RU1805403C - Method for prognostication course of ophthalmology herpes at men - Google Patents

Abstract

Scope of application; medicine, namely ophthalmologists for predicting the course of ophthalmic herpes. SUMMARY OF THE INVENTION: serum concentrations of testosterone and prostoglandin E are determined and, with testosterone values of 2.4 ng / mg or more and 1.1 ng / mg or less, a favorable and unfavorable course of the disease is predicted; in patients with testosterone indicators in the range of 1.2-2.3 ng / mg, prostaglandin E is determined, at values of which 340 ng / ml or more, an unfavorable course is predicted, 270 ng / mg or less is favorable. The application of the method in medical practice will increase the number of simultaneously conducted forecasts.

Description

The invention relates to the field of medicine, in particular to ophthalmology.

The purpose of the invention is to increase the number of simultaneously performed forecasts of ophthalmic herpes in patients.

The method is as follows

5-7 ml of blood are taken from the patient’s vein (up to 43 in the presence of one kit for radioimmunological determination), after retraction of the clot, the serum is taken in three test tubes: in two 0.1 ml for determination of testosterone and one - 1 ml for the detection of prostaglandin E.

When determining testosterone for extraction, 3 ml of diethyl ether are added to each of the two test tubes containing 0.1 ml each, and they are blocked by stoppers, vigorously shake the samples on a vortex mixer for 1 min, centrifuge the samples under acceleration, 1500-2000d for 2 min, transferring 1 ml

ethereal extract into assay tubes, which are then placed in a water bath and the ether is completely evaporated at 37 ° C. Before the extraction, the background testosterone in the assay provided by the solvent used is determined. To do this, determine the parameters of antiserum binding in a calibration sample without testosterone in the absence of (Bo) m in the presence of (Vef) solids after evaporation of 1 ml of solvent. Then 6 calibration testosterone samples are prepared using the appropriate serum testosterone radioimmunoassay reagent kit human blood STERON-1-125 J.

To this end, the contents of the concentrated buffer vial are poured into a glass and 30 ml of distilled water are added, and stirred. . Then, 11 ml of buffer is added to the 125J testosterone vial.

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solution, mix until completely dissolved. To obtain a solution of antisorum in a vial with this preparation 11 ml of buffer solution is added, mix. From a vial with a solution of testosterone in alcohol (640 ng / ml), 0.1 ml was collected and added to 9.9 ml of buffer solution. The resulting testosterone solution is a test sample of testosterone containing 6.4 ng / ml. All other testosterone calibration sample solutions containing 3.2 ng / ml; 1.6 ng / ml; 0.8 ng / ml; 0.4 ng / ml; 0.2 ng / ml was prepared by appropriate dilution with a buffer solution of the first sample with 5.4 ng / ml.

Label tubes for total activity (T), background testosterone (1.2), calibration curve (5-16) and for analysis (17-102), B tubes for determination of total activity (T), background testosterone (1-4) containing a dry residue of the ether extract from the analyzed sample (17-102), 0.1 ml of buffer solution is added.

In test tubes 5-16, 0.1 ml of a solution of the corresponding testosterone calibration samples are added. 0.2 ml of solution 2bJ and 0.2 ml of antiserum solution are added to all tubes. The contents of all tubes are thoroughly mixed using a vortex mixer ,

Then all tubes are incubated at room temperature 18.25 ° C for 2 hours. All tubes except T are poured with 1 ml of solution of polyethylene glycol, thoroughly mixed on a vortex mixer, then the tubes are placed in a centrifuge with a horizontal rotor and centrifuged , all samples at room temperature for 15 minutes with an acceleration of 1500-2000 d. After this, the supernatant is aspirated and the count rate of each precipitate is measured, and the tubes are measured for 1 minute in a gamma counter such as Gamma-1, Gamma-12. The arithmetic average of the counting rates of and-) testosterone is found for each of the 43 pairs of tubes. Then, calculations and graphical construction are carried out according to well-known instructions for using the CTEPOH-T-1 / 5J reagent kit for the radioimmunological determination of testosterone in human blood serum (approved by the USSR Ministry of Health on 16 November 1984). When translating the volume-weight concentration of testosterone into molar, the following ratio was used: nmol of testosterone / l ng / ml of testosterone x 3.46.

