Animal Health Diagnostic Center

Sign in | Register

Clinical Pathology  

Clinical Pathology


Clinical Pathology

Immunophenotyping Tests

General Information

Immunophenotyping is a term applied to the technique of identifying the specific lineage of cells through the use of antibodies that detect antigens or markers on the cell (hence the prefix “immuno”). The antigen can be expressed on the cell surface, in the cytoplasm or the nucleus of the cells. Some antigens are lineage-specific (found only on one type of cell) whereas others can be found on various cells. Few antibodies are completely lineage-specific. The term “immunophenotyping” is most frequently used in reference to phenotyping hematopoietic cells but this technique can be and is used to identify cells of non-hematopoietic origin.

Immunophenotyping is typically used for the following conditions involving hematopoietic cells:

  • Differentiation between B and T cell lymphoid neoplasms, i.e. lymphoma, chronic lymphocytic leukemia
  • Prognostication in lymphoma, e.g. a T helper phenotype (CD4-positive) has been associated with a poorer prognosis in peripheral T cell lymphoma, decreased MHCII expression has been associated with a poorer prognosis in peripheral B cell lymphoma
  • Differentiation between acute myeloid and lymphoid leukemia
  • Differentiation between reactive and neoplastic expansions of lymphocytes
  • Identification of specific subsets of lymphocytes, e.g. helper:cytotoxic T cell ratio

back to top

Tests

The type of test we use for immunophenotyping (flow cytometry or immunocytochemistry) depends on the sample type, i.e. whether it consists of suspended cells in fluid samples or cells adhered to slides.

  • For suspended cells, we generally use flow cytometry.
  • For slides, we can only perform immunocytochemistry and are limited by the number and quality of slides.

Please refer to our sample submission page on tips on collection and submission of samples for these tests. Also, please call the laboratory if unsure of which test to request.

back to top

Flow cytometry: This is performed on blood, bone marrow, body cavity fluids (peritoneal, pleural), and aspirates of solid tissues (e.g. lymph node) placed into liquid media. This is generally used for:

  1. Determination of cell lineage in lymphoma and leukemia
    We offer two flow panels in dogs for this purpose. We only offer a single flow panel for cats, due to fewer available antibodies (see table below).

    • Flow lymphoma: We recommend this more limited panel for lymphoid neoplasms (lymphoma, lymphocytic leukemia) or when a lymphoid origin is suspected for a leukemia based on morphologic features of the cells in Wright’s-stained smears.  This panel would also be used for prognostication in these neoplasms.

    • Flow leukemia: We recommend this more extensive panel on leukemias of uncertain lineage in dogs, i.e. the leukemia could be lymphoid or myeloid.

  1. Differentiation between reactive and neoplastic expansions of lymphocytes
    We recommend the flow lymphoma panel for this purpose, e.g. differentiating thymoma from lymphoma in an aspirate from a mediastinal mass or a reactive from a neoplastic lymphocytosis in blood (Figures 1 and 2).

    • A uniform phenotype would be supportive of neoplasia, particularly if combined with evidence of clonality (via PARR testing).

    • Aberrant phenotypes are more conclusive of neoplasia.

  1. Determination of lymphocyte subsets. This is usually done for research purposes for which we create customized panels.

Peripheral blood dog with marked lymphocytosis Dotplot of fluorescence staining for CD3 and CD8
Figure 1: Representative image of peripheral blood from a dog with a marked lymphocytosis (60,000 lymphocytes/uL). The lymphocytes were small with clumped nuclear chromatin and small amounts of light blue cytoplasm containing red cytoplasmic granules (arrows). Morphologic findings were compatible with a diagnosis of a leukemia of granular lymphocytes. A neutrophil and a monocyte are also present in this field (Wright’s stain, 1000x magnification). The dog had a concurrent neutrophilia but was not anemic or thrombocytopenic. Figure 2: Dotplot of fluorescence staining for CD3 (T cell marker) and CD8 (cytotoxic T cell marker) of the lymphocytes in question from Figure 1.  Most of the lymphocytes are double positive (*, upper right quadrant) for CD3 and CD8, indicating a leukemia of cytotoxic T cells. This is the most common phenotype of this type of leukemia, which usually arises in the spleen and behaves in an indolent fashion in dogs. A lymphocytosis of these T cells can be seen in dogs with Ehrlichia canis infection, however usually not to this degree. The dog was negative for Ehrlichia canis on serologic testing.

 

back to top

Immunocytochemistry: This involves immunostaining of smears prepared from tissue aspirates or body cavity fluids. We use this technique for identifying the cell of origin in smears prepared from aspirates of tissues with suspected hematopoietic neoplasia (e.g. lymphoma, histocytic sarcoma) (Figures 3 and 4). It also can be used for confirming or identifying the cell of origin with non-hematopoietic neoplasia (see table below). Since one antibody is applied to one smear, the main limitations to this technique are the number and quality of the smears. Examination of a Wright’s-stained smear is an essential component to interpreting these samples. Please call the laboratory if you need help determining which test to perform on these samples.

Photomicrograph of cytospin smears Immunostaining for Pax-5 and CD3
Figure 3: Representative photomicrograph of cytospin smears prepared from an aspirate of a popliteal lymph node from a dog with marked peripheral lymphadenopathy  (the aspirate was placed in physiologic saline with some added serum). Intermediate lymphocytes with deeply cleaved nuclei, fine chromatin and no nucleoli dominate, with low numbers of small lymphocytes (arrow) (Wright’s stain, 1000x magnification) Figure 4: Immunostaining for Pax-5 (B cell marker) and CD3 (T cell marker) was performed on the cytospin smears prepared from the aspirate. The small lymphocytes were positive for Pax-5 whereas the intermediate lymphocytes (the dominant population) were positive for CD3 (brown membraneous and cytoplasmic staining).  The results were diagnostic for a T cell lymphoma. (1000x magnification)

 

back to top

Results

Results are generally available 48 hours from the day of analysis. We are routinely performing flow cytometry on Tuesday and Thursday and immunocytochemistry on Friday. We provide a complete cytology report (description and interpretation) and also a pdf of our analysis of the flow cytometric results. Interpretation will be hampered by sample deterioration with storage (fresh is best!!) or if samples are poorly cellular.

