Flowcytometry in the diagnosis of ALL

Gayathri K
Lifeline Tapadia Specialty Lab for Blood
Disorders
Hyderabad. gaya3k2002@yahoo.co.in
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
Suspecting/ Recognising Acute Leukemia
Specific request for a clinical suspicion
Unexpected or Routine !
Known case ?
______________________
Blasts
How to suspect, be certain ?
When DD ??
Is a marrow evaluation necessary ?
How to be certain ?
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
FAB classification of Acute Leukemia
Why 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
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
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
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
*Burkitts
lymphoma
(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
Hematolymphoid malignancies Acute Leukemias
(WHO 2008)
Acute Myeloid
Leukemias and
related precursor
neoplasms
Precursor
lymphoid
neoplasms
AL of Ambiguous
lineage
Precursor
Lymphoid
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
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 options
Do not permit mistaken identity
Not possible to undo mistake at diagnosis
2008 WHO classification of Hematolymphoid
Neoplasms
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
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
B-CELL MATURATION ANTIGENIC EXPRESSION
STEM CELL
PRE-PRE
B-CELL
IMMATURE
-B-CELL
TdT
CD79a
CD19
CD10
CD20
CD22
PRE-B-CELL
MATURE
-B-CELL
ACTIVATED
-B-CELL
PLASMA
CELL
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
DOUBLE
CD4+
CD8+
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
Flow cytometer
Study of properties on single cells
Multiparametric analysis at a single cell
Co expression of antigens
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
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,
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
Acute Leukemia Diagnosis
Blast/Immature cell
Myeloid
morphology
Cytochemistry
Markers
Lymphoid
morphology
Markers
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
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 - cytoCD3
Myeloid lineage specific - Anti-MPO
Other Markers TdT, CD99, CD41, CD61, SmIg & CD56
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
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
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
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
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
LAIP & MRD
Detection of asynchronous antigen
Antigen over expression
Cross lineage antigen
_____________________________________
CD13, CD 33, CD 15 on B lymphoblasts
Cells co expressing TdT, CD 34with T lineage
Round cells in pediatric marrow
Blasts : lymphoblasts, myeloblasts
Hematogones
Non Hodgkin Lymphoma cells
Non hemapoietic neoplasms : neuroblastoma
_______________________________________
CD 10, CD 19, CD 20, CD 34, TdT; CD38, CD22
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