Laboratory Features of PID

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Initial Screening and Level 1 Testing

The initial diagnosis of primary immune deficiency diseases (PIDDs) requires a high index of suspicion and is frequently initiated when a patient presents with recurrent, severe, or unusual infections, autoimmunity, or failure to thrive,.
Basic screening must begin with a complete blood count (CBC) with differential, which is essential to identify baseline hematologic abnormalities such as severe neutropenia, persistent lymphopenia, or marked leukocytosis,,,.
A peripheral blood smear should be reviewed for distinct morphological clues, such as the presence of giant lysosomal granules characteristic of Chédiak-Higashi syndrome, or Howell-Jolly bodies indicative of isolated congenital asplenia,.
Newborn screening programs facilitate the early detection of severe combined immunodeficiency (SCID) by utilizing a quantitative polymerase chain reaction (PCR) assay to measure T-cell receptor excision circles (TRECs),.
TRECs are DNA byproducts formed 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.
Many newborn screening programs concurrently assay kappa excision circles (KRECs), which are generated during B-cell development, allowing for the simultaneous identification of infants with severe B-cell defects like congenital agammaglobulinemia.

Evaluation of Suspected Humoral (B-Cell) Deficiencies

Quantitative Immunoglobulins

Initial humoral evaluation requires the quantitative measurement of serum immunoglobulin levels, specifically IgG, IgA, IgM, and IgE, which must be compared to age-matched normal values,,,,.
A profile demonstrating very low or absent IgG, IgA, and IgE alongside normal or elevated IgM strongly suggests a class switch recombination defect, such as hyper-IgM syndrome,.
An isolated profound decrease in IgA (less than 5 mg/dL) with normal IgG and IgM confirms the diagnosis of selective IgA deficiency, though this diagnosis cannot be definitively established until approximately 4 years of age,.
The classic immunoglobulin profile in Wiskott-Aldrich syndrome demonstrates low IgM, elevated IgA and IgE, and normal or slightly decreased IgG.

Specific Antibody Responses

Evaluating specific antibody titers in response to previous routine vaccinations is critical for assessing functional humoral immunity,,.
Functional antibody capacity is tested by measuring titers against protein antigens (e.g., diphtheria and tetanus) and polysaccharide antigens (e.g., Haemophilus influenzae and pneumococcus),,.
In children over 2 years of age with normal total immunoglobulin levels but recurrent sinopulmonary infections, specific antibody deficiency (SAD) is evaluated using the 23-valent pneumococcal polysaccharide vaccine (PPV23),.
Protective antibody titers against pneumococcal polysaccharides are analyzed using an enzyme-linked immunosorbent assay (ELISA), with a titer of 1.3 mg/mL generally considered the protective threshold against mucosal infections.
SAD is classified into four phenotypes based on the PPV23 response: a severe phenotype lacks protective titers to most serotypes, a moderate phenotype depends on age-specific cutoffs, a mild phenotype involves failure to multiple serotypes, and a memory phenotype shows an adequate initial response that rapidly wanes within 6 months,.

B-Cell Enumeration and Phenotyping

Flow cytometry is mandatory for identifying, enumerating, and characterizing circulating B-cell populations using surface markers such as CD19 and CD20,,,.
The complete absence of CD19+ B-cells in the peripheral blood confirms the diagnosis of congenital agammaglobulinemias, such as X-linked agammaglobulinemia (XLA),.
Advanced flow cytometric analysis is utilized to enumerate the specific percentages of naive B-cells, memory B-cells, and class-switched memory B-cells,.
A specific deficiency or paucity of class-switched memory B-cells is a hallmark diagnostic feature in patients with Common Variable Immunodeficiency (CVID) or hyper-IgM syndrome,.

Evaluation of Suspected Cell-Mediated (T-Cell) Deficiencies

T-Cell Enumeration

When a T-cell or combined immunodeficiency is suspected, flow cytometry is used to precisely quantify total T-lymphocytes and their subsets utilizing markers such as CD3 (total T cells), CD4 (T-helper cells), and CD8 (cytotoxic T cells),,,.
The ratio of CD4+ to CD8+ T cells is analyzed, as significant alterations can indicate an underlying immunodeficiency or immune dysregulation.
Flow cytometry is also utilized to differentiate naive T cells from memory T cells by evaluating the surface expression of specific CD45 isoforms.

T-Cell Functional Assays

Functional T-cell competence is evaluated in vitro via lymphocyte blast transformation and proliferation assays,,.
Isolated peripheral blood mononuclear cells are stimulated in cell culture with specific mitogens, such as phytohemagglutinin (PHA) or concanavalin A, or with specific recall antigens like tetanus toxoid,,,.
A proliferative response to the mitogen PHA that is less than 10% of a normal control is a definitive diagnostic criterion confirming a severe combined immunodeficiency (SCID).
For infants presenting with SCID, fluorescence in situ hybridization (FISH) targeting the X and Y chromosomes can be performed in males to detect the presence of transplacentally engrafted maternal (XX) T cells.

