Automatic biochemical analyzer is a kind of biochemical analysis in the sampling, add reagents, remove interferences, mixing, constant temperature reaction, automatic monitoring, data processing, and cleaning after the experiment and other steps of the automated operation of the instrument, which completely imitates and replaces manual operation, and has become one of the must be not less than the instrument for clinical diagnosis of the medical institutions. Its application greatly improves the accuracy, precision and efficiency of biochemical testing, adapted to the development of clinical medicine on the requirements of laboratory medicine, however, all of this not only requires the technical basis of biochemical analyzers, but also requires a set of analytical parameters optimized for each project within the instrument. And most of the current biochemical analyzers for the open, closed instrumentation will generally be left in addition to some of the detection of the project's blank channel by the user to set up their own analytical parameters, so it is necessary to understand the biochemical analyzers of the basic meaning of each analytical parameter as well as setup methods.
1. Test name is often set to the project's acronym, such as total protein set to TP, albumin set to ALB and so on. 2. Method type biochemical analyzer commonly used methods such as endpoint method, continuous monitoring method, turbidimetric method, etc., according to the detection principle of the substance being examined to choose one of the analytical methods.
2.1 The endpoint method, also known as the equilibrium method, is based on the reaction to reach equilibrium when the reaction products of the absorption spectral characteristics and the magnitude of the intensity of the light absorption of a class of quantitative analysis of the substance, there is a one-point endpoint and two-point endpoint method of two types. One-point endpoint method is characterized by the use of one or two reagents, when the reaction between the substance to be measured and the reagent reaches the end point, the absorbance of the mixed solution is measured to calculate the concentration of the substance to be measured, the method is commonly used in the total protein bis(2-hydroxyurea), albumin bromo(cresol green), glucose oxidase method, etc., and the majority of the methods operated manually are one-point endpoint method. Two-point endpoint method, also known as the fixed-time method, if a single reagent analysis, when the wavelength of the measurement overlaps with the absorption spectrum of the interfering substances, through the selection of the two-point endpoint method can eliminate the interference caused by the sample blank, the analytical process is mixed in the sample and the reagent after a period of delay read a point A1, a certain amount of time and then read A2, and then compare the standard and the determination of the ΔA (ΔA = A2-A1) value, to find the concentration of the substance to be measured. The concentration of the substance to be measured is found. The creatinine picric acid method is a typical example of a single-reagent two-point method. If it is a two-reagent analysis, the choice of two-point endpoint analysis method can eliminate the interference caused by the sample blank in addition to the interference of endogenous interfering substances can also be eliminated, the analysis process is to add reagent 1 read A1, add reagent 2 read A2, A1 is equivalent to read out the value of the sample blank, A2 is the actual coloring reaction, and then compared to the standard and the determination of the ΔA (ΔA = A2-A1) value, to find the concentration of the substance to be measured. The concentration of the substance to be measured is obtained by comparing the ΔA (ΔA=A2-A1) values of the standard and the measurement. In order to improve the accuracy of the endpoint method of detection, the choice of the method should be set when the endpoint method of zero reading, sample blank and other two analytical parameters, the former is in the reaction before the beginning of the readings, can be deducted before the reaction of the reagent and the sample mixture of blank readings; the latter is the sample plus the absorbance obtained by the blank reagent, the reaction needs to take up a colorimetric cup.
