KR102317339B1 - reagent composition for detecting hemoglobin and detection method of hemoglobin using thereof - Google Patents

Abstract

The present invention relates to a hemoglobin measuring reagent composition capable of diagnosing the presence or absence of anemia, and a hemoglobin measuring method using the same, and more particularly, to a hemoglobin that hemolyzes blood to release hemoglobin in red blood cells into a fluid, and blue for increasing measurement sensitivity It relates to a hemoglobin measuring reagent composition including a series dye and a hemoglobin measuring method using the same.
The method for measuring hemoglobin according to the present invention comprises the steps of: (a) putting whole blood into a hemoglobin measuring reagent composition comprising a hemoglobin buffer and a blue dye and reacting; (b) administering the reactant to the measurement pad of the cartridge; and (C) calculating the ratio of hemoglobin by measuring the optical reflectivity of the measuring pad with an optical instrument.
In the reagent composition for measuring total hemoglobin according to the present invention, the amount of hemoglobin can be corrected by using a blue dye having a complementary absorbance to hemoglobin as a background. That is, by simultaneously irradiating a plurality of light sources to measure and correct optical reflectance with high sensitivity, the effect of blood and reagent loading can be minimized.

Description

Hemoglobin measuring reagent composition and hemoglobin measuring method using same {reagent composition for detecting hemoglobin and detection method of hemoglobin using thereof}

The present invention relates to a hemoglobin measuring reagent composition capable of diagnosing the presence or absence of anemia, and a hemoglobin measuring method using the same, and more particularly, to a hemoglobin that hemolyzes blood to release hemoglobin in red blood cells into a fluid, and blue for increasing measurement sensitivity It relates to a hemoglobin measuring reagent composition including a series dye and a hemoglobin measuring method using the same.

Hemoglobin is a red protein containing iron in red blood cells. It plays a role in supplying oxygen to human tissues. When hemoglobin is insufficient in the blood, it cannot supply sufficient oxygen necessary for metabolism, resulting in tissue hypoxia (anemia). The causes of anemia are very diverse, such as a decrease in the size, shape, and production of red blood cells, and among various test parameters, the presence or absence of anemia can be determined first by a decrease in the hemoglobin concentration. Measurement of the concentration of hemoglobin is very important for the classification of the cause.

Currently, it has been reported that 8-64% of children, 20% of adult females, and 50% of pregnant women are in iron-deficiency anemia, so it is necessary to monitor the efficacy of intravenous iron therapy as a sensitive indicator of iron deficiency. In almost all hospitals, hemoglobin test is a basic test item and is implemented using automatic equipment, so it has great marketability. However, since expensive inspection methods are used as large automatic equipment, it is necessary to miniaturize the equipment and reduce costs. As miniaturized products, Hemo control, Hemocue Hb201/Hb301, and Hemochroma are representative, but all are methods for measuring hemoglobin by converting it into azide-methemoglobin, and the development of various hemoglobin measuring products is still insufficient. In addition, since commercially available products require an excessive amount of blood, equipment that can easily measure with a small amount of blood is needed.

As a principle of measuring hemoglobin, most products use an optical method. In the optical method, when blood is diluted with a solution of potassium ferricyanide and potassium cyanide, potassium ferricyanide oxidizes hemoglobin and changes it to hemiglobin (Hi: methemoglobin). , When cyanide ion (CN-) of potassium cyanide binds to Hi to form hemiglobincyanide (HiCN: Cyanmethemoglogin), the principle of measuring absorbance at 540 nm is used. The photometric method is simple and accurate as a standard measurement method, but it requires an excess of blood and has the disadvantages of using toxic cynaide.

Accordingly, as a result of the present inventors' efforts to solve the above problems, when using a reagent composition comprising a 'hemolysis buffer' for hemolysis of blood to release hemoglobin in red blood cells into a fluid and a 'blue dye' for increasing measurement sensitivity, After administering a small amount of reactant to the cartridge without using toxic cynaide, it was confirmed that total hemoglobin can be accurately measured simply with an optical analyzer without a separate washing process, and the present invention was completed.

