CN107941741B - Method for detecting the release of methylene blue from plasma inactivation bags using near infrared spectroscopy - Google Patents
Method for detecting the release of methylene blue from plasma inactivation bags using near infrared spectroscopy Download PDFInfo
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- 230000002779 inactivation Effects 0.000 title claims abstract description 81
- RBTBFTRPCNLSDE-UHFFFAOYSA-N 3,7-bis(dimethylamino)phenothiazin-5-ium Chemical compound C1=CC(N(C)C)=CC2=[S+]C3=CC(N(C)C)=CC=C3N=C21 RBTBFTRPCNLSDE-UHFFFAOYSA-N 0.000 title claims abstract description 75
- 229960000907 methylthioninium chloride Drugs 0.000 title claims abstract description 75
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- G—PHYSICS
- G01—MEASURING; TESTING
- G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
- G01N21/00—Investigating or analysing materials by the use of optical means, i.e. using sub-millimetre waves, infrared, visible or ultraviolet light
- G01N21/17—Systems in which incident light is modified in accordance with the properties of the material investigated
- G01N21/25—Colour; Spectral properties, i.e. comparison of effect of material on the light at two or more different wavelengths or wavelength bands
- G01N21/31—Investigating relative effect of material at wavelengths characteristic of specific elements or molecules, e.g. atomic absorption spectrometry
- G01N21/35—Investigating relative effect of material at wavelengths characteristic of specific elements or molecules, e.g. atomic absorption spectrometry using infrared light
- G01N21/359—Investigating relative effect of material at wavelengths characteristic of specific elements or molecules, e.g. atomic absorption spectrometry using infrared light using near infrared light
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Abstract
本发明涉及一种近红外光谱检测仪，包括一透射平台，透射平台包括一基板，基板上竖直设有两块相互平行的竖板，两块竖板上各开设有相对应的透射窗，透射窗覆盖有透明的光学窗片，两块竖板之间设有一载物台，待测物位于两个光学窗片之间，至少一个竖板可沿基板的表面滑动，使得待测物夹持在两个光学窗片之间。本发明还涉及一种利用近红外光谱检测血浆灭活袋中亚甲蓝释放量的方法，包括：将两个光学窗片之间的光程固定为2.0‑5.0mm；将血浆灭活袋放在载物台上，对多个血浆标准溶液进行近红外光谱检测，利用PLS法建立亚甲蓝释放量的多变量分析模型；对未知样品进行近红外光谱检测，利用分析模型分析亚甲蓝的释放量。
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
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 utilizing near infrared spectrum.
Background
In order to prevent blood transfusion from infecting viruses, the clinical plasma is widely used for inactivating plasma viruses by a methylene blue light irradiation method, which is the only single blood component virus inactivation technology approved to be clinically used in China. The inactivation process is carried out in a plasma inactivation bag, and the release of the inactivating agent methylene blue in the plasma inactivation bag is that the plasma enters the inactivation bag together with the plasma through gradual dissolution when the plasma flows through a methylene blue storage under the aseptic condition, and the process is the release process of the methylene blue, and the release amount is a key parameter for determining the virus inactivation degree. However, the amount released is dependent on a variety of factors including flow rate, ambient temperature, the degree of moisture absorption of the methylene blue, and the material of the methylene blue immobilized in the reservoir, the manufacturing process, etc. If the release amount of the methylene blue in the plasma inactivation bag is too high, the residual methylene blue in the plasma is too high, which is easy to cause accumulated toxicity, and if the release amount is too low, the virus inactivation effect cannot be ensured. The vast data indicate that the viral inactivating agent methylene blue must be guaranteed to be released in a narrow range of amounts, prescribed in our country to 0.9-1.3. Mu. Mol/L. In order to detect the released amount as conveniently, rapidly and sensitively as possible, a number of methods based on sample detection have emerged, including solid phase extraction-spectrophotometry, sensitized fluorescence spectroscopy, HPLC, chemiluminescence, resonance rayleigh scattering spectroscopy. The sampling detection needs to use the extraction device to extract the sample from the plasma inactivation bag for detection, and as the extraction operation will damage the plasma inactivation bag, the method for detecting the damage is extremely easy to cause secondary pollution to the plasma during sampling, including bacteria, chemical pollutants and particulate matters, so that the plasma in the plasma inactivation bag can not be used for a human body, and the waste of the plasma is caused. Also based on this, the plasma with the release amount measured cannot be used in clinic anymore. Thus, for each bag applied to clinical plasma, such quality control of inactivation based on a lossy assay is not possible. Indeed, the quality of the plasma in the plasma inactivation bag is merely a statistic. Nondestructive testing of the release amount of methylene blue in the inactivation bags is a key problem for realizing the inactivation quality control of plasma in each bag.
