Clinical chemistry and Immunoassay Flashcards
1
Q
Early automation
First instruments were in
Many manual techniques were
A
✦ First instruments were in
hematology & chemistry
✦ Many manual techniques were
adaptable to automation, making
those areas of the laboratory
easy targets for automation
2
Q
Why Automation
Increase the number of tests blank
expansion of
Minimize blank
Minimize errors that blank
Improves blank
use less
reduction in
A
- Increase the number of tests by a operator in a
given time - Expansion of laboratory testing
- Minimize operator variables
- Minimize errors that could occur in manual
analyses - Improve laboratory safety
- Use less sample and reagent for each test
[Reduction of cost] - Reduction in turn around time
3
Q
Common task of automation
A
- Measurement and Proportion of sample and
reagents - Mixing
- Incubation
- Sensing
- Computation
- Readout
4
Q
Proportioning
Bulk and unit reagents
A
✦ Bulk (Stock) Reagents: pre-made
▪ as concentrates requiring dilution
▪ dry reagents(lyophilized) requiring reconstitution
✦ Unit Reagents: more expensive, less error
▪ Sufficient reagent is present for single test
▪ Dry Film: uses paper or a series of thin films impregnated
with desired reagents
▪ Reagent will be ready by wetting the paper or film with water,
buffer, or sample.
▪ Container/test tube with pre-measured liquids or powders
5
Q
addition of sample to reagent
A
✦ Unit Reagent: simply add exact amount of sample
✦ Aliquoting: aliquot proper amounts of sample & reagent
✦ Sample: introduced into the analyzer with a thin,
stainless steel probe
6
Q
Sample carryover
A
✦ Because the same probe is used repeatedly for
sequential samples, there is a potential for contamination
of a specimen by a preceding one.
7
Q
Sample carryover is reduced by
A
1) Aspiration of a wash liquid (saline, water or wash buffer)
between samples
2) A back flush of the probe: wash liquid flows through the
probe in an opposite direction to that of the aspiration,
into wast container.
3) Dispensing reagent through the same probe using the
greater volume to flush the probe
8
Q
Percent carry over
A
measured by placing and testing
low concentration samples before and after a high
concentration sample
9
Q
Percent carryover calculations
A
% carryover= (L2-L1)/H * 100%
L1= 1st low concentration reagent
L2= 2nd low concentration reagent
H= is high concentration reagent
10
Q
Calculations should be made on
A
each analyte to develop
repeat policy on high values
11
Q
Mixing
A
✦ (may be) accomplished by peristaltic pumps with
mixing coils, magnets, centrifugal force or
mechanical devices.
✦ Computer program determines how long, vigorous, etc.
✦ Mixing varies, dependent upon the automation system
12
Q
Mixing types
A
✦ Continuous flow systems
▪ mixing coils
✦ Discrete batch analyzers
▪ centrifugal force or flow
turbulence
✦ Unit reagent analyzers
▪ agitation of the pack
✦ Dry film technique
▪ liquid spreads via diffusion
through matrix of test area
13
Q
Incubation
A
✦ Required Time & Temperature for incubation must be
regulated
▪ Time is usually regulated by holding the reaction in
one step of the instrument (the cuvette or tubing) for a
period of time
▪ Temperature is controlled by heating blocks or baths
14
Q
Sensing devices
A
✦ Sensors include 1) spectrophotometers, 2) nephelometers,
3) fluorometers, 4) ISE/potentiometers
✦ May have one or many, depending on analyzer
✦ Most are capable of both single-point (endpoint) and
multiple-point measurements (kinetic-rate reactions)
15
Q
Computation
Beers Lambert law
A
✦ Automated computation: Analog & Digital.
✦ Many computations are based on Beer’s Law, with the
instrument comparing two readings, the voltage output of
the reaction and the blank (Analog)
✦ Electrometer converts voltage signal to digital form
(Digital)
✦ Internal computer uses stored calibration curve to derive
result
A=abc A = absorbance (no units)
a = molar absorptivity (L/mol/cm) or (L/gm/cm)
b = path length of cuvette (cm)
c = concentration (mol/L) or (gm/L)
16
Q
Readout devices
A
✦ Readouts
▪ Generally on a screen
▪ (may be) Printed
Results must be evaluated by MLS before release to
patient chart (QC evaluated, etc.): “Verification”
17
Q
Additional capabilities
Most automation now also have
Performed by what or what
Many have the ability to inference with blank and blank
A
✦ Most automation now also have
▪ Patient specimen identification capabilities
▪ Test ordering capabilities
➢(may be) Performed manually or by bar code
May have ability to interface with laboratory and hospital
information systems (many bi-directional)
18
Q
Computer interface.
