Method of analyzing liquid samples using attenuated total reflection far-ultraviolet spectroscopy

Abstract

To provide a technique that enables simple and high-sensitivity analysis of hemoglobin that are present outside erythrocytes in a liquid sample without requiring removal of erythrocytes.SOLUTION: Researchers of the present invention have discovered that use of attenuated total reflection spectroscopy using far-ultraviolet light enables simple and high-sensitivity analysis of hemoglobin that are present outside erythrocytes in a liquid sample without requiring removal of erythrocytes.SELECTED DRAWING: None

Description

[Technical field]

[0001] The present disclosure relates to a method for analyzing a liquid sample using attenuated total reflection far-ultraviolet spectroscopy, and a method for quality control of a liquid sample using attenuated total reflection far-ultraviolet spectroscopy. [Background technology]

[0002] Hemolysis is the rupture (dissolution) of red blood cells. Hemolysis can occur both in vivo and in vitro. Known causes of in vivo hemolysis include bacterial infection, ingestion of certain antibiotics and antifungals, autoimmune diseases such as autoimmune hemolytic anemia, and genetic diseases such as sickle cell disease and G6PD deficiency. Causes of in vitro hemolysis include osmotic shock, mechanical stress such as pressure and centrifugal force, freeze-thawing, and microbial contamination.

[0003] When hemolysis occurs, the contents of red blood cells, such as hemoglobin, are released into the surrounding fluid (e.g., plasma). Therefore, the amount of hemoglobin present outside of red blood cells is an indicator of the degree of hemolysis. Free hemoglobin (hemoglobin in plasma that is not bound to haptoglobin) is toxic, so blood products that have been found to be hemolyzed cannot be used by patients.

[0004] Known methods for measuring hemoglobin with high sensitivity include colorimetry, adsorption quantification, electrophoresis, etc. These methods cannot distinguish between hemoglobin present outside red blood cells and hemoglobin encapsulated in red blood cell membranes, etc., or components other than hemoglobin, such as cell membranes, affect the measurement accuracy. For this reason, when analyzing hemoglobin present outside red blood cells using the above methods, it is common to remove red blood cells by centrifugation or the like before analysis. However, as mentioned above, mechanical stress such as centrifugal force can cause hemolysis, so the possibility that the operation of removing red blood cells before analysis affects the results of the hemoglobin analysis cannot be excluded. Furthermore, the above methods have a problem in terms of the complexity of the operation. [Prior art documents] [Patent documents]

[0005] [Patent Document 1] Special Publication No. 2018-533012 [Patent Document 2] Special Publication No. 2019-518941 Summary of the Invention [Problem to be solved by the invention]

[0006] For the above reasons, techniques capable of analyzing hemoglobin present outside red blood cells without removing red blood cells have been studied. For example, in Patent Document 1, an attempt is made to separate the plasma phase from a whole blood sample by capillary action using a porous mirror with small holes, and to measure / detect hemoglobin by irradiating it with visible light. However, the porous mirror used in the disclosure of Patent Document 1 has a complex structure, which causes problems such as cumbersome washing and drying operations. Furthermore, Patent Document 1 describes that hemoglobin at about 4 mg / dL was detectable, but does not consider detection of hemoglobin at a lower concentration.

[0007] In addition, in Patent Document 2, an attempt is made to measure hemoglobin by measuring the absorption spectrum in the visible light region of a very thin acellular layer, mainly composed of plasma, which is generated at the edge of a channel by flowing blood in the channel using a flow cell. However, shear stress (i.e., mechanical stress) occurs when blood flows through a narrow channel, and as described above, mechanical stress can cause hemolysis, so the possibility that this affects the hemoglobin analysis results cannot be excluded. Furthermore, Patent Document 2 describes that 50 mg / dL of hemoglobin was detectable in a sample with hemolysis, but does not consider the detection of hemoglobin at a lower concentration.

