Flow Cytometry in the Diagnostic Laboratory Workup of Acute Lymphoblastic Leukemias
CC BY 4.0 · Indian J Med Paediatr Oncol 2023; 44(05): 474-481
DOI: DOI: 10.1055/s-0043-1772204
Abstract
Acute lymphoblastic leukemias (ALLs) are hematological neoplasms characterized by clonal proliferation of lymphoid blasts, which can be B- or T-cell type. Flow cytometric immunophenotyping is an integral component in establishing blast lineage during the diagnostic workup of ALLs, aiding in appropriate therapy, prognostication, and monitoring of the disease. The current review focuses on the utility of flow cytometry in the workup of ALLs, including the usefulness of various antibodies and pitfalls in diagnosis.
Keywords
acute lymphoblastic leukemia - flow cytometry - B-ALL - T-ALL
Publication History
Article published online:
04 November 2023
© 2023. The Author(s). This is an open access article published by Thieme under the terms of the Creative Commons Attribution License, permitting unrestricted use, distribution, and reproduction so long as the original work is properly cited. (https://creativecommons.org/licenses/by/4.0/)
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Abstract
Acute lymphoblastic leukemias (ALLs) are hematological neoplasms characterized by clonal proliferation of lymphoid blasts, which can be B- or T-cell type. Flow cytometric immunophenotyping is an integral component in establishing blast lineage during the diagnostic workup of ALLs, aiding in appropriate therapy, prognostication, and monitoring of the disease. The current review focuses on the utility of flow cytometry in the workup of ALLs, including the usefulness of various antibodies and pitfalls in diagnosis.
Keywords
acute lymphoblastic leukemia - flow cytometry - B-ALL - T-ALL
Introduction
Multiparametric flow cytometry is an indispensable tool for the diagnosis and subclassification of acute lymphoblastic leukemia (ALL). Accurate classification of ALLs into B- or T-cell types is crucial for the optimal choice of therapeutic regimens that varies based on the ALL subtype. The antigenic expression profile, particularly the immunophenotypic aberrancies by the blasts deviating from those encountered during normal hematopoiesis, aids in the differentiation of the blasts from their normal benign counterparts. The panel of antigens for clinical testing has evolved from 4 to 13 colors or more thanks to the substantial development in antibody clones, the fluorochrome conjugate options, and a wide variety of lasers that have dramatically increased the number of antigens that can be simultaneously studied. In this article, we attempt to discuss the strategy and approach to the classification of ALL into B- or T-cell subtypes and the evolution of consensus groups for antigen/antibody/fluorochrome selection, choice of reagents, sample processing methodology for appropriate diagnosis, and classification.
B-Acute Lymphoblastic Leukemias
B-lymphoblastic leukemia/lymphoma or B-cell precursor acute lymphoblastic leukemia (BCP-ALL) is the most common malignancy seen in childhood. Approximately 75-% of BCP-ALL cases occur in children under 6 years of age.[1] However, it shows a bimodal age distribution, with a small peak occurring during the fifth decade of life.[2] BCP-ALL is diagnosed by morphology combined with immunophenotyping, typically done by multiparametric flow cytometer (MFC). Immunophenotyping is essential for differentiating BCP-ALL from acute leukemias of other lineages, like T-lymphoblastic leukemia (T-ALL), acute myeloid leukemia (AML), mixed phenotype acute leukemia (MPAL), etc.
The flow cytometric diagnosis of BCP-ALL is based on identifying an expanded population of immature B cells showing immunophenotypic aberrancies that help distinguish the leukemic blasts from normal B-cell precursors, or hematogones.[3] These aberrancies are in the form of increased or decreased intensity of expression of an antigen on the leukemic blasts compared to the normal counterparts or gain of antigen expression, which are not specific to B-lineage.[4] Knowledge of the spectrum of antigenic expression on the B-progenitor cells during development is essential to differentiate them from B-leukemic blasts.[5] The normal B-progenitors show the expression of certain antigens in a sequential, tightly regulated manner.[3] The B-progenitor cells are derived from common lymphoid progenitor cells in the bone marrow, and they undergo three stages of maturation to become mature B-lymphoid cells. These three stages of cells are called hematogones type I (early/pre-B-I), hematogones type II (intermediate/pre-B-II), and hematogones type III (late/transitional B-cells).[6] The three stages of hematogones show a stepwise increase in the intensity of expression of CD45 and CD20 while showing a stepwise decrease in intensity of CD34 and CD10, which are finally lost at the late stage. CD19 expression is the lowest in stage I, and increases significantly in stage II, with an eventual mild reduction in mature B cells. Mature B cells show loss of CD34, CD10, and TdT, bright expression of CD20 and CD45, and a surface expression of polytypic immunoglobulins.[5] [Fig. 1] shows flow cytometric dot plots of a BCP-ALL patient.
