Device for hemolyzing and diluting puncture blood, and glycosylated hemoglobin analysis method using the same

Abstract

To provide a device and a method for easily hemolyzing / diluting puncture blood even is the device is an A1c measuring device using the HPLC method.SOLUTION: Glycosylated hemoglobin (A1c%) is measured by a chip set that is formed of a conical or pyramid-shaped chip made of resin that can suck up puncture blood by a capillary phenomenon, a container that can store the chip, and a lid body that can seal the container, and can collect the puncture blood and hemolyze and dilute the blood, the chip set capable of collecting puncture blood and hemolyzing and diluting the blood, wherein the container has an inner diameter larger than the maximum outer diameter of the chip by 0.5 to 5 mm, and the container has therein a flange that has a columnar shape having a height allowing storage of the chip and is at a position 5 to 15 mm from a top face of the container, and a method for collecting puncture blood and hemolyzing / diluting the blood by using the chip.SELECTED DRAWING: Figure 7

Description

[Technical Field]

[0001] Hemoglobin A1c (hereafter abbreviated as A1c or SA1c) is a value used as a diagnostic indicator for diabetes. There are several methods for measuring A1c, depending on the number of tests (scale) and the required accuracy. Broadly speaking, there are two methods: immunoassay and HPLC. Immunassay is an immunochemical method that uses antibodies for measurement. HPLC (High Performance Liquid Chromatography) is a method in which a sample is passed through a device called a column at high pressure to separate and quantify blood components based on differences in affinity and charge state. It is considered the most accurate method. In both methods, venous blood is collected from the patient, hemolyzed, and diluted before being submitted to the measurement system. [Background technology]

[0002] Simple immunoassays typically involve using a disposable "lancing device" to collect a few to several tens of microliters of blood from a finger or other sample. On the other hand, HPLC uses blood collected in a 5-10 mL blood collection tube. In this case, the whole blood in the blood collection tube is automatically processed using a hemolysis / dilution mechanism built into the measuring device before being analyzed.

[0003] Furthermore, in the HPLC method, as mentioned above, blood collection tubes are often used, but "punctured blood" is not directly handled as in the simple immunoassay.

[0004] There are two methods for measuring A1c using HPLC: one based on the principle of ion exchange due to differences in charge, and the other based on the principle of affinity due to specific adsorption and desorption. Figure 1 shows a schematic representation of the resulting chromatogram. Figure 1a shows the results from the ion exchange method, and Figure 1b shows the results from the affinity method. The left image shows a case where A1c is low, and the right image shows a case where A1c is high. In either case, A1c%, an indicator of diabetes, is calculated as the ratio of glycated hemoglobin (A1c) to total hemoglobin. In other words, it is calculated as the ratio of the glycated hemoglobin (A1c) peak area (filled peak) to the total peak area.

[0005] Therefore, unlike typical HPLC quantification, slight variations in the injection volume into the column have little effect on the quantitative value. In other words, when measuring blood samples, dilution accuracy is not required, and slight variations do not affect the resulting A1c%. Summary of the Invention [Problem to be solved by the invention]

[0006] The present invention has been made to solve the above-mentioned problems, and provides a device and method for easily hemolyzing / diluting punctured blood, even in an A1c measurement device using the HPLC method. [Means for solving the problem]

[0007] When measuring A1c using the HPLC method, multiple samples are placed on a "rack" that can hold multiple samples, which are then moved to the sampling position and injected sequentially into the analytical column for analysis. Figure 2 shows an example of the configuration of an A1c measurement device using the HPLC method.

[0008] The HPLC method is roughly composed of a pump (3) that can switch between multiple eluents (1) with different elution strengths, a sample injection valve (31) for injecting the sample into the analytical column (6), and a visible light detector (7) for detecting the degree of separation. The sample injection valve (31) has a sample loop (32) that can hold a fixed amount of sample.

[0009] In addition to the injection valve (31), the sample injection mechanism includes a dilution tank (35) for hemolyzing / diluting the whole blood sample, and a metering pump (37) for adding a hemolyzing / diluting solution and aspirating the sample.

