Automation & Immunochemical Techniques Flashcards
3 phases of analytic process
Preanalytic, analytic, post-analytic
The first continuous flow, single-channel, sequential batch analyzer capable of providing a single test result on approximately 40 samples/hr
AutoAnalyzer (AA)
It was the first noncontinuous flow,
discrete analyzer as well as the first instrument to have random access
capabilities, whereby stat specimens could be analyzed out of sequence on an as-
needed basis.
Automatic Clinical
Analyzer (ACA) (DuPont [now Siemens]).
This instrument was the first to use microsample
volumes and reagents on slides for dry chemistry analysis and to incorporate
computer technology extensively into its design and use.
Kodak Ektachem (now VITROS) Analyzer (now Ortho- Clinical Diagnostics)
The most recent milestone in chemistry analyzer development has been the
combination of chemistry and immunoassay
Modular analyzer
liquids (reagents, diluents, and samples) are pumped
through a system of continuous tubing.
Continuous flow analyzer
3 basic approaches with instruments
continuous flow,
centrifugal analysis, and discrete analysis.
uses the force generated by centrifugation to transfer
and then contain liquids in separate cuvettes for measurement at the perimeter of
a spinning rotor.
centrifugal analysis
Major advantage of centrifugal analysis
Batch analysis
the separation of each sample and accompanying
reagents in a separate container.
discrete analysis.
They are the most popular and versatile analyzers
discrete analysis.
Light source used in Reflectance spectrometry
Tungsten-halogen lamp
It has been and remains a manual process in most laboratories
Preparation of the sample for analysis
The most sophisticated approach that is commonly used
today in specimen identification
Employing a bar code
These slides have microscopically thin layers of dry
reagents mounted on a plastic support. The slides are approximately the size and
thickness of a postage stamp.
dry
chemistry slide
Most automated wet chemistry analyzers use ________ that dip into
the reaction container for a few seconds to stir sample and reagents, after which
they return to a wash reservoir
stirring paddles
causes major interference in many analyses.
Proteins
In the older continuous flow systems, a _____ was the separation or
filtering module. It performed the equivalent of the manual procedures of
precipitation, centrifugation, and filtration, using a fine-pore cellophane
membrane.
dialyzer
are attached from multiple remote stations where the reaction
mixtures reside to a centralized monochromator/detector unit that, in conjunction
with the computer, sequences and analyzes a large volume of light signals from
multiple reactions.
fiberoptic cables, or “light pipes”
refers to automated devices and robots
integrated with existing analyzers to perform all phases of laboratory testing.
Most attention to date has been devoted to development of the front-end systems that can identify and label specimens, centrifuge the specimen and prepare
aliquots, and sort and deliver samples to the analyzer or to storage.
Total Laboratory Automation
Developed RadioImmunoAssay (RIA)
Dr. Rosalyn Yallow
Immunoprecipitation techniques: Gel-Passive
Double Diffusion (Ouchterlony technique) Single Diffusion (Radial Immunodiffusion)
Immunoprecipitation techniques: Gel-Electrophoresis
Counterimmunoelectrophoresis
Immunoelectrophoresis
Immunofixation Electrophoresis
Rocket electrophoresis
Immunoprecipitation techniques: Soluble Phase
Turbidimetry & Nephelometry
The Ag diffuses from the well in all directions, binds to the soluble Ab
in the agarose, and forms a complex seen as a concentric precipitin ring. The
diameter of the ring is related to the concentration of the Ag that diffused from
the well.
Radial Immunodiffusion (RID)
2 variations in RID
Fahey-McKelvey (kinetic) & Mancini (endpoint)
an immune precipitation method that
uses an electrical field to cause the Ag and Ab to migrate toward each other.
Counterimmunoelectrophoresis
2 methods used in the clinical lab to characterize monoclonal proteins in serum & urine
Immunoelectrophoresis (IEP) and immunofixation electrophoresis (IFE)
In this quantitative technique, reagent Ab is
mixed with agarose; Ag is placed in the well and electrophoresed. As the Ag
moves through the agarose, it reacts with the reagent Ab and forms a “rocket,”
with stronger precipitation along the edges.
rocket technique (Laurell technique, or electroimmunoassay)
enhances the Ag–Ab interaction in both turbidimetry & nephelometry
Polyethylene glycol (PEG)
If the binding
reagent is an Ab, it is an ______
immunoassay
If the binding
reagent is a receptor, it is a _____
receptor assay
If the binding
reagent is a transport protein, it is a _______
Competitive protein-binding assay
the first chemiluminescent label used in immunoassays, is a cyclic
diacylhydrazide that emits light energy under alkaline conditions in the presence
of peroxide and peroxidase.
Luminol
compounds that absorb
radiant energy of one wavelength and emit radiant energy of a longer wavelength
in less than 10
−4 seconds.
