In summary, regulatory agencies ensure that new devices intended for the diagnosis, treatment, or prevention of disease are safe and effective. On the other hand, quality assessment programs are designed to regulate laboratories that perform testing on patient specimens to ensure accurate and reliable test results. focus on discussing the issues of nonfunctional immunoassays (flow cytometry, enzyme-linked immunosorbent assays, and turbidimetry/nephelometry, among others), as defined by the real quantification of proteins or cell subsets without cell activation or cell culture-based methods. Keywords: inborn errors of immunity, primary immunodeficiencies, quality assessment (QAS), standardization, reference range Introduction Primary immunodeficiency diseases (PID) constitute a large and fast-growing heterogeneous group of genetic diseases, mostly (but not exclusively) caused by loss or gain of function germline mutations. Although PID are classically manifested as increased PGF susceptibility to infections, recently, an increasing variety of autoimmune, autoinflammatory, allergic, and malignant phenotypes has also been acknowledged (1). This comprehensive concept was closely accompanied by a broader pathophysiological understanding of such disorders, which are now grouped in the category of inborn errors of immunity (IEI) (2). Despite individual rarity, IEI collectively represent a significant proportion of patients, with an estimated overall prevalence of 1 1:1,200-2,000 (3, 4). They now comprise 406 distinct disorders with 430 different gene defects subdivided into categories listed in the 2019 International Union of Immunological Societies (IUIS) classification (5, 6), approximately two-thirds of which were recognized in the past decade ( Table?1 ). As evidence of dynamic development in the area, 26 additional monogenic gene defects have recently been reported and may soon be included in the IEI portfolio (8). Table?1 IEI categories and clinical prototypes according to the 2019 IUIS update of the phenotypical classification (6). capsular polysaccharide vaccine)B cell immunophenotypingIntracellular protein expression C BTKCombined immunodeficiencies affecting cellular and humoral immunityChromosomal instabilityT cell immunophenotyping (flow cytometry)CD40/CD40L binding assayCell surface protein expression C CD132 (IL-2R), CD127 (IL-7R), MHC I and IIIntracellular protein expression C WASP (Wiskott-Aldrich protein)Lymphoproliferation in response to mitogens, alloantigens and recall antigensTCR repertoire analysis: TCR-CDR3 spectratyping and flow cytometry-based TCR V frequencyAdenosine deaminase and PNP activity cytokine production in cell culture supernatantDiseases of immune dysregulationNK cytotoxic activity assay and CD107a degranulationSoluble CD25Intracellular protein expression (PRF1, SAP/SH2DIA, XIAP)Double unfavorable TCR/ circulating T cellsLymphocyte apoptosis assaySoluble mediators: IL-10, IL-18, soluble FASL and vitamin B12T regulatory cells PBIT PBIT (CD4+/CD25+/CD127low/Foxp3+) number and functionSTAT1 phosphorylation assayCTLA-4 functional testingDefects in intrinsic and innate immunityIL-12/IFN axis functional assayIntracellular protein expression: IFN-R1 and IFN-R2Cell surface protein expression: CD18, CD11a/CD11b/CD11c, CD15IB degradationTLR functional assays (CD62L shedding)Autoinflammatory disordersType 1 interferon signatureSerum IgDUrinary mevalonic acidComplement deficienciesSpecific complement components Genetic and molecular assessments Karyotype, FISH, MLPA, copy number variation analysisSpecific single gene-sequencing (Sanger)Next-generation sequencing (panels, whole-exome or genome sequencing) Open in a separate window A list of the main nonfunctional IEI diagnostic assessments is provided and should be individually considered according to the stage of investigation. AH50, total hemolytic complement (alternative pathway); ALPS, autoimmune lymphoproliferative syndrome; CH50, total hemolytic complement (classic pathway); FISH, fluorescence in situ hybridization; IEI, inborn errors of immunity; MLPA, multiplex ligation-dependent probe amplification; TCR, T cell receptor; TLR, Toll-like receptors; TREC, T cell receptor excision circle. Despite the striking importance of laboratory data for clinical immunologists, several IEI relevant immunoassays still lack standardization, including standardized protocols, reference materials, and external quality assessment programs. Moreover, well-established reference values mostly remain to be decided, especially for early ages, when the most severe conditions manifest and diagnosis is critical for patient survival (10). Compared to biochemical assessments, standardization and quality controls in immunoassays are rudimentary, partially due to the particular complexity of analytes. Immunoassays usually assess heterogeneous molecules, such as serum polyclonal antibodies, that share common characteristics, but are in fact distinct analytes with individual features. The above-described issues reinforce the necessity of a parallel healthy control blood sample in some IEI diagnostic-driven nonfunctional immunoassays, assuming a high number of uncontrolled variables. This is particularly problematic in young patients whose PBIT blood is usually compared with adult control samples. Although challenging, assessments for the.