JP6605990B2 - Management substance for accuracy control of blood analyzer and manufacturing method thereof - Google Patents

Description

  The present invention relates to a quality control substance used for calibration of a blood analyzer used for measurement of hemoglobin A1c, blood glucose level, and hemoglobin concentration, or quality control, and a method for manufacturing the same.

  Glycohemoglobin in blood, which is an important management index for diabetes, blood sugar level, and hemoglobin concentration, which is an important index for anemia diagnosis, can be measured using a blood analyzer.

  Here, it is regularly inspected and confirmed that the blood analyzer is properly calibrated to give an accurate value and that the blood analyzer can be measured stably and accurately. It is important that management is carried out.

  Particularly, hemoglobin A1c, which is a kind of glycohemoglobin, is required to be standardized internationally and to calibrate the measurement result strictly.

  In recent years, with the diversification of clinical tests, tests performed next to patients called POCT (point of care testing) are easy to measure and are attracting attention as real-time tests. Other than laboratories such as wards and clinics Use in is widespread. On the other hand, an increase in the population of lifestyle-related diseases including diabetes has become a social issue, and in March 2014, part of the law on clinical laboratory technicians was revised to realize self-medication to promote self-health management. In the specimen measurement room, examinations using the POCT device by the examinee himself / herself have been accepted.

  In the inspection using the POCT equipment, it is necessary to control the accuracy as in the case of the inspection performed in a normal laboratory, and to guarantee the accuracy of the apparatus. Also, the accuracy management is required in the guidelines of the specimen measurement room. It is stated that there is.

  At present, freeze-dried products such as JCCRM423 are used as quality control substances for hemoglobin A1c.

  In addition, an ultra-low temperature storage product such as JCCRRM411 (JDS Lot 5) that requires ultra-low temperature storage can also be used as a quality control substance.

  Therefore, the freeze-dried product and the ultra-low temperature storage product are used for calibration and accuracy control of the conventional blood analyzer. In addition, the use of residual blood after a blood sampling test is also being studied.

International Publication WO2010 / 101206

Effects of whole blood storage on hemoglobin a1c measurements with five current assay methods, CLINICAL CHEMISTRY, Vol. 29, No. 6, 1983 1113: Randie R. Little, et al.

  Usually, in order to keep the hemoglobin solution stable for a long period of time, it must be stored at an ultra-low temperature of −70 ° C. or lower, and in the case of refrigerated storage at about 2 to 8 ° C. or frozen storage at about −20 ° C. in a general refrigerator-freezer, Therefore, it is said that long-term stable storage is not possible.

  This is because proteins are generally said to be able to be stored more stably as they are placed at lower temperatures, but they are not stable in a frozen state, and as shown in Non-Patent Document 1, at insufficient temperatures. Even in a frozen state, it is also grounded from reports that freeze denaturation may occur, such as an environment in which pH and hydrogen peroxide concentration increase and it is easily oxidized.

  In contrast, the freeze-dried product can be stored in a refrigerator or a freezer for home use (stored at -20 ° C), but since the storage state is dry, the lyophilized product is liquefied in order to use it. Is required. When this liquefaction operation is performed, laboratory instruments such as a micropipette, a tip, and purified water for diluting the lyophilized product are required. Further, since the liquefaction operation of the freeze-dried product is usually performed manually, skill is required to obtain an accurate control substance.

  In many cases, a sample measurement room installed in a pharmacy or the like does not have equipment such as the experimental instrument. Furthermore, since the liquefaction work is performed by dissolving the lyophilized product in a fixed volume of solution, precise work is required and work time is required. Therefore, the accuracy of the blood analyzer using the lyophilized product should be managed daily. It is difficult. Furthermore, since the processing is performed manually, there may be a problem in terms of quality deterioration and work accuracy.

  Further, the freeze-dried product is generally used after being dissolved in a solution of several hundred μL. With POCT equipment, it is possible to measure with a small amount of sample of about several μL. However, once lyophilized product is melted, it can be discarded without being used because it is refrigerated for about 2 weeks. .

  On the other hand, it is necessary to store in an environment of −70 ° C. for an ultra-low temperature storage product, and handling in a general examination room or specimen measurement room is difficult because it is too costly to maintain equipment.

  In addition, the accuracy control method using residual blood after a blood sampling test causes the hemoglobin solution to oxidize at room temperature or in a refrigerated environment, which increases measurement error due to an increase in methemoglobin concentration. In order to store the hemoglobin solution for a long period of time, an ultra-low temperature freezer is required after all. Then, it is difficult to handle residual blood after a blood collection test in a general laboratory or specimen measurement room as in the case of the ultra-low temperature storage product.