2.2 Continuous monitoring method, also known as dynamic analysis, rate method, etc., the basic principle is in the optimal conditions of the enzymatic reaction, physical, chemical or enzymatic reaction analysis methods, in the reaction rate constant period (zero-level reaction period) within a certain reaction time for continuous observation and recording of the amount of substrate or product changes to the initial speed of the enzyme reaction per unit of time calculated enzyme activity size and metabolite concentration. Specific methods include two-point rate method and multi-point rate method: two-point rate method is to observe the absorbance at two time points during the zero-stage reaction period, and calculate the absorbance change per minute by dividing the difference between the absorbance of the two points (ΔA) by the time (minutes); multi-point rate method is to monitor the reaction rate at certain intervals (2-30s) during the zero-stage reaction period, and then monitor it for several times in succession, to calculate the reaction rate per unit time. This method can be further divided into the least squares method, multi-point δ method, regression method, band rate time method, etc. This method has the obvious advantage that it is greatly improved. This method has the obvious advantage of greatly improving the analytical speed and accuracy, and is mainly applicable to the determination of enzyme activity and its metabolites. In the process of continuous monitoring method, even without the addition of samples, the substrate in the reagent will be automatically degraded to get a result, so the reagent blanking rate should be set, the reagent blanking rate of different batch reagents is not the same, and its value is the results of the project measured in water instead of the sample, the sample determination should be deducted from the value of the reagent blanking rate.
2.3 Turbidimetric method of automated biochemical analyzers can generally only do transmission immunoturbidimetric analysis, when the light through a certain volume of solution containing immune complexes, due to the presence of antigen-antibody complex particles in the solution on the reflection and absorption of the light, resulting in a reduction of the transmitted light, the determination of the luminous flux and antigen-antibody complexes inversely proportional to the amount of light. It is commonly used in end-point assays and is currently used for the detection of serum specialty proteins, such as apolipoproteins, microproteins, acute time-phase reactive proteins, immunoglobulins, and certain drug monitoring. However, if the concentration of the antigen to be tested in the sample is too high, the molecules of the immune complex formed by the antigen and antibody will
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To become smaller, and prone to dissociation, so that the turbidity instead of decreasing, so the immunoturbidimetric analysis process must be set up before the zone check, compare the analysis process after the difference between the two readings point, if the latter point than the former point of the absorbance is low, it indicates that the antigen has been excess, the sample should be diluted and retested.
3. reaction temperature automatic biochemical analyzer through the temperature control system to maintain a constant temperature to ensure that the reaction is carried out properly, the way to maintain a constant temperature there are three kinds of: dry thermostat heating, water bath circulation heating, thermostat circulation indirect heating. Thermostat controller can be 25 ℃, 30 ℃, 37 ℃ three temperatures for constant temperature, according to the needs of any choice, semi-automatic biochemical analyzer thermostat belongs to this kind. Automatic biochemical analyzer temperature controller can generally only control 37 ℃ a temperature, a few can also control 30 ℃ and 37 ℃ two temperatures.
4. Reaction wavelength when the determination of only one component of the system or a mixed solution of the component to be measured in the absorption peak and other ****existing material absorption wavelengths do not overlap, a single wavelength can be selected, if the substance to be measured has several absorption peaks, you can choose the absorbance of the largest one wavelength, or choose the absorption peak absorbance with the wavelength of the wavelength of the smaller change in the wavelength of a certain wavelength. When the examined solution is turbid or there are more interfering substances, light scattering and non-specific light absorption will occur during the determination process, thus affecting the accuracy of the results, at this time, dual-wavelength or even multi-wavelength determination can be used to improve the accuracy of the results, and in practical applications to select the auxiliary wavelength is mainly used to eliminate lipids, hemolysis, jaundice, the interference. As lipids, hemoglobin, bilirubin in a wide range of wavelengths have a strong light absorption, often with the determination of wavelength overlap, the measured absorbance contains the absorbance of the substance to be measured and the absorbance of the interfering substances, so it is necessary to choose the appropriate auxiliary wavelength to eliminate the absorbance of the interfering substances. Auxiliary wavelength setting principle is based on the determination of wavelength selection of auxiliary wavelengths, the requirement of interfering substances in the determination of wavelengths with the auxiliary wavelengths have the same absorbance.
5. The reaction direction has two kinds of positive and negative reactions, the increase in absorbance during the reaction is a positive reaction, and the decrease in absorbance is a negative reaction.
