Immunophenotyping

Also Known As

Immunophenotyping by Flow Cytometry or Immunohistochemistry

Formal Name

Immunophenotyping

This article was last reviewed on

December 29, 2014.

This article waslast modified on December 30, 2017.

At a Glance

Why Get Tested?

To help diagnose and classify a leukemia or lymphoma; to help guide treatment; to detect and evaluate leukocyte cancer cells that remain after treatment or disease relapse

When To Get Tested?

When you have signs and symptoms that a health practitioner thinks may be due to leukemia or lymphoma; when you have been diagnosed with leukemia or lymphoma but the specific subtype is unknown; sometimes to evaluate the effectiveness of treatment or to evaluate for recurrent disease

Sample Required?

A blood sample drawn from a vein in your arm; sometimes a bone marrow, tissue biopsy, or fluid sample collected by a health practitioner

Test Preparation Needed?

None

You may be able to find your test results on your laboratory's website or patient portal. However, you are currently at Lab Tests. You may have been directed here by your lab's website in order to provide you with background information about the test(s) you had performed. You will need to return to your lab's website or portal, or contact your healthcare practitioner in order to obtain your test results.

This Website is an award-winning patient education website offering information on laboratory tests. The content on the site, which has been reviewed by laboratory scientists and other medical professionals, provides general explanations of what results might mean for each test listed on the site, such as what a high or low value might suggest to your healthcare practitioner about your health or medical condition.

The reference ranges for your tests can be found on your laboratory report. They are typically found to the right of your results.

If you do not have your lab report, consult your healthcare provider or the laboratory that performed the test(s) to obtain the reference range.

Laboratory test results are not meaningful by themselves. Their meaning comes from comparison to reference ranges. Reference ranges are the values expected for a healthy person. They are sometimes called "normal" values. By comparing your test results with reference values, you and your healthcare provider can see if any of your test results fall outside the range of expected values. Values that are outside expected ranges can provide clues to help identify possible conditions or diseases.

While accuracy of laboratory testing has significantly evolved over the past few decades, some lab-to-lab variability can occur due to differences in testing equipment, chemical reagents, and techniques. This is a reason why so few reference ranges are provided on this site. It is important to know that you must use the range supplied by the laboratory that performed your test to evaluate whether your results are "within normal limits."

For more information, please read the article Reference Ranges and What They Mean.

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. Typical groupings of antigens are present on normal WBCs and are unique to specific cell types. Atypical but characteristic groupings are seen with leukemias and lymphomas. This allows immunophenotyping to be useful in helping to diagnose and classify these blood cell cancers.

Leukemias and lymphomas are caused by an abnormal cell that begins to clone itself uncontrollably. The abnormal lymphocytes or myeloid (granular) monoclonal cells proliferate, yet do not fight infections or perform other functions like normal WBCs. Because they do not die at a normal rate, they accumulate in the bone marrow, in a lymph node, or in other tissues, where they grow in numbers. As the number of cells increases in the bone marrow, they may crowd out and inhibit the production of normal red and white blood cells. Eventually, the abnormal cells will also be released into the bloodstream.

CBC (complete blood count) and differential tests performed on a sample of blood from someone with leukemia or a lymphoma may reveal an increased number of white blood cells with a predominance of one type. These tests may suggest lymphoma or leukemia, but more information is generally needed to confirm a diagnosis. CBC and differential testing cannot confirm the presence of monoclonal WBCs or detect the subtle differences that may exist between different types of blood cell cancers.

With immunophenotyping, a blood, bone marrow, or other tissue sample can be tested to gather this information – information that is then used to identify a specific type of leukemia or lymphoma and, where possible, used to predict its likely aggressiveness and/or responsiveness to certain treatment. The identification of different types of leukemias and lymphomas is based upon the presence or absence of antigens and a typical pattern that has been established with each leukemia/lymphoma.

Immunophenotyping detects the presence or absence of antigens found on the surface or interior of blood cells. Atypical or abnormal cells can demonstrate characteristic antigen groupings that are consistent with specific types of leukemia and lymphoma. The identifications 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 (see the table in "What does the test result mean?"). CD numbers represent a naming convention that is based upon international consensus. While hundreds of antigens have been identified and have received a unique CD number, only a small number of these are routinely used.

How is the sample collected for testing?

A blood sample is obtained by inserting a needle into a vein in the arm. A bone marrow aspiration and/or biopsy procedure is performed by a trained health practitioner. Fluid samples are obtained through collection of the fluid in a container or by inserting a needle into the body cavity and aspirating a portion of the fluid with a syringe. Sometimes, a tissue sample, such as from a lymph node, is obtained using a biopsy procedure.

Is any test preparation needed to ensure the quality of the sample?

No test preparation is needed.

Common Questions

How is it used?

Immunophenotyping is used primarily to help diagnose and classify the blood cell cancers, leukemias and lymphomas, and to help guide their treatment. It may be ordered as a follow-up test when a complete blood count (CBC) and differential show an increased number of lymphocytes and the presence of immature blood cells or when there is a significant increase or decrease in the number of platelets (thrombocytosis or thrombocytopenia).