  In this way, with existing managed substances, the cost for accuracy control and the difficulty of work are high, which becomes a factor for raising the inspection unit price. In particular, in a sample measurement room scheduled to be installed in a general pharmacy, such a burden is not preferable for the spread of accuracy management of blood analyzers.

  On the other hand, if the accuracy control of the blood analyzer is not thoroughly performed, poor test values are passed to the examinee, which may have a harmful effect on the health and welfare of the examinee, and in general on the medical treatment.

  Due to these problems, there has been a high demand for a quality control substance that can stably store hemoglobin even in a frozen state.

  Therefore, as a means for solving the above-mentioned problems, the control substance for blood analyzer accuracy control according to the present invention is to wash erythrocytes using a phosphate buffer solution that is isotonic with blood osmotic pressure or a hypertonic phosphate buffer solution. In addition, a hemoglobin extract obtained by hemolyzing red blood cells after replacing the internal solution of red blood cells with a phosphate buffer solution that is isotonic with blood and osmotic pressure, and a diluted solution of the phosphate buffer solution and the cryoprotectant It is a hemoglobin solution characterized by comprising as follows.

  The hemoglobin solution is a quality control management substance used for calibration of a blood analyzer used for measuring hemoglobin A1c, blood glucose level, and hemoglobin concentration, or for quality control.

  It is preferable that the quality control substance is sealed in a container having a certain capacity. Thereby, the quality control substance can be used without waste, and the cost required for the measurement by the analyzer can be reduced.

Further, as a means for solving the above problems, as a method for producing a quality control substance according to the present invention, it is used for calibration of a blood analyzer used for measuring hemoglobin A1c, blood glucose level, and hemoglobin concentration, or for quality control. A method for producing a controlled substance for quality control, comprising:
(A) centrifuge blood to remove plasma and buffy coat;
(B) a step of washing erythrocytes using a phosphate buffer solution that is isotonic with the osmotic pressure of the blood or a hypertonic phosphate buffer solution;
(C) adding a phosphate buffer solution with isotonicity of blood and isotonicity to the washed erythrocytes, and allowing to stand to replace the inside of the erythrocytes with the phosphate buffer solution; and
(D) centrifuging and removing red blood cells;
(E) after freezing the extracted red blood cells, lysing the red blood cells by thawing at room temperature, and creating a hemoglobin extract;
(F) adjusting the hemoglobin concentration by diluting the hemoglobin extract with a diluent containing the phosphate buffer and a cryoprotectant;
(G) a step of precipitating and removing an erythrocyte membrane using a centrifuge at a low temperature.

  The phosphate concentration in the phosphate buffer used in steps (B) and (C) is preferably 10 mM or more and 154 mM or less. A pH of 10 mM or more is preferable because the pH of the hemoglobin solution is kept neutral. Moreover, if the phosphate concentration is higher than 154 mM, the osmotic pressure of the phosphate buffer solution is likely to be higher than the osmotic pressure of blood.

  In the step (B), a phosphate buffer having a higher tension than the osmotic pressure of blood can be used. The hypertonic phosphate buffer solution causes erythrocytes to contract during deformation, and can cause stress on the erythrocytes. Therefore, it should be noted that if the osmotic pressure of the phosphate buffer is too high, hemolysis may occur from the stress. If an isotonic phosphate buffer is used in the step (B), the red blood cells are not contracted and deformed, so that the red blood cells that are hemolyzed can be minimized.

  Also in the step (C), by making the osmotic pressure of the phosphate buffer isotonic with the osmotic pressure of blood, the phosphate buffer can be introduced into the erythrocytes without hemolysis of the erythrocytes.

  Moreover, it is preferable that the density | concentration of the antifreezing agent contained in the quality control substance is 20-40 weight% with respect to the weight of the whole quality control substance. This is because if the concentration is lower than 20% by weight, a sufficient antifreezing effect cannot be obtained, and if it is 50% by weight or more, the hemoglobin in the quality control substance is denatured.

  The quality control management substance obtained by the present invention can be used for quality control of either a glycated hemoglobin measuring device or a blood glucose level measuring device.

  ADVANTAGE OF THE INVENTION According to this invention, even if it is a freezer of about -20 degreeC, it can preserve | save without impairing quality in a liquid state, and can provide the management substance for quality control which can be used in the state taken out from the freezer. .

