JP2007240500A - Liquid chromatograph - Google Patents

Abstract

<P>PROBLEM TO BE SOLVED: To suppress properly an influence of dissolved oxygen in a sample, exerted on an analysis result. <P>SOLUTION: In this liquid chromatograph equipped with a column for holding a filling material, and a sample preparation means for preparing the sample to be introduced into the column, the sample preparation means is constituted so that the sample for preparation is collected from an upper layer part of a layer including many corpuscles in the sample including the corpuscles. Preferably, the sample preparation means is constituted so that the sample for preparation is collected from the upper layer part of a corpuscle layer 13B when a blood sample 13 including erythrocyte is separated into the corpuscle layer 13B having rich erythrocyte and a plasma layer 13A having poor erythrocyte, and that the sample for introduction to be introduced into the column is prepared by using the sample for preparation. <P>COPYRIGHT: (C)2007,JPO&INPIT

Description

  The present invention relates to a liquid chromatography apparatus capable of reducing the influence of dissolved oxygen in a sample introduced into a column.

  When a biological component is separated and analyzed using a biological sample such as blood, a high performance liquid chromatography device (HPLC device) using high performance liquid chromatography (HPLC) is widely used (see, for example, Patent Documents 1 and 2). . In a general HPLC apparatus, as shown in FIG. 8, a sample containing a biological component is introduced into an analysis column 91 via an injection valve 90, and the biological component is adsorbed on the filler of the analytical column 91. On the other hand, the biological component adsorbed on the filler is desorbed by the eluent sent from the eluent bottle 92 to the analysis column 91 by the power of the liquid feed pump 93. The absorbance of the desorption solution from the analysis column 91 is continuously measured by the photometric mechanism 94, whereby the biological component is separated and analyzed.

  On the other hand, as a sample introduced into the analysis column 91, for example, when measuring glycohemoglobin, a sample prepared from red blood cells in whole blood is used. The sample is prepared by collecting blood cell components from whole blood, lysing red blood cells in a dilution tank 95, and then diluting the hemolyzed blood with distilled water or a diluent.

JP 2001-133445 A JP 2000-275229 A

  However, the sample is supplied from the dilution tank 95 to the injection valve 90 immediately after the completion of the preparation of the sample in the dilution tank 95 without taking any time. Therefore, since the dissolved oxygen concentration in the sample is low, the measured glycohemoglobin concentration may deviate from the true value. This is because when measuring the glycated hemoglobin concentration in the blood sample, glycated hemoglobin is grasped as the proportion of glycated hemoglobin in the total amount of hemoglobin, while the oxygen saturation (dissolved oxygen concentration) in the sample is This is because it may vary when stored blood is stored for a long time or separated into plasma and blood cells by centrifugation. That is, when blood is stored for a long period of time, dissolved oxygen is consumed over time, and the oxygen saturation (dissolved oxygen concentration) decreases. On the other hand, in the centrifuged blood, the lower layer portion in the blood cell layer has lower oxygen saturation (dissolved oxygen concentration) than the upper layer portion, and the oxygen saturation (dissolved oxygen concentration) in the blood cell layer depends on the position of the blood cell layer (depth). It will be different. Therefore, when the sample is prepared from blood cells collected from the upper layer in the blood cell layer, and when the sample is prepared from blood cells collected from the lower layer, the oxygen saturation (dissolved oxygen concentration) in the sample is different. .

  Thus, when oxygen saturation (dissolved oxygen concentration) differs, the ratio of oxyhemoglobin and deoxyhemoglobin in hemoglobin varies. On the other hand, since the blood sample is diluted and introduced into the analysis column 91 with a relatively large amount of oxygen, the photometric mechanism 94 uses 415 nm, which is the maximum absorption wavelength of oxyhemoglobin, as the measurement wavelength. Therefore, when using samples with different oxygen saturation levels (dissolved oxygen concentrations), the ratio between oxyhemoglobin and deoxyhemoglobin will be different, making accurate measurement difficult when measuring them at the same wavelength. It becomes.

  This invention makes it a subject to suppress appropriately the influence which the dissolved oxygen in a sample has on an analysis result.