Currently, near infrared spectroscopy is commonly used for nondestructive rapid detection of samples to be suitable for quality control of production processes. However, this method is generally applicable to solid, powder and paste, but is difficult to be applied to liquid samples, and since only extremely weak diffuse reflection light is generated after light is irradiated into liquid, most of the light is transmitted, quantitative detection of a certain substance in the liquid is difficult to be performed according to the diffuse reflection near infrared spectrum.
For the existing near infrared spectrum detection method for rapidly quantifying substances in liquid, sampling from a detected container is generally required for detection, and mainly two methods are included. The liquid sample is placed in a cuvette and then is placed in a cuvette frame of an instrument for detection, and the liquid fiber probe with a slit is inserted into liquid in a detected container, the detected liquid directly flows into the slit of the fiber probe serving as the cuvette for realizing sampling operation, and the fiber transmits signals again to a spectrometer for realizing rapid detection. Either way, a lossy detection method, the drawbacks of which have been detailed above. The existing infrared spectrometer is used for directly detecting the plasma bag, firstly, the plasma inactivation bag contains more bubbles, and the bubbles can cause great errors of measurement results if being positioned in a detection area, and secondly, when the plasma inactivation bag is directly detected, the bag body is softer, the shape is irregular, the optical path of spectrum detection is difficult to fix, and the content of an object to be detected is difficult to calculate by utilizing a quantitative formula. Of course, the plasma in the inactivation bag contains a large amount of biological macromolecules, and hydrocarbon bonds in the inactivation bag can generate a large amount of near infrared spectrum signals and interfere with detection results, so that a certain difficulty is brought to a detection method.
Disclosure of Invention
In order to solve the technical problems, the invention aims to provide a method for detecting the release amount of the methylene blue in the plasma inactivation bag by utilizing the near infrared spectrum, which overcomes the defect that the prior art is difficult to accurately perform near infrared quantification on irregularly-shaped samples, and realizes nondestructive, rapid and accurate analysis of the release amount of the methylene blue in the plasma inactivation bag.
In one aspect, the invention provides a near infrared spectrum detector, which comprises a light source, a monochromator, a detector, a computer processing information system, a transmission platform and a light source, wherein the transmission platform comprises a base plate, two first vertical plates and two second vertical plates which are parallel to each other are vertically arranged on the base plate, corresponding transmission windows are respectively arranged on the first vertical plates and the second vertical plates, the transmission windows are covered with transparent optical window sheets, the optical window sheets are positioned on opposite side surfaces of the two vertical plates, an objective table is arranged between the first vertical plates and the second vertical plates and used for bearing a soft bag containing an object to be detected, the objective table is positioned below the lower edge of the optical window sheets, the soft bag containing the object to be detected is positioned between the two optical window sheets, and at least one vertical plate can horizontally move along the surface of the base plate, so that the soft bag containing the object to be detected is clamped between the two optical window sheets, and the optical path between the two optical window sheets can be adjusted.
Further, the stage is positioned at least 2cm below the lower edge of the optical window.
Further, the first vertical plate is fixedly connected with the base plate, the objective table is fixedly connected with the first vertical plate, the second vertical plate is provided with a strip-shaped hole which is opposite to the objective table, the strip-shaped hole is matched with the shape of the objective table, and the objective table can be inserted into the strip-shaped hole in a sliding mode.
Further, a guide rail is fixedly connected to the base plate, and the second vertical plate can horizontally slide along the guide rail and can be locked at any position of the guide rail, so that the distance (namely, the optical path) between the two optical window sheets is fixed.
