Method for predicting the flow of rheumatoid uveitis

FIELD: medicine, ophthalmology.

SUBSTANCE: in patients with rheumatoid uveitis in acute period of disease, that is within the 7th to the 10th d one should detect the concentration of tumor necrosis factor (TNF-α) concentration in blood serum. At its content being 150 pcg/ml and higher the flow of rheumatoid uveitis is considered to be favorable, and at concentration ranged 50-100 pcg/ml - unfavorable. The innovation increases the accuracy in predicting the severity degree of the flow of rheumatoid arthritis and provides the chance for conducting adequate medicinal therapy.

EFFECT: higher efficiency of therapy.

6 ex

 

The invention relates to medicine, in particular to ophthalmology, and can be used in medical practice for prognosis of rheumatoid uveitis (RU).

Methods of prognosis of RU based on the study of this disease immunopathological changes, accompanied by abnormalities in the number and range of subpopulations of lymphocytes in the blood, changes in the level of antibodies to immunoglobulin G, Undenatured DNA (Weerasuriya. Clinical rheumatology, 2001, pp.52-54) and the local cytokine status (Lietuviska et al. Inflammatory diseases of the organ of vision, Chelyabinsk, 2004, p.9-11).

About the nature of the course of uveitis and the prediction of outcomes judged on functional activity of blood cells detected by the level of their synthesis of cytokines, one of which is tumor necrosis factor (TNF-α). This cytokine determines the acute phase of inflammation and affects the induction of autoimmune reactions.

Closest to the technical essence is the article Ossipova et al."A comparative study of the role of cytokines in various forms of eye diseases. Message 1. The tumor necrosis factor alpha (Ossipova et al., Journal of ophthalmology, 1998, No. 3, pp.28-32), which set out the possibility of predicting the course of endogenous uveitis while determining the content of TNF-α in the serum and after the Noah liquid.

The disadvantage of this method is the simultaneous intake with 2 samples (serum and tear fluid) to study the need for calculations of the ratio of the concentrations of cytokines in the serum and tear fluid that requires additional time, in addition to this, you should increase the consumption of expensive reagents.

The technical result of the invention is to improve the accuracy and reduce the time of prognosis of PN.

The technical result is achieved by way of forecast, which consists in determining the concentration of tumor necrosis factor alpha in acute period of the disease in the serum, wherein the tumor necrosis factor alpha is determined in serum from 7 to 10 days from the beginning of the disease, when we celebrate the more significant accumulation of this cytokine in the serum, and its contents 150 PCG/ml and above for rheumatoid uveitis assessed as favourable, and at a concentration in the range from 50 to 100 PCG/ml as negative.

The advantage of the proposed method of predicting the course of RU is a single definition of the content of TNF-α only in the serum during the acute period on the 7th-10th day of the disease, saving more than 2 times, time for preparation of the samples (no need to collect tear fluid and flow rate on roostaei reagents. Confirmation of the proposed method of prediction is the coincidence of the concentration indices of the studied cytokine in the serum with a predictable clinical course of RU in 85.7% of cases.

The method is as follows.

Blood samples without anticoagulant to obtain serum exercise on an empty stomach from the cubital vein of the subject in a strictly sterile conditions.

Determining the level of Pro-inflammatory TNF-α in the serum of patients spend enzyme-linked immunosorbent method with the use of horseradish peroxidase as indicator enzyme using test systems ProCon (LLC "Protein component", St. Petersburg).

In the microplate with immobilized antibodies contribute 100 ál of standards TNF-α and 100 μl of the samples of blood serum. 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. The tablet is dried by tapping the surface and on the laboratory bench, covered with 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 levels of the studied cytokines in the blood serum of healthy persons (control group), as a rule, does not exceed 50 PCG/ml

In 28 patients RU was studied the contents of the proinflammatory cytokine TNF-α in the serum during the acute period of the disease. Blood samples without anticoagulant to obtain serum was carried out on an empty stomach from the cubital vein of the subject in a strictly sterile conditions.

Determining the level of TNF-α in the serum of patients spend enzyme-linked immunosorbent method with the use of horseradish peroxidase as indicator enzyme.