  In the present invention, a column holding a filler and a sample preparation means for preparing a sample for introduction to be introduced into the column are provided, and the sample preparation means includes a large amount of blood cells in a sample containing blood cells. A liquid chromatographic apparatus is provided, wherein the preparation sample is collected from an upper layer portion of a layer containing the liquid chromatography device.

  The sample preparation means, for example, collects a preparation sample from the upper part of the blood cell layer when a blood sample containing red blood cells is separated into a blood cell layer rich in red blood cells and a plasma layer in which red blood cells are poor, and an adjustment sample Is used to prepare a sample for introduction.

  The sample preparation means comprises, for example, a detection means for detecting the interface between the blood cell layer and the plasma layer, and a preparation sample sampling nozzle for collecting a preparation sample from the upper layer of the blood cell layer Is done. In this case, the preparation sample sampling nozzle is operated so as to collect the preparation sample from the upper part of the blood cell layer based on the detection result by the detection means.

  The sample preparation means, for example, collects a sample for preparation from an area where the distance from the interface in the blood cell layer is in the range of 5 to 30% with respect to the thickness of the blood cell layer, or in the range of 0.5 to 5.0 mm. Configured to do. In this case, the adjustment sample is collected, for example, using a preparation sample sampling nozzle.

  The liquid chromatography apparatus of the present invention is configured to measure, for example, a glucohemoglobin concentration in a blood sample.

  Hereinafter, specific examples of the present invention will be described with reference to FIGS.

  The HPLC apparatus X shown in FIG. 1 is configured to automatically measure the glycated hemoglobin concentration in whole blood by setting a blood collection tube 11 held in a rack 10 on a table 20. The HPLC apparatus X includes a plurality of eluent bottles 12A, 12B, 12C (three in the drawing) and an apparatus main body 2.

  Each eluent bottle 12 </ b> A, 12 </ b> B, 12 </ b> C holds an eluent to be supplied to an analysis column 60 described later, and is arranged in the holder portion 21 in the apparatus main body 2. As eluents, for example, buffers having different pH and salt concentrations are used.

  The apparatus main body 2 includes a deaeration unit 4, a sample preparation unit 5, an analysis unit 6, and a photometric unit 7 housed in the housing 3 in addition to the table 20 and the holder unit 21 described above. .

  The table 20 is configured to move the blood collection tube 11 held in the rack 10 to a position where it can be collected by a nozzle 51 in the sample preparation unit 5 described later by moving the rack 10 set at a predetermined site. ing.

  The casing 3 is provided with an operation panel 30 and a display panel 31. The operation panel 30 is provided with a plurality of operation buttons 32. By operating the operation buttons 32, signals for performing various operations (analysis operation, printing operation, etc.) are generated, or various operations are performed. Can be set (analysis condition setting, subject ID input, etc.). The display panel 31 is for displaying an analysis result or an error, and for displaying an operation procedure or an operation status at the time of setting.

  As shown in FIG. 2, the deaeration unit 4 is for removing dissolved gas from the eluent before supplying the eluent to the analysis unit 6 (analysis column 60). The eluent bottles 12A, 12B, and 12C are connected to the eluent bottles 12A, 12B, and 12C via 80C and the manifold 61 of the analysis unit 6 is connected to the eluent bottles 12A, 12B, and 81C.

  The sample preparation unit 5 is for preparing a sample to be introduced into the analysis column 60 from blood cell components collected from the blood collection tube 11. The sample preparation unit 5 includes an interface detection mechanism 50, a nozzle 51, a preparation liquid tank 52, and a dilution tank 53.

  As shown in FIGS. 3 to 5, the interface detection mechanism 50 is for detecting the interface 13C between the plasma layer 13A and the blood cell layer 13B in the blood sample 13 of the blood collection tube 11 by an optical method, and transmits Type photosensor. In this interface detection mechanism 50, a light irradiation unit 55 and a light receiving unit 56 are arranged in a state of facing each other with respect to the U-shaped holder 54. The blood collection tube 11 is moved across the space between the light irradiation unit 55 and the light receiving unit 56 on the table 20 while being held by the rack 10.