Further, a positioning table is fixedly connected to one surface, far away from the first vertical plate, of the second vertical plate, the second vertical plate is in sliding connection with the guide rail through the positioning table, a screw is in threaded connection with the positioning table, the axial direction of the screw is perpendicular to the second vertical plate, a fixed baffle is fixedly connected to the guide rail, a through hole is formed in the fixed baffle, and the screw penetrates through the through hole.
Further, the optical window is made of quartz or optical glass.
Further, the transmission window is rectangular.
Further, the area of the optical window is larger than the area of the transmission window.
In another aspect, the invention also provides a method for detecting the release amount of methylene blue in a plasma inactivation bag by using near infrared spectrum, which comprises the following steps:
(1) Adjusting the distance between the first riser and the second riser to fix the optical path between the two optical window sheets to be 2.0-5.0mm, preferably 3.5mm;
(2) Placing the plasma inactivation bag on a stage of a near infrared spectrum detector, positioning the plasma inactivation bag between two optical window sheets, slightly pressing the plasma inactivation bag with force above the bag, enabling the plasma inactivation bag to be tightly contacted with the two optical window sheets, forming a regular plane after the plasma inactivation bag is pressed, and pressing the surface of the plasma inactivation bag into the plane, wherein the plasma inactivation bag is filled with a plasma standard solution with known methylene blue content;
(3) Performing near infrared spectrum detection on the plasma standard solution under the condition of wave number 4000-10000cm -1, repeatedly testing a plurality of samples, and establishing a multivariate analysis model of the release amount of the methylene blue by using a PLS method according to near infrared spectrum detection results of the plurality of samples;
(4) Placing a plasma inactivation bag containing methylene blue to be detected on the object stage, slightly pressing the plasma inactivation bag with force above the bag, so that the plasma inactivation bag is tightly contacted with the two optical window sheets, and a regular plane is formed after the plasma inactivation bag is pressed;
(5) Near infrared spectrum detection is carried out under the condition of wave number 4000-10000cm -1, and the release amount of the methylene blue in the plasma inactivation bag is determined by using a multivariate analysis model of the release amount of the methylene blue.
Further, in the step (2), the concentration of methylene blue in the plasma standard solution is 0.312. Mu. Mol/L to 2.959. Mu. Mol/L.
Further, in the step (3), the number of plasma standard solutions is 52.
Further, in step (3) and step (5), the near infrared light detected by near infrared spectrum has a scanning resolution of 8cm -1, a scanning number of 32 times, and a scanning speed of 0.3165.
Further, in step (3) and step (5), the scanning aperture for near infrared spectrum detection is 22, and the gain is 1.
Further, in the step (3) and the step (5), after near infrared spectrum detection, the absorbance average value in the interval 3999.7-4265.8cm -1 is selected as the drift amount, and the detected near infrared spectrum is subjected to drift elimination.
Further, in the step (3) and the step (5), after the near infrared spectrum detection, the absorbance in two intervals of 5350-6600cm -1 and 7200-10001cm -1 is selected as the rich information spectrum. Preferably, after near infrared detection, the absorbance in two intervals of 5827.9-6464.3cm -1 and 7467.1-9009.9cm -1 is selected as the rich information spectrum.
Further, in the step (3), a multivariate analysis model of the release amount of methylene blue is established by a PLS method by selecting a principal component number of 11, randomly selecting 11 samples according to the concentration distribution as a prediction set and the remaining 41 samples as a correction set.
Further, the multivariate analysis model of the release of methylene blue has the formula c=k 1A1+k2A2+···+knAn, wherein c represents the concentration of methylene blue in the sample, a 1、A2···An is the absorbance at wavelength λ 1、λ2···λn corresponding to the information-rich spectral interval selected from the detection spectrum of the sample, and k 1、k2···kn is the corresponding PLS regression coefficient.
Further, before the step (2), the method further comprises the step of placing an air-filled unused plasma inactivation bag on a stage of the near infrared spectrum detector for near infrared spectrum detection to serve as a spectrum background.
By means of the scheme, the invention has at least the following advantages:
The transmission platform of the near infrared spectrum detector can realize fixed optical path during nondestructive testing of the plasma inactivation bag, and can effectively solve the problem that bubbles in the plasma inactivation bag exist in a detection optical path, so that the quantitative result is accurate and reliable. Due to the extrusion effect of the optical window sheets, the plasma inactivation bag between the two optical window sheets is extruded into a regular plane, and meanwhile, due to the low density of bubbles, the bubbles in the plasma inactivation bag between the two optical window sheets are naturally driven into the plasma inactivation bag outside the extrusion part of the optical window sheets under the extrusion force of the optical window sheets, so that the bubbles cannot appear in a detection area.
