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  Oct 19, 2018

What is Immunophenotyping?

Immunophenotyping is a test used to identify cells on the basis of the types of markers or antigens present on the cell’s surface, nucleus, or cytoplasm. This technique helps identify the lineage of cells using antibodies that detect markers or antigens on the cells, hence the “immuno-” prefix.

While some antigens are found only on one type of cell, others are found on different types. This process is widely used to diagnose different types of lymphoma and leukemia by comparing normal cells and cancer cells. It has become a common technique for the identification and classification of acute leukemias, particularly acute myeloid leukemia (AML).

Uses of Immunophenotyping

Immunophenotyping is widely used for the following reasons:

  • To differentiate between:
    • Acute myeloid and lymphoid leukemia
    • B and T cell lymphoid neoplasms such as chronic lymphocytic leukemia and lymphoma
    • Reactive and neoplastic expansions of lymphocytes
  • Predicting prognosis in lymphoma
  • Identification of lymphocyte subsets

Immunophenotyping Tests

Two types of tests are used in immunophenotyping:

  • Flow cytometry
  • Immunocytochemistry

The choice of test is based on the type of sample:

  • Fluid suspensions (sample): flow cytometry (test method)
  • Cells on slides (sample): immunocytochemistry (test method)

Here’s a brief overview of the two types of test methods:

Flow Cytometry

In flow cytometry, the sample may range from blood, fluids in the body cavity (such as peritoneal or pleural fluids), bone marrow, or solid tissues in liquid media. Flow cytometry is generally used to determine cell lineage in leukemia and lymphoma.

Flow lymphoma is used in the case of lymphoid neoplasms or when a lymphoid origin is suspected on the basis of cell morphology after staining. This technique helps in prognostication and is also used to differentiate between neoplastic and reactive expansions of lymphocytes.

Flow leukemia can be used in the case of an extensive range of leukemias that could be myeloid or lymphoid.


This technique involves immunostaining of smears of fluids from body cavities or aspirates of tissues. It can be used for identifying the lineage of the cell in smears of tissues with suspected lymphoma or histocytic sarcoma.

This technique also helps identify or confirm the cell of origin in non-hematopoietic neoplasia. Immunocytochemistry is, however, limited by the quality and number of smears as one antibody is applied to one smear.

The results of flow cytometry or immunocytochemistry should always be interpreted along with the available medical history, clinical signs, imaging findings, and pathologic results of individual cases.

Immunophenotyping in AML

Immunophenotyping is widely used to identify and classify AML. Immunophenotype is a key parameter that is very valuable in predicting response to treatment as well as survival rates.

Several studies have identified a relationship between AML prognosis and antigens such as CD7, CD9, CD11b, CD13, CD14, CD15, CD33, CD34, and CD56, though some other studies report conflicting results.

Although the World Health Organization classification of AML takes into account immunophenotypic features, the criteria for the same in monocytic AML is not clearly defined.

According to the European Group for the Immunological Classification of Leukemias (EGIL), AML can be immunologically defined by the expression of atleast two of the following myeloid markers:

  • CD13
  • CD33
  • CDw65
  • CD117
  • Myeloperoxidase

Based on this classification, one study researched the prognostic significance of various immunophenotypic subgroups in 177 adult AML patients. The results of this study were compared with other clinical and biological features. None of the tested antigens were linked to treatment outcome.

Patients with full expression of panmyeloid phenotype expressed all five myeloid markers, had a higher complete remission rate, and were significantly different in overall and disease-free survival than those whose expressed <5 of the myeloid markers. Leukemic myeloblasts expressed many leukocyte differentiation antigens, thus reflecting association with myeloid lineage and maturation level.