  The light irradiation unit 55 can irradiate light in a certain range in the vertical direction of the blood collection tube 11 and can irradiate light having a peak wavelength in a wavelength range (500 to 570 nm) in which light absorption by red blood cells is large, for example. A linear light source is used. As the light irradiation part 55, what comprised the point light source so that an up-down direction can be scanned is also employable, for example. On the other hand, the light receiving unit 56 is for receiving light transmitted through the blood collection tube 11 and is capable of receiving light in a certain range in the vertical direction of the blood collection tube 11. As such a light receiving unit 56, for example, a line sensor or an area sensor can be used.

  Of course, the interface detection mechanism 50 may have, for example, a reflection type photosensor that detects light reflected on the surface of the blood collection tube 11, and is not limited to an optical method. 11 may be configured to detect the interface 13C between the plasma layer 13A and the blood cell layer 13B by a change in insertion resistance when inserted into the tube 11 or a change in electrical resistance.

  As shown in FIGS. 2 and 5, the nozzle 51 is for collecting various liquids including the blood sample 13 of the blood collection tube 11. The nozzle 51 can aspirate and discharge the liquid, and It is possible to move in the direction and horizontal direction. The operation of the nozzle 51 is controlled by a control means (not shown). When a blood sample 13 is collected from the blood collection tube 11, the interface 13C detected by the interface detection mechanism 50 is used as a reference and the interface 13C Also slightly below, it is operated to collect blood cell components from the blood cell layer 13B.

  The preparation liquid tank 52 shown in FIG. 2 holds a preparation liquid for preparing a sample for introduction to be introduced into the analysis column 60 based on the blood sample 13. In the preparation liquid tank 52, a dilution liquid containing a hemolytic agent for hemolyzing red blood cells is held as a preparation liquid. As the diluting liquid, it is preferable to use one having high oxygen saturation, for example, oxygen saturation of 85% or more. The nozzle 51 is used for collecting the preparation liquid from the preparation liquid tank 52.

  Although the preparation liquid tank 52 holds a hemolyzing agent-containing diluent, the preparation liquid tank 52 may separately hold a diluting liquid not containing the hemolytic agent and the hemolyzing agent.

  The dilution tank 53 is a place for preparing a sample for introduction by hemolyzing red blood cells in the blood sample 13 and diluting the blood sample 13, and bringing the sample for introduction into contact with the atmosphere to determine the oxygen saturation ( This is to increase the dissolved oxygen concentration. The dilution tank 53 is connected to an injection valve 63 in the analysis unit 6 to be described later via a pipe 83 so that an introduction sample prepared in the dilution tank 53 can be introduced into the analysis column 60 via the injection valve 63. It is configured. The dilution tank 53 is also open at the top to bring the sample for introduction into contact with the atmosphere.

  As shown in FIG. 2, the analysis unit 6 controls the adsorption / desorption of biological components with respect to the packing material of the analytical column 60, and supplies various biological components to the photometric unit 7. The temperature is controlled by the adjustment mechanism. The set temperature in the analysis unit 6 is about 40 ° C., for example. The analytical column 60 holds a filler for selectively adsorbing hemoglobin in a sample. As the filler, for example, a methacrylic acid acrylic copolymer is used.

  In addition to the analysis column 60, the analysis unit 6 has a manifold 61, a liquid feed pump 62, and an injection valve 63.

  The manifold 61 is for selectively supplying the eluent from the specific eluent bottles 12A, 12B, 12C among the plurality of eluent bottles 12A, 12B, 12C to the injection valve 63. The manifold 61 is connected to the deaeration unit 4 through pipes 81A, 81B, and 81C, and is connected to the injection valve 63 through a pipe 84.

  The liquid feed pump 62 is for applying power for moving the eluent to the injection valve 63, and is provided in the middle of the pipe 84. The liquid feed pump 62 is operated so that the flow rate of the eluent becomes 1.0 to 2.0 ml / min, for example.

