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CLS 790 > Quiz #2 - Autoimmmunity and Serological Techniques > Flashcards

Quiz #2 - Autoimmmunity and Serological Techniques Flashcards

1
Q

What produces immunoglobulins?

A

B cells or plasma cells

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2
Q

Where are immunoglobulins found in Serum Protein Electrophoresis (SPE)?

A

Gamma region

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3
Q

What does a gamma-globulin “spike” indicate?

A

Myeloma

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4
Q

What test determines existence of immunoglobulin abnormality?

A

Immunofixation Electrophoresis (IFE)

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5
Q

What activates the complement system?

A

Antibodies

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6
Q

What occurs during complement system activation?

A

Complement acts as opsonin and increases vascular permeability to recruit phagocytes, leading to lysis of invading cells

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7
Q

How is complement inactivated?

A

Heat at 56C

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8
Q

What does heat-labile mean?

A

Heat sensitive

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9
Q

What does total hemolytic complement assay measure?

A

Measures the ability of a sample to lyse 50% of sensitized sheep RBC

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10
Q

How many ways are there for labs to detect complement activity?

A

4

  • CH50 Assay
  • ELISA
  • Release of Hgb
  • Titration of complement’s hemolytic activity in immune response
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11
Q

What are the four serology technique fundamentals?

A
  1. Describe the immunologic theory and concepts in laboratory differentiating tests.
  2. Describe the difference between sensitivity vs. specificity.
  3. Perform laboratory procedures accurately and precisely as stated in SOP
  4. Calculate serial dilutions.
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12
Q

What is sensitivity?

A

The % of sick people who test positive

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13
Q

What does high sensitivity mean?

A

Few false negatives

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14
Q

What is specificity?

A

The % of well people who test negative

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15
Q

What does high specificity mean?

A

Few false positives

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16
Q

What are the 3 tests for infectious agents or immune status?

A
  • PCR, for quick Ag and Ab identification
  • Culture, for antibiotic susceptibility
  • Antibody response, for qualitative, semi-quantitative, or quantitative titers
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17
Q

How to determine a titer?

A

A patient’s serum is diluted in a series to allow best antibody concentration to be read as an endpoint.

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18
Q

What are titers?

A

Indicators of strength of an antibody response

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19
Q

What antibody is found in the initial titer?

A

IgM (acute phase antibody)

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20
Q

What antibody is found during the recovery phase?

A

IgG or IgA

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21
Q

What antibody is found in the convalescent phase?

A

IgG (provides immunity)

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22
Q

What is the best approach to diagnosis of an acute infection?

A

Find the presence of specific IgM tests vs detection of IgG during convalescence

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23
Q

What are the two parts to a dilution?

A
  1. Sample to be diluted

2. Diluent used to perform the dilution

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24
Q

What is the dilution formula?

A

Sample volume / (sample volume + diluent volume)

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25
Q

What is a dilution factor?

A

The reciprocal of the dilution (typically provided in procedures)

e.g. 1:5 dilution has a dilution factor of 5, 1:100 diltuion has a DF of 100

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26
Q

What tells how much sample plus how much diluent is necessary for dilution?

A

Dilution ratio

e.g. ratio 1:4 = 1 part sample to 4 parts diluent means the dilution was a 1:5 dilution (DF = 5)

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27
Q

What are serial dilutions?

A

a series of sequential dilutions, with a dilution in each tube

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28
Q

What’s important to remember regarding the last tube in serial dilutions?

A

to discard from the last tube to maintain the sample volume for all tubes in the series. When all reagents have been added, all tubes have the same volume.

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29
Q

What is the last tube that shows the antibody response?

A

Endpoint titer reading

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30
Q

What type of immune response do we look for evidence for?

A

A humoral response

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31
Q

At what point do Ag+Ab form immune complexes?

A

Zone of equivalence

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32
Q

What do we typically use to detect antibodies?

A

Antigens

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33
Q

What is aggregation of soluble test antigens?

A

Precipitation

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34
Q

What is aggregation of particulate test antigens?

A

Agglutination

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35
Q

What is seen at prozone because of too much antibody present?

A

False negatives

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36
Q

What occurs during prozone?

