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Basic Techniques In ClinicalChemistry Laboratory
临床生物化学基本技术
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Computerization and the use of automated methods of
analysis allow a high degree of productivity and improve the
quality of services.
Understanding the basic principles of techniques and the
theory of instrumental analysis will provide a working
knowledge of instruments, applications to patients testing in
the clinical chemistry laboratory.
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A variety of techniques have been used in clinical chemistrylaboratory for sample testingMost Fundamental Methods Include:
Spectrophotometry 分光光度技术
Nephelometry 散射比浊法Turbidimetry 比浊法Fluorometry 荧光测定法
Electrophoresis 电泳
Chromatography 层析法Mass spectrometry 质谱法Biochip(Protein and DNA Chip/Array) 生物芯片Biosensor 生物传感技术
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Photometry
Photometry光度测定
is defined as the
measurement of the luminous intensity of light or the amount of luminous light falling
on a surface from such a source.
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Photometry
Many determinations made in the clinical
laborotory are based on radiant energy emitted,
transmitted, absorbed, or reflected undercontrolled conditions. The principles invovled insuch measurements are considered in thefollowing spectrophotometry, nephelometry,turbidimetry, fluorometry.
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SPECTROPHOTOMETRY
分光光度技术
The absorbance吸光率of a solution is the amount
of light absorbed by that solution. According to
Beer ’ s Law the absorbance(A) varies directly with the
concentration of the solution(c) in question. It is
equals to the concentration of a substance in solution
multiplied by the length of the path(b) that the lightmust pass through the solution, multiplied by the
molar absorptivity摩尔吸收率(a)of the substance of
interest. A=abc(formula)
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Diagram of internal parts of aspectrophotometer
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Schematic diagram ofSpectrophotometer
In practice, a beam of
light is passed through a
monochromator单色光仪
that provides selection of
the desired region of the
spectrum to be used for
measurements.
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The light next passes throughan absorbance cell, where a
portion of the radiant energy
is absorbed, depending on
the nature and the
concentration of the solution.
Any light not absorbed is
transmitted to a photo-
detector which converts light
energy to electrical energy
that can be registered on a
galvanometer.
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In operation, an opaque block is substituted for
the cuvette, so that no light reaches the photocell,
and the meter is adjusted to read
0%T(transmittance). Next, a cuvette containing a
reagent blank 空白 is inserted and the meter is
adjusted to read 100%T (i.e., zero absorbance).
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Calibrating solutions containing various known
concentrations of the substance are inserted,
and readings are recorded. Finally a reading is
made of the unknown solution, and its
concentration is determined by comparison with
the readings obtained on the calibrator.
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Determination of the concentration ofthe unknown using the standard curve
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TURBIDIMETRY
Turbidity causes the decrease of the
intensity of the incident beam of light入射光
束
as it passes through a solution of particles.
The measurement of this decrease in
intensity of the incident light beam that iscaused by scattering, reflectance
反射
, and
absorption of the light is called turbidimetry
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NEPHELOMETRY
Nephelometry is defined as the detection of light
energy scattered or reflected toward a detector
that is not in the direct path of the transmitted
light.
Common nephelometers measure scattered light
at right angles to the incident light.
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Spectrophotometry has been successfullyapplied to the analysis of different kinds of
enzymes in serum, such as ALT, AST, ALP,GGT,and quantitative analysis of total protein andalbumin in serum.
Turbidimetry can be used to determine the
concentration of apolipoprotein,immunoglobulin,alexin and rheumatoidfactors.
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FLUOROMETRY 荧光测定法
The interaction of radiant energy with molecules
or particles in solution can result in either
fluorescence荧光
or light scattering散射光
.Fluorescence occurs when a molecule absorbs light at
one wavelength波长 and remits light at a longer
wavelength.
Light scattering occurs when radiant energy passing
through a solution meets a molecule in an elastic
collision, which results in the light being dispersed分散
in all directions.
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Several automated fluorometric analyzers have been
developed for special applications in clinicallaboratory, because of their sensitivity, speed,
simplicity,and reliability.These include the flow
cytometer, hematofluorometer, fluorescence
microscopy.
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The relationship of concentration to intensity of
fluorescence emission may be derived from the
Beer- Lambert law: the fluorescence intensity is
directly proportional to the concentration of the
fluorophore荧光基团
and the excitation intensity.
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Electrophoresis
Electrophoresis refers to the migration(移动) of
charged solutes or particles in a liquid or a poroussupporting medium, such as cellulose acetate乙酸纤维
素sheets or agarose gel film琼脂糖凝胶 , under the
influence of an electrical field电场
.
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Theory of electrophoresis
Definitions
1) anode阳极: the positively charged electrode电极
in electrophoresis system.2) cathode
阴极
: negative electrode.