Samples are analyzed using various panels of antibodies that have been established for various types of leukemia or lymphoma. For example, each of these cancers would have a pre-defined panel of antibodies that would be consistent with their diagnosis: acute lymphoblastic leukemia, acute myeloid leukemia, hairy cell leukemia, erythroleukemia, B-cell lymphoma, or T-cell lymphoma.

Typically, a health practitioner will provide information about an individual who they suspect has leukemia or lymphoma. Basic testing of a CBC, differential, and platelet count would be performed in addition to immunophenotyping. The antigen selection, or panel, is made based upon that information.

Testing may sometimes be performed to evaluate the effectiveness of leukemia or lymphoma treatment and to detect residual or recurrent disease by observing the continued presence of abnormal cells.

See below for detailed information on methods used for immunophenotyping.
When is it ordered?
Immunophenotyping may be ordered when a person has an increased number of lymphocytes (or sometimes an increase in another type of white blood cell (WBC)), an increased or decreased platelet count, or has immature WBCs that are not normally seen in blood. These are usually findings from a complete blood count (CBC) and differential and may be the first indication that a person might have a blood cell cancer. Symptoms of early leukemia and lymphoma may be unremarkable, mild, or nonspecific.

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 when a person has been treated for a leukemia or lymphoma to evaluate the effectiveness of treatment and detect residual or recurrent disease.

What does the test result mean?

The presence of certain antigens that are identified by immunophenotyping require expertise to interpret. 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 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 characterize the abnormal cells present (if any). 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" results and to known antigen associations with leukemias and lymphomas, each person's condition will also be unique. A person may have (or lack) certain antigens that are typically seen, yet still be diagnosed with a specific type of leukemia or lymphoma.

Abnormal immunophenotype profiles are usually present in: acute myelogenous leukemia (or acute myeloid leukemia), acute lymphoblastic leukemia, chronic lymphocytic or myelocytic leukemias, B-cell and T-cell non-Hodgkin lymphomas, erythroleukemia (RBC leukemia), megaloblastic leukemia (platelets), and multiple myeloma.

Markers that are often expressed in certain type of cells:

Cell	Markers
Immature precursor cells	HLA-DR, TdT, CD34, CD38, CD117
B-lymphocytes	CD19, CD20, CD22, CD79a, immunoglobulin heavy (gamma, alpha, mu or delta) and light chains (kappa or lambda) CD10 (pre-B cell)
T-lymphocytes	CD2, CD3, CD5, CD7, and either CD4 or CD8
Myeloid cells (granulocytes)	MPO (myeloperoxidase), CD11, CD13, CD15, CD16b, CD33, CD66
Natural killer (NK) cells	CD11b, CD16, CD56

Markers that suggest certain types of cell differentiation:

Cell	Markers
Megakaryocytic differentiation; Platelets	CD31, CD36, CD41, CD42, CD61
Red blood cell (erythroid) differentiation	CD235a
Monocytic differentiation	CD14, CD33, CD64, CD68
Hairy cell leukemia	CD11c, CD103

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 and CD8.
Is there a reason to choose one type of sample (blood, bone marrow, or tissue) over another for testing?

Which sample to be tested is up to your health practitioner and must be representative of your cancer. If abnormal cells are present in the bloodstream, a blood sample is often used for 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 would require other collection techniques.
Can immunophenotyping be done in my doctor's office?

No. The test requires specialized 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?

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 the pattern of antigens present and their established association with specific cancers, a health practitioner may determine how likely a cancer will respond to treatment and how aggressive the treatment might be. The course of treatment for that cancer will be determined by the health practitioner and their team based on immunophenotyping and other tests that might be performed.
Will my antigens change?

The antigens on specific monoclonal cancer cells will generally remain the same; however, treatment with chemotherapy and/or radiation will eliminate the abnormal cells. 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.
What are some methods used for immunophenotyping?

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 fluorescence or a peroxidase indicator that would make these abnormal cells visible when observed under a microscope. Immunohistochemistry is based upon immunologic cellular properties and has proven to be particularly valuable in evaluating tissue samples that help in establishing a diagnosis or identifying relapse.

Another technique is flow cytometry and is performed by processing a blood, bone marrow, tissue, or fluid sample by adding specific antibodies that have been tagged with fluorescent markers. These antibodies will bind to corresponding antigens on the white blood cells (WBCs), if present, and are often referred to as cell markers. The WBCs are suspended in a physiologic solution and passed through a flow cytometer. The cell suspension is forced through a fluid stream that passes multiple laser beams causing deflection or absorption of the laser light. These light changes are identified by very sensitive detectors that analyze individual cells based on various physical properties.

The flow cytometer rapidly measures characteristics about each cell, such as its size and granularity (internal cellular structures), and evaluates the type and quantity of fluorescent antigen-antibody complexes that are present. The advantage of flow cytometry over Immunohistochemistry is that thousands of cells are evaluated during the test. Based on the physical characteristics of the abnormal cells and the presence (or absence) of fluorescence, the investigator can quickly determine the type of leukemia or lymphoma that may be present.

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