Quiz #2 - Autoimmmunity and Serological Techniques Flashcards
(102 cards)
1
Q
What produces immunoglobulins?
A
B cells or plasma cells
2
Q
Where are immunoglobulins found in Serum Protein Electrophoresis (SPE)?
A
Gamma region
3
Q
What does a gamma-globulin “spike” indicate?
A
Myeloma
4
Q
What test determines existence of immunoglobulin abnormality?
A
Immunofixation Electrophoresis (IFE)
5
Q
What activates the complement system?
A
Antibodies
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
7
Q
How is complement inactivated?
A
Heat at 56C
8
Q
What does heat-labile mean?
A
Heat sensitive
9
Q
What does total hemolytic complement assay measure?
A
Measures the ability of a sample to lyse 50% of sensitized sheep RBC
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
11
Q
What are the four serology technique fundamentals?
A
- Describe the immunologic theory and concepts in laboratory differentiating tests.
- Describe the difference between sensitivity vs. specificity.
- Perform laboratory procedures accurately and precisely as stated in SOP
- Calculate serial dilutions.
12
Q
What is sensitivity?
A
The % of sick people who test positive
13
Q
What does high sensitivity mean?
A
Few false negatives
14
Q
What is specificity?
A
The % of well people who test negative
15
Q
What does high specificity mean?
A
Few false positives
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
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.
18
Q
What are titers?
A
Indicators of strength of an antibody response
19
Q
What antibody is found in the initial titer?
A
IgM (acute phase antibody)
20
Q
What antibody is found during the recovery phase?
A
IgG or IgA
21
Q
What antibody is found in the convalescent phase?
A
IgG (provides immunity)
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
23
Q
What are the two parts to a dilution?
A
- Sample to be diluted
2. Diluent used to perform the dilution
24
Q
What is the dilution formula?
A
Sample volume / (sample volume + diluent volume)
25
What is a dilution factor?
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
26
What tells how much sample plus how much diluent is necessary for dilution?
Dilution ratio
e.g. ratio 1:4 = 1 part sample to 4 parts diluent means the dilution was a 1:5 dilution (DF = 5)
27
What are serial dilutions?
a series of sequential dilutions, with a dilution in each tube
28
What's important to remember regarding the last tube in serial dilutions?
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.
29
What is the last tube that shows the antibody response?
Endpoint titer reading
30
What type of immune response do we look for evidence for?
A humoral response
31
At what point do Ag+Ab form immune complexes?
Zone of equivalence
32
What do we typically use to detect antibodies?
Antigens
33
What is aggregation of soluble test antigens?
Precipitation
34
What is aggregation of particulate test antigens?
Agglutination
35
What is seen at prozone because of too much antibody present?
False negatives
36
What occurs during prozone?
When the antibody concentration is too high, so it interferes with immune complex formation
37
What does it mean when the titer initially reads negative then becomes positive?
The antibody concentration has been sufficiently diluted to allow for immune complexes to form (dilutions are at zone of equivalence)
38
What occurs during postzone?
When the antigen concentration is too high, so it interferes with immune complex formation
39
What is seen at postzone because of too much antigens present?
False negatives
40
What occurs during zone of equivalence?
When antigen and antibody concentrations are in optimal proportions so immune complexes can be formed
41
What tests do we use for immune complex detection?
5
1. Precipitation tests
2. Agglutination tests
3. Complement fixation tests
4. Immunoassays (ELISA)
5. Molecular diagnostic assays (PCR)
42
What involves soluble antigen reacting with soluble antibody?
Precipitation reaction
43
What occurs when soluble antigen reacts with soluble antibody?
Production of insoluble Ag-Ab immune complexes, where lattice formation occurs resulting in visible reactions
44
What measures amount of light as it directly passes through the immune complexes?
Turbidimetric
45
What happens when immune complexes are formed at a high rate?
Allow for measurement of turbidity (cloudiness)
46
What measures the amount of light scattered by the immune complexes?
Nephelometry
47
What are the ouchterlony formation patterns?
Identity, nonidentity, partial identity (shared Ag epitope)
48
What does it mean when there's an increased light scatter?
Increase IgG
49
What happens when antigens have similar epitopes?
They'll fuse in a serological identity reaction
50
What does a ouchterlony formation of a single, smooth precipitin band indicate?
Identity reaction
51
What does a ouchterlony formation pattern of two crossed precipitation lines indicate?
Nonidentity reaction
52
What does a line crossed with a curved line ouchterlony formation pattern indicate?
Partial Identity reaction (shared Ag epitope)
53
What are the 3 immunodiffusion techniques for immunoproliferative diseases?
- RID
- IEP
- IFE
54
What's the procedure for RID?
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
What is the purpose of IFE?