the blank directional interface allows the input and output of results of results to the patient chart
Blank directional interface allows only reporting a result
A
✦ Many analyzers have capability to interface with LIS or
HIS
✦ Bi-directional interface allows input (ordering of
procedure from remote site or laboratory accessioning
site) and output of result to patient chart
✦ Unidirectional interface, in general, allows reporting a
result
19
Q
Quality control
many analyzers will blank, blank, and analyze blank
Alert of possible what
Require or automatically perform what
A
✦ Many analyzers will collect, store and analyze quality
control data, applying applicable rules
✦ Alert user of out-of-control data
✦ Alert user of possible problem
✦ Require or automatically perform calibration
20
Q
Archive information
analyzers store and retrieve what Information
Many include what information
A
Analyzers store and retrieve archived information
✓ May include calibration, quality control, and patient results for
designated periods of time
✓ May include maintenance and repair records
21
Q
Types of automation
A
✦ Total Laboratory Automation (TLA)
✦ Modular Integrated Systems (MIS)
✦ Stand-alone systems
22
Q
Total lab automation
Common in what labs
connected by what
what coded specimens
A
✦ “Cradle to grave” automation
✦ Common in Chemistry, Hematology, Coagulation
✦ Connected by track system
▪ Bar coded specimens
▪ Sorters
▪ Aliquoters
▪ Centrifuges
▪ Analyzers
▪ Archiving
23
Q
TLA components
A
✦ Centrifuge
▪ Often rate limiting step
✦ Decapper
✦ Aliquoter
▪ Clot sensor
▪ Ample quantity
▪ Proportioning into additional tubes (barcode)
24
Q
TLA benefits
A
Full Time Equivalent: the ratio of the total number of paid hours during a period by the number of working hours
✦ Low handling
▪ Less error (27% reduction)
▪ Safety
✦ Increased productivity
▪ Fewer FTEs (20% reduction)
✦ Decreased turn-around time
✦ Adaptable
▪ Additional units as needed
✦ Automated reporting
in that period
25
TLA drawbacks
✦ Substantial financial
investment
▪ $2-5 million w/o space
renovation
✦ Laboratory space
▪ Construction costs
✦ Integration of other vendor
instruments
26
Modular integrated systems.
Separate what for various departments
blank for different manufacturers
Easier to interface with the
Drawbacks
✦ Front-end specimen processing similar to TLA
✦ Separate tracks for various departments
✦ Increased handling over TLA
✦ Benefits
▪ Less cost, space
▪ Interface multiple modules from disparate manufacturers
▪ Easier to interface with the LIS.
✦ Drawback”
* Decrease mean turn around time vs, TLA system (50 min
- 2 hr)
* Lower productivity vs, TLA system
27
Modular integrated systems benefits
✦ MIS provides a more attractive approach for smaller labs.
✦ MIS are smaller, require less investment and less
planning than TLA.
✦ MIS can be configured to include several different
platforms, i.e. hematology and immunochemistry.
http://www.youtube.com/watch?v=TC_esYj4VIM
28
Continuous flow analyzers.
Continuously pumped what and separated by what and what
blank type of analyzer capable of providing a single test result on how many samples/hr
The blank is critical in determining flow rates and sample volumes
problems
❖ Continuous Flow (Early analyzers)
▪ tubing and coils
▪ flow stream
▪ continuously pump sample - separated by air bubbles and
wash solutions
✦ Single-channel, sequential batch analyzer capable of providing
a single test result on approximately 40 samples / hr
✦ The “diameter of the tubing” is critical in determining flow rates
and sample volume.