[0008] In view of the above circumstances, the inventors aimed to provide a technique capable of analyzing hemoglobin present outside red blood cells in a liquid sample simply and with high sensitivity, without the need to remove red blood cells. [Means for solving the problem]

[0009] The present inventors have found that attenuated total reflection spectroscopy using far-ultraviolet light can easily and sensitively analyze hemoglobin present outside red blood cells in a liquid sample without the need to remove red blood cells, and have made further improvements to the method, which has led to the completion of the present disclosure.

[0010] The present disclosure includes, for example, the subject matter described in the following sections: Section 1. A method for analyzing a liquid sample, comprising the step of analyzing extra-red blood cell hemoglobin in the liquid sample by attenuated total reflectance far-ultraviolet spectroscopy. Section 2. Item 1. The analytical method according to item 1, comprising a step of detecting and / or measuring extra-erythrocyte hemoglobin in a liquid sample by attenuated total reflection far-ultraviolet spectroscopy. Section 3. Item 3. The analytical method according to item 1 or 2, wherein the liquid sample contains a blood-derived component. Section 4. Item 3. The analytical method according to item 1 or 2, wherein the liquid sample contains red blood cells. Section 5. Item 3. The analytical method according to item 1 or 2, wherein the liquid sample is a blood product. Section 6. Item 3. The analytical method according to item 1 or 2, wherein the liquid sample is a whole blood sample. Section 7. A method for quality control of a liquid sample comprising a step of analyzing extra-erythrocyte hemoglobin in a liquid sample containing red blood cells by attenuated total reflection far-ultraviolet spectroscopy. Section 8. Item 8. The quality control method according to item 7, comprising a step of detecting and / or measuring extra-erythrocyte hemoglobin in a liquid sample containing red blood cells by attenuated total reflection far-ultraviolet spectroscopy. Section 9. Item 9. The quality control method according to Item 7 or 8, wherein the red blood cell-containing liquid sample is a blood product. Section 10. Item 9. The quality control method according to Item 7 or 8, wherein the red blood cell-containing liquid sample is a whole blood sample. Effect of the Invention

[0011] According to the present disclosure, there is provided a technique capable of analyzing hemoglobin present outside red blood cells in a liquid sample simply and with high sensitivity, without the need to remove red blood cells. [Brief description of the drawings]

[0012] [Figure 1] The results of Test 2-1 are shown below. 40 μL of the Hb standard solutions (0.1 mg / dL, 1 mg / dL, 10 mg / dL, and 50 mg / dL) prepared in Test Example 1-2, and PBS as a control, were applied to the measurement section of the ATR-FUV far-ultraviolet spectrometer, and the ATR-FUV spectrum was measured. The blank spectrum was subtracted from the measured spectrum. The absorption near 191 nm is attributed to the π-π* transition of the amide bond in hemoglobin. The absorption near 155 nm is attributed to water molecules. [Diagram 2] The results of Test 2-2 are shown below. 40 μL of the Hb leakage model sample prepared in Test Example 1-3 was applied to the measurement section of the ATR-FUV far-ultraviolet spectrometer, and the ATR-FUV spectrum was measured. The blank spectrum was subtracted from the measured spectrum. DETAILED DESCRIPTION OF THE PREFERRED EMBODIMENTS

[0013] Each embodiment included in the present disclosure will be described in more detail below. The present disclosure preferably includes a method for analyzing a liquid sample including a step of analyzing hemoglobin present outside of red blood cells, a method for quality control of a liquid sample containing red blood cells including a step of analyzing hemoglobin present outside of red blood cells, etc., but is not limited thereto, and the present disclosure includes all that is disclosed in the present specification and that can be recognized by a person skilled in the art.

[0014] In the present disclosure, the term "extracellular hemoglobin" refers to hemoglobin that is not encapsulated in red blood cells. Therefore, the extracellular hemoglobin in the present disclosure includes hemoglobin that is released from red blood cells by hemolysis.

[0015] In the technology of the present disclosure, the liquid sample to be analyzed may contain extra-erythrocytic hemoglobin, but does not necessarily have to actually contain extra-erythrocytic hemoglobin. The liquid sample may or may not contain red blood cells. The liquid sample preferably contains blood-derived components, and more preferably contains red blood cells. The liquid sample is preferably a blood product and / or a whole blood sample.

