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analytical_techniques

The document outlines various analytical techniques used in clinical laboratories, including spectrophotometry, light scattering, electrophoresis, and immunoassays. It explains the principles of these techniques, such as Beer’s Law in spectrophotometry and the migration of charged molecules in electrophoresis. Additionally, it highlights the significance of serum protein electrophoresis in diagnosing conditions like multiple myeloma and other lymphoproliferative diseases.

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Dalia Abdelkawy
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5 views

analytical_techniques

The document outlines various analytical techniques used in clinical laboratories, including spectrophotometry, light scattering, electrophoresis, and immunoassays. It explains the principles of these techniques, such as Beer’s Law in spectrophotometry and the migration of charged molecules in electrophoresis. Additionally, it highlights the significance of serum protein electrophoresis in diagnosing conditions like multiple myeloma and other lymphoproliferative diseases.

Uploaded by

Dalia Abdelkawy
Copyright
© © All Rights Reserved
We take content rights seriously. If you suspect this is your content, claim it here.
Available Formats
Download as PPTX, PDF, TXT or read online on Scribd
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Analytical

Techniques
Analytical techniques
 Spectrophtometric techniques
 Light scattering techniques
 Electrophoresis
 Immunoassays
 Molecular &Nucleic acid techniques
 Objectives

 Explain the basic principles and components of the following


analytic techniques

 Spectrophometry
 Nephelometry
 Turbidimetry
 Ion Selective Electrodes ( Potentiometry )
 Electrophoresis
 Immunoflourescence
 Flowcytometry
 ELISA
 RIA
Analytical techniques
 Many determinations in the clinical lab are
based on measurements of radiant energy which
is :

• Emitted ,

• Transmitted,

• Absorbed , or

• Reflected under controlled conditions.


Spectrophotometry

A narrow wavelength of light is isolated from the light source and


directed
thru a curvet containing patient’s molecules and reagents. Chemical
reactions
between the patient’s molecules and reagents produce new molecules
that absorb this wavelength of light energy. Light that is not absorbed
( transmitted ) passes thru the curvet and strikes the photodetector
and converted into an electrical signal. Absorbance (A) is a
mathematical relationship between the initial light intensity and the
transmitted light measured by the photodetector.
Spectrophtometry
Beer’s Law

1/v 
ABSORBANCE

CONCENTRATION
Beers law expresses a proportional or linear relationship between the
absorbance, and the concentration of the substance. This makes it a
very convenient way to measure unknown concentrations. Plots of
Absorbance versus concentration are referred to as calibration
curves . Beers law predicts a linear trend in these plots
Spectophotometry ( cont )

 Because Beer’s Law states that absorbance (A) is directly


proportional to concentration ( it’s a linear relationship ), the
following mathematical relationships can be established:

Cunknown = Concentration of the Unknown


Cstandard = Concentration of the Standard
Aunknown = Absorbance of the Unknown
Astandard = Absorbance of the Standard

Cunk Aunk
 This can be rewritten as:
C std Astd

Aunk
Cunk  x Cstd
Astd
Light
Scattering
Techniques
Nephelometer

Light is scattered from


insoluble complexes
( often Ab-Ag complexes )

Light “bounces” off insoluble complexes and hits a photodetector that


has
been placed at an angle off from the initial direction of the light. This
is a
measurement of Transmitted light
Turbidimetry is similar, except that the
photodetector is placed in the same angle of the
initial light path. This is a measurement of
Absorbance (A) - light that has been blocked by the
insoluble complexes
Nephelometry vs.
Turbidimetry

0°-90°
Light
Emission
Technique
s
Light Emission Techniques
The following are types of luminescence:
o Fluorescence, absorption of photons
causing re-radiation of photons of lower
energy .
o Chemiluminescence resulting of a
chemical reaction
o Radioluminescence : produced in a
material by the bombardment of
ionizing radiation
Fluorescence
The emission of light by an atom after
absorption of exciting photons or X-rays .
The emitted light is of lower energy or
longer wavelength.
Thus; Fluorescence is a property of some
molecules in which light of one colour is
absorbed that results in emitting a light of
a different colour.
Fluorophore : A molecule that can fluoresce
.
Fluorescence
Fluorometry Detection of fluorescent
light emitted by fluorescent
molecules

