Unit 21 - Immunodiagnostics

Question 1

What tools do you need to evaluate lymphocyte numbers?

Answer 1

Tagged Ab
Scope
FACS = fluoro activated cell sorting

Question 2

How are B-cell numbers counted?

Answer 2

Use surface immunoglobulin (Ab)!

Target CD19 and CD20 with a monoclonal Ab tagged with a fluorescent molecule to prevent false positives from other cells.

Question 3

How are T-cell numbers counted?

Answer 3

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.

Question 4

What cells are located in the upper right quadrant of a graph with the results of flow cytometry?

Answer 4

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.

Question 5

What are the axis of a graph with flow cytometry results?

Answer 5

y-axis = CD8, x-axis = CD4

Question 6

What cells are in the lower left quadrant on a flow cytometry graph?

Answer 6

Non-t-cells. Have neither CD8 nor CD4.

Question 7

How are Treg detected by flow cytometry?

Answer 7

Tag internal Ag, such as non-secreted cytokines or transcription factors. These can be specific to Treg and show up when they fluoresce.

Question 8

How are B-cells detected?

Answer 8

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.

Question 9

Where is the gamma peak for a normal blood sample?

Answer 9

It is the last, 5th peak!
Albumin peak is large,
alpha 1, alpha 2, beta peaks are small,
gamma peak is medium.

Question 10

What is happening if a blood sample has no gamma peak?

Answer 10

No Ig! Agammaglobulinemia

Ex: bruton’s disease

Question 11

What is happening if a blood sample has no gamma peak?

Answer 11

No Ig! Agammaglobulinemia

Ex: bruton’s disease

Question 12

What is happening if a blood sample has a really wide gamma peak?

Answer 12

Too much Ag! Hypergammaglobulinemia

Ex: leukemia
Ex: bacterial infection - polyclonal

Question 13

What is happening if a blood sample has a spiked gamma peak?

Answer 13

Only one type of Ig = monoclonal
Hypergammaglobulinemia

Ex: myeloma

Question 14

How are class and subclass of the antibodies identified?

Answer 14

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Question 15

How are class and subclass of the antibodies identified?

Answer 15

Use immunodiffusion or ELISA Assay.

Ask: “Does patient have an Ab to a given Ag?”

Question 16

How does immunodiffusion work?

Answer 16

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)

Question 17

How does the ELISA Assay work?

Answer 17

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.

Question 18

How does a titer assay work?

Answer 18

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.

Question 19

How are class and subclass of the antibodies identified?

Answer 19

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?”

Question 20

How does a titer assay work?

Answer 20

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.

Question 21

How does the cryoglobulins test work?

Answer 21

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.

Question 22

What is the word for no T-cell function?

Answer 22

Anergy

Question 23

What is the best way to test for T-cell function?

Answer 23

Skin test!

Apply candida, stretokinase/streptodornase, trichoplytin, mumps, tetnus, TB

Read after 24 - 48 hours

This is delayed-type immunopathology! T-cells!

Question 24

What are the other 4 tests for T-cell function?

Answer 24

1. Challenge DTH test: like intentional poison ivy
2. Add a mitogen: check for T-cell division
3. Check for product after proliferation: IL-2, IL-4, INF-gamma
4. 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

Question 25

What are the exact steps that happen with a skin test? There are 9!

Answer 25

1. Ag endocytosed by dendritic cells
2. Ag is digested within the endosome by lysosomal enzymes
3. Ag is loaded onto MHC Class II (eaten, not made)
4. MHC Class II cycles to the dendritic cell’s surface
5. Th1 anti-Ag (specific to the Ag used) come along
6. Th1 anti-Ag recognize the Ag + MHC Class II
7. Th1 cells are activated and secrete IFN_gamma
8. Macrophages are attracted
9. Macrophages are activated and become M1s

SO, shortly after administration of the test, would find dendritic cells and Th1s.

BUT, 48 hours after administration of the test, find Macrophages.