[0016] An embodiment of the liquid sample of the present disclosure that may contain extra-erythrocytic hemoglobin but does not contain red blood cells includes, for example, a blood product in which all contained red blood cells are hemolyzed. Another example includes a blood product from which red blood cells have been removed. If hemolysis occurs before the removal of red blood cells, the blood product contains hemoglobin released outside the red blood cells by hemolysis (i.e., extra-erythrocytic hemoglobin) but does not contain red blood cells.

[0017] In the present disclosure, the term "analysis" includes detecting a target substance and measuring a target substance. Thus, in the present disclosure, "analyzing extra-erythrocytic hemoglobin" includes both detecting the presence or absence of extra-erythrocytic hemoglobin in a liquid sample and measuring the concentration of extra-erythrocytic hemoglobin in the liquid sample.

[0018] When detecting whether extra-erythrocytic hemoglobin is present in a liquid sample using the technology of the present disclosure, for example, the absence of absorption at wavelengths assigned to hemoglobin may be used as an indicator, and the absorption spectrum of a control that is clearly free of extra-erythrocytic hemoglobin may be compared with that of the liquid sample to be analyzed. In addition, when measuring the concentration of extra-erythrocytic hemoglobin in a liquid sample using the technology of the present disclosure, a calibration curve may be created from the absorption spectra of multiple hemoglobin solutions with known concentrations, and the concentration of extra-erythrocytic hemoglobin in the liquid sample to be analyzed may be calculated.

[0019] The concentration of extra-erythrocyte hemoglobin in the liquid sample of the present disclosure is not particularly limited, and may be, for example, 0 to 100 mg / dL, preferably 0 to 50 mg / dL, and more preferably 0 to 10 mg / dL. The upper or lower limit of the range may be 0.01, 0.02, 0.03, 0.04, 0.05, 0.06, 0.07, 0.08, 0.09, 0.1, 0.15, 0.2, 0.25, 0.3, 0.35, 0.4, 0.45, 0.5, 0.55, 0.6, 0.65, 0.7, 0.75, 0.8, 0.85, 0.9, 0.95, 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 20, 30, 40, 50, 60, 70, 80, or 90 mg / dL. An extra-erythrocytic hemoglobin concentration of 0 mg / dL means that no extra-erythrocytic hemoglobin is detectable.

[0020] In the attenuated total reflection method (ATR), an internal reflection element (IRE) is placed in contact with a measurement sample, and light is incident on the IRE at an angle equal to or greater than the critical angle. The light that passes through the IRE is totally reflected at the interface with the measurement sample. During total reflection, a small amount of light seeps out from the IRE side to the measurement sample side (evanescent wave). The ATR method uses the evanescent wave that occurs during total reflection to obtain the absorption spectrum of molecules present near the interface (in other words, near the surface of the measurement sample).

[0021] In the ATR method, the penetration depth of light into the measurement sample depends on the wavelength of the incident light, the angle of incidence, the refractive index of the IRE, and the refractive index of the measurement sample. The relationship between the penetration depth of light and the wavelength of the incident light, the angle of incidence, the refractive index of the IRE, and the refractive index of the measurement sample is shown below.

[0022]

number

[0023] As is clear from the above formula, the shorter the wavelength of the incident light, the shallower the light seepage depth, and the longer the wavelength, the deeper it becomes. Infrared light (IR) is often used as the incident light in the ATR method. The ATR method using IR is generally called ATR-IR spectroscopy. The seepage depth in ATR-IR spectroscopy is about 1 to 10 μm. Therefore, in ATR-IR spectroscopy, the absorption spectrum of molecules present in a range of about 1 to 10 μm from the surface of the measurement sample is detected.

[0024] Patent Document 2 also discloses that light of about 420 nm is used as the incident light. The penetration depth of light under the conditions disclosed in Patent Document 2 is about 280 nm.

