The study developed three multicolor panels for peripheral blood and selected solid-tissue samples, combining markers including CD45, MIL-4, panT, CD1b3, CD14, CD4 and CD8, with intracellular CD79α used to confirm B-cell identity. Analysis was performed on a BD LSRFortessa flow cytometer with BD FACSDiva software, followed by data analysis in FlowJo. The investigators used forward- and side-scatter characteristics together with fluorescence and viability information to distinguish leukocyte populations. A fluorescence-minus-one control in which the panT primary antibody was omitted was retained for routine work because the MsGp7 panT antibody produced a relatively low staining index, while an isotype control supported interpretation of intracellular CD79α staining.

Technical gating was central to the approach. In blood, CD45 was not essential, whereas it helped establish the total leukocyte compartment in solid tissues. The MIL-4-containing panel separated granulocytes and monocytes from lymphocytes and allowed neutrophils and eosinophils to be distinguished largely by side-scatter intensity. Eosinophils were provisionally identified as MIL-4-positive, high-scatter cells, an assignment supported by the larger, more abundant cytoplasmic granules visible on blood smears. Monocytes were identified as MIL-4-positive and CD14-positive cells, while the lymphocyte gate was defined as MIL-4-negative cells with lymphoid scatter characteristics. Because the panels were not always used together, CD4 and CD8 expression was interpreted within the gated lymphocyte fraction rather than across the entire leukocyte population.

Several cross-reactive reagents made the protocol implementable without custom antibody conjugation. These included guinea pig-specific anti-CD45 and anti-CD1b3 antibodies, an anti-pig CD11R1/MIL-4 reagent that also recognized guinea pig cells, cross-reactive anti-human CD14 and CD79α antibodies, and guinea pig-specific CD4 and CD8 antibodies. The MsGp7 panT antibody also replaced a previously used CT5 clone that was no longer commercially available. B-cell identification nevertheless required caution because CD1b3 was not exclusive to B lymphocytes and was also expressed by monocytes. CD14 or MIL-4 was therefore needed to exclude monocytes, and intracellular CD79α provided additional confirmation of the B-cell gate.

In 13 clinically healthy client-owned guinea pigs aged 2–3 years, both routine hematology and flow cytometry showed a lymphocyte-dominant peripheral blood profile. The median total white blood cell count was 10.9 × 10^9 cells per liter, with a range of 6.8–13.8 × 10^9 per liter. By flow cytometry, lymphocytes represented a median 62.5% of viable leukocytes, compared with 8.0% neutrophils, 8.5% eosinophils and 3.0% monocytes. Routine hematology produced broadly similar results: lymphocytes accounted for a median 67.8%, neutrophils 10.5%, eosinophils 5.3% and monocytes 2.3%. These observations establish baseline distributions for the particular client-owned crossbred cohort, not universal reference intervals for every guinea-pig population.

Flow cytometry further showed that T cells predominated within the healthy lymphocyte compartment, making up a median 78.9% of lymphocytes, while B cells accounted for 6.3%. The largest defined T-cell subset was CD4-negative, CD8-positive cells, which represented a median 50.7% of T cells. A further population lacking panT, CD1b3 and CD14 markers made up a median 5.0% of gated lymphoid cells; it was heterogeneous and may include Kurloff cells, although the study did not characterize it further. Comparisons with an independent experimental cohort found no significant difference in T/B lymphocyte ratios among guinea pigs aged 2 months, 9 months and 2–3 years: the respective medians were 15.3, 9.7 and 9.1, with a Kruskal–Wallis p-value of 0.2611.

The nine clinical animals entered the study because they had generalized peripheral lymphadenopathy, a palpable cervical mass considered consistent with lymphoid enlargement, leukocytosis driven mainly by lymphocytes, or combinations of these findings. Histopathology identified five cases of high-grade lymphoma, two thyroid gland adenocarcinomas, one fibrolipoma and one lymphadenitis. All five lymphomas occurred in animals with marked B-cell-predominant lymphocytosis, ranging from 62.8% to 95.3% of circulating lymphocytes, and low T/B ratios of 0.006 to 0.432. The five lymphoma cases had a mean age of 3.7 years and included three females and two males. By contrast, the four cases without histological evidence of lymphoid neoplasia lacked a uniform B-cell-predominant pattern, showing either T-cell predominance or a mixed lymphocyte population.

A detailed example illustrated how blood and tissue findings could be integrated. A 2.5-year-old female guinea pig presented with apathy, anorexia, generalized lymphadenopathy and a white blood cell count of 126.5 × 10^9 per liter. Flow cytometry found that 86.8% of viable blood leukocytes were lymphocytes, of which 86.2% were B cells and 11.0% were T cells, producing a T/B ratio of 0.13. The submandibular lymph node contained 96.8% B cells and 1.9% T cells, while the mesenteric lymph node and spleen each contained 91.8% B cells. Histopathology showed a monomorphic population with cellular atypia and mitotic figures, consistent with high-grade lymphoma, and PAX5 immunohistochemistry supported B-cell lineage. The marked blood lymphocytosis meant that lymphoma with peripheral blood involvement and primary B-cell leukemia both remained possible diagnoses.

The results demonstrate the panel’s potential as a clinical adjunct, but the evidence comes from a deliberately small and selected series. Tissue flow cytometry was performed in only three of the nine clinical cases, and the experimental age comparison used a separate untreated cohort. Peripheral blood may not represent the full cellular composition of an affected lymph node, while the absence of bone-marrow examination prevented definitive separation of lymphoma with a leukemic phase from primary leukemia. Histopathology remained necessary to establish the nature of the lesion, and the study did not establish prognostic thresholds, treatment responses or population-wide reference intervals. Larger studies incorporating paired blood, marrow and tissue samples would be needed to validate those applications, but the panels provide a practical and reproducible starting point for immunophenotyping guinea pig leukocytes.

Subject of Research: Flow-cytometric immunophenotyping of guinea pig leukocytes and lymphoid neoplasia

Article Title: A practical flow cytometric approach to leukocyte immunophenotyping in guinea pigs and its potential applications in lymphoid neoplasia

Article References: Štěpánová, H., Jeklová, E., Škorič, M., Jarošová, R., & Jekl, V. (2026). A practical flow cytometric approach to leukocyte immunophenotyping in guinea pigs and its potential applications in lymphoid neoplasia. Frontiers in Veterinary Science, 13, Article 1926791. https://doi.org/10.3389/fvets.2026.1926791

Image Credits: AI Generated

DOI: 10.3389/fvets.2026.1926791

Keywords: guinea pigs, flow cytometry, leukocyte immunophenotyping, B-cell lymphoma, lymphocytosis, veterinary diagnostics, CD79α, lymphoid neoplasia, practical, flow, cytometric, approach