A device for hemolysis and dilution of puncture blood, and a method for analyzing glycated hemoglobin 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 (hereinafter also abbreviated as A1c and SA1c) is a value used as a diagnostic index for diabetes. There are several methods for measuring A1c depending on the number of tests (scale) and the required accuracy, etc. Broadly speaking, there are two methods: the immunoassay method and the HPLC method. The immunoassay method is a measurement by an immunochemical technique using an antibody, and the HPLC (High Performance Liquid Chromatography) method is a technique in which a sample is flowed through a device called a column at high pressure, and blood components are separated and quantified according to differences in affinity and charge state, and it is regarded as the most accurate method. In any method, venous blood of a patient is collected, and after performing a hemolysis / dilution operation, it is used for the measurement system.

Background Art

[0002] In a simple immunoassay method, it is common to collect several microliters to several tens of microliters of blood from a finger or the like using a disposable "puncture instrument". On the other hand, in the HPLC method, a sample collected in a blood collection tube of about 5 to 10 mL is used. In this case, the whole blood in the blood collection tube is automatically processed by a hemolysis / dilution mechanism provided in the measuring instrument and then subjected to analysis.

[0003] Also, in the HPLC method, as described above, blood collection tubes are often handled, but "puncture blood" like in a simple immunoassay method is not directly handled.

[0004] The measurement of A1c by the HPLC method includes a method based on the principle of ion exchange due to differences in charge and a method based on the principle of affinity due to specific adsorption and desorption. Fig. 1 schematically shows the obtained chromatogram. Fig. 1a shows the results of the ion exchange method, and Fig. 1b shows the results of the affinity method. Also, the left figure shows the case where A1c is low, and the right figure shows the case where A1c is high. In any case, the A1c% which is an index for diabetes is calculated as the ratio of glycated hemoglobin (A1c) to total hemoglobin. That is, it is calculated as the ratio of the peak area of glycated hemoglobin (A1c) (filled peak) to the total peak area.

[0005] Therefore, unlike quantitative analysis using general HPLC, 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 a major requirement, and slight variations do not affect the resulting A1c%. [Overview of the project] [Problems that the invention aims to solve]

[0006] The present invention was made to solve the aforementioned problems and provides a device and method for easily lysing / diluting blood from a puncture site, even with an A1c measuring 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, moved to the sampling location, and then sequentially injected into the analysis column for analysis. Figure 2 shows an example of the configuration of an A1c measuring device using the HPLC method.

[0008] In the HPLC method, the system broadly consists of a pump (3) capable of switching between and delivering multiple eluents (1) with different eluent strengths, a sample injection valve (31) for injecting the sample into an 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 certain amount of sample.

[0009] The sample injection mechanism includes, in addition to the injection valve (31), a dilution tank (35) for hemolysis / dilution of whole blood samples, and a metering pump (37) for adding hemolysis / diluent and aspirating samples.

[0010] In the field of blood analysis, venous blood is often collected in 2-10 mL blood collection tubes and measured directly in an analyzer. The same applies to A1c measurement using HPLC; the blood collection tubes are placed in a rack, automatically pre-processed, and then introduced into the analyzer. For A1c measurement, a small sample of whole blood is taken, hemolysis and dilution are performed automatically, and a portion of this sample is injected into the analytical column for analysis.

[0011] However, in the following cases, the sample should be manually hemolyzed and diluted before being subjected to analysis. (1) Patients or specimens for whom blood collection using blood collection tubes is difficult, (2) Lipo-dried specimens (calibration samples, control samples, etc.) In this case, measurement is possible by adjusting the volume to about 0.5 mL. The sample is then placed in a "dilution container" of about 2 mL, mounted on a "rack," and run through the analyzer. In this case, the aforementioned "pretreatment" is skipped. Therefore, analyzers that perform A1c measurement by HPLC are required to be able to automatically identify whether the sample is in a "collection tube" or a "dilution container."

