Pathology > Basic Hematology > White Cell Disorders > Leukemia: Laboratory Evaluation - Immunophenotype

Leukemia: Laboratory Evaluation - Immunophenotype

Immunophenotyping is an important means of identifying normal cells and leukemic blasts through the use of antibodies (usually monoclonal) to various cell surface and cytoplasmic proteins.

Combinations of antibodies allow for 1) identification of specific cell types, 2) determination of the degree of cell differentiation, and 3) recognition of abnormal cells.

Many monoclonal antibodies have been grouped together in Clusters of Differentiation (CD).

The use of immunologic markers to determine the lineage and degree of differentiation of a leukemia has become an essential part of the diagnostic evaluation. This information is invaluable for selecting therapy and for determining prognosis and can help in the detection of relapse or of secondary leukemia.

Most of these antibodies are against surface proteins that are not only often associated with particular cell lineages, but vary in expression with maturation, and thus are referred to as differentiation antigens. For instance, in the diagram below, you can see that CD19 is the earliest B specific protein expressed on B cells, but that it is lost as the activated B cell becomes a plasma cell. HLA-DR is not specific to B cells.

Note that with careful selection of markers you can distinguish different maturational stages of the B lymphocyte.

****2YMS do not need to know all the different antigens. You should recognize CD3, CD5, and CD7 as pan T markers; CD19 as a pan B marker; CD4 as T-helper, and CD8 as T-suppressor.****

For instance, if the cells of a lymphocytosis are positive only for CD19, CD10, and HLA-DR, at what stage of development would you place them?

If they were positive for CD19, CD20, CD21, weak for CIg, and strong for SIg where would you place them?

**** 2YMS do not need to know all the different antigens. You should recognize CD19 as a pan B marker and of course, you should know SIg or CIg indicates a B lymphoid lineage.****

CD2, CD3, CD5, and CD7 are considered "pan" T cell antibodies in that they are present on most normal mature T cells. CD2 and CD3 are the most specific for T cells.

CD5, although strongly associated with T cells, is expressed on a small subset of normal B lymphocytes and on B-chronic lymphocytic leukemia.

CD7, while an excellent marker of T cells, may occasionally be seen on early myeloid cells, especially in leukemia.

CD1 is referred to as the "thymic" marker in that it marks T cells at the narrow window of the cortical thymic stage of differentiation.

**** 2YMS do not need to know all the different antigens. You should recognize CD3, CD5, and CD7 as pan T markers; CD4 as T-helpers, and CD8 as T-suppressors.****

Note that observation of multiple markers allows distinction of different maturational stages of the T lymphocyte.

What is the stage of a population of T lymphocytes positive for CD 7, 2, and 5, but CD3 and CD1 negative?

What if they were CD 7, 2, 5, 3, 4, and 8 positive?

There are few markers specific for myeloid or monocytic cells, nor are current antigenic markers particularly useful for distinguishing between the various subtypes of nonlymphocytic leukemia.

**** 2YMS do not need to know all the different myeloid and monocytic antigens. **** CD13 and CD33 are the two most important.

Myelo/Mono Associated







CD13 and CD33 are the two most useful and specific markers for myelomonocytic cells.

CD14 is also helpful in that it is almost exclusive for monocytic cells.

The following are some of the more useful and commonly used markers not clearly associated with one of the B, T, or myelomonocytic categories.


Glycophorin A


CD45 or the leukocyte common antigen (LCA) is present on most hematopoietic cells. The several different forms of CD45 are a result of alternate splicing of a single gene causing variation of the extracellular region. The intracellular portion is a protein tyrosine phosphatase and is involved in cell activation.

CD10 or the common acute lymphoblastic leukemia antigen (CALLa) is expressed selectively on immature B lymphocytes. CD10 is a zinc metaloprotease which inactivates bradykinin, glucagon, etc.

The CD11 family or leukocyte function antigens (LFA) are linked to CD18 forming intercellular adhesion molecules. CD54 (ICAM-1) is the ligand to CD11a.

CD34 labels stem cells and blasts of varying lineage (B, T, and myeloid cells), making it useful for the I.D. of early cells..

HLA-DR is part of the MHC and is found on B cells, monocytes, and activated T cells.

CD42 (von Willebrand factor receptor) is found on platelets.

Glycophorin A is a marker of erythroid cells.

CD16, CD56, and CD57 are surface receptors associated with natural killer cells (NK), T-NK cells, and cytotoxic T cells.

CD71 is the transferrin receptor found on nucleated red blood cells and reticulocytes, and on activated T and B cells, monocytes, and proliferating cells.

The finding of aberrant antigen expression, ie. the expression or lack of expression of antigens in settings or combinations not normally encountered, is especially valuable as an indicator of abnormal, usually malignant cells.

For example, normal peripheral T lymphocytes express either CD4 or CD8, but not both. The finding of both CD4 and CD8 is abnormal and suggests a thymic stage of maturation as is frequently seen in T-ALL.

Although most common in T-malignancies (75%), B-lymphoid malignancies also aberrantly express antigens. For instance, 15% of B-malignancies have lost one normally expressed antigen; 7% have lost two, and 3% have lost three.

Practice Questions

A 21-year-old man complains of SOB. You discover he has a fever of 101 and pneumonia. His spleen is enlarged and he has several enlarged lymph nodes. His WBC was 15.9 x10 /L with 66% lymphocytes. Flow cytometric immunologic studies were as follows:

















What is the best diagnosis based on the immuno-marker studies of the peripheral blood?

A. c/w reactive viral lymphocytosis
B. c/w pertussis lymphocytosis
C. c/w a pro-B cell ALL
D. c/w a T-cell ALL, loss of usual pan T cell antigen
E. c/w a T-cell ALL, aberrant expression of CD7

A 6-year-old girl is brought to you because of a persistent "cold". No lymphadenopathy or hepatosplenomegaly was noted. Her WBC was 31.0 x10 /L with 75% blasts. The marrow aspirate contained >80% blasts.



















What is the best diagnosis based on the immuno-marker studies of the peripheral blood?

A. c/w a B-cell Chronic Lymphocytic Leukemia
B. c/w a pro-B-cell Acute Lymphoblastic Leukemia
C. c/w a pre-B-cell Acute Lymphoblastic Leukemia
D. c/w Acute Lymphoblastic Leukemia, L3 (Burkitt's)
E. c/w T-cell Acute Lymphoblastic Leukemia


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