06 flow cytometry in diagnosis of all
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06 Flow Cytometry in Diagnosis of ALLTRANSCRIPT
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Flowcytometry in the diagnosis of ALL
Gayathri K
Lifeline Tapadia Specialty Lab for Blood Disorders
Hyderabad. [email protected]
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Some interesting perspectives..
Acute Leukemia : Heterogenous group of diseases
Clonal proliferation and maturation arrest
Defining a blast- the diagnostic cell in Acute leukemia
Assigning Lymphoid, Myeloid and APML
Rapid precise diagnosis at presentation is critical to management
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Suspecting/ Recognising Acute Leukemia
Specific request for a clinical suspicion
Unexpected or Routine !
Known case ?
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Blasts
How to suspect, be certain ?
When DD ??
Is a marrow evaluation necessary ?
How to be certain ?
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Acute Leukemia Diagnosis
PB examination including smear
Followed by Marrow aspiration
Unusually other tissue Lymph node, soft tissue or other sites, first target organs
FAB:30% blasts.WHO 20% blasts on marrow
Enumeration of blasts (FAB typeI & typeII)
Distinguishing ALL from AML/ANLL (3%MPO positivity- FAB)
Need to establish type: B, T for ALL & minimal differentiation in AML, Biphenotypic
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FAB classification of Acute LeukemiaWhy we should know
First crucial advance that defined and classified Acute Leukemias.1976.
Revised. 1985
Addition. 1991
Criteria: 30% blasts. Marrow cytology and PB diff
Not to be used as default WHO categories
Still the basis of diagnosis
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Acute Leukemia Diagnosis- step by step
CBC, flags on printout -> Smear examination
Marrow aspiration cytology : 500 cell count; all nucleated cells, blasts as a percent of non erythroid cells
Cytochemistry :MPX, SBB; NSE
Immune markers by Flowcytometry
Chromosomal studies : Karyotyping,FISH, PCR
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WHO classification of Acute Leukemia (2001)
WrightGiemsa morphology, cytochemistry
Immuno markers
Specific chromosomal defects/translocations
Transformation from pre-existing conditions
Criteria: Blasts minimum 20% either in blood or marrow
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Hematolymphoid malignancies Acute Leukemias
(WHO 2001)
Acute Myeloid
Leukemias
B-cell Neoplasms
Precursor B-cell neoplasm
Precursor B-Lymphoblastic
leukemia/ lymphoma
(FAB-ALL-L1 & L2)
Mature B-cell neoplasms
*Burkittslymphoma
(FAB-ALL-L3)
T-cell Neoplasms
Precursor T cell neoplasm
Precursor T -Lymphoblastic
leukemia/ lymphoma
(FAB-ALL-L1 & L2) #Blastic NK cell
leukemia
AL of Ambiguous
lineage
Undifferentiated AL
Bilineal AL
Biphenotypic AL
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Hematolymphoid malignancies Acute Leukemias
(WHO 2008)
Acute Myeloid Leukemias and
related precursor neoplasms
Precursor lymphoid
neoplasms
AL of Ambiguous lineage
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PrecursorLymphoid neoplasms
B-Lymphoblastic leukemia/ lymphoma
B-LL/L, NOS
BLL/L with recurrent genetic
abnormalities
BLL/L with t(9;22); BCR/ABL1
BLL/L with t(v;11q23); MLL
rearranged
BLL/L with t(12;21);
TEL/AML1; ETV6/RUNX1
BLL/L with hyperdiploidy
BLL/L with hypodiploidy
BLL/L with t(5;14); IL3/IGH
BLL/L with t(1;19); E2A/PBX
T-Lymphoblastic leukemia/ lymphoma
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A. Myeloid neoplasms
B. Precursor lymphoid neoplasms
C. Mature B cell neoplasms
D. Mature T- and NK- cell neoplasms
E. Hodgkin lymphoma
F. Immunodeficiency associated LPD
G. Histiocytic and dendritic cell neoplasms
Different treatment optionsDo not permit mistaken identity
Not possible to undo mistake at diagnosis
2008 WHO classification of HematolymphoidNeoplasms
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From WHO 2001 to WHO 2008
Precursor B- / T- lymphoblastic leukemia/ lymphoma
Sub categorization of B-lymphoblastic leukemia/ lymphoma based on molecular abnormalities
AML 7 major categories
Significant risk stratification and prognostication
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CD markers
Large number of antibodies are available
Most of the leukocyte surface antigens are lineage associated, and not specific to a single lineage or stage of cellular maturation
Lineage specific markers - some
Blasts, Maturation patterns
Lymphoid markers of significance cytoplasmic (less mature)
Clonality Lymphoid cells in peripheral blood and also marrow to be differentiated from normal/ reactive lymphoid cells
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B-CELL MATURATION ANTIGENIC EXPRESSION
STEM CELLPRE-PRE B-CELL
IMMATURE-B-CELL
TdT
CD79a
CD19
CD10
CD20
CD22
PRE-B-CELLMATURE-B-CELL
ACTIVATED -B-CELL
PLASMA CELL
