Method for predicting ophthalmoherpes

FIELD: medicine.

SUBSTANCE: method involves concurrently examining anti-inflammatory IL-4 level in blood serum and lacrimal fluid. The value being within the limits of 60-70 pg/l in blood serum and 5-15 pg/l in lacrimal fluid, disease prognosis is considered to be unfavorable. The IL-4 concentration being within the limits of 90-100 pg/l in blood serum and 20-30 pg/l in lacrimal fluid, disease prognosis is considered to be favorable.

EFFECT: high accuracy of diagnosis.

 

The invention relates to medicine, in particular to ophthalmology, and can be used in medical practice for predicting the course of ophthalmic herpes.

Methods of prediction of ophthalmic herpes based on the study of immunopathological changes in this disease involving abnormalities in the number and range of subpopulations of lymphocytes and cytokine changes the status of the patient (Webmachine, 1997; A.Biglino, 1996).

There is a method of prognosis of ophthalmic herpes (Webmachine, Tonkonogov, Anabuki, "Ophthalmological journal, 1981, No. 3, p.142-143) by defining subpopulations of lymphocytes method rosethorne with sheep erythrocytes. The disadvantages of this method are its complexity, subjectivity in the evaluation of the results and the small number of simultaneously ongoing research.

More modern methods for the determination of lymphocyte subpopulations based on their immunophenotyping. The functional activity of blood cells, associated with the nature of the flow of ophthalmic herpes, determine the level of synthesis of cytokines by them.

The closest in technical essence is "the Way to prevent postoperative complications of penetrating corneoscleral wounds of the eye" (Ossipova et al., Journal of ophthalmology, 1998, No. 3, pp.28-32). The essence of the method consists in a single definition with the holding of tumor necrosis factor α in the serum and tear fluid of patients with ophthalmic herpes, with corneoscleral wounds, cataract, glaucoma, retinal detachment. The authors have shown pathogenetic role of cytokines - tumor necrosis factor α ophthalmic herpes and corneoscleral wounds, cataract, glaucoma, retinal detachment.

The disadvantage of this method of prediction is that the pathogenetic role of other cytokines and the possibility of using them as indicators of severity of herpes of the eye has not been studied. In addition, studies of the content of tumor necrosis factor α defined only once.

The cytokine IL-4 is a key mediator of immune response, provides a manifestation of the helper function of T-lymphocytes and an increase in its level, as a rule, is connected with its hyperproduction or regulatory effects of other cytokines.

The technical result of the invention is increasing the frequency and reducing the time of predicting the course of ophthalmic herpes on the basis of the determination of interleukin (IL) 4 in serum and tear fluid of patients with herpes of the eye in the acute period of the disease.

The technical result is achieved by determining the content of interleukins in the acute period of the disease, characterized in that both in the serum and is useful liquid investigate the level of anti-inflammatory IL-4, when its content in the blood serum within 60-70 PCG/ml and in the lacrimal fluid - 5-15 PCG/ml, the prognosis of the disease is assessed as unfavourable, and the IL-4 concentration in the range 90-100 PCG/ml in serum and 20-30 PCG/ml in tear fluid as favorable.

The advantage of the proposed method for the prognosis of herpes of the eye is the simultaneous determination of anti-inflammatory IL-4 in serum and tear fluid in the acute phase of ophthalmopathy flowing hard and most often leading to slobovian and blindness. Confirmation of the proposed method is a coincidence of contents of the studied cytokines with clinical course of ophthalmic herpes more than 90% of cases.

The method is as follows.

From the cubital vein of the patient, blood is taken on an empty stomach in a strictly sterile conditions in a 5 ml Vial with patient blood centrifuged to obtain serum.

Lacrimal fluid is picked up by the introduction of the conjunctival SAC in the amount of 1 ml.

Determining the level of anti-inflammatory IL-4 in serum and tear fluid of patients spend enzyme-linked immunosorbent method with the use of horseradish peroxidase as indicator enzyme using test systems Loop (OOO Protein loop”, With a weight is).

In the microplate with immobilized antibodies contribute 100 ál of standards IL-4 and 100 μl of the samples of serum and tear fluid of patients. The tablet is incubated for 1 hour at +37°With continuous shaking, then washed three times with buffer and twice with distilled water. Conducts a full aspiration of the remaining fluid.

In each of the wells of the microplate make 100 ál conjugate with streptavidin horseradish peroxidase, diluted with buffer. Tablet incubated for 30 min at +20-25°With continuous shaking. Fluid from the cells were removed and washed three times with buffer, then twice with distilled water. Tablet oosevelt by tapping the surface of the lab table covered with a filter paper.

All wells contribute 100 ál of substrate solution with the dye. The microplate incubated for 10-15 minutes at room temperature protected from the direct rays of the place on the shaker. There is the development of blue color. The reaction is stopped by adding 50 μl of sulfuric acid to each well. The total time of production research 2.5-3 hours.

