RU2646800C2 - Method for differential diagnosis of thyroid gland diseases - Google Patents

Abstract

FIELD: medicine.

SUBSTANCE: invention is used in pathological anatomy, endocrinology, and surgery. Method for differential diagnosis of thyroid gland diseases comprises histoenzymatic study of the thyreperoxidase (TPO) enzyme in the thyroid gland and based on the activity of TPO, diffuse toxic goiter, multi-site toxic goiter, follicular adenoma in the state of euthyroidism, adenocarcinoma are diagnosed.

EFFECT: aim of invention is to ensure reliability of results obtained in differential diagnosis of thyroid diseases.

1 cl, 4 ex, 2 dwg

Description

Scope: pathological anatomy, endocrinology, surgery, the invention can be used for the differential diagnosis of thyroid diseases (thyroid gland).

Thyroid peroxidase thyroid peroxidase (TPO) is a key enzyme in the synthesis of thyroid hormones. It is synthesized in a granular endoplasmic reticulum and then packed into exocytotic vesicles accumulates in the apical part of thyrocytes.

It is believed that TPO is built into the apical membrane of the thyroid follicular thyrocytes and catalyzes the peroxidation of iodine followed by tyrosine iodination in thyroglobulin. Thus, TPO catalyzes at least two reactions in the synthesis of thyroid hormones: iodination of tyrosine residues of thyroglobulin and fusion of iodothyrosines in the synthesis of thyroxine (T4) and triiodothyronine (T3). It must be considered unique that the finished hormones in the composition of thyroglobulin accumulate in the extracellular medium, and then, as necessary, enter the circulation by endocytosis.

From the above data it follows that the intensity of the synthesis of thyroid hormones in follicular thyrocytes is directly dependent on the activity of TPO. From this position the conclusion suggests itself that the functional state of the thyroid gland can be judged by the level of TPO activity.

It is important, first of all, for the reason that the pathomorphological conclusion in the study of postoperative material does not imply the establishment of the functional state of the organ.

Currently, the following methods are used to determine the functional state of the thyroid gland, which are described in the Instructions for the use of a reagent kit for quantitative enzyme-linked immunosorbent assay. “Approved” by Order of Roszdravnadzor dated November 18, 2011 No. 7492 - Pr / 11.

- Instructions for the use of a kit of reagents for the quantitative enzyme-linked immunosorbent assay of free thyroxine in human serum. ("Thyroid IFA-free T4").

- Instructions for use of the reagent kit for the quantitative enzyme-linked immunosorbent assay of free triiodothyronine in human serum ("Thyroid IFA-free T3").

- Instructions for the use of a set of reagents for the quantitative enzyme-linked immunosorbent assay of thyroid-stimulating hormone in human serum ("Thyroid IFA-TTG").

- Instructions for the use of a reagent kit for the quantitative enzyme-linked immunosorbent assay of thyroid peroxidase autoantibodies in human blood serum (“Thyroid IFA-atTPO”).

Used in practice, methods for determining the content of thyroid hormones and autoantibodies to autoantigens in the blood have the following disadvantages.

1. The need to use expensive reagents.

2. The length of time upon receipt of a response. It is due to the fact that hormones are determined not by each patient individually, but by a group of patients, accumulating them to a certain amount, it is understandable with the loss of time.

3. Only with the initial treatment of patients, the results can be reliable. In the vast majority of cases, patients who are registered and in need of re-examination are referred. In the study of hormones in this group of patients who most often take the appropriate drugs, the reliability of the analysis results is doubtful in the sense that the ability of thyrocytes to increased production of hormones is preserved. In particular, patients with diffuse toxic goiter (DTZ), taking thyrostatic drugs, are mostly in a state of medical euthyroidism and, accordingly, the hormone content is within normal limits, and clinical signs are preserved to some extent.

As a prototype, we took the method described in the Instructions for use of the reagent kit for the quantitative enzyme-linked immunosorbent assay of free thyroxine in human blood serum (“Thyroid IFA-free T4”).

The disadvantages of the prototype include the following:

- the need to use expensive reagents;

- the duration of the analysis.

The aim of the invention is to ensure the reliability of the results obtained in the differential diagnosis of thyroid disease by histochemical study of the activity of a key enzyme in the synthesis of thyroid hormones - TPO.

The essence of the invention is as follows.