Antigen |
Aberrancy |
---|---|
TdT |
Negative/uniform expression |
CD34 |
Negative/uniform expression |
CD45 |
Negative/uniform expression |
CD10 |
Negative/over[removed]uniform bright) |
CD20 |
Uniform expression or negative |
CD22 |
Negative/under expression |
CD38 |
Under expression |
CD19 |
Under/overexpression |
CD73 |
Overexpression |
CD58 |
Overexpression |
CD86 |
Overexpression |
CD123 |
Overexpression |
CD200 |
Overexpression |
CD81 |
Under expression |
CD304 |
Aberrant expression |
CD9 |
Overexpression |
CD44 |
Overexpression |
CD13, CD33, CD66c, CD15, CD7, and CD56 |
Lineage aberrant markers |
Subclassification |
CD10 |
Cytoplasmic IgM |
Surface IgM |
---|---|---|---|
B I (pro-B) ALL |
Negative |
Negative |
Negative |
B II (pre-pre-B or common B) ALL |
Positive |
Negative |
Negative |
B III (pre-B) ALL |
Positive |
Positive |
Negative |
B IV (mature B) ALL |
Negative/positive |
Negative/positive |
Positive |
Tubes |
Fluorochromes |
|||||||
---|---|---|---|---|---|---|---|---|
PacB |
AmCyan |
FITC |
PE |
PerCPCy5.5 |
PECy7 |
APC |
AF700 |
|
ALOT Tube |
CyCD3 |
CD45 |
CyMPO |
CyCD79a |
CD34 |
CD19 |
CD7 |
SmCD3 |
BCP-ALL Tube 1 |
CD20 |
CD45 |
CD58 |
CD66c |
CD34 |
CD19 |
CD10 |
CD38 |
BCP-ALL Tube 2 |
SmIgk |
CD45 |
CyIgμ |
CD33 |
CD34 |
CD19 |
SmIgM and CD117 |
SmIgλ |
BCP-ALL Tube 3 |
CD9 |
CD45 |
NuTdT |
CD13 |
CD34 |
CD19 |
CD22 |
CD24 |
BCP-ALL Tube 4 |
CD21 |
CD45 |
CD15 and CD65 |
NG2 |
CD34 |
CD19 |
CD12 3 |
CD81 |
Markers |
Pro-T-ALL |
Pre-T-ALL |
Cortical T-ALL |
Medullary T-ALL |
---|---|---|---|---|
CD1 |
– |
– |
+ + |
– |
CD2 |
+ |
+ + |
+ + |
+ + |
SmCD3 |
– |
– |
– (except in SmCD3+ subtypes, ++) |
+ + |
CyCD3 |
+ + |
+ + |
+ + |
+ + |
CD4–/CD8– |
+ + |
+ |
– |
– |
CD4–/CD8+ |
– |
± |
± |
± |
CD4+/CD8– |
– |
± |
± |
+ |
CD4+/CD8+ |
– |
– |
+ |
± |
CD5 |
– |
+ + |
+ + |
+ + |
CD7 |
+ + |
+ + |
+ + |
+ + |
T-ALL tubes |
Fluorochromes |
|||||||
---|---|---|---|---|---|---|---|---|
PacB |
AmCyan |
FITC |
PE |
PerCPCy5.5 |
PECy7 |
APC |
AF700 |
|
Tube 1 |
CyCD3 |
CD45 |
NuTdT |
CD99 |
CD5 |
CD10 |
CD1a |
SmCD3 |
Tube 2 |
CyCD3 |
CD45 |
CD2 |
CD117 |
CD4 |
CD8 |
CD7 |
SmCD3 |
Tube 3 |
CyCD3 |
CD45 |
TCRgd |
TCRab |
CD33 |
CD56 |
CyTCRb |
SmCD3 |
Tube 4 |
CyCD3 |
CD45 |
CD44 |
CD13 |
HLADR |
CD45RA |
CD123 |
SmCD3 |
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