  The injection valve 63 collects a predetermined amount of the introduction sample and enables the introduction sample to be introduced into the analysis column 60, and includes a plurality of introduction ports and discharge ports (not shown). An injection loop 64 is connected to the injection valve 63. The injection loop 64 can hold a fixed amount (for example, several μL) of liquid, and the injection loop 64 communicates with the dilution tank 53 by appropriately switching the injection valve 63 so that the injection loop 64 is connected to the dilution tank 53. The injection sample is supplied to the analysis column 60 via the pipe 85 and the introduction sample is introduced into the analysis column 60 from the injection loop 64, or the injection loop 64 is not shown in the figure. The state in which the cleaning liquid is supplied from the cleaning tank can be selected. As such an injection valve 63, for example, a six-way valve can be used.

  As shown in FIG. 6, the photometric unit 7 is for optically detecting hemoglobin contained in the desorbed liquid from the analysis column 60, and includes a photometric cell 70, a light source 71, a beam splitter 72, and a light receiving for measurement. A system 73 and a reference light receiving system 74 are provided.

  The photometric cell 70 is for defining a photometric area. This photometric cell 70 has an introduction flow path 70A, a photometry flow path 70B, and a discharge flow path 70C, and these flow paths 70A, 70B, and 70C communicate with each other in series. The introduction channel 70A is for introducing the desorbed liquid from the analysis column 60 (see FIG. 2) into the photometric channel 70B, and is connected to the analysis column 60 via a pipe 86. The photometric flow path 70B circulates the desorbed liquid to be measured and provides a field for measuring the desorbed liquid, and is formed in a straight line. The photometric flow path 70B is open at both ends and closed at both ends by the transparent cover 75. The discharge flow path 70C is for discharging the desorbed liquid from the photometric flow path 70B, and is connected to a waste liquid tank 88 via a pipe 87 (see FIG. 2).

  The light source 71 is for irradiating light to the desorbed liquid flowing through the photometric flow path 70B. The light source 71 is arranged in a state of facing the end face 70Ba (transparent cover 75) of the photometric flow path 70B so that the optical axis L passes through the center of the photometric flow path 70B. As the light source 71, a light source capable of emitting light in a wavelength range including light having a maximum absorption wavelength of 415 nm of oxyhemoglobin and a reference wavelength of 500 nm, for example, a halogen lamp is used. Of course, as the light source 71, other than the halogen lamp, for example, one provided with one or a plurality of LED elements can be used.

  The beam splitter 72 divides the light emitted from the light source 71 and transmitted through the photometric flow path 70B so as to be incident on the measurement light receiving system 73 and the reference light receiving system 74, and has an optical axis L. Above, it is arranged in a state inclined by 45 degrees. As the beam splitter 72, various known ones such as a half mirror can be used.

  The measurement light receiving system 73 selectively receives 415 nm light, which is the maximum absorption wavelength of oxyhemoglobin, out of the light transmitted through the beam splitter 72, and is disposed on the optical axis L. The measurement light receiving system 73 includes an interference filter 73A that selectively transmits light of 415 nm, and a light receiving element 73B for receiving the light transmitted through the interference filter 73A. A photodiode can be used as the light receiving element 73B.

  The reference light receiving system 74 selectively receives light having a reference wavelength of 500 nm out of the light reflected by the beam splitter 72 and changed in optical path. The measurement light receiving system 74 includes an interference filter 74A that selectively transmits light of 500 nm and a light receiving element 74B that receives the light transmitted through the interference filter 74A. A photodiode can be used as the light receiving element 74B.

  Next, the operation of the HPLC apparatus X will be described.

  When measuring glycated hemoglobin using the HPLC apparatus X, the rack 10 is first set on a predetermined portion of the table 20 with the blood collection tube 11 containing the blood sample 13 held in the rack 10. The blood sample 13 in the blood collection tube 11 is separated into a plasma layer and a blood cell layer in advance. Such separation can be performed by using a centrifuge or by spontaneous sedimentation of blood cell components.

  Separation of the plasma layer 13A and the blood cell layer 13B may be performed in the HPLC apparatus X by incorporating a centrifugal separator into the HPLC apparatus X. Also, the blood collection layer 11 is set on the table 20 and allowed to stand for a certain period of time. It may be done by doing.