1. The essence of the differential diagnosis of diffuse toxic goiter is as follows.

When stating the reaction for TPO, the reaction is carried out as follows: slices are prepared from fresh (postoperative) material, the resulting slices with a thickness of 15-20 microns are transferred to glass slides and stored for 2 hours for drying. Slice slides were coated with 0.3% ammonium molybdate solution for 5 minutes. After draining the previous solution onto the glass, cover the incubation medium for 15 minutes. The incubation medium is prepared as follows: 1 ml of a saturated solution of benzidine is applied to buksik and 1 drop of a 0.3% solution of hydrogen peroxide is added (the solution is designed for 3-4 glasses). After the incubation time has elapsed, the glass with the incubation medium is transferred to the microscope stage with a photo attachment and the pictures are taken immediately. The optimal increase at which it is possible to clearly see the colored structures is 400. When using the proposed method in the places of localization of the enzyme, dark blue granules are formed on the bluish background of the cytoplasm of follicular thyrocytes. In the overview micropreparations (Fig. 1, a) there are signs of macro- and microfollicular goiter, which is combined with the presence of adenomatous zones (Fig. 1, a). This allows you to make a diagnosis of diffuse toxic goiter.

2. The essence of the differential diagnosis of multinodular toxic goiter is as follows.

The abundance of adenomatous foci is characteristic, i.e. forming new follicles, most of which have no visible lumen. The activity of the enzyme in thyroid cells is moderate and high. Only single, fully formed follicles are visible; the content of colored reaction products in the wall is also high (Fig. 1, b). It is possible to diagnose multinodular toxic goiter.

3. With follicular adenoma, the state of euthyroidism is characterized by the presence of numerous adenomatous foci. However, in contrast to the previous case, the number of follicles formed is greater. In the follicle wall, the activity of the enzyme is moderate or high. Interfollicular connective tissue layers are clearly delineated; in their structures there are no colored products of histochemical reaction (Fig. 2, a).

4. Adenocarcinoma.

In histochemical preparations, there are entirely adenomatous and parenchymal lesions, mainly without clearance. The enzyme activity in them is very high. Only single follicles have a lumen. The epithelium in their wall is unevenly layered with moderate and high TPO activity. Often, adenomatous and parenchymal foci merge, forming solid colored areas.

In the interfollicular connective tissue layers, the products of the histochemical reaction are absent (Fig. 2, b).

Examples of specific performance of the method.

1. Patient R. Was admitted to the endocrine surgery department of the Republican Clinical Hospital on 10/25/15 with a diagnosis of "Diffuse toxic goiter of the IV st." Complaints about a tumor formation on the front surface of the neck, shortness of breath during exercise, tachycardia, irritability, tearfulness, headache, weakness, sweating.

The content of thyroid hormones in the blood: T4 St. - <5.15 pmol / L,

TTG - 0.039 honey / l.

Clinical diagnosis: Diffuse toxic goiter IV Art. The operation was performed - subfascial thyroidectomy.

Histoconclusion: Bazedofitsirovanny micro-macrofollicular diffuse toxic goiter.

Reaction to TVET. In the cytoplasm of follicular thyrocytes, the enzyme activity is high. Epithelial protrusions protrude into the cavity of the follicles in the form of papillae with an enlightened middle part. In the interfollicular spaces adenomatous foci with high enzyme activity.

Histochemical conclusion: Diffuse toxic goiter severe. The state of thyrotoxicosis.

2. Patient S. was admitted to the endocrine surgery department of the Republican Clinical Hospital on November 16, 2015 with a diagnosis of multinodular toxic goiter of the III st. Medical euthyroidism. ”Complaints of a tumor formation on the front of the neck, difficulty breathing during physical exertion, fast walking, tachycardia, irritability, tearfulness, headache, weakness.

The content of thyroid hormones in the blood: T3 St. - 3.7 pmol / l, T4 St. - 10.0 pmol / L, TSH - 11.3 mIU / L, AT to TPO - 625 IU / ml.

Clinical diagnosis: Multinodular toxic goiter III art. gravity. Medical euthyroidism. The operation was performed - subfascial thyroidectomy.

Histological conclusion: Against the background of colloidal goiter, nodes of the follicular adenoma with focal lymphocytic infiltration.

Reaction to TPO: In the field of view are numerous adenomatous lesions (proliferating thyrocytes) with high enzyme activity; only single formed follicles with a lumen are visible.

Histochemical conclusion: Nodular toxic goiter severe. The state of thyrotoxicosis.

3. Patient Sh. Was admitted to the endocrine surgery department of the Republican Clinical Hospital on 10.9.15 with a diagnosis of diffuse-nodal euthyroid goiter of the III degree. Complaints about tumor formation on the front surface of the neck, headaches, discomfort in the neck.

The content of thyroid hormones in the blood: T4 vol. - 21.8 nmol / L, T3 vol. - 13.7 ng / ml, TSH - 0.1 μMU / ml.

Clinical diagnosis: Multinodal euthyroid goiter III tbsp.