[0025] When detecting hemoglobin in a liquid sample by the conventional ATR method using light of the above wavelengths, it is not possible to distinguish between hemoglobin present inside red blood cells and hemoglobin present outside red blood cells. In other words, since the seepage depth is longer than the thickness of the cell membrane containing the glycosylated portion of the red blood cells (length of the glycan + thickness of the lipid bilayer, about 55 nm), the evanescent wave reaches the inside of the red blood cells, and as a result, the absorption spectrum obtained by the ATR method reflects both the absorption by hemoglobin present inside red blood cells and the absorption by hemoglobin present outside red blood cells.

[0026] For the above reasons, when analyzing hemoglobin present outside red blood cells using the conventional ATR method, in order to eliminate the effect of hemoglobin present inside red blood cells on the absorption spectrum, it is necessary to either remove red blood cells from the sample beforehand, or to use a porous mirror as disclosed in Patent Document 1 or a flow cell as disclosed in Patent Document 2.

[0027] In the technology of the present disclosure, far-ultraviolet light is used as the incident light in the ATR method. In the present disclosure, far-ultraviolet light (FUV) refers to electromagnetic waves with a wavelength of 100 to 300 nm. The ATR method using the far-ultraviolet light is sometimes called attenuated total reflection far-ultraviolet spectroscopy (ATR-FUV spectroscopy).

[0028] The wavelength of the far ultraviolet light used in the technology of the present disclosure is not particularly limited as long as the desired effect can be obtained. For example, it may be 100 to 300 nm, preferably 120 to 280 nm, and more preferably 140 to 260 nm. The upper or lower limit of the range may be 110, 120, 130, 140, 150, 160, 170, 180, 190, 200, 210, 220, 230, 240, 250, 260, 270, 280, or 290 nm.

[0029] Although not particularly limited, since hemoglobin has an absorption near 191 nm, it is preferable that the far-ultraviolet light used in the technology of the present disclosure includes a wavelength of 180 to 200 nm. The absorption near 191 nm is attributed to the π-π* transition of an amide bond in a protein. Furthermore, the cell membrane constituting red blood cells contains lipids, which have an absorption near 160 to 165 nm. Therefore, when the far-ultraviolet light used in the technology of the present disclosure includes a wavelength near 150 to 170 nm, red blood cells can be analyzed together with hemoglobin present outside red blood cells in a sample. That is, from the viewpoint of being able to analyze red blood cells together with hemoglobin present outside red blood cells in a sample, it is particularly preferable that the far-ultraviolet light used in the technology of the present disclosure includes wavelengths of 150 to 170 nm and 180 to 200 nm. Furthermore, it is preferable that the far-ultraviolet light used in the technology of the present disclosure includes a wavelength near 155 nm, where water molecules have absorption.

[0030] The material of the internal reflection element (IRE) used in the technology of the present disclosure is not particularly limited, but from the viewpoint of reducing the critical angle determined by the following formula, a material with a large refractive index is preferable.

[0031]

number

[0032] Examples of materials for the IRE include ZnSe, diamond, Ge, Si, and sapphire glass (aluminum oxide), with sapphire glass being particularly preferred.

[0033] In the technology of the present disclosure, the incidence angle of the far ultraviolet light is not particularly limited as long as it is equal to or greater than the critical angle. As described above, the light seepage depth is determined by the wavelength of the incident light, the incidence angle, the refractive index of the IRE, and the refractive index of the measurement sample. Therefore, the incidence angle can be appropriately adjusted according to the material of the IRE and the measurement sample so that the light seepage depth becomes a desired value. For example, when the material of the IRE is sapphire glass, the incidence angle of the far ultraviolet light may be 35 to 75°, preferably 50 to 75°, more preferably 65 to 75°, and particularly preferably 70°. The upper or lower limit of the range may be 35, 36, 37, 38, 39, 40, 41, 42, 43, 44, 45, 46, 47, 48, 49, 50, 51, 52, 53, 54, 55, 56, 57, 58, 59, 60, 61, 62, 63, 64, 65, 66, 67, 68, 69, 70, 71, 72, 73, 74, or 75 degrees.