It is an object of the present invention to provide a reagent composition capable of measuring a highly sensitive total hemoglobin amount with a small amount of whole blood without using a toxic substance and a method for measuring hemoglobin using the same.

Another object of the present invention is to provide a simple and miniaturized hemoglobin measurement cartridge.

In order to achieve the above object, the present invention provides a hemoglobin measurement reagent composition comprising a hemolysis buffer and a blue dye.

In the present invention, the hemolysis buffer is Hepes (HEPES), glycine (Glycine), hemolytic agent, sodium chloride (NaCl), magnesium chloride (MgCl ₂ ), sodium azide (NaN ₃ ), zinc chloride (ZnCl ₂ ) and water (water).

In the present invention, the hemolytic agent is selected from the group consisting of Triton X-100, sodium deoxycholate, sodium dodecyl sulfate (SDS) and saponin. .

In the present invention, the hemolysis buffer is 0.4 to 4.4% by weight of HEPES, 0.1 to 0.5% by weight of glycine, 0.1 to 0.5% by weight of a hemolytic agent, 0.2 to 2.4% by weight of sodium chloride (NaCl), magnesium chloride (MgCl ₂ ) 0.1 to 0.5% by weight, sodium azide (NaN ₃ ) 0.01 to 0.05% by weight, zinc chloride (ZnCl ₂ ) 0.1 to 0.5% by weight and the remaining amount of water (water).

In the present invention, the blue dye is Azure A (Azure A), brilliant blue (Brilliant blue), bromocresol green (Bromocresol green), bromophenol blue (Bromophenol blue), Coomassie blue (Coomassie Blue G-) 250), Fast Green FCF, Indigo blue, Indigo carmine, Methylene blue, and Xylene cyanol FF characterized in that it is selected from the group consisting of do it with

In the present invention, the blue-based dye is characterized in that 0.01 to 0.05% by weight of the total reagent composition.

The present invention also provides a hemoglobin measurement cartridge comprising the hemoglobin measurement reagent composition.

The present invention also includes the steps of: (a) putting whole blood into a hemoglobin measuring reagent composition comprising a hemolysis buffer and a blue dye and reacting; (b) administering the reactant to the measurement pad of the cartridge; and (C) measuring the optical reflectivity of the measuring pad with an optical device to calculate a hemoglobin ratio.

In the present invention, the measurement pad is characterized in that it comprises at least one selected from the group consisting of glass fiber, cellulose, polyester sulfone and nylon membrane.

In the present invention, the optical device is characterized in that the optical reflectance is measured by simultaneously irradiating blue and red light sources.

In the present invention, the blue light source is 450 ~ 490 nm, the red light source is characterized in that the range of 600 ~ 700 nm.

In the reagent composition for measuring total hemoglobin according to the present invention, the amount of hemoglobin can be corrected by using a blue dye having a complementary absorbance to hemoglobin as a background. That is, by simultaneously irradiating a plurality of light sources to measure and correct optical reflectance with high sensitivity, the effect of blood and reagent loading can be minimized.

In addition, it is safe because it does not need to use toxic cyanide, which is generally used for optical measurement, and has the advantage of being able to simply measure total hemoglobin with an optical analyzer without a separate washing process after administering the reactant to the cartridge.

1 is a flowchart illustrating a method for measuring hemoglobin using a reagent composition for measuring hemoglobin and an optical device of the present invention.
2 is a result of measuring absorbance spectra of various blue-based dyes.
3 and 4 are results of measuring hemoglobin samples according to concentrations using various blue-based dyes.
5 is a comparison result of the correction effect according to the presence or absence of a blue dye and the loading volume of a reagent for hemoglobin measurement.
6 is a result of comparing the correlation between the XP-300 analyzer of Sysmex, which is an upper device, and the hemoglobin measurement value according to the present invention.

In the present invention, when hemoglobin is eluted from whole blood components using a reagent composition for measuring hemoglobin containing a dye serving as a background and administered to a cartridge, it was attempted to confirm that total hemoglobin measurement is possible without using toxic substances.