A

When the antibody concentration is too high, so it interferes with immune complex formation

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37
Q

What does it mean when the titer initially reads negative then becomes positive?

A

The antibody concentration has been sufficiently diluted to allow for immune complexes to form (dilutions are at zone of equivalence)

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38
Q

What occurs during postzone?

A

When the antigen concentration is too high, so it interferes with immune complex formation

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39
Q

What is seen at postzone because of too much antigens present?

A

False negatives

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40
Q

What occurs during zone of equivalence?

A

When antigen and antibody concentrations are in optimal proportions so immune complexes can be formed

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41
Q

What tests do we use for immune complex detection?

A

5

  1. Precipitation tests
  2. Agglutination tests
  3. Complement fixation tests
  4. Immunoassays (ELISA)
  5. Molecular diagnostic assays (PCR)
42
Q

What involves soluble antigen reacting with soluble antibody?

A

Precipitation reaction

43
Q

What occurs when soluble antigen reacts with soluble antibody?

A

Production of insoluble Ag-Ab immune complexes, where lattice formation occurs resulting in visible reactions

44
Q

What measures amount of light as it directly passes through the immune complexes?

A

Turbidimetric

45
Q

What happens when immune complexes are formed at a high rate?

A

Allow for measurement of turbidity (cloudiness)

46
Q

What measures the amount of light scattered by the immune complexes?

A

Nephelometry

47
Q

What are the ouchterlony formation patterns?

A

Identity, nonidentity, partial identity (shared Ag epitope)

48
Q

What does it mean when there’s an increased light scatter?

A

Increase IgG

49
Q

What happens when antigens have similar epitopes?

A

They’ll fuse in a serological identity reaction

50
Q

What does a ouchterlony formation of a single, smooth precipitin band indicate?

A

Identity reaction

51
Q

What does a ouchterlony formation pattern of two crossed precipitation lines indicate?

A

Nonidentity reaction

52
Q

What does a line crossed with a curved line ouchterlony formation pattern indicate?

A

Partial Identity reaction (shared Ag epitope)

53
Q

What are the 3 immunodiffusion techniques for immunoproliferative diseases?

A
  • RID
  • IEP
  • IFE
54
Q

What’s the procedure for RID?

A

Agar with antibody incorporated with Anti-IgG, M, or A. Dilute the patient sample and inoculate into well. Patient IgG diffuses across the agar to zone of equivalence and precipitin ring (which is proportional to concentration) is formed and measured.

55
Q

What is the purpose of IFE?

A

Separation of proteins into discrete bands for easier interpretation yielding distinct immune complex because it’s more sensitive

56
Q

What is the procedure of IFE?

A

Ab applied directly onto the surface after electrophoresis; Ab contacts the Ag immediately

57
Q

What is the purpose of IEP

A

Separation of proteins as Ab is placed in a through running parallel to electrophoresis

58
Q

What is the procedure of IFE?

A
  • Diffusion of antigen & antibody and precipitin arc is formed
  • Interpretation dependent on comparison of control arc vs. patient arc
59
Q

The maturation of B cells produces what?

A

Plasma cells

60
Q

What do plasma cells produce?

A

Antibodies

61
Q

How do we screen for immunoglobulins?

A
  • SPE (Serum Protein Electrophoresis)
  • Immunodiffusions
  • ELISA
62
Q

What is monoclonal gammopathy?

A

an immunoproliferative disease

63
Q

What happens during monoclonal gammopathy?

A

the clonal proliferation of transformed plasma cells leads to overproduction of a single immunoglobulin

64
Q

What happens during polyclonal gammopathy?

A

more than one immunoglobulin response

65
Q

What are the 3 immunoglobulin abnormalities?

A
  • Hypogammaglobulinemia
  • A-gammaglobulinemia
  • Hypergammopathy
66
Q

What is the procedure for immunodiffusion?

A

Antibody is uniformly added to gel; antigen applied to a well cut in the gel; Ag-Ab combination occurs by diffusion

67
Q

What does an identity reaction indicate?

A

A fusion between antibody & two test antigens; this indicates the two test antigens are identical, which means the test is positive

68
Q

What does a nonidentity reaction indicate?

