• Flow cytometry is a means of precisely identifying the immunophenotype of cells in liquid suspension by the use of antibody markers specific for surface or intracytoplasmic molecules expressed by those cells.   
• This is a specialized procedure that is used in immunological research and occasionally in clinical diagnosis where the appropriate equipment and technical support is available.  
• Flow cytometry is generally performed in a university setting; however, some larger commercial clinical diagnostic laboratories may have access to the technique.  

Alone

• Flow cytometry is used to characterize populations of cells in suspension. These are generally leukocytes obtained by separating these cells from a blood sample (by density gradient centrifugation) or by aspirating from lymphoid tissue (ie a peripheral lymph node or bone marrow) or disaggregating cells from fresh tissue samples (mostly for research purposes – usually lymphoid tissues such as lymph nodes or mucosal, eg gut, lymphoid populations).   
• The most common clinical application of flow cytometry is in the immunophenotyping of lymphoid Lymphoproliferative disorders or myeloid leukemias Myeloproliferative disorders and occasionally of tissue lymphoma (via fine-needle aspiration Cytology: fine needle aspirate).   
• Flow cytometry may also be used to identify and quantify the major subsets of lymphocytes in the blood, ie B lymphocytes and CD4+ and CD8+ T lymphocytes. Of particular value is determining the CD4 to CD8 ratio as an index of immune status, as this ratio may increase or decrease with particular diseases. 
• Flow cytometry may also be used to detect the presence of antibody on the surface of red blood cells or platelets in immune-mediated hemolytic anemia Anemia: overview (and neonatal isoerythrolysis of foals) or immune-mediated thrombocytopenia Thrombocytopenia: overview, respectively. 
• Flow cytometry may be used to assess the phagocytic capability of neutrophils derived from a blood sample. Neutrophils incubated with fluorescently labeled particles, eg latex beads or staphylococcal bacteria, will phagocytose these particles and the proportion of neutrophils containing particles is measured by the flow cytometer. 
• Flow cytometry has also been used to detect the content of the enzyme myeloperoxidase in equine neutrophils. This molecule is recognized as a biomarker in equine intestinal, orthopedic and pulmonary diseases. 
• Flow cytometry is used in the characterization of stem cells that are now used in various applications in regenerative medicine. 
• Flow cytometry has been used for the detection of Cryptosporidium parvum oocysts Cryptosporidium spp in equine feces and was shown to be more sensitive than acid-fast staining or using immunofluorescent antibody testing. 

In combination

• Flow cytometry is a specialized test that would only be indicated after performing more routine diagnostics: 
  • For example, standard hematological Blood: overview and bone marrow examinations Bone marrow: biopsy would provide clinical evidence of leukemia and flow cytometry would then be applied to determine the immunophenotype of the neoplastic population. 
  • Similarly, cytology Cytology: fine needle aspirate, tissue biopsy Biopsy: overview and immunohistochemistry Immunostaining: immunohistochemistry / immunocytochemistry – direct and indirect would more likely be used in the diagnosis of lymphoma Lymphosarcoma: overview and flow cytometry would be an additional means of further refining the immunophenotype of the neoplastic cells.  
  • For example, tissue immunohistochemistry is readily able to identify T lymphocytes, but characterizing these cells as CD4+ or CD8+ would require either flow cytometry or immunohistochemistry on fresh-frozen tissue samples.   

• Assessment of blood leukocyte differential counts (identifying lymphopenia or lymphocytosis) would generally precede further evaluation of lymphocyte subsets by flow cytometry. 
• Hematological examination Blood: overview to characterize immune-mediated hemolytic anemia (IMHA) Anemia: overview or thrombocytopenia Thrombocytopenia: overview would be performed before flow cytometry. Flow cytometry provides an alternative to the traditional Coombs test Indirect Coombs test for detection of RBC-bound antibody in IMHA, but the Coombs test is much more widely available.  

