JP6467182B2 - Control sample of hemoglobin F - Google Patents

Description

  The present invention relates to a control sample used as a standard when quantifying a specific substance, and more specifically, relates to a control sample of hemoglobin F (HbF) having excellent stability.

  Human hemoglobin is a tetramer composed of two α chains and two non-α chains (β chain, γ chain, δ chain), and one heme is bound to each polypeptide chain. Depending on the type of polypeptide chain, there are three types of normal human hemoglobin: HbA (α2β2), HbA2 (α2δ2), and HbF (α2γ2). The α chain is made up of 141 amino acids, and the non-α chains (β chain, γ chain, δ chain) are all made up of 146 amino acids. This amino acid sequence is called the primary structure of hemoglobin. The β chain and δ chain differ in 10 amino acid sequences, and the β chain and γ chain differ in 39 amino acid sequences.

  There is a blood-related disease caused by this congenital abnormality of hemoglobin. For example, β thalassemia is a disease that causes abnormal production of normal adult HbA (HbA = α2β2) due to a synthesis defect of the β chain of globin. In this case, synthesis of δ chain and γ chain instead of β chain, and increase in the values of HbA2 and HbF are observed. However, this increase in production of HbA2 and HbF is insufficient to complement the production defect of HbA, and erythrocytes become microcytic hypochromic. Furthermore, anemia due to severe thalassemia is caused not only by abnormal hemoglobin synthesis, but also by erythroblast formation failure and peripheral hemolysis in the bone marrow. When excess α chain comes into contact with the red blood cell membrane, the red blood cells become brittle due to sedimentation.

  From the above-mentioned disease principle, HbF is measured as an index for an auxiliary test for diagnosis of thalassemia syndrome, abnormal hemoglobinosis, congenital hemolytic anemia, or the like. In addition, a high value of HbF may be observed in hematopoietic neoplastic diseases such as myelodysplastic syndrome and leukemia, and aplastic anemia, which may be measured as a means of grasping the disease state.

  HbF is measured clinically using a high performance liquid chromatography method (hereinafter also referred to as HPLC).

Japanese Patent No. 3686482

  When measuring biological materials, especially when quantifying them, it is desirable to use as many control samples (standard products) as possible for taking standards. In that case, it is required that the behavior of the control sample at the time of analysis is equivalent to that of the actual sample.

  Furthermore, long-term stability of individual lots is required for control samples. In addition, these freeze-dried products are required to have not only the stability of the freeze-dried product itself but also the stability after reconstitution with purified water during use.

  The same applies to the measurement of hemoglobin F. In the Hb standard and control samples, the actual specimen and fresh blood must have the same behavior at the time of analysis, and long-term stability. In addition, the restoration stability of the freeze-dried product is required.

  It is known that hemoglobin is mainly denatured by oxidation with the addition of time, temperature and the like. This is because oxidized methemoglobin and non-oxidized oxyhemoglobin have different charges. On the other hand, the HPLC method currently used for the measurement of hemoglobin performs separation based on the difference in the charge of hemoglobin. When blood containing methemoglobin is analyzed by oxidation, the chromatographic pattern is disturbed and separated from fresh blood. Are known to be different.

  Under such circumstances, provision of a management sample of hemoglobin F that is stable over time is also required for measurement of hemoglobin F, particularly for quantification.

  The inventors of the present invention have already invented a control sample of hemoglobin A1c used for quantification by HPLC of HbA1c, in which hemoglobin A1c (HbA1c) is stabilized with sucrose, and has already been patented. (Patent Document 1).

  However, when considering a stabilizer for stabilizing hemoglobin F, there are a number of problems.

(1) It is known that hemoglobin has a slightly different state of molecular charge depending on its type. The measurement of hemoglobin by HPLC uses a cation ion (cation) exchange column having a negative ion exchange group. Therefore, the electric charge accompanying the hemoglobin molecule is an important factor in the separation of hemoglobin. For example, hemoglobin having a larger positive charge than hemoglobin A1c is eluted more slowly than HbA1c. Conversely, hemoglobin having a smaller positive charge than hemoglobin A1c, eg, hemoglobin F, is eluted faster than HbA1c. One of the conditions for the stabilizer added in such a place where the difference in charge is large and affects the detection state of HPLC is that it is difficult to affect the charge state of such hemoglobin. It is difficult to predict from the correlation between the state of charge and the sugar molecules used as stabilizers.

(2) In the management sample of hemoglobin F, naturally, a considerable amount of hemoglobin F is required. However, how to secure hemoglobin F, which is hardly observed in erythrocytes of healthy adults, as an aspect that can be used as a control sample, and how to stabilize hemoglobin F in the secured content of hemoglobin F It is even more difficult to predict whether the problem (1) exists.