3) isoelectric point (pI)等电点of a molecule:
is the pH at which it has no net charge净电荷
and
will not move in an electrical field.
4) ampholyte or zwitterion两性离子 : is a molecule that can be
either positively or negatively charged; example: proteins,
amino acids.
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Chemical species carrying an electrical charge
move either to the cathode or the anode in an
electrophoresis system, depending on the kind of
charge they carry.
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In a solution more acid than the isoelectric point of
the solute, an ampholyte takes on a positive charge
and migrates toward the cathode. In the reverse
situation, it migrates toward the anode.
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The rate of migration 迁移率is dependent on factors
such as:
1) the net electrical charge净电荷
of the molecule,
2) the size大小 and shape 形状 of the molecule,
3) the electric field strength电场强度 ,
4) the characteristics of the supporting medium支持物,
5) and the operation temperature.
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Description of Electrophoresis
A schematic diagram of an
electrophoresis system shows:
two buffer boxes缓冲液箱(l)
with baffle plates隔板 containthe buffer used in the process.
In each buffer box is an
electrode电极(2) of either
platinum铂 or carbon, the
polarity极性 of which is fixed
by the mode of connection to
the power supply.
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The electrophoresis support支持物
(3) on which separation take place
is in contact with buffer by meansof wicks (strips) 条子 (4).
The entire device is covered 覆盖
(5) to minimize evaporation蒸发
and protect the system. The direct current power supply
may be either constant current 恒
流 or constant voltage or both.
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Automated Electrophoresis Systems
Although electrophoresis was traditionally a manual
technique, it has been improved by the introduction of
prepackaged gels预先包装的凝胶 and electronic
systems电子系统 that incorporated all the necessary
components and reagents to perform the procedureeasily.
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Example of rapid electrophoresis (REP) analyzer, (Helena Laboratories).
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Different types of electrophoresis
Starch Gel Electrophoresis淀粉凝胶电泳
Starch gel electrophoresis separates macromolecular
ions on the basis of both surface charge表面电荷 and
molecular size.
Because proper preparation of gels is relatively
difficult and requires considerable skill, this technique
is now rarely used in the clinical laboratory.
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Agarose Gel Electrophoresis琼脂糖凝胶电泳
It is a convenient method of electrophoresis that uses
a purified, essentially neutral fraction of agar called
agarose as a medium.
It has been successfully applied to the analysis of
serum proteins, hemoglobin variants, isoenzymes,
lipoproteins fractions and other substances.
The advantages of agarose gel include its lower affinity
低亲合力 for proteins and its native clarity after drying,
which permits excellent densitometric examination光密
度测量 (扫描).
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Cellulose Acetate Electrophoresis(CAE)乙(醋 酸纤维 素 电泳
Cellulose acetate is a thermoplastic resin热塑树脂 of
cellulose that is made by treating cellulose with acetic
anhydride乙酸酐to acetylate the hydroxyl groups to form
the raw material for membranes contain about 80% air
space in the form of pockets.
An advantage of CAE is the speed of separation (20min-1h) and the ability to store the transparent
membranes for long periods.
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Cellulose Acetate Electrophoresis
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Disc Electrophoresis 圆盘电泳
Serum protein zones determined by ordinary
electrophoretic techniques contain several proteins
with the same electrophoretic mobility电泳迁移率
and they tend to be large because proteins diffuse
during electrophoresis. Disc electrophoresis was
introduced in 1964 to overcome these deficiencies.
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Polyacrylamide Gel Electrophoresis(PAGE)
聚丙烯酰胺凝胶电泳
In this technique, individual gels are prepared in situ原位
in glass tubes by polymerizing a gel monomer凝胶单体
and a cross-linking agent交联物 with the aid of an
appropriate catalyst. The first gel to be poured into the
tubular-shaped electrophoresis cell is the small-pore
separation gel小孔分离胶
. After 30 min, during
which gelation takes place, a large pore gel, the spacer
gel 浓缩胶 is thrown on top of the separation gel.
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Then a large-pore monomer solution containing a
small amount of sample (serum) is polymerized聚合
above the spacer gel so that the finished product iscomposed of three different layers of gel.
When electrophoresis begins, all protein ions
migrate through the large-pore gel and amass on
the separation gel in a very fine zone.
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This process improves the resolution清晰度 and
concentrate protein components at the border zone,
so that pre-concentration of specimens with low
protein content may not be necessary. Separation
then takes place in the bottom separation gel with
the retardation of some proteins due to the
molecular sieve分子筛 phenomenon.
