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ANTINUCLEAR ANTIBODY (ANA) TESTING

A sample of diluted patient serum is added to a monolayer of cells on a glass slide. If there are antibodies in the patient’s serum to an antigen in the nuclei of the cells on the slide, they will become bound. A fluorescent-labeled anti-IgG antibody is then added to detect antibody from the patient serum that has become bound to the cell nuclei. The specimen is reviewed by fluorescence microscopy. Information about the presence of antinuclear antibody is obtained in this test, and specific autoimmune disorders may be identified from the pattern of nuclear staining observed microscopically. Common nuclear staining patterns include rimmed (peripheral), homogeneous (diffuse), speckled, and nucleolar, along with others.

PROTEIN ELECTROPHORESIS (PEP)

Samples containing protein subjected to electrophoresis include serum, concentrated urine, and concentrated cerebrospinal fluid. The samples are applied to a gel, and the proteins in the specimen are separated into distinct bands based upon charge and size. After the protein bands are separated, the gel is stained, and the relative concentrations of the bands determined by densitometry. If there is a monoclonal protein identified, the sample is further evaluated to identify that particular protein in high concentration, with a primary concern for excess production of antibody associated with a disorder such as multiple myeloma.

IMMUNOFIXATION TO IDENTIFY MONOCLONAL IMMUNOGLOBULINS

Immunofixation can identify proteins through the use of antibodies which bind to specific proteins. A common use is to identify monoclonal immunoglobulins in the serum of a patient with a high concentration of a monoclonal protein, often as a result of multiple myeloma or a related disorder. Immunofixation is typically a reflex test which is performed for those patients who have previously been shown to have a monoclonal band on serum, urine, or cerebrospinal fluid protein electrophoresis. Solutions containing antibodies to the heavy and light chains that make up the immunoglobulin protein are used to identify the type of antibody that is present. The antibodies used in this setting bind to the short chain immunoglobulin components (lambda and kappa) and the heavy chain components of immunoglobulins (gamma, mu, and alpha). The antibodies are added as an overlay to the proteins already separated by electrophoresis on the gel. The binding of the antibody to the protein results in its identification. If the monoclonal protein is an IgA lambda, there will be a band visible for light chain lambda and the heavy chain alpha. Immunofixation essentially replaced immunoelectrophoresis because of a shorter turnaround time and greater clarity of results.

FLOW CYTOMETRY

Flow cytometry is used to identify and count individual cells. Single cells ...

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