  As a result, it is possible to provide a quality control substance that can be stored at a lower cost than conventional ultra-low temperature storage products and can be used more easily than conventional freeze-dried products.

The composition for quality control by the present invention and the whole blood as a control sample are analyzed by cation exchange chromatography on the day of preparation of the quality control material and after storage at −20 ° C. for 5 weeks. It is a figure which shows the analysis result performed. The left figure is blood prepared by the production method according to the present invention, and the right figure is a chromatogram of control blood. The upper part of the figure shows the blood on the day of preparation, and the lower part shows the chromatogram of the blood after 5 weeks of frozen storage. HbSSG is glutathionyl Hb, which is a type of modified hemoglobin in which reduced glutathione inside erythrocytes is oxidized to become oxidized glutathione and modified into hemoglobin.

  Embodiments according to the present invention will be described below. The embodiments described below are preferred embodiments of the present invention, and thus various technically preferable limitations are given. However, the scope of the present invention is particularly limited in the following description. Unless otherwise described, the present invention is not limited to these embodiments.

<Manufacturing method of quality control substances>
1. Red blood cell extraction step 25 mL of fresh whole blood (oxyhemoglobin) added with anticoagulant (EDTA-2k) was dispensed into a 50 mL centrifuge tube and 3000 rpm for 15 minutes using a centrifuge set at 4 ° C. Centrifuge and remove supernatant plasma and buffy coat to obtain residual red blood cells. Here, the buffy coat refers to a milky white layer obtained above the red blood cell layer when whole blood is centrifuged, and includes white blood cells and platelets.
2. Erythrocyte washing step The erythrocyte inside the centrifuge tube is 12 mL, and a washing buffer solution (50 mM phosphate, 104 mM sodium chloride, pH 7.5) that is 3 times the volume of the erythrocyte and is made isotonic with whole blood is added to the centrifuge tube. In addition, after stirring, the red blood cells are separated by centrifugation at 3000 rpm for 15 minutes using a centrifuge, and the red blood cells are washed by removing the supernatant (washing buffer and plasma components). This washing operation is repeated once more, and the washing operation is performed twice in total.
3. Step of Replacing Solution Components in Red Blood Cells with Phosphate Buffer Solution Three times the amount of washing buffer solution is added to the red blood cells after the washing step, and left in a temperature environment of 4 ° C. for 12-14 hours. This is because, if it is allowed to stand for 12 to 14 hours in a temperature environment of 4 ° C., hemoglobin methation can be minimized and a phosphate buffer solution can be sufficiently introduced into erythrocytes. Red blood cells in which the internal solution components are filled with a phosphate buffer are called substituted red blood cells.
4). Substituted red blood cell extraction step After the replacement step with phosphate buffer, the centrifuge tube is centrifuged at 3000 rpm for 15 minutes to separate red blood cells, and the supernatant (washing buffer) is removed to extract substituted red blood cells.
5. Freezing step The substituted red blood cells are frozen in an ultra-low temperature freezer at -60 ° C or lower.
6). Thawing process Replaced red blood cells frozen in an ultra-low temperature freezer are thawed at room temperature. During this thawing process, the erythrocyte membrane of the substituted erythrocyte is destroyed, and hemoglobin in the erythrocyte is eluted. The eluted hemoglobin solution is referred to as a concentrated hemoglobin solution.
7. Dilution step Immediately after the thawing step, the concentrated hemoglobin solution is diluted with a diluent containing a phosphate buffer and a cryoprotectant, and adjusted to the concentration required as a controlled substance. Glycerin is used for the cryoprotectant. Examples of the composition of the diluent include the following.
(1) Low value control diluent:
50 mM phosphoric acid, 12.2 mM glucose, 74 mM NaCl, 30 mM NaN ₃ , 70% glycerin, pH 7.5
(2) Diluent for high value control:
50 mM phosphoric acid, 22.2 mM glucose, 74 mM NaCl, 30 mM NaN ₃ , 70% glycerin, pH 7.5
NaN ₃ is sodium azide and is added as a preservative.
8). Erythrocyte membrane removal step The diluted concentrated hemoglobin solution is centrifuged at 3000 rpm for 15 minutes using a centrifuge set at 4 ° C. to precipitate and remove the erythrocyte membrane, and the resulting substance is used as a quality control substance. .
9. Dispensing process The obtained quality control substances are divided into parts to be used for one calibration or quality control, and dispensed into containers such as eye drops. The dispensed quality control substances are stored in a liquid state in a freezer at about -20 ° C.

