Method for detecting the release of methylene blue from plasma inactivation bags using near infrared spectroscopy

Abstract

The present invention relates to a near-infrared spectrometer, comprising a transmission platform, the transmission platform comprising a substrate, two vertical plates parallel to each other are vertically arranged on the substrate, the two vertical plates are each provided with a corresponding transmission window, the transmission window is covered with a transparent optical window, a stage is arranged between the two vertical plates, an object to be tested is located between the two optical windows, at least one vertical plate can slide along the surface of the substrate, so that the object to be tested is clamped between the two optical windows. The present invention also relates to a method for detecting the release of methylene blue in a plasma inactivation bag using near-infrared spectroscopy, comprising: fixing the optical path between the two optical windows to 2.0-5.0 mm; placing the plasma inactivation bag on the stage, performing near-infrared spectroscopy detection on multiple plasma standard solutions, and establishing a multivariate analysis model of the release of methylene blue using the PLS method; performing near-infrared spectroscopy detection on unknown samples, and analyzing the release of methylene blue using the analysis model.

Description

Technical Field

[0001] The invention relates to the field of biological detection, in particular to a method for detecting the release amount of methylene blue in a plasma inactivation bag by using near infrared spectroscopy. Background Art

[0002] In order to prevent virus infection from blood transfusion, clinical plasma widely uses methylene blue irradiation to inactivate plasma viruses. This is the only single-person blood component virus inactivation technology approved for clinical use in my country. The inactivation process is carried out in the plasma inactivation bag, and the release of the inactivator methylene blue in the plasma inactivation bag is when the plasma flows through the methylene blue storage under sterile conditions and gradually dissolves and enters the inactivation bag together with the plasma. This process is the release process of methylene blue, and its release amount is the key parameter that determines the degree of virus inactivation. However, the release amount is related to many factors, including flow rate, ambient temperature, hygroscopicity of methylene blue, and the material and production process of methylene blue immobilized in the storage device. If the release amount of methylene blue in the plasma inactivation bag is too high, the residual amount of methylene blue in the plasma will be too large, which is easy to cause cumulative toxicity; if the release amount is too low, its virus inactivation effect cannot be guaranteed. A large amount of data shows that the virus inactivator methylene blue must be guaranteed to be in a very narrow release range, which is stipulated in my country as 0.9-1.3μmol / L. In order to detect the release amount as conveniently, quickly and sensitively as possible, a large number of methods based on sampling detection have emerged, including solid phase extraction-spectrophotometry, sensitized fluorescence spectroscopy, HPLC, chemiluminescence, and resonance Rayleigh scattering spectroscopy. Sampling detection requires the use of an extraction device to extract samples from the plasma inactivation bag for detection. Since the extraction operation will damage the plasma inactivation bag, this destructive detection method is very likely to cause secondary contamination of plasma during sampling, including bacteria, chemical pollutants, and particulate matter, causing the plasma in the plasma inactivation bag to no longer be used for the human body, resulting in a waste of plasma. It is also based on this that the plasma detected by the release amount can no longer be used in clinical practice. Therefore, it is impossible to conduct such destructive detection-based inactivation quality control for each bag of plasma used in clinical practice. In fact, whether the quality of plasma in the plasma inactivation bag is reliable or not is only a statistical result. Non-destructive detection of the release amount of methylene blue in the inactivation bag is the key issue to achieve quality control of the inactivation of each bag of plasma.

[0003] At present, near-infrared spectroscopy is usually used for non-destructive rapid detection of samples for quality control in the production process. Among them, diffuse reflectance near-infrared spectroscopy is the most widely used for non-destructive detection of samples, but this method has certain limitations: first, this method is generally applicable to solids, powders, and pastes, but it is difficult to apply to liquid samples. After the light is irradiated into the liquid, only very weak diffuse reflection light is generated, and most or even all of it is transmitted light. Therefore, it is difficult to perform quantitative detection of a substance in the liquid based on its diffuse reflectance near-infrared spectrum.