A

No antigenic determinants in common in the sample; this indicates the antibody is reacting with both antigens, but the antigens are not the same, which means the test is negative

69
Q

What does a partial identity reaction indicate?

A

The antigen is not identical but posses common determinants/epitopes/antigens; so the antigens have something in common but are not the same; inconclusive result

70
Q

What are the most common dysproteinemia?

A
  • Multiple myeloma
  • Neoplastic proliferation of single clone of plasma cells
  • Waldenstrom’s Macroglobulinemia
71
Q

What is happens during agglutination test?

A

Aggregation of particulate test Ags that have been absorbed onto latex beads. When Ag/Ab/latex particle complex forms, agglutination is seen as visible clumps

72
Q

What are examples of agglutination tests?

A

Assays for RA, Rubella, CRP

73
Q

What agglutination test uses RBCs?

A

Hemagglutination test, a viral testing

74
Q

What happens during a flocculation test?

A

precipitate of fine particles that is microscopic or macroscopic

75
Q

What are examples of flocculation tests?

A
  • VDRL

- RPR

76
Q

What happens during a complement fixation test?

A

Complement is used as a reagent & complement is “fixed” within Ag-Ab formation

77
Q

What does uptake of complement indicate?

A

Ag-Ab formation

78
Q

Does hemolysis occur when complement has reacted with Ag-Ab complex?

A

No, complement is fixed which means Ab is present

79
Q

Does hemolysis occur when complement has NOT reacted with Ag-Ab complex?

A

Yes, complement is not fixed which means Ab is not present

80
Q

What are 3 labeling techniques/immunoassays?

A
  • RIA
  • EIA/ELISA
  • FIA
81
Q

What test do we use radioisotopes to label Ag-Ab complex?

A

RIA

82
Q

What test do we use enzymes as labels for colormetric measurement of Ag-Ab complexes?

A

EIA/ELISA

83
Q

What occurs during molecular diagnostics procedure (PCR)?

A

Low levels of specific DNA sequences are amplified using oliginucleotides or primers (small portions of a single DNA strand) and enzymes

84
Q

What are 2 immunoblot techniques?

A
  • Western Blot

- Southern Blot

85
Q

What do we look for in a Western Blot?

A

Proteins

86
Q

What do we look for in a Southern Blot?

A

DNA

87
Q

What is the procedure for Western Blot?

A

Antigen material

88
Q

What is the procedure for Western Blot?

A

Antigen material is electrophoresed for separation and blotted to a nitrocellulose membrane

89
Q

What is the procedure for Southern Blot?

A

Specimen DNA is fragmented using restriction enzymes and separated by electrophoresis & blotted onto nitrocellulose membrane.

90
Q

What forms a band in a Western Blot?

A

Ag-Ab complex

91
Q

What test is used to detect the band in a Western Blot?

A

EIA for HIV testing

92
Q

What is used to detect the fragments in a Southern Blot?

A

Radioisotope (ss-DNA fragments and radiography)

93
Q

What is antigen capture tests?

A

Waived tests that are used for pregnancy testing, drug screening, etc

Sensitivity and specificity of kits determines appropriateness of retesting

94
Q

What are heterophile antibodies?

A

Antibodies which have the ability to react with antigens that are not responsible for their production

95
Q

What is a Forssman antibody?

A

An antibody against guinea pig kidney antigen, which is able to react with sheep red blood cells (an unrelated species)

96
Q

What is an example of a heterophile antibody?

A

Forssman antibody

97
Q

What type of antibodies are heterophile antibodies?

A

Heterophile antibodies are IgM antibodies with affinity for sheep and horse red blood cells

98
Q

What test is used to detect antibody production?

A

EIA

99
Q

What test is used as a genetic counseling tool?

A

HLA testing (people have common HLA that are associated with autoimmune diseases)

100
Q

What is responsible for graft rejection and transplantation reaction?

A

MHC I and II

101
Q

What is essential in activation of the T cells (CD4+/CD8+)?

A

HLA class I and II

102
Q

How do you diagnose Lyme Disease on a Western Blot?

A

To diagnose Lyme Disease, you must see the 41kD band and two more specific bands that react to the spirochete

CLS 790 (2 decks)

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