What contains a wide variety of safety hazards, many capable of producing serious injury or life-threatening disease? The clinical laboratory Why must clinical laboratory personnel what hazards exist and the basic safety precautions associated with them, and they must apply the basic rules of common sense required for every day safety? To work safely in this environment What are unique to the healthcare environment, and others are encountered routinely throughout life? hazards In which laboratory does the most significant hazard exists in obtaining and testing patient specimens? Immunology laboratory What is the transmission of microorganisms called? the of infection Why is an understanding of the transmission—the chain of infection—of microorganisms is necessary? to prevent infection The chain of infection requires a continuous link between what three elements? a source, a method of transmission, and a susceptible host. What is the most likely source of infection is through? contact with patient specimens What is the main concern when handling patient specimens? The exposure to viruses such as the hepatitis viruses and human immunodeficiency virus (HIV) What are designed to protect health-care workers from exposure to potentially harmful infectious agents? safety precautions What is the ultimate goal of biological safety? to prevent completion of the chain by preventing transmission What are designed to protect healthcare workers from exposure to potentially harmful infectious agents? Safety precautions Preventing the transmission of microorganisms from infected sources to susceptible is critical in? controlling the spread of infection What includes handwashing, wearing personal protective equipment (PPE), isolating highly infective or highly susceptible patients, and properly disposing contaminated materials? Procedures to prevent transmission of microorganisms Strict adherence to guidelines published by what organizations is essential? the Centers for Disease Control and Prevention (CDC) and the Occupational Safety and Health Administration (OSHA)
hypersensitivity reactions resembling poison ivy that appear 24 to 48 hours following exposure? True immediate hypersensitivity reactions often characterized by facial flushing and respiratory difficulty following contact with what? Latex Handwashing immediately removal of gloves and avoiding powdered gloves may aid in preventing the development of what? Latex allergy Replacing latex gloves with what type of gloves provides an acceptable alternative. nitrile or vinyl Any signs of a reaction should be reported to a supervisor, because what type of allergy can be life-threatening? A true latex allergy resistant laboratory coats with wrist cuffs are worn to protect the skin and clothing from contamination by patient specimens in which laboratories? immunology laboratory What is worn at all times when working with patient specimens? Laboratory coats What must be completely buttoned with gloves pulled over the cuffs? Laboratory coats What should be changed as soon as possible if they become visibly soiled, and they must be removed when leaving the laboratory? gloves and laboratory coats What must be protected from specimen splashes and aerosols? The mucous membranes of the eyes, nose, and mouth What can be used to protect the mucous membranes of the eyes, nose, and goggles, full-face plastic shields, and Plexiglas countertop shields Particular care should be taken to avoid what when removing container tops and when transferring and centrifuging specimens? splashes and aerosols centrifuge specimens in what type of tube? uncapped Never centrifuge specimens in what type of centrifuge? An uncovered centrifuge When specimens are received in containers with contaminated exteriors, what must be done to the exterior of the container? It must be disinfected or, if necessary, a new specimen may be requested.
What is the visible aggregation of particles caused by combination with specific antibody called? Agglutination Antibodies that combine with particles are called? Agglutinins Antigens must be exposed and able to bind with the antibody because this reaction takes place where? On the surface of the particle What are the two steps in agglutination? Sensitization followed by lattice formation In order to form antibody bridges or lattices, each particle must have? Multiple antigenic/determiniant sites What are the different categories of agglutination reactions? Direct, passive, reverse passive, agglutination inhibition, and coagglutination In agglutination, what is the first reaction involving antigen-antibody combination through single antigenic determinants on the particle surface? Sensitization step The initial reaction in agglutination is rapid and reversible, it follows which law? Law of mass action The second step in agglutination is the formation of cross-links that form? Visible aggregates Sensitization can be affected by? the nature of the antibody molecule What determines how much an antibody remains attached? Affinity and avidity _____ with a potential valence of 10 is over 700 times more efficient in agglutination that is ______ with a valence of 2. IgM, IgG What is a key factor in the initial sensitization process? The nature of antigen-bearing surface What happens if epitopes are sparse or obscured by other surface molecules? They are less likely to interact with an antibody The second stage of agglutination is dependent on __________ and the relative _________of antigen and antibody. Environmental conditions, concentrations Lattice formation is governed by what factors? Ionic strength, pH, and temperature Antibodies must be able to bridge the gap between cells by? One molecule being able to bind to two different cells such as erythrocytes or bacterial cells are difficult to bring together into lattice formation because? They have a slight negative surface charge