Histological conclusion: Macro-microfollicular colloid goiter with fibrotization fields and the formation of nodular complexes.

Reaction to TPO: Numerous adenomatous lesions with moderate to high enzyme activity. Some foci contain formed small follicles. In their wall, TPO activity is also moderate or high. Interfollicular connective tissue layers without colored reaction products are clearly delineated.

Histochemical conclusion: Follicular adenoma. The state of euthyroidism.

4. Patient I. was admitted to the laboratory and diagnostic center "Astramed" 12/22/15, with a diagnosis of Multinodular toxic goiter II Art. Medical euthyroidism. ”Complaints of a tumor formation on the front of the neck, headaches, tachycardia, weakness, sweating.

The content of thyroid hormones in the blood: T4 St. - 42.3 pmol / L, TSH - <0.004 nME / L.

Clinical diagnosis: Multinodular toxic goiter II tbsp.

Histological conclusion: Polymorphic adenoma of the thyroid gland with areas of papillary and follicular structure and signs of malignancy and hyperfunction of the epithelium.

Reaction to TPO: Adenomatous and parenchymal lesions with very high activity. In the wall of the formed adenomatous follicles, the uneven multilayer epithelium is of moderate, sometimes high enzyme activity. Many adenomatous and parenchymal foci are fused together.

Histochemical conclusion: Adenocarcinoma. The state of thyrotoxicosis.

Signs of the invention, distinctive from the prototype.

According to the proposed method, a histochemical study of the thyroid thyroid postoperative material is carried out by determining the activity of TPO, which allows to add a functional histological conclusion of pathomorphologists.

The positive effect of using the invention.

1. Used cheap domestic reagents.

2. From the moment the postoperative material is received until the histochemical reaction is set up and the response is received, 2-3 hours elapse (in the prototype, from several days to several weeks).

3. The histochemical picture of the thyroid gland, revealed by determining the activity of the main enzyme for the synthesis of thyroid hormones - TPO, is not affected by therapeutic thyrostatic drugs and reflects the true state of the functional activity of follicular thyroid cells and their potential (in the prototype, in cases of increased functional activity of the thyroid gland, thyreostatic drugs inhibit the release of hormones into the circulation and create a picture of artificial medical euthyroidism.

4. The method will allow doctors to conduct differential diagnosis of thyroid diseases, will make the final clinical diagnosis and conduct further postoperative treatment measures.

The technical result.

The proposed method of histo-enzymatic differential diagnosis of thyroid diseases by determining the activity of TPO will allow pathologists to supplement the functional morphological conclusion. This will help surgeons and therapists in making the final clinical diagnosis and conducting adequate postoperative treatment.

Information taken into account

- Instructions for use of the reagent kit for the quantitative enzyme-linked immunosorbent assay of free thyroxine in human serum (“Thyroid IFA-free T4”).

“Approved” by Order of Roszdravnadzor dated November 18, 2011 No. 7492 - Pr / 11 - prototype.

- Instructions for use of the reagent kit for the quantitative enzyme-linked immunosorbent assay of free triiodothyronine in human serum ("Thyroid IFA-free T3").

- Instructions for the use of a set of reagents for the quantitative enzyme-linked immunosorbent assay of thyroid-stimulating hormone in human serum ("Thyroid IFA-TTG").

- Instructions for the use of a reagent kit for the quantitative enzyme-linked immunosorbent assay of autoantibodies to thyroid peroxidase in human serum (“Thyroid IFA-atTPO”).

Claims (1)

A method for the differential diagnosis of thyroid diseases, consisting in a quantitative enzyme-linked immunosorbent assay of thyroid hormones, characterized in that they conduct a histo-enzymatic study of the thyroid peroxidase (TPO) enzyme in the thyroid gland, in order to detect colored reaction products localized only in the thyroid cytoplasm both in the follicle wall and in the interfollicular adenomatous zones, the identification of signs of macro- and microfollicular goiter, which is combined with the presence of adenomatous zones, di agnostic diffuse toxic goiter; if the activity of the enzyme in thyrocytes is moderate or high, only single fully formed follicles are visible, the content of colored reaction products in the follicle wall is high, a multi-site toxic goiter is diagnosed; if adenomatous foci are detected, the enzyme activity is moderate or high in the follicle wall, interfollicular connective tissue layers are clearly outlined and their products do not have stained histochemical reaction products, they diagnose follicular adenoma in a state of euthyroidism; when histochemical preparations reveal completely adenomatous and parenchymal foci without a lumen with very high enzyme activity, merging with each other into solid stained areas, the epithelium in the follicle wall is unevenly layered with moderate and high TPO activity, the products of the interfollicular connective tissue layer are absent, and there are no diagnostic .

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