[0012] One method for distinguishing between samples is by their height. This will be explained using Figure 3. Typically, blood collection tubes are 80 mm or longer, so the explanation will assume the use of a rack approximately 60 mm high. In this case, about 25 mm of the blood collection tube will protrude from the top surface of the rack. The "dilution container" will have a "flange" and will protrude about 10 mm from the top surface of the rack. Two "sensors" with different detection heights will be placed. The first sensor will be placed at a height that is "unreacted" for the dilution container and "reacted" for the blood collection tube, and the second sensor will be placed at a height that is "reacted" for both the dilution container and the blood collection tube. The combination of these responses will allow for the identification of the sample type. The sensors that detect this height can be contact sensors or optically discriminative 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 and the second sensor both "react," the sample is determined to be a "blood collection tube," and it moves to the sampling position to sample a small amount of blood. This sample is then automatically lysed / diluted, and a portion of it is injected into the analysis column for A1c measurement.

[0014] As shown in Figure 6, when the sample is mounted on a rack and moved to the height detection position, if the first sensor shows "no reaction" and the second sensor shows "reaction", the sample is determined to be a "diluted sample (dilution container)", moves to the sampling position, a certain amount is sampled, injected into the analysis column, and A1c is measured.

[0015] Furthermore, if both the first and second sensors show "no reaction," it is determined that no sample is loaded in the rack, and the measurement is skipped. If the first sensor shows "reaction" and the second sensor shows "no reaction," it is determined to be an "error."

[0016] Furthermore, in the field of clinical diagnosis, barcodes and QR codes (registered trademarks) are often used to electronically record patient information and to save labor and prevent sample mix-ups. In A1c measurement using the HPLC method, barcodes are often attached to the sample and automatically read by the measuring device. When using blood collection tubes, the length is about 100-130 mm, providing ample space to attach a barcode (see Figures 11a and 11b). On the other hand, in the case of diluted samples, the container is small and does not provide enough space to attach a barcode. As mentioned earlier, when measuring A1c in a blood sample using HPLC, it is necessary to perform the measurement manually, as shown in Figure 5.

[0017] First, a lancet is used to puncture a finger or other area, and approximately 1-50 μL of blood is drawn. 10 μL is then drawn up using a capillary tube, and dispensed into a dilution cup containing a predetermined amount of hemolysis / diluent. Hemolysis / dilution is performed by pipetting, and the sample is placed on a measurement rack for A1c measurement using the HPLC method. This process is very time-consuming, and because barcode management is not possible, human errors such as sample mix-ups cannot be completely eliminated. 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 details of the present invention will be described below. First, the first embodiment will be described. A resin conical or pyramidal tip capable of drawing up puncture blood by capillary action, A container capable of storing the aforementioned chip, A tip set comprising a lid that can seal the aforementioned container, capable of collecting, lysing, and diluting puncture blood, The inner diameter of the container is 0.5 to 5 mm larger than the maximum outer diameter of the chip. The container has a cylindrical shape with a height that allows the chip to be stored inside, The container has a flange located 5 to 15 mm from the top surface. A tip set characterized by its ability to collect, lyse, and dilute blood from a puncture. First, use a lancet to puncture a finger or other body part to draw blood.

[0019] As the first step, The aforementioned tip draws up a certain amount of 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 determine this considering the amount of hemolysis / diluent, with about 15 μL being preferable.

[0020] As the second step, Insert a chip that has aspirated puncture blood into a hemolysis / dilution container that has been pre-dispensed with a certain amount of hemolysis / dilution liquid, and then cover it with a lid. It is desirable to determine the amount of hemolysis / dilution liquid in consideration of the blood collection volume, and it is preferable to achieve a final dilution ratio of about 1 / 150. When the blood collection volume in step 2 is 15 μL, a hemolysis / dilution liquid volume of 2500 μL is preferable. The hemolysis / dilution liquid only needs to be compatible with the analytical instrument used for measurement, and there is no limitation on its composition, etc.

