RU2314535C1 - Method for diagnosing initial stage of open angle glaucoma - Google Patents

Abstract

FIELD: medicine.

SUBSTANCE: method involves determining autoantibodies to antigens to native and denatured DNA (n- and d-DNA) and circulating immune complexes content in lacrimal fluid of a patient. Auto antibodies to (n- and d-DNA) being greater than 0.7 and 0.8 conditional units, respectively, and circulating immune complexes content being greater than 33.0 conditional units, initial stage of open angle glaucoma is to be diagnosed.

EFFECT: high accuracy and sensitivity of the method.

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Description

The invention relates to ophthalmology and is intended for the diagnosis of the initial stage of open-angle glaucoma and, accordingly, the appointment of timely treatment.

Today in the world there are more than 750 thousand patients with glaucoma. The frequency of developing blindness from glaucoma in our country and in the world stably holds at the level of 14-15% of the total number of all blind, which allows us to classify the problem of glaucoma as not only important medical, but also medical and social.

With pronounced manifestations of glaucoma, a clinical and ophthalmic examination is enough to make an accurate diagnosis. However, there is a category of patients for whom the diagnosis of glaucoma, based on clinical and ophthalmic examination data, is difficult and often impossible. The problem of early diagnosis of the initial stage of open-angle glaucoma (OAG) and, accordingly, the correct and timely treatment of glaucoma remains one of the most urgent in modern ophthalmology, in this regard, the setting of an accurate, justified diagnosis is of particular importance.

At present, destructive processes in trabecula, leading to a decrease in its permeability to aqueous humor, are considered to be the key link in the pathogenesis of primary OAG (Nesterov D.P. Glaucoma. - M., 1995, p. 104, 115-117). Modern ideas about the nature of destructive injuries are based on the postulate of the leading role of changes in cell membranes up to their destruction due to lipid peroxidation (LPO) and free radical oxidation. These processes are controlled by an antioxidant system that protects the integrity of cell membranes from the damaging effects of free radicals (Bunin A.Ya. et al. Vestnik Ophthalmology, 1985, No, pp. 13-16).

Diagnosis of the initial stage of OAG is carried out most often by the value of intraocular pressure (IOP) and the state of visual functions (Nesterov D.P. - Glaucoma, 1995, p.165-170). Known, for example, is a method for diagnosing the initial stage of OAG by determining visual functions, specifically the state of visual fields (Listopadova N.A. et al. Vestnik Ophthalmology, 1989, No. 3, pp. 3-7). The disadvantage of this method is its low diagnostic accuracy, because a change in the field of view depends on many factors, such as time of day, general fatigue, fitness, noise, poor lighting in the room where the study is being conducted, etc.

Closest to the claimed prototype method is a method for predicting the development of glaucoma, including determining the total antioxidant activity of the tear and, when this indicator decreases below 70 μM, predicting the progression of glaucoma (RF Patent No. 2139538, CL G01N 33/53, publ. 10.10.99). The disadvantages of this method are its limited functionality, since its use is already provided for glaucoma patients.

The technical problem to which the claimed invention is directed is to increase the accuracy and ensure early diagnosis of the glaucomatous process at the stage of preclinical manifestations.

The technical task is achieved by the claimed method, which consists in the following.

A tear is taken from a patient with suspicion of the initial stage of glaucoma and the levels of autoantibodies (AAT) to antigens (AH) of native and denatured DNA (n- and d-DNA) and the level of circulating immune complexes (CEC) in the lacrimal fluid and at values of indicators are determined levels of autoantibodies to n- and d-DNA antigens are higher than 0.7 and 0.8 conv. respectively, and when the value of the indicator level of the CEC above 33.0 srvc diagnose the initial stage of OAG.

AAT to antigens of native and denatured DNA (n- and d-DNA) in tear fluid was determined using DNA-TEST kits manufactured by PMC Sibmedpribor according to the manufacturer's instructions. An enzyme-linked immunosorbent assay was recorded on a Multiscan MCC 340 vertical photometer at a wavelength of 492 nm. The test results were considered positive if the optical density of the analyzed tear fluid was greater than the critical optical density, which was the optical density of the negative control × 2. The level of AAT to antigens of native and denatured DNA was determined as the ratio (index) of optical density of the test sample and critical optical density.

Determination of CEC in the tear fluid was carried out by the method of liquid precipitation in 7.5% polyethylene glycogel with m.m. 6000 per 0.01 M borate buffer pH 8.4. 0.3 ml of 0.01 M borate buffer pH 8.4 (control) and 0.3 ml of 7.5% dilution of PEG-6000 per 0.01 were added to nearby wells of parallel strips of 96-well flat-shaped plates for immunological reactions M borate buffer (pH 8.4) (test). 0.005 ml of the investigated human tear fluid was added to the control and experimental wells. Incubated for 1 hour at room temperature. The optical density was read against the control at a wavelength of 450 nm on a vertical Multiscan MMC / 340 spectrophotometer. The number of circulating immune complexes was expressed in arbitrary units: optical density value · 1000 / (conventional units).