  Hereinafter, each process will be described based on the technical significance.

  The reason for removing the buffy coat in “1. Erythrocyte extraction step” is that the buffy coat is unnecessary for the measurement of hemoglobin A1c, glucose, and Hb concentrations. This is because there is a possibility that the pH and the like may be changed by metabolism or the like, and the influence is removed.

  In “2. Red blood cell washing step”, the use of a washing buffer solution comprising a phosphate buffer as a washing solution for red blood cells could prevent hemoglobin methation (heme oxidation) in red blood cells. On the other hand, when physiological saline is used for washing erythrocytes, the pH of the hemoglobin solution cannot be controlled, and hemoglobin is denatured, so that it may not be possible to produce a substance that can be stored for a long time as a quality control substance. Therefore, a phosphate buffer having a pH buffering ability was used as a washing solution. The phosphate concentration in the washing buffer is preferably 10 to 154 mM. As a washing buffer, a neutrally prepared one was used to prevent hemoglobin in red blood cells from being oxidized.

  By selecting phosphoric acid with a molecular weight of 98 as the buffer solution, it is possible to easily pass through the erythrocyte membrane, and the influence on the living body is kept low because phosphoric acid is a substance that is relatively contained in the living body. Can do.

  According to “3. Step of Replacing Solution Components Inside Red Blood Cells with Phosphate Buffer Solution”, the solution components inside red blood cells are discharged out of the cell membrane of red blood cells, and hemoglobin and the solution components are separated, followed by “4. The solution component can be efficiently removed after the hemoglobin in the erythrocytes remains by the “replacement erythrocyte extraction step”.

  After that, the concentrated hemoglobin solution extracted in “6. Thawing process” from the red blood cells frozen in “5. Freezing process” maintains the salt concentration in which the phosphate buffer solution is permeated into the substituted erythrocytes. . Since the salt concentration of the phosphate buffer used in the present embodiment is almost the same as the salt concentration of whole blood, it can be a quality control substance suitable for calibration and precision control of the blood analyzer.

  The antifreezing agent added in “7. Dilution step” can keep the prepared control material for accuracy control in a liquid state without being frozen even at a low temperature of about −20 ° C. Antifreezing agents are added in order to make it possible to store in a liquid form a control substance for quality control that could not be stored for a long time unless it was in a dry state in a conventional storage environment of about −20 ° C. Is. In the present embodiment, glycerin is used, but ethylene glycol can also be used as another antifreeze agent.

  In the “8. erythrocyte membrane removal step”, erythrocyte membranes unnecessary for the quality control substance can be removed.

  Since the control substance for quality control can be stored in liquid form, an appropriate amount can be weighed at the time of use, but it is optimal for calibration and precision control of blood analyzers in advance in “9. Dispensing process”. By dispensing a sufficient amount of quality control substances into the container, it is possible to save the trouble of weighing each use. In addition, by dispensing, it is not necessary to take out an unused amount of the quality control substance from the storage environment, so that it is possible to prevent deterioration in quality.

<Stability verification by composition analysis of controlled substances for accuracy control>
As the control material for quality control according to the present invention produced based on the “method for manufacturing quality control material” and a control sample, “1. Red blood cell extraction step” of “Method for manufacturing quality control material” The whole blood used in the above was subjected to composition analysis by cation exchange chromatography on the day of preparation of the quality control substance and after storage at −20 ° C. for 5 weeks. The chromatogram of the analysis result is shown in FIG.

  The verification is performed by changing the component ratio of hemoglobin A1c (HbA1c) indicated by the peak appearing on the horizontal axis 11 to 12 (min) in FIG. According to FIG. 1, the component ratio of HbA1c on the day of preparation was 3.3% for both the quality control substance and the whole blood.

  Subsequently, the component ratio after storage at −20 ° C. for 5 weeks is examined. After storage, the component ratio of HbA1c in the whole blood decreased to 2.8%, and glutathionyl hemoglobin (HbSSG) appeared clearly on the horizontal axis 19 to 20 (min) in FIG. This is a kind of modified hemoglobin that is bound to hemoglobin by oxidizing the reduced glutathionin inside the erythrocytes to become oxidized glutathionin, and suggests deterioration of the test sample.

  On the other hand, in the quality control substance, the component ratio of HbA1c was 3.3% even after storage at −20 ° C. for 5 weeks, and the value at the time of production was maintained. Moreover, the peak derived from HbSSG was not confirmed, and the stability of the quality control substance according to the present invention was shown.

