Immunophenotyping by Flow Cytometry

Flow cytometry immunophenotyping is used primarily to help diagnose and classify blood cell cancers (leukemias and lymphomas) and to help guide their treatment. It may be used in follow up to a complete blood count (CBC) and WBC differential that show an increased number of lymphocytes or the presence of immature blood cells or other abnormal cell counts.

Flow cytometry immunophenotyping may also be used:

• To predict how aggressive the cancer will be
• To predict whether the cancer will respond to certain treatment
• To help determine whether treatment of leukemia or lymphoma has been successful
• To determine whether the disease remains despite treatment (residual disease) or has come back after successful treatment (recurrent disease)

There are some other uses of this testing that are less common, but they are not addressed in this article.

Flow cytometry immunophenotyping may be ordered when you have an increased number of lymphocytes (or sometimes an increase in another type of white blood cell, WBC), anemia, a decreased platelet count, or immature WBCs that are not normally seen in the blood. These may be the first indication of a possible blood cell cancer.

Testing may be done when you have signs and symptoms of leukemia and lymphoma, though they may be unremarkable, mild, or nonspecific early in the disease.

Examples of signs and symptoms of a blood cell cancer include:

• Feeling tired or rundown, weakness
• Unexplained loss of weight or appetite
• Shortness of breath during normal physical activity
• Pale skin
• Bleeding or bruising easily
• Fever
• Bone and joint pain
• Enlarged lymph nodes, spleen, liver, kidneys, and/or testicles
• Headaches
• Vomiting
• Night sweats

Testing may also be ordered after you have been treated for leukemia or lymphoma.

A pathologist, often one specializing in the study of blood diseases and/or blood cell cancers (a hematopathologist), will consider the results from the complete blood count (CBC), differential, blood smear, bone marrow findings, and flow cytometry 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 (antigens) that are present on the cells as detected by flow cytometry immunophenotyping will help characterize the cells present. A normal cell will display a pattern of antigens that correlates with the type and maturity of the cell. (Blood cells normally mature in the bone marrow and are released into circulation when they are mature or nearly mature.) The results from your immunophenotyping are compared to the pattern of antigens for “normal” cells as well as to patterns that are associated with abnormal cells (e.g., cells present with leukemias and lymphomas).

Your health care practitioner will consider the flow cytometry immunophenotyping results together with your clinical history, physical examination, signs and symptoms, as well as all laboratory tests to help make a diagnosis.

Each person’s condition will be unique. You may have (or lack) certain antigens that are typically seen, yet you may still be diagnosed with a specific type of leukemia or lymphoma.

Here is one example:

• The blood of an older child or adult normally contains some mature B cells, but circulating immature B cells are not normally present.
• Both mature and immature B cells are normally positive for the CD19 marker.
• Mature B cells are normally positive for CD20 but not CD34. CD20 is a marker of maturity and CD34 is a marker of immaturity. In fact, these two markers are not normally expressed together.
• Now, if an adult has a small number of mature B cells but also has a large number of immature B cells which are positive for CD19 (remember, CD19 is a B-cell marker) and also positive for both CD34 and CD20 (which identifies those cells are both immature and abnormal), then the person has an immature B-cell leukemia known as B-lymphoblastic leukemia.
• Interestingly, some of the other antigens present might suggest a specific genetic subtype of B-lymphoblastic leukemia, which also might have a certain prognosis.
• More importantly, there are newer classes of treatment options like CAR-T therapy, bispecific T-cell engagers, and monoclonal antibodies that selectively target molecules like CD19 or CD20. These newer treatments may have reduced side effects compared to conventional chemotherapy (newer targeted therapies are usually added to traditional chemotherapy).

Abnormal immunophenotype profiles are usually present in:

• Acute myeloid leukemia
• Acute lymphoblastic leukemia (ALL)
• Chronic lymphocytic leukemia (CLL)
• B-cell and T-cell non-Hodgkin lymphomas
• Multiple myeloma

The following summarizes markers that are often expressed in certain types of cells:

The following summarizes markers that suggest certain types of cell differentiation:

T-lymphocyte subset analysis based on CD3, CD4 and CD8 expression is performed separately to monitor people with HIV/AIDS, for example.

The type of sample to be tested is up to your healthcare practitioner and must be representative of your cancer. If abnormal cells are present in the bloodstream, a blood sample is often used for flow cytometry immunophenotyping as it is easy to obtain and less invasive than other collection methods. However, lymphoma cells may or may not find their way to the bloodstream and might require other collection techniques.

It depends. It is not offered in every laboratory, but many larger hospitals and academic medical centers perform the testing or your sample may be sent to a reference laboratory.

Diagnosis of leukemia or lymphoma is based on the visual examination of a blood smear and/or bone marrow biopsy and aspiration for the presence of certain cell types. Depending upon flow cytometry immunophenotyping results, a healthcare practitioner may determine how likely your cancer will respond to treatment and how aggressive the treatment might be. The course of treatment for your cancer will be determined by your health care practitioner and their team based on flow cytometry immunophenotyping and other tests that might be performed.

It depends. The antigens on specific leukemia or lymphoma cells may remain the same over time. However, treatment with chemotherapy may eliminate the abnormal cells, and if treatment is successful, normal white blood cells (WBCs) will replace abnormal cells. Because of this, immunophenotyping results will be different by reflecting the current population of WBCs that would be present in an individual in remission. Sometimes, however, the cancer cells adapt to evade the therapy by not expressing anymore an antigen that they expressed earlier, which might have been targeted by a monoclonal antibody or other therapy, like CAR T-cells.

Originally, glass slides with fixed tissue sections were treated with an antibody that was specific for a type of antigen typically found on certain abnormal cells associated with a particular leukemia or lymphoma. These antibodies were often linked with a fluorescent or a chemical indicator that would make these abnormal cells visible when observed under a microscope. This approach, called immunohistochemistry, is used every day for some leukemia and lymphoma markers and other types of cancer. It may be because the markers of interest are not available for flow cytometry or because fresh cells or tissue are not available (a requirement for flow cytometry immunophenotyping).