[0004] For the existing near-infrared spectroscopy detection method for rapid quantitative analysis of substances in liquids, it is usually necessary to take samples from the container to be tested for detection, which mainly includes two methods. The first is to place the obtained liquid sample in a cuvette and then put it into the cuvette holder of the instrument for detection; the second is to insert a liquid optical fiber probe with a slit into the liquid in the container to be tested, and the liquid to be tested directly flows into the slit of the optical fiber probe as a cuvette to achieve the sampling operation, and the optical fiber retransmits the signal to the spectrometer to achieve rapid detection. No matter which of the two methods is used, it is a destructive detection method, and the defects of this method have been described in detail above. However, using the existing infrared spectrometer to directly detect the plasma bag will encounter great difficulties. First, there are many bubbles in the plasma inactivation bag. If these bubbles are in the detection area, the measurement results will have a large error; secondly, when the plasma inactivation bag is directly detected, because the bag body is relatively soft and its shape is irregular, the optical path of the spectrum detection is difficult to fix, and it is difficult to calculate the content of the object to be tested using the quantitative formula. Of course, the plasma in the inactivated bag contains a large number of biological macromolecules, the carbon-hydrogen bonds in which will produce a large number of near-infrared spectral signals, which will also interfere with the detection results and bring certain difficulties to the detection method. Summary of the invention

[0005] In order to solve the above technical problems, the purpose of the present invention is to provide a method for detecting the release amount of methylene blue in a plasma inactivation bag using near-infrared spectroscopy. The method overcomes the defect that it is difficult to accurately perform near-infrared quantification on samples of irregular shapes in the prior art, and realizes non-destructive, rapid and accurate analysis of the release amount of methylene blue in a plasma inactivation bag.

[0006] In one aspect, the present invention provides a near-infrared spectrometer, comprising a light source, a monochromator, a detector and a computer information processing system, and also comprising a transmission platform, the transmission platform comprising a substrate, on which two mutually parallel first and second vertical plates are vertically arranged, the first and second vertical plates are each provided with a corresponding transmission window, the transmission window is covered with a transparent optical window sheet, and the optical window sheet is located on the opposite sides of the two vertical plates, a loading platform is provided between the first and second vertical plates for carrying a soft bag containing an object to be tested, and the loading platform is located below the lower edge of the optical window sheet, the soft bag containing the object to be tested is located between the two optical windows, and at least one vertical plate can be moved horizontally along the surface of the substrate, so that the soft bag containing the object to be tested is clamped between the two optical windows and the optical path between the two optical windows can be adjusted.

[0007] Furthermore, the stage is located at least 2 cm below the bottom edge of the optical window.

[0008] Furthermore, the first vertical plate is fixedly connected to the base plate, the loading platform is fixedly connected to the first vertical plate, the second vertical plate is provided with a strip hole facing the loading platform, the strip hole is adapted to the shape of the loading platform, and the loading platform can be slidably inserted into the strip hole.

[0009] Furthermore, a guide rail is fixedly connected to the base plate, and the second vertical plate can slide horizontally along the guide rail and can be locked at any position of the guide rail, thereby fixing the distance between the two optical windows (ie, the optical path).

[0010] Furthermore, a positioning platform is fixedly connected to a surface of the second vertical plate away from the first vertical plate, the second vertical plate is slidably connected to the guide rail through the positioning platform, a screw is threadedly connected to the positioning platform, the axial direction of the screw is perpendicular to the second vertical plate, a fixed baffle is fixedly connected to the guide rail, the fixed baffle has a through hole, and the screw is passed through the through hole.

[0011] Furthermore, the optical window is made of quartz or optical glass.

[0012] Furthermore, the transmission window is rectangular.

[0013] Furthermore, the area of ​​the optical window is larger than the area of ​​the transmission window.