Natural Killer (NK) Cell Evaluation

NK cells are evaluated first by enumerating their absolute count and percentage within the lymphocyte population using flow cytometry targeting surface markers CD16 and CD56,,.
Flow cytometry can further delineate functionally distinct NK cell subsets, such as CD56-dim and CD56-bright populations, to calculate specific ratios useful for diagnosing conditions like GATA2 deficiency.
NK cell functional cytotoxicity is measured utilizing either a flow cytometry-based killing assay or the traditional radioactive chromium release (51CR) cytotoxicity assay.
Because NK cells utilize lytic granules to destroy targets, activation-induced degranulation can be functionally assessed by using flow cytometry to measure the upregulation of CD107a (lysosomal-associated membrane protein-1) on the NK cell surface.

Evaluation of Suspected Phagocytic Defects

Functional Assays for Oxidative Burst

Patients presenting with deep tissue abscesses, osteomyelitis, or infections with catalase-positive organisms require functional evaluation of the neutrophil respiratory burst to screen for Chronic Granulomatous Disease (CGD),.
The dihydrorhodamine (DHR) reduction assay, performed via flow cytometry, is the gold standard test; it measures oxidant production through increased fluorescence when DHR is oxidized by intracellular hydrogen peroxide,,,.
The DHR flow cytometric assay is highly sensitive and has largely replaced the older nitroblue tetrazolium (NBT) slide test,,.
In cases of severe myeloperoxidase (MPO) deficiency, the DHR assay for neutrophils may yield a false-positive result mimicking CGD; however, evaluating the patient's eosinophils, which still reduce DHR normally in MPO deficiency, reliably differentiates the two conditions.
Flow cytometry via DHR testing is also highly effective for identifying the X-linked carrier state of CGD in mothers by demonstrating a bimodal response to neutrophil stimulation.

Assays for Leukocyte Adhesion and Motility

When Leukocyte Adhesion Deficiency (LAD) is suspected due to severe omphalitis, extreme neutrophilia, and absent pus formation, flow cytometry is used to evaluate specific surface adhesive glycoproteins,,,.
The diagnosis of LAD type 1 is established by flow cytometric measurements demonstrating the absence or severe reduction of CD11b and CD18 (beta-2 integrins) on the surface of stimulated and unstimulated neutrophils,,.
The diagnosis of LAD type 2, a distinct defect in fucosylation, is established by flow cytometric measurement demonstrating the absence of the sialyl Lewis X antigen (CD15) on neutrophils,,.
While specialized biological assays exist for evaluating neutrophil and monocyte adherence, aggregation, and chemotaxis, they possess high variability and are generally restricted to specialized research laboratories,,.

Evaluation of Suspected Complement Deficiencies

Global Pathway Screening

The functional integrity of the complement system is initially assessed using global hemolytic assays,,.
The CH50 assay evaluates the total functional activity of the classical complement pathway by measuring the complement-mediated lytic destruction of antibody-sensitized sheep erythrocytes,,.
The AP50 or AH50 assay specifically screens the functional activity of the alternative complement pathway,,,.
Alternatively, an enzyme-linked immunosorbent assay (ELISA) can screen pathway activation by detecting the full assembly of the terminal MAC complex (C5b-9) in microtiter wells coated with specific activating agents (e.g., IgM for the classical pathway or mannan for the lectin pathway).

Component Quantification and Activation Profiling

If initial screening by CH50 or AH50 reveals an abnormality, specific immunochemical tests, such as radial immunodiffusion, ELISA, or nephelometry, are utilized to definitively quantify single complement components like C3, C4, properdin, or mannose-binding lectin (MBL),,.
Low CH50 results might reflect a primary genetic deficiency, or they could indicate acquired complement consumption secondary to active immune complex diseases, such as systemic lupus erythematosus,.
To distinguish between a primary genetic deficiency and active acquired consumption, specific complement activation products and split fragments (such as C3a, C4d, Ba, Bb, and the soluble terminal complement complex sC5b-9) are quantified.
The evaluation of Paroxysmal Nocturnal Hemoglobinuria (PNH) relies on flow cytometry to detect abnormally reduced surface levels of specific complement regulatory proteins, namely CD55 and CD59, on blood cells.

Advanced Molecular and Genetic Testing

The most definitive method for diagnosing the vast majority of primary immunodeficiency disorders is establishing the precise genetic etiology through mutation analysis,,.
Targeted gene sequencing utilizing specific SCID panels or broader primary immunodeficiency (PID) gene panels is routinely employed following abnormal functional laboratory results,.
Genetic diagnosis is strictly necessary in conditions categorized as primary immune regulatory disorders (PIRDs), which present predominantly with autoimmunity or hyperinflammation, because functional immunological testing is frequently normal,.
Definitive identification of specific pathogenic variants dictates feasibility and conditioning regimens for definitive cellular therapies, provides guidance for targeted biologic agents, and is essential for accurate prenatal genetic counseling,.