Screening for Primary Immunodeficiency

Clinical Red Flags (Clinical Screening)

• Primary immune deficiency diseases (PIDDs) encompass over 450 distinct disorders that impair the development or function of the immune system, with diagnosis frequently delayed due to a low index of clinical suspicion.
• Clinical screening relies on identifying warning signs or "red flags," which include recurrent or sentinel infections, fever without a clear focus, periodontitis, poor formation of pus at infection sites, and unusual autoimmune or inflammatory conditions.
• Patients with PIDDs often experience severe infections requiring hospitalization and prolonged intravenous antibiotics, or present with infections caused by opportunistic pathogens or live attenuated vaccines.
• Clinical suspicion for specific immune defects is guided by distinct patterns of infection:
  • Antibody (B-cell) defects: Characterized by recurrent bacterial sinopulmonary infections (e.g., Streptococcus pneumoniae, Haemophilus influenzae) and gastrointestinal infections (e.g., Giardia, Salmonella, enteroviruses).
  • Cell-mediated (T-cell) defects: Suggested by infections with Pneumocystis jirovecii, Mycobacterium avium-intracellulare, severe viral infections (e.g., Epstein-Barr virus, cytomegalovirus), chronic mucocutaneous candidiasis, and failure to thrive.
  • Phagocytic defects: Manifest as recurrent skin, liver, and lymph node abscesses (commonly involving Staphylococcus aureus, gram-negative bacteria, and fungi), severe periodontitis, and poor wound healing.
  • Complement defects: Screened for clinically when patients present with early-onset systemic lupus erythematosus or recurrent sepsis and meningitis caused by blood-borne encapsulated bacteria (e.g., Neisseria, Pneumococcus).

Newborn Screening

• Newborn screening programs facilitate the early detection of severe combined immunodeficiency (SCID) and other immunodeficiencies associated with severely low T-cells, enabling treatment before the onset of symptomatic infections.
• The primary screening method utilizes a quantitative polymerase chain reaction (PCR) assay to measure T-cell receptor excision circles (TRECs).
• TRECs are DNA byproducts generated during the V(D)J rearrangement of T-cell receptor chains; because they do not replicate during cell division, they serve as an accurate marker for quantifying recent thymic emigrants.
• A low or absent TREC count on a newborn screen raises high suspicion for SCID, necessitating prompt evaluation of lymphocyte subsets and functional testing.
• In certain regions, kappa excision circles (KRECs)—which are generated during early B-cell development—are assayed simultaneously with TRECs to identify infants with profound B-cell defects, such as agammaglobulinemia.

Laboratory Screening Approach

• Initial laboratory evaluation for suspected immunodeficiency begins with a complete blood count (CBC) with differential to identify baseline abnormalities such as lymphopenia, neutropenia, or marked leukocytosis.
• Screening for Humoral (B-cell) Defects:
  • Quantitative measurement of serum immunoglobulin levels: IgG, IgA, IgM, and IgE.
  • Assessment of specific antibody production by measuring titers in response to protein vaccines (e.g., diphtheria, tetanus) or polysaccharide vaccines (e.g., pneumococcus).
  • Flow cytometry to identify and enumerate circulating B-cells and memory B-cell subsets using surface markers such as CD19 and CD20.
• Screening for Cellular (T-cell) Defects:
  • Flow cytometry to quantify total T-cells, helper T-cells (CD4), cytotoxic T-cells (CD8), and natural killer (NK) cells.
  • Analysis of naïve versus memory T-cell populations by evaluating CD45 isoform expression.
  • Functional screening utilizing lymphocyte proliferation assays to evaluate T-cell responses to mitogens (e.g., phytohemagglutinin) or specific antigens.
• Screening for Phagocyte Defects:
  • The dihydrorhodamine (DHR) reduction assay by flow cytometry or the nitroblue tetrazolium (NBT) slide test is employed to evaluate the neutrophil oxidative burst, serving as the primary screen for chronic granulomatous disease (CGD).
  • Flow cytometry to assess the surface expression of adhesion molecules, such as CD11b/CD18, for the screening of leukocyte adhesion deficiency type 1 (LAD-1) or CD15 for LAD-2.
• Screening for Complement Defects:
  • The total hemolytic complement assay (CH50) is the optimal screening test to evaluate the integrity of the classical complement pathway.
  • The AH50 assay is utilized to screen the functional activity of the alternative complement pathway.
  • If CH50 or AH50 screening results are abnormal, further immunochemical quantification of single complement components (e.g., C3, C4) is indicated.
• Advanced Genetic Screening:
  • Following abnormal functional or quantitative screening, definitive diagnosis is established through genetic mutation analysis using targeted primary immunodeficiency gene panels, whole-exome sequencing, or whole-genome sequencing.