RU2216023C2 - Method for predicting hypercortisolemia - Google Patents

Abstract

FIELD: medicine, laboratory diagnostics. SUBSTANCE: patient's blood serum should be applied upon a slide and covered with a cover glass, dried at 37-38 C, kept for 2.5-3 h at open air and microscoped in transmitted light. At the presence of skeletal fern-like crystals in the preparation mentioned one should detect hypercortisolemia. The present innovation enables to simplify prediction technique and increase information value of the results obtained. EFFECT: higher accuracy of diagnostics. 7 ex, 10 dwg

Description

 The alleged invention relates to medicine, physiology and can be used to diagnose situations associated with a high content of cortisol in the human body. The diagnostic value of the determination of cortisol in various departments of medicine increases dramatically.

 Cortisol is a steroid hormone secreted by the adrenal cortex. It accounts for 75-90% of corticoids circulating in the blood, and regulates all physiological and biochemical processes in the body.

The content of cortisol in the blood increases:
- in diseases of the hypothalamic-pituitary system associated with increased release of adrenocorticotropic hormone (ACTH), which contributes to an increase in the function of the adrenal cortex;
- with diseases of the adrenal cortex (hyperplasia, hypertrophy, adenoma, cyst, tumor),
- with hypothyroidism, cirrhosis, asthma, diabetes mellitus;
- with ACTH-ectopic syndrome;
- during pregnancy;
- with continuous use of glucocorticoids in order to increase the protective properties of the immune system.

In the clinic, hypercortisolism syndrome is observed in the form of:
- Itsenko-Cushing's disease;
- Itsenko-Cushing's syndrome (corticosteroma - benign or malignant, bilateral small-node dysplasia of the adrenal cortex);
- in the presence of tumors of the bronchi, pancreas, liver, ovaries, secreting ACTH and corticotropin-releasing hormone (KRG);
- feminization and virilization syndrome;
- for cancer of the thyroid gland, mammary glands, stomach, colon (Nazarenko G.I., Kishkun A.A. Clinical evaluation of the results of laboratory tests. M., 2000. - P. 404-407; Bates SE et al., 1985 )

 The concentration of cortisol in the blood rises with stress, pain, fever, meningitis, central nervous system tumors, acromegaly, right ventricular failure, renal hypertension, mental depression, taking synthetic glucocorticoid analogues (prednisone, prednisone), estrogens, amphemin.

 The clinical use of synthetic glucocorticoids containing cortisol (corticosterone) for a long time in diseases such as diffuse connective tissue diseases, asthma, acute leukemia, neurodermatitis, eczema, allergic diseases (anaphylactic shock. Serum sickness, angioedema) can lead to the syndrome overdose, to the development of the pharmacological syndrome of hypercortisolism (P. V. Sergeev, P. A. Galenko-Yaroshevsky, N. P. Shimanovsky. Essays on biochemical pharmacology. M., 1996).

The concentration of cortisol in serum is usually evaluated by immunochemical methods (radioimmunological, enzyme immunoassay). A set of reagents Steron-K- ¹²⁵ 1-M is used (Instructions for using a set of reagents for radioimmunological determination of cortisol in human blood serum using cortisol labeled with J ¹²⁵ and precipitating reagent Steron-K- ¹²⁵ 1-M. Minsk .: Flame, 1988: .

 For the enzyme immunoassay method, the test kit "Steroid IHLA CORTIZOL" of CJSC "Alkor-Bio", Russia is used. St. Petersburg, st. Cabinet, 28a.

 The above methods along with the undoubted advantages (high sensitivity and specificity) have limitations in use. The progress of the study is laborious, requires a long time.

So, for example, for the radioimmunological determination of human serum cortisol, a kit is required, which includes:
1) J ¹²⁵ cortisol, lyophilized preparation, 1 bottle, total activity 90-180 kBq;
2) 7 calibration serum samples containing known amounts of cortisol - 0, 20, 50, 150, 400, 1000, 2000 nmol / L after dissolution, lyophilized preparations or liquids;
3) buffer solution;
4) antiserum;
5) precipitating reagent.

