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Lab ops > Clinical chemistry and Immunoassay > Flashcards

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

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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
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3
Q

Common task of automation

A
  1. Measurement and Proportion of sample and
    reagents
  2. Mixing
  3. Incubation
  4. Sensing
  5. Computation
  6. Readout
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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

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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

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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.

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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

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8
Q

Percent carry over

A

measured by placing and testing
low concentration samples before and after a high
concentration sample

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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

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10
Q

Calculations should be made on

A

each analyte to develop
repeat policy on high values

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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

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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

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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

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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)

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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)

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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”

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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)

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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

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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

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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

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21
Q

Types of automation

A

✦ Total Laboratory Automation (TLA)
✦ Modular Integrated Systems (MIS)
✦ Stand-alone systems

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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

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23
Q

TLA components

A

✦ Centrifuge
▪ Often rate limiting step
✦ Decapper
✦ Aliquoter
▪ Clot sensor
▪ Ample quantity
▪ Proportioning into additional tubes (barcode)

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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

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25
Q

TLA drawbacks

A

✦ Substantial financial
investment
▪ $2-5 million w/o space
renovation
✦ Laboratory space
▪ Construction costs
✦ Integration of other vendor
instruments

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26
Q

Modular integrated systems.

Separate what for various departments

blank for different manufacturers

Easier to interface with the

Drawbacks

A

✦ 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

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27
Q

Modular integrated systems benefits

A

✦ 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

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28
Q

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

A

❖ 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

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29
Q

Non-selective analyzers

A

▪ Perform the entire battery of tests on each sample

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30
Q

Batch analyzers

A

▪ Perform the same test simultaneously on all
samples
▪ Decreased use

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31
Q

Random access analyzers

A

▪ Perform specified multiple tests in any order

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32
Q

Multiple Parameter (Analytes) Analyzer

A

▪ Perform multiple tests per sample either sequentially
or simultaneously

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33
Q

Random access.

blank test possible

Not every blank performed on every blank

Less blank waste

Tests can be blank or blank

replaced what

A

✦ Multiple tests possible
✦ Not every test performed on every sample
▪ Programmable
▪ Less reagent waste
▪ Tests can be sequential or simultaneous
✦ Mostly replaced batch analyzers

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34
Q

Discrete analyzers

A

✦ Instruments that compartmentalize each test reaction
✦ Most current analyzers are discrete

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35
Q

Discrete batch analyzers

A

– Perform only one type of assay at a time on
compartmentalized samples

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36
Q

Discrete multi-sequential analyzers

A

– Discrete: each specimen separate from others
– Sequential: tests performed one at a time
– Multiple: numerous tests can be performed on each
sample

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37
Q

Discrete Multiple Simultaneous Analyzers

A

– 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

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38
Q

Terminology

A

✦ Test repertoire
▪ Immediate
▪ Total
✦ Dwell time
▪ Time specimen is in instrument
▪ Seconds to days (vitamin assay)
✦ Throughput
▪ Max tests per hour, shift, day

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39
Q

Current automation

A

✦ 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

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40
Q

Results of automation in the lab

A

✦ 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

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41
Q

Changing roles

A

✦ Manual methods in a direct, hand-on production of data
▪ Highly complex tests
▪ New test development
▪ PPM
✦ Data analyzer/ manager
▪ Routine tests
▪ Moderately complex

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42
Q

examples of changing roles

A

✦ Operate instruments
✦ Analyze and interpret data
✦ Perform preventive maintenance
✦ Basic instrument repair
✦ Sophisticated problem-solving skills
✦ Educator (POC, novel technologies)

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43
Q

Immunochemical techniques

Most what

Add blank to the sample containing what

Detects what

Ex

A

✴ 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

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44
Q

Antibodies in immunoassays

A

Characteristics: 1) Specificity, 2) Affinity, 3) Cross-linking

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45
Q

A1antitrypsin

A

remove trypsin
trypsin- is a proteolytic enzyme

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46
Q

Prozone

A

zone of antibody excess

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47
Q

zone of equivalence

A

where you can read reaction

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48
Q

Post zone

A

Zone of antigen excess

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49
Q

Enhancing precipitation

blank effect

polymer types

A

✦ Polymer Effect
‣ Linear polymers enhance immune complex
precipitation
✦ Polymer Types
* Dextran
* PVA
* PEG

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50
Q

Methods for ag-ab detection.

