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  • Notes On Flow Cytometry

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    alibayaty1
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    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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    Notes on Flow Cytometry

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    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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    85 vues16 pages

    Notes On Flow Cytometry

    Transféré par

    alibayaty1
    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.

    Droits d'auteur :

    Attribution Non-Commercial (BY-NC)
    Téléchargez comme PPT, PDF, TXT ou lisez en ligne sur Scribd
    Signaler comme contenu inapproprié
    sur 16

    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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