Diagnostic workflow for X-linked agammaglobulinemia integrating BTK expression and B-cell maturation analysis by flow...

X-linked agammaglobulinemia (XLA) is most often caused by impaired Bruton's tyrosine kinase (BTK) function. Rapid laboratory support is needed when genetic findings are inconclusive or pending. We developed a flow-cytometric assay to quantify intracellular BTK protein in monocytes, aiding the diagnosis of XLA. A stain index (SI) reference range was established using healthy donors (n = 25; SI median 1.14, range 1.01-1.66). ROC analysis defined a diagnostic cutoff of SI = 1.79, providing 95% sensitivity and 100% specificity compared to patients with pathogenic BTK variants (n = 40). Using this cutoff, abnormal BTK expression was detected in 38/40 genetically confirmed XLA patients. All patients with variants of uncertain/likely pathogenic significance (n = 17) had SI values above the cutoff. Bone marrow analysis in a patient subset showed a developmental arrest at the Pre-BI stage. The assay offers a rapid, clinically applicable readout to support XLA diagnosis and carrier evaluation in females, effectively complementing genetic testing.