L5: Immunological Methods Flashcards
How do you deduce the absolute count of a particular cell type from a CBC?
Using the percentage of cells from the differential count, back-calculate the absolute value from the total number of cells
How does flow cytometry work?
Measures characteristics of cells as they flow in a single file past a laser beam → the amount of light emitted from fluorescently labeled cells is measured and quantitated by photomultiplier tubes → information on the size and complexity (granularity) of a cell is also obtained by the way the light is scattered
In flow cytometry, what does light scatter gating tell you?
Forwards scatter tells you cell size: the larger the cell, the further up on the scale
Side scatter tells you on complexity or granularity
There are characteristics patterns on side scatter vs. forward scatter graph for the different cell types
How can flow cytometry be used to determine the types of markers and receptors are on the surface of cell?
A fluorescent dye is attached to antibodies or receptor ligands → these cells can then be subjected to flow cytometry → the amount of receptor on their surface is detected by the level of fluorescence
These experiments can be designed to incorporate more than one fluorescent marker at a time, giving the ability to detect multiple cell-surface markers simultaneously, e.g. CD4 and CD8
HIstogram (flow cytometry)
If a single fluorescent marker is used, the readout is a histogram
Display 1 fluorescence at a time
Cells also have auto-fluorescence, so it will still have a signal
Fluorescence intensity vs. Number of cells
Scattergram (flow cytometry)
When 2 or more fluorescent markers are used
What are some applications of flow cytometry?
Quantitative analysis of lymphocytes subsets, using surface markers CD antigens
Immunophenotyping of leukemias and lymphomas
Define early stages in the development and lineage of T and B lymphocytes
DNA analysis (ploidy): useful for diagnosis of malignancy since malignant cells acquire lots and lots of DNA
Intracellular flow for cytokines and receptors
Monitoring of HIV infected and immunocompromised pts: virus will initially cause a lot of CD4+ T cell death
Fluorescence-activated cell sorting (FACS) for isolating different cell population
What is produced when antigens combine w/ antibodies?
Precipitate
Agglutination
Smaller antigens are adsorbed on to particles (RBCs, latex beads, charcoal) to visualize the reaction)
What do primary interactions b/w antigen and antibody depend on?
Dependent on pH, temperature, and the relative concentrations of antigen and antibody
What are applications of antigen-antibody reactions?
Blood typing
Coombs
Direct antiglobulin test (for fetal Rh reactions)
Measurement of rheumatoid factor
Measurement of C reactive protein
Measurement of antibodies to Treponema pallidum (reagin)
When does Rh type mother-fetus incompatibility occur?
When an Rh+ man (DD or Dd) fathers a child with an Rh-mother (dd)
Only the Rh+ children (Dd) are likely to have medical complications
When both the mother and her fetus are Rh- (dd) the birth will be normal
What type of antibody is anti-Rh antibody?
IgG
Describe the direct Coombs test for anti-Rh antibody in a Rh- mother pregnant w/ Rh+ child
Take blood directly from the fetus → if mother has Rh antibodies, the RBCs of the fetus will already be coated with maternal antibodies → add a secondary reagent (rabbit anti-human antibody) →if the antibodies are present, there will be agglutination
Describe the indirect Coombs test for anti-Rh antibody in a Rh- mother pregnant w/ Rh+ child
Take a sample from maternal serum → mix these antibodies w/ another source of RBCs that are known to be Rh+ → if mother has antibodies, they will form a complex w/ the RBCs → add secondary reagent (rabbit anti-human antibody → agglutination
How are antibodies measured in a lab?
Immunoassays, such as ELISA (enzyme linked immunosorbent assay)
Describe process of ELISA
Bind antigen to wells → take pt’s serum and through serial dilutions add it to the wells → if pt has antibodies for this antigen, they will bind → do several washes to remove any non-specific bindings → add secondary antibody that comes covalently linked w/ an enzyme (this enzyme catalyzes a reaction that produces a color change) → add substrate → look for color change
Why are serial dilutions of pt’s serum done in ELISA?
So you get a range of responses if pt has the the antibody specific for that particular antigen
What is a Western blot?
Immunblotting
Another way to test for presence of antibodies
Describe Western blot for detection of anti-HIV antibodies?
Take a mixture of HIV proteins and run them in an electrophoresis gel → proteins that remain stuck in the gel are transferred to nitrocellulose membrane → can blot this membrane w/ control antibodies and serum antibodies from pt → utilize a secondary antibody that is labeled w/ another enzyme that allows you to measure the amount of binding using a radiograph → get a pattern of bands → can see how many antibodies are specific for many than one HIV antigen
What is the advantage of a Western blot over ELISA?
In Western blot, can blot entire serum
In ELISA, have to do one antigen at a time; would have to do multiple ELISAs
What does a positive HIV immunoblot show?
