar24(3)512-20
bull Swerdlow SH Campo E Pileri SA Harris NL Stein H Siebert R Advani RGhielmini M Salles
GA Zelenetz AD Jaffe ES The 2016 revision of the World Health Organization classification of
lymphoid neoplasms Blood 2016 May 19127(20)2375-90
bull 3 Moench L Sachs Z Aasen G Dolan M Dayton V Courville EL Double- and triple-hit
lymphomas can present with features suggestive of immaturity including TdT expression and
create diagnostic challenges Leuk Lymphoma 2016572626-35
bull Sutherland DR Keeney M Illingworth A Practical guidelines for the high-sensitivity detection
and monitoring of paroxysmal nocturnal hemoglobinuria clones by flow cytometry Cytometry B
Clin Cytom 2012 Jul82(4)195-208
69
Case 6 PB
Case 6 BM
Case 6
bull BM aspirate ndash Hypocellular relative lymphocytosis
marked reduction in myeloid lineages
hypoplasticaplastic anemia
bull Flow Cytometry ndash less than 3 blasts maturing
myeloid 68 lymphocytes polyclonal B helper
suppressor T
bull Unusual pattern of CD13CD16 staining noted
Case 6 flow cytometry Aplastic Anemia
Normal Patient
Normal
Patient
Patient
Normal
Case 6 RBC Test for PNH CD235a v s CD59
Case 6 Neutrophil test for PNH clone
FLAER CD24 CD45CD15
18 PNH clone
Case 6
bull Follow up
bull Patient on steroids acyclovir and anti-thymocyte
globulin
bull PNH clone Neutrophils 96 RBC 1 Nov 2015
bull In remission as of April 2017
- Lkc 5500uL (Neuts 23)
- Hb 129gL
- Plt 208 X10e9
Summary
bull B cell malignancies are the most common
hematological disorders seen in flow cytometry
bull Many have complex overlapping phenotypes
bull Important to look at ungated plots as well as gating on
specific subpopulations
bull Level of expression of markers is as important as
positive or negative (think CD13 vs CD16)
68
References
bull Craig FE Foon KA Flow cytometric immunophenotyping for hematologic neoplasms Blood
2008 Apr 15111(8)3941-67
bull Wood BL Principles of minimal residual disease detection for hematopoietic neoplasms by flow
cytometry Cytometry B Clin Cytom 2015 Apr 23
bull Shanafelt TD Ghia P Lanasa MC Landgren O Rawstron AC Monoclonal B-cell
lymphocytosis (MBL) biology natural history and clinical management Leukemia 2010
Mar24(3)512-20
bull Swerdlow SH Campo E Pileri SA Harris NL Stein H Siebert R Advani RGhielmini M Salles
GA Zelenetz AD Jaffe ES The 2016 revision of the World Health Organization classification of
lymphoid neoplasms Blood 2016 May 19127(20)2375-90
bull 3 Moench L Sachs Z Aasen G Dolan M Dayton V Courville EL Double- and triple-hit
lymphomas can present with features suggestive of immaturity including TdT expression and
create diagnostic challenges Leuk Lymphoma 2016572626-35
bull Sutherland DR Keeney M Illingworth A Practical guidelines for the high-sensitivity detection
and monitoring of paroxysmal nocturnal hemoglobinuria clones by flow cytometry Cytometry B
Clin Cytom 2012 Jul82(4)195-208
69
Case 6 BM
Case 6
bull BM aspirate ndash Hypocellular relative lymphocytosis
marked reduction in myeloid lineages
hypoplasticaplastic anemia
bull Flow Cytometry ndash less than 3 blasts maturing
myeloid 68 lymphocytes polyclonal B helper
suppressor T
bull Unusual pattern of CD13CD16 staining noted
Case 6 flow cytometry Aplastic Anemia
Normal Patient
Normal
Patient
Patient
Normal
Case 6 RBC Test for PNH CD235a v s CD59
Case 6 Neutrophil test for PNH clone
FLAER CD24 CD45CD15
18 PNH clone