In order to determine prostagledm E, 3 ethers are added to 1 ml of patients serum, after removal of which 3 ml of a solution containing ethyl acetate is added.

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isopropanol, 0.2 HCI in a ratio of 3: 3: 1. The mixture is stirred and 2 ml of ethyl acetate and 3 ml of distilled water are added. Stirred, centrifuged, the solution was separated into phases. The organic phase, 3 ml, is transferred to a polypropylene tube and dried in a stream of air under the hood. A selica gel column is used to separate fractions of prostaglandins. Next, paired tubes are numbered as follows:

TL Ta - total amounts of 1,2 non-specific binding complement (NSB)

.3,4-containing PGBi standard, ng / ml (Wo)

5, 6 - 1/243 standard (8.2 ng / ml) 7, 8 - 1/81 standard (25 ng / ml) 9; 10 - 1/27 standard (74 ng / ml) 11-12 - 1/9 standard (222 ng / ml) 13, 14 - 1/3 standard (667 ng / ml) 15-16-1 / 1 standard (2000 ng / ml) 17-18 - samples of patient samples. In order to prepare samples of the PGBi standard (40 ng / ml), the vial containing it, add 1.0 ml of distilled water, settle for 15 minutes, mix. In tubes numbered 1/3, 1/9, 1/27, 1/81. 1/243, 0.6 ml of gel Tris buffer are added (10 g of gelatin are added to 100 ml of Tris buffer, heated by stirring until the gelatin is dissolved, cooled to room temperature and the pH is adjusted to 7.4 , added 0.1 P NaOH or HCI),

The original PGBi standard is given the number 1/1, transfer 0.3 ml of this standard to 1/3 tubes, mix and 0.3 ml standard 1/3, transfer to 1/9 tube, mix. Continue this dilution to the last 1/243 tube.

Then, 1.1 ml of gel Tris buffer is added to the tubes with NSB, 1 ml of gel Tris buffer is added to test tubes N 3-16 and 0.6 ml of it each to test tubes with patient samples. Add to the corresponding paired tubes 50 μl of each standard PGBi and 0.4 MKL patient samples. 50 μl of 3H PGBi, which is part of a reagent kit for radioimmunological determination of prostaglandins and dissolved in 10 ml of Tris gel buffer, is added to all tubes. 50 MCL of rabbit anti-PGBi serum, also included in this kit and dissolved in 10 ml of room temperature Tris gel buffer, was added to all tubes. The contents of the tubes are mixed on a vortex mixer. Incubated in a water bath at 37 ° C for 60 minutes. In all tubes except P and Ta, 100 MCL of normal is added.

rabbit serum and 100 MCL of goat aichi-rabbit serum included in the reagent kit for radioimmunological determination of prostaglandins, mixed on a vortex mixer and incubated for 18-20 hours at 2-8 ° C, then all tubes except Ti and T2 are centrifuged. 2-8 ° C for 30 minutes at 1600 d. From all tubes except for Ti and T2, the precipitate was completely removed. Dry the tube externally and internally without touching the pellet / filter paper. 1.0 ml of 0.1 P #OH was added to all tubes, mixed and the pellet was dissolved. Pour the solutions from all tubes into the corresponding numbered vials, then add 11 ml of scintillation fluid thereto, mix. The total activity of prostoglandins in each sample is considered. Then, 0.01 ml of radioactive prostaglandim 3H PGB are added to all bottles, including Ti and Ta, all are mixed and the results are counted again. Normalized CPM should be used in all calculations according to the following formula: the normalized amount of prostaglandins in a standard or sample (CPM) is calculated by the formula

.SRMP SRM x (T1-T),

(SRM, -SRMP)

where T is the average of Tch and T; T1 is the average of Ti1 and T2, i.e. T - after measurement with H; CPM - amount of prostaglandins after 3H addition.

