Assays Flashcards
Important considerations for evaluating patients for possible immune deficiencies:
–Complete history: including details of any / all infections, age of onset of infections, severity / frequency, management
–Family history / social history: any indications of family history of problems with infections / autoimmune disease, early deaths, etc. Full exposure history / risks of infection
–Immunization history
–Complete PE
What does a CBC with differential tell you?
- number of cells
- morphology of cells
What are important initial screening labs to determine if someone has an immune deficiency?
- Complete blood count (CBC) with differential
- General markers of inflammation / inflammatory markers (i.e. erythrocyte sedimentation rate, C reactive protein)
- Consider: Chemistry panel
–Check organ systems (e.g. liver, kidney)
–Can include urinalysis -evaluate kidney disease / protein loss.
•Consider: Targeted evaluation for specific infections (imaging, labs) if indicated
Next step: Decision on evaluation for specific immune defects
Need to be familiar with options for diagnostic testing
Many immune disorders present with abnormal levels of serum ___
Ig
What immune deficiency might be a possible diagnosis for a patient who has low levels of ALL Igs?
SCID
Check serum IgD only if patient presents with ____
periodic fever
If any antibody deficiency is confirmed, consider _____ quantitation
B cell quantitation
Consider ____ as a cause for low Ig levels
protein loss
Describe methods to test antibody function:
- Check titer to a vaccine (e.g. diptheria or tetanus titer)
- Check repsonse to protein antigens and polysaccharides
- For polysaccharides: pneumococcal titers in older children given the polysaccharide vaccine
- Isohemagglutinins: antibodies generated in response to polysaccharides of gut flora which cross-react with A or B blood group erythrocyte antigens
How can you check response to polysaccharide antigens?
Test pneumococcal titers in children who were given the polysaccharide vaccine
How can you evaluate antibody response to protein antigens?
Isohemagglutinins: Check for antibodies generated in response to polysaccharides of gut flora which cross-react with A or B blood group erythrocyte antigens
Common T cell function tests use _____ such as (name 3 examples)
Mitogen tests:
- phytohemagglutinin
- concanavalin A
- pokeweed mitogen
Describe the technical process of mitogen tests:
–Incubate patient’s lymphocytes & monocytes with test substance(s) or cells 3-6 days
–Last 24 hours add tritiated thymidine
–Dividing lymphocytes incorporate thymidine into their DNA
–Extent of proliferation - measure the radioactivity taken up by cells
–Result compared with control - negative, partial, or normal
What is the classic in vivo test for cellular immunity?
Cutaneous Delayed-Type Hypersensitivity test (e.g. a skin TB PPD test)
What is a Cutaneous Delayed Type Hypersensitivity Test?
•Intradermal injection of antigen to which the individual has been exposed
–E.g. Candida, tetanus
- Positive response requires: uptake and processing of antigen by antigen-presenting cells, interaction with CD4 cells, cytokine production, and recruitment and activation of monocytes and macrophage
- Positive response is instructive; many factors can produce a negative response (e.g. recent infection with some pathogens)
A positive response of a cutaneous DTH test requires uptake and processing of antigen by _______, interaction with ___ cells, ______ production, and recruitment and activation of ______ and _______
A positive response of a cutaneous DTH test requires uptake and processing of antigen by ANTIGEN-PRESENTING CELLS, interaction with CD4 cells, CYTOKINE production, and recruitment and activation of MONOCYTES and MACROPHAGES
Which is more informative - a positive or negative cutaneous DTH result?
Positive - a positive test result in informative. Many factors can produce a negative response (e.g. recent infection with some pathogens)
How is neutrophil oxidative burst potential tested?
