known to interfere with the reactions of certain syphilis tests and complement components (e.g.,C1q). Complement may also cause lysis of the indicator cells in hemagglutination assays. In body fluids can be inactivated by heating to 56 degrees C for 30 mins.
the process that destroys complement activity.
used in the immunology/serology lab for the quantitative transfer of reagents and the preparations of serial dilution of specimens such as serum.
allow fast, repetitive measurement and delivery of solutions of equal volumes. Micropipettors -automatic devices allow rapid, repetitive measurements and delivery of predetermined volumes of reagents or specimens.
All dilutions are a form of ratio. reducing the concentration of a chemical constituent in a solution.
the stepwise dilution of a substance in solution
a representative portion of a larger sample or specimen
the concentration or strength of an antibody expressed as the highest dilution of the serum that produces agglutination
when the disease is first discovered or suspected. Then another sample drawn during the convalescent phase, usually about two weeks later.
A written procedural protocol should contain the following information in this order:
1. Specimen collection and storage; (2) Reagents, supplies, and equipment; (3) Procedural method; (4) Reference values
Factors that can denature, coagulate, or alter protein molecules include:
Heat, Strong acid solution, and Strong alkali solution
A serum specimen should be frozen at -20 degrees C, If testing cannot be done within how many hours?
Complement can be inactivated in human serum by heating to:
56 degrees C
A specimen should be reinactivated when more than _____ hour(s) has/have elapsed since inactivation.
A graduated pipette can be used when
Extreme accuracy is not needed
A meniscus is the:
Curvature in the top surface of a liquid.
Automatic pipettes have the advantage of:
Being fast, allowing repetitive measurement of solutions, and delivering equal volumes of solutions.
A dilution is a(n):
Ratio of volume or number of parts of the substance to be diluted in the total volume, or parts, of the final solution; Indication of relative concentration; Frequently used measure in serologic testing.
Serum for detection of antibodies should be drawn during the:
Acute and convalescent phases of illness
A central concept of serologic testing is:
Manifestation of a rise in antibody titer.
Point of Care Testing (POCT)
defined as laboratory assays performed near the patient. Development of new POCT assays is increasing at a rapid rate; testing can include home tests kits and handheld monitors. The major advantage is the speed of obtaining the results. Major drawback is cost, particularly if a large volume of testing is done. Other areas of concern include maintenance of quality control (QC) and quality assurance (QA). POCT falls with the "waived" or "moderately complex" category.
Simple procedures with little chance of negative outcomes if performed inaccurately. Any over the counter test approved by the FDA is automatically place into the waived category.
Moderately complex tests
More complex than waived tests but usually automated.
Highly complex tests
Usually nonautomated or complicated test requiring considerable judgement.
Provider-performed microscopy tests
Slide examination of freshly collected body fluids.
A major advantage of POCT is:
Faster turnaround time.
POCT assays are usually in the _____ CLIA category.
Important characteristics to consider when selecting a POCT kit are:
Rapid turnaround time; Easy to perform protocol; Storage of temperature reagents; Length of time until expiration.
Formation of a solid mass from previously soluble components. An alternate definition is to occur suddenly or unexpectedly. Also the term for the aggregation of soluble test antigens.
Process of particulate antigens aggregating (clumping) to form a larger complex in the presence of a specific antibody. (Agglutination of particles to which soluble antigen has been absorbed produces a serum method of demonstrating precipitins.)
A technique similar to hemaggluntination except smaller, antigen-coated latex particles are substituted for erythrocytes for the detection of antibodies. Antibodies can be absorbed into the latex particles by binding to the Fc region of antibodies, leaving the Fab region free to interact with antigens present in the patient specimen. Coagglutination and liposome-enhanced testing are variations of latex agglutination. (Latex Beads)
Clumping together of particles to form visible masses. For antibody detection are based on the interaction of soluble antigen with antibody, which results in the formation of a precipitate of fine particles. Can be used in syphilis serologic testing; these test are the classic Venereal Disease Research Laboratories (VDRL) and the rapid plasma reagin (RPR) test.
