JP3569401B2 - Preparation of hemolysis sample - Google Patents

Description

[0001]
TECHNICAL FIELD OF THE INVENTION
The present invention relates to a method for preparing a hemolyzed sample.
[0002]
[Prior art]
Clinical tests, for example, measurement of hemoglobin are important for diagnosis of diabetes and the like. In the measurement of hemoglobin, blood is collected using a blood collection device such as a syringe or a reduced-pressure blood collection tube, and after transferring once from the blood collection device to a test tube, or directly from the blood collection device, a predetermined amount of blood is micropipetted or collected. The sample is injected into another test tube using a dropper such as a dropper, and a hemolysis sample having a predetermined concentration is prepared using the hemolysis liquid previously stored in the test tube in a fixed amount, and the solution sample is subjected to high performance liquid chromatography (HPLC). The hemoglobin value is obtained by measurement according to the method.
[0003]
The reduced-pressure blood collection tube, which is a blood collection tool, is, for example, a tubular body sealed in a decompressed state as described in Japanese Patent Publication No. 7-16484, and has a bottomed tube and its opening. And a closing plug. The blood collection tube is an auxiliary tool for collecting blood into a reduced-pressure blood collection tube. A blood collection needle whose both ends are pierced is attached to the distal end of the blood collection tube, and the distal end of the blood collection needle outside the cylinder (blood collection). After puncturing the blood vessel with the tip of the blood collection needle, the decompression blood collection tube is inserted into the blood collection tube with the plug side facing the front end, and the other end inside the cylinder of the blood collection needle (the rear end of the blood collection needle) is pierced through the plug to reduce the pressure. Collect blood into blood collection tube.
[0004]
[Problems to be solved by the invention]
However, regardless of the blood collection method, when preparing a hemolyzed sample, for example, a method using a micropipette dispenses while paying attention to the scale, so that the operator is burdened. In addition, the drop of the dropper causes a variation in the amount of liquid depending on the degree of pressing at the time of dropping.
[0005]
In addition, in preparing a hemolyzed sample when blood is collected using a reduced-pressure blood collection tube, the operation is more complicated because the plug of the reduced-pressure blood collection tube is first removed from the tube, and a micropipette or the like is inserted into the tube to aspirate the blood. In addition, there is a problem that blood is scattered during operation and the surroundings are contaminated, and there is a problem that the collected blood itself is exposed to outside air and contaminated.
[0006]
It is an object of the present invention to provide a method for preparing a hemolyzed specimen which solves the above-mentioned problems and is simple in operation and does not expose blood to outside air from blood collection to preparation.
[0007]
[Means for Solving the Problems]
In the blood collection method using a reduced pressure blood collection tube and a blood collection tube, which is a known technique, the blood remains in the blood collection needle that has been removed from the blood collection tube after use and has been discarded without being sucked into the reduced pressure blood collection tube. Focusing on the fact that the amount of blood remaining in the blood collection needle has a specific average value for each specification of the blood collection needle, as a result of research based on the idea of using this blood, In addition, the inventors have clarified that the method can be used for preparing a hemolyzed sample, thereby completing the present invention.
[0008]
The present invention has the following features.
(1) The blood collection needle of the following (A) is used, and the blood collection needle is pierced through the plug of the second reduced-pressure blood collection tube in which the hemolytic liquid is sealed, so that the blood collection needle remains inside the blood collection needle. A method for preparing a hemolyzed sample, wherein the blood thus obtained is transferred into a second reduced-pressure blood collection tube and dissolved in a liquid for hemolysis to obtain a hemolyzed sample.
(A) A blood collection needle whose both ends are pierced at both ends penetrates the inside and outside of the cylindrical body at the closed side of the cylindrical body having one end surface opened and the other end surface closed. A blood collection tube having a structure mounted in a state, wherein a distal end (a blood collection needle tip) outside the cylinder among both ends of the blood collection needle is punctured into a blood vessel, and a first reduced-pressure blood collection tube is inserted into the blood collection cylinder. The other end of the blood collection needle inside the cylinder (the rear end of the blood collection needle) was pierced through the plug of the reduced-pressure blood collection tube, blood was collected into the reduced-pressure blood collection tube, and then the reduced-pressure blood collection tube was removed from the blood collection tube. A blood collection tube in which blood remains inside the blood collection needle.