The method of the invention realizes nondestructive, rapid and accurate analysis of the release amount of the methylene blue in the plasma inactivation bag outside the plasma inactivation bag in a non-sampling mode. The correlation coefficient of the calculated concentration of the model to the actual concentration of each sample is 98.16 percent (correction set) and 98.93 percent (prediction set), and the average recovery rate of the samples in the high-medium-low concentration group is 103.6 percent to 110.8 percent.
The foregoing description is only an overview of the present invention, and is intended to provide a better understanding of the present invention, as it is embodied in the following description, with reference to the preferred embodiments of the present invention and the accompanying drawings.
Drawings
FIG. 1 is a schematic perspective view of a transmissive platform of the present invention;
FIG. 2 is a schematic view of the front side structure of the transmission platform of the present invention;
FIG. 3 is a schematic view of the left side structure of the transmissive platform of the present invention;
FIG. 4 is an enlarged schematic view of the structure of circle A in FIG. 3;
FIG. 5 is a schematic top view of a transmissive platform of the present invention;
FIG. 6 is a near infrared spectrum after baseline wander elimination in example 2;
FIG. 7 is a residual of the PLS model based on leave-one-out interactive verification;
FIG. 8 is a graph of a multivariate analysis model of the release of methylene blue;
reference numerals illustrate:
1-second vertical plate, 2-quartz plate, 3-plasma inactivating bag, 4-transmission window, 5-base plate, 6-first vertical plate, 7-objective table, 8-positioning table, 9-screw rod, 10-guide rail and 11-fixed baffle.
Detailed Description
The following describes in further detail the embodiments of the present invention with reference to the drawings and examples. The following examples are illustrative of the invention and are not intended to limit the scope of the invention.
Example 1
Referring to fig. 1-5, the near infrared spectrum detector of the invention comprises a light source, a monochromator, a detector, a computer processing information system, a transmission platform, a base plate 5, wherein the base plate 5 is movably connected with a first vertical plate 6 and a second vertical plate 1 which are parallel to each other, and the first vertical plate 6 and the second vertical plate 1 are perpendicular to the base plate 5. Corresponding rectangular transmission windows 4 are respectively arranged on the two vertical plates, square transparent quartz plates 2 are arranged on the upper covers of the transmission windows 4, the quartz plates 2 are positioned on the opposite side surfaces of the two vertical plates, and the area of the quartz plates 2 is larger than that of the transmission windows 4. The first vertical plate 6 is fixedly connected with the base plate 5, the first vertical plate 6 is fixedly connected with an objective table 7, the objective table 7 is located at least 2cm below the lower edge of the quartz plate 2, and the objective table 7 is used for placing a soft bag containing objects to be detected. The upper surface of the objective table 7 is parallel to the substrate 5, the second vertical plate 1 is provided with a strip-shaped hole facing the objective table 7, and the strip-shaped hole is matched with the shape of the objective table 7, so that the objective table 7 can be slidably inserted into the strip-shaped hole. Two guide rails 10 are fixedly connected to the base plate 5, the guide rails 10 are perpendicular to the second vertical plate 1, and the second vertical plate 1 can slide horizontally along the guide rails 10. One side of the second vertical plate 1 far away from the first vertical plate 6 is fixedly connected with a positioning table 8, the positioning table 8 is in threaded connection with a screw rod 9, the axial direction of the screw rod 9 is perpendicular to the second vertical plate 1, the positioning table 8 is in sliding connection with a guide rail 10, a fixed baffle 11 is fixedly connected to the guide rail 10, a through hole is formed in the fixed baffle 11, and the screw rod 9 penetrates through the through hole.