The photodetector must be


placed at a 90° angle from
the initial light source

This eliminates any


interference from the initial
light source and detects only
fluorescent light.
Fluorometry ( cont )

Definition of fluorescence :

Certain molecules absorb light and a given frequency, and then re-emit that
light at a different and longer frequency

Advantages of fluorescence: Very specific and sensitive

Disadvantages of fluorescence: Few molecules fluoresce and very susceptible


to pH and temperature changes
Electrophoresi
s
Electrophoresis
 Molecules from patient specimens are treated with buffer solutions to give them
electrical charge and are placed onto the surfaces of semi-solid supporting media

 The media must be able to conduct electrical current and connects a cathode (=)
to an anode (+)

 When electrical current flows through the media, electrically charged molecules
“migrate”, or move along the supporting media

 The rate at which different molecules move along the supporting media
( strip ) will vary depending on the physical characteristics of the molecules and
the methodology of the electrophoresis

 After migration, the strip is removed and stained with an appropriate stain -
“bands” of stained molecules will be visible

 A densitometer scans the stained strip and reports a graphical representation of


the bands
Electrophoresis ( cont )

 Each peak represents a different band of molecules that migrated together


during electrophoresis

 Peaks with narrow bases reflect homogeneous molecules that migrated


closely together

 Peaks with wide bases reflect heterogeneous molecules that spread out
during migration

 Factors that affect migration rates of molecules:

 Molecular weight
 Molecular shape
 Molecular electrical charge in the buffer ( buffer pH )
 Supporting media
 Temperature
 Electrical voltage
 Migration time
Electrophoresis
Illustration of Electrophoresis chamber
Most commonly used are:
-Serum protein electrophoresis
-Hb electrophoresis
Electrophoresis ( continued )

The densitometer scans the


stained electrophoresis strip
and converts the intensity of
the stain into graphical form
Electrophoresis ( continued )

Graphical representation of the


densitometer’s scan of the electrophoresis
strip.

The densitometer calculates the area under


the curves and expresses each as a
percentage of the total
Serum Protein Electrophoresis
 Serum electrophoresis (SPE) is a sensitive screening
test for the detection of monoclonal immunoglobulin
bands of greater than 0.5 g/L.
 Serum and urine EP should be requested initially on
all patients suspected of having myeloma, as 20% of
myeloma patients secrete monoclonal light chains
only, and the light chains will only appear in serum in
the presence of renal dysfunction.
 A distinct narrow band may be visible on EP,
suggestive of a paraprotein. However,
immunoglobulin clonality can only be confirmed by
immunoelectrophoresis (IEP) or isoelectric
focusing/immunofixation (IEF-IF).
 The serum EP may also indicate immune paresis, and
investigation for light-chain-only myeloma should be
undertaken.
 Progress measurement of the paraprotein using EP
analysis is usually the best way to monitor progress
ELECTROPHORESIS
S.P.E
M-Components in Plasma Cell
Neoplasia
Serum protein electrophoresis (SPEP). In the bottom plot, a sharp
spike has replaced a diffuse hump in the gamma region, because
normal gammaglobulins are decreased and large amounts of a
single, monoclonal immuglobulin have taken their place. Because
the protein is monoclonal, each molecule has identical
electrophoretic qualities that move it to the same place in the
electric field, creating a narrow, intense band or spike. Discovery of
an M-component suggests the presence of multiple myeloma,
monoclonal gammopathy of undetermined significance,
Waldenstrom's macroglobulinemia, other lymphoproliferative
disease, or primary systemic amyloidosis.
Serum Protein Electrophoresis.
Identification of Monoclonal
Immunoglobulin

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