Lymphomas account for approximately 15% of pediatric neoplasms, ranking behind brain tumors and leukemia. These diseases may progress rapidly; therefore, fast and accurate diagnostic methodologies, such as flow cytometric immunophenotyping, are essential to establish a precise diagnosis and enable appropriate treatment.

To evaluate the clinical impact of the introduction of flow cytometry in a diagnostic workflow for pediatric lymphomas.

Prospective case consecutive study, conducted between February 2023 and June 2025. Inclusion criteria: 0 to 18 years, in diagnostic investigation for lymphadenopathy and/or tumor masses (TM). Exclusion criteria: bone marrow (BM) infiltration by acute leukemia. Samples: TM (including lymph nodes), biological fluids (BF) and BM. TM underwent manual disaggregation. Samples were labeled with a lymphoma tube (CD3, CD4, CD8, CD10, CD19, CD20, CD45, TCRab, TRBC1, kappa, lambda). From June 2024, a Hodgkin Lymphoma (HL) tube was incorporated (confidential). When lymphoblastic lymphoma (LL) was suspected, a leukemia screening tube (cyCD3, CD3, CD19, CD34, CD45, cyCD79a, CD117, MPO) was added. Immunophenotyping findings were compared to those obtained by reference methodologies.

46 samples from 38 patients between 1 and 17 years (mean 10) were analyzed. 34 TM, 9 BM, and 3 pleural fluids. Results concorded in 30 samples (65%): 13 without hematologic malignancies, 7 B or T LL, 7 mature B or T lymphomas and 3 HL. Results discorded in 16 samples (35%): 10 HL (1 with the targeted panel applied); 3 T lymphomas; 1 BM with Burkitt lymphoma; 1 with low cellular viability; and 1 TM showed no infiltration in histopathology but B-LL by immunophenotyping. Twenty-five patients had concordant diagnoses between the diagnostic methodologies. Of these, 11 were samples without neoplastic infiltration. Among the 14 samples with infiltration by hematologic neoplasms, one did not undergo histopathological examination (a pleural fluid sample infiltrated by T-cell lymphoblastic lymphoma). The interval between the start of treatment and the date of the histopathological report ranged from –22 to +14 days, with a mean of 4 days. Seven patients began treatment before the histopathological report was available (–22 to –4 days), including 3 with T-cell lymphoblastic lymphoma, 2 with Burkitt lymphoma, 1 with B-cell post-transplant lymphoproliferative disorder (PTLD), and 1 with T-cell PTLD.

The diagnostic workflow is useful for the screening of pediatric lymphomas and enables the early initiation of treatment, particularly in cases of aggressive diseases. The implementation of a targeted panel for the identification of HL is essential to achieve more accurate results.