  In the HPLC apparatus X, when an instruction to start measurement is confirmed, the rack 10 is moved on the table 20 and the blood sample 13 is collected from the target blood collection tube 11. The measurement start instruction is performed when the user operates a predetermined operation button 32 of the HPLC apparatus X.

  Collection of the blood sample 13 from the blood collection tube 11 is performed in a region separated downward from the interface 13C by a fixed distance D after the interface detection mechanism 50 detects the interface 13C between the plasma layer 13A and the blood cell layer 13B. More specifically, in the interface detection mechanism 50, light is irradiated from the light irradiation unit 55 to the blood collection tube 11, and light transmitted through the blood collection tube 11 is received by the light receiving unit 56. Here, when the light irradiation unit 55 is irradiated with light having a peak wavelength in a wavelength range where light absorption by red blood cells is large, light absorption in the blood cell layer 13B is larger than that in the plasma layer 13A. Therefore, in the interface detection mechanism 50, the interface 13C between the plasma layer 13A and the blood cell layer 13B can be detected by detecting a portion where the light absorption changes significantly based on the amount of light received by the light receiving unit 56. it can.

  When the detection of the interface 13C is completed in the interface detection mechanism 50, the blood sample 13 is collected from the upper layer portion of the blood cell layer 13B by operating the nozzle 51 based on the detection result of the interface detection mechanism 50. In this case, the nozzle 51 has a region in which the distance D from the interface 13C between the plasma layer 13A and the blood cell layer 13B is within a range of, for example, 5 to 30% with respect to the thickness of the blood cell layer 13B, or the distance D is 0.1. The tip is located in a region in the range of 5 to 5.0 mm, and the blood sample 13 is collected from the blood collection tube 11 by performing a suction operation in this state.

  Here, when the blood cell component is separated at the bottom of the blood collection tube 11 by centrifugation or the like, the upper layer of the blood cell layer 13B has higher oxygen saturation (dissolved oxygen concentration) than the lower layer, and the blood sample Even when 13 is allowed to stand, the upper layer portion of the blood cell layer 13B having a small distance from the gas phase has higher oxygen saturation (dissolved oxygen concentration) than the lower layer portion. Therefore, if the operation of the nozzle 51 is controlled based on the detection result of the interface 13C in the interface detection mechanism 50 and the blood sample 13 is collected from the upper layer portion of the blood cell layer 13B, the oxygen saturation (dissolved oxygen concentration) is increased. A high blood sample 13 can be collected. In addition, when the blood sample 13 is collected from a portion where the distance D from the interface 13C is in the previous region, the blood sample 13 can be reliably collected from the upper layer portion of the blood cell layer 13B.

  The blood sample 13 collected by the nozzle 51 is supplied to the dilution tank 53 by operating the nozzle 51. The dilution tank 53 is further supplied with a dilution liquid from the preparation liquid tank 52, and a sample for introduction is prepared by mixing the liquid in the dilution tank 53 by pipetting operation using the nozzle 51. In addition, when using together the dilution liquid which does not contain a hemolytic agent, and a hemolytic agent, a dilution liquid and hemolysis are supplied simultaneously to the dilution tank 53, dilution of the blood sample 13 and the blood cell of the blood sample 13 are carried out. Hemolysis may be performed simultaneously, or they may be supplied individually with time, and dilution of blood sample 13 and hemolysis of blood cells of blood sample 13 may be performed as separate steps.

  The sample for introduction prepared in the dilution tank 53 is brought into contact with the atmosphere in the dilution tank 53 for a certain period of time, and then supplied to the injection loop 64 and held in the injection loop 64.

  When the introduction sample is brought into contact with the atmosphere for a certain period of time, the dissolved oxygen concentration of the introduction sample is increased. When the oxygen saturation of the sample for introduction is increased in this manner, the sample for introduction having a large dissolved oxygen concentration can be supplied to the injection loop 64 and, consequently, the analytical column 60. That is, the proportion of oxyhemoglobin in the hemoglobin contained in the sample for introduction can be increased.

