CN106706519A - Spectrometry method for simultaneous rapid qualitative analysis of activities of two aminotransferases in serum - Google Patents

Abstract

The invention provides a spectral method for rapid and non-destructive qualitative analysis of the activities of two transaminases in serum at the same time, belonging to the field of biochemical detection. The method comprises the following steps: collecting blood samples with different activities of alanine aminotransferase (ALT) and aspartate aminotransferase (AST); preparing corresponding serum samples; measuring the activities of ALT and AST in the serum samples by biochemical method ; measure the spectrum of serum samples; preprocess the spectral data with chemometric techniques; combine the normal range of ALT and AST activity with the appropriate spectral modeling area, and establish a qualitative discrimination model for the normal and abnormal activities of ALT and AST; The performance of the built model was evaluated; the built model was used to qualitatively analyze whether the activities of ALT and AST in unknown serum samples were normal or not. This method is suitable for qualitative analysis of ALT and AST activities in serum, and quickly finds out subjects with abnormal detection indicators.

Description

Rapid and simultaneous qualitative analysis of the activities of two transaminases in serum based on spectrometry

technical field

The present invention relates to a rapid and non-destructive analysis method for alanine aminotransferase (alanine aminotransferase, ALT) and aspartate aminotransferase (aspartate aminotransferase, AST) activities in serum, specifically involving the use of spectroscopic methods combined with chemometric techniques , and simultaneously qualitatively analyze the activities of ALT and AST in serum, belonging to the field of biochemical detection.

Background technique

Aminotransferase (aminotransferase) is a class of enzymes that catalyze the amino transfer between amino acids and keto acids, and participate in the synthesis and decomposition of amino acids. Among them, alanine aminotransferase (alanine aminotransferase, ALT) and aspartate aminotransferase (aspartate aminotransferase, AST) are important diagnostic markers. ALT mainly exists in liver cells, and its intracellular concentration is significantly higher than that in serum. When the liver cells are damaged, the concentration of ALT in the serum will increase significantly. Therefore, the World Health Organization (WHO) recommends ALT serum activity as the most sensitive evaluation index of liver function damage. AST is widely distributed, especially in the heart and liver. When the heart, liver and other organs are damaged, the concentration of AST in the serum will increase significantly. Therefore, AST serum activity was used as another evaluation index of liver function damage. In addition, AST serum activity is also used as a sensitive evaluation index of cardiac function affected by drug toxicity.

The activities of ALT and AST in serum are currently detected by automatic biochemical analyzers in hospitals, blood centers and other medical institutions. This method has many disadvantages. It is particularly worth noting that blood samples are biological samples obtained through destructive collection methods. Traditional biochemical testing methods destroy blood samples and cannot continue to use the same sample for testing other items. In addition, the pretreatment is cumbersome and time-consuming, the use of detection reagents is required, and it is easy to cause environmental pollution. These are the defects of traditional biochemical detection methods. Therefore, it is very necessary for clinical detection to invent a method for rapid and non-destructive qualitative analysis of the activities of two transaminases in serum.

The spectral analysis of chemical substances is commonly used in the ultraviolet-visible, near-infrared and infrared spectral regions to measure delocalized electronic transitions, double frequency and combined frequency transitions of molecular vibrations, and molecular vibrational transitions, respectively. It can be seen that the spectrum carries a wealth of sample information, combined with chemometric techniques, it can achieve high-throughput, rapid, and non-destructive analysis of complex systems. Spectral analysis technology is convenient and fast, and is suitable for medical and inspection institutions at all levels.

Contents of the invention

The purpose of the present invention is to analyze the activity of two kinds of transaminases in the serum quickly and nondestructively, mainly comprising the following steps:

(1) Collect blood samples with different activities of ALT and AST;

(2) Prepare the collected blood samples into corresponding serum samples;

(3) Determination of ALT and AST activities in serum samples by biochemical methods;

(4) Determination of the spectrum of the serum sample;

(5) Preprocessing the spectral data of the measured serum samples by using chemometric techniques;

(6) Establish a qualitative discrimination model for normal and abnormal activities of ALT and AST with the appropriate spectral modeling area of serum samples, combined with the normal range of ALT and AST activities;

(7) Evaluate the performance of the established ALT and AST activity qualitative discrimination model;

(8) Use the established model to qualitatively analyze whether the activities of ALT and AST in unknown serum samples are normal or not.