✦ Significant carry-over problems and wasteful use of
continuously flowing reagents
29
Non-selective analyzers
▪ Perform the entire battery of tests on each sample
30
Batch analyzers
▪ Perform the same test simultaneously on all
samples
▪ Decreased use
31
Random access analyzers
▪ Perform specified multiple tests in any order
32
Multiple Parameter (Analytes) Analyzer
▪ Perform multiple tests per sample either sequentially
or simultaneously
33
Random access.
blank test possible
Not every blank performed on every blank
Less blank waste
Tests can be blank or blank
replaced what
✦ Multiple tests possible
✦ Not every test performed on every sample
▪ Programmable
▪ Less reagent waste
▪ Tests can be sequential or simultaneous
✦ Mostly replaced batch analyzers
34
Discrete analyzers
✦ Instruments that compartmentalize each test reaction
✦ Most current analyzers are discrete
35
Discrete batch analyzers
– Perform only one type of assay at a time on
compartmentalized samples
36
Discrete multi-sequential analyzers
– Discrete: each specimen separate from others
– Sequential: tests performed one at a time
– Multiple: numerous tests can be performed on each
sample
37
Discrete Multiple Simultaneous Analyzers
– All analyzers of this type have random access
capability
– Discrete: each specimen separate from all others
– Multiple: numerous tests can be performed on each
sample
– Simultaneous: many tests are performed at same
time
38
Terminology
✦ Test repertoire
▪ Immediate
▪ Total
✦ Dwell time
▪ Time specimen is in instrument
▪ Seconds to days (vitamin assay)
✦ Throughput
▪ Max tests per hour, shift, day
39
Current automation
✦ Many discrete, random access analyzers that incorporate
multiple chemical, electronic, immunologic, and optic
technologies.
✦ Contain and use sophisticated computer technology for
data handling
✦ Offer large test menus
40
Results of automation in the lab
✦ Users of laboratory services have pressed for increased
efficiency, productivity, precision, accuracy
✦ Technology changes rapidly: A new ‘generation’ of
instruments appears approximately every 5 years
41
Changing roles
✦ Manual methods in a direct, hand-on production of data
▪ Highly complex tests
▪ New test development
▪ PPM
✦ Data analyzer/ manager
▪ Routine tests
▪ Moderately complex
42
examples of changing roles
✦ Operate instruments
✦ Analyze and interpret data
✦ Perform preventive maintenance
✦ Basic instrument repair
✦ Sophisticated problem-solving skills
✦ Educator (POC, novel technologies)
43
Immunochemical techniques
Most what
Add blank to the sample containing what
Detects what
Ex
✴ Most common
‣ Add labeled antibody to
the patient’s sample
(analyte) contains
antigen
‣ Detecting patient’s
antibody
Ex) Infectious disease, serology,
allergy testing, &
autoimmune testing
44
Antibodies in immunoassays
Characteristics: 1) Specificity, 2) Affinity, 3) Cross-linking
45
A1antitrypsin
remove trypsin
trypsin- is a proteolytic enzyme
46
Prozone
zone of antibody excess
47
zone of equivalence
where you can read reaction
48
Post zone
Zone of antigen excess
49
Enhancing precipitation
blank effect
polymer types
✦ Polymer Effect
‣ Linear polymers enhance immune complex
precipitation
✦ Polymer Types
* Dextran
* PVA
* PEG
50
Methods for ag-ab detection.
* Precipitation or Agglutination
* Hemagglutination and Hemagglutination
inhibition
* Passive Gel Diffusion
* Radio-immunoassays
* ELISA
* Immunofluorescence
* Immmunoblotting
* Immunochromatography
51
Precipatation
‣ Antibodies react with soluble substances
ex) proteins, carbohydrates, etc
‣ Reaction visible with naked eye - particles
52
Agglutination
‣ Antibodies react with insoluble substances
ex) RBCs, bacterial cells, latex particles coated with antigen
‣ Reaction visible with naked eye – larger clumps
(aggregates)
‣ If agglutination target is RBCs, called hemagglutination
53
Direct agglutination.
This particle antigen may contain blank, blank, and blank
๏ Test patient serum against large, cellular antigens to
screen for the presence of antibodies in pt. serum.
Antigen is naturally present on the surface of the cells.
In this case, the Ag-Ab reaction forms an agglutination,
which is directly visible.