[0009]
(2) the hemolysis liquid is a reagent liquid for the measurement of hemoglobin, process for the preparation of hemolysis sample (1).
(3) The method for preparing a hemolyzed sample according to (1) above, wherein the hemolyzed sample is a hemolyzed sample for measuring hemoglobin concentration, measuring hemoglobin A1c concentration, or measuring glycohemoglobin.
[0010]
BEST MODE FOR CARRYING OUT THE INVENTION
Hereinafter, the present invention will be described in detail with reference to the drawings.
FIG. 1 is an explanatory diagram showing an example of a specific procedure of the method for preparing a hemolyzed sample according to the present invention. All the devices in the figure are schematically shown. Also, the device is shown in cross section, and the cross section is hatched to clarify the section.
[0011]
FIG. 1A shows a state in which blood is collected using a blood collection tube and a reduced-pressure blood collection tube. The outline of the blood collection tube and the reduced-pressure blood collection tube is as described in the related art. More specifically, the blood collection tube A has a structure in which a blood collection needle 2 is mounted on the end surface 1a of the cylindrical body 1 in which one end surface 1b is open and the other end surface 1a is closed. The blood collection needle 2 has both ends 2a and 2b sharply finished so that both can be punctured, and is attached to the end 1a of the tubular article 1 while penetrating the inside and outside of the tubular article. The decompressed blood collection tube is a tubular member sealed in a state where the inside 5 is decompressed, as shown as a first decompressed blood collection tube B in the figure, and has a bottomed tube 3 and a plug 4 for closing its opening. Consists of
[0012]
In the method for preparing a hemolyzed sample according to the present invention, first, as shown in FIG. 1A, of both ends 2 a and 2 b of the blood collection needle 2, the tip (a blood collection needle tip) 2 a on the outside of the tube is moved from the living body surface S into the blood vessel. (Not shown), and then insert the first decompressed blood collection tube B into the cylinder from the opening side 1b of the cylindrical body 1 and insert the other end (the rear end of the blood collection needle) 2b inside the blood collection needle 2 into the cylinder. By piercing the plug 4 of the vacuum blood collection tube B into the tube, blood is sucked into the vacuum blood collection tube and collected.
[0013]
In the method for preparing a hemolyzed sample according to the present invention, after the suction by the reduced-pressure blood collection tube B is completed, the reduced-pressure blood collection tube B is taken out from the blood collection tube A, and the blood collection tube is pulled out from the surface of the living body. As shown in (1), the tip 2a of the blood collection needle is pierced into the plug of the second reduced-pressure blood collection tube in which the hemolytic liquid 6 is sealed. As a result, the blood remaining inside the blood collection needle is transferred into the second reduced-pressure blood collection tube C and dissolved in the hemolysis liquid 6 to obtain a hemolysis sample. FIG. 1B is a diagram illustrating an example of a posture when the tip of the blood collection needle is pierced into the second decompression blood collection tube, and, for example, is upside down from the state of FIG. The posture and direction of the blood collection tube and the reduced-pressure blood collection tube at the time of puncturing are not limited, such as piercing the blood collection needle tip downward with the second reduced-pressure blood collection tube as the lower side.
[0014]
Known blood collection tubes and reduced-pressure blood collection tubes used in the present invention may be used. As described with reference to FIG. 1, any type of blood collection tube may be used as long as it has a structure in which a blood collection needle whose both ends are pierced and pierced is mounted in a state penetrating the inside and outside of the cylindrical object. Although it may be provided, it is preferable that the blood collection needle be detachably attached to the cylindrical object in order to make only the blood collection needle disposable.
[0015]
The specification of the blood collection needle is not limited, but is preferably a specification such that the amount of residual blood is appropriate for the measurement purpose and the variation between blood collections is smaller. (Outer diameter φ0.80 mm) and 22G (outer diameter φ0.70 mm) are preferable.
