What are the methods for “taking points” for IVD response curves?
1. END POINT METHOD
A method of quantitatively analyzing substances based on the absorption spectral characteristics of the reaction product and its absorbance when the reaction reaches equilibrium. For general chemical reactions, the reaction end point is when the reaction is complete (or the forward and reverse reactions are dynamically balanced) and the reaction products are stable. For the antigen-antibody reaction, the end point is when the antigen and antibody react completely and form the largest and stable immune complex. On the reaction time course curve is a line segment parallel to the X-axis. In terms of measurement and calculation methods, there are generally two types: one-point method and two-point method.
1. One point method (ONE POINT) uses the absorbance value of the air blank (GB), water blank (WB) or reagent blank (RB) before the reagent and sample are mixed as the measurement calculation base point, and subtracts the blank reading from the absorbance reading at the end of the reaction to obtain Reaction absorbance. The measurement results are obtained by comparing the absorbance with the reaction absorbance of the calibration solution under the same conditions. It is often used in conjunction with the one-point calibration method, that is, using a calibration concentration, the calibration curve passes through the zero point and becomes linear. Multi-point calibration is also applied.
2. The two-point end point method (TWO POINT END), that is, the end point-start point method, uses a certain time point after the reagent and sample are mixed as the starting point, and subtracts the starting point reading from the absorbance reading at the end point of the reaction. Under certain conditions, the specificity and interference of the sample to the reaction or the reaction itself can be reduced (mainly referring to chromaticity interference). Double reagents are often used, and a point before adding R2 is often used as the starting point for determination; in some cases, a point after adding R2 can also be used as the starting point for determination. If a single reagent is used, it is difficult to use when the main reaction starts too fast or the initial reading point of the instrument is limited.
The only difference between the fixed time method (FIXED METHOD) and the two-point end point method is that the end point of the measurement reading is not in the reaction equilibrium section, but is selected according to the methodology. Such as serum creatinine (picric acid method) determination.
3． The three-point endpoint method is the dual-endpoint method, which measures two reaction-related endpoints at one time in one channel. For example, it also measures free fatty acids and triglycerides. Some instruments (such as the HITACHI series) set this method.
2. CONTINUOUS MONITORING METHOD
Also called rate method (RATE ASSAY). That is, a method of continuously monitoring the reaction process and performing quantitative analysis based on the measured rate of product formation or substrate consumption. The reaction time course curve is a section where the reaction shows a constant speed (the slope remains unchanged), which is often used to determine the linear reaction period of enzyme activity.
1. The continuous monitoring method is the zero-order reaction rate method, also known as the slope method. In a longer reaction time period (at least 90-120 seconds), read the absorbance value at a certain time (usually 2 to 30 seconds), read at least 4 points, and get 3 △A; generally, continuous Multiple readings are processed by the least square method. If the reading interval is too short, the rate time (TR) multi-point method is used. The readings of the linear reaction part are all taken to calculate the reaction rate ΔA/MIN per unit time. This method must be based on zero-order reaction as the basis for measurement and calculation, because only under zero-order reaction, the absorbance change per unit time (reaction rate ΔA/MIN) is directly proportional to the enzyme activity. This method relatively reduces analysis errors and greatly improves analysis speed and accuracy. However, semi-automatic biochemical analyzers use single sample continuous monitoring, which is quite time-consuming. To apply the continuous monitoring method, you should first prepare high, medium and low concentration samples within the linear range, and make reaction time process curves respectively to understand the entire reaction process at different concentrations, taking into account the determination of the delay time and linear monitoring period.
2. The two-point rate method is the so-called pseudo-first-order rate method. Select two time points T 1 and T 2 in the reaction, read the absorbance A 1 and A2, and calculate (A2-A1), (T2-T1) = △A, △T. There are two main differences between this method and the two-point endpoint method: the reaction at the latter reading point does not reach the endpoint, and the result is calculated based on the rate. Compared with the continuous monitoring method, its disadvantage is that T 1 and T 2 are manually determined, and there are many uncertain factors. It cannot guarantee that the reaction will be linear during the period from T 1 to T 2, which affects the accuracy of the results; a pre-test should be done before routine measurement. to determine the linear time period. If the reaction is not linear within the selected time period (for example, the zero-order reaction period is short and the instrument cannot be set or measured), the end-point method can only be used instead. The advantage is that the method is simple; when the enzyme activity is low and the measured absorbance value is small, the measurement time period can be increased without being limited by the continuous monitoring time points of the instrument, thereby reducing reading errors.
3． The rate B method performs two reaction-related rate method measurements in one channel at one time. It can be used for two test measurements, or it can also be used for automatic compensation of interference or/and sample blank. The principle of the latter use is: using the microcomputer of the instrument for automatic processing, on the premise that the first reaction (interference reaction) always maintains linearity, The carryover effect of the first reaction rate can be subtracted from the rate of the second reaction (the primary reaction). For example, it is used to eliminate the decrease in absorbance when bilirubin is converted into biliverdin, and the negative interference in the determination of creatinine picric acid method, etc. Some instruments (such as the HITACHI series) set this method.
3. BLANK correction
In spectrophotometry, blank solutions are often used to adjust the absorbance zero point of the instrument, or to offset interference factors in certain measurements. In biochemical analyzer measurement, in addition to using dual or multi-wavelength, two-point methods, etc. to eliminate background interference, special blank measurement is often used to deduct its influence from the measured absorbance of the sample. Proper selection of blank correction plays an important role in improving accuracy.
1. Reagent blanks are generally divided into two categories: with or without reagent blanks in method type and calibration mode. The reagent blank is measured alone or in conjunction with calibration, and the deionized water sample cup or reagent blank rack needs to be pre-selected. The absorbance of each measurement point of the calibration or patient sample must be deducted from the reagent blank absorbance or blank rate value of the corresponding measurement point. For methods without reagent blank, the water blank in the reaction cup is often used as the measurement reference value.
In many instruments, the reagent blank measurement is similar to the calibration measurement and is not measured in real time when the patient sample is measured. Therefore, attention should be paid to its measurement frequency to avoid calculation errors caused by changes in reagent blank due to changes in reagent batch number or quality.
2. Sample blank is mainly to eliminate the interference of turbidity or chromaticity of the sample itself. The blank channel method is often used, and the measurement calibration result = the color reaction channel result - the blank channel result. Most instruments must occupy additional measurement channels and the analysis speed is halved, but the accuracy of interference removal should be higher than the two end points.