Separation of proteins into discrete bands for easier interpretation yielding distinct immune complex because it's more sensitive
56
What is the procedure of IFE?
Ab applied directly onto the surface after electrophoresis; Ab contacts the Ag immediately
57
What is the purpose of IEP
Separation of proteins as Ab is placed in a through running parallel to electrophoresis
58
What is the procedure of IFE?
- Diffusion of antigen & antibody and precipitin arc is formed
- Interpretation dependent on comparison of control arc vs. patient arc
59
The maturation of B cells produces what?
Plasma cells
60
What do plasma cells produce?
Antibodies
61
How do we screen for immunoglobulins?
- SPE (Serum Protein Electrophoresis)
- Immunodiffusions
- ELISA
62
What is monoclonal gammopathy?
an immunoproliferative disease
63
What happens during monoclonal gammopathy?
the clonal proliferation of transformed plasma cells leads to overproduction of a single immunoglobulin
64
What happens during polyclonal gammopathy?
more than one immunoglobulin response
65
What are the 3 immunoglobulin abnormalities?
- Hypogammaglobulinemia
- A-gammaglobulinemia
- Hypergammopathy
66
What is the procedure for immunodiffusion?
Antibody is uniformly added to gel; antigen applied to a well cut in the gel; Ag-Ab combination occurs by diffusion
67
What does an identity reaction indicate?
A fusion between antibody & two test antigens; this indicates the two test antigens are identical, which means the test is positive
68
What does a nonidentity reaction indicate?
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
What does a partial identity reaction indicate?
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
What are the most common dysproteinemia?
- Multiple myeloma
- Neoplastic proliferation of single clone of plasma cells
- Waldenstrom’s Macroglobulinemia
71
What is happens during agglutination test?
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
What are examples of agglutination tests?
Assays for RA, Rubella, CRP
73
What agglutination test uses RBCs?
Hemagglutination test, a viral testing
74
What happens during a flocculation test?
precipitate of fine particles that is microscopic or macroscopic
75
What are examples of flocculation tests?
- VDRL
| - RPR
76
What happens during a complement fixation test?
Complement is used as a reagent & complement is "fixed" within Ag-Ab formation
77
What does uptake of complement indicate?
Ag-Ab formation
78
Does hemolysis occur when complement has reacted with Ag-Ab complex?
No, complement is fixed which means Ab is present
79
Does hemolysis occur when complement has NOT reacted with Ag-Ab complex?
Yes, complement is not fixed which means Ab is not present
80
What are 3 labeling techniques/immunoassays?
- RIA
- EIA/ELISA
- FIA
81
What test do we use radioisotopes to label Ag-Ab complex?
RIA
82
What test do we use enzymes as labels for colormetric measurement of Ag-Ab complexes?
EIA/ELISA
83
What occurs during molecular diagnostics procedure (PCR)?
Low levels of specific DNA sequences are amplified using oliginucleotides or primers (small portions of a single DNA strand) and enzymes
84
What are 2 immunoblot techniques?
- Western Blot
| - Southern Blot
85
What do we look for in a Western Blot?
Proteins
86
What do we look for in a Southern Blot?
DNA
87
What is the procedure for Western Blot?
Antigen material
88
What is the procedure for Western Blot?
Antigen material is electrophoresed for separation and blotted to a nitrocellulose membrane
89
What is the procedure for Southern Blot?
Specimen DNA is fragmented using restriction enzymes and separated by electrophoresis & blotted onto nitrocellulose membrane.
90
What forms a band in a Western Blot?
Ag-Ab complex
91
What test is used to detect the band in a Western Blot?
EIA for HIV testing
92
What is used to detect the fragments in a Southern Blot?
Radioisotope (ss-DNA fragments and radiography)
93
What is antigen capture tests?
Waived tests that are used for pregnancy testing, drug screening, etc
Sensitivity and specificity of kits determines appropriateness of retesting
94
What are heterophile antibodies?
Antibodies which have the ability to react with antigens that are not responsible for their production
95
What is a Forssman antibody?
An antibody against guinea pig kidney antigen, which is able to react with sheep red blood cells (an unrelated species)
96
What is an example of a heterophile antibody?
Forssman antibody
97
What type of antibodies are heterophile antibodies?
Heterophile antibodies are IgM antibodies with affinity for sheep and horse red blood cells
98
What test is used to detect antibody production?
EIA
99
What test is used as a genetic counseling tool?
HLA testing (people have common HLA that are associated with autoimmune diseases)
100
What is responsible for graft rejection and transplantation reaction?
MHC I and II
101
What is essential in activation of the T cells (CD4+/CD8+)?
HLA class I and II
102
How do you diagnose Lyme Disease on a Western Blot?
To diagnose Lyme Disease, you must see the 41kD band and two more specific bands that react to the spirochete