[0021] As a third step, Invert and stir the sealed container to hemolyze / dilute the puncture blood. Since the inner diameter of the container is about 0.2 to 5 mm larger than the maximum outer diameter of the chip, there is a gap between the inner surface of the container and the chip. Since the chip for blood collection is made of resin and has a low specific gravity, it will move up and down in the liquid by inversion and stirring, and hemolysis / dilution can be efficiently performed.

[0022] Also, the "diluted sample" prepared up to the above steps As a fourth step, Remove the lid, and with the chip for blood collection inside, place the container as it is on a rack for A1c measurement, and perform A1c measurement by HPLC method. The container processed above is placed on the rack and sequentially moved to a position for determining the specimen type. Here, since the container has a "flange", about 10 mm or so of the part above the flange protrudes. The height sensor 1 is in the "unreacted" state, and the height sensor 2 is in the "reacted" state, and it is discriminated as a diluted specimen. Therefore, the sampling needle only descends about 20 mm from the upper surface of the rack, aspirates the diluted specimen, and is injected into the analysis column "without pretreatment" for analysis.

[0023] During the descent of the sampling needle, the blood collection chip floating in the container is pushed down by the tip of the needle, but there is no hindrance to the aspiration of the specimen. At this time, due to the tumbling agitation, the blood collection chip in the container often adheres to the inner surface of the container or becomes inclined. However, since it is pushed down together during the descent of the needle and reaches the central position, there is no hindrance to the aspiration of the specimen, which is also a major feature of the present invention (see Fig. 10).

[0024] In addition, the container of the present invention has a length of about 45 mm extending downward from the rack, and as shown in Fig. 11c, it is also possible to attach a barcode, and the fact that it can be automatically read / managed on the side of the analytical instrument is also a major feature of the present invention.

[0025] The hemolysis / dilution container of the present invention is not limited to the above-described form, and any form that can achieve the same effect is acceptable. For example, as shown in Fig. 12c, even if it has the same container shape as described above but without a "flange" structure. In that case, if a spacer is inserted between the rack and the container so that the height of the container from the upper surface of the rack reacts with the height sensor 2 and does not react with the height sensor 1, the same effect can be obtained (mode 2). Also, as shown in Fig. 12d, if the container has a length that reaches the bottom surface of the rack similar to a blood collection tube and the height of the portion above the upper surface of the rack reacts with the height sensor 2 and does not react with the height sensor 1, it may have a structure without a "flange", and the same effect can be obtained (mode 3).