The levels of autoantibodies to AH of native and denatured DNA, as well as the level of circulating immune complexes, which are indicators of destructive degenerative processes leading to the destruction of cell membranes and the release of autoantigens and in the occurrence and development of autoimmune reactions, are quite informative prognostic tests to determine the initial stage of AS.

We tested the levels of AAT for AH of native and denatured DNA and the levels of CEC in the lacrimal fluid in two groups of patients.

The main study group was selected 82 patients with suspected glaucoma.

As normative values of AAT levels for AH of native and denatured DNA and CEC levels in the lacrimal fluid were taken data obtained in the Novosibirsk branch of the Federal State Institution IRTC Eye Microsurgery in 2000.

In all patients, a tear was aspirated by a microcannula from the lower conjunctival fornix of both eyes into a dry, sealed tube without prior anesthesia in an amount of 0.5 ml. Stimulation of tear production was carried out by mechanical stimulation of the receptor endings of the trigeminal nerve in the lacrimal membrane of the eye.

The levels of AAT to AH of n-DNA and AH of d-DNA, as well as the level of CEC in patients of the study group and are normally presented in the table.

Indicators CEC level, conv.sd The level of AAT to AG n-DNA, conventional units The level of AAT to AG d-DNA, usled Normative values 25.13 ± 3.99 0.47 ± 0.03 0.57 ± 0.04 Study group 35.32 ± 2.62 (p <0.01) 0.77 ± 0.10 (p <0.01) 0.82 ± 0.06 (p <0.01)

As can be seen from the table, the average level of AAT to antigens of native and denatured DNA significantly exceeded the standard values by 0.3 srvc, which allows us to consider their determination as a fairly informative test at the preclinical stage of glaucoma development. The analysis of individual values obtained in patients at the initial stage of OAG allows us to note the presence of high AAT levels for both nA DNA AG in 60.9% and AT d DNA in 62.2% of cases.

The “average” CEC levels in the study group was 35.32 ± 2.63 conv. units, which significantly exceeded the "average" standard values equal to 25.13 ± 3.99 st. units, for 10.2 conventional units

Thus, significantly high levels of AAT to AH of n- and d-DNA in the lacrimal fluid were revealed in patients with a diagnosis of “initial stage of OAG”, which indicates the development of destructive processes with damage to cell membranes leading to the release of nuclear AH at the preclinical stage of the disease to n- and d-DNA in the lacrimal fluid.

It was shown that the initial stage of OAG is also accompanied by activation of the humoral link of the immune system, which manifests itself as a significant increase in the level of CICs, which are the product of the antigen-antibody reaction.

Studies on patent and scientific and technical sources of information showed that the proposed method is not known and does not follow explicitly from the prior art, i.e. meets the criteria of "novelty" and "inventive step".

The proposed method is distinguished by diagnostic accuracy, simplicity and accessibility and can be applied in any ophthalmology department equipped with appropriate equipment.

The invention is illustrated by the following examples of specific performance.

Example 1

Patient K., 62 years old, was admitted to the NF FSI MNTK Eye Microsurgery with suspected glaucoma in both eyes.

An examination revealed an increase in the IOP of both eyes to 27 mm. According to the results of a clinical and ophthalmological study, data on glaucoma was not found.

When immunobiochemical examination of the lacrimal fluid of the claimed method revealed: the level of AAT to AH n-DNA = 0.72 srvc .; the level of AAT to AG d-DNA = 0.87 srvc .; CEC level = 37 conventional units

The diagnosis was made: the initial stage of open-angle glaucoma in both eyes. Upon re-examination after 7 months, the first clinical manifestations were revealed in the form of an expansion of DZN excavation to 0.5, and also according to static perimetry, the appearance of scotomas characteristic of the initial stage of glaucoma in the field of view in the Bierrum zone.

The diagnosis of "initial stage of OAG" is confirmed, timely treatment is prescribed.

Example 2

Patient T., 52 years old, was admitted to the NF FSI MNTK Eye Microsurgery with suspicion of open-eyes of both eyes.

When immunobiochemical examination of the lacrimal fluid of the claimed method revealed: the level of AAT to AH n-DNA = 0.52 srvc .; the level of AAT to AG d-DNA = 0.58 srvc .; CEC level = 25 conv. units

Since the levels of AAT to AH of n- and d-DNA, as well as the level of CEC were below critical values, the diagnosis “initial stage of OAG” was not made, the patient was not treated.

During the follow-up examination after 6 and 12 months, no negative dynamics were detected both in clinical and in immunobiochemical parameters, the diagnosis of “suspected glaucoma” was removed, the patient is under observation, without treatment.

Using the proposed method will allow with high accuracy to carry out early diagnosis of the initial stage of OAG, conduct timely, adequate treatment and prevent the transition of the glaucomatous process from the initial to the developed stage.

Claims (1)

A method for diagnosing the initial stage of open-angle glaucoma by examining tear fluid, characterized in that the levels of autoantibodies to antigens of native and denatured DNA (n- and d-DNA) and the level of circulating immune complexes (CIC) are determined and when the levels of autoantibodies to antigens n - and d-DNA above 0.7 and 0.8 srvc respectively, and with the value of the CEC level above 33.0 srvc. diagnose the initial stage of open-angle glaucoma.