Presence of p24 AND either gp41, gp120, or gp160
What do rapid HIV test measure antibodies to?
gp41
Acid-fast sputum microscopy test for TB
An old test
TB-infected person would cough up → take sputum sample from that pt → smear it on a slide → stain for presence of TB
What is the limitation of an acid-fast sputum microscopy test for TB?
Pt would have to have active infection and an active cough for you to recognize presence of the bacteria
TB skin test
Indirectly tests for TB by looking for immune response
Are injected w/ TB antigens → if you have been exposed to TB, you get inflammation at the area of injection
What is the limitation of TB skin test?
Not very specific
Foreigners who have been vaccinated w/ BCG will respond
What to tests for the measurement of cell-mediated immunity do?
Measure delayed type hypersensitivity responses
Antigens against which people are exposed and develop memory cells
Lymphocyte proliferation in response to specific antigens
Cytokine production by CD4+ T cells
What are some applications for test for measurement of cell-mediated immunity?
Quantiferon Gold test for TB
Measurement of IFN-γ due to specific stimulation of T cells sensitized to TB
Lymphocyte proliferation for (immunosuppressed) transplant recipients
What test can be employed to identify activation of immune response?
Flow cytometry (FACS) to look at activation surface markers
What test can be employed to identify clonal expansion of immune response?
Assays on DNA synthesis
What test can be employed to identify effector function of immune responses?
Quantiferon measured cytokine production by CD4+ T cells once they get activated
What forms the basis of quantiferon gold test?
Detection and subsequent quantification of IFN-γ
Recognition of mycobacterial antigens ESAT-6 and CFP-10 from M. tuberculosis organisms → generation and secretion of IFN-γ from the CD4+ T cells
Why can pts w/ BCG vaccinations be tested w/ the Quantiferon test?
The proteins ESAT-6 and CFP-10 are absent from all BCG strains and from most non-tuberculosis mycobacteria w/ a few exceptions
Describe how Quantiferon is performed
Blood culture of heparinized whole blood → transfer undiluted whole blood into wells of a culture plate and add antigens → Culture overnight at 37 deg. C (TB infected individuals respond by secreting IFN-gamma)
Harvest plasma from above settled cells and incubate 60 min in ‘sandwich’ ELISA → wash, add substrate, incubate 30 minutes, then stop reaction → Measure OD, determine IFN-gamma levels and interpret test
What does an Elispot assay do?
Allows you to quantitate exactly how many T cells producing cytokine there are in a sample
In ELISA, can only say they are there, not how many
Describe Elispot assay
In plate where T cells are being placed, it is coated w/ antibodies against IFN-gamma → the antibodies capture the cytokines as the T cells release them → remove the T cells → add secondary antibody conjugated w/ an enzyme that allows you to measure color → add substrate → in every spot where there was a T cell that made cytokine, will end up w/ color in that spot (very localized color spot) → can count the number of dots to know the number of T cells
How can Elispot assay be used for B cells?
Same process, but the well would be coated w/ antigen instead of antibody
B cells would get activated and if they are specific to the antigen there would be localized deposition of antibody
Lymphocyte proliferation assay
Simple and old test to look at proliferatic burst of T cells
Take blood and PBMC and put them in culture media → give the T cells radioactive thymidine (which will be used in DNA synthesis) → immobilize cells on glass fibre filter → quantitate by measuring radioactive signaling → know how much DNA was synthesizes; if they start to proliferate, get more signaling
Cytotoxicity assay by chromium release
To specifically look at cytolytic activity of CD8+ cells Activate T cells → take MHC class I cells and load them up w/ a particular antigen (these will serves as the target cells) and also radioactive molecule (such as 51-Cr)→ if CD8+ T cells are specific for this target, they will start lysing them → if target cells are lysed, there will be release of radioactivity into the supernatant → measure the amount of radioactivity released into the supernatatn
Serum electrophoresis
Serum proteins can be separated in an electrical field, in which most proteins (being negatively charged) migrate toward the positive electrode → Transfer proteins from the gel to a membrane and do a stain →This is quantitated → Get a characteristic pattern
What would serum electrophoresis look like in a pt w/ myeloma?
Will get an increase in presence of γ spike
Myeloma protein spike (M spike) in the γ globulin region
There are some other rare cases where there is alpha-2 region M-spike
What are some applications for serum electrophoresis?
Monoclonal gammopathies
Immunoelectrophoresis or immunofixation uses antibodies to identify the type of immunoglobulin and the light chain utilized
Immunofixation
When you know that a person has a malignancy, can now test for what kind of malignancy that is (IgG, IgM, IgA)
Would again do a serum electrophoresis, but would then blot w/ secondary antibodies that are specific for one of the isotypes
In Waldenstrom’s macroglobulinemia, what antibody is secreted by the cancerous clone?
IgM
How could immunofixation be used for light chain?
Electrophoresed serum proteins can be analyzed w/ immunofixation w/ antibody to kappa chains