The principle of operation of the kit is as follows:
1) the components of the kit and a sample of blood serum J ¹²⁵ - cortisol and endogenous cortisol enter the reverse immunochemical reaction with rabbit antibodies against cortisol;
2) simultaneously with this reaction, the interaction of antibodies against cortisol with the second antibodies (against rabbit immunoglobulins) proceeds and as a result a triple complex forms;
3) the amount of antibody bound J ¹²⁵ -cortisol is inversely related to the concentration of cortisol in the system:
4) the concentration of cortisol in the patient’s blood serum sample is determined from the graph of the activity of the precipitated bound J ¹²⁵ -cortisol on the concentration of cortisol in the calibration samples.

The main disadvantages of this analogue:
- the presence of a gamma radiation source in the form of J ¹²⁵ -cortisol;
- compliance with the rules for working with radioactive substances in the third class:
- the presence of a measurement counter type "Gamma-1":
- the complexity of the course of determination:
- high cost of equipment.

 We used an immunometric method for the quantitative determination of cortisol in human serum as a prototype (test kit Steroid IHLA CORTIZOL - CJSC Alkor-Bio, St. Petersburg, 28a Korpusnaya St. Tel. 7 (812) 230-78-56.

 The method is based on a competitive method based on enhanced luminescence (competition of endogenous cortisol contained in the sample with cortisol labeled with horseradish peroxidase, competition for binding to antibodies immobilized on the inner surface of the wells).

The method is as follows:
1) prepare a working solution (conjugate cortisol peroxidase) by mixing a concentrated solution of "Conjugate E" and "Buffer A" in a ratio of 1:25 in accordance with the table:
- strips - 2-8;
- conjugate E - 320-400-520-600-680-800-920 μl;
- buffer A-8-10-13-15-17-20-20-23 ml;
2) reconstructed calibration samples are prepared by adding 0.5 ml of double-distilled water to vials with calibration samples “1” - “6”;
3) prepare a signal solution, for which signal reagents A and B in a ratio of 1: 1 are added to a 35 ml bottle adapted to the Amerlight dispenser and mixed;
4) a wash buffer is prepared, for which 450 ml of double-distilled water is added to a vessel with a capacity of 1000 ml and 50 ml of concentrated “Buffer P” is added;
5) carry out the following sequence of operations:
- install in the holder a sufficient number of cells for analysis of all necessary samples and samples;
- 50 μl of calibration samples of the control and test sera are pipetted into the appropriate wells:
- a dispenser contribute 200 μl of conjugate to each antibody well;
- cover the wells with a lid, shake for 1 hour in the incubator-shaker Amerlight at 37 ^o C;
- washed the wells in the device for washing the Amerlight,
- add 250 μl of LIANA signal solution to all wells with antibodies;
- 2-20 minutes after the introduction of the signal solution, the wells are read on the Amerlight analyzer, having previously entered into the analyzer the values of the cortisol content in the calibration samples:
- print out analyzes.

The disadvantages of the method:
1. The complexity of execution.

 2. The high cost of the method.

 3. The use of individual devices, expensive equipment.

 4. The presence of potentially infected material (human blood products).

Tasks:
1. Simplify the method of sample preparation.

 2. Isolate microtypes of crystals according to the pattern of specific crystallization of cortisol by increasing the level of its concentration in blood serum.

 3. Identify typical crystallograms of blood serum with hypercortisolemia due to various diseases.

 4. Reduce the cost of determining the concentration of cortisol.

 5. Reduce the complexity.

 6. To increase informational content.

The essence of the invention lies in the fact that to determine hypercortisolemia, the patient's blood serum is applied to a glass slide, covered with coverslips, dried at T = + 37-38 ^o C, kept in the open air for 2.5-3 hours, then microscopied in transmitted light and in the presence of skeletal fern-like crystals in the preparation, hypercortisolemia is determined.

