Immunodiagnosis (complete) Flashcards
Describe the procedure used in serum protein electrophoresis. What are the underlaying principles?
- Serum (urine, CSF) is placed on a gel
- Turn on voltage
- Measure peaks of albumin, alpha1, alpha2, beta, and gamma
What does no gamma peak indicate in an electrophoresis?
Agammaglobulinemia
- No IgG
What does a high, broad gamma peak indicate in an electrophoresis?
Polyclonal hypergammaglobulinemia
or severe pyogenic (pus-producing) infections
What does a high, single gamma peak indicate in an electrophoresis?
Monoclonal hypergammaglobulinemia
What does a normal serum protein electrophoretic pattern look like?
- Large albumin peak
- Baby peaks for alpha1&2, beta, gamma
Go google it NOW!
What does a serum protein electrophoretic pattern look like for a person with selective IgA deficiency?
Same as a normal peak
You can’t pick up this deficiency with serum electrophoresis
What does a serum protein electrophoretic pattern look like for a person with multiple myeloma?
Large spike in gamma region (AKA M spike) — remember the M protein in MM?
Not as sharp as the monoclonal hypergammaglobulinemia
What is single radial immunodiffusion?
Measures individual immunoglobulin (sub)classes — or just Ag
What are the types of Ag that can be quantified in single radial immunodiffusion?
Multivalent Ag
- Can form a precipitate w/ an appropriate Ab
How is the quantization done in single radial immunodiffusion?
You compare patient’s serum to the controls
What are the advantages of passive agglutination over precipitation?
A lot quicker and more sensitive
Describe passive agglutination test
- Couple small Ags to RBCs or latex beads
- Add dilutions of pt serum
- Look for agglutination
The agglutination titer is the reciprocal of the highest dilution that produces agglutination
What is the difference between direct and indirect immunofluorescence techniques?
Direct:
pt swab smeared on slide, fluorescent-labelled Abs added — a test for ANTIGEN
Indirect:
Known bacteria on slide, add pt’s serum, wash and added fluorescent-labelled anti-human Ig — a test for ANTIBODY
Describe the reactions involved when the ELISA is used to measure Ag
Enzyme-linked immunosorbent assay
- works well if Ag is at least divalent
- Need 2 monoAbs
- Put one on the bottom => add pt’s serum
- Wash, then add 2nd monoAb (will stick to other epitope)
- 2nd Ab has enzyme coupled to it (peroxidase)
- Add colorless peroxidase substate => produces color if peroxidase bound to 2nd Ab
Describe the reactions involved when the ELISA is used to measure Ab
- Ag is at bottom of plate, sticks there
- Put on pt’s serum
- Wash everything that doesn’t stick
- Add anti-Ab Ig (has enzyme on the end)
- Add enzyme substrate => produces color if testing Ab is present
Describe a test which can be used to evaluate T cell immunocompetence in a clinic or on the ward
Think of a TB test
1) Ag is endocytosed by DCs
2) Ag digested w/in endosomes by lysosomal enzymes
3) Ag loaded onto MHC Class II
4) MHC Class II cycles to the DCs surface
5) Th1 anti-TB cells come by
6) Th1 anti-TB cells recognize Ag + MHC Class II.
7) Th1 cells are activated and secrete IFNγ
8) Macrophages are attracted
9) Macrophages are activated and become M1
What are tests used to evaluate T cell numbers and function in the lab?
You can stain for CD3, CD4, or CD8 in serum
Describe flow cytometry
- Light scattered according to CD markers
- Increasing intensity of CD8 on y-axis
- Increasing intensity of CD4 on x-axis
Where would you find both CD4 and CD8 cells?
Only the thymus
- This is where double positives are weeded out
Where would you find cells without CD4 or CD8 markers?
Lymph nodes
- Primarily B cells
- These have no CD4 or 8 markers
