Immunophenotyping – a flow cytometry based method now available to Veteo clients

The Veteo Laboratory, in cooperation with a team of veterinary oncologists and scientists, offers immunophenotyping of cells using flow cytometry.

The founder and supervisor of the project is Dr. Dariusz Jagielski.

“At the beginning of my work with oncology patients, almost twenty years ago, I became interested in immunophenotyping and flow cytometry and was fascinated by the potential of this method. In the end, we managed to gather a group of people who offer cytometry as a standard, commercially available test for patients with cancers of the hematopoietic system.
The key to success is contact with the attending veterinarian – this leads to obtaining additional useful clinical insights from the cytometric examination.”

Our team

Our team consists of qualified staff under the supervision of an experienced leader, dr n. med. lek. wet. Marta Idziak, a specialist in veterinary laboratory diagnostics.

Scientific support is provided by dr. Aleksandra Pawlak from the Faculty of Veterinary Medicine at the University of Environmental and Life Sciences in Wrocław.

“Flow cytometry is one of the fundamental scientific methods I use in my work. Cytometric immunophenotyping of canine lymphomas and leukemias was one part of my doctoral thesis, and since then I have been a true enthusiast of this diagnostic method, continually developing my expertise in its use.
In my opinion, the commercial availability of this examination is the pinnacle of my scientific work — because science should serve practice!”

We invite veterinarians with an interest in veterinary oncology who wish to advance their hematologic and oncologic diagnostic capabilities to collaborate with us.

We are also open to cooperation with research centers.

Flow cytometry

Flow cytometry is a method widely used in laboratory diagnostics. By detecting laser light scanning a cell suspension, it enables precise assessment of both the number of cells and their morphological characteristics. Since the beginning of our activity, we have been using the most advanced veterinary flow cytometers available on the market, offering professional analyses of hematological parameters in many animal species.

Currently, as the only laboratory in Poland, we combine flow cytometry technology with monoclonal antibodies and fluorescence, allowing us to perform advanced immunophenotyping of cells. This approach enables the detection of differentiation antigens (clusters of differentiation, CD) on the surface and inside cells, providing precise determination of their origin and characteristics.

Indications for immunophenotyping

Immunophenotyping in veterinary medicine is used primarily in small animal hemato-oncology.
In the early years of our activity, this test was offered exclusively to canine patients. It is now also available for cats.
In addition, we have expanded our offer to include Ki-67 antigen testing in dogs – a marker of cell proliferation that is helpful in assessing tumor aggressiveness.

The main indications for immunophenotyping by flow cytometry in dogs and cats include:

differentiation between reactive and neoplastic lymphoproliferative processes (lymphocytosis/reactive lymph node vs. leukemia/lymphoma)

differentiation between acute and chronic leukemias (note: dogs only)

differentiation of lymphoma cell origin (T-cell vs. B-cell)

differentiation of leukemia cell origin (lymphoid vs. myelomonocytic)

assessment of prognostic parameters in lymphoma/leukemia (phenotype, aberrations, MHC II molecule expression)

differentiation of low- and high-grade lymphomas (Ki-67; note: dogs only)

definitive confirmation of T-zone lymphoma (note: dogs only)

differentiation between thymoma and mediastinal lymphoma (note: dogs only)

The panel of monoclonal antibodies we use includes:

in dogs, antibodies against all of the following markers
in cats, only antibodies directed against the markers indicated

CD45 / CD18

leukocyte marker

CD34

stem cell marker

CD3

T-cell marker (surface)

CD3c

T-cell marker (intracellular)

CD4

T-cell helper marker

CD8

cytotoxic T-cell marker

CD5

T-cell marker

CD21

mature B-cell marker (surface)

CD79α

B-cell marker (intracellular)

CD14

monoidal line marker

MHCII

major histocompatibility complex class II marker (present on lymphocytes and macrophages)

