Functional Clinical Flow Cytometry Assays to Assess Lymphocyte Proliferation, Neutrophil Oxidative Burst, and Leukocyte Adhesion Deficiency Immune Defects.

Inborn errors of immunity (IEI) are a diverse group of inherited disorders caused by detrimental variants in the genome that compromise the immune system and lead to an increased susceptibility to infections, autoimmunity, and/or malignancy. Although the diagnostic process traditionally begins with a clinical workup and laboratory screening, such as complete blood counts, serum immunoglobulin measurements, and lymphocyte subset enumeration, functional assays serve as a vital bridge to diagnosis by providing a real-time assessment of immune system performance. Such assays include lymphocyte proliferation assays for evaluating T cell and B cell defects along with dihydrorhodamine-123 (DHR-123) and leukocyte adhesion deficiency testing to assess neutrophil function. These assays facilitate rapid screening and provide evidence for therapeutic intervention while helping to validate the pathogenicity of novel genetic variants and variants of unknown significance. Ultimately, functional assay results are indispensable in both the diagnosis and monitoring of treatment efficacy for patients with IEIs. © 2026 Wiley Periodicals LLC. Basic Protocol 1: Evaluation of lymphocyte proliferation by EdU flow cytometry Basic Protocol 2: Dihydrorhodamine-123 (DHR-123) assessment of neutrophil oxidative burst Basic Protocol 3: Evaluation for leukocyte adhesion deficiency.