Case 6
bull Follow up
bull Patient on steroids acyclovir and anti-thymocyte
globulin
bull PNH clone Neutrophils 96 RBC 1 Nov 2015
bull In remission as of April 2017
- Lkc 5500uL (Neuts 23)
- Hb 129gL
- Plt 208 X10e9
Summary
bull B cell malignancies are the most common
hematological disorders seen in flow cytometry
bull Many have complex overlapping phenotypes
bull Important to look at ungated plots as well as gating on
specific subpopulations
bull Level of expression of markers is as important as
positive or negative (think CD13 vs CD16)
68
References
bull Craig FE Foon KA Flow cytometric immunophenotyping for hematologic neoplasms Blood
2008 Apr 15111(8)3941-67
bull Wood BL Principles of minimal residual disease detection for hematopoietic neoplasms by flow
cytometry Cytometry B Clin Cytom 2015 Apr 23
bull Shanafelt TD Ghia P Lanasa MC Landgren O Rawstron AC Monoclonal B-cell
lymphocytosis (MBL) biology natural history and clinical management Leukemia 2010
Mar24(3)512-20
bull Swerdlow SH Campo E Pileri SA Harris NL Stein H Siebert R Advani RGhielmini M Salles
GA Zelenetz AD Jaffe ES The 2016 revision of the World Health Organization classification of
lymphoid neoplasms Blood 2016 May 19127(20)2375-90
bull 3 Moench L Sachs Z Aasen G Dolan M Dayton V Courville EL Double- and triple-hit
lymphomas can present with features suggestive of immaturity including TdT expression and
create diagnostic challenges Leuk Lymphoma 2016572626-35
bull Sutherland DR Keeney M Illingworth A Practical guidelines for the high-sensitivity detection
and monitoring of paroxysmal nocturnal hemoglobinuria clones by flow cytometry Cytometry B
Clin Cytom 2012 Jul82(4)195-208
69
Case 6
bull BM aspirate ndash Hypocellular relative lymphocytosis
marked reduction in myeloid lineages
hypoplasticaplastic anemia
bull Flow Cytometry ndash less than 3 blasts maturing
myeloid 68 lymphocytes polyclonal B helper
suppressor T
bull Unusual pattern of CD13CD16 staining noted
Case 6 flow cytometry Aplastic Anemia
Normal Patient
Normal
Patient
Patient
Normal
Case 6 RBC Test for PNH CD235a v s CD59
Case 6 Neutrophil test for PNH clone
FLAER CD24 CD45CD15
18 PNH clone
Case 6
bull Follow up
bull Patient on steroids acyclovir and anti-thymocyte
globulin
bull PNH clone Neutrophils 96 RBC 1 Nov 2015
bull In remission as of April 2017
- Lkc 5500uL (Neuts 23)
- Hb 129gL
- Plt 208 X10e9
Summary
bull B cell malignancies are the most common
hematological disorders seen in flow cytometry
bull Many have complex overlapping phenotypes
bull Important to look at ungated plots as well as gating on
specific subpopulations
bull Level of expression of markers is as important as
positive or negative (think CD13 vs CD16)
68
References
bull Craig FE Foon KA Flow cytometric immunophenotyping for hematologic neoplasms Blood
2008 Apr 15111(8)3941-67
bull Wood BL Principles of minimal residual disease detection for hematopoietic neoplasms by flow
cytometry Cytometry B Clin Cytom 2015 Apr 23
bull Shanafelt TD Ghia P Lanasa MC Landgren O Rawstron AC Monoclonal B-cell
lymphocytosis (MBL) biology natural history and clinical management Leukemia 2010
Mar24(3)512-20
bull Swerdlow SH Campo E Pileri SA Harris NL Stein H Siebert R Advani RGhielmini M Salles
GA Zelenetz AD Jaffe ES The 2016 revision of the World Health Organization classification of
lymphoid neoplasms Blood 2016 May 19127(20)2375-90