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Polyacrylamide Gel Electrophoresis
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Polyacrylamide Gel Electrophoresis
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Isoelectric Focusing (IEF) Electrophoresis等电聚焦电泳
Amphoteric compounds两性化合物
,
such as proteins, can be separated
by virtue of migration in a mediumpossessing a stable pH gradient
using isoelectric focusing
electrophoresis(IEF)等电聚焦 . The
protein moves to a zone in the
medium where the pH is equal to its
isoelectric point (pI). At this pH, the
charge becomes zero and migration
ceases.
+ –
pH 3 pH 7.5 pH 10
+ –
pH 3 pH 7.5 pH 10
+ –
pH 3 pH 7.5 pH 10
+ –
pH 3 pH 7.5 pH 10
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Two-Dimensional (2D) Electrophoresis双向电泳
In the first dimension, it uses charge-dependent电
荷依赖的 IEF electrophoresis in a large-pore medium
such as agarose or large-pore polyacrylamide gel;
and in the second dimension, molecular weight-
dependent 分 子 量 依 赖 的 electrophoresis in
polyacrylamide.
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+ – pH 3 pH 7.5 pH 10
+ –
pH 3 pH 7.5 pH 10
+ –
pH 3 pH 7.5 pH 10
+ –
pH 3 pH 7.5 pH 10
第一向:等电聚焦
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+ –
pH 3 pH 7.5 pH10
charge
size
第二向:
SDS-PAGE电泳
Proteins migrate
through the gel at
a rate proportional
to their size.
Smallest proteins
travel the furthest
distance
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Capillary Electrophoresis (CE)
毛细管电泳
In CE, the classic techniques of electrophoresis are
carried out in a small-hollow小洞(10 - 100μm of
diameter) fused silica熔融石英
capillary tube of 20 to 200 cm in length. This capillary tube is
connected to a detector at its terminal end, via
buffer reservoirs to a high-voltage power supply高
压电.
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Two distinct advantages of the capillary format
include the ease of automation and the efficient
heat dissipation(热分散 that permits the
application of much high voltages (25 to 30 kV)
than in traditional electrophoresis. This high
voltage enhances separation efficiency andreduces separation time in some cases to less
than 1 min.
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CHROMATOGRAPHY 层析法,色谱法
Chromatography is a physical method of
separation in which the components to be separatedare distributed between two phases两相, one of which
is stationary (stationary phase固定相) while the other
(mobile phase流动相
) moves in the definite direction.
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Basic concepts and definitions
The primary goal of the chromatographic process
is to separate a mixture混合物into its individual
components成分
, which are called solutes oranalytes.
A chromatographic separation requires a sample
to be introduced into a flowing stream流动的
of gas or liquid (mobile phase) that passes through
a bed, layer, or column containing the stationary
phase.
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If the mobile phase is a gas, the technique is
known as gas chromatography (GC)气相层析,
if a liquid, it is called liquid chromatography (LC)
液相层析.
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The stationary phase may be particles of a solid or
gel or a liquid.
As the mobile phase carries the sample through
the stationary phase, the solutes with lesser
affinity亲合力
for the stationary phase remain inthe mobile phase and travel faster and separate
from those that have a greater affinity for it.
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Separation mechanisms
Adsorption, affinity, ion exchange, partition,
and steric exclusion chromatography describe
the predominant chemical or physicalmechanisms used to separate solutes.
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Gel-Filtration Chromatography凝胶过滤层析
It is also known as steric exclusion chromatography空
间排阻层析,gel-permeation, size exclusion, molecular
exclusion, molecular sieve chromatography分子排阻层
析 and separate solutes on the basis of their molecular
size.
A variety of materials have been used asstationary phases including cross link dextran交联葡聚
糖(Sephadex), polyacrylamide聚丙烯酰胺凝胶 (Bio-
Gel), agarose (Sepharose), etc.
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Molecular size chromatography
Molecules too large to
enter the pores remain
exclusively in the mobile
phase and rapidly elude洗脱 from the column.
Molecules that are
intermediates in size (and
small molecules) haveaccess to various fractions
of the pore volume and
elude slowly.
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In addition to preparative applications (制备应用),
gel-filtration chromatography has been used in the
clinical laboratory to :
1. to determine molecular weights of
macromolecules,
2. to remove low-molecular-weight salts or buffer
ions from protein solutions.
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Adsorption chromatography吸附层析
Adsorption chromatography exploits the polarity极性,
or the related tendency for hydrogen binding氢键 of
molecules in order to partition between a polar
sorbent and a less polar solvent, or vice-versa, as the
mobile phase moves through the stationary sorbent.
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Affinity chromatography亲和层析
The term affinity chromatography describes a
number of separation mechanisms with interactions
that occur between biochemical species (enzyme-substrate, hormone-receptor, or antigen-antibody
complexes).