[0014] On the other hand, the present invention also provides a method for detecting the amount of methylene blue released from a plasma inactivation bag using near infrared spectroscopy, using the above-mentioned near infrared spectroscopy detector, comprising the following steps:

[0015] (1) adjusting the distance between the first vertical plate and the second vertical plate so that the optical path between the two optical windows is fixed at 2.0-5.0 mm; preferably, the optical path is 3.5 mm;

[0016] (2) placing the plasma inactivation bag on the stage of the near-infrared spectrometer so that it is located between two optical windows, and slightly squeezing the bag so that the plasma inactivation bag is in close contact with the two optical windows, and the plasma inactivation bag is squeezed to form a regular plane and its surface is pressed into a plane, wherein the plasma inactivation bag contains a plasma standard solution with a known methylene blue content;

[0017] (3) At wave number 4000-10000cm -1 The plasma standard solution was tested by near infrared spectroscopy under the conditions of , and multiple samples were tested repeatedly. According to the near infrared spectroscopy test results of multiple samples, a multivariate analysis model of methylene blue release was established by PLS method;

[0018] (4) placing a plasma inactivation bag containing methylene blue to be tested on the stage, and slightly squeezing the bag from above so that the plasma inactivation bag is in close contact with the two optical windows and the plasma inactivation bag is squeezed to form a regular plane;

[0019] (5) At wave number 4000-10000cm -1 Near infrared spectroscopy detection is performed under conditions of , and the release amount of methylene blue in the plasma inactivation bag is determined using the multivariate analysis model of the methylene blue release amount.

[0020] Furthermore, in step (2), the concentration of methylene blue in the plasma standard solution is 0.312 μmol / L-2.959 μmol / L.

[0021] Furthermore, in step (3), the number of plasma standard solutions is 52.

[0022] Furthermore, in step (3) and step (5), the near infrared light scanning resolution of the near infrared spectrum detection is 8 cm -1 , the number of scans is 32 times, and the scan speed is 0.3165.

[0023] Furthermore, in step (3) and step (5), the scanning aperture of the near-infrared spectrum detection is 22 and the gain is 1.

[0024] Furthermore, in step (3) and step (5), after near infrared spectroscopy detection, 3999.7-4265.8 cm -1 The mean absorbance value of the interval is taken as the drift amount, and the drift of the detected near-infrared spectrum is eliminated.

[0025] Furthermore, in step (3) and step (5), after near infrared spectroscopy detection, 5350-6600cm -1 and 7200-10001cm -1 The absorbance in the two intervals is used as the information-rich spectrum. Preferably, after near-infrared detection, 5827.9-6464.3 cm -1 and 7467.1-9009.9cm -1 The absorbance in the two intervals is used as the information-rich spectrum.

[0026] Furthermore, in step (3), the number of principal components was selected as 11, 11 samples were randomly selected as the prediction set according to the high and low concentration distribution, and the remaining 41 samples were used as the correction set. The PLS method was used to establish a multivariate analysis model for the release of methylene blue.

[0027] Furthermore, the formula of the multivariate analysis model of methylene blue release is: c = k 1 A 1 +k 2 A 2 +···+k n A n , where c represents the concentration of methylene blue in the sample, A 1 , A 2 ···An The wavelengths corresponding to the information-rich spectral intervals selected from the detection spectrum of the sample are λ 1 , 2 ···λ n The absorbance at k 1 , k 2 ···k n is the corresponding PLS regression coefficient.

[0028] Furthermore, before step (2), the method also includes placing an air-filled and unused plasma inactivation bag on the stage of the near-infrared spectrometer to perform near-infrared spectroscopic detection and use it as a spectral background.

[0029] By means of the above scheme, the present invention has at least the following advantages:

[0030] The structural setting of the transmission platform of the near-infrared spectrometer of the present invention can realize the fixed optical path during the non-destructive detection of the plasma inactivation bag, and can effectively solve the problem that the bubbles in the plasma inactivation bag exist in the detection optical path, so that the quantitative result is accurate and reliable. Due to the extrusion effect of the optical window, the plasma inactivation bag between the two optical windows is squeezed into a regular plane. At the same time, due to the low density of the bubbles, under the extrusion force of the optical window, the bubbles in the plasma inactivation bag between the two are naturally driven into the plasma inactivation bag outside the extrusion part of the optical window, so that the bubbles will not appear in the detection area.