  Here, the contact time (atmospheric release time) between the sample for introduction and the atmosphere is, for example, 1 to 2 minutes. This is because when the open time to the atmosphere is too short, a sufficient amount of oxygen cannot be dissolved in the sample for introduction, whereas when the open time to the atmosphere is too long, the injection loop 64 is prepared after the preparation of the sample for introduction. This is because the time until the introduction of the sample for introduction is increased and the measurement time becomes longer.

  In addition, when a diluent having a high oxygen saturation (dissolved oxygen concentration), for example, one having an oxygen saturation of 85% or more is used, it is not always necessary to open the diluted introduction sample to the atmosphere. That is, in the case of using a diluting solution having a high oxygen saturation (dissolved oxygen concentration), even after using a closed diluting tank 53, it is allowed to stand for a certain period of time (for example, 1 minute or longer), and after dilution. Thus, the oxygen saturation (dissolved oxygen concentration) of the sample for introduction can be increased, and the proportion of oxyhemoglobin in hemoglobin can be increased.

  Further, in the HPLC apparatus X, when an instruction to start measurement is confirmed, an eluent is supplied to the injection valve 63. The eluent is supplied from the eluent bottles 12A, 12B, and 12C to the injection valve 63 through the deaeration unit 4 and the manifold 61 by the power of the liquid feed pump 62, and is also supplied to the plurality of eluent bottles 12A, 12B, and 12C. Which of the eluent bottles 12 </ b> A, 12 </ b> B, and 12 </ b> C is supplied is selected by controlling the manifold 61.

  The eluent supplied to the injection valve 63 is supplied to the analysis column 60 via the pipe 85. On the other hand, by performing the switching operation of the injection valve 63, the sample for introduction of the injection loop 64 is introduced into the analysis column 60 together with the eluent. When a certain time has elapsed since the introduction of the introduction sample, the eluent is continuously supplied to the analysis column 60 and the injection loop 64 is washed by switching the injection valve 63. On the other hand, simultaneously with the cleaning of the injection loop 64, the introduction sample is prepared from the blood sample 13 of the blood collection tube 11 different from the previous one, and after the injection loop 64 is cleaned, The introduction sample is again introduced into the injection loop 64. Such preparation, introduction, and washing of the sample for introduction are repeated according to the number of blood collection tubes 11 (blood samples 13) to be measured while appropriately switching the injection valve 63.

  On the other hand, in the analytical column 60, glycohemoglobin is adsorbed to the filler by introducing the introduction sample. After glycated hemoglobin is adsorbed to the filler, the type of eluent supplied to the analysis column 60 is appropriately switched by the manifold 61, and the glycated hemoglobin adsorbed to the filler is desorbed.

  The desorption liquid containing glycohemoglobin discharged from the analysis column 60 is supplied to the photometric cell 70 of the photometric unit 7 through the pipe 86. A desorption liquid is introduced into the photometric cell 70 via the pipe 86 and the introduction flow path 70A. The desorption liquid passes through the photometry flow path 70B and the discharge flow path 70C and then passes through the pipe 87 to the waste liquid tank 88. Led to.

  In the photometric unit 7, the light is continuously irradiated to the desorbed liquid by the light source 71 when the desorbed liquid passes through the photometric channel 70 </ b> B. On the other hand, the light transmitted through the photometric flow path 70B is split by the beam splitter 72 and then received by the measurement light receiving system 73 and the reference light receiving system 74. In the measurement light receiving system 73, light of 415 nm, which is the maximum absorption wavelength of oxyhemoglobin transmitted through the interference filter 73A, is selectively received by the light receiving element 73B. On the other hand, in the reference light receiving system 74, light having a reference wavelength of 500 nm transmitted through the interference filter 74A is selectively received by the light receiving element 74B.

  The results of light reception by the light receiving elements 73A and 74A are output to an arithmetic circuit (not shown), and the hemoglobin chromatogram and glycohemoglobin concentration (the ratio of glycohemoglobin in the total amount of hemoglobin) are calculated. The calculation result in the calculation circuit is displayed on the display panel 31 and is printed out automatically or by the user's button operation.