6. Sample volume and reagent volume The determination of sample and reagent volume is generally in accordance with the proportion of the reagent manual, and combined with the characteristics of the instrument to set up, can also be based on the manual method of proportional scaling or redesign, but take into account the detection sensitivity, linear range, as far as possible, will be the sample dilution times larger, in order to reduce the impact of other components in the sample. In the process of setting the amount of samples and reagents should pay attention to the following aspects: ① dilution water, add sample dilution water is to wash out the trace serum adhering to the inner wall of the sampling needle to reduce the sampling error, add reagent dilution water is to avoid cross contamination between the reagents, the amount of two kinds of dilution water should be deducted proportionately in the reagent when the reagents are re-solubilized. If the use of liquid reagent kits due to no longer use water, can not deduct the amount of dilution water, so the two dilution water should be minimized to avoid excessive dilution of reagents. ② minimum sample size, that is, the analyzer injection needle can be drawn within the specified error range of the minimum sample size, with the continuous improvement of technology, the minimum sample size of the instrument is gradually reduced, and there are currently as little as 1.6μl. In the case of samples containing high concentrations of metabolites or high concentrations of active enzymes often need to use the analyzer's minimum sample size as a reduction parameter, so that the upper limit of the detection range of the instrument can be expanded. ③Total reaction volume, in different analyzers have a different prescribed range, in the setting of the sample volume and reagent volume and can not exceed this range. This value is affected by the optical system of the instrument, the direct light path is difficult to reduce the volume of the reaction liquid tested due to the wide beam, the cluster light path is narrowed by a lens, which can detect the reaction liquid mixture as low as 180μl, and in recent years there is a point light source technology, which has a much smaller beam, and irradiates to the sample cup as a single point, which can reduce the volume of the reaction liquid to 120μl.③ Total reaction volume, do not try to save money on sample volume ratio. Sample volume ratio, do not excessively reduce the amount of reagent to save reagents, because in the endpoint colorimetric method, reduce the reagent volume / sample volume ratio will reduce the linear range, when the high concentration of samples will be due to insufficient reagent volume and make the results low.
7. Analysis timeThe analysis time includes incubation time, delay time, monitoring time, etc., and the corresponding analysis time should be chosen for different analytical methods.
7.1 Incubation time is set when choosing the endpoint method, in one-point endpoint method is the time from the beginning of the sample and reagent mixing to the end point of the reaction, and in the two-point endpoint method is the time from the beginning of the first absorbance selection point to the end of the second absorbance selection point. In setting the incubation time, some analytical methods should pay special attention to, such as the choice of bromocresol green method for the determination of serum albumin, due to the serum α1-globulin, transferrin, etc. can also be colored with the bromocresol green, although the reaction rate is slower than that of the albumin, but in fact, when the serum is mixed with the albumin, the "slow reaction" has already occurred, so in order to reduce the non-specific binding of the reaction, it is necessary to set the time to the end of the reaction. Therefore, to minimize non-specific binding reactions, the absorbance should be read 30 s after mixing the bromocresol green with the serum. When the enzymatic Trinder reaction is chosen for the determination of glucose, total cholesterol, glycerol
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When triglycerides are determined, the time taken to reach the endpoint of the enzymatic reagent reaction must be determined because the enzymatic reaction at 37°C is slow. Enzyme reagent reaction to reach the end point of time, the automatic analyzer with the kit can generally be in all the addition of samples within 5 minutes after the reaction is complete, so you should choose the maximum reaction time of the analyzer.
7.2 Delay time is set when selecting the continuous monitoring method or the two-point endpoint method, i.e., the time between the start of mixing of the sample with the reaction reagent (second reagent) and the first absorbance selection point. During the continuous monitoring method, when the enzyme is mixed with the substrate it takes some time for the enzyme to activate until the linear reaction period before monitoring can begin, and some items require the use of instrumental enzymes to deplete endogenous metabolites and eliminate interference. The general single-reagent method requires only 30s, and the commonly used items of glutamate aminotransferase and aspartate aminotransferase require special attention, but for double-substrate reactions or coenzyme participants are required, it is usually 1-3min.7.3 Monitoring timeAfter the delayed enzyme reaction period, the rate of the reaction is accelerated and stabilized to the stage of the presence of an excess of substrate, i.e., the enzyme reaction is carried out at a constant rate, which is not influenced by the substrate Concentration of the substrate, this period of time is called the linear reaction period or zero-level reaction period, automatic biochemical analyzer monitoring time that is this period. Continuous monitoring method in the zero-level reaction period should be monitored at least 90-120s or at least 4 points (3 ΔA), less than 3 ΔA can not be called continuous monitoring method, because it can not be calculated linearity; monitoring time is too long, then it is easy to substrate depletion, the measurable range becomes narrow. Medical education network collects and organizes.