[0031] The method of the present invention realizes non-destructive, rapid and accurate analysis of the methylene blue release amount in the plasma inactivation bag outside the plasma inactivation bag in a non-sampling mode. The correlation coefficients of the model calculated concentration to the true concentration of each sample are 98.16% (calibration set) and 98.93% (prediction set), respectively, and the average recovery rate of samples in the high, medium and low concentration groups is 103.6%-110.8%.

[0032] The above description is only an overview of the technical solution of the present invention. In order to more clearly understand the technical means of the present invention and implement it according to the contents of the specification, the following is a detailed description of the preferred embodiments of the present invention in conjunction with the accompanying drawings. BRIEF DESCRIPTION OF THE DRAWINGS

[0033] Figure 1 It is a schematic diagram of the three-dimensional structure of the transmission platform of the present invention;

[0034] Figure 2 It is a schematic diagram of the front side structure of the transmission platform of the present invention;

[0035] Figure 3 It is a schematic diagram of the left side structure of the transmission platform of the present invention;

[0036] Figure 4 yes Figure 3 A schematic diagram of the enlarged structure of the middle circle A;

[0037] Figure 5 1 is a schematic diagram of the top view of the transmission platform of the present invention;

[0038] Figure 6 is a near infrared spectrum after the baseline drift is eliminated in Example 2;

[0039] Figure 7 is the residual of the PLS model based on leave-one-out interaction test;

[0040] Figure 8 is the multivariate analysis model diagram of methylene blue release;

[0041] Description of reference numerals:

[0042] 1-second vertical plate; 2-quartz plate; 3-plasma inactivation bag; 4-transmission window; 5-substrate; 6-first vertical plate; 7-stage; 8-positioning stage; 9-screw; 10-guide rail; 11-fixed baffle. DETAILED DESCRIPTION

[0043] The specific implementation of the present invention is further described in detail below in conjunction with the accompanying drawings and examples. The following examples are used to illustrate the present invention, but are not intended to limit the scope of the present invention.

[0044] Example 1

[0045] See also Figure 1-5A near infrared spectrometer of the present invention comprises a light source, a monochromator, a detector and a computer information processing system, and also comprises a transmission platform, the transmission platform comprises a substrate 5, on which two mutually parallel first vertical plates 6 and second vertical plates 1 are movably connected, and the first vertical plates 6 and second vertical plates 1 are arranged perpendicular to the substrate 5. A corresponding rectangular transmission window 4 is provided on each of the two vertical plates, and a square transparent quartz plate 2 is provided on the transmission window 4, and the quartz plate 2 is located on the opposite sides of the two vertical plates, and the area of ​​the quartz plate 2 is larger than the area of ​​the transmission window 4. The first vertical plate 6 is fixedly connected to the substrate 5, and a stage 7 is also fixedly connected to the first vertical plate 6, and the stage 7 is located at least 2 cm below the lower edge of the quartz plate 2, and the stage 7 is used to place a soft bag containing an object to be tested. The upper surface of the stage 7 is parallel to the substrate 5, and a strip hole facing the stage 7 is provided on the second vertical plate 1, and the strip hole is adapted to the shape of the stage 7 so that the stage 7 can be slidably inserted into the strip hole. Two guide rails 10 are fixedly connected to the base plate 5. The guide rails 10 are arranged perpendicular to the second vertical plate 1. The second vertical plate 1 can slide horizontally along the guide rails 10. A positioning platform 8 is fixedly connected to the side of the second vertical plate 1 away from the first vertical plate 6. The positioning platform 8 is threadedly connected to a screw 9. The axial direction of the screw 9 is perpendicular to the second vertical plate 1. The positioning platform 8 is slidably connected to the guide rails 10. A fixed baffle 11 is fixedly connected to the guide rails 10. The fixed baffle 11 has a through hole, and the screw 9 is passed through the through hole.

