Reference no: EM132383215
Primary Immunodeficiencies (PIDs) are malfunctions of the immune response, which are caused by inherited genetic defects. The environment may still be playing a role in the phenotype observed but the key causal factor of the immune malfunction is genetic mutations/alleles a patient is born with. This is in contrast to secondary immunodeficiency, which is acquired (from environmental factors like an HIV infection, or malnutrition).
The exact symptoms and course of an immunodeficiency will depend on what aspect of the immune response is missing/damaged. As many genes and cells are involved in the immune response, and environment conditions can impact the immune system as well, there are a large number of immunodeficiencies.
Nitroblue-tetrazolium (NBT) is a yellowish, soluble compound that when reduced is converted to Formazan, a blue insoluble compound. Dihydrorhodamine-123 (DHR-123) is a colourless compound that upon oxidation is converted into rhodamine-123 (R-123), a fluorescent compound.
NADPH Oxidase is activated in neutrophils during the oxidative burst response. The oxygen radicals formed during these responses can reduce NBT and oxidize DHR-123. Therefore the presence of the blue, insoluble Formazan deposits or the fluorescent R-123 indicates a working oxidative burst mechanism.
Normally NADPH Oxidase is only activated when neutrophils detect an infectious microorganism (such as when their TLR receptors bind a bacterial ligand). In the lab, rather than use a whole infectious organism, a variety of mechanisms exist for stimulating neutrophils to activate NADPH Oxidase (including using solutions of latex toxin or bacterial filtrate [pieces]).
Brief Procedure: The principles behind the DHR-123 test and the NBT Reduction test are similar. Whole blood is collected from the patient and some blood is combined with NBT solution or with DHR-123.
The blood-NBT or blood-DHR-123 mixture is mixed and incubated. For the NBT Reduction test the mixture is then smeared on a slide and cells are stained for identification. The appearance of cells is observed; the numbers of neutrophils with and without formazan deposits are counted. The DHR-123 test uses FACS (Fluorescence-activated cell sorting) analysis to measure the number of cells with and without the fluorescent R-123 compound.
Both stimulated and unstimulated tests can be performed. In the unstimulated test the procedure above is followed. In the stimulated test both NBT or DHR-123 and a stimulation solution (such as a solution containing latex toxin) are combined with the blood. Incubation, smearing, staining, and counting of cells (NBT test), or FACS analysis (DHR-123 test) is the same in both the stimulated and unstimulated tests.
- If you performed the NBT reduction test on normal individuals whose neutrophils were capable of performing the oxidative burst response would you expect to see more blue cells (cells with Formazan) deposits in the stimulated or unstimulated NBT test? Explain your answer.
- If you were to perform the stimulated and unstimulated DHR-123 test on individuals whose oxidative burst responses did not work what would you expect to see? Explain your answer.
Note: these results are relative to "normal" (normal = functional oxidative burst) so would you expect to see more cells with fluorescent R-123 than normal or less? You should have an answer for both the stimulated and unstimulated test.
If a patient has abnormal results in either the CH50 or AH50 test of complement function further testing of individual complement components can be done.
If you were treating a patient whose AH50 test came back abnormal (significantly lower level of cell lysis) but whose CH50 test was normal, would you order the following follow up tests? For each of the three tests answer whether you would order it and explain why or why not.
Test of the _______ level