  In light of the technical difficulties described above, the present inventor has repeatedly studied that umbilical cord blood is appropriate as a supply source of hemoglobin F, and that sucrose is appropriate as a stabilizer for a management sample of hemoglobin F. I found it. That is, the present invention is a hemoglobin F control sample (hereinafter also referred to as a control sample of the present invention), characterized in that sucrose is contained in a mixed hemolysate of human erythrocytes and human umbilical cord blood. It is the invention which provides. As described later, “mixed hemolysate” is obtained by adding human umbilical cord blood to human erythrocyte lysate as it is, and the erythrocytes of human umbilical cord blood naturally lyse due to the hypotonicity of the lysate. Is a hemolysate. As will be described later, even if hemolysis is performed on umbilical cord blood together with normal human erythrocytes, a composition that can be used as a management sample of hemoglobin F as desired cannot be prepared. That is, the basic part of the management sample of hemoglobin F of the present invention is “prepared by mixing umbilical cord blood before disrupting blood cells into hemolyzed material”, and existing hemoglobin prepared only from the original hemolysate. This is completely different from the control sample of A1c.

  The management sample of the present invention has a special configuration in which human umbilical cord blood as described above is added as it is, and further has a molecular structure closer to hemoglobin A1c than hemoglobin F, that is, hemoglobin A1A having a charge state approximate to hemoglobin A1c, Furthermore, for hemoglobin LA1c +, which is an aspect of hemoglobin A1c, a stabilizing effect by sucrose that can be used practically was not recognized (Examples described later). The inventor had never expected.

  The content of sucrose in the management sample of the present invention is preferably 5 to 20% by mass, more preferably 6 to 18% by mass, and more preferably 10 to 10% by mass with respect to the entire management sample having a hemoglobin concentration of 10 mg / ml. It is very preferably ˜15% by mass. Although the hemoglobin concentration in the control sample can be variously set, in this specification, the content of the sucrose is defined based on “10 mg / ml” which is the hemoglobin concentration of a standard product. . Naturally, in the case of a product having a different hemoglobin concentration, the sucrose content varies depending on the hemoglobin concentration. Moreover, it is very preferable to use human umbilical cord blood without dilution, and the amount of undiluted human umbilical cord blood added to the lysate of human erythrocytes is the mixture obtained by diluting human erythrocytes in a 2-fold volume dilution. It is preferably 0.05 to 0.5% by mass, more preferably 0.05 to 0.2% by mass, and extremely preferably 0.05 to 0.1% by mass. preferable.

  Regardless of the purpose, the control sample of the present invention can be used as a control sample for quantifying hemoglobin F in a blood sample. Typically, hemoglobin as an auxiliary test for thalassemia syndrome such as β-thalassemia It is used as a control sample when F is quantified.

  Although the control sample of the present invention can be used in a liquid state, it is usually used as a lyophilized product. In the control sample of the present invention as this freeze-dried product, very good temporal stability is observed.

  As described above, the present invention uses the management sample of the present invention as a standard for hemoglobin F when measuring, in particular, quantifying hemoglobin F in a blood sample using HPLC. The invention provides a method for stabilizing hemoglobin F in a mixture of human erythrocyte hemolysate and human umbilical cord blood containing sucrose.

  Furthermore, the present invention is characterized in that human umbilical cord blood is mixed with a lysate of human erythrocytes to prepare a mixture of the lysate and umbilical cord blood, and sucrose is contained in the mixture. It is invention which provides the production method of this control sample. In the production method of the present invention, a lyophilization treatment can be performed on a mixture of human erythrocyte hemolysate and human umbilical cord blood containing sucrose.

  According to the present invention, there is provided a control sample that serves as a standard when measuring hemoglobin F having excellent stability over time. The present invention also provides a method for using the control sample, a method for stabilizing hemoglobin F, and a method for producing the control sample. The control sample includes both liquid form and lyophilized form.

It is drawing which shows the result of the acceleration test about hemoglobin F. FIG. It is drawing which shows the result of the acceleration test about hemoglobin A1c. It is drawing which shows the result of the acceleration test about hemoglobin A1A. It is an enlarged view which shows the result of sucrose addition in drawings of FIGS. 1-3.

(1) Production of control sample of the present invention (a) Preparation of lysate of human erythrocytes Red blood cells are separated from human whole blood that is allowed to contain components for storage as appropriate by a conventional method such as centrifugation. This step may be omitted and only red blood cells may be obtained and used. The erythrocytes obtained in this manner are washed with physiological saline, etc., and then the hypotonic solution, typically purified water is added to perform hemolysis, and the lowermost red fraction obtained by centrifugation (purification) Lysed blood) is “lysed human erythrocyte”. The addition amount of hypotonic solution such as purified water is in accordance with a conventional method for hemolysis and is not particularly limited, but is generally 0.5 to 1.5 volumes with respect to the total volume of red blood cells, Preferably it is 0.7-1.2 capacity.

