Serology Exam 1

Label the immune response curve

A-Prozone B-Zone of Equivalence C-Postzone

Zone of equivalence

Lattice formation: Ag-Ab together in approximately equal proportions

Prozone

Antibody excess. It appears false-negative

Postpone

Antigen excess

Lag phase

Time period between the encounter with the antigen and the production of a detectable antibody

Label

A- Fab Region

B- Fc Region

Label

A- CH1

B- VH

C- VL

D- CL

E- CH2

F-CH3

G- Light chain

H- Heavy chain

How do primary and secondary response differ?

Primary response has a longer lag phase, secondary response has a shorter lag phase

Which immunoglobulin is produced first in an immune response?

IgM

What is the predominant antibody in a secondary response?

IgG

Where does complement attach to the immunoglobulin molecule?

Fc region

What role does complement play in the immune response?

Enhance phagocytosis by opsonization and lyses certain bacteria and other cell types

Last steps in complement activation result in the formation of _

Membrane Attack Complex (MAC)

How is complement on a patient’s sample inactivated?

Heat

What is the monoclonal antibody, why are they important, how are they made?

Contain a single antibody that recognizes only 1 epitope on a multivalent antigen and cannot cross react.

Delivery of therapeutic agents in diseases (Continuos source)

Developed based on knowledge that B cells are genetically preprogrammed to synthesize very specific antibody.

What is an antibody titer?

Indication of antibody strength

What is considered a diagnostic increase in titer?

A four fold rise in a titer

What is seroconversion?

Change of a serological test result from antibody negative to antibody positive from samples obtained during the course of an immune response

When should the convalescent specimen be drawn for serological testing?

2 weeks after presentation of symptoms

In serological testing what is meant by the “window period”?

The time between infection and the detection of antibodies in the blood, during which tests may yield false negatives.

When an antibody is present in the blood of a newborn, how can one determine if it is passively acquired maternal antibody or antibody produced by the baby?

Testing for specific immunoglobulin types, such as IgG or IgM, can help distinguish between maternal antibodies and those produced by the infant.

Which antibody class may require the use of an enhancement technique to visualize the reaction in the lab?

IgG

What is a hapten?

A simple chemical group that can bind to antibody once it is formed but that cannot stimulate antibody formation unless bound to a larger carrier molecule

Active vs passive immunity

Active- Individual produces antibodies

Passive- Antibodies transferred to individual

What is the difference between quality control and quality assurance?

Quality control is a system that helps us detect/prevent errors that would impact the accuracy and precision of tests results; Quality assurance establishes standards and documentation to ensure quality requirement is met

List and give examples of the three phases of quality assurance. Pre-Analytical

Orders test, patient preparation, sample drawn, sample transported, sample delivered, sample processed

List and give examples of the three phases of quality assurance. Analytical

Sample analyzed for accuracy and precision, ensuring that test results are reliable before being reported.

List and give examples of the three phases of quality assurance. Post-Analytical

Results verified, results reported, and physician sees patient

In which phase of testing do most errors occur?

Pre-analytical

Positive predictive value

The percentage of all positives in a serological test that are TRUE positives

Negative predictive value

The probability that a person with a negative screening test does not have the disease being tested for

Analytical sensitivity

The lowest measurable amount of analyte that can be reliably detected by a test.

Clinical/diagnostic sensitivity

The proportion of people who have a specific disease or condition and who have a positive result

Analytical specificity

An assay’s ability to generate a negative result when the analyte is not present

Clinical/diagnostic specificity

The proportion of people who do not have a specific disease or condition and who have a negative result

Accuracy

The ability of a test to measure what it claims to measures

Precision

The ability to consistently reproduce the same result upon repeated testing of the same sample

Solute, solvent, solution

A solute is a substance that is dissolved in a solvent to create a solution, which is a homogeneous mixture of the two.

Cross reactivity

When the antibody combines with an antigen that is structurally similar to the immunogen that stimulated the antibody reaction

According to CAP, how often must quality control statistics be calculated?

At least once every 24 hours or at the beginning of each shift

What is the name of a sample that is chemically and physically similar to the unknown and can be tested in the same manner as the unknown to monitor the precision of the test system?

Control or standard

Who should run controls?

Laboratory personnel or technicians responsible for testing.

When should results of control be verified for acceptability?

Before patient’s results are reported

Which part of a quality management program involves the use of external controls?

Quality assurance

What is an antigen?

Macromolecule that is capable of eliciting formation of immunoglobulins or sensitized lymphocytes in an immunocompetent host

What is an epitope?

The key portion of the immunogen against which the immune response is directed

What is an antibody?

Glycoprotein produced by B lymphocytes and plasma cells in response to foreign substance exposure

Define affinity

The initial attraction force between a single antibody binding site and a single epitope

Define avidity

The sum of the attractive fore’s between a multivalent antigen and an antibody that keeeps the molecule together

Direct agglutination

A clumping of cells that occurs when antiibodies react with antigens that are naturally found on the surface of the cells

Hemagglutination

An antigen-antibody reaction that results in the clumping of RBC (direct)

Passive agglutination

A reaction in which particles coated with antigens not normally found on their surfaces clump together because of their combination with antibody

Agglutination inhibition

Based on competition between antigen-coated particles and soluble patient antigens for a limited number of antibody-combining sites. Lack of agglutination = positive reaction

Reverse passive agglutination

Antibody is attached to carrier particle. Agglutination occurs if antigen is present in patient’s sample.