Fluorescent labels (fluorochromes or fluorophores)
atoms with unstable
nuclei that spontaneously emit radiation are radioactive and referred to as:
Radionuclides
Radiolabeled antigen is also called:
tracer
The earliest immunoassay was a ________ in which the radiolabeled Ag competed with unlabeled Ag for a
limited number of binding sites (Ab)
Competitive immunoassay
use a labeled reagent Ab to detect the Ag. Excess labeled Ab is required to
ensure that the labeled Ab reagent does not limit the reaction.
Noncompetitive immunoassays/Immunometric assays
physical separation is necessary and is
achieved by adsorption, precipitation, or interaction with a solid phase
Heterogeneous assays
the activity or expression of the label depends on whether the labeled reactant is
free or bound.
Homogeneous assays
______ is porous and
readily combines with small molecules to remove them from solution; ______
prevents nonspecific protein binding to the charcoal.
Charcoal; dextran
use particles to trap small Ags, labeled or unlabeled.
Adsorption techniques
occurs when the environment is altered, affecting the
solubility of protein.
Nonimmune precipitation
Soluble Ag–Ab complexes can be precipitated by a second Ab that
recognizes the primary Ab in the soluble complex. The result is a larger complex
that becomes insoluble and precipitates.
Immune precipitation/Double Ab/2nd Ab method
immobilize reagent Ab or Ag provides a method to
separate free from bound labeled reactant after washing.
Use of a solid phase
Advantage of sandwich-type immunoassays
production of linear
calibration curves
Disadvantage of sandwich-type immunoassays
Subjected to false (+) and false (-) interferences
an engineered antibody whereby the
antigen recognition site (Fab) originates from the mouse, while the constant
portion contain sequences that are from the human species.
Chimeric antibody
contains small segments from the mouse but sufficient in number
to recognize the foreign antigen.
Humanized antibody
a homogeneous
competitive immunoassay in which low molecular weight Hps bound to particles
compete with unlabeled analyte for the specific Ab.
Particle-enhanced turbidimetric inhibition immunoassay
the unlabeled
Ag in the sample competes with the labeled Ag for the Ab-binding sites; as the
concentration of unlabeled Ag increases, less enzyme-labeled Ag can bind to the
Ab. Therefore, more labeled Ag is free, and the enzymatic activity is greater.
enzyme-multiplied immunoassay technique (EMIT),
are competitive,
homogeneous assays in which the genetically engineered label is β-galactosidase
Cloned enzyme donor immunoassays (CEDIAs)
is an automated assay
available on the AxSYM Analyzer (Abbott Laboratories). The microparticles
serve as the solid phase, and a glass fiber matrix separates the bound labeled
reagent.
Microparticle capture enzyme immunoassay (MEIA)
fluid-phase unlabeled Ag is captured by Ab on the
solid phase; after washing, the detector Ab (with a fluorescent label attached)
reacts with the solid-phase captured Ag.
Solid-phase fluorescence immunoassays
a heterogeneous,
competitive immunoassay in which particles are used to localize the reaction and
concentrate the fluorescence.
Particle concentration fluorescence immunoassay
Fluorescein-labeled Ag and unlabeled Ag compete for rhodamine-
labeled Ab. More unlabeled Ag lessens the amount of fluorescein-labeled Ag
that binds; therefore, more fluorescence is present
Fluorescence excitation transfer immunoassay
This homogeneous immunoassay uses polarized light to
excite the fluorescent label. Polarized light is created when light passes through
special filters and consists of parallel light waves oriented in one plane.
Fluorescence polarization immunoassay (FPIA)
is an
automated system (Thermo Fisher Scientific) that measures time-delayed
fluorescence from the label europium.
Dissociation-enhanced lanthanide fluoroimmunoassay (DELFIA)
heterogeneous, competitive assay with a tracer.
1 When
bound tracer is measured, the signal from the label (CPM) is inversely related to
the concentration of the unlabeled Ag in the sample.
RIA
When used to microscopically identify bacteria or
constituents in tissue (e.g., immune complexes deposited in vivo), the method is
called
direct immunofluorescence (DIF) or direct fluorescence assay (DFA).
The
substrate is placed on a microscopic slide, serum is overlaid and allowed to react
with the Ag, and the bound Ab is detected by the labeled antihuman globulin
reagent.
indirect
immunofluorescence (IIF) or an indirect immunofluorescence assay (IFA).
is used to classify cell lineage and identify the stage of cell
maturation.
immunophenotyping
is based on cells transported under fluidic pressure passing one by
one through a laser beam.
Flow
cytometry
is related to the size of the cell
Forward light scatter
related to the
granularity of the cell.
Side light scatter
are oligonucleotides that have been designed and engineered to
bind to specific analytes and offer an alternative to Abs used in immunoassays.
Aptamers