In addition, the measured value of the amount of hemoglobin varies depending on the amount of blood through the step of administering the reaction solution to the cartridge and measuring the concentration (g/dL) of total hemoglobin by comparing the optical reflectivity of hemoglobin and blue dye with an optical device. I wanted to check if it was corrected.

In the present invention, a reagent composition including a dye serving as a correction of a hemoglobin measurement value was prepared in a buffer composition for eluting hemoglobin from red blood cells to measure hemoglobin. Next, a whole blood sample was added to the prepared total hemoglobin reagent composition, reacted, administered to the measurement pad, and the amount of hemoglobin was measured by measuring the optical reflectance of the measurement pad with an optical instrument without a separate washing process. As a result, it was confirmed that anemia could be diagnosed simply and quickly through the ratio of hemoglobin.

That is, in one embodiment of the present invention, a hemoglobin measurement reagent composition including a hemoglobin buffer and a blue-based dye is prepared, reacted with a whole blood sample, administered to the measurement pad, and then the measurement pad is applied with an optical device to a red and blue light source It was confirmed that the amount of hemoglobin could be measured simply and quickly by measuring the optical reflectance of each hemoglobin and blue-based dye.

Accordingly, in one aspect, the present invention relates to a hemoglobin measurement reagent composition comprising a hemolysis buffer and a blue dye.

In order to measure the concentration of hemoglobin in a blood sample, hemoglobin contained in red blood cells must be eluted by first hemolysis of the blood sample.

In the present invention, the hemolysis buffer may be used without limitation as long as it contains a reagent capable of hemolysis of red blood cells, but HEPES, glycine, hemolytic agent, sodium chloride (NaCl), magnesium chloride (MgCl ₂ ), ah Sodium azide (NaN ₃ ), zinc chloride (ZnCl ₂ ) and water (water) can be used.

The hemolytic agent may be exemplified by Triton X 100 (Triton X-100), sodium deoxycholate, sodium dodecyl sulfate (SDS), saponin, and the like, but is not limited thereto.

The hemolysis buffer is Hepes (HEPES) 0.4 to 4.4% by weight, glycine 0.1 to 0.5% by weight, hemolytic agent 0.1 to 0.5% by weight, sodium chloride (NaCl) 0.2 to 2.4% by weight, magnesium chloride (MgCl ₂ ) 0.1 ~0.5% by weight, sodium azide (NaN ₃ ) 0.01 ~ 0.05% by weight, zinc chloride (ZnCl ₂ ) 0.1 ~ 0.5% by weight, and preferably containing the remaining amount of water (water). When each component is out of the above content range, the error of the hemoglobin measurement result may increase.

In the present invention, the dye is characterized in that it uses a blue-based dye that can be distinguished from hemoglobin absorbance. The blue-based dye has an absorption wavelength of 600 to 700 nm, and the blue-based dye includes Azure A, Brilliant blue, Bromocresol green, and Bromophenol blue. ), Coomassie Blue G-250, Fast Green FCF, Indigo blue, Indigo carmine, Methylene blue, Xylene cyanol FF (Xylene cyanol) FF, acid Blue 147) and the like can be exemplified.

The blue-based dye is preferably contained in an amount of 0.01 to 0.05% by weight of the total reagent composition. When the amount of the blue dye is less than 0.01% by weight, the background signal of the membrane by the dye is insufficient, and when it exceeds 0.05% by weight, the signal strength of the dye is too strong, so the correction effect of the amount of hemoglobin using the blue dye disappears. may occur.

1 is a flowchart illustrating a method for measuring total hemoglobin using a reagent composition for measuring hemoglobin and an optical device of the present invention.

As shown in FIG. 1 , when a whole blood sample is reacted with a hemoglobin measurement reagent composition containing a hemolysis buffer and a blue dye and then administered to a cartridge containing a membrane, the reflectance and dye of total hemoglobin without a separate membrane washing process The amount of total hemoglobin can be calculated from the ratio of reflectance of

Accordingly, in another aspect, the present invention relates to a hemoglobin measurement cartridge comprising the hemoglobin measurement reagent composition.