➡ The particle antigen may be a bacterium.
ex) Serotyping of E. coli, Salmonella
➡ The particle antigen may be a parasite.
ex) Serodiagnosis of Toxoplasmosis
➡ The particle antigen may be a red blood cell.
ex) Determination of blood groups
54
Passive agglutination
Particle carriers include
* An agglutination reaction that employs particles that
are coated with antigens not normally found in the
cell surfaces
* Particle carriers include:
– Red blood cells
– Polystyrene latex
– Bentonite
– charcoal
55
Reverse passive agglutination
✴Principle
‣ Antigen (in serum) binds
to antibody (from kit)
coated on carrier particles
and results in
agglutination
ex) detecting cholera toxin
Reverse passive agglutination
56
Passive gel diffusion
Performed on what
✦ Performed on semisolid (agarose)
✦ Precipitin band dependent on:
* Solubility of antigen/antibody
* Relative concentration of each
* Temperature
* Time
* Gel viscosity
57
Simple diffusion
blank suspended uniformly in gel
blank added to the gel
blank results in the blank ring
❖ Antibody: suspended uniformly in gel
❖ Antigen: applied to well cut in gel
‣ Diffusion away from well dilutes antigen
‣ Zone of equivalence results in precipitin ring
‣ Area of zone can be compared to known antigen concentrations
(standards)
Passive Gel Diffusion: Simple Diffusion (Radial Immunodiffusion)
58
Ouchterlony
Identity
NonIdentity
Partial Identity
✦ Both Antibody and antigens added to separate wells
of gel
✦ Position of precipitin bands indicates identity
1. Identity (common epitopes)
2. Non-identity (different epitopes)
3. Partial identity (some epitopes in common)
59
V pattern
share all ag determinants
60
X pattern
different Ag determinants
61
full line + half line
share some Ag determinants
62
Immunoelectrophoesis
Serum proteins are
Blank is placed in a tough running blank
Diffusion to
✦ Immunoelectrophoresis (IEP)
‣ Serum proteins are
electrophoretically separated
‣ Reagent antibody is placed in
a trough running parallel
‣ Diffusion to precipitin arc
63
Immunoelectrophoresis
Blank crossed IE aka
serum Separation by blank first
Turn what
✦ Two dimensional crossed IE (aka 2D IE, CRIE)
* Serum separation by charge/size first
* Turn 90°, electrophorese into gel containing ab
Immunoelectrophoresis
64
Electroimmunoassay (Rocket Electrophoresis)
* Antigen applied to wells in the lower gel
* Upper gel contains antibody
* Electrophorese – quantitative when compared
to calibrators
* Rapid detection & quality
65
Immunofixation electrophoresis
✦ Electrophorese proteins
✦ Antibody placed on gel
– precipitation
✦ All other proteins washed
out
✦ Stain gel with dye
(Coomassie blue, etc)
66
Western blot
67
Indicator labeled Immunoassay
Addition of blank or blank
rarely what
Common types of enzymes
✦ Addition of “labeled” antigen or antibody
✦ Labels
‣ Radioactive isotopes (rarely)
‣ Enzymes (common)
* HRP, ALP, glucose oxidase
* Substrate ! Product (colored)
* If cleavage of substrate produces photon of light
➡ “Chemiluminescent”
‣ Fluorescent labels (common)
68
Competitive immunoassay
Typically use what
Carried out in the presence of what
competition between what
Inversely what
Typically use labeled antigen (reagent)
Carried out in presence of “excess antigen”
Patient’s unknown analyte & the labeled analyte
compete for binding sites on a known/limited amount of
antibody
Value of patient analyte is inversely proportional to the
signal generated by the bound labeled antigen
69
Noncompetitive immunoassay
Typically use an what
Carried out in presence of
Patients unknown analyze Is held between what
The value of blank is directly proportional to blank
Labeled entity is antibody
Typically use an unlabeled capture antibody and a labeled
indicator antibody (reagent)
Carried out in presence of “excess antibody”
Patient unknown analyte antigen is held between the two
antibodies (‘sandwich’)
Value of patient analyte is directly proportional to the signal
generated by the bound labeled antibody
70
Labeled immunoassay
✦ Heterogeneous immunoassays
‣ Usually require washing step to separate antibody-
bound antigen from remaining free antigen.
