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Flow cytometry applications include confirmation and categorization of lymphomas and leukemias,1 diagnose cases of immune-mediated hemolytic anemia (IMHA) and immune-mediated thrombocytopenia (ITP),2 and assess sperm quality in breeding animals.3 Flow cytometry uses fluorochrome-labeled antigen-antibody complexes to identify specific cellular markers associated with disease states. Canine diagnostics offer a greater repertoire of species-specific antigen-antibody complexes than feline diagnostics.1.4 Feline diagnostic capabilities continue to improve over time. In human medicine, flow cytometry has applications in immunology, molecular biology, bacteriology, virology, cancer and infectious diseases.4
In veterinary medicine, clinicians use flow cytometry mainly for the diagnosis of lymphomas, leukemias,5 and immune-mediated blood dyscrasias. Clinicians who suspect lymphoma based on clinical examination and cytology may perform fine needle aspiration on an enlarged lymph node. The cell sample is injected into a white top tube (WTT). Next, 1 ml of sterile saline and 0.1 ml of serum from the patient, or serum from an individual of the same species, are added to the WTT. The clinician gently swirls the solution, so as not to generate foam or bubbles, by removing and reinserting the sample until the solution appears cloudy. The clinician sends the sample on ice overnight to a flow cytometry laboratory.2.6
Note that the tube type may vary for different samples. A whole blood sample for suspected leukemia, abdominal fluid aspirates for ascites, and pleural fluid aspirates for pleural effusion are submitted in a lavender top tube (LTT) EDTA. A cell sample obtained with pleural fluid in a LTT is appropriate for cases with pleural effusion, and a cell sample obtained with a 211 gauge needle from an abdominal or thoracic mass is added to a WTT with solution saline and patient serum.2.6
Once the sample containing live cells arrives at the flow cytometry laboratory, it is separated into wells. The chances of an accurate diagnosis decrease if the cells are dead, whether due to rough handling, poor sample preparation, or cell fragility associated with aspirates from animals on steroid treatment. Next, fluorochrome-labeled antibodies, which bind to specific cell surface or intracellular antigens, are added to the sample in each well.2 Each colored sample is then passed through a tube and hydrodynamic focusing is performed. With hydrodynamic focusing, colored cells flow through a tube, one cell at a time.2 Each cell is hit with a laser beam and optics are used to measure the scattering created when the laser hits the fluorochrome-labeled antigen-antibody complexes.
The optics communicate with a computer program that generates a diagnostic printout. The print is interpreted by a qualified veterinary clinical pathologist, who generates a diagnostic report indicating whether the sample was identified as neoplastic or inflammatory. If the diagnosis is neoplasia, the clinical pathologist can determine the cancer type or lymphoma subtype,7 by assessing the types of markers on cells.7 Distinct types of lymphomas can be identified. For example, high cell surface CD21 expression on lymphocytes is indicative of B-cell lymphomas. Once the type is identified, an oncologist can determine the patient’s prognosis and the likelihood of a beneficial response to chemotherapy.
If there is any doubt regarding the diagnosis, stained slides or paraffin-embedded tissue samples, which contain dead cells, can be analyzed by polymerase chain reaction for antigen receptor rearrangement (PARR) analysis. In cases where the histopathologic features of a tissue sample are indistinct, immunohistochemical staining can be used to discern the cell type of origin (sarcoma or carcinoma) or to differentiate B-cell lymphoma from T-cell lymphoma. Immunohistochemical staining for cell proliferation markers such as Ki67 can aid in the diagnosis of canine mast cell tumors. Many diagnostic laboratories offer diagnostic panels combining immunohistochemistry, flow cytometry and PARR analysis when lymphoma is suspected.6
Flow cytometry is limited by the number of cell surface and intracellular antigens that have been identified and associated with various diseases. It is also limited by the number of antibodies that can be labeled with fluorochromes and bind to said antigens.5,7,8 Canine medicine offers a more complete range of identifiable antigen-antibody complexes than feline medicine. Feline medicine researchers are working to expand the repertoire of antigen-antibody complexes for identifying diseases. In feline medicine, flow cytometry, PARR analysis, and immunohistochemical staining have improved the ability of diagnosticians to differentiate feline inflammatory bowel disease from small cell intestinal lymphoma.6.9 Flow cytometric analysis of cytologic aspirates from affected mesenteric lymph nodes has improved clinicians’ ability to differentiate these 2 common feline gastrointestinal diseases, as have immunohistochemistry and PARR analysis of intestinal biopsies.
In summary, flow cytometry is useful in the diagnosis of distinct types of lymphomas, leukemias, and immune-mediated hematologic diseases such as IMHA and ITP, and it facilitates the assessment of sperm viability. As veterinary scientists discover more disease-specific cellular antigens, which can be detected using fluorochrome-labeled antibodies, veterinary clinicians will be able to use flow cytometry to diagnose more diseases.
Ann M. Brown, DVM, is a small animal veterinarian certified in veterinary medical acupuncture and diagnostic abdominal ultrasound for pets. She is a member of the American Medical Writers Association (AMWA) and has earned an Essential Skills (ES) certificate through AMWA. She is currently working toward the Medical Writer Certified (MWC) designation. A 1995 graduate of Michigan State University, Brown has practiced veterinary medicine for 28 years. She lives in Colorado with her husband, 2 children, 4 dogs and 2 snakes. A member of local fiction writing groups, she has written and published 3 novels and a short story.
The references
- Martini V, Bernardi S, Marelli P, Cozzi M, Comazzi S. Flow cytometry for feline lymphoma: a retrospective study regarding pre-analytical factors that may affect sample quality. J Feline Med Surg. 2018;20(6):494-501. doi:10.1177/1098612X17717175
- Garden OA, Kidd L, Mexas AM et al. ACVIM consensus statement on the diagnosis of immune-mediated hemolytic anemia in dogs and cats. J Vet Intern Med. 2019;33(2):313-334. doi:10.1111/jvim.15441
- Niżański W, Partyka A, Rijsselaere T. Use of fluorescent stains and flow cytometry for the evaluation of canine sperm. Reproduce Domest Anim. 2012;47(supplement 6):215-221. doi:10.1111/rda.12048
- Riondato F, Colitti B, Rosati S, Sini F, Martini V. A method for testing antibody cross-reactivity toward animal antigens for flow cytometry. Cytometry A. 2023;103(5):455-457. doi:10.1002/cyto.a.24691
- Comazzi S, Riondato F. Flow cytometry in the diagnosis of canine T-cell lymphoma. Veterinarian before Sci. 2021;8:600963. doi:10.3389/fvets.2021.600963
- Ehrhart EJ, Wong S, Richter K, et al. Polymerase chain reaction for antigen receptor rearrangement: comparative analysis of the performance of a lymphoid clonality assay in various types of canine samples. J Vet Intern Med. 2019;33(3):1392-1402. doi:10.1111/jvim.15485
- Parys M, Bavcar S, Mellanby RJ, Argyle D, Kitamura T. Use of multicolor flow cytometry for characterization of canine immune cells in cancer. PLoS One. 2023;18(3):e0279057. doi:10.1371/journal.pone.0279057
- McKinnon, K.M. Flow cytometry: an overview. Curr Protoc Immunol. 2018;120:5.1.1-5.1.11. doi:10.1002/cpim.40
- Avery PR, Burton J, Bromberek JL et al. Flow cytometric characterization and clinical outcome of CD4+ T-cell lymphoma in dogs: 67 cases. J Vet Intern Med. 2014;28(2):538-546. doi:10.1111/jvim.12304