[0046] When performing sample testing, such as sample testing in a plasma inactivation bag 3, first adjust the screw to drive the second vertical plate 1 to move along the guide rail 10 so that it slides to the required distance, and the sliding direction is perpendicular to the plane where the vertical plate is located. Then adjust the screw 9 to fix the second vertical plate 1 in this position, that is, the optical path of the transmission detection is fixed. And during the sliding process of the second vertical plate 1, the stage 7 can be slidably inserted into the bar-shaped hole provided thereon to facilitate the adjustment of the optical path and ensure that the quartz plate 2 can clamp the plasma inactivation bag 3 under different optical paths. Place the plasma inactivation bag 3 on the stage 7, and squeeze the bag slightly above the bag so that the plasma inactivation bag is in close contact with the two optical windows, so that the plasma inactivation bag 3 is clamped between the two quartz plates 2. When the quartz plates 2 squeeze the plasma inactivation bag 3, the plasma inactivation bag 3 between the two quartz plates 2 forms a regular shape, thereby fixing the exact optical path (such as Figure 4 The distance indicated by the mark “5” in the figure is the detection optical path), and the bubbles in the plasma inactivation bag 3 migrate to the plasma bag portion above the upper edge of the quartz plate 2, so that there are no bubbles between the quartz plates 2 to prevent them from affecting the detection results.

[0047] Example 2

[0048] In the following examples, the materials used include: non-inactivated human plasma provided by Suzhou Red Cross Central Blood Station, using a filter in a disposable virus inactivation supporting device to filter out white blood cells to obtain plasma without methylene blue (i.e., blank plasma). A disposable blood collection and transfusion device (transfer bag, specification: 35ml, production batch number: 170428, Sichuan Nanger Biotechnology Co., Ltd.) containing a plasma inactivation bag. Methylene blue hydrochloride (analytical grade, based on the content of the dried product> 97%, Sigma-Aldrich Company), and the experimental water is triple distilled water. A near-infrared spectrometer (model: NEXUS, Thermo Company, USA), whose transmission platform is modified according to Example 1.

[0049] This embodiment is a method for establishing a multivariate analysis model for methylene blue release, which is specifically as follows:

[0050] (1) First, prepare a methylene blue stock solution: weigh 1.3953 g of methylene blue (analytical grade, loss on drying 10.6%), place it in a 500 mL volumetric flask, and dilute to volume with PBS solution. Accurately measure 1.00 ml of methylene blue solution, and then dilute to 1000 ml with PBS solution to obtain a 7.8 μmol / L methylene blue stock solution. Store in a sealed container away from light.

[0051] (2) Prepare the samples to be tested: First, use the methylene blue stock solution and PBS buffer (pH = 7.4) to prepare the primary and secondary diluents by stepwise dilution. Then, use the stock solution, diluent and plasma to accurately prepare a total of 52 methylene blue plasma solutions with a concentration of 0.312-2.959 μmol / L. Finally, use a disposable sterile syringe to add the methylene blue plasma solution to each blood collection and transfer bag to obtain the samples to be tested.

[0052] (3) Near infrared spectroscopy of samples

[0053] Wipe the outer surface of the plasma inactivation bag to be tested and the quartz plate on the transmission platform with alcohol, adjust the distance between the first vertical plate 6 and the second vertical plate 1 according to the method of Example 1, and fix the optical path between the two optical windows to 3.5 mm. After that, place the plasma inactivation bag on the transmission platform according to the method of Example 1, and align the left and right sides of the plasma inactivation bag as much as possible each time, and measure the sample spectrum. Instrument parameters are: wave number 4000-10000cm -1 , optical path 3.5mm, resolution 8cm -1 , scan number 32, scan speed 0.3165, aperture 22, gain 1. Before testing samples on the same day, an unused plasma inactivation bag filled with air was used as the spectral background.

[0054] (4) The near infrared transmission spectrum of the sample in the plasma bag obtained by the test was 3999.7-4265.8 cm -1The spectrum of the interval is used as the standard for baseline drift calibration. Select 5827.9-6464.3cm -1 and 7467.1-9009.9cm -1 In order to find the information-rich interval, the mean-centered spectrum was used as the model variable, the number of principal components was selected as 11, 11 samples were randomly selected as the prediction set according to the high and low concentration distribution, and the remaining 41 samples were used as the correction set. The multivariate analysis model of methylene blue release was established using the PLS method.

