Immunodiagnostics

Immunoassays are plate-based techniques that can detect and quantify many types of molecules through antibody-antigen reactions. An immunoassay typically involves an analyte, a targeted antibody, and labels. Classification of immunoassays is based on the type of label utilized, which includes enzymes (ELISA), light-emitting molecules/tracers (e.g., chemiluminescence and fluorescence immunoassays), and radioactive isotopes (radioimmunoassays). These specialized immunoassays are relatively sensitive, specific, inexpensive, and rapid, and are widely used in a clinical setting. Immunoassays are used in the diagnosis of infectious diseases, identification of tumor markers, allergy testing, and monitoring drug levels.

Blotting techniques involve the separation (via electrophoresis) and transfer of DNA, RNA, or proteins onto a blotting membrane. This separation is generally followed by complexing of the target with a labeled molecule for detection. Southern blotting is used to evaluate for specific DNA sequences and may be used in identification of genetic mutations and in forensics. Northern blotting focuses on RNA sequences and is helpful in assessing gene expression. Western blotting identifies proteins and antibodies and has applications in diagnosing infectious diseases, protein abnormalities (such as prion disease), and autoimmune conditions. Although these tests have good specificity, they have significant disadvantages owing to their expense, labor-intensiveness, and slow turnaround time.

Chromosomal testing can be accomplished using several techniques, all of which can identify chromosomal abnormalities. Karyotyping is the staining, organization, and visualization of chromosomes, which can help identify aneuploidy and major structural changes. Karyotyping is not sensitive in identifying small abnormalities and is a labor-intensive process. Chromosomal microarray analysis is a comparative technique that utilizes fluorescence to identify and quantify specific genetic sequences and is much more sensitive in identifying copy number variants, such as microdeletions or microduplications. However, chromosomal microarray analysis is not useful in identifying certain variations such as balanced translocations. Fluorescence in situ hybridization utilizes fluorescent probes to identify and locate specific genes on chromosomes. Compared with karyotyping, FISH is much more sensitive and specific in determining several abnormalities (except for point mutations) but is limited by the currently available gene probes.

Polymerase chain reaction (PCR) is a technique that amplifies DNA fragments exponentially for analysis. The process is highly specific, allowing for the targeting of specific genomic sequences, even with minuscule sample amounts. The PCR cycles multiple times through 3 phases: denaturation of the template DNA, annealing of a specific primer to the individual DNA strands, and synthesis/elongation of new DNA molecules. From there, the DNA can be visualized with analysis techniques such as gel electrophoresis. The speed, inexpensiveness, ease of use, and high sensitivity and specificity make this technique highly useful for the basic and biomedical sciences, and it has become instrumental in many applications, including forensic analysis, diagnosis of infectious diseases, and diagnosis and screening of genetic abnormalities.

B lymphocytes, also known as B cells, are important components of the adaptive immune system. In the bone marrow, the hematopoietic stem cells go through a series of steps to become mature naive B cells. The cells migrate to secondary lymphoid organs for activation and further maturation. The process entails antigen stimulation, with or without the help of T cells. The T-cell–independent activation generates a short-lived immune response (via plasma cells), and this is seen with antigens such as bacterial lipopolysaccharides. T-cell–dependent activation, on the other hand, produces both plasma cells and memory cells. Activated B cells then proliferate in the germinal centers, but not all become effector B cells. Through somatic hypermutation, B cells undergo additional mechanisms to increase the affinity of the antibody to the antigen. Only those with high-affinity B-cell receptors subsequently advance for terminal differentiation. B cells then go through class switching (from IgM to another class of Ig) under the influence of cytokines. After class switching, the B cells become plasma cells (which produce antibodies) or memory cells (which mount a robust secondary immune response).

T cells, also called T lymphocytes, are important components of the adaptive immune system. Production starts from the hematopoietic stem cells in the bone marrow, from which T-cell progenitor cells arise. These cells migrate to the thymus for further maturation. A functional mature T cell develops from a step-by-step process creating a T-cell receptor (TCR) complex, selecting T cells with appropriate affinity to self-antigens associated with major histocompatibility molecules (positive selection), and expressing either CD4 or CD8. In this series, cells predisposed to autoimmunity undergo apoptosis (negative selection). When released from the thymus, the naive mature T cells travel to the secondary lymphoid organs for activation. Two signals, an antigen-specific binding of TCR and costimulation, are required to be activated (ready to mount an immune response). In the case of CD8+ T cells, additional cytokine stimulation is necessary. Depending on the cytokines they are exposed to during antigen stimulation, the undifferentiated mature T cell (Th0) develops into cells with different functions: CD4+ become T helper (Th) cells and CD8+ become cytotoxic, or cytolytic, cells. Th cells have other subtypes; the most characterized are Th1, Th2, Th17, follicular Th cells, and regulatory T cells. Other types include natural killer T cells and memory T cells. These mature differentiated T cells ensure effective surveillance and immediate response to pathogens, tumor cells, and foreign tissues and provide immunologic memory.