[0010] In the field of hematology, venous blood is often collected in 2-10 mL collection tubes and directly measured on an analyzer. The same is true for A1c measurement using the HPLC method, where the collection tubes are placed on a rack, pre-processed automatically, and then introduced into the analyzer. For A1c measurement, a small amount of whole blood is sampled, automatically hemolyzed and diluted, and a portion of it is injected into an analytical column for analysis.

[0011] However, in the following cases, the sample should be manually hemolyzed and diluted before being analyzed. (1) Patients or specimens for which blood collection using a blood collection tube is difficult, (2) specimens in a freeze-dried state (calibration specimens, control specimens, etc.) In this case, measurement is possible with an adjustment of about 0.5 mL, which is placed in a 2 mL "dilution container," placed on a "rack," and then run through the analyzer. In this case, the aforementioned "pretreatment" step is skipped. Therefore, analyzers that perform A1c measurement using the HPLC method are required to be able to automatically distinguish between a "collection tube" and a "dilution container."

[0012] One such method is to distinguish specimens by their height. This will be explained based on Figure 3. Since blood collection tubes are typically 80 mm or longer, we will assume that a rack approximately 60 mm high is used. In this case, approximately 25 mm of the blood collection tube will protrude from the top of the rack. The "dilution container" has a flanged shape and protrudes approximately 10 mm from the top of the rack. Two "sensors" with different detectable heights are placed. The first sensor is placed at a height position indicating "unreacted" for the dilution container and "reacted" for the blood collection tube, while the second sensor is placed at a height position indicating "reacted" for the dilution container and "reacted" for the blood collection tube. The combination of their responses allows the specimen type to be identified. The height-detecting sensors can be contact-type sensors or optically identifiable sensors, and are not particularly limited.

[0013] As shown in Figure 4, when the device is mounted on a rack and moved to the height detection position, if the first sensor indicates a "reaction" and the second sensor indicates a "reaction," the sample is determined to be a "blood collection tube," the device is moved to the sampling position, a minute amount of blood sample is sampled, and the sample is automatically hemolyzed and diluted, and a portion of it is injected into the analytical column to measure A1c.

[0014] As shown in Figure 6, when the sample is loaded onto a rack and transported to the height detection position, if the first sensor indicates "unreacted" and the second sensor indicates "reacted," the sample is determined to be a "diluted sample (dilution container)," and the sample is moved to the sampling position, a certain amount is sampled, and injected into the analytical column to measure A1c.

[0015] If both the first and second sensors are "unreacted," it is determined that no sample is loaded on the rack and the measurement is skipped. If the first sensor is "reacted" and the second sensor is "unreacted," it is determined as an "error."

[0016] In addition, in the field of clinical diagnosis, barcodes and QR codes (registered trademarks) are often used to electronically record patient information, reduce labor, and prevent sample mix-ups. Even in A1c measurement using HPLC, barcodes are often attached to samples and automatically read by the measuring device. When using blood collection tubes, the length is approximately 100 to 130 mm, providing ample space for attaching a barcode (see Figures 11a and 11b). On the other hand, for diluted samples, the containers are too small to provide sufficient space for attaching a barcode. As mentioned above, when measuring A1c from punctured blood using HPLC, manual measurement is required, as shown in Figure 5.

[0017] First, a finger or other part is pricked with a lancing device to extract approximately 1 to 50 μL of blood. 10 μL is drawn up using a capillary or other device capable of collecting minute amounts of blood, and then pushed into a dilution cup into which a fixed amount of hemolysis / dilution solution has been dispensed in advance. Hemolysis / dilution is performed by aspirating / dispensing with a pipette, and the sample is then placed on a measurement rack for A1c measurement using HPLC. This is extremely time-consuming, and since barcode management is not possible, it is not possible to completely eliminate human error such as sample mix-up. If barcode management is absolutely necessary, a barcode can be attached to a cylindrical adapter, as shown in Figure 11b, and the diluted sample can be placed on top of it.

