Lymphoma is one of the most prevalent cancers in dogs . Diagnostic testing and prognosis is based on clinical signs and degree of spread, morphological features of the lymph node and lymphocytes, and other cytopathologic features such as mitotic rate, and clonality of antigen-receptor rearrangement or of cluster of differentiation (CD) antigens.
Immunophenotyping CD antigens has contributed significantly to both diagnosis and prognosis of lymphoid neoplasia. This approach measures the binding of labelled, monoclonal antibodies to specific intracellular or surface CD antigens. It is well-established and has long been used in cell analysis, particularly in the fields of haematology and immunology [2–4]. For lymphoma, it can be accomplished using either immunohistochemistry of tissue-biopsy sections  or by immunocytochemistry of fine needle aspirates. Cytologic analysis can be done manually on smears using microscopy  or on cell suspensions using automated, flow cytometry.
Immunophenotyping is most easily and rapidly accomplished by flow cytometry. Flow cytometry of blood, lymph node and bone marrow samples may improve evaluation and prognosis of dogs with lymphoma [4, 7–9]. However, in veterinary medicine this technique is mainly available only as a research tool, rather than for widespread diagnostic use as occurs in human medicine . There are only a few European laboratories that routinely provide immunophenotyping by flow cytometry for veterinary patients. The main barriers associated with the expansion of flow cytometry in veterinary medicine are the substantial cost of the analyser, reagents, and facilities, and the need for advanced training of the instrument operators. In addition, analysis and interpretation of results requires understanding and knowledge of flow cytometry and its principles.
It is well documented that immunophenotype of neoplastic lymphocytes correlates significantly with the survival time of dogs with lymphoma and is of significant value in prognosis [10–15]. In 175 dogs with lymphomas, T-cell phenotype had shorter relapse-free time (52 versus 160 days, p<0.001) and shorter survival times (153 versus 330 days, p<0.001) than B-cell phenotype . Dobson, Blackwood et al. 2001 found that the T- cell phenotype is associated with a significantly shorter recurrence-free interval and reduced survival times. Hazard ratio for T-cell versus B-cell immunophenotype lymphoma was 4, with 95% confidence interval from 1.4 to 11.3, p=0.035. However, the correlation of immunophenotype with prognosis is not perfect, and exceptions have been identified. For example, the small and clear T-cell lymphoma has one of the best prognoses . Thus for prognosis, immunophenotyping data must be used along with other clinical and cytological assessments of degree of spread, morphologic features, and mitotic rate.
In recent years, multiple research studies involving immunophenotyping by flow cytometry have been published in the veterinary literature [2–4, 6, 15–20]. However, limited veterinary availability of flow cytometers, expertise, and reagents is a major limitation to the diagnostic use of immunophenotyping . Also, the current protocols used in veterinary diagnostics are labour intensive and time-consuming, involve standard technology, and have not been customised for widespread diagnostic use in the clinical pathology laboratory.
The recent development of the personal flow cytometer may overcome the barriers previously associated with diagnostic use of flow cytometry in veterinary medicine. The personal flow cytometer is a miniaturised, user-friendly, and affordable version of the standard instrument  found in dedicated facilities and operated by dedicated personnel. Different manufacturers name the instruments they produce “personal”. Such instruments include the different Guava platforms produced by Millipore, the Accuri C6 from BD Biosciences, the Attune flow cytometer by Applied Biosystems and the HPC-100 system by Handyem. The technical bulletin for the Accuri C6 analyzer from BD Biosciences is entitled “Making Flow Cytometry Personal” They can be used in any laboratory and by any laboratory technologist, and seem ideal for the veterinary clinical pathology laboratory.
The Guava flow cytometer (Guava Technologies, Hayward, CA, US) is the first, such personal flow cytometer, and has several advantageous features for veterinary use. It is an automated, easy-to-use, bench-top, single-cell, analyser that can perform a wide range of multi-parameter, cell-based assays using light scatter and multiple fluorescence measurements and also direct measurement of cell counts without the need for reference beads . It requires far less sample and reagents volume , making it ideal for veterinary and diagnostic cytology applications. It requires far less operator time and expertise, less maintenance and is automated for multiple tube or microtiter plate sampling. It requires far less bench space than the standard analyser. The purchase cost is considerably lower than conventional flow cytometers. Also, it does not produce voluminous waste, because it does not use sheath fluid to move the cells through the analyser .
The aim of this study was to adapt the immunochemical protocol used for immunophenotyping of canine lymphoma to the personal flow cytometer for rapid, effective and user-friendly application to diagnosis and prognosis of canine lymphoma and to demonstrate its practicality for widespread veterinary application. Parts of this study have also been presented in the 10th and 13th Annual Congresses of the European Society of Veterinary Clinical Pathology in Barcelona, Spain and Dublin, Ireland [25, 26].