[0055] For the low wavenumber region of the near infrared spectrum, there will usually be a spectral baseline. It should be a horizontal line, and the spectra of samples with different concentrations in this interval should completely overlap, have no statistical differences, and have an absorbance mean of 0. Figure 6 In the middle, between 3999.7-4265.8cm -1 The interval is the spectral baseline of each sample. However, in the original spectrum, the baseline of samples with different concentrations drifted by about 7. This concentration-independent drift will affect the accuracy of the methylene blue release. The detection spectrum of each sample is between 3999.7-4265.8cm -1 The mean absorbance value of the interval is taken as the drift amount, and the drift is eliminated to obtain the drift-eliminated spectra of all samples.

[0056] The spectrum after baseline drift removal ( Figure 6 ) It can be found that the plasma sample of methylene blue appears in the entire spectral range of 5350-6600cm -1 and 7200-10001cm -1 The obvious absorption peaks in the two intervals are the absorption peak spectral region. For the former, it is composed of two shoulder peaks, high and low, at 5350-5828cm -1 In the low wave number region, the spectrum has low absorbance, which is basically a horizontal line without peaks, and has wavy burrs. This kind of spectral signal with low signal-to-noise ratio will affect the precision of the quantitative analysis model. -1 The spectrum of 5827.9-6464.3cm in the absorption peak spectrum region is selected. -1 The spectrum of 7200-10001cm is a signal-rich spectrum. -1 Absorption peak spectral region, select 7467.1-9009.9cm -1 The spectrum is a signal-rich spectrum.

[0057] 5827.9-6464.3cm -1 and 7467.1-9009.9cm -1The spectrum is the information-rich spectrum, the mean-centered spectrum is used as the model variable, and the calibration set samples are tested by leave-one-out interaction to calculate the residual error of the PLS model predicting the methylene blue concentration when the number of principal components is different ( Figure 7 ). We can find that as the number of principal components increases, the predicted concentration residual tends to decrease gradually, indicating that the accuracy of the model gradually increases. When the number of principal components is ≥11, the degree of reduction of the predicted residual becomes significantly smaller, indicating that the contribution rate of the continued increase in the number of principal components to the accuracy of the model is low. Of course, too large a number of principal components will also lead to overfitting of the model, so 11 is selected as the number of principal components.

[0058] The information-rich spectra of the two selected intervals were used as identification variables for the methylene blue release. Based on the principal component number determined above, the spectral mean was centered and the multivariate analysis model of the release was established using the PLS method. According to the high and low distribution of methylene blue concentration, 11 samples were randomly selected as the prediction set, and the remaining 41 samples were used as the prediction set. The results are shown in Figure 8 , ▲ in the figure represents the calibration set sample, and ● represents the prediction set sample. It can be found that in the multivariate analysis model, the calibration concentration of the calibration sample can correspond to the true concentration well. In the entire detection concentration interval, they are all distributed near the true concentration (diagonal line), and the correlation coefficient Rcorr=98.16%. Utilizing the established model, the methylene blue release in the independent prediction set samples is predicted (i.e., sample detection), and it is not difficult to find that the predicted concentrations of these samples still show a good corresponding relationship, and they can also be distributed near the true concentration, and the correlation coefficient Rpred is as high as 98.93%. This shows that the model established by the present invention has good accuracy.

[0059] In order to illustrate the accuracy of the model, samples of high, medium and low concentration groups were designed to conduct recovery rate tests, and the results are shown in Table 1. As can be seen from Table 1, regardless of the concentration, each concentration group has a good recovery rate, ranging from 103.6% to 110.8%, and there is no correlation between high and low concentrations, which is random, indicating that the established non-destructive testing method is accurate and reliable. Of course, the lowest recovery rate is as low as 96%, while the highest is as high as 119%, which is a completely acceptable accuracy for a non-destructive and rapid detection method, especially for the detection of biological samples.