In the sample detection, for example, the sample detection in the plasma inactivation bag 3, the screw is firstly adjusted to drive the second vertical plate 1 to move along the guide rail 10 so as to slide to a required distance, the sliding direction is perpendicular to the plane where the vertical plate is located, and then the screw 9 is adjusted to fix the second vertical plate 1 at the position, namely, the optical path of the transmission detection is fixed. And in the sliding process of the second vertical plate 1, the objective table 7 is slidably inserted into the strip-shaped hole arranged on the objective table, so that the optical path is convenient to adjust, and the quartz plates 2 can clamp the plasma inactivation bag 3 under different optical paths. The plasma deactivation bag 3 is placed on the stage 7 and pressed with gentle force over the bag, bringing the plasma deactivation bag into close contact with the two optical louvers, so that the plasma deactivation bag 3 is clamped between the two quartz plates 2. When the quartz plates 2 squeeze the plasma inactivation bags 3, the plasma inactivation bags 3 between the two quartz plates 2 form a regular shape, so that an exact optical path (the distance marked by a mark line '5' in fig. 4 is a detection optical path) is fixed, and bubbles in the plasma inactivation bags 3 migrate to the upper edge of the quartz plates 2 along the plasma bag part above, so that no bubbles exist between the quartz plates 2, and the influence on the detection result is prevented.
Example 2
In the following examples, materials were used including non-inactivated human plasma provided from the red cross center blood station in Suzhou, and white blood cells were filtered off using a filter in a disposable virus inactivation kit to obtain plasma free of methylene blue (i.e., blank plasma). A disposable blood sampling device (transfer bag, 35ml, manufacturing lot number: 170428, sichuan south Grail Biotechnology Co., ltd.) containing a plasma inactivation bag. Methylene blue hydrochloride (analytically pure, >97% based on dry product content, sigma-Aldrich company) and the water for the experiments was triple distilled water. A near infrared spectrometer (model: NEXUS, thermo Co., U.S.A.) whose transmission platform was modified as in example 1.
The present embodiment is a method for establishing a multivariate analysis model of the release amount of methylene blue, which is specifically as follows:
(1) Firstly, preparing methylene blue stock solution, namely weighing 1.3953g (analytically pure, weight loss on drying is 10.6%) of methylene blue, placing the methylene blue stock solution into a 500mL volumetric flask, and fixing the volume by using PBS solution. Precisely measuring 1.00ml of methylene blue solution, and then using PBS solution to fix the volume to 1000ml to obtain 7.8 mu mol/L methylene blue stock solution. And (5) keeping in a sealed manner in a dark place.
(2) The sample to be tested was prepared by first preparing primary and secondary dilutions using a stepwise dilution method using methylene blue stock solution and PBS buffer (ph=7.4). Then, 52 methylene blue plasma solutions with the concentration of 0.312-2.959 mu mol/L are precisely prepared by stock solution, diluent and plasma. And finally, filling methylene blue plasma solution into each blood collection and transfer bag by using a disposable sterilization syringe to obtain a sample to be tested.
(3) Near infrared spectrum detection of samples
The outer surface of the plasma inactivation bag to be tested and the quartz plate on the transmission platform were wiped with alcohol and the distance between the first riser 6 and the second riser 1 was adjusted as in example 1 so that the optical path between the two optical louvers was fixed at 3.5mm. Then, the plasma inactivation bag was placed on the transmission platform as in example 1, and the positions of each plasma inactivation bag were aligned as far as possible on the left and right sides, and the spectrum of the sample was measured. The instrument parameters are 4000-10000cm -1 wave number, 3.5mm optical path, 8cm -1 resolution, 32 scan times, 0.3165 scan speed, aperture 22 and gain 1. Prior to testing the samples on the same day, an air-filled, unused plasma inactivation bag was used as a spectroscopic background.
(4) And (3) carrying out baseline drift calibration on the near infrared transmission spectrum of the sample in the plasma bag obtained by detection by taking the spectrum in the interval 3999.7-4265.8cm -1 as a standard. The method comprises the steps of selecting 5827.9-6464.3cm -1 and 7467.1-9009.9cm -1 as rich information intervals, taking a spectrum centered by a mean value as a model variable, selecting a principal component number of 11, randomly selecting 11 samples according to concentration distribution as a prediction set, taking the rest 41 samples as a correction set, and establishing a multivariate analysis model of methylene blue release by a PLS method.