[0016]
The amount of blood remaining in the blood collection needle indicates a unique value for each specification (inner diameter, length) of the needle. For example, the amount of the remaining blood is about 15 μl (microliter) in the blood collection needle of the designation number 21G, and the amount of the remaining blood is about 10 μl in the blood collection needle of the designation number 22G. However, these values show variations within an allowable range. For example, even when blood is collected using the same needle repeatedly, an error of about ± 0.3 μl to ± 0.4 μl occurs for each trial. In addition, since the inner diameter and length vary due to manufacturing errors of the needle, the error in the amount of blood when a new needle is replaced for each trial and blood is collected is further ± 0.1 μl to ± 0.2 μl. Consider that an error of up to
[0017]
The hemolysis liquid previously sealed in the second reduced-pressure blood collection tube may be selected according to the purpose of measurement. Examples include a hemolytic reagent for Liquitec HbA1c, an ammonium chloride solution, a surfactant-containing solution, and water.
[0018]
The usefulness of the present invention is particularly remarkable when hemoglobin is measured using the obtained hemolyzed sample. Examples of the measurement of hemoglobin include, for example, measurement of hemoglobin A1c concentration, measurement of hemoglobin concentration, and measurement of hemoglobin A1c% and glycated hemoglobin obtained therefrom, which are important for diagnosis of diabetes. The measuring method may use a known technique.
[0019]
For example, when measuring the hemoglobin A1c concentration, the hemoglobin concentration, and the hemoglobin A1c% obtained therefrom, the hemolytic liquid to be sealed in the second reduced-pressure blood collection tube includes a hemolytic reagent for Liquitec HbA1c, an ammonium chloride solution. , A surfactant-containing solution, and a liquid reagent such as water.
[0020]
【Example】
Hereinafter, the present invention will be specifically described with reference to examples.
In this example, hemoglobin A1c concentration and hemoglobin concentration were actually measured using the hemolyzed sample obtained according to the present invention, and hemoglobin A1c% was obtained therefrom. Measurement was performed on the hemolyzed sample obtained by the conventional preparation method. By comparing with the result, the reliability of the preparation method according to the present invention was confirmed, and the operability was evaluated.
[0021]
According to the procedure shown in FIG. 1, blood is collected from the first vacuum blood collection tube B using the combination of the blood collection tube A and the vacuum blood collection tubes B and C according to the preparation method of the present invention, and blood is collected from the second vacuum blood collection tube C. The blood remaining in the needle was transferred to obtain a sample of a hemolysis specimen.
The number of specimens was 49, and the first decompressed blood collection tube in each blood collection was No. 1. Nos. B1 to B49, and the second decompressed blood collection tube containing the hemolyzed sample according to the present invention is No. B1 to B49. As C1 to C49, the same suffix numbers are given to the containers for blood and hemolyzed samples obtained from the same sample.
[0022]
In the second reduced-pressure blood collection tube (No. C1 to C49), a hemolytic reagent for Liquitec HbA1c (manufactured by Boehringer Mannheim Co., Ltd.) was previously prepared as a liquid reagent for use as a hemolyzed specimen capable of measuring hemoglobin A1c concentration. , Product number 844574) was enclosed in an amount of 1 ml. The blood collection needle used was designated 22G (0.7 × 38 mm), and the blood collection needle was replaced with a new one for each subject.
[0023]
For the blood in the first reduced-pressure blood collection tubes (Nos. B1 to B49), hemolysis samples are prepared according to a conventionally known method for preparing a hemolysis sample (hereinafter, a basic method), and the hemoglobin A1c concentration and the hemoglobin concentration of each sample are prepared. Was measured, and the ratio was taken as hemoglobin A1c%. Briefly explaining the basic method in this example, 1 ml of a hemolytic reagent and 0.01 ml of whole blood were accurately weighed by a micropipette using a micropipette, and dispensed into a test tube to obtain a 101-fold diluted hemolyzed sample. It is to be produced.
[0024]
Sample No. of the hemolyzed specimen obtained by the preparation method of the present invention. The measurement results of hemoglobin A1c concentration, hemoglobin concentration, hemoglobin A1c% obtained therefrom, and blood sample No. C1 to C49. Analysis of the relationship between each hemoglobin A1c concentration, hemoglobin concentration, and the measurement results of hemoglobin A1c% obtained from the hemolyzed specimen samples obtained by the basic method from B1 to B49 resulted in FIG. 2 (analysis relating to hemoglobin A1c concentration). 3 (analysis related to hemoglobin concentration) and a very strong linear correlation as shown in the graph of FIG. 4 (analysis related to hemoglobin A1c%). From these results, it was found that the method for preparing a hemolytic sample according to the present invention is a stable and useful method that can replace the conventional method for preparing a hemolytic sample.