[0034] In the technology of the present disclosure, the light seepage depth is 80 nm or less. From the viewpoint of selectively analyzing hemoglobin present outside red blood cells, the light seepage depth is preferably 70 nm or less, more preferably 60 nm or less. The lower limit is not particularly limited, and may be, for example, 10 nm or more, or 20 nm or more. Therefore, the light seepage depth in the technology of the present disclosure may be, for example, 10 to 80 nm, preferably 20 to 70 nm, and more preferably 20 to 60 nm. The upper or lower limit of the range may be 10, 15, 20, 25, 30, 35, 40, 45, 50, 55, 60, 65, 70, or 75 nm.

[0035] As described above, the thickness of the cell membrane containing the glycosylated portion of the red blood cell (the length of the sugar chain + the thickness of the lipid bilayer) is about 55 nm. Therefore, according to the technique of the present disclosure using ATR-FUV spectroscopy, the evanescent wave does not reach the inside of the red blood cell, or if it does reach the inside of the red blood cell, it reaches only a very small amount. In addition, the evanescent wave has a characteristic that the intensity decreases the farther it is from the interface. Therefore, even if the evanescent wave reaches the inside of the red blood cell, the effect on the absorption spectrum of the hemoglobin present in the red blood cell is very small. Although not particularly restricted, it is presumed that the technique of the present disclosure can easily and sensitively analyze the hemoglobin present outside the red blood cell in the liquid sample by the above mechanism without requiring a process of removing the red blood cells or a porous mirror or flow cell used in Patent Documents 1 and 2.

[0036] Due to the above-mentioned features, the technique disclosed herein can be suitably used to detect the presence or absence of hemolysis using the presence of extra-erythrocyte hemoglobin as an indicator. When the liquid sample to be analyzed is blood, the technique can be an indicator for diagnosing whether or not an abnormality causing hemolysis has occurred in a subject from which the blood is derived.

[0037] As described above, extra-erythrocytic hemoglobin is toxic. Therefore, when the liquid sample to be analyzed by the technique of the present disclosure is a blood product, it can be an indicator for determining whether the blood product is suitable for transfusion. In other words, when extra-erythrocytic hemoglobin is detected by the technique of the present disclosure, the blood product can be determined to be unsuitable for transfusion.

[0038] Furthermore, as described above, the technology disclosed herein can detect extra-erythrocyte hemoglobin with high sensitivity, and is therefore expected to contribute to advanced quality control of blood products and, ultimately, to extending the usable life of blood products.

[0039] In addition, in this specification, the term "comprising" includes "essentially consisting of" and "consisting of" in addition to "containing." In addition, the present disclosure includes all arbitrary combinations of the constituent elements described in this specification.

[0040] In addition, the various characteristics (properties, values, functions, etc.) described in each embodiment of the present disclosure above may be combined in any way to specify the subject matter included in the present disclosure. In other words, the present disclosure includes all subject matter consisting of all combinations of the combinable characteristics described in this specification. EXAMPLES

[0041] Hereinafter, the embodiments of the present disclosure will be described more specifically with reference to examples, but the embodiments of the present disclosure are not limited to the following examples.

[0042] 1. Sample Preparation 1-1. Red blood cell (RBC) purification After collecting blood from the rat heart, 0.5 M EDTA-phosphate buffered saline (PBS) was mixed to a final concentration of 0.1 M to prevent coagulation. The blood was then centrifuged at 15,000 rpm, 10 min, and 4° C. to remove the supernatant (plasma components) and buffy coat (white blood cells and platelets). The remainder (red blood cells, sometimes referred to as red blood cells: RBC in this disclosure) was stored at 4° C.

[0043] 1-2. Preparation of hemoglobin (Hb) standard solution 400 μL of the RBC purified in 1-1 was taken, and 400 μL of ultrapure water was added and pipetted to perform hemolysis. Then, the mixture was centrifuged at 15,000 rpm, 10 min, and 4° C. to collect the supernatant. This supernatant is sometimes called hemoglobin (Hb) stock solution.