Note, that we never interpret results of flow cytometry or immunocytochemistry in isolation.  We always use the provided signalment, clinical signs, history, imaging findings, and clinical pathologic results to help with our interpretation. It is imperative that we have Wright’s-stained smears of the submitted sample to evaluate concurrently so that we can use the morphologic features of the cells, along with their immunophenotyping results, to make the best diagnosis.  For this reason, we ask that the following tests are requested along with these samples for immunophenotyping (see our sample submission page for more information):

  • Blood: Hemogram or blood smear examination
  • Bone marrow: Bone marrow examination
  • Body cavity fluids: Cytology smear examination or body cavity fluid analysis
  • Solid tissue aspirates: Cytology smear examination

back to top

Flow cytometric panels and immunocytochemical stains for immunophenotyping hematopoietic neoplasia

Antigen

Flow lymphoma

Flow leukemia

Immunocyto-chemistry

Comments

Panleukocyte

CD45

+

+

T lymphocyte

CD3

+

+

+*

Can be false positive on flow

CD5

+*

+

CD4

+*

+

+

Helper T cell, neutrophil, reactive histiocyte

CD8

+*

+

+

Cytotoxic T cell

TCRαβ

+

+

CD1c

+

+

Monocyte, B lymphoma, dendritic cell

CD90

+

+

Monocyte, dendritic cell, stem cells

CD11d

+

+

Monocyte, macrophage

B lymphocyte

CD21

+*

+

CD22

+

+

Pax-5

+*

Neutrophil

Neutrophil specific antigen

+

Monocyte

CD14

+*

+

Only mature cells

Neutrophil/
monocyte

CD11b

+*

CD11c

+

Platelet

CD61

+

vWf

+

Endothelial cells

Stem cell

CD34

+

+

Other

MHCII

+

+

Lymphocytes, monocytes

* Available for cats.

back to top

Immunocytochemical stains for identification of hematopoietic and non-hematopoietic neoplasia (only for cytology smears)

Neoplasm

Available markers

Species

T cell lymphoma

CD3

All

B cell lymphoma

Pax-5

Canine, feline, bovine

B cell lymphoma

BLA-36

All (also stains dendritic cells)

Carcinoma

Cytokeratin

All

Hemangiosarcoma

Von Willebrand factor

All

Histiocytic tumors

CD4, CD8, CD11c, CD11d, CD1c, CD90

Canine

Sarcoma

Vimentin

All

 

back to top

Important tips on immunophenotyping

  • Animals should ideally not have been treated with any chemotherapeutic agents, including corticosteroids, prior to the first immunophenotyping test when using flow cytometry. Antigens can be downregulated on lymphocytes after treatment.
  • Samples will not be diagnostic if there are insufficient numbers of the neoplastic cells in question or the cells have deteriorated or are lysed. This particularly applies to cytology smears (where cell quality cannot be assured or predicted) and aged fluid samples.
    • Samples of cerebrospinal fluid are usually of too low cellularity to perform immunophenotyping using flow cytometry.
    • In our experience, flow cytometry does not work well on samples with suspected histiocytic sarcoma (the cells lyse readily during the procedure).
    • Before application of the antibodies for flow cytometry, we usually check the sample for sufficient preservation and number of cells in question. If there are insufficient cells or the cells are mostly lysed, we will not proceed with the full flow cytometric evaluation. However, substantial effort and time has been expended to get the sample to this point and, thus, a minimum charge of $40 will be applied to the sample, regardless of whether the complete analysis is done.
  • In our experience, acute myeloid leukemias are frequently negative for most of the markers that we test for (even in our leukemia flow panel), other than the stem cell markers, CD34 and CD90. Under these circumstances, we will recommend additional testing – specifically, cytochemical staining for cytoplasmic enzymes – to determine cell lineage and obtain a definitive diagnosis.
    • Note that we lack markers for cells of erythroid lineage (acute erythroid leukemias) and natural killer cells.
  • Immunophenotyping is not recommended for diagnosis of chronic myeloid leukemias.
  • Some lymphoid tumors express B and T cell markers. To determine cell lineage in these cases, we recommend testing for antigen receptor re-arrangements in B or T cell receptors by polymerase chain reaction (PARRs). Note that PARRs are not recommended for differentiating between acute myeloid and lymphoid leukemias. Clonal rearrangements in lymphoid receptor genes can be seen in acute myeloid leukemias. 
  • A disadvantage of flow cytometry is that you cannot see the location of the positive reaction in the cells in question. False positive reactions may occur, especially if the cells are permeabilized to access nuclear or cytoplasmic antigens.
  • The advantage of immunocytochemical over immunohistochemical staining (done on formalin-fixed tissue) is that more antigens are preserved in fresh tissue. The main disadvantage is that cell morphology is inferior in cytology smears and the cells may wash off during staining.
  • Certain infectious diseases can cause clonal or restricted expansions of lymphocytes, which can mimic lymphoid neoplasia. This includes Ehrlichia canis (expansion of cytotoxic T cells) and Lyme disease (expansion of B cells). Infectious disease testing is always recommended in animals with an unexplained or persistent lymphocytosis.

back to top