JP2023014502A - Method for assisting diagnosis of basedow disease - Google Patents

Abstract

To provide a method for assisting the diagnosis of Basedow disease by distinguishing Basedow disease from painless thyroiditis with the use of a specimen separated from a living body.SOLUTION: A method for assisting the diagnosis of Basedow disease includes measuring thyroglobulin concentration in a specimen separated from a living body. The concentration being equal to or more than a predetermined cut-off value indicates a high possibility of not painless thyroiditis but Basedow disease. It is preferable that the specimen is the blood specimen. The cut-off value is, e.g., within the range of 20ng/mL-190ng/mL.SELECTED DRAWING: Figure 1

Description

TECHNICAL FIELD The present invention relates to a method for assisting diagnosis of Graves' disease, and more particularly to a method for assisting diagnosis of Graves' disease by distinguishing it from painless thyroiditis.

Basedow's disease is a representative disease of hyperthyroidism, and TSH receptor antibody (TRAb) that stimulates the thyroid gland causes an increase in blood free T4 and free T3. Clinical findings include thyrotoxicosis, including tachycardia, hand tremors, increased sweating, and weight loss. A similar form of thyrotoxicosis is observed with the release of thyroid hormones into the blood due to destruction of the thyroid gland. A typical disease of such destructive thyrotoxicosis is painless thyroiditis.

Graves' disease and painless thyroiditis both show symptoms of thyrotoxicosis, but they have different causes and treatment methods, so it is important to distinguish between the two. However, painless thyroiditis is often triggered by excessive iodine intake and stress (mental, surgery, childbirth, etc.) in patients with Hashimoto's disease (chronic thyroiditis). It may also occur during remission (when activity subsides) and be mistaken for a relapse of Basedow's disease.

In Graves' disease, which is a form of hyperthyroidism, blood TSH receptor antibody (TRAb) is generally increased, whereas in painless thyroiditis, the blood TRAb level is low. Both are discriminated against each other (Non-Patent Document 1). However, some patients with Graves' disease have low TRAb levels, and some patients with painless thyroiditis also have high TRAb levels, and there were cases in which TRAb alone could not distinguish between the two. .

Imaging tests such as ultrasonography and scintigraphy have been performed for differential diagnosis between the two, but there has been a demand for a method that can more accurately and easily differentiate between the two using only blood markers.

As a method for measuring blood thyroglobulin, a method has been reported in which a more accurate thyroglobulin level can be measured by a single test without being affected by the interference of anti-thyroglobulin antibodies (Patent Document 1). Blood thyroglobulin is used as a marker for surgical evaluation of differentiated thyroid carcinoma and the presence or absence of postoperative recurrence and metastasis. In addition, it is used as an index of therapeutic effect and remission in Graves' disease, but its use in diagnosis of Graves' disease, particularly in differentiation from painless thyroiditis, was not known.

International publication WO 2018/047792

Journal of the Japanese Society of Internal Medicine Vol.97 No.3 March 10, 2008 pp.558-563

An object of the present invention is to provide a method for assisting diagnosis of Graves' disease by distinguishing between Graves' disease and painless thyroiditis using a specimen isolated from a living body.

As a result of intensive research, the inventors of the present application found that if the thyroglobulin concentration in the specimen is equal to or higher than a predetermined cutoff value, there is a high possibility that it is not painless thyroiditis but Graves' disease, and completed the present invention. bottom.

That is, the present invention provides the following.

(1) Including measuring the concentration of thyroglobulin in a specimen isolated from a living body, and if the concentration is equal to or higher than a predetermined cutoff value, there is a high possibility of Graves' disease rather than painless thyroiditis. A method for assisting in the diagnosis of Graves' disease.
(2) The method according to (1), wherein the sample is a blood sample.
(3) The method according to (2), wherein the cutoff value is in the range of 20ng/mL to 190ng/mL.
(4) The method according to (3), wherein the cutoff value is in the range of 30ng/mL to 150ng/mL.

According to the method of the present invention, a specimen isolated from a living body can be used to distinguish Graves' disease from painless thyroiditis, thereby assisting diagnosis of Graves' disease.

FIG. 2 is a diagram showing serum thyroglobulin concentrations of Graves' disease patients and painless thyroiditis patients measured in the following examples.

The specimen used in the method of the present invention is not particularly limited as long as it is a bodily fluid, but a blood specimen is preferred. As a blood sample, whole blood, serum or plasma is preferable, and serum or plasma is particularly preferable. Dilutions of these can also be used.

