Basic Techniques In Clinical Chemistry Laboratory 临床生物化学基本技术 Jian Cheng Tu Dept. of Clinical Laboratory Medicine Zhongnan Hospital, Wuhan University Tele: 13507119120 E-mail: znyyjyk@sina. com
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 theory of instrumental analysis will provide a working knowledge of instruments, applications to patients testing in the clinical chemistry laboratory.
A variety of techniques have been used in clinical chemistry laboratory for sample testing Most Fundamental Methods Include: Electrophoresis 电泳 Chromatography 层析法 Spectrophotometry 分光光度技术 Mass spectrometry 质谱法 Fluorometry 荧光测定法 Nephelometry 散射比浊法 Turbidimetry 比浊法 Biochip(Protein and DNA Chip/Array) 生物芯片 Biosensor 生物传感技术
Electrophoresis Refers To the Migration(移动) of Charged Solutes or Particles in A Liquid or A Porous Supporting 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.
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 (p. I)等电点of a molecule: is the p. H 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.
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.
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.
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.
Description of Electrophoresis Schematic diagram
Description of Electrophoresis A schematic diagram of an electrophoresis system shows: ¡ two buffer boxes缓冲液箱(l) with baffle plates隔板 contain the 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.
¡ The electrophoresis support 支持物(3) on which separation take place is in contact with buffer by means of 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.
Automated Electrophoresis Systems Although electrophoresis was traditionally a manual technique, it has been improve 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 procedure easily. Example of rapid electrophoresis (REP) analyzer, (Helena Laboratories).
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.
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).
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 (20 min-1 h) and the ability to store the transparent membranes for long periods.
(+) Albα 1α 2β γ (-) 正常人血清（γ区弥漫性分布，多克隆） Ig. G型MM (γ区狭窄深染区带，单克隆) Ig. A型M蛋白血症 7 s-Ig. M+2 Ig. G 三M蛋白血症 λ-LCD ( S ) λ-LCD ( U )
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.
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.
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 is composed 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.
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.
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 medium possessing a stable p. H gradient using isoelectric focusing (IEF)等 电 聚 焦. The protein moves to a zone in the medium where the p. H is equal to its isoelectric point (p. I). At this p. H, the charge becomes zero and migration ceases.
等电聚焦 – + p. H 3 p. H 7. 5 p. H 10 – + p. H 3 p. H 10 p. H 7. 5 p. H 10
Two-Dimensional (2 D) 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 weightdependent分 子 量 依 赖 的 electrophoresis in polyacrylamide.
第一向：等电聚焦 – + p. H 3 p. H 7. 5 p. H 10 – + p. H 3 p. H 10 p. H 7. 5 p. H 10
第二向：SDS-PAGE电泳 – + p. H 3 p. H 7. 5 p. H 10 Proteins migrate through the gel at a rate proportional to their size. Smallest proteins travel the furthest distance size charge
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 and, via buffer reservoirs to a high-voltage power supply高 压电.
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 k. V) than in traditional electrophoresis. This high voltage enhances separation efficiency and reduces separation time in some cases to less than 1 min.
2 - CHROMATOGRAPHY 层析法，色谱法 Chromatography is a physical method of separation in which the components to be separated are 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.
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 or analytes. 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.
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. 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 in the mobile phase and travel faster and separate from those that have a greater affinity for it.
Separation mechanisms Adsorption, affinity, ion exchange, partition, and steric exclusion chromatography describe the predominant chemical or physical mechanisms used to separate solutes.
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 as stationary phases including cross link dextran交 联 葡 聚 糖 (Sephadex), polyacrylamide聚丙烯酰胺凝胶 (Bio-Gel), agarose (Sepharose), etc.
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) have access to various fractions of the pore volume and elude slowly.
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.
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.
Affinity chromatography 亲和层析 The term affinity chromatography describes a number of separation mechanisms with interactions that occur between biochemical species (enzymesubstrate, hormone-receptor, or antigen-antibody complexes). The stationary phase in affinity chromatography is prepared by immobilizing a ligand配体 on particles of a support either directly or via a spacer.
Adjustments of p. H and ionic strength are required "to achieve optimum binding of the analyte to the ligand. If this interaction is specific, the analyte may be removed in a single step by addition of a substrate or an inhibitor, or by a p. H 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 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 p. H and/or ionic strength of the mobile phase.
Cation-exchange resins阳离子交换 树 脂 contain covalently bound, negatively charged functional groups, such as sulfonate ions, carboxylate ions or carboxy-methyl (CM) groups. This technique is most useful for separation of organic and inorganic ions, amino acids, nucleotides, and proteins.
Anion-exchange resins阴 离 子 交 换 树 脂 are characterized by the presence of strongly basic quaternary amines (triethylamino-ethyl groups) or weakly basic groups (aminoethyl, diethylaminoethyl) which can bear a positive charge. Ion-exchange chromatography is widely used to separate and remove inorganic ion from aqueous mixtures.
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.
Thin-layer chromatography, showing set-up and separation of constituents and the calculations for Rf value
FIG： Two-dimensional thin layer or paper chromatography
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 pressures.
The effluent洗脱液from the column carries the separated sample constituents to the detector which produces a signal that is displayed as a series of peaks高峰 The volume and time at which an unknown solute elutes 洗脱from a column are used to identify the unknown compound. These values are compared and matched with those obtained from reference compounds.
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).
High-Performance Liquid Chromatography (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 a liquid 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 high pressure is required to pump liquid through an efficient column.
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 glycosylated hemoglobin
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).
The analysis is qualitative, quantitative, and extremely useful for determining the elemental composition and structure of both inorganic and organic 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.
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 Chromatography/ Mass Spectrometry Compared to gas chromatographs, it has been more difficult to interface liquid chromatographs with mass spectrometers because the analytes are dissolved in a liquid, rather than a gas phase.
SPECTROPHOTOMETRY 分光光度技术 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. The absorbance吸光率of a solution is the amount of light absorbed by that solution.
According to Beer’s Law the absorbance varies directly with the concentration of the solution in question. It is equals to the concentration of a substance in solution multiplied by the length of the path that the light must pass through multiplied by the molar absorptivity摩尔吸收率of the substance of interest.
Diagram of internal parts of a spectrophotometer
Schematic diagram of Spectrophotometer 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.
The light next passes through an 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.
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. Next, a cuvette containing a reagent blank空白is inserted and the meter is adjusted to read 100%T (i. e. , zero absorbance).
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.
Determination of the concentration of the unknown using the standard curve
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.
Several automated fluorometric analyzers have been developed for special applications in clinical laboratory, because of their sensitivity, speed, simplicity, and reliability. These include the flow cytometer, hematofluorometer, fluorescence microscopy. 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.
TURBIDIMETRY Turbidity causes the decrease of the intensity of the incident beam of light入射波束as it passes through a solution of particles. The measurement of this decrease in intensity of the incident light beam that is caused by scattering, reflectance反 射 , and absorption of the light is called turbidimetry比浊法.
NEPHELOMETRY Nephelometry比浊法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.
SUMMARY METHODS APPLICATIONS
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