The blood sample collection method in the step (1) is as follows: collect an appropriate amount of venous blood from fasting volunteers, and place it in a blood collection tube containing a coagulant.

The preparation method of the serum sample in the step (2) is as follows: wait for the blood to coagulate and separate into layers, centrifuge, and take the supernatant as the serum sample.

The method for determining the reference value of ALT and AST activity in the step (3) is a method commonly used in medical institutions at present, such as using a fully automatic biochemical analyzer for detection.

The spectrum in the step (4) can be measured by Fourier transform near-infrared spectrometer, ultraviolet-visible spectrophotometer and other instruments. The measurement parameters of the instrument include but are not limited to spectral measurement range, resolution and scan times.

The preprocessing method of spectral data in the described step (5) can be but not limited to one or more in unprocessed, first derivative, second derivative, Norris smoothing, Savitzky-Golay smoothing, mean centralization, calibration .

The reasonable modeling spectral region in the step (6) can be manually selected, automatically selected by modeling software and combined automatically and manually; qualitative modeling algorithms include but are not limited to discriminant analysis and artificial neural network methods.

The parameters for evaluating the performance of the established qualitative model in the step (7) include but are not limited to the correct judgment rate of the calibration set and the correct judgment rate of the verification set.

When judging the unknown sample with the built calibration model in the step (8), the spectral measurement method used by the unknown sample, the spectral measurement parameter, the preprocessing method and the spectral region of the spectral data should be consistent with the calibration set sample; Data processing software includes but not limited to TQ Analyst and MATLAB.

The method is suitable for rapid and nondestructive qualitative analysis of ALT and AST activities in serum at the same time, and rapid screening of blood samples with abnormal detection indicators. During the detection process, the sample is non-destructive, no complicated sample pretreatment is required, no detection reagent is used, and no pollution. It is a high-throughput, convenient and fast analysis technology that is worthy of promotion.

Description of drawings

Figure 1 DA model results of male ALT and AST activity.

Fig. 2 Results of DA model of female ALT and AST activity.

detailed description

The present invention will be further described below in conjunction with the accompanying drawings and examples, which should not be construed as limiting the present invention.

Example

The purpose of this example is to establish a qualitative model of discriminant analysis (DA) by Fourier transform near-infrared transmission spectroscopy combined with chemometrics technology to preliminarily determine whether the activities of ALT and AST in serum samples are normal.

The instrument involved in this embodiment is Antaris II Fourier transform near-infrared spectrometer (Thermo Company, USA), the sampling device is a transmission analysis module, the signal acquisition software is RESULT 3.0, and the data analysis and DA modeling software is TQ Analyst 8.0. The enzyme activity assay instrument is BS-800M automatic biochemical analyzer (Shenzhen Mindray Company).

The method of this embodiment mainly includes the following steps:

1. Preparation of Serum Samples

Collect venous blood from volunteers on an empty stomach, put it in a blood collection tube containing a coagulant, place it at room temperature until the blood coagulates and separate layers, and centrifuge to get the supernatant, which is the serum sample. Samples: 90 serum samples, 45 blood samples from men and 45 blood samples from women.

2. Determination of ALT and AST activities in serum samples by biochemical method

The activities of ALT and AST in serum samples were measured by automatic biochemical analyzer. The upper limit of ALT activity in serum of healthy people: 41U/L for men and 31U/L for women; the upper limit of AST activity: 37U/L for men and 31U/L for women.

3. Determination of near-infrared transmission spectrum

Take an appropriate amount of serum sample and inject it into the clean liquid sample tube equipped with the instrument. The injection volume is about 2/3 of its volume, and put it into the liquid sample holder correctly. The spectral signal acquisition software is RESULT 3.0. The spectral scanning range is 10000-4000cm ^-1 , the resolution is 8cm ^-1 , and the number of scanning is 32 times. Before each scan of the sample, scan with the same parameters and subtract the background.

4. Establishment of qualitative model and performance evaluation

Because the upper limit of normal value is different for men and women, qualitative models of DA for men and women were established respectively. The activities of ALT and AST were within the upper limit of the normal value as normal, and the rest as abnormal. The ratio of randomly selected calibration set to validation set is about 3:1, and the serum sample information is shown in Table 1. Unknown samples are 3 males and 3 females. The performance of the model is evaluated by the following indicators: the correct judgment rate of the calibration set and the correct judgment rate of the verification set.