Hemagglutination inhibition

A test for detecting antibodies to certain viruses, based on lack of agglutination that results from antibody neutralizing the virus

Compare precipitation vs agglutination

Both are immunological reactions used to detect and quantify antigens or antibodies, but precipitation involves soluble antigens and antibodies to produce visible insoluble complexes, whereas agglutination involves specific antigens.

Compare nephelometry and turbidimetry

Nephelmetry measures light scattered, turbidimetry measures light absorbed

Compare Ouchterlony vs Radial immunodiffusion

Ouchterlony is a double diffusion technique with both Ag and Ab diffusing in two dimensions. Radial immunodiffusion is a single-diffusion technique where only Ag migrates. Both are end-point reactions

Determine result

Serological Identity

Serological identity

If antigens are identical, they will react with the same anti-body and precipitate to form a continuous arch

Determine result

Non-identity

Non-identity

If antigen share no identical determinants, they will react least with different antibodies and two cross lines are form

Determine result

Partial identity

Partial identity

Antibodies react with two similar antigens, forming two line and a spur that point to the simpler antigen (react least with antibody)

What are some sources of error in the ouchterlony/radial immunodiffusion technique?

Improper dilutions, overfilling wells, nicking the side of the well when filling, contaminating samples, and inconsistent agarose thickness.

Know how to determine concentration of unknown from Radial immunodiffusion

Measuring the diameter of the precipitin ring formed in the agarose and comparing it to a standard curve created using known concentrations.

What type of antibodies can cause false positive reactions in hemagglutination tests?

Cold agglutins

What must be present in the serum for tests based on hemolysis of RBCs?

Antibodies

Which is more sensitive precipitation or agglutination?

Agglutination is more sensitive than precipitation.

What happens if you rotate your agglutination card for more than the recommended time?

False-positive

Rank the following methods in order of lowest to highest sensitivity (Agglutination, Precipitation, Immunoassay)

Precipitation, agglutination, immunoassay

Compare labeled and unlabeled immunoassays

Labeled immunoassays utilize conjugated markers, enhancing sensitivity and enabling detection of lower analyte concentrations, whereas unlabeled assays depend on visual methods that may require higher concentrations for effective analysis.

Compare competitive vs non-competitive, which is most sensitive?

Competitive immunoassays are generally more sensitive than non-competitive immunoassays, as they consist of limited antibody and labeled antigen.

Compare homogeneous v heterogeneous, which is most sensitive?

Heterogenous immunoassays require physical separation of bound from free analyte, making them generally more sensitive than homogeneous assays that do not require this step.

In a competitive immunoassay the amount of signal is inversely or directly proportional to the amount of patient's antibody?

Inversely

In a non-competitive immunoassay the amount of signal is inversely or directly proportional to the amount of patient's antibody?

Directly

What step is required in a heterogeneous immunoassay that is not required in a homogeneous immunoassay?

Centrifugation, binding solid-phase material or magnetic separation

ELISA

Direct

ELISA

Indirect

ELISA

Sandwhich

ELISA

Competitive

Fluorescent immunoassay

Direct

Fluorescent immunoassay

Indirect

Sources of error in ELISA testing

Contamination, pipetting errors, reagents, and improper incubation.

First types of immunoassays used what label?

Radioisotope

Why are enzyme immunoassays (EIA) replacing most radioimmunoassays?

Due to their safety, stability, ease of use, and the absence of hazardous radioactive materials, which makes them more suitable for routine diagnostic testing.

What enzyme labels are commonly used in EIA procedures?

Horseradish peroxidase (HRP), alkaline phosphatase (ALP), B-D-galactosidase, and G6PDH

What is measured in enzyme immunoassays?

Visible light, UV light, fluorescent light and luminescence

What can cause a false positive result in enzyme immunoassays?

Heterophiles, anti-animal Ab, auto-antibodies (RF), and biotin

Bands in serum protein electrophoresis, which band travels farthest toward the anode?

Albumin

What band do antibodies migrate to in serum protein electrophoresis?

Gamma globulin band

Explain immunofixation electrophoresis (IFE), what is it useful for

A qualitative technique that can be used to visualize increases or decreases in immunoglobulin production ant to differentiate monoclonal from polyclonal immunoglobulins

Compare monoclonal vs polyclonal gammopathy

Monoclonal gammopathy is a disorder in which a clone of lymphoid cell causes overproduction of a single immunoglobulin component towards a same epitope. Polyclonal gammopathy is a disorder in which a clone of lymphoid cell causes over production of multiple immunoglobulin components towards different episodes.

Waldenström's primary macroglobulinemia vs Multiple Myeloma including types of immunoglobulins commonly seen.

Both are monoclonal gammopathy. Multiple myeloma causes an overproduction of IgAs and IgGs, while Waldenström cause overproduction of IgMs

Explain “flow-through” membrane-bound EIA

A type of enzyme immunoassay where the sample flows through a membrane impregnated with capture antibodies, allowing for the detection of specific antigens or antibodies in a continuous manner.

What type of immunoassay would be best used for point of care?

A lateral flow immunoassay is best used for point of care due to its simplicity, rapid results, and ease of use.

What additional quality control is required for tests with built-in controls?

Running positive and negative controls

Which methods are most often used on automated immunoassay analyzers?

Turbidimentry and nephelometry are commonly used methods on automated immunoassay analyzers.

What is chemiluminescence?

The production of light energy by a chemical reaction

Name a disease that can cause false positive results in latex agglutination tests because of an antibody in the serum that reacts with IgG?

Rheumatoid arthritis