Brief Explanation of Drawings

[0026] [Figure 1] It is a diagram schematically showing the pattern of the chromatogram in the HPLC method. Fig. a shows the ion exchange mode, and Fig. b shows the affinity mode. In both cases, the shaded component indicates the component of A1c. [Figure 2] It is a diagram showing an example of the configuration of an A1c measuring device in the HPLC method. [Figure 3] It is a diagram schematically showing the types of specimen containers to be handled and the descent position of the sampling needle in an A1c measuring device in the HPLC method. [Figure 4] It is a diagram schematically showing the operation when it is determined as a "blood collection tube" in an A1c measuring device in the HPLC method. [Figure 5] This diagram schematically illustrates the procedure for handling puncture blood samples when using an A1c measurement device with the HPLC method. [Figure 6] This diagram schematically illustrates the operation of an A1c measuring device using the HPLC method when it is determined to be a "dilution container." [Figure 7] This diagram schematically illustrates the process from collecting puncture blood to hemolysis / dilution according to the present invention. [Figure 8] This flowchart illustrates the procedure for analyzing hemolyzed / diluted samples using HPLC according to the present invention. The thick solid line in the diagram shows the procedure for measuring blood samples obtained by puncture. The dashed line in the diagram shows the procedure for measuring blood collected from a blood collection tube (whole blood). [Figure 9] This figure schematically illustrates the determination of sample type and the descent position of the sampling needle when analyzing a hemolyzed / diluted sample by HPLC according to the present invention. [Figure 10] This diagram schematically illustrates the movement of the tip used for sample collection when analyzing a hemolyzed / diluted sample by HPLC according to the present invention. [Figure 11] This diagram schematically illustrates the method of measuring by attaching a barcode to a hemolysis / dilution container according to the present invention. [Figure 12] This diagram schematically shows another form of the hemolysis / dilution container according to the present invention and its positional relationship when mounted on a rack. [Figure 13] This diagram shows the dimensions of the blood collection tip and hemolysis / diluent container used in the example. [Figure 14] This diagram shows the steps from sample collection to dissolution / dilution in the example. [Figure 15] The results of hemolysis / dilution using the tip according to the present invention, obtained in Example 1, are shown. Figure a shows the visible absorption spectrum, and Figure b shows the absorbance at a specific wavelength (calibration curve). [Figure 16] The results of the reproducibility of the hemolysis / dilution method using the tip according to the present invention, obtained in Example 1, are shown. Figure a is a superimposed visible absorption spectrum, and Figure b shows the change in absorbance at a specific wavelength. [Figure 17]This figure shows the Tosoh "Glycohemoglobin Analysis System GHbVIII," the A1c measurement device used in Example 2 for the HPLC method. Figure a is an overall view, and Figure b is a magnified view of the sample transport area. [Figure 18] This diagram shows the configuration of the Tosoh "Glycohemoglobin Analysis System GHbVIII," which is the A1c measurement device used in Example 2 for the HPLC method. [Figure 19] This diagram shows the sample rack and sample types in the Tosoh "Glycohemoglobin Analysis System GHbVIII," which is the A1c measurement device used in Example 2 for the HPLC method. [Figure 20] This diagram schematically shows the operation of the sample rack in the Tosoh "Glycohemoglobin Analysis System GHbVIII," which is the A1c measurement device used in Example 2 for the HPLC method. [Figure 21] This diagram shows the dimensions and sensor positional relationship when a blood collection tube is mounted on the Tosoh "Glycohemoglobin Analysis System GHbVIII," which is the A1c measurement device using the HPLC method used in Example 2. [Figure 22] This diagram shows the dimensions and sensor positional relationship when a conventional "dilution sample container" is mounted on the Tosoh "Glycohemoglobin Analysis System GHbVIII," which is the A1c measurement device for the HPLC method used in Example 2. [Figure 23] This figure shows the dimensions and sensor positional relationship when the "diluted sample container" of the present invention is mounted on the Tosoh "Glycohemoglobin Analysis System GHbVIII," which is the A1c measurement device for the HPLC method used in Example 2. [Figure 24] This figure compares a conventional "diluted sample container" with 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] This figure shows the results of measurements taken in Example 2 after diluting / hemolyzing whole blood (difference in measurement method). Figure a shows the raw data, and Figure b shows the results normalized by the height of the A1c peak. [Figure 26]This figure shows the results (reproducibility) obtained in Example 2 after diluting / hemolyzing whole blood. Figure a shows the raw data, and Figure b shows the results normalized by the height of the A1c peak. [Figure 27] This figure shows the reproducibility in Example 2. [Figure 28] This shows the results of measurements taken by attaching a barcode to the container of the present invention. [Figure 29] This diagram schematically shows the needle drop when a diluted sample is identified (determined) as a dilution of an A1c sample using the Tosoh "Glycohemoglobin Analysis System GHbGX," an A1c measurement device using the HPLC method. [Figure 30] This diagram schematically illustrates the flow of applying the container and hemolysis / dilution procedure of the present invention to A1c measurement. The lower right diagram shows the application to liquid chromatography, while the lower left diagram shows the application to POCT instruments using immunoassay, enzymatic methods, electrophoresis, etc. [Modes for carrying out the invention]

[0027] The present invention will be described in detail below. However, the present invention can be implemented in different forms and is not limited to the embodiments and examples shown below. [Examples]

[0028] In this example, the following blood collection tips and hemolysis / dilution containers were used (see Figure 13). The blood collection tips were made of resin and were conical in 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 and an inner diameter of 13.2 mm, with a flange (outer diameter 13.2 mm) located approximately 45 mm from the base. The maximum container capacity was 3 mL. When a blood collection tip is inserted into this hemolysis / dilution container, there is a clearance of approximately 1.6 mm. For hemolysis / diluent, we used reagents specifically designed for the glycated hemoglobin analyzer manufactured by Tosoh Corporation. (Example 1) First, we verified the accuracy of the blood collection and hemolysis / dilution method using the chip in this invention. Here, instead of using blood from a puncture, we dripped approximately 50 μL of whole blood collected in a blood collection tube into a microcontainer to simulate blood from a puncture (see Figure 14).

