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Immunology Block 2 > Assays > Flashcards

Assays Flashcards

1
Q

Important considerations for evaluating patients for possible immune deficiencies:

A

–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

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2
Q

What does a CBC with differential tell you?

A
  • number of cells
  • morphology of cells
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3
Q

What are important initial screening labs to determine if someone has an immune deficiency?

A
  • 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

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4
Q

Many immune disorders present with abnormal levels of serum ___

A

Ig

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5
Q

What immune deficiency might be a possible diagnosis for a patient who has low levels of ALL Igs?

A

SCID

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6
Q

Check serum IgD only if patient presents with ____

A

periodic fever

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7
Q

If any antibody deficiency is confirmed, consider _____ quantitation

A

B cell quantitation

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8
Q

Consider ____ as a cause for low Ig levels

A

protein loss

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9
Q

Describe methods to test antibody function:

A
  • 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
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10
Q

How can you check response to polysaccharide antigens?

A

Test pneumococcal titers in children who were given the polysaccharide vaccine

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11
Q

How can you evaluate antibody response to protein antigens?

A

Isohemagglutinins: Check for antibodies generated in response to polysaccharides of gut flora which cross-react with A or B blood group erythrocyte antigens

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12
Q

Common T cell function tests use _____ such as (name 3 examples)

A

Mitogen tests:

  1. phytohemagglutinin
  2. concanavalin A
  3. pokeweed mitogen
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13
Q

Describe the technical process of mitogen tests:

A

–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

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14
Q

What is the classic in vivo test for cellular immunity?

A

Cutaneous Delayed-Type Hypersensitivity test (e.g. a skin TB PPD test)

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15
Q

What is a Cutaneous Delayed Type Hypersensitivity Test?

A

•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)
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16
Q

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

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

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17
Q

Which is more informative - a positive or negative cutaneous DTH result?

A

Positive - a positive test result in informative. Many factors can produce a negative response (e.g. recent infection with some pathogens)

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18
Q

How is neutrophil oxidative burst potential tested?

A

Nitroblue tetrazolium test (NBT) or flow cytometry

19
Q

Giant azurophilic granules are seen in granulocytes of persons with ______

A

Chediak-Higashi Syndrome

20
Q

Sensitivity:

A

The extend to which the test is accurate for those who have the disease in questions, avoiding “false negative” errors

21
Q

Specificity:

A

The extend to which the test is accurate for those who do not have the disease in question, avoiding “false positive” errors

22
Q

Positive predictive value:

A

The extent to which a positive test indicates presence of disease

23
Q

Negative predictive value:

A

The extend to which a negative test indicates absence of disease

24
Q

Pros and cons of light microscopy:

A

-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

25
Q

Culturing organisms:

A
  • 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
26
Q

The sensitivity of culturing organisms has a _____ sensitivity than microscopy and a ____ sensitivity than nucleic acid amplification tests.

A

The sensitivity of culturing organisms has a HIGHER sensitivity than microscopy and a LOWER sensitivity than nucleic acid amplification tests.

27
Q

Immunoassays involve the use of ______ and _____

A

antigens and antibodies

28
Q

Formats of Immunoassays include (4):

A
  • ELISA
  • Western Blots
  • Rapid immunochromatographic strip tests (e.g. OTC pregnancy test; can be generated to detect antibody or antigens)
  • Particle agglutination tests
29
Q

Epitope vs. antigen

A

An epitope is the smallest part of an antigen than an antibody can recognize

30
Q

In antigen detection tests, the primary detection antibody will recognize the same _____ but will not recognize the same ______

A

In antigen detection tests, the primary detection antibody will recognize the same ANTIGEN but will not recognize the same EPITOPE

31
Q

Describe the process of ELISA testing when detecting ANTIGEN:

A
  • 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.

32
Q

Antigen detection tests are indicators of ____

A

current infection

33
Q

Antibody detection tests are indicators of _____

A

past infection

34
Q

Not all infections result in _____ production

A

antibody

35
Q

Which test property (sensitivity vs. specificity) is more important for testing a sample with low organism burden?

A

Sensitivity

(want a test that can detect a low number)

36
Q

Which test property (sensitivity vs. specificity) is more important for testing a sample needing detection of closely related organisms?

A

Specificity

37
Q

Which test property (sensitivity vs. specificity) is more important for testing a sample with little specimen?

A

Sensitivity

38
Q

Which test property (sensitivity vs. specificity) is more important for testing a low prevalence population?

A

Specificity

39
Q

Where are the majority of errors made in the testing process?

A

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.)

40
Q

What does a highly specific and sensitive test tell you in a high prevalence population?

A

Good, basically with a 99% sensitivity and 99% specificity with 20% population prevalence, have a PPV of 96%

41
Q

What does a highly specific and sensitive test tell you about test results in a low prevalence population?

A

Enh. With low population incidence, can start with 99% sensitive and 99% specific test but end up with a PPV of 50%

42
Q

What are nucleic acid amplification tests?

A

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

43
Q

Consequences of NAAT sensitivity:

A

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)

44
Q

Rank the sensitivity of these tests:

Culture

Nucleic Acid Amplification

Microscopy

Antigen Test

A

Microscopy < Culture < Antigen Test < Nucleic Acid Amplification

Immunology Block 2 (9 decks)

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