A laboratory technique for the detection of antibodies thatt involves the agglutination of red blood cells. (Blood Clot)
Human chorionic gonadotropin (hCG)
A glycoprotein hormone secreted by the trophoblast of the developing embryo that rapidly increases in the urine or serum during the early stages of pregnancy. Helps to maintain the corpus luteum. Stimulates production of progesterone. Reaches peak levels at 2 to 3 months after the last menstrual period.
Strongest reaction in Agglutination testing the Ig responsible is:
Immunoglobulin M (IgM) antibodies are more efficient at agglutination because their large size and multivalency permit more effective bridging of the space between cells caused by zeta potential.
Methods of enhancing Agglutination
Centrifugation, Treatment with proteolytic enzymes, use of colloids, antihuman globulin (AHG) tests
Pregnancy Test (hCG); RPR (rapid plasma reagin, syphilis test); ABO Blood Grouping (Reverse Grouping) are just a few examples
The quality of test results in an agglutination reaction depends on all the following:
Duration of incubation; Amount of antigen conjugated to the carrier; Avidity of antigen conjugated to the carrier.
Flocculation procedures differ from latex agglutination procedures because:
Soluble antigen reacts with antibody.
In the hemagglutination technique, antihuman globulins are used to enhance medium to detect which antibodies?
The prozone phenomenon can result in a (an):
The effect of competing antibodies seeking to attach to antigen sites is called:
All of the following are methods that can be used to enhance agglutination of IgG antibodies:
Centrifugation; Treatment with proteolytic enzymes; Using colloids
A classic technique for the detection of viral antibodies is:
Aggregation of soluble test antigens
Aggregation of particulate test antigens
Uses antibodies bound to a particle to enhance visibility of agglutination.
Based on the interaction of soluble antigen with antibody, resulting in formation of a precipitate of fine particles.
Agglutination of erythrocytes in test for antibody detection.
Artifical or biologic carriers that can be used in an agglutination reaction include:
Latex particles; Colloidal charcoal; Erythrocytes coated with antigen in a constant amount.
Is the first phase of agglutination and Represents the physical attachment of antibody molecules to antigens on the RBC membrane.
Agglutination can be used to enhance reactions by all the following:
Decreasing ionic strength of the reaction; centrifugation; Using colloids and antihuman globulin.
Human Pregnancy testing
Tests detect human chorionic gonadotropin (hCG); The hCG is secreted by the trophoblast of the developing embryo; Presence of hCG rapidly increases in urine or serum.
The most common laboratory method for detecting hCG is
In the latex agglutination method for the detection of hCG, no agglutination indicates:
Presence of hCG
Can a urine specimen for pregnancy testing be frozen?
A false positive reaction in a latex agglutination test for hCG can be caused by all the following:
Chorioepithelioma, Hydatidiform mole, Excessive ingestion of aspirin.
the migration of charged solutes or particles in an electrical field. A method of separating macromolecules such as proteins on the basis of net electrical charge and size(MFW)
A positively charged particle in a solution
A negatively charged particle in a solution
When a favorable antigen/antibody ratio exists; the antigen antibody complex becomes visible as precipitin lines or bands.
involves the electrophoresis of serum or urine followed by immunodiffusion. Two step procedure involving the electrical separation of proteins, followed by the linear diffusion (immunofixation) of antibodies into the electrophoretic gel from a trough that extends through the length of the gel adjacent to the electrophoretic path. The reactions produce preciptin arcs at positions of equivalence.
confirms the presence or absence of major protein fractions.
specific individual immunoglobulins identify only the corresponding proteins. If the nonspecific antisera have combining sites for H and L chains, the combining sites will react with L chains of other immunoglobulins or with the free L chains of BJ protein (Bence Jones protein). H chain specific sera do not cross react with other proteins.
Immunofixation electrophoresis (IFE); immunofixation
specific antibodies are used to produce sensitive and specific qualitative visual identification of paraproteins by electrophoretic position. Has replace IEP in the evaluation of monoclonal gammopathies because of its rapidity and ease of interpretation. IFE is a two stage procedure: (1) agarose gel protein electrophoresis and (2) immunoprecipitation. Test specimen may be serum, urine, cerebrospinal fluid or other body fluids.