bull 3 Moench L Sachs Z Aasen G Dolan M Dayton V Courville EL Double- and triple-hit
lymphomas can present with features suggestive of immaturity including TdT expression and
create diagnostic challenges Leuk Lymphoma 2016572626-35
bull Sutherland DR Keeney M Illingworth A Practical guidelines for the high-sensitivity detection
and monitoring of paroxysmal nocturnal hemoglobinuria clones by flow cytometry Cytometry B
Clin Cytom 2012 Jul82(4)195-208
69
Case 6 flow cytometry Aplastic Anemia
Normal Patient
Normal
Patient
Patient
Normal
Case 6 RBC Test for PNH CD235a v s CD59
Case 6 Neutrophil test for PNH clone
FLAER CD24 CD45CD15
18 PNH clone
Case 6
bull Follow up
bull Patient on steroids acyclovir and anti-thymocyte
globulin
bull PNH clone Neutrophils 96 RBC 1 Nov 2015
bull In remission as of April 2017
- Lkc 5500uL (Neuts 23)
- Hb 129gL
- Plt 208 X10e9
Summary
bull B cell malignancies are the most common
hematological disorders seen in flow cytometry
bull Many have complex overlapping phenotypes
bull Important to look at ungated plots as well as gating on
specific subpopulations
bull Level of expression of markers is as important as
positive or negative (think CD13 vs CD16)
68
References
bull Craig FE Foon KA Flow cytometric immunophenotyping for hematologic neoplasms Blood
2008 Apr 15111(8)3941-67
bull Wood BL Principles of minimal residual disease detection for hematopoietic neoplasms by flow
cytometry Cytometry B Clin Cytom 2015 Apr 23
bull Shanafelt TD Ghia P Lanasa MC Landgren O Rawstron AC Monoclonal B-cell
lymphocytosis (MBL) biology natural history and clinical management Leukemia 2010
Mar24(3)512-20
bull Swerdlow SH Campo E Pileri SA Harris NL Stein H Siebert R Advani RGhielmini M Salles
GA Zelenetz AD Jaffe ES The 2016 revision of the World Health Organization classification of
lymphoid neoplasms Blood 2016 May 19127(20)2375-90
bull 3 Moench L Sachs Z Aasen G Dolan M Dayton V Courville EL Double- and triple-hit
lymphomas can present with features suggestive of immaturity including TdT expression and
create diagnostic challenges Leuk Lymphoma 2016572626-35
bull Sutherland DR Keeney M Illingworth A Practical guidelines for the high-sensitivity detection
and monitoring of paroxysmal nocturnal hemoglobinuria clones by flow cytometry Cytometry B
Clin Cytom 2012 Jul82(4)195-208
69
Case 6 RBC Test for PNH CD235a v s CD59
Case 6 Neutrophil test for PNH clone
FLAER CD24 CD45CD15
18 PNH clone
Case 6
bull Follow up
bull Patient on steroids acyclovir and anti-thymocyte
globulin
bull PNH clone Neutrophils 96 RBC 1 Nov 2015
bull In remission as of April 2017
- Lkc 5500uL (Neuts 23)
- Hb 129gL
- Plt 208 X10e9
Summary
bull B cell malignancies are the most common
hematological disorders seen in flow cytometry
bull Many have complex overlapping phenotypes
bull Important to look at ungated plots as well as gating on
specific subpopulations
bull Level of expression of markers is as important as
positive or negative (think CD13 vs CD16)
68
References
bull Craig FE Foon KA Flow cytometric immunophenotyping for hematologic neoplasms Blood
2008 Apr 15111(8)3941-67
bull Wood BL Principles of minimal residual disease detection for hematopoietic neoplasms by flow
cytometry Cytometry B Clin Cytom 2015 Apr 23
bull Shanafelt TD Ghia P Lanasa MC Landgren O Rawstron AC Monoclonal B-cell
lymphocytosis (MBL) biology natural history and clinical management Leukemia 2010
