Flow
Flow
Flow
Acute Leukemia
• Leukaemia is a disease resulting from the neoplastic
proliferation of haemopoietic or lymphoid cells
Clinical Molecular
Information + Morphology +Immunophenotyping+ Cytogenetics + genetics
MOLECULAR MORPHOLOGY &
STUDIES IMMUNOPHENOTYPING
•Immunoenzymatic techniques
•Using Peroxidase or ALP
•Utilise cytospin/ paraffin
embedded/ fresh frozen tissue
• Flow cytometry - preferred method for the lineage
assignment and maturational analysis of hematological
malignancies
Biopsies from solid organs and bone marrow can also be used after
manual disaggregation and filtration of large particles
• Volume – 2 - 5 ml
• Anticoagulant
– EDTA - reduces leukocytes and platelets adhesivity and
aggregation
Proerythroblast
Myeloblast Monoblast
Gp A
Megakaryoblast CD 13, 15, 33, 34 CD 13, 15, 33, (11c)
Gp IIIa (CD 61),
Gp Iib (CD 41) Erythroblast
Gp A Promyelocyte Promonocyte
CD 13, 15, 33,(11c) CD 13, 14, 33, 11c
Megakaryocyte
Gp IIIa, Gp IIb Myelocyte Monocyte
CD 13, 15, 33, 11c CD 13, 14, 33, 11c
Granulocyte Macrophage
Thrombocyte Erythrocyte
CD 13, 15, 11c CD 13, 14, 33, 11c, 1
Gp IIIa, Gp IIb Gp A
(Lineage
assignment)
(Definite phenotype,
Maturation)
• B cell – CD 19
• T cell – CD 3, CD 7
• Myeloid – CD 13, CD 33, CD 117
• Monocytic lineage – CD 14
• Megakaryocytic – CD 61
• CALLA – CD 10
The CD45/Side Scatter “Blast Gate”
G
T
B
HLA –DR +,
CD 34+,
MPO +, CD
13 +, weak
CD 33
Acute Promyelocytic Leukemia With
t(15;17)(q22;q12); PML-RARA
Acute Promyelocytic Leukemia With
t(15;17)(q22;q12); PML-RARA
• Hypogranular variant –
– Frequent expression of CD34, CD2, at least on a
fraction of cells
Immunophenotyping in AML, NOS
AML M0
AML, M1 HLA DR- +, CD 34+
(70%) , CD 13+, CD
117 +, CD 33 +,
MPO + (fraction)
CD 11b, CD 15,
CD 14, CD 64 -
NEG
AML, M1
AML, M2
AML, M4
CD 33 (bright), HLA –DR (intermediate), CD 117 (intermediate)
CD 34 neg
CD 13 (intermediate),
CD 15 (intermediate)
CD11b (low to intermediate), CD64 (bright) and CD36 (intermediate to
bright), CD14 (low)
AML, M5
This CD45 vs SSC plot corresponds This CD45 vs SSC plot corrponds to
to the above acute monocytic the above acute monoblastic
leukemia case. Abnormal monocytic leukemia case. Abnormal monocytic
population is green. Note the higher population is green. Note the lower
SSC. AML M5 b SSC. AML M5a
AML, M5
AML, M6
AML, M6
AML, M6
AML, M7
Immunophenotyping in ALL
Precursor B cell lymphoblastic leukemia
Precursor B cell lymphoblastic leukemia
T-lymphoblastic leukemia
T-lymphoblastic leukemia
Additionally, some nonhematolymphoid tumors such as small cell
carcinoma or pediatric small round cell tumors can resemble leukemic
blasts on smears but are readily distinguished from leukemic blasts by flow
cytometry, where they appear as CD45-negative cells lacking specific
markers of B cell, T cell, or myeloid lineage, often with higher side scatter
than might be expected for a cell with so little cytoplasm (Chang, 2003)
Biphenotypic, bilineal, ambiguous or mixed lineage: strange
leukemias!
Requirements for assigning more than one lineage to a single
blast population
• LAIP:
– Absence of expected ags
– Overexpression of ags
– Asynchronous expression
– Lineage infidelity
– Aberrant light scatter property
Minimal Residual Disease
• Drawbacks
– lack of antigen specificity for malignant cells as these
cells represent the counterparts of normal cells with, in
many cases, identical or similar antigen profiles
B- ALL - anti-CD22
(epratuzamab) and
CD52 (alemtuzamab)
CD 13, CD 33 + Blasts
AML, M7
HLA DR+, CD 34+
AML, M4
CD 71, GPA CD14 +, CD 64+
AML, M6