[0018] The present invention will be described in detail below. First, a first embodiment will be described. This tip set is capable of collecting, hemolyzing, and diluting punctured blood, and is composed of a conical or pyramidal resin tip that can suck up punctured blood by capillary action, a container that can store the tip, and a lid that can seal the container. The tip set is characterized in that the inner diameter of the container is 0.5 to 5 mm larger than the maximum outer diameter of the tip, the container is cylindrical in shape and has a height that allows the tip to be stored within the container, and the container has a flange located 5 to 15 mm from the top surface. First, a finger or the like is punctured with a puncture device, and the punctured blood is withdrawn.

[0019] As the first step, The tip sucks up a certain amount of the punctured blood by capillary action. The amount of blood to be collected is preferably in the range of 10 to 50 μL, but it is desirable to decide this taking into consideration the amount of hemolysis / dilution solution, and approximately 15 μL is suitable.

[0020] As the second step, The tip that has sucked up the punctured blood is inserted into a hemolysis / dilution container into which a predetermined amount of hemolysis / dilution solution has been dispensed in advance, and the container is then capped. The amount of hemolysis / dilution solution is desirably determined taking into consideration the amount of blood collected, and it is preferable to use a solution that results in a final dilution ratio of approximately 1 / 150. If the amount of blood collected in step 2 is 15 μL, then the amount of hemolysis / dilution solution should preferably be 2500 μL. The hemolysis / dilution solution should be compatible with the analytical equipment used for measurement, and there are no limitations on its composition, etc.

[0021] As the third step, The sealed container is then inverted to agitate the punctured blood, causing it to hemolyze and dilute. The inner diameter of the container is approximately 0.2 to 5 mm larger than the maximum outer diameter of the tip, leaving a gap between the inner surface of the container and the tip. Since the blood collection tip is made of resin and has a light specific gravity, the tip moves up and down in the liquid when inverted, allowing for efficient hemolysis and dilution.

[0022] In addition, the "diluted sample" prepared up to the above steps is As the fourth step, The lid is removed, and the container with the blood collection tip inserted is placed on a rack for A1c measurement, where A1c is measured by HPLC. The processed containers are placed on the rack and moved sequentially to the position where the sample type is determined. Here, the container has a "brim," so the portion above the brim, approximately 10 mm, protrudes. Height sensor 1 indicates "unreacted," while height sensor 2 indicates "reacted," indicating that the sample is diluted. Therefore, the sampling needle descends only approximately 20 mm below the top of the rack, aspirating the diluted sample and injecting it into the analytical column without pretreatment, where it is subjected to analysis.

[0023] As the sampling needle descends, the blood collection tip floating in the container is pushed down by the tip of the needle, but this does not interfere with the aspiration of the sample. During this process, the blood collection tip inside the container often becomes stuck to the inner surface of the container or tilted due to the inversion and agitation. However, as the needle descends, it is pushed down and returns to its central position, which does not interfere with the aspirating of the sample, which is a major feature of the present invention (see Figure 10).

[0024] Furthermore, the containers of the present invention have a length of approximately 45 mm extending below the rack, and as shown in Figure 11c, it is possible to attach a barcode, which can be automatically read and managed by the analytical equipment, which is another major feature of the present invention.

[0025] The hemolysis / dilution container of the present invention is not limited to the above-described configuration, and any configuration that can achieve the same effect is acceptable. For example, as shown in Figure 12c, a container with the same shape as the above-described container but without a "brim" is also acceptable. In this case, the same effect can be achieved by inserting a spacer between the rack and the container so that the height of the container from the top of the rack reacts to height sensor 2 but not height sensor 1 (Mode 2). Furthermore, as shown in Figure 12d, if the container is long enough to reach the bottom of the rack like a blood collection tube, and the height of the portion above the top of the rack reacts to height sensor 2 but not height sensor 1, the container can also have a "brim"-less configuration, and the same effect can be achieved (Mode 3). [Brief explanation of the drawings]