to produce breakdown products that may be chromogenic, fluorogenic or luminescent? The following are examples of what? Horseradish peroxidase, dehydrogenase, alkaline phosphatase, and β-D-galactosidase. Typical enzymes used in labeled immnoassays Why are alkaline phosphatase and horseradish peroxidase so often used in labeled immunoassays? They have the highest turnover (conversion of substrate), high sensitivity and are easy to detect Enzyme assays are classified as either ____ or _____? Heterogeneous or homogeneous Enzyme assays are classified as heterogeneous or homogeneous on the basis of what? Whether a separation step is necessary What type of enzymatic immunoassay requires a step to physically separate free from bound analyte? Heterogeneous enzyme immunoassays What type of enzymatic immunoassay requires no separation step? Homogeneous enzyme immunoassays Why do homogenous enzyme immunoassays not require a separation step? Because enzyme activity diminishes when binding of antibody and antigen occurs The enzyme immunoassays (EIAs) were competitive assays based on the principle of what type of assay? RIA (radioimmunoassay) Enzyme activity is inversely proportional to the concentration of test substance. What does this mean? The more patient antigen is the less antigen can attach Currently type of immunoassays are more common? Competitive or enzyme immunoassays? Non-competitive enzyme immunoassays Although competitive have high specificity, why are non-competitive enzyme immunoassays more common? Non-competitive enzyme immunoassays high sensitivity and specificity, simplicity and low cost. Non-competitive assays are often referred to as what? ELISA (indirect enzyme-linked immunosorbent assays) In labeled immunoassays, either antigen or antibody may be bound to solid phases such as? Microtiter plates, nitrocellulose membranes, and magnetic latex beads When antigen is bound to solid phase, what is done with patient serum? Unknown antibody is added to it and it is incubated In labeled immunoassays, after a wash step, what is added? An enzyme-labeled antiglobulin. phosphate
What happens with active site on the enzyme, when antibody binds to specific determinant sites on the antigen, resulting in a measurable loss of activity? the active site on the enzyme is blocked Enzyme activity is directly in proportion to what? concentration of patient antigen Enzyme activity is directly in proportion to what? hapten present in the test solution Why sensitivity of homogeneous immunoassays is far less than that achievable by heterogeneous enzyme assays? because the amplification properties of enzymes are not utilized Enzyme immunoassays have achieved a sensitivity similar to what assay without attendant health hazards or waste disposal problems? RIA In what assays there is no need for expensive instrumentation, and reagents are inexpensive and have a long shelf life? Enzyme immunoassays What is disadvantage of enzyme immunoassays? some specimens may contain natural inhibitors The size of the enzyme label may be a limiting factor in the design of what assays? Enzyme immunoassays Nonspecific protein binding is potential difficulty in what assays? Enzyme immunoassays In what immunoassay fluorophores or fluorochromes absorb energy from an incident light source and emit light of a longer wavelength and lower energy as the excited electrons return to the ground state? fluorescent immunoassay techniques What two compounds most often used in fluorescent immunoassay? fluorescent immunoassay techniques Fluorescein and rhodamine are usually used in what form? isothiocyanates Why fluorescein and rhodamine are usually used in the form of isothiocyanates? because these can be readily coupled with antigen or antibody Why fluorescein and rhodamine are used together? Because their absorbance and emission patterns differ What compound can be used in fluorescent immunoassay? phycoerythrin What compound can be used in fluorescent immunoassay? europium (β-naphthyl trifluoroacetone)
How does the intensity of the fluorescent signal maintain a particular location? Intensity is proportional to the sequence homology at a particular locus. How are (SNPs) single-nucleotide polymorphisms detected? They are identified with the set of SNPs that are directly related to or that cause disease. How can diseases such as leukemias, tumors, and characterizing microbial agents be distinguished? Detection of point mutations can be used for classifying these diseases. How is it determined on low sensitive and specific hybridization techniques? Techniques are determined by conditions of the reaction itself. How must two nucleic acid strands keep a perfect complementarity to form a stable hybrid? Nucleic acid strands must share at least 16-20 consecutive bases. What must be considered with amplification? Conditions such as stringency, or correct pairing. How is stringency affected? Stringency is affected by salt concentration temperature, and the concentration of D’s stabilizing agents such as urea. What is DNA sequencing? DNA sequencing is considered the “gold standard” for many molecular applications. How do you provide greater accurancy for both DNA strands? The sequence should be analyzed on both DNA strands. How are mutations detected? Patient sequences are compared to known reference sequences. Why is a PCR amplification include most sequence strategies? It includes a first step to amplify the region of interest to be sequenced. What is the name of the reaction to sequencing? The reaction is on dideoxy chain termination reaction developed by Sanger and collegues in 1977. What happens when one of the four dideoxynucleotides are incorporated into a growing DNA Synthesis is halted. What type of ring does these nucleotides lack the 3’ and 2’ hydroxyl (OH) group? The pentose ring. What happens when you try to incorporate dideoxynucleotide to elongate the DNA chain? It terminates the length What is the most commonly used for DNA sequencing, similar to PCR? sequencing What are the steps that are involved in cycle sequencing? The steps involve denaturing, annealing of a primer, chain extending, and terminating by varying the temperature of the reaction. What are generated fragments tagged with? They are tagged with fluorescent dye and separated, based on size, by denaturing gel or capillary electrophoresis. What are separated fragments spotted by? They are spotted by fluorescence detectors as the fragments pass through the detector.
