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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?


- 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?

pt swab smeared on slide, fluorescent-labelled Abs added --- a test for ANTIGEN

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