<Verification of the effect of quality control substances on blood glucose measurement>
A quality control material is prepared based on the above-mentioned “Production method of quality control material”, and in “7. Dilution step”, the concentrated hemoglobin solution and (1) the low value control dilution solution are mixed in an equal weight. Thus, a sample corresponding to a healthy subject corresponding to the blood hemoglobin concentration (HbA1c value 6.0%) was prepared. Similarly, in the “7. Dilution step”, an equivalent weight of blood hemoglobin concentration (HbA1c value 8.0%) is obtained by mixing an equal weight of the concentrated hemoglobin solution and (2) the high-value control dilution solution. Samples were prepared. A plurality of quality control substances consisting of a healthy subject equivalent sample or a diabetic patient equivalent sample were prepared as test samples and stored in a liquid state in a freezer at about -20 ° C.

[Measurement of hemoglobin A1c concentration]
The hemoglobin A1c concentration (HbA1c value) of each test sample was measured with a measurement apparatus (Sakae A1c iGear Co., Ltd.) that was calibrated in advance at the time of manufacture and after 3 to 6 weeks from the date of manufacture. From the change, the stability of the quality control substance according to the present invention was verified. Table 1 shows the measured concentrations of each hemoglobin A1c.

  From Table 1, the coefficient of variation of the healthy subject equivalent sample and the diabetic patient equivalent sample is 2.3% for the healthy subject equivalent sample and 1.0% for the diabetic subject until the storage period of 6 weeks elapses. Since the coefficient of variation was suppressed to 3.0% or less, it was found that sufficient quality stability as a quality control substance for a hemoglobin concentration measuring device can be maintained over a long period of storage. As the coefficient of variation, a value obtained by dividing the average value of the data for 6 weeks in each sample by the standard deviation was expressed as a percentage (the same applies hereinafter).

(Blood glucose measurement)
Furthermore, the blood glucose level of each test sample is measured with a pre-calibrated measuring device (Panasonic Healthcare Inc., Glutest Mint) at the date of manufacture and at the time of 3 to 6 weeks from the date of manufacture. From these changes, the stability of the quality control substance according to the present invention was verified. The measured blood glucose levels are shown in Table 2.

According to Table 2, the coefficient of variation of the healthy subject equivalent sample and the diabetic patient equivalent sample is 2.3% for the healthy subject equivalent sample and 0.9% for the diabetic subject until the storage period of 6 weeks elapses. Since the coefficient of variation was suppressed to 3.0% or less, it was found that the treatment performed by the production method according to the present invention has no factor that affects the blood glucose level measurement result. In addition, since the blood glucose level obtained by measuring the blood glucose levels of the quality control substances of multiple concentrations was stable, there may be no factor that affects the blood glucose level without depending on the concentration. I understood.

Claims (4)

Erythrocytes are washed with a phosphate buffer solution that is isotonic with blood osmotic pressure or with a hypertonic phosphate buffer solution, and the internal solution of the red blood cells is replaced with a phosphate buffer solution that is isotonic with blood osmotic pressure. A hemoglobin extract obtained by hemolyzing red blood cells after,
A hemoglobin solution comprising the phosphate buffer and a cryoprotectant as a diluent. A control substance used for calibration of a blood analyzer used for measuring hemoglobin A1c, blood glucose level, and hemoglobin concentration, or for quality control,
A quality control substance comprising the hemoglobin solution according to claim 1. A quality control agent, wherein the quality control substance according to claim 2 is sealed in a container having a certain capacity. A method of manufacturing a quality control substance used for calibration of a blood analyzer used for measurement of hemoglobin A1c, blood glucose level, and hemoglobin concentration, or for quality control,
(A) centrifuge blood to remove plasma and buffy coat;
(B) a step of washing erythrocytes using a phosphate buffer solution that is isotonic with the osmotic pressure of the blood or a hypertonic phosphate buffer solution;
(C) adding a phosphate buffer solution with isotonicity of blood and isotonicity to the washed erythrocytes, and allowing to stand to replace the inside of the erythrocytes with the phosphate buffer solution; and
(D) centrifuging and removing red blood cells;
(E) after freezing the extracted red blood cells, lysing the red blood cells by thawing at room temperature, and creating a hemoglobin extract;
(F) adjusting the hemoglobin concentration by diluting the hemoglobin extract with a diluent containing the phosphate buffer and a cryoprotectant;
(G) A method for producing a control substance for quality control, comprising a step of precipitating and removing an erythrocyte membrane using a centrifuge at a low temperature.

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