Clinical Assays II Flashcards
What is the principle of immunofluorescence?
Describe direct fluorescence?
- exploits ability of certain compoungs to emit light of restricted wavelength during UV radiation.
- compounds are linked to antibodies that directly bind antigens or indirectly bind Ig that was previously bound to antigens. document with microscopic visualization during UV excitation.
Describe indirect immunofluorescence.
Patient serum containing possible antibodies is layered on a substrate rich with nuclei and anti-human Ig-fluorochrome is added.
If there are anti-nuclear antibodies they would bind to nuclear antigens, which would react with the Ig, and UV light passing through the sample emits fluorescence at the site of binding.
What type of cells can flow cytometry get information for?
How does flow cytometry work?
- single cells in a mixed cell population.
- Cell population being studied is led into thin stream of single cells that have been probed. Flows past laser that emits light. If a cell has the antibody, the emitted wavelength is then photo multiplied and quantitated by computer software.
How is a monoclonal made?
TEST TEST
- Take spleen cells producing antibody from mouse immunized with antigen A and myeloma cells without antibody secretion and enzyme HGPAT
- Mix and fuse cells with PEG
- Transfer to HAT medium. Immortal hybridomas proliferate; mortal spleen cells and unfused HGPRT- myeloma cells die
- Select hybridoma that makes antibody specific for antigen A
- Clone selected hybridoma.
How would you determine WBC cell lineage with flow cytometry?
How would you determine WBC phenotype with flow cytometry?
- Use markers for granulocytes, lymphocytes, and monocytes.
- Select CD3 and B cell markers to sort out the 2 populations, then use markers for the specific chains to determine the clonality.
How could you determine white blood cell clonality?
- separate serum proteins with an electric field. Igs will migrate to the gamma region, albumin will go the other way.
- Numerous clones of plasma cells will make antibodies to infecting organism and each plasma cell antibody will migrate to a unique spot because each clone is producing a slightly diferent antibody in response to the infection.
What pole does albumin migrate towards in electrophoresis?
What pole do Igs migrate toward in electrophoresis?
- positive pole
2. negative pole
If a single clone of B cells is expanding, the immunoglobulin the clone produces will be:
- homogenous with respect to size and charge
What is the Coombs test?
If there is an antibody binding to an antigen on the rbc, it can be detected by adding an antibody that reacts with human Ig. (anti-human antibody)
What controls are needed for a Coombs test?
- erythrocytes with no antibody, antisera raised against some serum protein other than human Ig
If you had a patient with hemolysis and you didn’t know what Ig is causing it, what would be the first test to do?
- use an anti human antibody that is specific for kappa and lambda chains.
If the test showed agglutination (positive), you could identify the specific isotype by repeating the assay
What is direct fluorescence?
What is indirect fluorescence?
- a specific fluorescinated antiserum to an antigen is used to probe tissue for the antigen. Direct fluorescence is used in the diagnosis of many infectious diseases
- the use of a fluorescinated anti-antibody to a human immunoglobulin that the clinician suspects is causing pathology.
- This is a common procedure and used routinely when studying renal biopsies