  In such an HPLC apparatus X, the oxygen saturation (dissolved oxygen concentration) in the sample for introduction to be introduced into the analytical column 60 is prepared based on the blood sample 13 collected from the upper part of the blood cell layer 13B. Later, before being introduced into the analytical column 60, the oxygen saturation (dissolved oxygen concentration) of the sample for introduction is increased in the dilution tank 53. Therefore, since the introduction sample introduced into the analytical column 60 is uniformized at a high oxygen saturation (dissolved oxygen concentration) for each measurement, the ratio of oxyhemoglobin and deoxyhemoglobin in the introduction sample is made constant. Can be supplied to the analysis column 60. As a result, with respect to the photometric unit 7, variation in the ratio of oxyhemoglobin and deoxyhemoglobin for each sample to be introduced is suppressed, and variation in measurement results due to this variation can be suppressed.

  The present invention is not limited to the embodiment described above, and can be variously changed. For example, as means for increasing the oxygen saturation (dissolved oxygen concentration) of the sample for introduction prepared in the dilution tank 53, for example, as shown in FIG. Alternatively, a bubbling mechanism 54 for bubbling with air may be employed.

  The collection of blood cell components from a blood sample is not necessarily performed by separating the plasma layer and the blood cell layer, and may be performed in a state where the plasma and blood cells are mixed. When preparing a sample for introduction using such a blood sample, the blood is collected in a region slightly below the liquid surface of the blood sample (upper layer portion of the blood sample), for example, in the range of 0.5 to 5.0 mm. By collecting, an adjustment sample having a high oxygen saturation (dissolved oxygen concentration) may be secured.

  The present invention is not limited to an HPLC apparatus for measuring the concentration of glycated hemoglobin in blood, but when a sample other than blood is used, a component other than the glycated hemoglobin concentration is measured, or a liquid chromatography apparatus other than an HPLC apparatus. Can also be applied.

(Reference example)
In this reference example, the measured value of glycohemoglobin in the upper layer and lower layer of the blood cell layer when whole blood was centrifuged was examined.

  Centrifugation of whole blood was carried out in a state where 2 ml of whole blood was retained in a blood collection tube (“VPDK052” manufactured by Terumo Corporation) having a diameter of 13.2 × 78 mm, Hitachi Koki Co., Ltd. For 10 minutes at 1850 G.

  The collection of blood cell components from the upper layer in the blood cell layer was performed by visually checking the interface between the plasma layer and the blood cell layer, and at a position 2 mm below the interface. On the other hand, collection of blood cell components from the lower layer in the blood cell layer was performed with the nozzle pressed against the bottom of the blood collection tube.

  Glycohemoglobin was measured using a glycated hemoglobin measuring device (“ADAMS A1c HA-8160”; manufactured by ARKRAY, Inc.). As the eluent, trade names “61A”, “61B” and “61C” (manufactured by ARKRAY, Inc.) were used, and the eluent was supplied at a flow rate of 1.7 ml / min. The measured values of glycohemoglobin in the upper layer portion and the lower layer portion in the blood cell layer are shown in Table 1 below. In Table 1, the measurement results of glycohemoglobin for six types of samples are shown.

  As can be seen from Table 1, the glycated hemoglobin concentration in the upper layer portion and the lower layer portion in the blood cell layer tends to increase as the lower layer portion in the blood cell layer, and there is a relatively large gap in measured values between the two. is there. That is, it can be seen that the measured value varies depending on the position where the blood cell component is collected in the blood cell layer. This is because the sample prepared from the blood cell component collected from the upper layer in the blood cell layer and the sample prepared from the blood cell component collected from the lower layer have different measured values due to the difference in oxygen saturation (dissolved oxygen amount). It is thought that there is.

