Utility of flow cytometric PAX5 protein detection for the diagnosis of B-cell acute lymphoblastic leukemia

OBJECTIVES: Accurate 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. METHODS: 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). RESULTS: 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). CONCLUSIONS: 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.