✦ Homogeneous immunoassays
‣ No separation between antibody-bound antigen with
free antigen
71
Free vs bound
✦ Relates to label at end of assay
‣ Free: floating in mixture, sometimes washed out,
sometimes precipitated or adsorbed
‣ Bound: stuck to surface in some way
✦ Know relationship of free to bound for each assay
✦ Know what you are measuring – free vs. bound
✦ Know relationship of signal to unknown concentration
(directly or indirectly proportional)
72
Seperation techneques
✦ Solid phase*
✦ Adsorption with dextran-treated charcoal or dextran
gel (Sephadex)
✦ Precipitated with ammonium sulfate
✦ Precipitated via double antibody technique (bound
fraction)
73
Standards
✦ Regardless of assay type, standards MUST BE USED to
convert signal to amount
✦ In case of saturation – dilute sample
74
Radioisotopes
Common Radio-labels
✦ Inexpensive, sensitive
✦ Requirements:
‣ Licensing
‣ Specialized equipment
‣ Speci alized handling and disposal
✦ Common Radiolabels:
‣ Iodine (gamma)
‣ Tritium (beta)
75
Radioimmunoassay uses
✦ Uses:
‣ Hormones in plasma
‣ Drug levels (digoxin, drugs of abuse)
‣ Hepatitis B Surface Antigen (HBsAg)
‣ anti-DNA Antibodies (SLE)
‣ Allergy Testing (RAST)
‣ Essentially any antibody test
76
Luminescent immunoassays
✦ Luminescent compounds emit a photon of light as
the result of an electrical biochemical or chemical
reaction
✦ “Highly sensitive”
✦ Often used as substrate, and coupled with enzymes
as immunoassay label
77
CHEMILUMINESCENT IMMUNOASSAYS
example
✦ Luminol - substrate
✦ Peroxidase - label
Luminol + 2H2O + OH ---> 3-aminophthalate + light
✦ Enhancer—luciferin
Peroxidase
CHEMILUMINESCENT IMMUNOASSAYS
78
RADIOISOTOPE METHODOLOGIES
* Competitive Protein Binding
Patient antibody competes with thyroglobulin.
79
Radioimmunoassay competitive binding
80
Immunoradiiometric assay
Can also use bound antigen and radio-labeled antigen to assay
for patient antibody
1.) solid surface coated with antibody and react with patients antigen then add radiolabel on antibody
81
Radioallergosorbent test (RAST)
82
Enzyme techniques
ELISA
* Enzyme Linked Immunosorbent Assay (ELISA)
– Competitive Binding ELISA
83
Elisa blank technique
sandwich
84
Elisa blank technique
sandwich
85
EMIT
* Enzyme Multiplied Immunoassay Technique
(EMIT)
86
Detection systems
RIA, Flame emission or atomic absorption Spec, Turbidimetry & Nepherometry,
Fluorometry & fluorescence polarization (fluoro immunoassay)
87
RADIOIMMUNOASSAY: RIA
Uses what as labels
how sensitive
Has been adapted to blank for the east of what
Requires what
* Uses radioactive isotopes as labels
* Very sensitive ~ 5 pg/ml
* Adapted to many procedures
‣ Has been adapted to solid phase immunoassay for easy
separation of bound and free components
* Requires licensure for handling radioactive substances
& special disposal
88
Emission- liquid scintillation
Highly colored compounds (blood)
may need dilution due to interference
(quenching)
* γ counter – similar, but use a crystal
instead of fluidβ Emission – Liquid Scintillation
β Emission – Liquid Scintillation
Scintillator
89
A=
A=2-log(%T)
90
Flame emmision
Principle
1.If energy (thermal, electrical, electromagnetic) is supplied to
anatom, its e-s can move to an orbit of higher energy
2.Excited atom releases a photon (of light) when its e-s fall back
into their original orbits (ground state)
The WAVELENGTH of emission gives the IDENTITY of the element
while the INTENSITY of the emitted light is PROPORTIONAL TO THE
# OF ATOMS IN THE ELEMENT.
91
Instrumentation of flame photometer
1. Radiation source (the excited atoms)
Each element or ion emits a גּ or color characteristic for
that element (SQB1)
