Notes on Flow Cytometry

8/2/2019 Notes on Flow Cytometry

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Immunophenoptyping of ALImmunop

henoptyping of AL

Indicated in all types of Leukemiathat are not clearly myeloid to :

1. Make a positive diagnosis ofALL.

2. Diagnose unequivocally cases ofAML particularly of M0 and M7subtypes; Sometimes M6 and M5a.


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Immunophenotyping Techniques

Immunoenzymatic techniques : appliedto fixed slides.

Immunoflourescent techniques :Antibodies are attached to flourochrome.Two techniques are used to detect areaction :

- Flourescent micrscope.

- Flow cytometry.


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Flow cytometryFlow cytometry

Is a technique by which a stream of cellsthat have been labelled with an antibody

conjugated to a flourescent dye flowpast a detector and can be counted andsized.

It is a rapid highly accurate and can

detect several antigens on the same cellssimultaneously and the strength of Agexpression.


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Samples that could be used

Anticoagulated whole blood or bonemarrow, in which red cells have been

lysed.

Antibodies are labeled byflourochromes.


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Is there anything I must know?

A laser beam is passed through a runningflow of cells, which have been treatedwith the labeled antibodies.

Scattering of light occurs, and it maybeeither :

Forward scatter (FSC) : related to cell

size. Side Scatter (SSC) : related tostructure of the cells includinggranularity.


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The most useful antibodies for acute

leukemia CD 45 : it is the common leucocyte

antigen present in all hemopoietic cellsexcept RBC.

CD 13, 33 and anti-cMPO : myeloidmarkers.

CD 14,64 : monocytoid antibodies.

CD 2, cCD3 : T cell. CD19, cCD22, CD10, cCD79a: B cell. TdT : Non-lineage specific.


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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 thisgroup of selected cells.


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M.G

Blasts

Monocytic

cells

Lymphocytes


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Approach to Acute leukemia Diagnosisby

immunological markers First Panel :- B Lymphoid : CD19; cCD22; CD10CD19; cCD22; CD10.

- T Lymphoid : cCD3; CD7; CD2.CD3; CD7; CD2.

- Myeloid : CD13*; CD33; Anti-CD13*; CD33; Anti-MPO.MPO.- Non-lineage related : Tdt.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).


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Immunological classification of ALLImmunological 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, goodprognosis) :

TdT +; CD10 +; CyIg -; SmIg -

- Pre-B ALL. TdT -; CD10 +; CyIg +; SmIg -- B-ALL (very bad prognosis). (FAB L3-ALL) TdT -; CD10 +/-; CyIg -/+; SmIg +


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Immunological classification of Acute

LeukemiaThe T lineage ALL (CD3, TdT & CD7 positive) isfurther subtyped into:

-Pre-T (Early T-precursor) ALL. CD2 negative.

-T-ALL.

CD2 Positive.-T-lineage ALL have generally good prognosis inadults, bad in children.


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Stages of Myeloid maturationStages of Myeloid maturation

Stem cellsMyeloblast (CD13,CD33,

CD34, HLA-DR)

Promyelocytes(CD13, CD33)

Myelocyte (CD13, CD33,

CD11b,CD14)Metamyelocyte(CD13,

CD11b, CD14)

Neutrophils (CD13, CD11b, CD14)

Monoblast (CD13, CD34,

CD33, HLA-DR)

Promonocyte (CD13,

CD33, CD11b, CD14,

HLA-DR)

Monocytes (CD13, CD33,

CD11b, CD14, HLA-DR)

Notes on Flow Cytometry

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