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Flow Cytometry in Acute Leukemia

Flow cytometry is a technique used to immunophenotype acute leukemias. It uses fluorescent antibodies attached to cells which are passed through a laser, detecting cell size, granularity, and antigen expression. This allows rapid and accurate identification of multiple antigens simultaneously. Key antibodies include CD45, CD13/33/MPO for myeloid cells, CD14/64 for monocytes, CD2/3 for T-cells, and CD19/22/10/79a for B-cells. Gating selects specific cell populations for further analysis, aiding leukemia diagnosis and classification into B-cell, T-cell, or myeloid subtypes with different prognoses.

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322 views16 pages

Flow Cytometry in Acute Leukemia

Flow cytometry is a technique used to immunophenotype acute leukemias. It uses fluorescent antibodies attached to cells which are passed through a laser, detecting cell size, granularity, and antigen expression. This allows rapid and accurate identification of multiple antigens simultaneously. Key antibodies include CD45, CD13/33/MPO for myeloid cells, CD14/64 for monocytes, CD2/3 for T-cells, and CD19/22/10/79a for B-cells. Gating selects specific cell populations for further analysis, aiding leukemia diagnosis and classification into B-cell, T-cell, or myeloid subtypes with different prognoses.

Uploaded by

alibayaty1
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© Attribution Non-Commercial (BY-NC)
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Download as PPT, PDF, TXT or read online on Scribd
  • Notes on Flow Cytometry: Title page introducing the topic of flow cytometry as applied to acute leukemia.
  • Immunophenotyping of AL: Describes the reasons and objectives of using immunophenotyping in diagnosing leukemia subtypes.
  • Immunophenotyping Techniques: Explains the techniques used in immunophenotyping, including immunoenzymatic and immunofluorescent methods.
  • Flow Cytometry: Details the process and advantages of using flow cytometry to analyze cell properties.
  • Samples that Could be Used: Lists the types of biological samples suitable for flow cytometry.
  • Is There Anything I Must Know?: Provides additional important information about the procedures and considerations when applying flow cytometry.
  • The Most Useful Antibodies for Acute Leukemia: Identifies and explains the function of antibodies critical to diagnosing acute leukemia.
  • What is Gating?: Introduces the concept of gating in flow cytometry to analyze specific cell populations.
  • Graphical Data Representations: Presents visual data examples from flow cytometry analysis, emphasizing key cell groups.
  • Approach to Acute Leukemia Diagnosis by Immunological Markers: Outlines the immunological markers used in the diagnostic approach of acute leukemia.
  • Immunological Classification of ALL: Discusses the classification of acute lymphoblastic leukemia using specific markers.
  • Immunological Classification of Acute Leukemia: Describes the further immunological classification within acute leukemia types.
  • Stages of Myeloid Maturation: Depicts the maturation stages of myeloid cells, providing marker information at each stage.

Notes on flow cytometry

In acute Leukemia

Immunophenoptyping of AL
Indicated in all types of Leukemia that are not clearly myeloid to : 1. Make a positive diagnosis of ALL. 2. Diagnose unequivocally cases of AML particularly of M0 and M7 subtypes; Sometimes M6 and M5a.

Immunophenotyping Techniques
Immunoenzymatic techniques : applied to fixed slides. Immunoflourescent techniques : Antibodies are attached to flourochrome. Two techniques are used to detect a reaction : - Flourescent micrscope. - Flow cytometry.

Flow cytometry
Is a technique by which a stream of cells that have been labelled with an antibody conjugated to a flourescent dye flow past a detector and can be counted and sized. It is a rapid highly accurate and can detect several antigens on the same cells simultaneously and the strength of Ag expression.

Samples that could be used


Anticoagulated whole blood or bone marrow, in which red cells have been lysed. Antibodies are labeled by flourochromes.

Is there anything I must know?


A laser beam is passed through a running flow of cells, which have been treated with the labeled antibodies. Scattering of light occurs, and it maybe either : Forward scatter (FSC) : related to cell size. Side Scatter (SSC) : related to structure of the cells including granularity.

The most useful antibodies for acute leukemia


CD 45 : it is the common leucocyte antigen present in all hemopoietic cells except RBC. CD 13, 33 and anti-cMPO : myeloid markers. CD 14,64 : monocytoid antibodies. CD 2, cCD3 : T cell. CD19, cCD22, CD10, cCD79a: B cell. TdT : Non-lineage specific.

What is Gating?
It is simply to select a single group of cells , e.g. by CD45 and SSC, and restrict further immuno-phenotyping to this group of selected cells.

M.G

Blasts

Monocytic cells Lymphocytes

First Panel - B Lymphoid : - T Lymphoid : - Myeloid :

Approach to Acute leukemia Diagnosisby immunological markers


: CD19; cCD22; CD10. CD10 cCD3; CD7; CD2. CD13*; CD33; Anti-MPO.

- Non-lineage related : Tdt. Second Panel : B- lymphoid : C ; SmIg. T-Lymphoid : CD1, CD5, CD4, CD 8. -If myeloid : CD14 (M4-5), anti-glycophorinA (M6), CD41 (M7).

Immunological classification of ALL


Using immunological markers (CD markers) it could be divided into two categories namely, those of B or T lineage. The B lineage ALL (CD19 and CD22 positive) could further subtyped into : - Early B-precursor ALL (bad prognosis). TdT +; CD10 -;CyIg -; SmIg -. - Common ALL.(most common ALL, good prognosis) : TdT +; CD10 +; CyIg -; SmIg - Pre-B ALL. TdT -; CD10 +; CyIg +; SmIg - B-ALL (very bad prognosis). (FAB L3-ALL) TdT -; CD10 +/-; CyIg -/+; SmIg +

Immunological classification of Acute Leukemia


The T lineage ALL (CD3, TdT & CD7 positive) is further subtyped into: -Pre-T (Early T-precursor) ALL. CD2 negative. -T-ALL. CD2 Positive. -T-lineage ALL have generally good prognosis in adults, bad in children.

Stages of Myeloid maturation


Myeloblast (CD13,CD33, CD34, HLA-DR) Promyelocytes (CD13, CD33) Myelocyte (CD13, CD33, CD11b,CD14) Metamyelocyte(CD13, CD11b, CD14) Neutrophils (CD13, CD11b, CD14) Stem cells Monoblast (CD13, CD34, CD33, HLA-DR) Promonocyte (CD13, CD33, CD11b, CD14, HLA-DR) Monocytes (CD13, CD33, CD11b, CD14, HLA-DR)

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