Table 1 Serum sample information

The male DA model adopts the original spectral modeling, the spectral range is 9881-7200cm ^-1 , 7000-5400cm ^-1 and 5200-4119cm ^-1 , the principal component is 3, the cumulative contribution rate is 98.2%, the positive The judgment rate is 100.0%. The female DA model is modeled using the original spectrum, the spectral ranges are 9881-7200cm ^-1 , 7000-6700cm ^-1 , 6600-5400cm ^-1 and 5200-4119cm ^-1 , the principal component score is 7, the cumulative contribution rate is 99.5%, corrected The correct judgment rate of both the set and the prediction set is 100.0%. See Figure 1 for the diagrammatic effect of the male DA model, and Figure 2 for the diagrammatic effect of the female DA model.

5. Application of Qualitative Models

Three male and three female unknown serum samples were used to predict the activities of ALT and AST by the model. The spectral measurement method, spectral measurement parameters, spectral data preprocessing method and spectral modeling area used for the unknown sample are all consistent with the calibration set samples. Judgment results: 3 male serum samples were abnormal; 2 female serum samples were normal, and 1 female serum sample was abnormal.

6 Conclusion

The present invention establishes a DA qualitative model through Fourier transform near-infrared transmission spectroscopy combined with chemometrics technology. The DA model can quickly judge whether the activities of ALT and AST in serum samples are normal or not. Compared with the traditional method, this method can measure multiple detection indicators at the same time. It is fast and non-destructive, easy to operate, and has high accuracy. It is suitable for medical and inspection institutions at all levels.

Claims (9)

1. A kind of spectroscopic method of two kinds of transaminase activities in the rapid non-destructive qualitative analysis serum simultaneously, mainly comprises the following steps: (1) collecting blood samples with different activities of alanine aminotransferase and aspartate aminotransferase; (2) Prepare the collected blood samples into corresponding serum samples; (3) Determination of the activity of alanine aminotransferase and aspartate aminotransferase in serum samples by biochemical method; (4) Determination of the spectrum of the serum sample; (5) Preprocessing the spectral data of the measured serum samples by using chemometric techniques; (6) Use the appropriate serum sample spectral modeling area, combined with the normal range of alanine aminotransferase and aspartate aminotransferase activities, to establish normal activity of alanine aminotransferase and aspartate aminotransferase Qualitative discriminant model with abnormality; (7) Evaluate the performance of the established alanine aminotransferase and aspartate aminotransferase activity qualitative discrimination model; (8) Use the established model to qualitatively analyze whether the activities of alanine aminotransferase and aspartate aminotransferase in unknown serum samples are normal or not. 2. The method according to claim 1, characterized in that: the blood sample collection method in the step (1) is as follows: collect an appropriate amount of venous blood from a fasting volunteer, and place it in a blood collection tube containing a coagulant. 3. The method according to claim 1, characterized in that: the preparation method of the serum sample in the step (2) is as follows: wait for the blood to coagulate and stratify, centrifuge, and take the supernatant to be the serum sample. 4. The method according to claim 1, characterized in that: the assay method of alanine aminotransferase and aspartate aminotransferase activity reference value in the described step (3) is the method commonly used in current medical institutions, Such as the use of automatic biochemical analyzer detection and so on. 5. the method for claim 1 is characterized in that: in described step (4), spectrum can adopt Fourier transform near-infrared spectrometer, instruments such as ultraviolet-visible spectrophotometer to measure. The measurement parameters of the instrument include but are not limited to spectral measurement range, resolution and scan times. 6. The method according to claim 1, characterized in that: the preprocessing method of spectral data in the step (5) can be but not limited to unprocessed, first derivative, second derivative, Norris smoothing, Savitzky-Golay One or more of smoothing, mean centering, and scaling. 7. method as claimed in claim 1, is characterized in that: in the described step (6), reasonable modeling spectral region can be selected manually, uses modeling software to automatically select and automatically and manually combine; Qualitative modeling algorithm comprises but Not limited to discriminant analysis and artificial neural network. 8. The method according to claim 1, characterized in that: the parameters for evaluating the performance of the established constructive model in the step (7) include but not limited to the correct judgment rate of the calibration set and the correct judgment rate of the verification set. 9. The method according to claim 1, characterized in that: when the unknown sample is judged by the built calibration model in the step (8), the spectral measurement method used by the unknown sample, the spectral measurement parameter, the prediction of the spectral data The processing method and spectral region should be consistent with the samples in the calibration set; the software used for data processing includes but is not limited to TQ Analyst and MATLAB.