Analysis is carried out with the use of automatic photometer “Multiscan” at a wavelength of 450 nm, set the zero absorbance in the hole with standard 0. The results are expressed in PCG/ml Normal level of the investigated C is Takenov in healthy people, as a rule, does not exceed 50 PCG/ml

In 86 patients with ophthalmic herpes studied the content of anti-inflammatory IL-4 in serum and tear fluid in the acute period of the disease. From the cubital vein of the patients blood samples were taken fasting in strictly sterile conditions in an amount of 5 ml test Tubes with the blood of the patients was centrifuged to obtain serum. Tear fluid was taken by the introduction of the conjunctival SAC in the amount of 1 ml.

Determining the level of anti-inflammatory IL-4 in serum and tear fluid of patients was carried out by enzyme-linked immunosorbent method with the use of horseradish peroxidase as indicator enzyme.

In the microplate with immobilized antibodies were made in 100 μl of standard IL-4 and 100 μl of the samples of serum and tear fluid of patients. Tablet incubated for 1 hour at +37°With continuous shaking, then washed three times with buffer and twice with distilled water. Undergoing a complete aspiration of the remaining fluid.

In each of the wells of the microplate made of 100 ál conjugate with streptavidin horseradish peroxidase, diluted with buffer. Tablet incubated for 30 min at +20-25°With continuous shaking. Fluid from the cells was removed and washed three times with buffer, then twice with distilled water. Tablet abusively by postalia the Oia on a laboratory bench, covered with filter paper.

All wells were made in 100 µl of substrate solution with the dye. The microplate was incubated for 10-15 minutes at room temperature protected from the direct rays of the place on the shaker. Observed the development of blue color. The reaction was stopped by adding 50 μl of sulfuric acid to each well.

Analysis was performed using an automatic photometer “Multiscan” at a wavelength of 450 nm, setting the zero absorbance in the hole with standard 0. The results were expressed in PCG/ml

Within 90-100 PCG/ml in serum and 20-30 PCG/ml lacrimal fluid it was detected in 51 patients. However, 47 (92.2 per cent) of people the disease was favorable, the inflammatory process was docked within 14 days. 4 (7,8%) patients for ophthalmic herpes was unfavorable for 3 weeks did not occur epithelization of the cornea remained inflammatory response. By the end of 4 weeks was marked subsidence of the inflammatory process in the eye.

The content of cytokines in the acute period of the disease in the serum within 60-70 PCG/ml and in the lacrimal fluid - 5-15 PCG/ml was detected in 35 patients with ophthalmic herpes, and unfavorable course of the disease (more than 30 days) was observed in 33 (94,3%), while 2 (5.7 per cent) was noted a short (up to 14 days) blahop etoe for herpes of the eye.

Thus the proposed method makes a prediction of ophthalmic herpes reliable in 92,2% and unfavorable in 94.3% of cases, it is generally correct in 80 patients from 86 (93%).

Example 1. Patient F. was hospitalized about herpes tree keratitis. Determination of cytokines in serum and tear fluid was carried out by the above described method. In the midst of disease, the content of Il-4 in serum was equal to 66.7 PCG/ml, in the tear fluid and 6.2 PCG/ml Despite ongoing antiviral treatment, it was noted defeat the deep layers of the cornea with involvement in the inflammatory process of the choroid. Was diagnosed materpiece keratitis, acute iridocyclitis. The patient was hospitalized for 21 days. Thus, low levels of IL-4 in serum and a significant decrease in tear fluid in the acute phase indicate an unfavorable prognosis, course of ophthalmic herpes.

Example 2. The patient s Diagnosis: the tree of herpetic keratitis. Determination of cytokines in serum and tear fluid was carried out by the above described method. In the midst of disease, the content of anti-inflammatory IL-4 in serum was equal to 94.7 PCG/ml, and in the tear fluid for 29.3 PCG/ml subsidence of the inflammatory process, resorption detail is litrato of the cornea, noted to 10 days after onset of the disease. In this period remained insignificant peritoneally injection of the sclera, deep optical medium were calm. Installed above the levels of IL-4 in serum and tear fluid indicate a favorable prognosis of the ophthalmic herpes.

Thus, the proposed method for prediction of ophthalmic herpes allows you to define the content of anti-inflammatory IL-4 in the acute period of the disease simultaneously in serum and tear fluid: when its content in the blood serum within 60-70 PCG/ml and in the lacrimal fluid - 5-15 PCG/ml, the prognosis of the disease is assessed as unfavourable, and the IL-4 concentration in the range 90-100 PCG/ml in serum and 20-30 PCG/ml in tear fluid as favorable.

The method of prediction of ophthalmic herpes by determining the content of interleukins in the acute period of the disease, wherein simultaneously in serum and tear fluid investigate the level of anti-inflammatory IL-4, while its content in the blood serum within 60-70 PCG/ml and in the tear fluid 5-15 PCG/ml, the prognosis of the disease is assessed as unfavourable, and the IL-4 concentration in the range 90-100 PCG/ml in serum and 20-30 PCG/ml in tear fluid as favorable.



 

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EFFECT: high accuracy of diagnosis.

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