For the low wavenumber region of the near infrared spectrum, a spectral baseline will typically occur. It should be horizontal, the spectra of samples of different concentrations should overlap completely in this interval, there is no statistical difference, and the absorbance mean is 0. In FIG. 6, the spectrum baseline of each sample is shown as the 3999.7-4265.8cm -1 interval. However, in the original spectrum, the baseline of samples of different concentrations drifts by about 7, which concentration-independent drift would have an effect on the accuracy of the methylene blue release. And taking the absorbance average value of the detection spectrum of each sample in the interval 3999.7-4265.8cm -1 as the drift amount, and carrying out drift elimination on the absorbance average value to obtain the spectrum of all samples after the drift elimination.
From the spectrum after baseline drift elimination (fig. 6), it was found that the plasma sample of methylene blue showed distinct absorption peaks in both the 5350-6600cm -1 and 7200-10001cm -1 regions throughout the spectral region, which were the absorption peak spectral regions. For the former, the spectrum is composed of high and low shoulders, in the low wave number region of 5350-5828cm -1, the absorbance is low, the horizontal line is basically without peaks, wave-shaped burrs appear, and the spectrum signals with low signal to noise ratio influence the precision of a quantitative analysis model. The spectrum of the high wave number range 6464-6600cm -1 is the end spectrum of the absorption peak, the absorption degree is lower, the signal is weaker, and the signal-to-noise ratio is also low. Thus, the spectrum of 5827.9-6464.3cm -1 of the absorption peak spectral region was chosen to be the rich signal spectrum. Similarly, in the spectral region of the absorption peak of 7200-10001cm -1, the spectrum of 7467.1-9009.9cm -1 is selected as the rich signal spectrum.
The PLS model predicted the residual of methylene blue concentration at different principal component numbers was calculated by cross-checking of the correction set samples by leave-one-out with the 5827.9-6464.3cm -1 and 7467.1-9009.9cm -1 spectra as rich information spectra, with the averaged centered spectra as model variables (FIG. 7). We can find that as the number of principal components increases, the predicted concentration residuals have a decreasing trend, indicating that the accuracy of the model increases gradually. When the principal component number is more than or equal to 11, the degree of prediction residual error reduction obviously becomes smaller, which indicates that the contribution rate of the continued increase of the principal component number to model accuracy is lower. Of course, too large a principal component number will also result in an overfitting of the model, and therefore 11 is chosen as the principal component number.
The rich information spectrums of the two selected intervals are taken as identification variables of methylene blue release, based on the determined principal component numbers, spectrum average centering is carried out, a multivariate analysis model of release is established by utilizing a PLS method, wherein 11 samples are randomly selected as a prediction set according to the high-low distribution of methylene blue concentration, the rest 41 samples are prediction sets, and as a result, see fig. 8, the square in the figure represents a correction set sample, and the ∈ represents a prediction set sample. It can be found that in the multivariate analysis model, the corrected concentration of the corrected sample and the true concentration can be better correlated, and they are distributed around the true concentration (diagonal line) throughout the detection concentration interval, with a correlation coefficient Rcorr = 98.16%. By using the established model to predict the release amount of methylene blue in the independent prediction set samples (i.e. sample detection), it is not difficult to find that the predicted concentrations of the samples still show a good correspondence, and they can also be distributed near the true concentration, and the correlation coefficient Rpred is as high as 98.93%. Therefore, the model established by the invention has good accuracy.
To illustrate the accuracy of the model, samples of the high, medium and low concentration groups were designed and the recovery rate test was performed, and the results are shown in table 1. As can be seen from Table 1, each concentration group has good recovery rate, the recovery rate is 103.6% -110.8%, the concentration group has no correlation, and the method has randomness, thus indicating that the established nondestructive testing method is accurate and reliable. Of course, the minimum recovery is as low as 96% and the maximum is as high as 119%, which is entirely acceptable accuracy for a non-destructive, rapid detection method, particularly for biological samples.