• The procedure involves isolating the required population of leukocytes (normally mononuclear cells, including lymphocytes and monocytes) and resuspending these cells in appropriate buffer on ice.   
• The cells are then incubated with antibody specific for the surface molecule of interest, eg CD21 for B cells, CD4 or CD8 for T helper and T cytotoxic cells, respectively. These antibodies are normally monoclonal and purchased commercially. Ideally, the antibodies are already labeled with a fluorescent dye (a ‘fluorochrome’ such as fluorescein isothiocyanate [FITC] or rhodamine). For example, an FITC-labeled monoclonal antibody specific for CD4 would be used to identify T helper cells.   
• The labeled cells are then taken up into the flow cytometer and pass through a vibrating chamber that releases individual cells into a sheath of buffer fluid: 
  • Each individual cell then passes through a beam of laser light (the wavelength of light is specific for each fluorochrome). As the cell does this, it will scatter the laser light in such a way that enables the machine to determine the size and cytoplasmic complexity (granularity) of that cell.  
  • Light scattered in a forward direction (forward scatter; FSC) is proportional to the size of the cell, while light scattered at a 90° angle to the direction of the laser beam (side scatter, SSC) is proportional to the cell granularity. Measuring these parameters enables the machine to select (‘gate’) particular populations for further analysis.  
  • The labeled cells will also emit a pulse of light if they have been bound by the particular antibody.  
  • The burst of light is recorded and after 10,000 individual cells have been examined, the proportion of the population carrying the label is determined. 
  • Labeling is depicted graphically either in a histogram or dot-plot format. 
• Sometimes, combinations of antibodies with different fluorochrome labels, eg FITC and rhodamine, can be added to the cell suspension and the machine will detect each population within the suspension (to calculate, eg the CD4 to CD8 ratio) and the presence of any cells carrying both target molecules (so called ‘double positive’ cells). 
• Sometimes the cells can be permeabilized to allow monoclonal antibodies to enter the cell and detect target molecules within the cytoplasm. This, for example, allows the detection of intracellular cytokine protein. Two-color labeling could then be applied to allow the machine to detect those CD4+ lymphocytes that had intracytoplasmic expression of interferon-ϒ, ie T helper 1 lymphocytes. Similarly, intracellular detection of the Foxp3 molecule enables characterization of those CD4+ T cells with a regulatory phenotype. Both techniques have been applied to studies of equine lymphocytes. 
• In research settings, some flow cytometers will not only identify cell subsets, but be able to sort them into separate test tubes – allowing experimental studies of the function of the different subpopulations. 
• In the case of detection of red cell or platelet bound immunoglobulins, the fluorochrome-labeled antisera would have specificity for these molecules, eg anti-equine IgG or IgM, and the starting cell suspensions would be of erythrocytes or platelets. 

• As described above, flow cytometric testing is rarely available outside of a university center where there is access to a flow cytometer and dedicated technical support. 

• The test is limited by the nature of the sample, transportation requirements and the availability of equine specific antibodies for the target molecule. 

• The test is limited by availability and cost. 

Refereed papers

• Recent references from PubMed and VetMedResource. 
• Tessier L, Bienzle D, Williams L B et al (2015) Phenotypic and immunomodulatory properties of equine cord blood-derived mesenchymal stromal cells. PLosOne 10 (40), e0122954 PubMed. 
• Wauters J, Franck T, Pille F et al (2011) Flow cytometric detection of myeloperoxidase in horse neutrophils: a novel technique in equine diagnostic research. Vet Immunol Immunopathol 144 (3-4), 417-422 PubMed. 
• Robbin M G, Wagner B, Noronha L E et al (2011) Subpopulations of equine blood lymphocytes expressing regulatory T cell markers. Vet Immunol Immunopathol 140 (1-2), 90-101 PubMed.
• Reggeti F & Bienzle D (2011) Flow cytometry in veterinary oncology. Vet Pathol 48 (1), 223-235 PubMed. 
• Bezdekova B, Faldyna M, Zapletal O et al (2009) Acute B-lymphoid leukemia in a mare: a case report. Veterinarni Medicina 54, 249-255. 
• Merant C, Bonnefont C, Desbos A et al (2003) Cross-species reactivity of seven monoclonal antibodies with equine lymphocytes by flow cytometry. Vet Res 34 (6), 791-901 PubMed. 
• Davis E G, Wilkerson M J & Ruxh B R (2002) Flow cytometry: clinical applications in equine medicine. J Vet Int Med 16 (4), 404-410 PubMed. 
• Nunez R, Gomes-Keller M A, Schwarzwald C et al (2001) Assessment of equine autoimmune thrombocytopenia (EAT) by flow cytometry. BMC Blood Disorders 1 (1), 1 PubMed. 
• Cole D J, Snowden K, Cohen N D et al (1999) Detection of Cryptosporidium parvum in horses: thresholds of acid-fast stain, immunofluorescence assay, and flow cytometry.  J Clin Microbiol 37 (2), 457-460 PubMed. 

Other sources of information

• Day M J & Schultz R D (2014) Veterinary Immunology: Principles and Practice. 2nd edn. CRC Press, USA.

• College of Veterinary Medicine (online) Phenotyping. Website: https://www.vet.cornell.edu/
• Kansas State Veterinary Diagnostic Laboratory (online) Clinical Immunology. Website: http://www.ksvdl.org