[0030] The verification was carried out using the following procedure. Dispense 2.5 mL of hemolysis / diluent into the hemolysis / diluent container mentioned above. Apply the blood collection tip to the side of the infused 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 invert and mix 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. Therefore, by inverting and mixing, the tip containing blood moves up and down, mixing with the liquid and allowing for hemolysis / dilution. A fixed amount of the diluted sample was dispensed into a cell for a spectrophotometer, and the visible absorption spectrum was measured. This operation was repeated 10 times to verify accuracy. In addition, to calculate the degree of dilution (dilution ratio), the same sample was manually diluted 50 times, 100 times, 150 times, and 200 times, and the visible absorption spectrum was measured in the same manner.

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

[0033] [Table 1]

[0034] [Table 2]

[0035] The spectrophotometer used was a Shimadzu UV-2600. From the calibration curve obtained by manual methods, it can be calculated that the blood collection and hemolysis / dilution method using the chip of the present invention achieves a dilution of approximately 150 times. Furthermore, it was found that the concentration reproducibility is approximately 6.5%.

[0036] In typical quantitative analysis using HPLC, a reproducibility of 6% is often considered too high and unacceptable. However, in A1c measurement, since it is evaluated in area percentage, a 6% variation in dilution does not affect the final A1c percentage. Therefore, the accuracy of hemolysis / dilution is acceptable. (Example 2) Here, the usefulness of the present invention was verified by actually using a glycated hemoglobin analyzer by the HPLC method. The "Glycated Hemoglobin Analyzer GHbVIII" manufactured by Tosoh Corporation was used as the glycated hemoglobin analyzer. Furthermore, the eluent, hemolysis / diluent, column, etc., were all the dedicated products sold by the same company.

[0037] This "Glycohemoglobin analyzer GHbVIII" can handle two types of samples: blood collection tubes (whole blood) and diluted samples. The device uses a rack (17) that can hold 10 samples, which are then placed on a rack loader (13). Upon instruction to analyze, the rack loader is driven, sequentially moving the samples to the sample ring position. A needle then samples the samples for analysis (see Figure 17). Two height sensors are located in front of the sample ring position to distinguish between sample types. The rack (17) is 65 mm high.

[0038] The first height sensor is positioned 70 mm from the bottom, and the second height sensor is positioned 80 mm from the bottom. As a sample passes through, the system distinguishes between a blood collection tube (whole blood) and a diluted sample based on the combination of responses indicating whether or not the sensor flags come into contact with the sample.

[0039] The system can accommodate blood collection tubes ranging from 75mm to 100mm in length. Diluted samples are dispensed into dedicated resin containers (approximately 40mm in length, 2mL in capacity), as shown in Figure 6. These diluted sample containers have a flange, and when mounted on the rack, approximately 10mm of the flange protrudes above the rack. In other words, when the diluted sample containers are mounted on the rack, the top surface of the flange is approximately 750mm from the bottom. On the other hand, when blood collection tubes are mounted on the rack, the top surface of the blood collection tubes is 20mm to 40mm.

[0040] In the case of a diluted sample container, only height sensor 2 responds, while in the case of a blood collection tube, both height sensors 1 and 2 respond. This difference allows the system to determine the sample type and the needle's descent position. If a diluted sample container is detected, the needle descends only to near the bottom of the container (45 mm from the bottom of the rack), aspirates approximately 100 μL of diluted sample, and introduces a portion of it (a few μL) into the analysis column. On the other hand, if a blood collection tube is detected, the needle descends to near the bottom of the tube (25 mm from the bottom of the rack), aspirates a few μL of whole blood, and after hemolysis / dilution by the built-in automatic dilution mechanism, introduces a portion of it (a few μL) into the analysis column.