The hemoglobin measurement cartridge according to the present invention may include a housing having a built-in membrane (measurement pad) that allows a mixed solution of a whole blood sample and a buffer composition to contact separately from the hemoglobin measurement reagent composition.

As the measurement pad, a commonly used one, such as glass fiber, cellulose, polyester sulfone, or nylon membrane, may be used without limitation.

In another aspect, the present invention provides a method comprising the steps of: (a) putting whole blood into a hemoglobin measuring reagent composition comprising a hemolysis buffer and a blue dye and reacting; (b) administering the reactant to the measurement pad of the cartridge; and (C) measuring the optical reflectivity of the measuring pad with an optical device to calculate a hemoglobin ratio.

In the present invention, the optical device can be used without any particular limitation as long as it can measure optical reflectivity using optical properties, and the light source of hemoglobin (blue) and the light source of the dye (red) are simultaneously irradiated and reflected light By measuring the signal with a photodetector, the amount of hemoglobin can be measured through an optical signal converter.

The blue light source is preferably in the range of 450 to 490 nm, and the red light source is preferably in the range of 600 to 700 nm.

In general, anemia can be diagnosed when the amount of hemoglobin is 10 g/dL or less.

[Example]

Hereinafter, the present invention will be described in more detail through examples. These examples are only for illustrating the present invention, and it will be apparent to those of ordinary skill in the art that the scope of the present invention is not to be construed as being limited by these examples.

Example 1: Preparation of Hemoglobin Measurement Reagent Composition

1-1: Preparation of Hemoglobin Measurement Reagent Composition

In a 1 L beaker, add 0.5 L of tertiary distilled water, 17 g of Hepes, 2 g of glycine, 1.5 g of Triton X 100, 10 g of sodium chloride, 2 g of magnesium chloride, 0.25 g of sodium azide, 1.5 g of zinc chloride, and 300 for 15 minutes. stirred at rpm. Finally, 0.25 g of brilliant blue was added and stirred.

1-2: Check absorbance of blue dye

A blue-based dye that is also used as a background for calibration in the hemoglobin measurement reagent composition (Xc: xylene cyanol FF, BB: brilliant blue, FG: pest green, BPB: bromophenol blue, IC: indigo carmine, CBB: coomassie) The absorbance of sea blue, BCG: bromocresol green, MB: methylene blue, Azure A: Azure A) was measured with a spectrophotometer, and the results are shown in FIG. 2 .

As shown in FIG. 2 , since blue-based dyes have absorbances of 600 to 700 nm, it can be seen that they can be suitably used as a background for correction.

Example 2: Measurement of total hemoglobin using a hemoglobin measurement reagent composition

2-1: Reaction between hemoglobin and reagent

500 uL of the hemoglobin measuring reagent composition prepared in Example 1 was placed in a transparent tube, and 5 uL of a whole blood sample in which the amount of hemoglobin (g/dL) was measured with an upper hematology analyzer, Sysmex XP-300 was added. Then, the mixture was shaken about 10 times. 25 uL of the reaction solution was placed on a cartridge measuring pad of an ^{optical instrument (Epithod ® 616, Dixgen) and absorbed.}

2-2: Hemoglobin measurement

The optical reflectance of the red hemoglobin and the optical reflectance of the blue dye were measured using a blue light source (470 nm) and a red light source (630 nm), respectively, in an optical device (Epithod ^{® 616, Dixgen).}

2-3: Hemoglobin measurement using K/S value

The concentration of hemoglobin (g/dL) was determined by comparing the total hemoglobin measured in 2-2 and the optical reflectance of the blue dye. The % reflectance (%R) measured from each light source was used by converting the K/S value, which is a quantitative indicator of how much colored material is present on the surface, and the % reflectivity as the K/S value. The conversion formula is as follows.

(K= 흡수계수, S= 산란계수)

(K = absorption coefficient, S = scattering coefficient)

Therefore, the % reflectance value obtained by irradiating the red light source representing the total amount of hemoglobin and the % reflectance value obtained from the blue light source representing the dye amount are calculated as K/S values, respectively, and by calculating their ratio, the amount of whole blood The value that varies depending on (K/S blue) was corrected with the dye (K/S red), so that the amount of total hemoglobin could be measured with high sensitivity.

