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 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

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

*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

Hematolymphoid malignancies Acute Leukemias

(WHO 2008)

Acute Myeloid Leukemias and

related precursor neoplasms

Precursor lymphoid

neoplasms

AL of Ambiguous lineage

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

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

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 CELLPRE-PRE B-CELL

IMMATURE-B-CELL

TdT

CD79a

CD19

CD10

CD20

CD22

PRE-B-CELLMATURE-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

DOUBLECD4+

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

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

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

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