Diagnostic utility of targeted flow cytometry in genetically confirmed inborn errors of immunity: a 20-year...

Inborn errors of immunity (IEIs) are a heterogeneous group of genetic disorders in which timely diagnosis is essential for appropriate treatment and improved clinical outcomes. Although next-generation sequencing provides definitive molecular confirmation, it is often time-consuming, costly, and may not identify all disease-causing variants. We evaluated the diagnostic performance and diagnostic contribution of targeted flow cytometry (FCM) in genetically confirmed IEIs.

We retrospectively analyzed 243 pediatric patients with genetically confirmed IEIs who underwent targeted flow cytometric evaluation at a single center between 2005 and 2025. Immunological evaluation included T-, B-, and NK-cell immunophenotyping, detailed T- and B-cell subset analysis, targeted protein expression assays, and neutrophil oxidative burst assessment using the dihydrorhodamine 123 assay. Molecular diagnoses were established by targeted gene panels, whole-exome sequencing, or Sanger sequencing.

The median age at symptom onset and molecular diagnosis was 12 and 70 months, respectively. Predominantly antibody deficiencies (31.7%) and syndromic combined immunodeficiencies (27.1%) were the most frequent IEI categories. Abnormal FCM findings were detected in 153 patients (63%) and predicted the underlying genetic category in 41 (27%). Diagnostic yield reached 100% in combined immunodeficiencies and 82.6% in immune dysregulation disorders, significantly exceeding that of syndromic combined immunodeficiencies and antibody deficiencies (p < 0.05). Importantly, FCM findings preceded molecular confirmation by a median of 12 months.

Targeted flow cytometry provides rapid functional evidence that complements molecular testing in patients with IEIs. Its integration into the initial diagnostic evaluation facilitates early disease stratification, guides targeted genetic investigations, and supports timely therapeutic decision-making.