For each standard, calculate% deviation (Vp) according to the formula

In 100 x

(SRMp MSB)

Be

where NSB is the arithmetic mean in tubes 1 and 2, Bc B0-NSB. where B0 is the arithmetic mean in tubes 3 and A.

Then, a graph is plotted on semilogarithmic paper, where% is plotted on the vertical axis. deviations of PGBi, and along the horizontal axis the concentration of PGBi, a standard curve is built and, knowing the corresponding% deviations of each control and sample, the PGBi concentration (ng / ml) is found.

With testosterone values of 2 A ng / ml or more and 1.1 ng / ml or less, a favorable and unfavorable course of the disease is predicted, respectively: in patients with testosterone values in the range 1.2-2.3 ng / ml, a determination is made

prostaglandin E, at values of which 240 ng / ml or more predict an unfavorable course, 270 ng / ml and less favorable,

5 PRI me R 1. In a patient M. with a diagnosis of Herpetic keratitis ra-. by the immunological method, a testosterone concentration of 0.4 ng / ml was detected in the blood serum. With this

0 purpose — a CTEPOH-1-125J testosterone reagent kit for radioimmunological assay was used. The study of testosterone uroon was carried out as described above. According to our proposed method, we predicted a prolonged course of infection. Indeed, the disease took a long course, did not stop with antiviral agents and lasted 45 days.

Example 2, Patient G. has relapsed superficial herpetic keratitis on a corneal transplant. The measured testosterone concentration at day 7 of the disease was 3.7 Ig / ml. The prognosis of ophthalmic herpes is a favorable course. On the 27th day of the disease, a favorable outcome occurred without a decrease in visual acuity.

Example 3. In patients N., B., C, s

With ophthalmic herpes, testosterone levels were 1.2 ng / ml, 2.3 ng / ml and 2.2 ng / ml. Testosterone was determined using the radio immunological method described above. K-centering this

5 hormones ranged from 1.2-2.3 ng / ml. In this regard, the concentration of prostaglandin E determined in parallel studies was analyzed using an appropriate reagent kit. Its indicators were 200 ng / ml, 350 yg / ml and 470 ng / mg in the aforementioned patients, respectively. Based on our proposed method for predicting ophthalmic herpes in the first case, the disease should have been

5 have a favorable course, and in 2 and 3 - an unfavorable, prolonged course. In accordance with the prognosis of patient N., herpetic keratouveitis proceeded favorably and after 20 days, recovery began, in patients B and C. with herpetic keratitis keratitis, the disease was prolonged.

the course proceeded with complications in the form of a layering of a secondary infection and lasted 87 and 42 days, respectively. In the group of patients examined by our proposed method, the prognosis of ophthalmic herpes was correct in 94% ± 6 cases. Moreover, the prophylaxis of ophthalmic herpes was simultaneously carried out while studying 43 serum samples taken from 26 patients, which significantly increased the productivity of the laboratory worker.

Claims (1)

SUMMARY OF THE INVENTION A method for predicting the course of ophthalmic herpes in men by examining a patient’s blood, characterized in that, in order to increase productivity by increasing the number of concurrent forecasts in a sample the blood concentration of testosterone is determined and with a value of this indicator of 2.4 kg / ml or more, a favorable course of the disease is predicted, with a value of 1.1 kg / ml or less, an unfavorable course of the disease, and with a value of 1.2-2.3 kg / ml additionally determine the content of prostoglandin E and, with its value of 340 mg / ml or more and 270 ng / ml or less, an adverse course of the disease is predicted.