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Also known as: Immunophenotyping by Flow Cytometry or Immunohistochemistry
Formal name: Immunophenotyping

At a Glance

Why Get Tested?

To help diagnose and classify a leukemia or lymphoma; to help guide treatment; to detect and evaluate residual cancer cells

When to Get Tested?

When a doctor thinks that you may have 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, or fluid sample collected by your doctor

Test Preparation Needed?


The Test Sample

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. Atypical but characteristic groupings are seen with specific leukemias and lymphomas. This allows immunophenotyping to be useful in helping to diagnose and classify these blood cell cancers.

Leukemias are caused by an abnormal lymphocyte or 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. They accumulate 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 leukemia or lymphoma cells may be released into the blood stream.

CBC (Complete Blood Count) and differential tests performed on a sample of blood from someone with leukemia 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 leukemia, but more information is generally needed to confirm a diagnosis. CBC 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 leukemia 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 analyzed 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 leukemias 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 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 doctor or other trained specialist. 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. Tissue is obtained using a biopsy procedure.

NOTE: If undergoing medical tests makes you or someone you care for anxious, embarrassed, or even difficult to manage, you might consider reading one or more of the following articles: Coping with Test Pain, Discomfort, and Anxiety, Tips on Blood Testing, Tips to Help Children through Their Medical Tests, and Tips to Help the Elderly through Their Medical Tests.

Another article, Follow That Sample, provides a glimpse at the collection and processing of a blood sample and throat culture.

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

No test preparation is needed.

The Test

Common Questions

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Article Sources

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NOTE: This article is based on research that utilizes the sources cited here as well as the collective experience of the Lab Tests Online Editorial Review Board. This article is periodically reviewed by the Editorial Board and may be updated as a result of the review. Any new sources cited will be added to the list and distinguished from the original sources used.

Craig, F. and Foon, K. (2008 April 15). Flow cytometric immunophenotyping for hematologic neoplasms. Blood Journal v111 (8) [On-line information]. Available online at through Accessed April 2011.

Jaffe, E. et. al. (2008 December 1). Classification of lymphoid neoplasms: the microscope as a tool for disease discovery. Blood. 2008 December 1; 112(12): 4384–4399. [On-line information]. Available online at through Accessed April 2011.

(Updated 2011 March 13). Blood Tests. Leukemia & Lymphoma Society [On-line information]. Available online at through Accessed April 2011.

Torpy, J. (2009 January 28). Acute Lymphoblastic Leukemia. JAMA Patient Page V301 (4) [On-line information]. PDF available for download at through Accessed April 2011.

Bahler, D. (Updated 2011 February). Lymphoma Phenotyping. ARUP Consult [On-line information]. Available online at through Accessed April 2011.

(© 1995–2011). Unit Code 3287: Leukemia/Lymphoma Immunophenotyping by Flow Cytometry. Mayo Clinic, Mayo Medical Laboratory [On-line information]. Available online at through Accessed April 2011.

(Reviewed 2010 December). Lymphoid Neoplasms Laboratory Support of Diagnosis and Management Test Guide. Quest Diagnostics [On-line information]. Available online at through Accessed April 2011.

Wittwera, C. and Brown, M. (2000). Flow Cytometry: Principles and Clinical Applications in Hematology Clinical Chemistry 46:8(B) 1221–1229 [On-line information]. Available online at through Accessed April 2011.

Mayo Clinic Staff (2010 November 24). Chronic lymphocytic leukemia. MayoClinic [On-line information]. Available online at through Accessed April 2011.

Acute Leukemia. Merck Manual for Healthcare Professionals [On-line information]. Available online at through Accessed April 2011.

Pagana, K. D. & Pagana, T. J. (© 2011). Mosby's Diagnostic and Laboratory Test Reference 10th Edition: Mosby, Inc., Saint Louis, MO. Pp 244-247.

Wu, A. (© 2006). Tietz Clinical Guide to Laboratory Tests, 4th Edition: Saunders Elsevier, St. Louis, MO. Pp 1633-1711.

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