(B) Addition of human umbilical cord blood Human umbilical cord blood is added to the lysate of human erythrocytes obtained in (a) above. Human umbilical cord blood is fetal blood contained in the umbilical cord (umbilical cord), which is a fetal side tissue connecting the fetus and the mother, and erythrocytes contained in the umbilical cord blood are rich in hemoglobin F. It is very preferable to add human umbilical cord blood to the above-mentioned lysate of human erythrocytes. That is, even if human whole blood and human umbilical cord blood are mixed in the step (a), it is difficult to prepare an intermediate of the control sample of the present invention containing the desired hemoglobin F. By adding human umbilical cord blood to the lysate of human erythrocytes as described above, the erythrocytes in the umbilical cord blood are naturally lysed due to the hypotonicity of the lysate. The amount of human umbilical cord blood added is described above.

(C) Addition of sucrose To the “mixture of human erythrocyte lysate and human umbilical cord blood” obtained in (b) above, sucrose as a stabilizer is added. The amount of addition is as described above. At the time of this addition, it is preferable to prepare an aqueous solution of sucrose having an appropriate concentration in advance and add it to the above mixture so as to obtain a desired sucrose concentration. Here, the hemoglobin concentration of the mixture is measured, and the concentration is adjusted to a desired concentration. The hemoglobin concentration in this example is 10 mg / mL, but is not limited thereto. The hemoglobin concentration is preferably adjusted using an aqueous solution having a desired sucrose concentration.

  The composition obtained through this process is the control sample (liquid agent stage) of the present invention.

(D) Freeze-drying In many cases, the control sample is used after being freeze-dried and adjusted for use. The same applies to the control sample of the present invention. The method of lyophilization follows conventional methods, and is specifically disclosed in the examples.

(2) Use of the control sample of the present invention As described above, the control sample of the present invention can be used as a standard for quantifying hemoglobin F in a blood sample. The method for quantifying hemoglobin F using the control sample of the present invention is high performance liquid chromatography (HPLC).

  In addition, a disease for which hemoglobin F in a blood sample is quantified using the control sample of the present invention as a standard is a hemoglobin F high value, elevation, or transient that is hardly recognized in adults due to the disease. If it is the disease by which an increase is recognized, it will not specifically limit. Examples of diseases in which a high level of hemoglobin F is recognized include thalassemia syndromes such as β thalassemia and δβ thalassemia, hereditary hyper-HbF disease, juvenile chronic myeloid leukemia and the like. Examples of diseases that are elevated include unstable Hb disease, congenital hemolytic anemia, acute myeloid leukemia, myelodysplastic syndrome, and the like. Examples of diseases that show a transient increase include aplastic anemia, PNH, and a marked bone marrow peak period after bone marrow transplantation. Typical examples among these are thalassemia syndromes such as β thalassemia and δβ thalassemia.

  The control sample of the present invention is very suitable as a standard to be used for quantification of hemoglobin F in the blood sample as described above, as it shows excellent temporal stability both as a solution and as a lyophilized product. .

  Examples of the present invention will be disclosed below.

[Example 1] Preparation of control sample The control sample of the present invention and a comparative example thereof were prepared as follows.

( Process 1 )
(1) 450 mL of human whole blood is mixed by inversion while in a pack (including citrate-phosphate-dextrose-adenine (CPDA) stock solution) to make it uniform.
(2) Approximately 45 mL of 450 mL of human whole blood is dispensed into a 50 mL centrifuge tube and applied to a centrifuge (3000 rpm × 5 min, 4 ° C.) to remove supernatant plasma, platelets, and white blood cells and separate red blood cells. For infectivity denial test, centrifuge in 15 mL centrifuge tube and remove supernatant for test.
(3) Add 1/2 volume of physiological saline to the separated erythrocytes, mix, and centrifuge (3000 rpm × 5 min, 4 ° C.) to remove the physiological saline. This operation is repeated 8 times. However, the eighth centrifugation time is 10 minutes.

( Process 2 )
(1) Add 1.0 equivalent volume of purified water to the washed red blood cells obtained in step 1 and shake to lyse the red blood cells.