[0060] Table 1 Accuracy of non-invasive detection of methylene blue release in plasma by near infrared spectroscopy

[0061]

[0062] The above results show that the present invention uses the good penetrability of near-infrared light and uses near-infrared transmission spectroscopy to establish a non-destructive detection model and method for the release of methylene blue, a plasma virus inactivator, in blood collection and transfer bags. The near-infrared transmission spectrum of the plasma bag sample obtained by non-destructive testing outside the plasma bag is used as the identification variable, with a wavelength of 3999.7-4265.8 cm -1 The spectrum of the interval was used as the standard for baseline drift calibration, and 5827.9-6464.3cm was selected. -1 and 7467.1-9009.9cm -1 The spectrum of the interval is the information-rich spectrum, the mean-centered spectrum is used as the model variable, the number of principal components is selected as 11, 11 samples are randomly selected as the prediction set, and the remaining 41 samples are used as the calibration set. The multivariate analysis model of methylene blue release is established using the PLS method. The formula of the model is as follows:

[0063] c=k 1 A 1 +k 2 A 2 +···+k n A n , where c represents the concentration of methylene blue in the sample, A 1 , A 2 ···A n The wavelengths corresponding to the information-rich spectral intervals selected from the detection spectrum of the sample are λ 1 , 2 ···λ n The absorbance at k 1 , k 2 ···k n is the corresponding PLS regression coefficient.

[0064] The correlation coefficients of the model-calculated concentration to the concentration of each sample were 98.16% (calibration set) and 98.93% (prediction set), and the average recovery rates of the high, medium and low concentration groups were 103.6%-110.8%. This shows that the near-infrared transmission spectroscopy method established by the present invention can realize non-destructive, rapid and accurate analysis of the release amount of methylene blue in the collection and transfer bag outside the plasma bag. It indicates that the quality of plasma virus inactivation in the collection and transfer bag can be monitored without secondary pollution.

[0065] Example 3

[0066] This embodiment provides a method for detecting the amount of methylene blue released in plasma using the multivariate analysis model of methylene blue release established above, as follows:

[0067] Wipe the outer surface of the plasma inactivation bag to be tested and the quartz plate on the transmission platform with alcohol, adjust the distance between the first vertical plate 6 and the second vertical plate 1 according to the method of Example 1, and fix the optical path between the two optical windows to 3.5 mm. Then put the plasma inactivation bag with unknown methylene blue content (i.e., the plasma inactivation bag that has released methylene blue) on the stage, and align the left and right sides of the plasma inactivation bag as much as possible each time, and measure the sample spectrum. The instrument parameters are: wave number 4000-10000cm -1 , optical path 3.5mm, resolution 8cm -1 , scan number 32, scan speed 0.3165, aperture 22, gain 1. Before testing the samples on the same day, an unused plasma inactivation bag filled with air was used as the spectral background. The near-infrared transmission spectrum was then measured at 3999.7-4265.8cm -1 The spectrum of the interval is used as the standard for baseline drift calibration. Select 5827.9-6464.3cm -1 and 7467.1-9009.9cm -1 The absorbance at each wavelength in this interval is substituted into the formula of the model established in Example 2 to calculate the methylene blue content in the plasma inactivation bag.

[0068] The above description is only a preferred embodiment of the present invention and is not intended to limit the present invention. It should be pointed out that a person skilled in the art can make several improvements and modifications without departing from the technical principles of the present invention, and these improvements and modifications should also be regarded as within the scope of protection of the present invention.