The stationary phase in affinity chromatography isprepared by immobilizing a ligand配体 on particles of
a support either directly or via a spacer.
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Adjustments of pH and ionic strength are required
to achieve optimum binding of the analyte to the
ligand. If this interaction is specific, the analytemay be removed in a single step by addition of a
substrate or an inhibitor, or by a pH change, an
ionic strength change, or addition of a hydrogen
bond-breaking agent, such as urea.
In the clinical laboratory, it has been used to
separate proteins and antibiotics.
Ion-exchange chromatography
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Ion-exchange chromatography
离子交换层析
In ion-exchange chromatography离子交换层析 ,
solutes in a sample are separated by their
differences in sign and magnitude of ionic charge.
In practice, ionic analytes are selectively eluted
from ion-exchange resins by varying the pH
and/or ionic strength of the mobile phase.
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Cation-exchange resins阳离子交换树脂 contain
covalently bound, negatively charged functional
groups, such as sulfonate ions, carboxylate ions orcarboxy-methyl (CM) groups.
This technique is most useful for separation of
organic and inorganic ions, amino acids,nucleotides, and proteins.
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Anion-exchange resins 阴 离 子 交 换 树 脂 are
characterized by the presence of strongly basic
quaternary amines (triethylamino-ethyl groups) orweakly basic groups (aminoethyl, diethylaminoethyl)
which can bear a positive charge.
Ion-exchange chromatography is widely used toseparate and remove inorganic ion from aqueous
mixtures.
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Partition chromatography分配层析
In partition chromatography (also called thin-layer
chromatography)分配层析, a thin film 薄膜of liquid is
adsorbed onto the surfaces of support particles.Separation is based on differences in the relative
solubility溶解度 of solute molecules in this film and the
mobile phase.
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FIG: Two-dimensional thin layer or paper chromatography
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Gas chromatography (GC)
GC is a process by which a mixture is separated
into its constituent components by forcing a gaseous
mixture of it and mobile phase (carrier gas运载气体)
through a column containing the stationary phase.
Separation of the solutes in the mixture is based on
the relative differences in their vapor pressures蒸汽
压 and their interaction with the stationary phase.
A compound with a high vapor pressure will be eluted
more rapidly than compounds with lower vapor
ressures.
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The effluent洗脱液 from the column carries the
separated sample constituents to the detector
which produces a signal that is displayed as aseries of peaks高峰
The volume and time at which an unknown solute
elutes 洗脱
from a column are used to identify theunknown compound. These values are compared
and matched with those obtained from reference
compounds.
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Peak size (area or height) is proportional to the
amount of the compound detected and can be used
to quantify the amount of analyte in the sample.
Depending on the nature of the stationary phase,
GC techniques are divided into two categories:
gas-solid chromatography (GSC) and gas-liquid
chromatography (GLC).
Hi h P f Li id
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High-Performance LiquidChromatography (HPLC)
In LC液相层析, separation is based on the distribution of
the solutes between a liquid mobile phase and a
stationary phase. When an efficient column is used in aliquid chromatograph, the technique is HPLC高效液相层
析. Because column efficiency is inversely related to the
particle size of the column packing, relatively highpressure is required to pump liquid through an efficient
column.
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Different components of HPLC
A basic liquid chromatograph consists of a solvent
reservoir 储溶剂器, a pump泵 to force the liquid
mobile phase through the system; an injector注射器
for introducing an aliquot of sample into the column;
a chromatographic column to separate the analytes
being measured; an on-line detector to detect the
separated analytes as they elute from the column;
and a computer to control the system and process
data.
Different components of HPLC
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Different components of HPLCglycosylated hemoglobin
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MASS SPECTROMETRY (MS)
A mass spectrometry 质 谱 法 is an analytical
technique that first ionizes a target molecule and
then separates and measures the mass of a
molecule or its fragments. Mass analysis is the
process by which a mixture of ionic species is
identified according to the mass-to-charge (m/z)
ratios (ions).
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The analysis is qualitative, quantitative, and
extremely useful for determining the elemental
composition and structure of both inorganic andorganic compounds.
Mass spectrometry when coupled with either gas or
liquid chromatography, the resultant technique is a
particularly powerful analytical technique that has
found extensive use for clinical applications.
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Coupled Techniques
Gas Chromatography/Mass Spectrometry
is a powerful analytical technique that combines
gas chromatograph resolving power with the
Excellent specificity and sensitivity of the mass
spectrometer.
High Performance Liquid
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Chromatography/ Mass
Spectrometry
Compared to gas chromatographs, it has been
more difficult to interface liquid chromatographswith mass spectrometers because the analytes are
dissolved in a liquid, rather than a gas phase.
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SUMMARY
METHODS
APPLICATIONS