* Hemoglobin amount = K/S (Blue) / K/S (Red)

3 and 4 are results of measuring hemoglobin samples by concentration with Azure A, Brilliant Blue, Methylene Blue, and Xylene Cyanol blue dyes. In this case, the control group measured the hemoglobin sample without using the blue dye.

Although it is possible to measure hemoglobin only with K/S (blue) without correction of the dye from FIGS. 3 and 4, when the hemoglobin value of K/S (blue) is corrected with the dye of K/S (red), the hemoglobin measurement value is It was confirmed that the correlation (R ² ) was further improved.

On the other hand, when a measurement reagent that does not contain a blue dye is used, there is a problem in that the hemoglobin measurement value also increases as the loading volume increases despite the blood having the same hemoglobin concentration. As shown in FIG. 5, when the K/S (red) value of the dye in the hemoglobin measurement reagent including the Brilliant blue dye is corrected with the K/S (blue) value of the hemoglobin, the K of 20, 25, 30 uL loading volume of the reagent reaction solution It was confirmed that the /S (Blue)/(Red) ratio value was constant.

As shown in FIG. 6 , it was confirmed that the hemoglobin measurement value according to the present invention was almost similar to that of Sysmex's XP-300 analyzer, which is an upper level hematology analysis equipment.

As the specific parts of the present invention have been described in detail above, for those of ordinary skill in the art, it is clear that these specific descriptions are only preferred embodiments, and the scope of the present invention is not limited thereby. will be. Accordingly, it is intended that the substantial scope of the present invention be defined by the appended claims and their equivalents.

Claims (11)

(a) hemolysis buffer and (b) Azure A, Brilliant blue, Bromocresol green, Bromophenol blue, Coomassie Blue G-250 ), Fast Green FCF, Indigo blue, Indigo carmine, Methylene blue, and Xylene cyanol FF, a blue-based dye selected from the group consisting of In the hemoglobin measuring reagent composition comprising a, the blue-based dye is a hemoglobin measuring reagent composition, characterized in that 0.01 to 0.05% by weight of the total reagent composition.
According to claim 1, wherein the hemolysis buffer is Hepes (HEPES), glycine (Glycine), hemolytic agent, sodium chloride (NaCl), magnesium chloride (MgCl ₂ ), sodium azide (NaN ₃ ), zinc chloride (ZnCl _{2 )} ) and a hemoglobin measurement reagent composition comprising water.
The method of claim 2, wherein the hemolytic agent is selected from the group consisting of Triton X-100, sodium deoxycholate, sodium dodecyl sulfate (SDS) and saponin. Hemoglobin measurement reagent composition.
The method of claim 2, wherein the hemolysis buffer is 0.4 to 4.4 wt% of HEPES, 0.1 to 0.5 wt% of glycine, 0.1 to 0.5% of a hemolytic agent, 0.2 to 2.4 wt% of sodium chloride (NaCl), chloride Magnesium (MgCl ₂ ) 0.1 to 0.5% by weight, sodium azide (NaN ₃ ) 0.01 to 0.05% by weight, zinc chloride (ZnCl ₂ ) 0.1 to 0.5% by weight and the remaining amount of water (water) characterized in that it contains Hemoglobin measurement reagent composition.
delete delete A hemoglobin measurement cartridge comprising the hemoglobin measurement reagent composition of any one of claims 1 to 4.
(a) adding whole blood to the hemoglobin measurement reagent composition of claim 1 and reacting;
(b) administering the reactant to the measurement pad of the cartridge; and
(c) measuring the optical reflectivity of the measuring pad with an optical device, and calculating the ratio of hemoglobin.
The method of claim 8, wherein the measuring pad comprises at least one selected from the group consisting of glass fiber, cellulose, polyester sulfone and nylon membrane. Methods for measuring hemoglobin.
The method of claim 8, wherein the optical device measures optical reflectivity by simultaneously irradiating red and blue light sources.
11. The method of claim 10, wherein the blue light source is 450 to 490 nm, and the red light source is 590 to 700 nm.

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