Claims

1. A method for detecting the amount of methylene blue released from a plasma inactivation bag using near infrared spectroscopy, characterized in that: The method uses a near infrared spectrometer, comprising the following steps: (1) adjusting the distance between the first vertical plate and the second vertical plate so that the optical path between the two optical windows is fixed at 2.0-5.0 mm; (2) placing a plasma inactivation bag on the stage of the near-infrared spectrometer and positioning it between two optical windows, so that the plasma inactivation bag is in close contact with the two optical windows, wherein the plasma inactivation bag contains a plasma standard solution with a known methylene blue content; (3) At wave numbers 4000-10000 cm -1 The plasma standard solution was tested by near infrared spectroscopy under the conditions of , and multiple samples were tested repeatedly. According to the near infrared spectroscopy test results of multiple samples, a multivariate analysis model of methylene blue release was established by PLS method; the formula of the multivariate analysis model of methylene blue release was: c = k1A1 + k2A2 + ... + k n A n , where c represents the concentration of methylene blue in the sample, A1, A2…A n The wavelengths corresponding to the information-rich spectral intervals selected from the detection spectrum of the sample are λ1, λ2…λ n The absorbance at k1, k2…k n is the corresponding PLS regression coefficient; (4) placing a plasma inactivation bag containing methylene blue to be tested on the stage so that the plasma inactivation bag is in close contact with the two optical windows; (5) At wave numbers 4000-10000 cm -1 Near infrared spectroscopy detection is performed under the conditions of , and the release amount of methylene blue in the plasma inactivation bag is determined by using the multivariate analysis model of the methylene blue release amount; The near-infrared spectrometer includes a light source, a monochromator, a detector and a computer information processing system. The near-infrared spectrometer also includes a transmission platform. The transmission platform includes a substrate. Two mutually parallel first and second vertical plates are vertically arranged on the substrate. The first and second vertical plates are respectively provided with corresponding transmission windows. The transmission windows are covered with transparent optical windows, and the optical windows are located on opposite sides of the two vertical plates. A stage is provided between the first and second vertical plates for carrying a soft bag containing an object to be tested. The stage is located below the lower edge of the optical window. The soft bag containing the object to be tested is located between the two optical windows. At least one vertical plate can move horizontally along the surface of the substrate so that the soft bag containing the object to be tested is clamped between the two optical windows and the optical path between the two optical windows can be adjusted.

2. The method for detecting the amount of methylene blue released in a plasma inactivation bag using near infrared spectroscopy according to claim 1, characterized in that: The first vertical plate is fixedly connected to the base plate, the loading platform is fixedly connected to the first vertical plate, the second vertical plate is provided with a strip hole facing the loading platform, the strip hole is adapted to the shape of the loading platform, and the loading platform can be slidably inserted into the strip hole.

3. The method for detecting the amount of methylene blue released in a plasma inactivation bag using near infrared spectroscopy according to claim 1, characterized in that: The base plate is fixedly connected with a guide rail, and the second vertical plate can slide horizontally along the guide rail and can be locked at any position of the guide rail.

4. The method for detecting the amount of methylene blue released in a plasma inactivation bag using near infrared spectroscopy according to claim 1, characterized in that: The optical window is made of quartz or optical glass.

5. The method for detecting the amount of methylene blue released in a plasma inactivation bag using near infrared spectroscopy according to claim 1, characterized in that: In step (2), the concentration of methylene blue in the plasma standard solution is 0.312 μmol / L-2.959 μmol / L.

6. The method for detecting the amount of methylene blue released in a plasma inactivation bag using near infrared spectroscopy according to claim 1, characterized in that: In step (3) and step (5), the near-infrared light scanning resolution of the near-infrared spectroscopy detection is 8 cm -1 , the number of scans is 32 times, and the scan speed is 0.3165.

7. The method for detecting the amount of methylene blue released from a plasma inactivation bag using near infrared spectroscopy according to claim 1, characterized in that: In step (3), after near-infrared spectroscopy detection, select 3999.7-4265.8 cm -1 The mean absorbance value of the interval is taken as the drift amount, and the infrared spectrum detection results are drift eliminated.

8. The method for detecting the amount of methylene blue released from a plasma inactivation bag using near infrared spectroscopy according to claim 1, characterized in that: In step (3) and step (5), after near infrared spectroscopy detection, select 5350-6600 cm -1 and 7200-10001cm -1 The absorbance in the two intervals is used as the information-rich spectrum.

9. The method for detecting the amount of methylene blue released from a plasma inactivation bag using near infrared spectroscopy according to claim 1, characterized in that: Prior to step (2), the method further includes placing an unused plasma inactivation bag filled with air on the stage of the near-infrared spectrometer to perform near-infrared spectroscopic detection and use the bag as the spectral background.

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