Mar24(3)512-20
bull Swerdlow SH Campo E Pileri SA Harris NL Stein H Siebert R Advani RGhielmini M Salles
GA Zelenetz AD Jaffe ES The 2016 revision of the World Health Organization classification of
lymphoid neoplasms Blood 2016 May 19127(20)2375-90
bull 3 Moench L Sachs Z Aasen G Dolan M Dayton V Courville EL Double- and triple-hit
lymphomas can present with features suggestive of immaturity including TdT expression and
create diagnostic challenges Leuk Lymphoma 2016572626-35
bull Sutherland DR Keeney M Illingworth A Practical guidelines for the high-sensitivity detection
and monitoring of paroxysmal nocturnal hemoglobinuria clones by flow cytometry Cytometry B
Clin Cytom 2012 Jul82(4)195-208
69
Case 6 Neutrophil test for PNH clone
FLAER CD24 CD45CD15
18 PNH clone
Case 6
bull Follow up
bull Patient on steroids acyclovir and anti-thymocyte
globulin
bull PNH clone Neutrophils 96 RBC 1 Nov 2015
bull In remission as of April 2017
- Lkc 5500uL (Neuts 23)
- Hb 129gL
- Plt 208 X10e9
Summary
bull B cell malignancies are the most common
hematological disorders seen in flow cytometry
bull Many have complex overlapping phenotypes
bull Important to look at ungated plots as well as gating on
specific subpopulations
bull Level of expression of markers is as important as
positive or negative (think CD13 vs CD16)
68
References
bull Craig FE Foon KA Flow cytometric immunophenotyping for hematologic neoplasms Blood
2008 Apr 15111(8)3941-67
bull Wood BL Principles of minimal residual disease detection for hematopoietic neoplasms by flow
cytometry Cytometry B Clin Cytom 2015 Apr 23
bull Shanafelt TD Ghia P Lanasa MC Landgren O Rawstron AC Monoclonal B-cell
lymphocytosis (MBL) biology natural history and clinical management Leukemia 2010
Mar24(3)512-20
bull Swerdlow SH Campo E Pileri SA Harris NL Stein H Siebert R Advani RGhielmini M Salles
GA Zelenetz AD Jaffe ES The 2016 revision of the World Health Organization classification of
lymphoid neoplasms Blood 2016 May 19127(20)2375-90
bull 3 Moench L Sachs Z Aasen G Dolan M Dayton V Courville EL Double- and triple-hit
lymphomas can present with features suggestive of immaturity including TdT expression and
create diagnostic challenges Leuk Lymphoma 2016572626-35
bull Sutherland DR Keeney M Illingworth A Practical guidelines for the high-sensitivity detection
and monitoring of paroxysmal nocturnal hemoglobinuria clones by flow cytometry Cytometry B
Clin Cytom 2012 Jul82(4)195-208
69
Case 6
bull Follow up
bull Patient on steroids acyclovir and anti-thymocyte
globulin
bull PNH clone Neutrophils 96 RBC 1 Nov 2015
bull In remission as of April 2017
- Lkc 5500uL (Neuts 23)
- Hb 129gL
- Plt 208 X10e9
Summary
bull B cell malignancies are the most common
hematological disorders seen in flow cytometry
bull Many have complex overlapping phenotypes
bull Important to look at ungated plots as well as gating on
specific subpopulations
bull Level of expression of markers is as important as
positive or negative (think CD13 vs CD16)
68
References
bull Craig FE Foon KA Flow cytometric immunophenotyping for hematologic neoplasms Blood
2008 Apr 15111(8)3941-67
bull Wood BL Principles of minimal residual disease detection for hematopoietic neoplasms by flow
cytometry Cytometry B Clin Cytom 2015 Apr 23
bull Shanafelt TD Ghia P Lanasa MC Landgren O Rawstron AC Monoclonal B-cell