Na+ = yellow K+ = red-violet Li+ = red Mg2+ = blue
2. Sample cell or cuvette (the flame!)
The greater the thermal energy of a flame, the greater the number of atoms
that will be excited. BUT increased background noise is often an unwanted
by product
92
Automizer
✦Mixes liquid sample into the flame
✦Sample introduced at a rapid, stable rate
Advantages: delivery of a completely representative aliquot of
sample into the flame
Disadvantages: wide variation of droplet size, aspiration
variance due to sample viscosity, turbulence (results in much
background noise)
93
Monochromator
‣ Isolates the characteristic wavelength of emitted light
‣ Accomplished via: prisms / diffraction gratings or filters
94
Detector
(photomultiplier tube or photocell)
‣ converts light emitted by excited atoms into electrical
energyInstrumentation of flame photometer
Instrumentation of flame photometer
95
Internal standards
* Internal standards are used to eliminate instrument variance
due to temperature, atomization, oxygen / fuel flow, and
voltage fluctuations
* Simultaneous analysis for both (the internal reference
standard & the element of interest)
* because both internal standard & element of interest are
affected in an identical manner
* Internal standard - if running sodium, use lithium or cesium
as internal standard
96
Atomic Absorption spectrophotometer
* Differences from Flame Emission Photometry:
1. Specimen is atomized in flame, not excited
2. Spectrally pure light source is also directed at flame
3. Atoms in flame that match wavelength of light
(spectral alignment) will absorb that light (atomic
absorption)
Benefits:
‣ 100 times more sensitive than FEP
‣ Highly specific for element being measured
‣ Used for Al, Ca, Cu, Pb, Li, Mg, and Zn
97
Atomic absorption method
1. Hollow cathode (exciter) lamp produces a beam of high spectral
purity (monochromatic)
2. Chopper transforms the specific incident light into a pulsing beam
(AC, alternating current)
3. Sample introduced to flame, vaporized (& reduced) to their ground
state
4. Reduced sample atoms absorb the light when bombarded by AC
from chopper
5. Reduced samples emit light that is not in a pulsing beam (DC2)
6. DC2 deflected by the Monochromator (grating) to the Detector
(photomultiplier tube)
4. Modulated Meter is synchronized to the chopper so that it
recognizes AC as well as DC2
98
Atomic absorption intereferes
1. Chemical interference (contaminating chemicals might ionize &
interfere or merely interfere with reading)
2. Ionization interference (elements other than elements of interest
might ionize similarly)
3. Matrix interference (other components of the sample matrix such as
buffer salts or other additives like detergent or solvents may ionize
& interfere or merely interfere with reading)
4. Emission interference (other elements might absorb & emit at same
wavelength
99
Turbidimetry & Nephelometry
✦ Turbidimetry: Measurement of loss in intensity (dynamic change) of a
light beam through a solution that contains suspended particulate matter
✦ Nephelometry: The measurement of the intensity of reflected light
100
Light scatter
* Nephelometry & turbidimetry are related
techniques that are dependent on measurement of
light scatter.
* Quantitative relationships exist between size &
number of suspended particles degree of scatter.
* When a light beam strikes a particle, its electric
field moves the particle’s electrons in one
direction and the nucleus in the opposite direction
(Oscillating dipole)
* The maximum amount of movement is
proportional to the electric field strength of the
light beam.Turbidimetry & Nephelometry
101
Turbidimetery
➡ Turbidimetry can be performed on any standard
spectrophotometer
✦ Wavelength selection criteria.
* Turbidimetric measurement is based on the size of
the particle.
* Particles not more than 40 nm in diameter can be
measured with light of a 400 nm wavelength,
* larger particles (300 nm) as might occur with latex
agglutination are probably going to be measured
with a light of a wavelength in the 500-600 nm
range
102
Nephelometry
* Nephelometry is one of the most commonly used
measurement principle for the immunochemical
determination of protein in serum, urine and other body fluids
103
Nephelometry
scattering and instrumentation
* Measurement of light scatter
* Scattering
– Size of particles
– Number of particles
* Instrumentation
– Similar to spectrophotometers, fluorometers
104
Small particles
Light scattered symmetrically but minimally at 90 degree (Rayleigh)
d< .1wavelength
D is size
105
Large particles
Light scattered preferentially forward ( Rayleigh-Debye)
D< wavelength
106
Very large particles
Light scattered mostly forward (Mie)
D> wavelength
107
Immunonephelometry
* Antigen
* Antibody
* Antigen-Antibody complexes
108
Nephelometer
Angle of detection
light source
Use
Angle of detection
– 5°-90° (depends on light source)
* Light source
– Monochromatic (filtered, laser, diffraction gradient)
– Polarized (collminating lens)
* Use
– Serum and CSF proteins, antibodies, drugs
109
Flourometry
✦ Principle
* When certain molecules are exposed to light energy,
they may absorb it & enter an excited state.
* The excited energy of the molecule may be given off in
an alternate energy form as the electrons return to their
original orbits & the molecule returns to the ground state.