CD11b

myelomonocytic line marker

Ki-67

cell proliferation activity marker

Important notice

Immunophenotyping of cells using flow cytometry requires maintaining high cell viability. For this reason, the analysis must be performed no later than 48–72 hours after collection of the cellular material.
In the case of flow cytometric analysis of lymph node biopsies and solid organs, preparation of a cell suspension using a dedicated medium is required (see Instructions). This requirement does not apply to blood, bone marrow, or body cavity fluids, which are ready-to-analyze cell suspensions.
Cellular material must not be frozen or fixed.
Samples for flow cytometric testing are accepted from Monday to Thursday (analyses are not performed on weekends or public holidays).
Medications, particularly cytostatics and glucocorticosteroids, can significantly affect immunophenotyping results. Therefore, it is recommended to perform the analysis prior to initiating therapy.
Immunophenotyping results cannot be interpreted without knowledge of the cytopathological features of the analyzed material and the patient’s clinical data. Therefore, it is necessary to complete the online questionnaire and submit fresh samples and/or cytological test results.

Material for examination

Any type of liquid suspension of viable cells can be used for immunophenotyping by flow cytometry.
If peripheral blood, bone marrow, or body cavity fluid is to be analyzed, the material should be collected into a tube with anticoagulant (as for a complete blood count) and sent to the laboratory.
In the case of biopsies from lymph nodes and other solid organs, it is necessary to suspend the cells in a specialized transport medium.

Instructions for preparing the transport medium for lymph node/solid organ biopsies

Collect peripheral blood from the patient undergoing biopsy into an EDTA tube.
Centrifuge the collected blood sample to obtain plasma.
Mix the plasma with sterile 0.9% NaCl solution at a ratio of 1:9. Approximately 2 ml of medium is sufficient for one patient sample (1.8 ml of 0.9% NaCl + 0.2 ml of plasma).
Place the cell sample in the prepared medium.

The minimum cellularity required to perform the analysis is approximately 3 million viable cells. The sample volume to be sent for analysis depends on the cell count in the tested tissue and is difficult to specify precisely (typically, 2–3 lymph node biopsy cores, about 2–3 ml of peripheral blood, or 1–2 ml of bone marrow are sufficient).

Along with the cellular material, it is advisable to provide blood smears/cytological slides and/or current cytology results.

Questionnaire for the referring veterinarian

Examination questionnaire

Interesting cases

Below are brief case reports that demonstrate the diagnostic potential of immunophenotypic testing of canine cells using flow cytometry.

Schnauzer with clinical suspicion of lymphoma

A 10-year-old Schnauzer with clinical suspicion of lymphoma confirmed by cytopathological examination of the lymph nodes.
To assess the clonality and origin of the neoplastic cells, a PARR test was performed, which surprisingly yielded a negative result (features of polyclonal proliferation of T and B lymphocytes were found, as seen in non-neoplastic stimulation of lymph nodes).
The results of the cytometric analysis showed that the dominant population in the examined lymph node consisted of medium to large cells with the immunophenotype CD45+ CD21+ CD79α+ MHC II+. This finding indicated a disruption of the physiological proportions between T and B lymphocytes in the lymph node and clearly suggested excessive proliferation of B lymphocytes.*

Taking into account the clinical data and the cytology results of the patient’s lymph nodes, immunophenotyping made it possible to resolve doubts about the final diagnosis — the presence of B-cell lymphoma was confirmed and appropriate therapy was initiated.

*Pawlak A, et al. Immunophenotypic characterization of canine malignant lymphoma: a retrospective study of cases diagnosed in Poland Lower Silesia, over the period 2011–2013. Veterinary and Comparative Oncology, 2014, 14, 52–59.

Shih Tzu with signs of generalized lymphadenopathy and marked lymphocytosis in peripheral blood

An 11-year-old female Shih Tzu with signs of generalized lymphadenopathy and marked lymphocytosis in peripheral blood persisting for about a year.
Histopathological examination of the excised submandibular lymph node performed at the beginning of the diagnostic process indicated reactive hyperplasia, which did not correlate with the clinical picture.
The immunophenotyping result indicated the presence of a clearly dominant population of cells in the examined lymph node with the immunophenotype CD45- CD3+ CD8+ CD5+ MHC II+. The presence of a large population of cells with the phenotype of cytotoxic T lymphocytes lacking expression of the lymphocyte marker (CD45) was indicative of their neoplastic transformation.