TABLE 1 accuracy of near infrared Spectroscopy for non-invasive detection of the amount of methylene blue released in plasma
The results show that the invention establishes a nondestructive testing model and a nondestructive testing method for the release amount of the plasma virus inactivating agent methylene blue in the blood collection and transmission transfer bag by utilizing the good penetrability of near infrared light and adopting near infrared transmission spectrum. And (3) taking near infrared transmission spectrum of a sample in the plasma bag obtained by nondestructive testing outside the plasma bag as an identification variable, carrying out baseline drift calibration by taking a spectrum in a 3999.7-4265.8cm -1 interval as a standard, selecting the spectrums in the 5827.9-6464.3cm -1 and 7467.1-9009.9cm -1 intervals as rich information spectrums, taking a spectrum centered by a mean value as a model variable, selecting a main component of 11, randomly selecting 11 samples as a prediction set, taking the rest 41 samples as a correction set, and establishing a multivariate analysis model of methylene blue release by a PLS method. The formula of the model is as follows:
c=k 1A1+k2A2+···+knAn, where c represents the concentration of methylene blue in the sample, a 1、A2···An is the absorbance at wavelength λ 1、λ2···λn corresponding to the rich information spectral interval selected from the detection spectrum of the sample, and k 1、k2···kn is the corresponding PLS regression coefficient.
The correlation coefficient of the calculated concentration to each sample concentration is 98.16 percent (correction set) and 98.93 percent (prediction set), and the average recovery rate of the samples in the high-medium-low concentration group is 103.6 to 110.8 percent. Therefore, the near infrared transmission spectrometry established by the invention can realize nondestructive, rapid and accurate analysis of the release amount of the methylene blue in the sampling transfer bag outside the plasma bag. The plasma virus inactivation quality in the collection and transfer bag can be monitored under the condition of no secondary pollution.
Example 3
The embodiment provides a method for detecting the release amount of the methylene blue in the plasma by adopting the established multivariate analysis model of the release amount of the methylene blue, which comprises the following steps:
the outer surface of the plasma inactivation bag to be tested and the quartz plate on the transmission platform were wiped with alcohol and the distance between the first riser 6 and the second riser 1 was adjusted in the same way as in example 1 so that the optical path between the two optical louvers was fixed at 3.5mm. And then placing a plasma inactivation bag with unknown methylene blue content (namely the plasma inactivation bag with the methylene blue released) on the object stage, aligning the left side and the right side of each placing position of the plasma inactivation bag as far as possible, and measuring the spectrum of the sample. The instrument parameters are 4000-10000cm -1 wave number, 3.5mm optical path, 8cm -1 resolution, 32 scan times, 0.3165 scan speed, aperture 22 and gain 1. Prior to testing the samples on the same day, an air-filled, unused plasma inactivation bag was used as a spectroscopic background. The measured near infrared transmission spectrum is then calibrated for baseline drift with the spectrum in the 3999.7-4265.8cm -1 interval as standard. 5827.9-6464.3cm -1 and 7467.1-9009.9cm -1 are selected as rich information intervals, absorbance at each wavelength of the intervals is substituted into a formula of a model established in example 2, and the methylene blue content in the plasma inactivation bag is calculated.
The above description is only of the preferred embodiments of the present invention and is not intended to limit the present invention, and it should be noted that it is possible for those skilled in the art to make several improvements and modifications without departing from the technical principle of the present invention, and these improvements and modifications should also be regarded as the protection scope of the present invention.
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| WO1998038961A1 (en) * | 1997-03-03 | 1998-09-11 | Cme Telemetrix Inc. | Method and apparatus for screening plasma for interferents in plasma from donor blood bags |
| US20050037505A1 (en) * | 2000-05-11 | 2005-02-17 | James Samsoondar | Spectroscopic method and apparatus for analyte measurement |
| CN1327812C (en) * | 2002-03-25 | 2007-07-25 | Tyt技研株式会社 | Noninvasive blood component value measuring instrument and method |
| US7842247B2 (en) * | 2005-08-19 | 2010-11-30 | Canadian Blood Services | Sample holder for dynamic light scattering |
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| US4522494A (en) * | 1982-07-07 | 1985-06-11 | The United States Of America As Represented By The Department Of Health And Human Services | Non-invasive optical assessment of platelet viability |
| US5510621A (en) * | 1994-10-03 | 1996-04-23 | Optical Solutions, Inc. | Apparatus and method for measuring components in a bag |
| CN104062257A (en) * | 2013-04-15 | 2014-09-24 | 山东东阿阿胶股份有限公司 | Method for determining total flavone content of solution based on near infrared spectroscopy |
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