[0041] On the other hand, when using the hemolysis / dilution container of the present invention, since it has a flange (outer diameter 13.2 mm) located about 45 mm from the bottom surface, when mounted on a rack, the flange catches on the rack, and about 13 mm protrudes above the rack. The height from the bottom of the rack will be approximately 75mm.

[0042] In other words, similar to the dedicated dilution sample container for this device mentioned above, when detecting the sample type, the height sensor is used. 2 Only the sample that reacts is recognized as a "diluted sample," and the needle descends to a position 45 mm from the bottom of the rack, drawing in approximately 100 μL of the diluted sample, with a portion of it (a few μL) being introduced into the analytical column.

[0043] Because the blood collection tip inserted into the container is made of resin, it will float in the hemolytic / diluent. Alternatively, it may adhere to the inner surface of the container, sticking higher than expected, or adhere to the inner surface at an angle, causing its center position to shift. However, as the needle descends, it pushes the tip down, so even if the center position is shifted, the tip will be in the center position at the lowest point of the needle's descent, allowing for normal sampling.

[0044] Similar to Example 1, blood was collected using the method of the present invention, and the hemolyzed / diluted container was loaded into the "Glycohemoglobin Analyzer GHbVIII" for measurement. 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 sampling directly from a blood collection tube and measuring after hemolysis / dilution by the automatic dilution mechanism of the device are also shown.

[0045] [Table 3]

[0046] In Figure 25a, *1 shows the result (chromatogram) of measurement after directly sampling from a blood collection tube and hemolysis / dilution using the device's automatic dilution mechanism, *2 shows the result (chromatogram) of the conventional manual method, and *3 shows the measurement result (chromatogram) of the method of the present invention. It can be seen that although the intensity of the method of the present invention (*3) is slightly lower, all measurement methods show similar patterns. To make it easier to understand, when the output of the A1c peak is normalized to "50" and compared, it can be seen that the three data sets 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 equivalent, suggesting that there are no problems with measuring using the method of the present invention.

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

[0049] [Table 4]

[0050] Furthermore, in the field of clinical diagnosis, patient information is often recorded electronically, and barcodes and QR codes (registered trademarks) are frequently used to save labor and prevent sample mix-ups. In A1c measurement using the HPLC method, barcodes are often attached to samples and automatically read by the measuring device.

[0051] When using blood collection tubes, their length is approximately 100-130 mm, providing ample space to attach a barcode (see Figure 11a). On the other hand, diluted samples use smaller containers, making it impossible to secure enough space for a barcode. Therefore, when a barcode is required for a diluted sample, a makeshift solution is used, as shown in Figure 11b, where the barcode is attached to a cylindrical adapter and the dilution container is placed on top of it. This method is time-consuming and costly, and is therefore often only used for lyophilized calibration samples or control samples.

[0052] The dilution and hemolysis container of the present invention has a total length of 55 mm and a length of 50 mm below the flange, allowing for the attachment of a barcode. This makes it useful even when measuring actual patient samples (see Figure 11c).

[0053] Figure 28 shows an example of a barcode being attached to this container and measured. 'a' is the barcode, and 'b' is the reading result (report). As you can see, the barcode can be read accurately.

[0054] In this embodiment, the GHbVIII glycated hemoglobin analyzer manufactured by Tosoh Corporation, which uses a sample rack capable of holding 10 samples, is described as an example. However, for example, the GHbX glycated hemoglobin analyzer also manufactured by Tosoh Corporation, which uses a rotating table as shown in Figure 29 to load samples and sequentially transports them to the sample aspiration position by rotation, can achieve a similar effect. Therefore, the structure of the sample rack does not limit the drive method.