* If the alternate energy form is a photon of light, and if
this emitted light is of lesser energy (longer
wavelength) that the impinging light, the phenomenon is
known as fluorescence.Fluorometry
Fluorometry
110
FLourometry is the
Example: a substance that absorbs at 380 nm & emits at
460 nm is fluorescing.
Only certain compounds (fluors or flurophors) are capable of
fluorescence.
Analyte of interest doesn’t fluoresce – it is tagged with a fluor
Fluorometry is the
‣quantitative measurement of the intensity of light produced
by an excited fluorescent substance
‣relationship between measured fluorescent intensity &
[substance].
111
Fluoremetry components
What lamp
what light is focused onto sample
* Usually) UV light generated by xenon, deuterium, mercury
vapor arc lamp directed onto excitation filter.
* This approximately monochromatic light focused onto sample.
* If sample is a fluorophor, emits light longer than the one it
received.
* Emission filter (at 90° angle)
* Only allows emitted light to hit detector
112
FLUORESCENT IMMUNOASSAYS
* Direct Fluorescent Immunoassay
– Same as ELISA Sandwich, 2° antibody fluorescent
labeled
* Indirect Fluorescent Immunoassay
– Same as above, except measure excess unbound
antibody
* Substrate Labeled Fluorescent Immunoassay
113
FLUORESCENT IMMUNOASSAYS
MEIA
* Microparticle Enzyme Immunoassay
114
MEIA
* Substrate: 4-methylumbelliferyl Phosphate (MUP)
* Enzyme Label: Alkaline Phosphatase (ALP)
* Reaction:
– MUP + ALP (Ab label on sandwich complex) g MU (fluorescent)
* Rate of production of MU is measured.
115
FPI
Fluorescence polarization immunoassay
116
Luminescent Immunoassays emit
✦ Luminescent compounds emit a photon of light as
the result of an electrical, biochemical or chemical
reaction
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Luminescent Immunoassays
Often used as
And highly what
✦ “Highly sensitive”
✦ Often used as a substrate, and coupled with enzymes
as immunoassay label
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Competitive protein binding reaction
T4(patient) ----> Bound Thyroglobulin + T4(patient) or Thyroglobulin+
Competes with
---> Free T4 patient
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Radioimmunoassay competitive binding reaction
T4(patient) + Anti T4 -----> bound T4(patient) with anti-T4 antibody
Competes with
Free T4 patient
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Immunoradiometric assay reaction
Step one- solid surface coated with antibodies (surface treated to minimize nonspecific binding); then add Wash and Add patients sample containing antigen.
Step two: reaction of antigen with immobilized antibody. Add wash and then add radioactively labeled antibody.
Step three- Reaction of Immobilized antigen with labeled antibody
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IRMA can also use
Can also use bound antigen and radio-labeled antigen to assay
for patient antibody
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Radioallergosorbent test
Used to detect IgE to a specific allergen in patient serum
Sensitivity can be altered by the type of label used. RAST assays use radioactive label on detection antibody to antigen-specific IgE. Other labels include enzymes, chemoluminescence and fluorescence
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Rast test reaction
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Enzyme-linked Immunosorbent assay
Competitive Binding ELISA
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Competitive binding ELISA reactions
Ag(patient) and antibody with a label such as HRP is added to well with antibody, and then Ag(patient) competes with Ag(label). Then free ag is washed away, and the reaction is measured.
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Enzymatic techniques
Enzyme-linked Immunosorbent assay is a what
reaction
Sandwich technique
step one- Solid surface coated with antibody ( surface treated to minimize nonspecific binding) and a wash happens and then you add patients sample containing antigen
Step two Reaction of antigen with immobilized antibody and then a washing step and then add enzyme-labeled antibody
Step three- Reaction of immobilized antigen with labeled antibody and then washing and then you add the substrate.
Step 4- color change measured ( enzyme converts substrate to produce P and product measured as color change)
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Atomic absorption benefits
Benefits:
‣ 100 times more sensitive than FEP
‣ Highly specific for element being measured
‣ Used for Al, Ca, Cu, Pb, Li, Mg, and Zn
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Test reportoire
The full supply of what a lab machine can do
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Dwell time
Covers the period that elapses between the application of the sample to the test slide and its insertion into the measurement device.
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What takes days
vitamin assays
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Throughput
Max test per hour, shift, or day