[0026] [Figure 1] These are schematic diagrams of chromatogram patterns obtained by HPLC. Panel a shows the ion exchange mode, and panel b shows the affinity mode. In both cases, the solid components indicate the A1c components. [Figure 2] FIG. 1 is a diagram showing an example of the configuration of an A1c measurement device using the HPLC method. [Figure 3] FIG. 1 is a diagram showing the types of specimen containers handled and the lowering position of the sampling needle in an A1c measurement device using the HPLC method. [Figure 4] FIG. 10 is a diagram showing a schematic diagram of the operation of an A1c measurement device using the HPLC method when a sample is determined to be a "blood collection tube." [Figure 5] FIG. 1 is a diagram showing a schematic diagram of the procedure for handling punctured blood in an A1c measurement device using the HPLC method. [Figure 6] FIG. 10 is a diagram showing a schematic diagram of the operation of the A1c measurement device using the HPLC method when it is determined to be a "dilution container." [Figure 7] FIG. 1 is a diagram showing a schematic diagram of the process from collecting punctured blood to hemolysis / dilution according to the present invention. [Figure 8] 1 is a flowchart showing the flow when analyzing a hemolyzed / diluted sample by HPLC according to the present invention. The thick solid line in the figure shows the flow when measuring puncture blood. The dashed line in the figure shows the flow when measuring a blood collection tube (whole blood). [Figure 9] FIG. 1 is a diagram showing a schematic diagram of specimen type determination and the lowering position of the sampling needle when analyzing a hemolyzed / diluted specimen by HPLC according to the present invention. [Figure 10] FIG. 1 is a diagram showing a schematic diagram of the movement of a tip for collecting a sample when a hemolyzed / diluted sample is analyzed by HPLC according to the present invention. [Figure 11] FIG. 10 is a diagram showing a schematic diagram of a case where a barcode is attached to a hemolysis / dilution container and measurement is performed according to the present invention. [Figure 12] 10 is a diagram showing a schematic diagram of another form of hemolysis / dilution container according to the present invention and its positional relationship when mounted on a rack. FIG. [Figure 13] FIG. 1 is a diagram showing the dimensions of the blood collection tip and hemolysis / dilution solution container used in the examples. [Figure 14] FIG. 1 is a diagram showing the steps from specimen collection to dissolution / dilution in an example. [Figure 15] 1 shows the results of hemolysis / dilution using the chip of the present invention obtained in Example 1. Panel a shows the visible absorption spectrum, and panel b shows the absorbance at a specific wavelength (calibration curve). [Figure 16] This shows the results of the reproducibility of the hemolysis / dilution method using the chip of the present invention obtained in Example 1. Figure a shows the visible absorption spectrum overlaid, and Figure b shows the change in absorbance at a specific wavelength. [Figure 17]1 shows a diagram illustrating the "Glycohemoglobin Analysis System GHbVIII" manufactured by Tosoh Corporation, which is the HPLC A1c measurement device used in Example 2. Figure a is an overall view, and Figure b is an enlarged view of the sample transport area. [Figure 18] FIG. 1 shows the configuration of the "Glycohemoglobin Analysis System GHbVIII" manufactured by Tosoh Corporation, which is the A1c measurement device for the HPLC method used in Example 2. [Figure 19] FIG. 1 shows the sample racks and sample types in the Tosoh "Glycohemoglobin Analysis System GHbVIII," an A1c measurement device for the HPLC method used in Example 2. [Figure 20] FIG. 10 is a diagram showing the operation of the sample rack in the Tosoh "Glycohemoglobin Analysis System GHbVIII," an A1c measurement device for the HPLC method used in Example 2. [Figure 21] FIG. 10 is a diagram showing the dimensions and positional relationship of the sensor when a "blood collection tube" is mounted on the "glycated hemoglobin analysis system GHbVIII" manufactured by Tosoh Corporation, which is the A1c measurement device by HPLC method used in Example 2. [Figure 22] FIG. 10 is a diagram showing the dimensions and positional relationship of the sensor when a conventional "diluted sample container" is installed on the Tosoh "Glycohemoglobin Analysis System GHbVIII," which is the HPLC A1c measurement device used in Example 2. [Figure 23] This figure shows the dimensions and positional relationship of the sensor when the "diluted sample container" of the present invention is installed on the Tosoh "Glycohemoglobin Analysis System GHbVIII," which is the HPLC A1c measurement device used in Example 2. [Figure 24] FIG. 10 is a diagram comparing a conventional "diluted sample container" and the "diluted sample container" of the present invention using the Tosoh "Glycohemoglobin Analysis System GHbVIII," an A1c measurement device for the HPLC method used in Example 2. [Figure 25] 1 shows the results of measurement of diluted / hemolyzed whole blood in Example 2 (difference in measurement method). Panel a shows the raw data, and panel b shows the results normalized by the height of the A1c peak. [Figure 26]1 shows the results (reproducibility) of measurement performed after diluting / hemolyzing whole blood in Example 2. Panel a shows the raw data, and panel b shows the results normalized by the height of the A1c peak. [Figure 27] FIG. 10 is a graph showing the reproducibility in Example 2. [Figure 28] The graph shows the results of measurements taken when a bar code was attached to a container of the present invention. [Figure 29] This is a diagram showing a schematic diagram of needle descent when a diluted sample is identified (determined) using the Tosoh "Glycohemoglobin Analysis System GHbGX," an A1c measurement device using the HPLC method. [Figure 30] This is a diagram showing a schematic diagram of the flow when the container and hemolysis / dilution procedure of the present invention are applied to A1c measurement. The diagram on the right in the lower part shows the case where it is applied to liquid chromatography, and the diagram on the left shows the case where it is applied to POCT equipment using immunoassay, enzymatic method, electrophoresis, etc. DETAILED DESCRIPTION OF THE INVENTION