[0055] Furthermore, in immunoassays, many POCT (Point of Care Testing) devices targeting blood samples have become commercially available. Although the measurement process varies depending on the model, it consists of three steps: "the first step of collecting puncture blood," "the second step of hemolysis and dilution," and "the 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 healthcare professional, or automatically by the device. The third step will be performed on the equipment side.

[0056] These operations should ideally be as simple and inexpensive as possible. Furthermore, the second step should be automated by the equipment as much as possible to prevent human error and reduce the risk of infection.

[0057] Up to this point, 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 method, and is not limited to the method of A1c measurement. The hemolysis / dilution step of the present invention is performed in a closed system, and when setting it in the measuring device, it is only necessary to remove the cap without removing the blood collection chip, thus reducing the risk of infection. [Explanation of Symbols]

[0058] 1. Eluent 2. Hemolytic lavage solution 3. Fluid delivery mechanism 4. Automated sample dilution mechanism 5. Specimen injection mechanism 6. Analysis Columns 7. Visible light detector 8. Specimen transport rack 9. Drain 10. Sample height sensor 1 11. Sample height sensor 2 12. Specimen aspiration location 13. Specimen transport mechanism 14. Blood collection tube 15. Container for diluted specimen 16.Measurement part 17. Hemolysis / Dilution Container 18. Lid 19. Puncture blood collection tip 20. Sampling needle 21.Puncture device 22. Spacer 23. Tsuba (projection) 24. Barcode reader 25. Barcode 26. Adapter 27. Turntable 28. Sample holder 29. Eluent Opening / Closing Mechanism 30. Liquid transfer pump 31. Injection valve 32. Sample holding loop 33. Hemolytic wash solution switching mechanism 34. Constant temperature bath 35. Hemolysis / Dilution Tank 36. Degassing device 37. Metering pump

Claims

1. A resin conical or pyramidal tip capable of drawing up puncture blood by capillary action, A container capable of storing the aforementioned chip, This chipset comprises a lid that can seal the aforementioned container, is capable of collecting, lysing, and diluting puncture blood, and can be mounted on the rack (8) of a glycated hemoglobin measuring device. The height of the aforementioned rack is 65 mm. The glycated hemoglobin measuring device is A height sensor 2 (11) for detecting a container is located 70 mm from the bottom surface of the rack, The rack is equipped with a height sensor 1 (10) that detects a container at a position 80 mm from the bottom surface of the rack, 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 with a height that allows the chip to be stored inside, The container has a flange located 5 to 15 mm from the top surface. A method for collecting, hemolyzing, and diluting puncture blood using the tip set, characterized in that when the container is mounted on the rack, the top surface of the container is positioned at a height of 70 to 80 mm from the bottom surface of the rack, The first step involves using the aforementioned tip to draw up the punctured blood by capillary action, The second step involves inserting the tip from which the puncture blood has been drawn into the container from which a certain amount of hemolytic and diluent solution has been dispensed in advance, and then closing the container with the lid. A method for collecting, lysing, and diluting puncture blood, comprising a third step of inverting and stirring the sealed container to lyse and dilute the puncture blood.

2. The method according to claim 1, wherein, after the third step, the container containing the hemolysis and diluent is mounted on the rack of a glycated hemoglobin measuring device based on the principle of liquid chromatography, and glycated hemoglobin (A1c%) is measured.

3. The method according to claim 1, wherein, after the third step, the container containing the hemolysis and diluent is mounted on the rack of a glycated hemoglobin measuring device based on the principle of immunoassay, and glycated hemoglobin (A1c%) is measured.

4. The method according to claim 1, wherein, after the third step, the container containing the hemolysis and diluent is mounted on the rack of a glycated hemoglobin measuring device based on the principle of enzymatic methods, and glycated hemoglobin (A1c%) is measured.

5. The method according to claim 1, wherein, after the third step, a container containing hemolysis and diluent is mounted on a rack of a glycated hemoglobin measuring device based on electrophoresis, and glycated hemoglobin (A1c%) is measured.

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