Post on 05-Apr-2018
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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)