The method of the present invention measures the concentration of thyroglobulin in a specimen isolated from a living organism. The measurement of the thyroglobulin concentration is not particularly limited as long as the method can measure the thyroglobulin concentration in the sample, and can be performed, for example, by immunoassay, mass spectrometry, or the like.

Well-known methods can be used as the immunoassay used in the method of the present invention. As described in Patent Document 1, various immunoassays such as sandwich method, indirect competition method, and direct competition method are known, and any of them can be used. Such immunoassays include chemiluminescence enzyme immunoassay (CLEIA), chemiluminescence immunoassay (CLIA), turbidimetric immunoassay (TIA), enzyme immunoassay (EIA) (e.g., direct competitive ELISA, indirect competitive ELISA, and sandwich ELISA), radioimmunoassay (RIA), latex agglutination assay, fluorescence immunoassay (FIA), and immunochromatographic methods. Since kits for measuring thyroglobulin by immunoassay and devices therefor are commercially available, thyroglobulin immunoassay can be easily performed using these commercially available products.

Well-known methods can be used as mass spectrometry used in the method of the present invention. Mass spectrometry is not particularly limited, but liquid chromatography-mass spectrometry (LC-MS), liquid chromatograph/tandem mass spectrometer (LC-MS/MS), and the like can be used. Since mass spectrometers and reagents capable of quantifying proteins such as thyroglobulin are commercially available, thyroglobulin can be easily quantified using these commercially available products.

As for the cut-off value, as specifically described in the examples below, specimens from patients with Graves' disease and specimens from patients with painless thyroiditis were collected, the thyroglobulin concentration was measured in them, and various numerical values were used as cut-off values. It can be set by calculating the positive predictive value and/or the positive likelihood ratio in each case and selecting a value that can achieve the desired positive predictive value and/or the positive likelihood ratio. As specifically shown in the examples below, in serum samples, if the cutoff value is 20 ng/mL, the positive predictive value is 84%, and if it is 30 ng/mL, the positive predictive value is 94%. Become.

A positive predictive value is a value defined as "probability of being a true positive when judged positive" in a certain test. Basedow's disease is positive, painless thyroiditis is negative, "the number of true positive patients who test positive a", "the number of true positive patients who test negative c", and "true negative" When the number of patients who are determined to be positive in the test is b ""the number of true negative patients who are determined to be negative in the test is d", the positive predictive value in the method of the present invention is "a / (a + b)". It can be calculated by In addition, the positive likelihood ratio indicates how many times more likely that a person with a disease is positive than a person without a disease when focusing on a person whose test result is positive in a certain test. In the method of the present invention, the positive likelihood ratio can be calculated by "(a/(a+c))/(b/(b+d))".

The cut-off value can be set as described above, but for blood samples the cut-off value is usually within the range of 20 ng/mL to 190 ng/mL, preferably within the range of 30 ng/mL to 150 ng/mL.

The method of the present invention focuses on the positive predictive value and/or the positive likelihood ratio.
The methods of the invention may be used in combination with known diagnostic methods. A preferred example of known diagnostic methods is a method based on the TRAb value. By combining with such a known method for diagnosing Graves' disease, it becomes possible to make a more accurate diagnosis than the known method.

EXAMPLES The present invention will be specifically described below based on examples. However, the present invention is not limited to the following examples.

Example: Differentiating Graves' Disease from Painless Thyroiditis Using Thyroglobulin Measurements To investigate whether it is possible to distinguish between Graves' disease and painless thyroiditis based on thyroglobulin concentration in blood samples, samples from patients diagnosed with Graves' disease Thyroglobulin concentrations were measured in blood samples from patients diagnosed with painless thyroiditis.

As measurement subjects, 179 serum specimens derived from patients diagnosed with Graves' disease and 115 serum specimens derived from patients diagnosed with painless thyroiditis were used. All samples were purchased from Trina Bioreactives, Inc., and these sera stored at -20°C were dissolved and used before measurement.

For the measurement, Lumipulse Presto (registered trademark) iTACT (registered trademark) Tg and calibrator (manufactured by Fujirebio), and reagents for Lumipulse Presto (substrate solution, washing solution, etc.) (manufactured by Fujirebio) were used, and the package insert and attachment were used. Thyroglobulin was measured in the samples according to the protocol of .