( Process 3 )
(1) Add 1/4 volume of toluene to the hemolyzed blood obtained in step 2, shake vigorously (about 5 minutes), centrifuge (3000 rpm x 30 min, 4 ° C), and lower layer part of the three layers Is collected in a test tube with a Pasteur or the like.
(2) The liquid collected in (1) is subjected to a second centrifugation (3000 rpm × 15 min, 4 ° C.), and only the transparent part is tested with a Pasteur etc. except for the insoluble matter that floats on the upper layer or settles on the bottom. Collect in tubes.
(3) Add 1 mL of human umbilical cord blood to this purified hemolyzed blood (about 500 mL) and mix.
(4) Purified hemolyzed blood of (3) is added, and 1/4 volume of stabilizer is added to a stabilizer with a final concentration of 10%, and 1% is added to a stabilizer with 5%. Add / 4 volume of 25% strength stabilizer and mix until uniform. “Stabilizer” is sucrose or other saccharide used as a comparative example.
(5) The hemoglobin concentration is measured, and the purified hemolyzed blood containing 10% stabilizer is diluted with a 10% stabilizer solution and adjusted to 10 mg / mL. Purified hemolyzed blood containing 5% stabilizer is diluted with 5% stabilizer solution and adjusted to 10 mg / mL. As a result, a control sample of the present invention at the liquid preparation stage or a comparative example thereof was prepared.

( Process 4 )
(1) Dispense 1 mL each of the liquid-phase management sample containing the 10% stabilizer prepared in (3) of step 3 in a 10 mL vial and perform freeze-drying.
(2) Freeze-drying consists of pre-freezing: -30 ° C for 4 hours, primary drying: -20 ° C for 10 hours, secondary drying: -10 ° C for 10 hours, tertiary drying: -4 ° C for 10 hours, Final drying: The process was performed at 4 ° C. for 60 hours.
(3) Replace with nitrogen and tighten the lip cap after rubber plugging.

  Thereby, the control sample of the present invention as a freeze-dried product or a comparative example thereof was prepared.

[Test Example 1] Sugar addition test In Example 1 above, (1) maltose, (2) sucrose, (3) sorbitol, (4) arabinose, (5) xylose were used as stabilizers. The lyophilized product of step 3 was prepared with an addition amount of 10% by mass with respect to each control sample, and (a) hemoglobin F (HbF), (b) hemoglobin A1c (HbA1c), (c) by HPLC. For the hemoglobin A1A (HbA1A), the abundance before lyophilization was compared with the abundance after lyophilization (purified water was added before lyophilization and after addition to the same volume). The results are disclosed in Table 1 (Tables 1-1, 1-2, 1-3).

  HbF is shown in Table 1-1, HbA1c is shown in Table 1-2, and HbA1A is shown in Table 1-3.

  Based on these results, the following accelerated test was conducted using sucrose and sorbitol as candidates for hemoglobin F stabilizers.

[Example 2] Thermal acceleration test In the thermal acceleration test, a test sample (lyophilized product: sucrose or sorbitol added at 5% or 10%) was allowed to stand for 4 weeks immediately after the test at 37 ° C. Immediately after the test, purified water was added to each volume before freeze-drying and at the same volume, and the amount of hemoglobin was quantified by HPLC. The results are shown in Table 2 (Tables 2-1, 2-2, 2-3). ) And FIG. 1 (FIGS. 1-1, 1-2, 1-3, 1-4). (A) Table 2-1 and FIG. 1-1 are hemoglobin F (HbF), (b) Table 2-2 and FIG. 1-2 are hemoglobin A1c (HbA1c), (c) Table 2-3 and FIG. 1-3. 1-4 shows the result about hemoglobin A1A (HbA1A). FIG. 1-4 is an enlarged view showing the result of addition of sucrose of FIG. 1-3.

  From these results, it is clear that sorbitol does not give a sufficient stabilizing effect over time to any hemoglobin, and sucrose is superior to hemoglobin A1c as conventionally recognized. It has become clear that the present invention exhibits a stabilizing effect and also exhibits an excellent stabilizing effect on hemoglobin F, which is the subject of the present invention. However, it has been clarified particularly in FIGS. 1-4 that hemoglobin A1A does not provide a stabilizing effect that can withstand practical use.

Claims (8)

A management sample of hemoglobin F, wherein sucrose is contained in a mixed hemolysate of human erythrocytes and human umbilical cord blood. The control sample according to claim 1, wherein the content of sucrose is 5 to 20% by mass with respect to the total control sample having a hemoglobin concentration of 10 mg / ml. The control sample according to claim 1 or 2, which is a control sample for an auxiliary test for thalassemia syndrome. The control sample according to any one of claims 1 to 3, wherein the control sample is a freeze-dried product. Use of the control sample according to any one of claims 1 to 4 , wherein the control sample is used as a standard for hemoglobin F when measuring hemoglobin F in a blood sample using high performance liquid chromatography. Method. A method for stabilizing hemoglobin F in a mixture, wherein sucrose is contained in a mixed hemolysate of human erythrocytes and human umbilical cord blood. Human umbilical cord blood is mixed with human erythrocyte hemolysate to prepare a mixture of the hemolysate and umbilical cord blood, and as a mixed hemolysate, sucrose is contained in the mixed hemolysate, A method for producing a hemoglobin F control sample. A method for producing a hemoglobin F control sample, comprising lyophilizing a lysed human erythrocyte lysate and human umbilical cord blood lysate containing sucrose.

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