Table 2.
Suggested IEI clinically guided laboratory investigation guidelines, according to three proposed main consecutive steps: screening, advanced tests, and molecular confirmation.
| Screening | |
| Cell blood count and peripheral smear Serum immunoglobulins (IgG, IgA, IgM and total IgE) Vaccine response against polysaccharide (e.g.: Streptococcus pneumoniae) and protein antigens (e.g.: tetanus toxoid); spontaneous specific antibodies (anti-blood group Abs, isohemagglutinins) Peripheral blood basic immunophenotyping: CD3, CD4, CD8, CD19, and CD16/CD56 Complement system assessment: CH50 and AH50 Phagocyte oxidative burst: dihydrorhodamine oxidation assay TREC copies | |
| Advanced tests | |
| Predominantly antibody deficiencies | IgG subclasses Vaccine response against neoantigens (bacteriophage, Salmonella typhi capsular polysaccharide vaccine) B cell immunophenotyping Intracellular protein expression – BTK |
| Combined immunodeficiencies affecting cellular and humoral immunity | Chromosomal instability T cell immunophenotyping (flow cytometry) CD40/CD40L binding assay Cell surface protein expression – CD132 (IL-2Rγ), CD127 (IL-7Rα), MHC I and II Intracellular protein expression – WASP (Wiskott-Aldrich protein) Lymphoproliferation in response to mitogens, alloantigens and recall antigens TCR repertoire analysis: TCR-CDR3 spectratyping and flow cytometry-based TCR Vβ frequency Adenosine deaminase and PNP activity In vitro cytokine production in cell culture supernatant |
| Diseases of immune dysregulation | NK cytotoxic activity assay and CD107a degranulation Soluble CD25 Intracellular protein expression (PRF1, SAP/SH2DIA, XIAP) Double negative TCRα/β circulating T cells Lymphocyte apoptosis assay Soluble mediators: IL-10, IL-18, soluble FASL and vitamin B12 T regulatory cells (CD4+/CD25+/CD127low/Foxp3+) number and function STAT1 phosphorylation assay CTLA-4 functional testing |
| Defects in intrinsic and innate immunity | IL-12/IFNγ axis functional assay Intracellular protein expression: IFNγ-R1 and IFNγ-R2 Cell surface protein expression: CD18, CD11a/CD11b/CD11c, CD15 IκBα degradation TLR functional assays (CD62L shedding) |
| Autoinflammatory disorders | Type 1 interferon signature Serum IgD Urinary mevalonic acid |
| Complement deficiencies | Specific complement components |
| Genetic and molecular tests | |
| Karyotype, FISH, MLPA, copy number variation analysis Specific single gene-sequencing (Sanger) Next-generation sequencing (panels, whole-exome or genome sequencing) | |
A list of the main nonfunctional IEI diagnostic tests 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.