A
  • Precipitation or Agglutination
  • Hemagglutination and Hemagglutination
    inhibition
  • Passive Gel Diffusion
  • Radio-immunoassays
  • ELISA
  • Immunofluorescence
  • Immmunoblotting
  • Immunochromatography
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51
Q

Precipatation

A

‣ Antibodies react with soluble substances
ex) proteins, carbohydrates, etc
‣ Reaction visible with naked eye - particles

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52
Q

Agglutination

A

‣ 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

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53
Q

Direct agglutination.

This particle antigen may contain blank, blank, and blank

A

๏ 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
Q

Passive agglutination

Particle carriers include

A
  • 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
Q

Reverse passive agglutination

A

✴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
Q

Passive gel diffusion

Performed on what

A

✦ Performed on semisolid (agarose)
✦ Precipitin band dependent on:
* Solubility of antigen/antibody
* Relative concentration of each
* Temperature
* Time
* Gel viscosity

57
Q

Simple diffusion

blank suspended uniformly in gel

blank added to the gel

blank results in the blank ring

A

❖ 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
Q

Ouchterlony

Identity

NonIdentity

Partial Identity

A

✦ 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
Q

V pattern

A

share all ag determinants

60
Q

X pattern

A

different Ag determinants

61
Q

full line + half line

A

share some Ag determinants

62
Q

Immunoelectrophoesis

Serum proteins are

Blank is placed in a tough running blank

Diffusion to

A

✦ Immunoelectrophoresis (IEP)
‣ Serum proteins are
electrophoretically separated
‣ Reagent antibody is placed in
a trough running parallel
‣ Diffusion to precipitin arc

63
Q

Immunoelectrophoresis

Blank crossed IE aka

serum Separation by blank first

Turn what

A

✦ Two dimensional crossed IE (aka 2D IE, CRIE)
* Serum separation by charge/size first
* Turn 90°, electrophorese into gel containing ab
Immunoelectrophoresis

64
Q

Electroimmunoassay (Rocket Electrophoresis)

A
  • Antigen applied to wells in the lower gel
  • Upper gel contains antibody
  • Electrophorese – quantitative when compared
    to calibrators
  • Rapid detection & quality
65
Q

Immunofixation electrophoresis

A

✦ Electrophorese proteins
✦ Antibody placed on gel
– precipitation
✦ All other proteins washed
out
✦ Stain gel with dye
(Coomassie blue, etc)

66
Q

Western blot

A
67
Q

Indicator labeled Immunoassay

Addition of blank or blank

rarely what

Common types of enzymes

A

✦ 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
Q

Competitive immunoassay

Typically use what

Carried out in the presence of what

competition between what

Inversely what

A

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
Q

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

A

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
Q

Labeled immunoassay

A

✦ 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
Q

Free vs bound

A

✦ 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
Q

Seperation techneques

A

✦ Solid phase*
✦ Adsorption with dextran-treated charcoal or dextran
gel (Sephadex)
✦ Precipitated with ammonium sulfate
✦ Precipitated via double antibody technique (bound
fraction)

73
Q

Standards

A

✦ Regardless of assay type, standards MUST BE USED to
convert signal to amount
✦ In case of saturation – dilute sample

74
Q

Radioisotopes

Common Radio-labels

A

✦ Inexpensive, sensitive
✦ Requirements:
‣ Licensing
‣ Specialized equipment
‣ Speci alized handling and disposal
✦ Common Radiolabels:
‣ Iodine (gamma)
‣ Tritium (beta)

75
Q

Radioimmunoassay uses

A

✦ 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
Q

Luminescent immunoassays

A

✦ 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
Q

CHEMILUMINESCENT IMMUNOASSAYS
example

A

✦ Luminol - substrate
✦ Peroxidase - label
Luminol + 2H2O + OH —> 3-aminophthalate + light
✦ Enhancer—luciferin
Peroxidase
CHEMILUMINESCENT IMMUNOASSAYS

78
Q

RADIOISOTOPE METHODOLOGIES
* Competitive Protein Binding

A

Patient antibody competes with thyroglobulin.

79
Q

Radioimmunoassay competitive binding

A
80
Q

Immunoradiiometric assay

A

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
Q

Radioallergosorbent test (RAST)

A
82
Q

Enzyme techniques
ELISA

A
  • Enzyme Linked Immunosorbent Assay (ELISA)
    – Competitive Binding ELISA
83
Q

Elisa blank technique

A

sandwich

84
Q

Elisa blank technique

A

sandwich

85
Q

EMIT

A
  • Enzyme Multiplied Immunoassay Technique
    (EMIT)
86
Q

Detection systems

A

RIA, Flame emission or atomic absorption Spec, Turbidimetry & Nepherometry,
Fluorometry & fluorescence polarization (fluoro immunoassay)

