Pootrakul T.Khiankaew B.Boonsakan P.Paisooksantivatana K.Mahidol University2026-02-072026-02-072026-01-01Clinical Chemistry and Laboratory Medicine (2026)14346621https://repository.li.mahidol.ac.th/handle/123456789/114836Accurate B-lineage assignment in acute leukemia is critical for therapeutic decisions. While nuclear expression of PAX5 protein (nPAX5) is a highly reliable marker for B-cell differentiation, its conventional assessment by time-consuming immunohistochemistry (IHC) is not ideal for rapid diagnosis. This study aimed to validate the utility of flow cytometric (FCM) detection of nPAX5 for the diagnosis of B-lymphoblastic leukemia (B-ALL), potentially enhancing diagnostic efficiency. A retrospective study was conducted, comparing nPAX5 expression by FCM and IHC in 125 bone marrow biopsies (57 B-ALL, 12 T-ALL, 56 AML). Further diagnostic performance analysis, using ROC analysis of the geometric mean fluorescence intensity ratio (Blast/Normal T-cell), was performed for nPAX5, cCD22, and cCD79a across a larger cohort of 538 acute leukemia cases (123 B-ALL, 29 T-ALL, 386 AML). FCM and IHC for PAX5 expression showed 100% concordance across the 125 cases (57/57 B-ALL positive; 0/12 T-ALL negative). The single PAX5-positive AML case harbored the t(8;21) translocation. ROC analysis demonstrated excellent diagnostic performance for both cCD79a (AUC 0.980) and nPAX5 (AUC 0.955). At optimal criteria, cCD79a achieved 100.00% specificity and 91.06% sensitivity. nPAX5 showed strong utility, matching the 91.06% sensitivity with 89.45% specificity (criterion >6.30). cCD22 exhibited moderate discriminatory power (AUC 0.807). Flow cytometric nuclear PAX5 (nPAX5) demonstrated 100% agreement with IHC and exhibited excellent diagnostic accuracy (AUC 0.955, 91.06% sensitivity). These compelling results validate nPAX5 as a reliable and powerful FCM marker, supporting its immediate adoption for rapid and efficient B-lineage assignment in acute leukemia diagnostics.Biochemistry, Genetics and Molecular BiologyMedicineArticleSCOPUS10.1515/cclm-2025-16472-s2.0-10502861965914374331