What is being tested?

Immunophenotyping detects the presence or absence of white blood cell (WBC) antigens. These antigens are protein structures found on the surface or interior of WBCs and are unique to specific cell types and stages of cell maturation. Atypical but characteristic groupings are seen with specific leukaemias and lymphomas. This allows immunophenotyping to be useful in helping to diagnose and classify these blood cell cancers.

Leukaemias are caused by an abnormal lymphocyte myeloid (granular) WBC that begins to clone itself; lymphomas by an abnormal lymphocyte. The monoclonal cells produced do not fight infections like normal WBCs, and they do not die at a normal rate thus accumulating in the bone marrow or in the lymph node where they originated. As the number of WBC clones increases, they may crowd out and inhibit the production of normal red and white blood cells and the leukaemia or lymphoma cells may be released into the blood stream.

FBC (Full Blood Count) and WBC differential tests performed on a sample of blood from someone with leukaemia or lymphoma will usually reveal an increased number of white blood cells with a predominance of one type of WBC. These tests may suggest lymphoma or leukaemia, but more information is generally needed to confirm a diagnosis. FBC and differential testing often cannot confirm monoclonal WBCs or detect the subtle differences that may exist between different types of blood cell cancers and they cannot distinguish between the different types of lymphocytes.

With immunophenotyping, a blood, bone marrow, or other sample can be tested to gather this information – information that is then used to identify a specific type of leukaemia or lymphoma and, where possible, used to predict its likely aggressiveness and/or responsiveness to certain treatment. The identifications and predictions made are based upon a "library" of antigen associations and patterns that have been established over time.

Most of the antigens that immunophenotyping detects are identified by a CD (clusters of differentiation or cluster designation) number, such as: CD1a, CD2, CD3, CD4, CD8, CD13, CD19, CD20, CD33, CD61, or CD235. CD numbers represent a naming convention that is based upon international consensus. Several hundred antigens have been identified and received CD designations, but only a small number of these are routinely tested for clinical use.

Traditionally, immunophenotyping was performed using immunohistochemistry; applying stains to cells that were fixed on a glass slide and then evaluating them under the microscope. While this technique is still used, most immunophenotyping is now performed using flow cytometry.

Flow cytometry is performed by processing a blood, bone marrow, tissue, or fluid sample and then adding specific antibodies that have been tagged with fluorescent markers. These antibodies attach to corresponding antigens on the white blood cells, when the antigens are present. The WBCs are then sent in a fluid stream past multiple lasers and detectors and each cell is analysed individually.

The flow cytometer rapidly measures characteristics about each cell, such as its size and granularity, and evaluates the type and quantity of fluorescent antigen-antibody complexes that are present. Thousands of cells are evaluated during the test. Results are then graphed and compared to "normal" results and to patterns that are known to be associated with different leukaemias and lymphomas. This process allows the person interpreting the test results to determine the types of WBCs present, their maturity, and to determine the types and quantities of antigens on or in these cells.

How is it used?

Immunophenotyping is ordered primarily to help diagnose and classify blood cell cancers (leukaemias and lymphomas). It may be ordered as a follow-up test, when a FBC and differential show an increased number of lymphocytes and the presence of immature WBCs or when there is a significant increase or decrease in the number of platelets (thrombocytosis or thrombocytopenia). Testing is most often performed on blood and/or bone marrow samples, but may also be done on body fluids or other biopsy tissue samples.

With immunophenotyping, testing proceeds from the general to the specific. Samples are analysed for panels or groups of specific antigens, and then, based upon the initial findings, additional antigens are analysed as deemed necessary. The number of antigens in a panel will vary from laboratory to laboratory, and the specific panel ordered will depend upon the person's clinical findings and the doctor's suspicions. Typically, a doctor will provide information about the person to the laboratory and antigen selection is made based upon that information. An example panel that could be ordered when a B-cell cancer is suspected might include: CD5, CD10, CD19, CD20, CD22, CD23, CD34, Kappa and Lambda (to determine whether the cells are monoclonal) and may include other antigens such as CD79a, TdT (terminal deoxynucleotidyl transferase) and CD103.

Testing may be performed to evaluate the effectiveness of leukaemia or lymphoma treatment. Either to detect the continued presence of abnormal cells (residual disease) or to detect if it has come back following successful treatment (recurrent disease).

When is it requested?

Immunophenotyping is ordered when a person has an increased number of lymphocytes (or sometimes an increase in another type of WBC, especially in the immature state), an increased or decreased platelet count, or has immature WBCs that are not normally seen in blood. These are usually findings from a FBC and differential, and they may be the first indication that a person might have a blood cell cancer - as symptoms of early leukaemia and lymphoma may be absent, mild, or nonspecific.

Testing may also be ordered when a person has been treated for a leukaemia or lymphoma to evaluate the effectiveness of treatment and detect residual or recurrent disease.

What does the result mean?

The patterns of antigens that are produced through immunophenotyping require expertise to interpret. A pathologist, often one specialising in the study of blood diseases and/or blood cell cancers, will consider the results from the FBC, differential, blood film, bone marrow findings, and immunophenotyping as well as other tests in order to provide a diagnostic interpretation. A laboratory report will typically include specific results from the tests as well as an analysis of what those results mean.

The markers that are present on the cells as detected by immunophenotyping will help characterise the abnormal cells present (if any) as being, for example, B-lymphocytes or T-lymphocytes. A normal cell will display a pattern of markers that correlates with the type and maturity of the cell. This information is considered together with the affected person's clinical history, physical examination, signs and symptoms as well as all laboratory tests to help make a diagnosis.

It must be kept in mind that while findings represent comparisons to "normal" findings and to known associations with leukaemias and lymphomas, each person's condition will also be unique. A person may have (or lack) antigens that are typically seen and still be diagnosed with a specific type of leukaemia or lymphoma.

Abnormal immunophenotype profiles are usually present in: acute myelogenous leukaemia (or acute myeloid leukaemia), acute lymphoblastic leukaemia, B-cell and T-cell non-Hodgkin lymphomas and multiple myeloma.

Markers that are often expressed in certain type of cells:

Markers that suggest certain types of cell differentiation:

Is there anything else I should know?

T-lymphocyte subset analysis based on CD3, CD4 and CD8 expression is performed separately to monitor people with HIV/AIDS. For more on this, see the article on CD4

Common questions

• Is there a reason to choose one type of sample over another for testing?

Which sample is tested is up to you and your doctor, but it must be representative of your cancer. If abnormal cells are present in the blood stream, a blood sample is often used for immunophenotyping as it is easy to obtain and less invasive than other collection methods.

• Can immunophenotyping be done in my doctor's office?

No. The test requires highly specialised equipment and expertise in interpretation. It is not offered in every laboratory, but many larger hospitals perform the testing or your sample may be sent to a reference laboratory.

• Can results of testing be used to determine the course of my cancer?

Only in a general way; depending upon the pattern of antigens present and their associations, a doctor may determine that a cancer is likely to be more aggressive or more resistant to treatment. Each person, their cancer, and the course of that cancer will be unique. New techniques examining protein expression may become more common. It is possible that these will allow determination of treatment types, though this is only used in research settings at present.

• Will my antigens change?

The antigens on specific monoclonal cancer cells will generally remain the same, but the overall population of white blood cells (WBCs) is constantly being renewed and replaced. Because of this, immunophenotyping results will always be slightly different – they will reflect the current population of WBCs.

More information

RCPA Manual: Flow cytometry

RCPA Manual: Lymphocyte immunophenotyping

RCPA Manual: Cell immunophenotyping