In this case, immunophenotyping enabled confirmation of a clinically significant lymphoma and, based on literature data, suggested its subtype as T-zone lymphoma (TZL).*

*Seelig DM, Avery P, et al. Canine T-zone lymphoma: unique immunophenotypic features, outcome, and population characteristics. J Vet Intern Med 2014; 28: 878-86.

Boxer with generalized lymphadenopathy and cytological suspicion of lymphoblastic lymphoma

A 7-year-old Boxer with generalized lymphadenopathy and cytological suspicion of lymphoblastic lymphoma
Cytometric analysis enabled the identification of a clearly dominant population of cells in the examined lymph nodes with the phenotype CD45+ CD3+ CD5+ CD4+, corresponding to helper T lymphocytes.Díky imunofenotypizaci byla přítomnost lymfomu rychle a jednoznačně potvrzena, navíc byl jeho původ stanoven na T buňky. According to the literature, the CD45+ CD4+ phenotype in T-cell lymphomas may predict an aggressive nature and rapid progression of the neoplastic disease, and Boxers are considered a breed with a high predisposition to this type of neoplasia*.**

*Avery PR, Burton JL, et al. Flow cytometric characterization and clinical outcome of CD4+ T-Cell Lymphoma in dogs: 67 cases. J Vet Intern Med 2014; 28: 538-546.
**Lurie DM, Milner RJ, et al. Immunophenotypic ad cytomorphologic subclassification of T-cell lymphoma in the boxer breed. Vet Immunology and Immunopathology 2008; 125: 102-110.

Mixed-breed dog in severe general condition with marked leukocytosis and the presence of atypical cells in a peripheral blood smear

A 9-year-old mixed-breed dog in severe general condition with marked leukocytosis, anemia, thrombocytopenia, and the presence of atypical cells in a peripheral blood smear
As a result of the blood cytometric analysis, a clearly dominant population of stem cells (CD34+) was identified, with concurrent expression of the B-lymphocyte marker (CD79α+).
The phenotyped cells did not express CD14 or CD4, which made it highly probable to exclude a myelomonocytic hyperplasia lineage. In this case, immunophenotyping formed the basis for the final diagnosis of acute B-lymphoblastic leukemia (ALL-B)*.

* Vernau W, Moore PF. An immunophenotypic study of canine leukemias and preliminary assessment of clonality by polymerase chain reaction. Vet Immunol Immunopathol 1999; 69: 145-164.

Miniature Schnauzer showing no clinical symptoms with marked lymphocytosis persisting for several months

A 15-year-old Miniature Schnauzer showing no clinical symptoms, with marked lymphocytosis persisting for several months and no atypical cells present in peripheral blood
In the cytometric blood analysis, the dominant cell population displayed the phenotype CD45+ CD21+ CD79α+ MHC II+, corresponding to B lymphocytes. At the same time, the lack of expression of the stem cell marker (CD34) in the examined cells excluded the presence of acute lymphoblastic leukemia. In this case, the immunophenotyping result (in the context of the clinical data and the peripheral blood smear findings) made it possible to consider the presence of chronic B-cell lymphocytic leukemia (B-CLL) or, alternatively, small B-cell lymphoma (B-SLL) with blood involvement*.

*Comazzi S, Gelain ME, Martini V. Immunophenotype Predicts Survival Time in Dogs with Chronic Lymphocytic Leukemia. JVIM 2011: 25; 100-106.

Cat with chronic lymphocytosis of unclear origin

A 12-year-old cat with lymphocytosis persisting for over 2 months and mild atypical features of lymphocytes observed on microscopic examination of the blood smear
Flow cytometric analysis of the patient’s blood sample allowed the identification of a clearly dominant subpopulation of lymphocytes with a CD18+CD5+CD4+ phenotype, corresponding to helper T lymphocytes. According to the literature*, such an immunophenotyping result is strongly suggestive of a neoplastic process of lymphoid lineage (leukemia or lymphoma in a leukemic stage).

*Rout E.D., et al. Immunophenotypic characterization and clinical outcome in cats with lymphocytosis. Journal of veterinary internal medicine 2020; 34: 105-116.