lymphocytosis (MBL) biology natural history and clinical management Leukemia 2010
Mar24(3)512-20
bull Swerdlow SH Campo E Pileri SA Harris NL Stein H Siebert R Advani RGhielmini M Salles
GA Zelenetz AD Jaffe ES The 2016 revision of the World Health Organization classification of
lymphoid neoplasms Blood 2016 May 19127(20)2375-90
bull 3 Moench L Sachs Z Aasen G Dolan M Dayton V Courville EL Double- and triple-hit
lymphomas can present with features suggestive of immaturity including TdT expression and
create diagnostic challenges Leuk Lymphoma 2016572626-35
bull Sutherland DR Keeney M Illingworth A Practical guidelines for the high-sensitivity detection
and monitoring of paroxysmal nocturnal hemoglobinuria clones by flow cytometry Cytometry B
Clin Cytom 2012 Jul82(4)195-208
69
Summary
bull B cell malignancies are the most common
hematological disorders seen in flow cytometry
bull Many have complex overlapping phenotypes
bull Important to look at ungated plots as well as gating on
specific subpopulations
bull Level of expression of markers is as important as
positive or negative (think CD13 vs CD16)
68
References
bull Craig FE Foon KA Flow cytometric immunophenotyping for hematologic neoplasms Blood
2008 Apr 15111(8)3941-67
bull Wood BL Principles of minimal residual disease detection for hematopoietic neoplasms by flow
cytometry Cytometry B Clin Cytom 2015 Apr 23
bull Shanafelt TD Ghia P Lanasa MC Landgren O Rawstron AC Monoclonal B-cell
lymphocytosis (MBL) biology natural history and clinical management Leukemia 2010
Mar24(3)512-20
bull Swerdlow SH Campo E Pileri SA Harris NL Stein H Siebert R Advani RGhielmini M Salles
GA Zelenetz AD Jaffe ES The 2016 revision of the World Health Organization classification of
lymphoid neoplasms Blood 2016 May 19127(20)2375-90
bull 3 Moench L Sachs Z Aasen G Dolan M Dayton V Courville EL Double- and triple-hit
lymphomas can present with features suggestive of immaturity including TdT expression and
create diagnostic challenges Leuk Lymphoma 2016572626-35
bull Sutherland DR Keeney M Illingworth A Practical guidelines for the high-sensitivity detection
and monitoring of paroxysmal nocturnal hemoglobinuria clones by flow cytometry Cytometry B
Clin Cytom 2012 Jul82(4)195-208
69
References
bull Craig FE Foon KA Flow cytometric immunophenotyping for hematologic neoplasms Blood
2008 Apr 15111(8)3941-67
bull Wood BL Principles of minimal residual disease detection for hematopoietic neoplasms by flow
cytometry Cytometry B Clin Cytom 2015 Apr 23
bull Shanafelt TD Ghia P Lanasa MC Landgren O Rawstron AC Monoclonal B-cell
lymphocytosis (MBL) biology natural history and clinical management Leukemia 2010
Mar24(3)512-20
bull Swerdlow SH Campo E Pileri SA Harris NL Stein H Siebert R Advani RGhielmini M Salles
GA Zelenetz AD Jaffe ES The 2016 revision of the World Health Organization classification of
lymphoid neoplasms Blood 2016 May 19127(20)2375-90
bull 3 Moench L Sachs Z Aasen G Dolan M Dayton V Courville EL Double- and triple-hit
lymphomas can present with features suggestive of immaturity including TdT expression and
create diagnostic challenges Leuk Lymphoma 2016572626-35
bull Sutherland DR Keeney M Illingworth A Practical guidelines for the high-sensitivity detection
and monitoring of paroxysmal nocturnal hemoglobinuria clones by flow cytometry Cytometry B
Clin Cytom 2012 Jul82(4)195-208
69