87
Q

RADIOIMMUNOASSAY: RIA

Uses what as labels

how sensitive

Has been adapted to blank for the east of what

Requires what

A
  • 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
Q

Emission- liquid scintillation

A

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
Q

A=

A

A=2-log(%T)

90
Q

Flame emmision

A

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
Q

Instrumentation of flame photometer

A
  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
Q

Automizer

A

✦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
Q

Monochromator

A

‣ Isolates the characteristic wavelength of emitted light
‣ Accomplished via: prisms / diffraction gratings or filters

94
Q

Detector

A

(photomultiplier tube or photocell)
‣ converts light emitted by excited atoms into electrical
energyInstrumentation of flame photometer
Instrumentation of flame photometer

95
Q

Internal standards

A
  • 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
Q

Atomic Absorption spectrophotometer

A
  • 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
Q

Atomic absorption method

A
  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)
  7. Modulated Meter is synchronized to the chopper so that it
    recognizes AC as well as DC2
98
Q

Atomic absorption intereferes

A
  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
Q

Turbidimetry & Nephelometry

A

✦ 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
Q

Light scatter

A
  • 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
Q

Turbidimetery

A

➡ 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
Q

Nephelometry

A
  • Nephelometry is one of the most commonly used
    measurement principle for the immunochemical
    determination of protein in serum, urine and other body fluids
103
Q

Nephelometry
scattering and instrumentation

A
  • Measurement of light scatter
  • Scattering
    – Size of particles
    – Number of particles
  • Instrumentation
    – Similar to spectrophotometers, fluorometers
104
Q

Small particles

A

Light scattered symmetrically but minimally at 90 degree (Rayleigh)
d< .1wavelength
D is size

105
Q

Large particles

A

Light scattered preferentially forward ( Rayleigh-Debye)
D< wavelength

106
Q

Very large particles

A

Light scattered mostly forward (Mie)
D> wavelength

107
Q

Immunonephelometry

A
  • Antigen
  • Antibody
  • Antigen-Antibody complexes
108
Q

Nephelometer
Angle of detection
light source
Use

A

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
Q

Flourometry

A

✦ 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
Q

FLourometry is the

A

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
Q

Fluoremetry components

What lamp

what light is focused onto sample

A
  • 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
Q

FLUORESCENT IMMUNOASSAYS
* Direct Fluorescent Immunoassay

A

– Same as ELISA Sandwich, 2° antibody fluorescent
labeled
* Indirect Fluorescent Immunoassay
– Same as above, except measure excess unbound
antibody
* Substrate Labeled Fluorescent Immunoassay

113
Q

FLUORESCENT IMMUNOASSAYS
MEIA

A
  • Microparticle Enzyme Immunoassay
114
Q

MEIA

A
  • 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
Q

FPI

A

Fluorescence polarization immunoassay

116
Q

Luminescent Immunoassays emit

A

✦ Luminescent compounds emit a photon of light as
the result of an electrical, biochemical or chemical
reaction

117
Q

Luminescent Immunoassays

Often used as

And highly what

A

✦ “Highly sensitive”
✦ Often used as a substrate, and coupled with enzymes
as immunoassay label

118
Q

Competitive protein binding reaction

A

T4(patient) —-> Bound Thyroglobulin + T4(patient) or Thyroglobulin+

Competes with

—> Free T4 patient

119
Q

Radioimmunoassay competitive binding reaction

A

T4(patient) + Anti T4 —–> bound T4(patient) with anti-T4 antibody

Competes with

Free T4 patient

120
Q

Immunoradiometric assay reaction

A

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

121
Q

IRMA can also use

A

Can also use bound antigen and radio-labeled antigen to assay
for patient antibody

122
Q

Radioallergosorbent test

A

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

123
Q

Rast test reaction

A
124
Q

Enzyme-linked Immunosorbent assay

A

Competitive Binding ELISA

125
Q

Competitive binding ELISA reactions

A

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.

126
Q

Enzymatic techniques

Enzyme-linked Immunosorbent assay is a what

reaction

A

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)

127
Q

Atomic absorption benefits

A

Benefits:
‣ 100 times more sensitive than FEP
‣ Highly specific for element being measured
‣ Used for Al, Ca, Cu, Pb, Li, Mg, and Zn

128
Q

Test reportoire

A

The full supply of what a lab machine can do

129
Q

Dwell time

A

Covers the period that elapses between the application of the sample to the test slide and its insertion into the measurement device.

130
Q

What takes days

A

vitamin assays

131
Q

Throughput

A

Max test per hour, shift, or day

Lab ops (2 decks)

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