[0027] The present invention will be described in detail below. However, the present invention can be embodied in various forms and is not limited to the following embodiments and examples. [Example]

[0028] In this example, the following blood collection tip and hemolysis / dilution container were used (see FIG. 13): The blood collection tip was made of resin and had a conical shape with a total length of 40 mm and a maximum outer diameter of 10 mm.

[0029] The hemolysis / dilution container used was cylindrical, with a total length of 53 mm, an inner diameter of 13.2 mm, and a flange (outer diameter 13.2 mm) located approximately 45 mm from the bottom. The maximum container capacity was 3 mL. When a blood collection tip is inserted into this hemolysis / dilution container, there is a clearance of about 1.6 mm. The hemolysis / dilution solution used was a reagent specifically designed for the glycohemoglobin analyzer manufactured by Tosoh Corporation. Example 1 First, we verified the accuracy of the blood collection and hemolysis / dilution method using the chip of the present invention. Here, instead of puncture blood, we dripped about 50 μL of whole blood collected in a blood collection tube into a small container to simulate puncture blood (see Figure 14).

[0030] The verification was carried out using the following procedure. Dispense 2.5 mL of hemolysis / dilution solution into the hemolysis / dilution container. Place the blood collection tip sideways against the dripped sample and allow the sample to be drawn up to approximately 10 mm from the tip. Insert the blood collection tip into the container, close the cap, and mix by inverting several times.

[0031] In this case, there is a clearance of approximately 1.6 mm between the inner diameter of the container and the outer diameter of the tip, so by inverting the container to mix, the tip containing the blood moves up and down, mixing with the liquid and causing hemolysis / dilution. A fixed amount of the diluted sample was dispensed into a spectrophotometer cell, and the visible absorption spectrum was measured. This procedure was repeated 10 times to verify accuracy. In addition, to calculate the degree of dilution (dilution rate), the same sample was manually diluted 50-fold, 100-fold, 150-fold, and 200-fold, and the visible absorption spectrum was measured in the same way.

[0032] Figure 16a shows the absorption spectrum in the visible light region obtained by the method of the present invention (10 data points are overlaid), and Figure 16b and Table 1 show the absorbance at 540 nm and 575 nm. Figure 15a shows the spectrum of a sample diluted manually, and Figure 15b and Table 2 show the relationship between the dilution rate and the absorbance (calibration curve).

[0033] [Table 1]

[0034] [Table 2]

[0035] The spectrophotometer used was UV-2600 manufactured by Shimadzu Corporation. From the calibration curve obtained by the manual method, it was calculated that the blood collection and hemolysis / dilution method using the chip of the present invention could achieve approximately 150-fold dilution. Furthermore, it was found that the reproducibility of the concentration was about 6.5%.

