Laboratory Studies
Characteristics of the Ags determine delayed-type hypersensitivity (DTH) skin test reactivity. Conjugation of the Ag to lipids facilitates DTH reaction. This explains the consistent response to mycobacteria in which Ag are isolated from the lipid cell wall. Size, valence, chemical composition, and dose are additional factors that are relevant to immunogenicity. Repetitive testing with the same Ag can cause an immediate IgE-mediated response and may diminish the DTH skin test reactivity. High doses of Ag that induce predominant Th2 responses, such as in miliary tuberculosis, abrogate the DTH responses by a negative feedback mechanism elicited by Th2 responses.
By convention, the Ags used for DTH skin testing are injected intradermally into the volar surface of the forearm with a volume of 0.1 mL each. Erythema and induration are measured at 24, 48, and 72 hours. A reaction at 24 hours does not represent DTH induced by CMI, or type IV reactivity. The Food and Drug Administration (FDA) –approved antigens for DTH skin testing are limited to PPD and Candida Ag.
Conventionally, children are tested with Candida and Dermatophytin in a 1:10 or 1:100 dilution and tested with tetanus in a 1:10 or 1:100 dilution of the diphtheria-tetanus (DT) vaccine. The higher dilution is used when the child has undergone a significant infection or unusually frequent immunization respectively.
Adults are initially tested with the 1:100 concentrations of these Ags.
When interpreting DTH skin reactivity, whether adequate exposure to the Ags has taken place prior to the procedure must be considered. A vigorous immune response to one Ag, such as in measles infection, leads to the abrogation of other DTH responses, for example, to PPD even though the patient is also infected with tuberculosis.
Ags that are poorly immunogenic in children and in some adults include mumps (no longer on the US market) and Trichophyton. Dinitrochlorobenzene (DNCB) and dinitrofluorobenzene (DNFB) have been superseded by in vitro assessments of CMI because of the risk of local tissue necrosis.
When an absent DTH reaction is noted, screening tests for a T-cell disorder should include enumeration of T and B cell subsets, and a chest radiograph to detect the thymus. Cell surface marker analysis of peripheral blood mononuclear cells (PBMCs) by flow cytometry and in vitro lymphocyte proliferation responses or production of T cell cytokins in response to mitogens (polyclonal stimulants) and recall antigens are also indicated.
Contact hypersensitivity to poison ivy and nickel is determined clinically; skin testing is not considered necessary. Nickel DTH reaction can be assessed by patch skin testing.
Adverse drug reactions to antibiotics, phenytoin, and carbamazepine may involve non-immune or immune-mediated mechanisms. The clinical setting of a reaction at 3 days or later with manifestation of a fixed rash with induration is more suspicious of involvement of a DTH response.
Imaging Studies
A chest radiograph to determine whether the thymus is present is an appropriate screening test for T-cell disorders only in the newborn period and early infancy; however, the thymus may involute in stressed infants in the context of overwhelming infection or severe congenital cardiac disease.
Other Tests
When DTH response is absent and a T-cell immunodeficiency is suspected, testing T-cell functions in vitro is indicated. Typically, lymphocyte proliferation responses against polyclonal stimulants such as mitogens (eg, phytohemagglutinins [PHA], concanavalin A [conA], pokeweed mitogen [PWM]) and specific antigens (eg, Candida, tetanus) are assessed. Measurement of production of IFN-γ, TNF-α, and IL-12 in response to various stimulants can also be helpful for screening severe T cell immunodeficiency as well as in mutations in IFNGR1, IFNGR2, STAT-1, IL12P40, or IL12RB1 when such mutations are suspected. Low levels of one or more of these cytokines increase the likelihood of these mutations.
In patients with severe eczema, recurrent skin abscesses, elevated IgE, and history of frequent bone fractures, assessment of IL-17 production or IL-17 expression in Th cells may be helpful. This is because patients with hyper-IgE syndrome caused by STAT3 mutation or DOCK8 deficiency have impaired development of Th17 cells, which is a major cellular source of IL-17. [17, 18]
Cell surface markers for monocytes, T-cells (CD4, CD8, CD28, TCR α/β, TCR γ/δ), and activated T cells (CD25, HLA-DR, and CD5) are reported to be normal in IFNGR1, IL12P40,STAT1,IL12RB1, STAT3 mutations. In profound primary T-cell deficiencies such as SCID, the pattern of cell surface marker expression of lymphocyte and natural killer (NK) cells may identify the type of T-cell defect in conjunction with the clinical manifestations.
Mutational analysis for IFNGR1, IFNGR2, STAT-1,STAT-3, IL12P40, and IL12RB1 is available in commercial laboratories and/or specific research laboratories.
Additional genes that control downstream immune responses initiated by IFN-γ in the DTH response are recognized; IFNGR2 does not bind IFN-γ but is needed for the activation of STAT-1 and its translocation to the nucleus.
Procedures
See the list below:
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When disseminated BCG or NTM is suspected, perform biopsy of infected sites in order to examine granuloma formation and detect acid-fast mycobacteria.
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Tissue culture to detect mycobacteria is also indicated when disseminated BCG or NTM is suspected.
Histologic Findings
See the list below:
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Granuloma formation in an intact DTH response shows predominant infiltrates of activated macrophages and lymphocytes that can be identified as CD4+ T cells by immunohistochemical staining.
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When NTM infection is present, multinucleated giant cells formed by fused activated macrophages are observed in the immunocompetent host.
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In the patient with a T-cell defect, the formed granuloma lacks CD4+ T cells and these giant cells (due to ineffective macrophage activation by T cells). Instead, granulomatous lesions are characterized by infiltrate of polymorphonuclear cells, vacuolated cells, and macrophages.
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Mycobacteria may be present in abundance but are not frequently stained, although they are isolated by culture techniques.