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T-CELL MATURATION ANTIGENIC EXPRESSION
PROTHYMOCYTE SUBCAPSULAR THYMOCYTE
CORTICAL THYMOCYTE
MEDULLARY THYMOCYTE
PERIPHERAL T-CELL
TdT
CD7
CD2/CD5
CD3
CD1a
CD4/CD8
CYTOPLASMIC SURFACE
DOUBLECD4+
CD8+
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Few useful facts
MPO negative by cytochemistry MUST be proven by IPT (CD13, CD33, CD1117)
Cytoplasmic: CD3 for T, CD79a for B
TdT positive in almost all B & T lymphoblasts, some AML blasts NOT seen in Lymphoma cells
Loss of CD10 associated with mature B cell neoplasm
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Flow cytometer
Study of properties on single cells
Multiparametric analysis at a single cell
Co expression of antigens
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T cell marker associations
Cytoplasmic CD3 +/- CD7 expressed by most immature T cells
CD1a positive in upto mature T lymphoid blasts and is negative in mature T lymphoid
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Common CD markers&
Acute Leukemia diagnosis
All lymphoid cells CD45+ (LCA)
B-cells CD19, CD10, cCD22
T-cells CD3, CD5, cCD3
Myeloid cells CD13, CD33, CD117, anti MPO
Megakaryocytic CD41, CD61
Blasts CD34, TdT, CD99
Others: HLA-DR, CD20, CD79a,
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Inherent Difficulties of morphologic evaluation
Diagnostic Dilemmas
Diagnostic dilemmas: RCT vs Ac Leuk M3v vs ALL Blasts vs post viral lymphoid proliferation Extramedullary deposits Extensive stromal change- gelatinous
transformation, myelonecrosis, myelofibrosis, Hypoplastic marrow MDS vs AML M7 & Pancytopenia with dry tap Blasts vs Hematogones
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Acute Leukemia Diagnosis
Blast/Immature cell
Myeloid
morphology
Cytochemistry Markers
Lymphoid
morphologyMarkers
B-lineage-CD19,CD20,CD21,cIg & sIg
T-linage-CD2,CD3,CD4,CD5 CD7 & CD6
NK lineage-CD56,CD57,CD16& CD3cyto
Myeloid CD13,CD33,CD15,CD11c &CD34
Magakaryocyte-CD41,CD61 & VIIIag
Chromosome study/ FISH
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Panels for Acute Leukemia
A) Primary panel:CD10, CD19, CD3, CD7, CD4, CD8, CD13, CD33, CD117, HLADR, CD34 CD 45 gating
B) Secondary panel: B-lineage specific - cytoCD22 / cytoCD79a T-lineage specific - cytoCD3Myeloid lineage specific - Anti-MPO
Other Markers TdT, CD99, CD41, CD61, SmIg & CD56
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Preferred primary panel
It is further recommended to do all lineage specific
cytoplasmic markers and Tdt with rest of the minimal panel.
B cell CD10, CD19, cCD22/cCD79a
T cell CD7, CD5, cCD3
Myeloid CD13, CD33, CD117, AMPO
Other CD34, HLA-DR, CD45, TdT
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Panels CD5 CD7 CD10 CD19 CD13 CD33 CD117 CD34 HLADR CD45 EXTRAS
US Canadian
1997 (12)
Y Y Y Y Y Y Y Y CD2, CD14
k,l
ISAC 2000
(> 14)
Y Y Y Y Y Y Y T, B,
Mye, Eryth,
Mega
BCSH
2002 (10)
Y Y Y Y cCD22, CD79,
cCD3, aMPO,
Tdt, CD2,
Second Latin
American
2005 (18)
Y Y Y Y Y Y Y Y Y cCD3, aMPO,
CD2, CD79a,
sIg, k, l,
CD15, Tdt
TMH
Mumbai 2008
(10)
Y Y Y Y Y Y Y Y Y Y
AL - Recommendation by various panels(n = 10-18) Gujral et al, IJPM
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AL OF AMBIGUOUS
LINEAGE
Acute Undifferentiated Leukemia
Mixed Phenotypic AL (MPAL) with t(9;22); BCR/ABL1
MPAL, B/Myeloid NOS
MPAL, T/Myeloid NOS
MPAL, NOS rare types
Mixed B/T
Others
Acute unclassified
NK lymphoblastic leukemia/ lymphoma
MPAL with t(v;11q23); MLL rearranged
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Minimal residual disease
It is the persistence of malignant cells in the bone marrow or other tissues of patients with hematologic malignancies after remission at levels below the limit of detection by conventional morphologic assessment
These residual malignant cells may be the source of disease relapse in many patients
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Morphologic evaluation, with an overall detection limit of approximately 5%, is clearly not suitable for the detection of MRD
FCM, FISH and PCR with detection limits of 10-2 to 10-4 cells have been applied
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LAIP & MRD
Detection of asynchronous antigen
Antigen over expression
Cross lineage antigen
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CD13, CD 33, CD 15 on B lymphoblasts
Cells co expressing TdT, CD 34with T lineage
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Round cells in pediatric marrow
Blasts : lymphoblasts, myeloblasts
Hematogones
Non Hodgkin Lymphoma cells
Non hemapoietic neoplasms : neuroblastoma
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CD 10, CD 19, CD 20, CD 34, TdT; CD38, CD22
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Purpose of FCM in ALL
Confirms presence of blasts
Assigns lineage
Sub classifies
Differentiates from lymphoma
Identifies high risk prognostic factors
Identifies aberrant leukemia associated immunophenotypes ( LAIP) for MRD monitoring