  Further, according to the experiments by the present inventors, the recovery rate of hemoglobin in the desorbed liquid from the column tends to decrease as the desorption time becomes longer, and the oxygen saturation is higher (the dissolved oxygen amount is higher). When the oxygen saturation is low (the amount of dissolved oxygen is small), the rate of decrease in the recovery rate is greater than when the oxygen concentration is large. According to this experimental result, when the oxygen saturation is low (the amount of dissolved oxygen is small), the recovery rate of HbA0 occupying most of the recovered hemoglobin is higher than the recovery rate of glycated hemoglobin (HbAlc). Since it becomes smaller, the measured value is considered to be higher than when the oxygen saturation is high (the amount of dissolved oxygen is large).

(Example)
In the present embodiment, the interface detection mechanism 50 described above is incorporated into a glycated hemoglobin measuring device (“ADAMS A1c HA-8160”; manufactured by ARKRAY, Inc.), and the distance from the detected interface 13C is 2 mm below. The concentration of glycohemoglobin in whole blood was measured using a device (see FIG. 5) that was modified to control the operation of the nozzle 51 so as to collect blood cell components at the position. The type and flow rate of the eluent were the same as those in the reference example, and glycohemoglobin was measured for six types of samples similar to the reference example. The measurement results are shown in Table 2 below when blood is collected from the upper layer of the blood cell layer in the reference example.

  As can be seen from Table 2, when the interface is detected by the interface detection mechanism and the operation of the nozzle is controlled according to the detection result, it is substantially the same as when the blood cell component is visually collected from the upper layer of the blood cell layer. Results were obtained. That is, in the improved apparatus, blood cell components can be reliably collected from the upper layer of a blood cell layer having a high oxygen saturation (a large amount of dissolved oxygen), and an increase in the measured value can be suppressed.

It is a whole perspective view which shows an example of the HPLC apparatus which concerns on this invention. It is a schematic block diagram of the HPLC apparatus shown in FIG. It is a perspective view for demonstrating the interface detection mechanism of the HPLC apparatus shown in FIG. It is sectional drawing which follows the IV-IV line of FIG. It is sectional drawing and the principal part enlarged view for demonstrating the sampling method. It is sectional drawing for demonstrating the photometry unit in the HPLC apparatus shown in FIG. It is a schematic diagram which shows the bubbling mechanism in the HPLC apparatus which concerns on this invention. It is a schematic block diagram which shows an example of the conventional HPLC apparatus (high performance liquid chromatography apparatus).

Explanation of symbols

X HPLC apparatus 13 Blood sample 13A Plasma layer 13B Blood cell layer 13C Interface 5 Sample preparation unit 50 Interface detection mechanism 51 Nozzle 7 Photometric unit (detection mechanism)

Claims (6)

A column holding a filler;
Sample preparation means for preparing a sample for introduction to be introduced into the column;
With
The liquid chromatographic apparatus characterized in that the sample preparation means is configured to collect the preparation sample from an upper layer portion of a layer containing a lot of blood cells in a sample containing blood cells.   The sample preparation means collects a preparation sample from the upper part of the blood cell layer when a blood sample containing red blood cells is separated into a blood cell layer rich in red blood cells and a plasma layer in which red blood cells are poor, and the adjustment The liquid chromatography apparatus according to claim 1, wherein the introduction sample is prepared using a sample for use. The sample preparation means includes a detection means for detecting an interface between the blood cell layer and the plasma layer,
A sample sampling nozzle for preparation for collecting the sample for preparation from the upper layer part of the blood cell layer, and
The liquid chromatography apparatus according to claim 2, wherein the preparation sample sampling nozzle is operated so as to collect the preparation sample from the upper layer of the blood cell layer based on a detection result by the detection means.   The sample preparation means is configured to collect the preparation sample from a region whose distance from the partial interface in the blood cell layer is in the range of 5 to 30% with respect to the thickness of the blood cell layer. Item 4. The liquid chromatography apparatus according to any one of Items 1 to 3.   The said sample preparation means is comprised so that the said sample for preparation may be extract | collected from the area | region which the distance from the said interface in the said blood cell layer exists in the range of 0.5-5.0 mm. A liquid chromatography apparatus according to claim 1. The liquid chromatography apparatus according to claim 1, wherein the liquid chromatography apparatus is configured to measure a glucohemoglobin concentration in a blood sample.

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