 The method is as follows.

 1. For the fence, peripheral blood is used (from the finger), for which a scarifier inflicts damage in the area of the human finger.

 2. 0.06-0.1 ml of blood is collected by the Panchenkov capillary.

 3. The resulting volume is centrifuged to obtain serum.

 4. Serum in the form of droplets with a volume of 0.01-0.02 ml each is applied to a glass slide and each drop is covered with a coverslip.

5. Dry in a thermostat at T = + 37-38 ^o C for 1.5-2 hours.

 6. Withstand outdoors 2.5-3 hours.

 7. Under a microscope, the pattern of crystallization is studied in transmitted light, and if skeletal fern-like crystals are present in the preparation, hypercortisolemia is determined.

 We previously created model reference crystallograms, for which a healthy person’s blood serum placed in a glass container was enriched with calibration samples containing a known amount of cortisol - 300, 400, 800 and 1000 mmol / l.

 Figure 1 presents the crystallogram of blood serum (SC) of a healthy person.

 Figure 2, 3 presents the reference crystallograms (KG) of blood serum enriched in a known concentration of cortisol.

 In FIG. 2a, b, small skeletal fern-like crystals are visible, the concentration of cortisol was 300 nmol / L (a) and 400 nmol / L (b).

 In FIG. 3a, b large skeletal fern-like crystals are visible, the concentration of cortisol was 800 nmol / l (a) and 1000 nmol / l (b).

 We conducted 1178 experimental studies.

Examples:
Example 1, FIG. 4. B-th P., medical history (IB) 416. Diagnosis: Itsenko-Cushing's disease Figure 4 shows the CG of blood serum of patient P., there are small skeletal fern-like crystals.

Technology. The former was damaged by a scarifier in the finger area, 0.06 ml of blood was taken by the Panchenkov capillary, it was centrifuged. Drops of blood serum (SC), each with a volume of 0.01 ml, were applied to a glass slide, each covered with a coverslip and dried in an thermostat at T = + 37 ^o C for 1.5 hours, then kept in the open air for 2.5 hours and microscopic in transmitted light. Small skeletal fern-like crystals were found. At the same time, the concentration of cortisol was determined in the SC. which turned out to be increased and amounted to 410 nmol / L (normal - 270.0 ± 1.3 nmol / L). Alleged hypercortisolemia was confirmed.

 Example 2, FIG. 5. B. B. IB 699. Diagnosis: Allergic reaction. Hives. Figure 5 shows the CG of serum b-th B., there are small skeletal fern-like crystals.

Technology. B. B. using a scarifier caused damage to the finger. Capillary Panchenkov took 0.08 ml of blood, it was centrifuged. Drops of SC, each 0.02 ml in volume, were applied to a glass slide, each was covered with a coverslip and dried in a thermostat at T = + 38 ^° C for 2 hours, then it was kept in the open air for 3 hours and microscopied in transmitted light. Small skeletal fern-like crystals were found. At the same time, the concentration of cortisol was determined in the SC, which turned out to be increased and amounted to 320 nmol / L (270.0 ± 1.3 nmol / L). Alleged hypercortisolemia was confirmed.

 Example 3, Fig.6. B-naya G. IB 998. Diagnosis: Type I diabetes mellitus with Moriak's syndrome. Photo 6 shows the serum CG of b-th G., there are small skeletal fern-like crystals.

Technology. Gth G. with a scarifier caused damage to the finger. Capillary Panchenkov took 0.08 ml of blood, it was centrifuged. Drops of SC, each with a volume of 0.01 ml, were applied to a glass slide, each was covered with a coverslip and dried in a thermostat at Т = + 38 ^° С for 1.5 hours, then it was kept in the open air for 3 hours and microscopied in transmitted light. Small skeletal fern-like crystals were found. At the same time, the concentration of cortisol was determined in the SC, which turned out to be increased and amounted to 350 nmol / L (normal 270.0 ± 1.3 nmol / L). Alleged hypercortisolemia was confirmed.