[0025]
The measurement of the hemoglobin A1c concentration, that is, HbA1c (g / dl) in FIG. 2 was performed using the immunoinhibition turbidimetric method. In the immunoinhibition turbidimetric method, hemoglobin A1c in a sample binds to an anti-hemoglobin A1c antibody in a reagent, and an anti-hemoglobin A1c antibody not bound to hemoglobin A1c binds to a polyhapten in a reagent to form a complex. The turbidity generated at this time was measured at 340 nm, and the concentration of hemoglobin A1c was determined from a calibration curve.
The hemoglobin concentration, that is, Hb (g / dl) in FIG. 3 was measured by colorimetry at 570 nm for the hemolyzed sample.
Further, hemoglobin A1c%, that is, HbA1c (%) in FIG. 4 is a ratio of hemoglobin A1c in total hemoglobin, and is calculated by [hemoglobin A1c concentration] ÷ [hemoglobin concentration] × 100.
[0026]
Further, the sample No. C1 to C49 can be prepared in an extremely simple operation, and can be prepared in a nearly closed state from blood collection to preparation of a hemolyzed sample, and it has been confirmed that blood can be efficiently performed without scattering blood around. .
[0027]
【The invention's effect】
By adopting the method for preparing a hemolyzed sample as described above, it is possible to transfer a fixed amount of blood peculiar to each specification of a blood collection needle to a reduced-pressure blood collection tube containing a hemolysis liquid without performing complicated work. And can be used as a hemolysis sample as it is. In addition, since there is no operation such as transfer from a reduced-pressure blood collection tube to a test tube, and a state in which blood is exposed to the outside air can be sufficiently avoided, contamination of the surroundings and contamination of the blood itself can be suppressed. Furthermore, the blood in the blood collection needle, which was conventionally discarded after the blood collection operation, can be effectively used without waste.
[Brief description of the drawings]
FIG. 1 is a diagram illustrating an example of a method for preparing a hemolyzed sample according to the present invention.
FIG. 2 is a graph showing the relationship between the method of the present invention and the basic method with respect to hemoglobin A1c concentration.
FIG. 3 is a graph showing the relationship between the method of the present invention and the basic method with respect to hemoglobin concentration.
FIG. 4 is a graph showing the relationship between the method of the present invention and the basic method with respect to hemoglobin A1c%.
[Explanation of symbols]
A blood collection tube B first decompression blood collection tube C second decompression blood collection tube 1 cylindrical object 2 blood collection needle 6 liquid for hemolysis

Claims (3)

The blood remaining in the blood collection needle is pierced by inserting the blood collection needle into the plug of the second reduced-pressure blood collection tube in which a hemolytic liquid is sealed, using the blood collection tube of the following (A). A method for preparing a hemolyzed sample, wherein the hemolyzed sample is transferred into a second reduced-pressure blood collection tube and dissolved in a hemolysis liquid to obtain a hemolyzed sample.
(A) A blood collection needle whose both ends are pierced at both ends penetrates the inside and outside of the cylindrical body at the closed side of the cylindrical body having one end surface opened and the other end surface closed. A blood collection tube having a structure mounted in a state, wherein a distal end of the outer side of the tube among the both ends of the blood collection needle is punctured into a blood vessel, a first decompressed blood collection tube is inserted into the blood collection tube, and a blood collection inside the cylinder is performed. After the other end of the needle is pierced into the plug of the decompression blood collection tube and blood is collected in the decompression blood collection tube, the decompression blood collection tube is removed from the blood collection tube, so that blood remains inside the blood collection needle. Blood sampling tube. The hemolysis liquid is a reagent liquid for the measurement of hemoglobin, process for the preparation of hemolysis sample according to claim 1, wherein. The method for preparing a hemolyzed sample according to claim 1, wherein the hemolyzed sample is a hemolyzed sample for measuring hemoglobin concentration, measuring hemoglobin A1c concentration, or measuring glycated hemoglobin.

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