[0044] The Hb stock solution was diluted with PBS, and the absorption spectrum from 400 to 600 nm was measured with a UV-visible spectrophotometer using a quartz glass cell with an optical path length of 1 cm.

[0045] The concentration (mg / dL) of the Hb stock solution was determined from the peak intensity at 414 nm at which oxygenated Hb has absorption, the molar absorption coefficient, and the molecular weight of Hb, and a dilution series of the Hb stock solution (Hb standard solutions) was prepared.

[0046] 1-3. Preparation of Hb leakage model sample 20μL of RBC purified in 1-1 and the Hb standard solution prepared in 1-2 were mixed to prepare Hb leakage model samples with Hb concentrations of 0.1mg / dL, 10mg / dL, and 50mg / dL. In addition, 20μL of PBS was mixed with 20μL of RBC instead of the Hb standard solution as a control. Mixing was performed as gently as possible to avoid hemolysis.

[0047] 2. Measurement of ATR-FUV spectrum The ATR-FUV spectrum was measured using an ATR-FUV far-ultraviolet spectrometer manufactured by Bunkoukeiki Co., Ltd. In this test example, unless otherwise specified, the measurement was performed using an IRE made of sapphire glass and with an incident angle of 70°. In addition, the ATR-FUV spectrum measured with the measurement unit empty is referred to as a blank in this test example.

[0048] 2-1.Spectral measurement of Hb standard solution 40μL of the Hb standard solutions (0.1mg / dL, 1mg / dL, 10mg / dL, and 50mg / dL) prepared in 1-2 and PBS as a control were applied to the measurement section of the ATR-FUV far-ultraviolet spectrometer, and the ATR-FUV spectrum was measured. The results obtained by subtracting the blank spectrum from the measured spectrum are shown in Figure 1.

[0049] 2-2.Spectral measurement of Hb leakage model sample 40μL of the sample prepared in 1-3 was applied to the measurement section of the ATR-FUV far-ultraviolet spectrometer, and the ATR-FUV spectrum was measured. Figure 2 shows the results of subtracting the blank spectrum from the measured spectrum.

[0050] 3. Discussion 1 and 2, the 191 nm band assigned to the π-π* transition of the amide bond in hemoglobin showed a tendency for the band intensity to increase as the hemoglobin concentration in the sample increased. This result suggests that the technique disclosed herein can measure the concentration of extra-erythrocyte hemoglobin in a liquid sample without the need to remove red blood cells.

[0051] It was also suggested that the technique disclosed herein makes it possible to detect extra-erythrocyte hemoglobin at extremely low concentrations of about 0.1 mg / dL in a liquid sample with high sensitivity, without the need to remove red blood cells.

Claims

1. A method for analyzing a liquid sample, comprising the step of analyzing extra-red blood cell hemoglobin in the liquid sample by attenuated total reflectance far-ultraviolet spectroscopy.

2. 2. The method of claim 1, comprising detecting and / or measuring extra-red blood cell hemoglobin in a liquid sample by attenuated total reflectance far-ultraviolet spectroscopy.

3. The analytical method according to claim 1 or 2, wherein the liquid sample contains a component derived from blood.

4. The analytical method according to claim 1 or 2, wherein the liquid sample contains red blood cells.

5. The method according to claim 1 or 2, wherein the liquid sample is a blood product.

6. The analytical method according to claim 1 or 2, wherein the liquid sample is a whole blood sample.

7. A method for quality control of a liquid sample comprising a step of analyzing extra-erythrocyte hemoglobin in a liquid sample containing red blood cells by attenuated total reflection far-ultraviolet spectroscopy.

8. 8. A quality control method according to claim 7, comprising detecting and / or measuring extra-erythrocyte hemoglobin in a red blood cell-containing liquid sample by attenuated total reflectance far-ultraviolet spectroscopy.

9. 9. The quality control method according to claim 7 or 8, wherein the red blood cell-containing liquid sample is a blood product.

10. 9. The quality control method according to claim 7 or 8, wherein the red blood cell-containing liquid sample is a whole blood sample.

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