 Example 4, Fig.7. B-th M. IB 39. Diagnosis: Asthma, mixed, severe. The patient is hormone-dependent, constantly takes 15 mg of prednisone. 7, there are large skeletal fern-shaped crystals.

Technology. G-th M. was inflicted with a scarifier on the finger. Capillary Panchenkov took 0.1 ml of blood, centrifuged. Drops of SC, each with a volume of 0.01 ml, were applied to a glass slide, each was covered with a coverslip and dried in a thermostat at T = + 37 ^° C for 1.5 hours, then it was kept in the open air for 2.5 hours and microscopied in the light. Large skeletal fern-like crystals were found. At the same time, the concentration of cortisol was determined in the SC, which turned out to be increased and amounted to 850 nmol / L. Alleged hypercortisolemia was confirmed.

 Example 5, Fig. 8. B-th R. IB 218. Diagnosis: Adrenal gland adenoma. In FIG. 8 large skeletal fern-like crystals are present.

Technology. B. R. was inflicted damage on the finger with a scarifier. Capillary Panchenkov took 0.08 ml of blood, centrifuged. Drops of SC, each with a volume of 0.01 ml, were applied to a glass slide, each was covered with a coverslip and dried in a thermostat at T = + 37 ^° C for 1.5 hours, then it was kept in the open air for 2.5 hours and studied in the light. Large skeletal fern-like crystals were found. At the same time, the concentration of cortisol was determined in the SC, which turned out to be increased and amounted to 750 nmol / L. Alleged hypercortisolemia was confirmed.

 Example 6, Fig.9. B-th L., IB 795. Diagnosis: Systemic lupus erythematosus, immunocomplex nephritis. Figure 9 shows large skeletal fern-like crystals.

Technology. B-th L. scarifier caused damage in the finger area. 0.09 ml of blood was taken by the Panchenkov capillary, centrifuged. Drops of SC, each 0.02 ml in volume, were applied to a glass slide, each was covered with a coverslip and dried in a thermostat at T = + 38 ^° C for 1.5 hours, then it was kept in the open air for 2.5 hours and microscopied in the light. Large skeletal fern-like crystals were found. At the same time, the concentration of cortisol was determined in the SC, which turned out to be increased and amounted to 600 nmol / L. Alleged hypercortisolemia was confirmed.

 Example 7, figure 10. B-th U., IB 795. Diagnosis: pituitary adenoma. Acromegaly. Hypercorticism Syndrome. Figure 10 shows small skeletal fern-like crystals.

Technology. B-th W. with a scarifier caused damage to the finger. Capillary Panchenkov took 0.1 ml of blood, it was centrifuged. Drops of SC, each with a volume of 0.01 ml, were applied to a glass slide, each was covered with a coverslip and dried in a thermostat at T = + 37 ^° C for 1.5 hours, then it was kept in the open air for 2.5 hours and microscopied in the light. Small skeletal fern-like crystals were found. At the same time, the concentration of cortisol was determined in the SC, which turned out to be increased and amounted to 370 nmol / L. Alleged hypercortisolemia was confirmed.

The proposed method when used allows you to:
1. Simplify the definition of hypercortisolemia.

 2. Eliminate the need for complex reagents and devices.

 3. Create economic benefits.

 4. To provide the possibility of rapid diagnosis of hypercortisolemia, high information content and reliability.

 The application of the method allows to increase the reliability of obtaining results, significantly reduce labor costs, simplify the method of determining hypercortisolemia.

Claims (1)

A method for determining hypercortisolemia, characterized in that the patient's blood serum is applied to a glass slide, covered with coverslips, dried at T = (37 - 38) ^o C, kept in the open air for 2.5-3 hours, then microscopic in transmitted light and at the presence of skeletal fern-like crystals in the preparation determines hypercortisolemia.

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