[0036] In general quantitative analysis using HPLC, a reproducibility of 6% is often unacceptable as it is a large value, but in A1c measurement, evaluation is performed using area %, so even if the dilution rate fluctuates by 6%, it does not affect the final A1c% obtained. There is no problem with the accuracy of hemolysis / dilution. Example 2 Here, the usefulness of the present invention was verified using an actual glycohemoglobin analyzer based on the HPLC method. The glycohemoglobin analyzer used was the "Glycohemoglobin Analyzer GHbVIII" manufactured by Tosoh Corporation. The eluent, hemolysis / dilution solution, column, etc. were the same as those sold by the same company.

[0037] This "Glycohemoglobin Analyzer GHbVIII" can handle two types of samples: blood collection tubes (whole blood) and diluted samples. In this device, samples are placed in a rack (17) that can hold 10 samples and then placed in the rack loader (13). The rack loader is driven by an analysis command, and the samples are moved one after another to the sampling position, where they are sampled with a needle and then submitted for analysis (see Figure 17). Two height sensors are placed in front of the sampling position to distinguish the type of sample. The height of the rack (17) is 65 mm.

[0038] The first height sensor is located 70 mm from the bottom, and the second height sensor is located 80 mm from the bottom. When a sample passes through, the sensor flag responds by detecting whether or not it comes into contact with the sample, and this determines whether the sample is a blood collection tube (whole blood) or a diluted sample.

[0039] Blood collection tubes ranging from 75mm to 100mm can be loaded. Diluted samples are dispensed into dedicated plastic containers (total length approximately 40mm, capacity 2mL) as shown in Figure 6. This diluted sample container has a flange and is designed so that when it is loaded on a rack, it extends approximately 10mm above the rack. In other words, when a diluted sample container is loaded on a rack, the top of the flange is approximately 750mm from the bottom. On the other hand, when a blood collection tube is loaded on a rack, the top of the blood collection tube is 20mm to 40mm.

[0040] In the case of a diluted sample container, only height sensor 2 responds; in the case of a blood collection tube, both height sensors 1 and 2 respond. This difference determines the type of sample and the position at which the needle descends. If the container is determined to be a diluted sample container, the needle descends only to near the bottom of the dilution container (45 mm from the bottom of the rack), aspirating approximately 100 μL of diluted sample, a portion of which (several μL) is introduced into the analytical column. On the other hand, if the container is determined to be a blood collection tube, the needle descends to near the bottom of the blood collection tube (25 mm from the bottom of the rack), aspirating several μL of whole blood, which is then hemolyzed and diluted by the built-in automatic dilution mechanism, after which a portion of which (several μL) is introduced into the analytical column.

[0041] On the other hand, when the hemolysis / dilution container of the present invention is used, the flange (outer diameter 13.2 mm) is attached approximately 45 mm from the bottom, so when it is mounted on a rack, the flange catches on the rack, causing approximately 13 mm to protrude above the rack, resulting in a height of approximately 75 mm from the bottom of the rack.

[0042] In other words, just like the diluted sample container dedicated to this device mentioned above, when detecting the sample type, only height sensor 1 responds, recognizing it as a "diluted sample," and the needle descends to a position 45 mm from the bottom of the rack, aspirating about 100 μL of the diluted sample, and a portion of it (about a few μL) is introduced into the analytical column.

[0043] Because the blood collection tip inserted into the container is made of resin, it floats in the hemolysis / dilution solution. It may also come into contact with the inner surface of the container, causing it to stick higher than expected, or it may come into contact with the inner surface of the container at an angle, causing the center position to shift. However, because the needle presses down on the tip as it descends, even if the center position shifts, the tip will be centered at the needle's lowest point, allowing normal sampling.

[0044] As in Example 1, blood was collected by the method of the present invention, and the container containing the hemolyzed and diluted blood was loaded into the "Glycohemoglobin Analyzer GHbVIII" and measurements were performed. Figure 25 and Table 3 show the measurement results using the method of the present invention and the results using the conventional manual method. For reference, the results of a sample taken directly from a blood collection tube, hemolyzed and diluted using the automatic dilution mechanism of the device, and then measured are also shown.

