By J. Lee Talbott, DVM, DACVIM (Medical Oncology)
Lymphoma is the most common hematopoietic malignancy diagnosed and treated in veterinary patients, representing 7-24% of all canine neoplasms.1,2 Canine lymphoma is a heterogeneous disease characterized by variable clinical signs and response to therapy.1,2 Initial diagnosis of lymphoma is most often made by cytology or histology of an enlarged peripheral lymph node, or other abnormality noted during physical exam and staging. Molecular diagnostic testing in canine lymphoma is useful to definitively diagnose hematopoietic malignancies, establish prognosis, and in some cases guide treatment options.1 The following is a review of flow cytometry and PARR, and the utility of each test for the diagnosis and molecular classification of canine lymphoma.
Flow Cytometry (FCM)
Flow cytometry consists of staining live cells with fluorescently labeled antibodies that bind to proteins expressed on the cell surface, through the use of sophisticated machines that utilize fluidics, optics (fluorescence), and electronics.2-5 The basic principle of FCM is a complex device that can rapidly analyze cells (up to 5,000 cells per second) in single file through fluid in front of lasers so they can be detected, counted and sorted.3-5 Cell components are fluorescently labeled and excited by the laser to emit light at varying wavelengths. The cell size (or volume) and inner complexity of the particle (i.e. shape of nucleus, amount and type of cytoplasmic granules) are measured. The basics of flow cytometry are depicted simplistically in Figure 1.
Figure 1: The basics of a flow cytometer
Immunophenotyping is the use of fluorescently labeled antibodies to identify cells of lymphoid and myeloid origin.1-3 In oncology, flow cytometry is the most commonly used immunophenotyping test. This has direct diagnostic and prognostic applications.1 Table 1 shows the list of commonly used cell surface antigens offered by most laboratories. The author recommends submitting FCM samples for immunophenotyping to the Clinical Immunology Laboratory at Colorado State University. When a sample is submitted for FCM (immunophenotypic analysis), it is divided into multiple tubes and subjected to analysis using a panel of anti-receptor antibodies. The results are interpreted by a flow cytometrist/clinical pathologist and a diagnosis made, such as B cell lymphoma (i.e. CD 21 lymphocytosis).3,4
Table 1: Canine leukocyte markers commonly used for immunophenotyping
||Antigen generally found on
||T helper cells
||T cytotoxic cells
||Hematopoietic progenitor cells/leukemia
||Part of B cell receptor, on immature cells
||Macrophages of spleen and bone marrow
FCM Key Points:
- FCM is the test of choice for immunophenotypic analysis in canine lymphoma.
- Sample requires LIVE cells suspended in fluid
- Commonly run samples: blood, lymph node aspirates, effusions, CSF, etc.
- The author recommends the clinical immunology laboratory at CSU.
- See clinical immunology laboratory specimen submission instructions for CSU.
- Send ALL samples to CSU overnight with a cold pack for delivery M-F. Samples received by 10am on Saturday will be viable on Monday (exception is bone marrow aspirates).
- CSU does not charge for FCM if cell viability test confirms the sample is dead (shipping charges still incurred).
- DO NOT FREEZE SAMPLE.
PCR for Antigen Receptor Rearrangement (PARR)
Polymerase chain reaction (PCR) for antigen receptor rearrangement (PARR) is a test used to detect clonality within a population of cells, where monoclonal represents lymphoma and polyclonal represents reactive lymphocytes (to some antigenic stimulation). PCR is a repetitive enzymatic reaction that generates 109 copies of a particular DNA sequence for 1 original copy.6 To review, lymphocytes have unique cell surface receptors that allow them to recognize a variety of antigens, part of normal lymphocyte maturation in early development. Different rearrangements of the VDJ (variable, joining, diversity) sequences in the genes that code for these cell surface receptors allows for this recognition. Therefore, reactive lymphocytes should be from multiple sources and have different VDJ sequences (i.e. polyclonal).6
It is important to note that nonlymphoid cells do not have unique DNA sequences amongst cells, therefore other tests must be utilized to assess for clonality.1
The sensitivity of PARR, or the ability of the test to detect a true positive in canine samples ranges from 72%-100%. The specificity of PARR, or the ability of the test to detect a true negative in canine samples is 96%-100%.7 It is important to remember that many factors affect the PARR assay (i.e. it is not a yes or no answer), including the quality of the DNA, inhibitors of the enzymatic reaction, primer choice, and the method of analysis.8 It is important to interpret PARR results in conjunction with history, clinical signs, cytology, biopsy, FCM, and immunohistochemistry.
PARR Key Points6-8:
- PARR detects clonality only
- PARR is NOT interchangeable with FCM for immunophenotyping
- Recommend to use when:
- Suspect lymphoproliferative disease
- Unable to obtain sample for FCM
- Monitoring disease status
- PARR can often determine B vs. T cell lineage
- Immunoglobulin gene rearrangements = B cell
- TCR (T cell receptor) gene rearrangements = T cell
- PARR can be performed on MANY samples
- Commonly run samples: blood, lymph node aspirates, bone marrow, effusions, and CSF.
If you ever have any questions regarding the most appropriate test or sample for that test (and method of collection/submission), we recommend contacting your local oncologist, clinical pathology lab used for submissions, or the clinical immunology laboratory at Colorado State University.
- Vail DM, Pinkerton M, Young K. Withrow and MacEwen’s Small Animal Clinical Oncology 6th St. Louis, MO. Elsevier 2020.
- Burkhard MJ, Bienzle D. Making Sense of Lymphoma Diagnostics in Small Animal Patients. Vet Clin Lab Med 2015 Sep: 35(3): 591-607.
- Thalheim L, Williams LE, Borst LB, Fogle JE, Suter SE. Lymphoma immunophenotype of dogs determined by immunohistochemistry, flow cytometry, and polymerase chain reaction for antigen receptor rearrangements. J Vet Intern Med 2013 Nov-Dec;27(6): 1509-16.
- Suter S. Basic Oncology Workshop: Molecular and Immunologic Diagnostics. Meeting of the Veterinary Cancer Society. October 2012.
- Fogle JE, English LB, Tompkins MB. Flow Cytometry for the Busy Oncologist. Veterinary Cancer Society Member News 2011 Vol 35, No.2.
- Abbas AK, Lichtman AH, Pillai S: Cellular and Molecular Immunology 8th 2015
- Vernau W, Moore PF. An immunophenotypic study of canine leukemias and preliminary assessment of clonality by polymerase chain reaction. Vet Immuno & Immunopathol 69 (1999).
- Schleis SE. Cancer Screening Tests for Small Animals. Vet Clin North Am Small Anim Pract 2014; 44: 871-881.