Nitroblue tetrazolium test (NBT) or flow cytometry
Giant azurophilic granules are seen in granulocytes of persons with ______
Chediak-Higashi Syndrome
Sensitivity:
The extend to which the test is accurate for those who have the disease in questions, avoiding “false negative” errors
Specificity:
The extend to which the test is accurate for those who do not have the disease in question, avoiding “false positive” errors
Positive predictive value:
The extent to which a positive test indicates presence of disease
Negative predictive value:
The extend to which a negative test indicates absence of disease
Pros and cons of light microscopy:
-PROS: relatively inexpensive, can give rapid results
CONS: sensitivity is often low
General: specimens (may be fresh or stained) examined directly to visualize bacteria, protozoa, or host cells. Specificity depends on organisms and specimins
Culturing organisms:
- Requires recovery of live organisms
- Requires specialised media, incubator, may include microscopy or more specialized equipment to identify microbes
- Sensitivity is higher than microscopy (biological amplification) but lower than nucleic acid amplification tests (NAATs)
- Some bugs can’t be cultured in vitro
The sensitivity of culturing organisms has a _____ sensitivity than microscopy and a ____ sensitivity than nucleic acid amplification tests.
The sensitivity of culturing organisms has a HIGHER sensitivity than microscopy and a LOWER sensitivity than nucleic acid amplification tests.
Immunoassays involve the use of ______ and _____
antigens and antibodies
Formats of Immunoassays include (4):
- ELISA
- Western Blots
- Rapid immunochromatographic strip tests (e.g. OTC pregnancy test; can be generated to detect antibody or antigens)
- Particle agglutination tests
Epitope vs. antigen
An epitope is the smallest part of an antigen than an antibody can recognize
In antigen detection tests, the primary detection antibody will recognize the same _____ but will not recognize the same ______
In antigen detection tests, the primary detection antibody will recognize the same ANTIGEN but will not recognize the same EPITOPE
Describe the process of ELISA testing when detecting ANTIGEN:
- Generate an antibody in lab (specificity determines how applicable it is to different strains).
- This antibody is pinned down on a plate or some surface.
- Add the specimen
- If the antigen is contained in the specimen, it will bind to the antibody.
- Everything else is washed away
- A primary detection antibody is added to recognize the same antigen (but will not recognize the same epitope). We can’t see this additional protein added, so we still need something to help visualize.
- Add a secondary antibody which has something added to it (could be a fluorophore, something that can cleave a substance and make a color change, etc.)
Can have a secondary antibody that recognizes the Fc region of an antibody - this can be much less specific.
Antigen detection tests are indicators of ____
current infection
Antibody detection tests are indicators of _____
past infection
Not all infections result in _____ production
antibody
Which test property (sensitivity vs. specificity) is more important for testing a sample with low organism burden?
Sensitivity
(want a test that can detect a low number)
Which test property (sensitivity vs. specificity) is more important for testing a sample needing detection of closely related organisms?
Specificity
Which test property (sensitivity vs. specificity) is more important for testing a sample with little specimen?
Sensitivity
Which test property (sensitivity vs. specificity) is more important for testing a low prevalence population?
Specificity
Where are the majority of errors made in the testing process?
The pre-analytical areas (important to ask whether this is the right test, right specimen, what a positive/negative test would really tell you, etc.)
What does a highly specific and sensitive test tell you in a high prevalence population?
Good, basically with a 99% sensitivity and 99% specificity with 20% population prevalence, have a PPV of 96%
What does a highly specific and sensitive test tell you about test results in a low prevalence population?
Enh. With low population incidence, can start with 99% sensitive and 99% specific test but end up with a PPV of 50%
What are nucleic acid amplification tests?
Molecular detection assays for bacteria, viruses, and eukaryotic pathogens.
Targets are microbial DNA or RNA
Enzymatic amplification of target molecules (e.g. PCR, rtPCR, TMA, SDA)
Don’t require live organisms
Consequences of NAAT sensitivity:
Detection of dead organisms impacts timing of test or cure (tests may remain positive for a few days after treatment)
Increased sensitivity may allow use with non-invasive specimens (esp. important with sexually transmitted pathogens that can be detected in urine)
Rank the sensitivity of these tests:
Culture
Nucleic Acid Amplification
Microscopy
Antigen Test
Microscopy < Culture < Antigen Test < Nucleic Acid Amplification