Introducing sample, adding reagent, mixing reagent and sample, incubating, detecting, calculating, and reporting read out or results are included during which stage? Analytical Which of these tasks may be automated on various immunoassay analyzers? All or some of the tasks Reagent used in automated immunoassay analyzers requires consideration of what factors? Handling, preparing and storing, and dispensing Some reagents come ready for use, but if not, the analyzer must be able to do what to reagents before they can be used? Properly dilute What do most reagents come with that is read by the analyzer to reduce operator so if the wrong reagent is loaded into the analyzer by mistake, the analyzer will detect the error and generate an error message? Bar After reagents have been added to the samples, the next concern is proper mixing to obtain what? Reliable results Magnetic stirring, rotation paddles, forceful dispensing, and vigorous lateral shaking are all analyzers use of which method? Mixing Whichever method is used, what is imperative to not do between sample wells to prevent erroneous results? That there be no splashing incubation is then carried out at what temperatures, or analyzers need to have built-in incubators for incubation? Ambient temperatures What does the detection of the final analyte depend on? The chemistry involved in the immunoassay In addition to fluorescence or chemiluminescence, both of which require fluorescence detectors, what other method can be used? Colorimetric absorption spectroscopy Regardless of the instrumentation considered, what must always be performed? Proper validation of new instrumentation or methodology The laboratory must determine how it will meet which standard regulations for verifying the manufacturer’s performance specifications? Clinical Laboratory Improvement Amendment (CLIA) As designated by CLIA, what are the required verifications to be determined for a new method? Accuracy, precision, analytic sensitivity, analytic specificity, interfering substances, reportable range, and reference intervals What validation method is referred to as the test’s ability to actually measure what it claims to measure? Accuracy
Parallel testing with an alternative method or technology is a form of which kind of testing? Accuracy testing What validation method is referred to as the ability to consistently reproduce the same result on repeated testing of the same sample? Precision What types of controls should be included in precision testing? At least one normal and one abnormal control What validation method is defined as the lowest measurable amount of an analyte? Analytic sensitivity What validation method is defined as the assay’s ability to generate a negative result when the analyte is not present? Analytic specificity What validation method is defined as the of values that will generate a positive result for the specimens assayed by the test procedure? Reportable range What validation method is defined as the range of values found in healthy individuals who do not have the condition that is detected by the assay? Reference interval Choosing an automated immunoassay analyzer is complicated and depends greatly on what? The main tasks to be performed in a particular laboratory What type of analyzers may work best if only one type of testing is performed on a large scale? analyzers What type of analyzer allows for more flexibility, including processing of stat samples? Random access analyzers In either case, what is required on any new instruments before patient results can be reported with confidence? Extensive validation Increased turnaround time for testing, remove the possibility of manual errors, and allow for greater sensitivity in determining the presence of low-level analytes is all made possible by what? Instrumentation What is defined as a heightened state of immune responsiveness? Hypersensitivity reactions What type of reaction is an exaggerated response to a harmless antigen that results in injury to the tissue, disease, or even death? Hypersensitivity reaction British immunologists P.G.H. Gell and R.R.A. Coombs devised a classification system for such reactions based on how many different categories? Four
Cell-bound antibody reacts with antigen to release physiologically active substance is which type of hypersensitivity reactions? Type I What is not involved in type I reactions? Complement Free antibody reacts with antigen associated with cell surfaces in which type of hypersensitivity reactions? Type II What plays a major role in producing tissue damage in type II reactions? Complement Antibody reacts with soluble antigen to form complexes that precipitate in the tissues in which type of hypersensitivity reaction? Type III Complement a major role in producing tissue damage in which type of hypersensitivity reactions? Type III type of hypersensitivity reaction differs from the other three because sensitized T-cells rather than antibody are responsible for the symptoms that develop? Type IV What is not involved in type IV hypersensitivity reactions? Complement Which hypersensitivity types have previously been referred to as immediate hypersensitivity because symptoms develop within a few minutes to a few hours? Types I-III type of hypersensitivity has been called delayed hypersensitivity because its manifestations are not seen until hours after contact with antigen? Type IV The short lag time, usually seconds to minutes, between exposure to antigen and the onset of clinical symptoms is the distinguishing feature of which type of hypersensitivity? Type I What is the key reactant present in type I, or immediate, hypersensitivity reactions? IgE In type I hypersensitivity, what are the antigens called that trigger the formation of IgE? Atopic antigens, or allergens An inherited tendency to respond to naturally occurring inhaled and ingested Atopy