basic tech in clinical chimistry

8/16/2019 Basic Tech in Clinical Chimistry

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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

basic tech in clinical chimistry

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