Unit 21 - Immunodiagnostics Flashcards
What tools do you need to evaluate lymphocyte numbers?
Tagged Ab
Scope
FACS = fluoro activated cell sorting
How are B-cell numbers counted?
Use surface immunoglobulin (Ab)!
Target CD19 and CD20 with a monoclonal Ab tagged with a fluorescent molecule to prevent false positives from other cells.
How are T-cell numbers counted?
Count cells with either CD3 (for total T-cells) or separate and count the cells with CD4 or CD8.
Use a monoclonal Ab tagged with a fluorescent molecule targeting CD3, CD4, or CD8.
What cells are located in the upper right quadrant of a graph with the results of flow cytometry?
Both CD8 and CD4 cells are in the upper right quadrant, so the sample must be from a thymus…only place where the cells have BOTH markers.
What are the axis of a graph with flow cytometry results?
y-axis = CD8, x-axis = CD4
What cells are in the lower left quadrant on a flow cytometry graph?
Non-t-cells. Have neither CD8 nor CD4.
How are Treg detected by flow cytometry?
Tag internal Ag, such as non-secreted cytokines or transcription factors. These can be specific to Treg and show up when they fluoresce.
How are B-cells detected?
Humoral! Ab! Use electrophoresis.
Cheap, easy, separate based on size and electricity (smaller move further). Can quantify the bands by sending through a mass spec.
Where is the gamma peak for a normal blood sample?
It is the last, 5th peak!
Albumin peak is large,
alpha 1, alpha 2, beta peaks are small,
gamma peak is medium.
What is happening if a blood sample has no gamma peak?
No Ig! Agammaglobulinemia
Ex: bruton’s disease
What is happening if a blood sample has no gamma peak?
No Ig! Agammaglobulinemia
Ex: bruton’s disease
What is happening if a blood sample has a really wide gamma peak?
Too much Ag! Hypergammaglobulinemia
Ex: leukemia
Ex: bacterial infection - polyclonal
What is happening if a blood sample has a spiked gamma peak?
Only one type of Ig = monoclonal
Hypergammaglobulinemia
Ex: myeloma
How are class and subclass of the antibodies identified?
asdf
How are class and subclass of the antibodies identified?
Use immunodiffusion or ELISA Assay.
Ask: “Does patient have an Ab to a given Ag?”
How does immunodiffusion work?
Drop the patient’s serum into a well.
The well is located in a gel containing the antiserum to the Ig in question.
Measure how far the serum containing the potential Ig diffuses out.
Compare the patient test to a set of standards to estimate the value.
Higher value = have the Ig in question.
Ag + Ab (DIRECT)
How does the ELISA Assay work?
Use a tube that already contains the Ab in question.
Add the patient’s serum containing the Ag…may attach to the Ab if present.
Add another Ab that is tagged –> check for fluorescence.
Ab + Ag + Ab (INDIRECT)
NOTE: the Ab must be divalent in order to be able to see it and have good results.
How does a titer assay work?
To check titer levels! Passive test!
Serial dilute the serum numerous times.
Add the tested Ag to the serum.
Check for agglutination at each dilution level.
The LAST dilution with agglutination = the titer value.
How are class and subclass of the antibodies identified?
Use the mechanism of Ab-Ag complexes.
Use immunodiffusion, ELISA Assay, titer assay, cryoglobulin test, etc.
Ask: “Does patient have an Ab to a given Ag?”
How does a titer assay work?
To check titer levels! Passive test!
Serial dilute the serum numerous times.
Add the tested Ag to the serum.
Check for agglutination at each dilution level.
The LAST dilution with agglutination = the titer value.
How does the cryoglobulins test work?
It is immune complexes of Ag-Ab that precipitate out in the cold.
Used to detect: Hep C, Lupus, Rheumatoid Arthritis
Next take a sample of the precipitate and analyze via electrophoresis/mass spec.
What is the word for no T-cell function?
Anergy
What is the best way to test for T-cell function?
Skin test!
Apply candida, stretokinase/streptodornase, trichoplytin, mumps, tetnus, TB
Read after 24 - 48 hours
This is delayed-type immunopathology! T-cells!
What are the other 4 tests for T-cell function?
- Challenge DTH test: like intentional poison ivy
- Add a mitogen: check for T-cell division
- Check for product after proliferation: IL-2, IL-4, INF-gamma
- Killer Cell Assay: Check to see if they can kill…remember that the T-cell must see the Ag presented on the correct (unique) MHC