Comparison of Techniques
Immunoelectrophoresis (IEP) is technically simpler and less subject to antigen excess phenomenon than immunofixation electrophoresis (IFE). If high concentrations of monoclonal protein iwth IFE give no visible reactions, IEP is considered to be a better technique for typing large monoclonal gammopathies. Immunofixation electrophoresis can be optimized to give both greater sensitivity and resolution than IEP. (page 155)
Easy to use; less sensitive; better for typing large monoclonal gammopathies; interpretation is challenging
Immunofixation Electrophoresis (IFE)
more complex to use; more sensitive; used for difficult to characterize anomalous proteins, interpretation is easier
Protein can be separated into how many fractions by use of serum electrophoresis?
What is the most common application of immunoelectrophoresis (IEP)?
Diagnosis of monoclonal gammopathies
Immunofixation electrophoresis (IFE) is best used in the :
workup fo monoclonal gammopathy
Comparion of IEP and IFE
IEP is technically simplier and less subject to antigen excess phenomenon than IFE; IFE can be optimized to give greater sensitivity and resolution than IEP; IFE should be reserved for anomalous proteins that are difficult to characterize by IEP.
Immunoelectrophoresis (IEP) involves:
Separation of proteins based on the rate of migration of individual components in an electrical field; Electrophoresis of serum or urine; double immunodiffusion following electrophoresis.
In IEP, proteins are differentiated by:
Electrophoresis; Diffusion coefficient; antibody specificity
IEP can divide the proteins of normal serum into how many distinct precipitation bands?
25 to 40
IEP is useful for clinically detecting:
Structural abnormalities; Concentration changes in proteins; cogenital deficiency of some complement components.
The most common application of IEP of serum is:
Diagnosis of monoclonal gammopathy
Immunofixation electrophoresis (IFE) can test:
Serum, urine and cerebrospinal fluid
The primary use of IFE is:
Characterization of monoclonal immunoglobulins
An older and less frequently used laboratory technique involving the use of radioactive substances to evaluate immunoglobulins. Traditional RIA is done with specific antibodies in liquid solution. Solid-phase RIA uses antibody bound to solid support.
Enzyme immunoassay (EIA)
designed to detect antigens or antibodies by producing an enzyme triggered color change; uses a nonisotopic label that offers the advantage of safety. A general term for quantitative testing of both antigens and antibodies. The method uses color changed products of enzyme substrate interaction or inhibition to measure the antigen antibody reactions. Also called ELISA.
refers to light emission produced during a chemical reaction and is used extensively in automated immunoassay. Two formats are used: competitive and sandwich immunoassays. Has excellent sensitivity and dynamic range and does not require sample radiation.
Antinuclear antibodies (ANAs)
directed against a variety of macromolecules occur in extraordinarily high frequency in systemic rheumatic disease. Many rheumatic diseases are characterized by the presence of one or more ANAs. The identification of the specific antibody is useful in the detection and diagnosis of the disease.
fluorescent labeling is another method used to demonstrate the complexing of antigens and antibodies. Basic methods: Direct immunofluorescent assay, Inhibition immunofluorescent assay, and Indirect immunofluorescent assay.
Indirect fluorescent assay (IFA)
Procedure used to detect homogeneous antigen plus antigen with antiimmunoglobulins using fluorescent microscopy.
Fixed amount of labeled antigen competes with unlabeled antigen from patient specimen for limited number of antibody binding sites.
Sample antigen binds to antibody fixed onto solid phase; second, chemiluminescent labeled antibody binds to antigen antibody complex.
Enzyme labels often used in indirect procedures are:
Alkaline phosphatase, Horseradish peroxidase, and Beta-galactosidase
Enzyme immunoassay (EIA)
uses a nonisotopic label
uses antibody labeled with fluorescein isothiocyanate (FITC)
Direct immunofluorescent assay
Uses conjugated antibody to detect antigen/antibody reactions
Inhibition immunofluorescent assay
Antigen first exposed to unlabeled antibody, then labeled antibody
Indirect immunofluorescent assay
based on antibodies acting as antigens and reacting with antiimunoglobulins
For an enzyme to be used in an EIA, it must meet all the following criteria:
High amount of stability, extreme specificity, and no alteration by inhibitor with the system.
A fluorescent substance is one that,
while absorbing light of one wavelength, emits light of another (longer) wavelength.