Diagnostic Testing Flashcards Preview

FDN3 By Nathan and Minnie > Diagnostic Testing > Flashcards

Flashcards in Diagnostic Testing Deck (22)
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1
Q

What is a monoclonal antibody?

A

An antibody that binds one specific epitope of one specific antigen

Epitope = site on the antigen

2
Q

What is immunofluorescence used for?

A

Evaluating for an autoimmune response, especially in evaluating kidney disease and autoimmunity

3
Q

What is flow cytometry used for?

A
  • White blood cell differential analysis
    • Can evaluate the presence and amounts of lymphocytes, monocytes, granulocytes
  • Count the type of a specific cell
    • Ex: evaluate immune status of an HIV patient by counting T-cells
  • Anything where you are looking at amounts/types of cells
4
Q

What is nephelometry used for?

What kind of information can you find out

A

Nephelometry is used to detect antigens or antibodies in the serum.

It can quantify the amount of antibody isotopes (IgG, IgM, IgA) in a patient sample

5
Q

Which assay would you use to evaluate the protein expression of lymphocyte subsets?

A

Flow cytometry

6
Q

What is nephelometry?

Describe the process of using monoclonal antibodies in nephelometry:

A

Nephelometry measures the amount of light that gets scattered while passing through a substance

  • Monoclonal antibodies specific for an antigen are put into solution with the patient sample
    • Note: antibodies can be antigens! Known monoclonal antibodies can be specific for human antibodies that you are looking for in the patient sample
  • If the antigen is present, it will bind to the monoclonal antibody and form a precipitate
  • The precepitate will cause the light to scatter
  • More antibody:antigen bindign = more scatter
7
Q

Why are monoclonal antibodies useful in diagnostic testing?

A

They can evaluate for the presence of a specific antigen by binding to it

Note: the “antigen” can be another antibody; monoclonal antibodies can also detect the presence or absence of specific antibodies

8
Q

What are two possible sources of error in tests that use antibodies?

A

Corss reactivity with very closely related antigens

Antibodies present in the patient’s blood can interfere with the assay

9
Q

Which test would you use to get a patients white blood cell count?

A

Flow cytometry

10
Q

In patients with hyper-IgM syndrome, which T-cells would you expect to be deficient?

Which protein is commonly deficient in these cells?

A

Th2 CD4+ Helper T-cells

These cells are required to form the germinal centers in which IgM would class-switch to other isotypes

Often, patients with IgM fail to express CD40L on the Th2 CD4+ Helper T-cells; without CD40L, the T-cell cannot form the CD40:CD40L interaction needed to produce cytokines and form the germinal center

11
Q

What are western blot assays used for?

A

Used to detect patient antibodies against particular proteins or antigens

Confirmation after positive ELISA screening test

12
Q

What is a westen blot assay?

Describe the process of using monoclonal antibodies in western blot assays

A

Western blot assays detect patient antibodies against specific antigens

  • Protein is separated using gel electrophoresis
  • Proteins are transferred to filter papter
  • The paper is exposed to a patient sample
  • If the patient has antibodies against the protiens, they will bind to the proteins on the filter paper. Wash
  • Expose the fliter paper to detection antibodies: enzyme-linked anti-human isotype antibodies (ex: anti-human IgG). Wash
  • Measure enzymatic signal from the detection antibodies
13
Q

What is immunofluorescence?

Describe the process of using monoclonal antibodies in immunofluorescence

A

Immunofluorescence is a method of using fluorescent probes bound to antibodies to detect the presense whatever that antibody is specific for (usually autoantibodies or complement bound to pt. tissue)

  • Monoclonal antibodies specific for the autoantibody or complement protein are labled with a fluorescent probe
  • The labeled antibodies are exposed to the patient sample. Wash
  • Expose sample to lights (laser) that will cause the fluorescent probes to emit a light of known wavelength
  • Visualize the fluorescence via microscope
    • The binding pattern will give information about the type of antibody or immune response that is present
14
Q

Which diagnostic tests can quantify the amount of IgG, IgM, IgA, and IgE isotypes in a patient sample?

A

Nephelometry (Use if large amounts are present as in IgG, IgM, IgA)

ELISA (Use if less abundant; IgE, specific subgroups of an isotype)

15
Q

What is flow cytometry?

Describe the process of using monoclonal antibodies in flow cytometry

A

Flow cytometry is a method of evaluating cells as they pass one by one in a fluid stream through a laser beam

To evaluate intrinsic properties

  • Cells pass through a laser
  • Intrinsic properties (size, complexity, nuclear features) will cause a specific pattern of forward and side scatter that is unique to each cell type

To evaluate protein markers (ex, presence of CD3, CD4, CD8)

  • Cells are reacted with monoclonal antibodies labeled with fluorescent probes
    • Can use different antibodies with different specificities labeled with their own probes at one time
  • Solution is passed through the flow cytometer
  • Each cell will give off a specific signal based on the combination of fluorescently-labeled antibodies that bind
    • Can use to identify very specific cell types (ex: CD4+ T-cells)
16
Q

Which assay would you use to evaluate your patient’s HIV progression?

A

Flow cytometry to count T-cells

17
Q

Your 3-week old patient has a family history of hyper-IgM syndrome

What is an appropriate assay to evaluate for the serum immunoglobulin levels?

A

Nephelometry to measure levels of IgM, IgG

(Could also use ELISA)

18
Q

Which assays are most commonly used to detect antibodies in a patient sample?

A

Nephelometry

ELISA

Western Blot

19
Q

Which assays can be used to evaluate cell function?

A
  • Functional flow cytometry assay
    • Evaluate for the presence of a fluorescent product
    • Ex: Chronic granulomatous disease = no oxidative burst to kill pathogens
      • Use permeable dye
      • Oxidative burst = cell will fluoresce
      • No oxidative burst = cell will not fluoresce
20
Q

What is ELISA?

Describe the process of using monoclonal antibodies in ELISA

A

ELISA = Enzyme-Linked Immunosorbent Assay

To measure an antibody in the patient serum

  • Known antigen is fixed on a well
  • Add patient serum. If antibody for the antigen is present, it will bind (Wash)
  • Monoclonal antibody attached to an enzyme is added. This monoclonal antibody is specific for the patient’s antigen that is bound to the known antibody (Wash)
  • Add enzyme substrate and measure enzyme activity
    • Color change = the monoclonal antibody w/attached enzyme is present, which means the patient’s antibody is present
    • Can quantify activity to quantify amount of antibody

To measure an antigen in the patient serum

  • Basically the same process, but you fix a known, monoclonal antibody on a well (instead of an antigen)
  • The enzyme-linked monoclonal antibody is specific for a different epitope of the antigen in question
  • Quantify binding by measuring enzyme activity (Wash steps are still important)
21
Q

What is ELISA used for?

A

To measure amounts of antigen or antibody present in a patient specimen

Can determine amounts of IgG, IgM, IgA, IgE etc

Can also determine amounts of specific subgroups of each isotype

22
Q

Suppose a flow cytometry assay confirms that a patient’s CD4+ helper T-cells express CD40L when stimulated.

Can you rule out hyper-IgM? Why or why not?

A

You cannot rule out hyper-IgM, especially if there is a high clinical suspicion

CD40L may be present, but nonfunctional

  • It may fail to bind to CD40
  • There may be a defect in cytokine production

The takeaway: Standard flow cytometry can evaluate the presence or absence of a protein, but not necessarily its function

(But there are functional flow cytometry assays that evaluate for the presence of a fluorescent product)