8. Calculate the factor (F value) and measured F value with continuous monitoring method for enzyme activity determination, do not need to make a standard tube or standard curve, according to the molar absorption coefficient is easy to carry out the calculation of enzyme activity concentration. The change in absorbance per minute (ΔA/min) in the linear range is measured first, and when the enzyme activity concentration is represented by U/L, it can be calculated according to the following formula: U/L, t℃ = ΔA/min × F = = ΔA/min × V × 106 / (ε × V × L)
Where V: total volume of the reaction system (ml); 106: converting mol to μmol; ε: molar absorption coefficient (cm2/mol); ε: molar absorption coefficient (cm2/mol); ε: molar absorption coefficient (cm2/mol). absorption coefficient (cm2/mol); v: sample volume (ml); L: optical diameter of the colorimetric cup (cm). When the conditions are fixed, theoretically V, v and L are fixed values, ε value is a constant, so the F value is constant.F value is very important for enzyme determination, too high although the determination of the linearity of the wider, but poor reproducibility, and vice versa, although the precision is good, but the detection of the linearity of the narrow, so it should be based on the actual situation of the reasonable settings and applications. However, in clinical practice, many factors of the instrument, such as the accuracy of wavelength, the size of the half-wave width, the optical diameter of the colorimetric cup and the wear and cleanliness, the accuracy of the temperature control, the condition of the filling system, etc., if they are not in accordance with the requirements or changes will affect the ε value of the indicator or the relevant terms in the above formula, so the measured ε and F values of the indicator should be regularly checked and practically determined under the specific conditions of the instrument. Because the pure NAD (P) H solution is not stable, so the ε value of NAD (P) H needs to be measured by glucose hexokinase method, the actual operation of a concentration of glucose solution repeated 5-10 times to get the corresponding set of absorbance A value, Aˉ ± s, note that the s must be lower than the specified permissible value of the batch, and then according to the following two formulas were calculated measured ε and NAD (P) H value and F value. F value:
Where C is the concentration of standard solution (mol/L). Some other pigment source indicators in different media environment, its ε value will occur in varying degrees of change, 5-thio-2-nitrobenzoic acid, p-nitrophenol, p-nitroaniline, etc. These can be obtained from a highly pure and stable indicator, can be prepared in a certain medium, according to the clinical specimens used in the field of the reagents and instrumentation for the determination of absorbance to find the measured ε value and F value.
9. Linearity is the boundaries of the non-linear ratio, commonly used as a %, which is calculated by the formula, where: k1 is the slope of the former reading time within 2/3 of the continuous monitoring time, k2 is the slope of the latter 2/3 of the reading time, and k3 is the total slope, which is generally set to 15%. For some enzyme activity items, it can be appropriately relaxed, when there is no need to set the check limit, set it to 0. A large percentage of linearity indicates that Δk is no longer linear between; exceeding the limit indicates that the substrate is insufficient, the test results will be low, should be diluted and retested. 10. The substrate depletion limit is set when selecting the continuous monitoring method or two-point endpoint method, the design of different models of analyzers is not the same, some are the difference between the zero point and the absorbance at the first point of monitoring, and some are the value of the absorbance rising or falling to the specified absorbance (MAX-OD/MIN-OD). Exceeding the limit indicates that the enzyme activity of the sample is very high, the substrate is consumed too quickly, the change in absorbance within the linear reaction period specified by the instrument program often does not represent the true enzyme activity, resulting in low results, the sample should be diluted by a certain number of times to re-measure.