Specifically, first, 90 μL of the pretreatment liquid attached to LUMIPULSE Presto iTACT Tg and 30 μL of the sample for measurement were mixed, and then reacted at 37° C. for 6.5 minutes. Next, after adding 80 μL of antibody-bound particles (anti-thyroglobulin monoclonal antibody (mouse)-bound ferrite particles) to which an anti-thyroglobulin monoclonal antibody (mouse) that specifically binds to thyroglobulin is bound, the mixture is reacted at 37° C. for 8 minutes. let me The residual reaction liquid was separated and removed from the magnetic particles by magnetic force, and washed with a washing liquid. ALP-labeled anti-thyroglobulin monoclonal antibody (mouse) was added to the washed particles, and after stirring, the particles were allowed to react at 37°C for 8 minutes. The magnetic particles and the residual reaction liquid were again separated and removed by magnetic force, and washed with a washing liquid. 3-(2′-spiroadamantane)-4-methoxy-4-(3″-phosphoryloxy)phenyl-1,2-dioxetane disodium salt (AMPPD), which is a chemiluminescent substrate for alkaline phosphatase, was added to the particles. 200 μL of the substrate solution containing this was added and reacted at 37° C. for 4 minutes. The amount of chemiluminescence (wavelength 463 mm) after the reaction was measured with a luminometer. A fully automatic chemiluminescence immunoassay device Lumipulse Presto II or Lumipulse L2400 (manufactured by Fujirebio) was used for the measurement.

The sample was measured according to the above measuring method to obtain the measured value (ng/mL) of thyroglobulin. The results are shown in FIG. As a result, the measured values of thyroglobulin tended to be distributed in the high value region in Graves' disease patients. A two-tailed t-test revealed a p-value <0.05, indicating a significant difference in the distribution of the measured values.

Number of positive and negative cases in 179 specimens from patients with Graves' disease and 115 specimens from patients with painless thyroiditis when the cutoff is set to a range of thyroglobulin measurement values from 0.001 ng/mL to 200 ng/mL or more. was calculated, and Table 1 shows the positive predictive value (PPV) and positive likelihood ratio (LR) at each cutoff value. Note that when the number of positives is 0, the positive predictive value and the positive likelihood ratio cannot be calculated, which is indicated by a dotted line in Table 1.

As shown in Figure 1 and Table 1, the positive predictive value (PPV) was 84% when the thyroglobulin measurement value was 20 ng/mL as the cutoff value, and Graves' disease and painless thyroiditis accounted for 84%. It became clear that it is possible to distinguish between In addition, the positive predictive value (PPV) is 92% to 100% when the thyroglobulin measurement value is 30 ng/mL or more as the cutoff value, and it is possible to differentiate between Graves' disease and painless thyroiditis at a very high rate. was found to be possible.

From these results, it was shown that Graves' disease and painless thyroiditis can be distinguished with high accuracy by measuring thyroglobulin in the specimen. According to the method of the present invention, it is possible to distinguish between Graves' disease and painless thyroiditis, which are difficult to distinguish only from clinical symptoms and blood thyroid hormone levels, from the measured thyroglobulin level. Specifically, when the thyroglobulin measurement value is equal to or higher than the cutoff value compared to the cutoff value, the specimen can be determined to have Graves' disease rather than painless thyroiditis. In other words, Graves' disease and painless thyroiditis can be differentiated simply and with high precision by a serum test without performing imaging tests such as color Doppler ultrasound and thyroid scintigraphy.

Furthermore, as shown in Table 1, the positive likelihood ratio (LR) was 3.4 when the thyroglobulin measurement value was set to a cutoff value of 20 ng/mL. When the thyroglobulin level is above the cutoff value, Graves' disease patients are 3.4 times more likely to be positive than painless thyroiditis patients by measurement of thyroglobulin. In addition, when the thyroglobulin measurement value was set to a cutoff value of 30 ng/mL, the positive likelihood ratio (LR) was even higher, and the thyroglobulin measurement increased the probability of distinguishing between Graves' disease and painless thyroiditis. shown.

Claims (5)

Including measuring the thyroglobulin concentration in the specimen isolated from the living body, the concentration is more than a predetermined cutoff value, indicating that Graves' disease rather than painless thyroiditis is highly likely, A method for assisting diagnosis of Graves' disease. 2. The method of claim 1, wherein said specimen is a blood specimen. 3. The method of claim 2, wherein said cut-off value is within the range of 20ng/mL to 190ng/mL. 4. The method of claim 3, wherein said cut-off value is within the range of 30ng/mL to 150ng/mL. 5. The method according to any one of claims 1 to 4, wherein the measurement is performed by immunoassay or mass spectrometry.

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