[0045] [Table 3]

[0046] In Figure 25a, *1 shows the results (chromatogram) of a sample taken directly from a blood collection tube, hemolyzed and diluted using the device's automatic dilution mechanism, *2 shows the results (chromatogram) obtained using the conventional manual method, and *3 shows the results (chromatogram) obtained using the method of the present invention. Although the intensity of the method of the present invention (*3) is slightly lower, the same pattern is observed regardless of the measurement method. For ease of comparison, the data are normalized so that the A1c peak output is "50," revealing that the three data have exactly the same pattern (Figure 25b).

[0047] Table 3 shows the area of ​​each component and the A1c% calculated from the total area. Although the total area differs depending on the measurement method, the A1c% values ​​are all the same, suggesting that there is no problem with measurement using the method of the present invention.

[0048] Furthermore, Figure 26 and Table 4 show the reproducibility of this method. Figure 26 shows chromatograms obtained by measuring 10 times using the method of the present invention, with the results superimposed. Table 4 shows the A1c peak area, total area, and A1c%. Note that Figure 26a shows the raw data, and Figure 26b shows the data normalized so that the A1c peak output is "50," as described above. As can be seen from these figures, although the reproducibility of the A1c peak and total area is somewhat poor at around 4%, the reproducibility of the finally calculated A1c% is a good value of 0.7%. A Cv of 1.0% or less, which is often required for A1c% measurement using HPLC, was achieved, demonstrating the usefulness of this invention.

[0049] [Table 4]

[0050] In addition, in the field of clinical diagnosis, barcodes and QR codes (registered trademarks) are often used to electronically record patient information, save labor, and prevent sample mix-ups. Even in A1c measurement using the HPLC method, it is becoming more common to attach a barcode to the sample and have it automatically read by the measuring device.

[0051] When using blood collection tubes, their length is approximately 100–130 mm, providing ample space for attaching a barcode (see Figure 11a). On the other hand, for diluted samples, the containers are small, making it impossible to provide sufficient space for attaching a barcode. Therefore, when a barcode is required for a diluted sample, a last resort is to attach the barcode to a round tube adapter and then place the diluted sample container on top of it, as shown in Figure 11b. Because this method is time-consuming and increases costs, it is often only used for freeze-dried calibration or control samples.

[0052] The dilution and hemolysis container of the present invention has a total length of 55 mm and a length below the flange of 50 mm, making it possible to attach a barcode, which makes it useful for measuring actual patient samples (see Figure 11c).

[0053] Figure 28 shows an example of an actual measurement using a barcode attached to a container. a is the barcode, and b is the reading result (report). As shown here, the barcode can be read accurately.

[0054] In this example, the glycohemoglobin analyzer GHbVIII manufactured by Tosoh Corporation is described as an example, using a sample rack capable of holding 10 samples. However, for example, the glycohemoglobin analyzer GHbX also manufactured by Tosoh Corporation can achieve the same effect even if the samples are placed on a rotating table as shown in FIG. 29 and are transported sequentially to the sample aspiration position by a rotating operation, and the structure of the rack that holds the samples and the drive method are not limited.

[0055] In addition, for immunoassays, many point-of-care testing (POCT) devices for puncture blood have become commercially available. Although the measurement process differs depending on the model, it consists of a "first step of collecting blood by puncturing," a "second step of hemolysis and dilution," and a "third step of measurement." The first step is performed manually by the patient or a healthcare professional. The second step is performed manually by the patient or a medical professional, or automatically by the device. The third step is carried out on the device side.

[0056] It is desirable that this series of operations be as simple and inexpensive as possible. In addition, it is desirable that the second step be performed automatically on the device side as much as possible to prevent human error and reduce the risk of infection.

