[Software Testing Limaye.pdf
Everardo Laboy
Defence against potentially harmful pathogens is achieved by physical barriers such as skin and mucous membranes, and the coordinated efforts of the innate and adaptive immune systems. Innate immune responses are carried out by macrophages, neutrophils and natural killer cells, together with cytokines, complement and acute phase reactants such as C-reactive protein. Adaptive immunity relies upon B and T lymphocytes which express antigen-specific surface receptors. It can be divided into humoral (antibody-mediated and dependent upon B lymphocytes) and cellular (coordinated by T lymphocytes) immunity. While this distinction is oversimplified and somewhat inaccurate in that both types of responses are dependent upon helper T lymphocytes, it provides a useful model for classifying and evaluating suspected immunodeficiency.
This occurs when failure of any part of the immune system leads to an increased predisposition to infection and associated sequelae such as autoimmunity and malignancy. Primary immunodeficiency results from genetic mutations of components intrinsic to the immune system. Clinical diagnosis should be accompanied by molecular identification of a genetic mutation wherever possible to confirm the diagnosis, identify genotype-phenotype correlation, assist with genetic counselling and identify suitable candidates for gene-specific therapy. Secondary immunodeficiency results from defective immune function as a consequence of another condition such as HIV infection. Drugs such as corticosteroids, azathioprine, methotrexate or cyclosporin can also cause secondary immunodeficiency. Subtle impairment of immune function can also accompany certain chronic medical conditions including diabetes and chronic renal failure.
Immunodeficiency can be classified functionally into humoral or cell-mediated arms, as dysfunction of either pathway is characterised by specific clinical presentations (Table 1). Possible investigations for suspected immunodeficiency are presented in Table 2. These tests should be performed when the patient is clinically well, and not during an acute infective illness.
Antibody deficiency, or hypogammaglobulinaemia, can occur as a result of intrinsic defects of humoral immunity (primary), or secondary to another pathological condition. It is the most common manifestation of primary immunodeficiency and encompasses a broad range of clinical diagnoses. Clinical presentation can range from asymptomatic, to recurrent, atypical or life-threatening infections. Encapsulated bacteria, such as Streptococcus pneumoniae, Neisseriae species and Haemophilus influenzae, pose a particular threat as well as other bacterial species including Staphylococcus aureus, Pseudomonas aeruginosa, Campylobacter fetus and Mycoplasmaspecies. Recurrent or unusually severe sinopulmonary infection, other infections (gastrointestinal, skin, joint or central nervous system), or evidence of end-organ damage such as bronchiectasis, should alert the doctor to the possibility of an underlying humoral immunodeficiency.
Defective T cell-mediated immunity predisposes patients to a broader range of infections than humoral immunodeficiency, including intracellular pathogens, persistent superficial candidiasis or recurrent viral, fungal or protozoal infections (Table 1). Defects can again be classified as either primary, or secondary to extrinsic factors. HIV infection resulting in progressive depletion of CD4T cells is a particular consideration. As helper T cells are required for B cell-mediated antibody production, T cell immunodeficiency can result in functional B cell defects, thus patients with cell-mediated immunodeficiency often have an accompanying hypogammaglobulinaemia. This is termed combined immunodeficiency.
Measurement of cell-mediated immunity can be undertaken by both in vitro and in vivo methods. It is, however, more problematic than humoral assessment as assays are plagued by difficulties in standardisation, biological variability, imprecision and technical complexity. Most tests are highly specialised and referral to a clinical immunologist is recommended.
The first step in the evaluation of cell-mediated immunity is to quantify circulating immune cells and their subsets by flow cytometric analysis. Patients' blood cells are incubated with fluorochrome-labelled monoclonal antibodies directed against cell surface molecules and analysed by a flow cytometer, which measures light scatter and fluorescence emission from individual cells. Different cell populations (B cells, and CD4/CD8T cells and natural killer cells) can be distinguished based on their scatter profile and surface molecule expression. Absolute cell numbers are calculated as a percentage of the total white cell count and results are compared to age-matched reference ranges. It is important to note, however, that analogous to immunoglobulin measurement, quantification of lymphocyte numbers does not give an indication of their functional capacity. Lymphocyte subset analysis aids in the diagnosis and classification of paediatric severe combined immunodeficiency syndromes, and is also recommended in the evaluation of hypogammaglobulinaemia in common variable immunodeficiency. Quantifying CD4T lymphocytes provides prognostic information and gives an indication of susceptibility to opportunistic infections in patients with HIV infection.
Skin testing identifies functional memory T cells to a particular antigen, or the presence of cutaneous anergy. The latter is defined as an impaired cutaneous hypersensitivity response to a panel of common antigens and is consistent with cellular immune dysfunction. Causes of cutaneous anergy are listed in Table 3.
These assays are technically complex and are only performed by specialist laboratories. As the investigation can be time-consuming, it is advisable to first discuss the appropriateness of testing and choice of assay with the laboratory. Results are affected by immunosuppressive drugs, severe nutritional deficiencies and intercurrent illness1 and these factors must be considered when interpreting results. As with skin testing, the patient must have been previously exposed to the antigen, thus antigen proliferation assays are not feasible in babies lessthan six months of age. Response to mitogens, however, can be performed at any age from birth onwards.2
Other functional in vitro measures of lymphocyte activation include determining changes in surface marker expression (CD25, CD69, CD71) following activation3 or measurement of intracellular cytokines of T lymphocytes.
T cell proliferation following stimulation can be measured by succinimidyl ester of carboxyfluorescein diacetate (CFSE) dilution techniques,4 or T cell cytokine production quantified by ELISPOT assays. These assays, however, are not in routine use and are confined to research or specialised reference immunology laboratories.
The recently introduced interferon gamma (IFNγ) release assays measure T lymphocyte production of IFNγ in response to antigen exposure thereby providing an assessment of cell-mediated immunity. As with delayed-type hypersensitivity skin testing, clinical application is currently confined to the domain of tuberculosis latency and exposure.
Assessing natural killer cells is indicated in patients suffering recurrent infection with herpes virus, or papillomavirus (associated with cutaneous warts). Natural killer cell cytotoxicity is assessed by a 51Cr-release assay in which patients' natural killer cells are incubated with 51Cr-labelled target cells. Lysis of the target cells by natural killer cells leads to the release of radioactivity which can be measured. Natural killer cell dysfunction may occur in patients with CD16 genetic mutations, chronic mucocutaneous candidiasis, severe combined immunodeficiency and other cellular immunodeficiency syndromes.5 These conditions need to be considered and excluded if natural killer cell dysfunction is confirmed. As with T and B lymphocytes, functional natural killer cell deficits can occur even when natural killer cell counts are normal. Natural killer cell assays are technically complex and are rarely performed in diagnostic immunology laboratories.
The evaluation of suspected immunodeficiency is guided by clinical presentation. Screening tests of humoral and cellular immune function are initially performed, followed by referral to a specialist for more advanced investigations if clinically indicated (Table 2). Secondary causes of immunodeficiency, including HIV infection, need to be considered and excluded. When interpreting results, confounding factors such as immunosuppressive drug therapy and patient comorbidities, as well as analytical variables such as assay precision and reproducibility, need to be considered.
795a8134c1