November 2018


By Dr. Nora Springer

Immunophenotyping is a technique of identifying cell lineage by utilizing antibodies that detect cell antigens or markers. The pattern of marker expression is unique to specific cell lineages, similar to a fingerprint. Most of the markers we evaluate are expressed on the cell surface, but they can also be expressed in the cytoplasm or nucleus of the cell. In clinical settings, immunophenotyping is frequently performed to identify cells of hematopoietic origin but can be used for numerous research applications.

At KSVDL we perform immunophenotyping of blood, bone marrow, body cavity fluids (peritoneal, pleural) and solid tissue aspirates, such as lymph nodes. This test is currently validated and available for samples from dogs, cats, and horses.

Immunophenotyping via flow cytometry is typically used for the following purposes:

  • Lymphoma prognostication: Immunophenotyping is used to differentiate B-cell from T-cell lymphoma, which have prognostic implications. Additionally, we can detect other indicators of prognosis via the lymph node immunophenotype panel at KSVDL. For example, low MHC class II molecule expression has been associated with a worse clinical prognosis in peripheral B cell lymphoma. T helper phenotype (CD4+) has been associated with a poorer prognosis in peripheral T cell lymphoma. Small cell T cell lymphoma lacking the pan-leukocyte marker (CD45) is consistent with T-zone lymphoma, an indolent lymphoma with good prognosis.
  • Differentiating lymphoma from acute leukemia: Expression patterns of the stem cell marker CD34 and MHC class II molecules can help differentiate a Stage V lymphoma from acute leukemia. This differentiation is essential as acute leukemia of either myeloid or lymphoid origin has a grave prognosis.
  • Differentiating reactive from neoplastic lymphocyte populations: In cases of lymphocytosis, a uniform phenotype would be supportive of neoplasia. Aberrant expression of cell surface molecules or lack of expression of an expected cell surface molecule would also be supportive of a neoplastic population.

Immunophenotyping by flow cytometry is a powerful diagnostic tool but the results should not be interpreted in isolation.Since the cells are not visualized during the procedure, we need to assess a cytology smear or blood smear of the submitted fluid to evaluate the morphology of the population of interest. Often, this step has been completed prior to flow cytometry as a KSVDL Clinical Pathologist may recommend immunophenotyping as an adjunctive test due to cytology or hematology findings.

Please include the relevant KSVDL accession number on the Immunophenotyping request form. Other helpful information is signalment, clinical signs, history, imaging finding, and additional pertinent diagnostic test results.

Sample collection and handling for immunophenotyping:

For peripheral blood or body cavity immunophenotyping, 2-3mL of whole blood or effusion in EDTA is required. This sample should be shipped overnight on a cold pack. It is important that the sample is analyzed within 48 hours of collection.

For lymph node aspirates, a minimum of 2-3 and up to 6-8 needle aspirate biopsies should be collected into sterile saline. Using a 22 guage needle, redirect several times in the affected lymph node. Attach to a 3mL syringe containing 1mL of sterile saline. Briefly puncture a red top tube (without clot activator) allowing just enough saline to pass through the needle to flush out the collected cells. Repeat the procedure until the entire 1mL of saline has been used, and is collected in the red top tube. Ship aspirated lymph node material overnight on a cold pack. If the lymph node sample cannot be shipped overnight, it can be stabilized for 2-day shipping by adding 10% autologous serum from the patient. Most lymph node aspirates retain acceptable viability if analyzed within 48 hours of collection.

Before application of the antibodies for flow cytometry, we check the sample for sufficient preservation (viability) and number of cells in question. If there are insufficient cells or the cells are not viable, we will not proceed with the full flow cytometric evaluation and you will be contacted by KSVDL client care to inform you the test has been canceled.

Importantly, immunophenotyping is only performed Monday through Friday. Please do not ship samples to KSVDL on Fridays as they will not be viable for analysis the following Monday.

The clinical pathology and clinical immunology team at KSVDL is here to help. Immunophenotyping can play a major role for managing challenging and confusing cases of hematopoietic neoplasia. Speaking to a clinical pathologist or clinical immunology staff member prior to selecting tests and submitting samples can help guide your choices and maximize your diagnostic results. Our goal is to give you the best service possible and assist you in your practice of veterinary medicine.

Nora Springer, DVM, Diplomate ACVP is the Director of Clinical Immunology at the Kansas State Veterinary Diagnostic Laboratory.

For more information, please contact KSVDL Client Care at 866-512-5650 or

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