[0057] So far, we have explained the usefulness of applying the method of the present invention to A1c measurement by HPLC, but it can also be applied to the second step in the aforementioned immunoassay (POCT), enzymatic method, and electrophoresis, and the A1c measurement method is not limited. The hemolysis / dilution step of the present invention is performed in a closed system, and when setting the blood collection tip in the measurement device, it is only necessary to remove the cap without removing the blood collection tip, thereby reducing the risk of infection. [Explanation of symbols]

[0058] 1. Eluent 2. Hemolysis Wash Solution 3. Liquid delivery mechanism 4. Automatic sample dilution mechanism 5. Sample injection mechanism 6.Analytical column 7. Visible Light Detector 8. Sample transport rack 9. Drain 10. Specimen height sensor 1 11. Specimen height sensor 2 12. Sample aspiration position 13. Sample transport mechanism 14. Blood collection tube 15. Container for diluted specimen 16.Measurement part 17. Hemolysis / Dilution Container 18. Lid 19. Blood collection chip 20. Sampling needle 21.Puncture device 22. Spacer 23. Tsuba (protrusion) 24. Barcode Reader 25. Barcode 26. Adapter 27. Turntable 28. Sample holder 29. Eluent opening and closing mechanism 30. Liquid transfer pump 31. Injection valve 32. Sample holding loop 33. Hemolytic washing solution switching mechanism 34. Constant temperature bath 35. Hemolysis / Dilution Tank 36. Degassing device 37. Metering pump

Claims

1. a conical or pyramidal resin tip that can suck up punctured blood by capillary action; a container capable of storing the chip; a tip set that can collect, hemolyze, and dilute punctured blood and that is configured with a lid that can seal the container; the container has an inner diameter that is 0.5 to 5 mm larger than the maximum outer diameter of the tip; The container has a cylindrical shape having a height that allows the chip to be stored in the container, The container has a flange located 5 to 15 mm from the top surface. A tip set capable of collecting punctured blood, hemolyzing and diluting the blood, characterized by:

2. The blood obtained by pricking a finger or the like with a puncture device is a first step of sucking up the punctured blood by capillary action using the tip; a second step of inserting the tip into the container into which a predetermined amount of hemolysis / dilution solution has been dispensed in advance, and then closing the container with the lid; a third step of agitating the sealed container by inversion to hemolyze and dilute the punctured blood.

3. A method for measuring glycohemoglobin (A1c%) by loading the hemolyzed / diluted specimen prepared as claimed in claim 2 into a glycohemoglobin measuring device based on liquid chromatography.

4. The method according to claim 3, characterized in that in the glycohemoglobin measuring device based on the liquid chromatography method, samples are loaded onto a rack capable of loading one or more samples, the rack is mechanically moved and transported to a sample sampling position, the device automatically identifies whether the sample is a "blood collection tube" or an "other container," and based on the identification result, if the sample is a "blood collection tube," the device aspirates the sample, automatically performs hemolysis / dilution operations, and injects the sample into an analytical column, and if the sample is an "other container," the device aspirates the sample, and injects it directly into the analytical column without undergoing hemolysis / dilution operations.

5. The method according to claim 3, characterized in that, in the glycohemoglobin measuring device based on the liquid chromatography method, a sample is placed on a rotary table capable of holding one or more samples, the rotary table rotates, the sample is transported to a sample sampling position, the device automatically identifies whether the sample is a "blood collection tube" or an "other container," and based on the identification result, if the sample is a "blood collection tube," the sample is aspirated, automatically subjected to hemolysis / dilution operations, and injected into an analytical column, and if the sample is an "other container," the sample is aspirated, and injected directly into the analytical column without undergoing hemolysis / dilution operations.

6. A method for measuring glycohemoglobin (A1c%) by loading the hemolyzed / diluted specimen prepared as claimed in claim 2 into a glycohemoglobin measuring device based on the principle of immunoassay.

7. A method for measuring glycohemoglobin (A1c%) by loading the hemolyzed / diluted specimen prepared as claimed in claim 2 into a glycohemoglobin measuring device based on an enzymatic method.

8. A method for measuring glycohemoglobin (A1c%) by loading the hemolyzed / diluted specimen prepared as claimed in claim 2 into a glycohemoglobin measuring device based on electrophoresis.

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