In the microplate with immobilized antibodies contribute 100 ál of standards TNF-α and 100 μl of the samples of blood serum ballnet 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 minutes at +20 - +25°With continuous shaking. Fluid from the cells were removed and washed three times with buffer, then twice with distilled water. The tablet is dried 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. See the development of blue color. The reaction is stopped by adding 50 μl of sulfuric acid to each well.

Analysis is carried out with the use of automatic photometer "Multiscan" at a wavelength of 450 nm, setting the zero absorbance in the hole with standard 0. The results are expressed in PCG/ml

In 22 (78.6 per cent) of 28 patients RU content of TNF-α in the serum at 7-10 days from the onset of the disease was detected in the range from 150 PCG/ml and above. However, 20 (90.9 per cent) of them clinically, the disease was progressing favorably, and by the end of the 2nd week of the inflammatory process was docked 2 (9,1%) patients during uveitis was characterized as adverse, because within 2 weeks of continued inflammatory response perikornealnoy injection and precipitates. Only towards the end of the 3rd week was marked subsidence of the inflammatory process in the eye.

In 6 (21.4 per cent) of 28 patients RU concentration of TNF-α serum in the same period of the disease ranged from 50 to 100 PCG/ml negative for RU was observed in 4 (66,7%) patients. Despite ongoing anti-inflammatory therapy, there was a further decrease in visual acuity, increased pericorneal injection, there were fresh precipitates on the endothelium of the cornea and posterior synechia, neovascularization of the iris. 2 (33.3%) patients the disease progressed favorably with the relief of the inflammatory process and good visual results to the end of the second week of treatment.

Thus, the proposed method of predicting the course of RU was correct in 24 of the 28 patients, which was significantly in 85.7% of cases. In addition, staged reactions carried out with one type of sample - blood, was reduced in 2 times the consumption of reagents, and the collection and preparation of the sample is greater than 2 times that in the end, it was possible to reduce the time of prognosis of uveitis more than 2 times.

Example 1. Patient X. the hospital treatment of rheumatoid uveitis. Determination of cytokines in serum was carried out n the above-described method. In the midst of disease for 8 days, the content of TNF-α serum was equal 59,7 PCG/ml on the basis of which predicted an unfavorable course. Indeed, in the development of the disease, in spite of anti-inflammatory treatment, it was noted the emergence of fresh precipitates, new adhesions and increased cell suspension of moisture in the front of the camera. The patient was hospitalized for more than 3 weeks. Thus, low levels of TNF-α in the serum (59,7 PCG/ml) in the acute stage indicates an unfavorable prognosis of PN.

Example 2. Patient S., 43 years old, was hospitalized about rheumatoid uveitis. Determination of cytokines in the serum was performed according to the above method. In the midst of disease on day 7, the content of TNF-α serum was equal 56,2 PCG/ml, was expected unfavorable course of uveitis. Anti-inflammatory treatment, carried out sick for 20 days did not improve the clinical picture of the disease, continued pain, low visual acuity, biomicroscopic revealed precipitates and adhesions, which confirmed the unfavorable prognosis of PN.

Example 3. Patient F., 56 years old, was admitted to our Department with a diagnosis of relapse of chronic uveitis rheumatoid etiology. On the 9th day of onset of sod is neigh TNF-α serum amounted to 91.4 PCG/ml, which assumed the adverse prognosis of RU. Despite ongoing for 17 days comprehensive treatment, to achieve positive clinical effect failed, which was a confirmation of the adverse prognosis of PN.

Thus, low levels of TNF-α in the serum (50-100 PCG/ml) in the acute stage indicates an unfavorable prognosis of PN.

Example 4. Patient O. Diagnosis of PN. Determination of cytokines in the serum was performed according to the above method. On the 7th day from the onset, the content of TNF-α serum - 178,5 PCG/ml on the basis set within the above interval (interval (150 PCG/ml and higher) values of TNF-α in the serum of the patient is predicted favorable for rheumatoid uveitis. Subsidence of the inflammatory process, the disappearance of pericorneal injection and partial resorption of precipitates on the endothelium of the cornea, were noted to 13-14 days after the onset of the disease, which confirmed the previously suggested favorable prognosis of PN.

Example 5. Patient S., 62. Diagnosis: RU. On the 7th day from the onset, the content of TNF-α serum - 1165,5 PCG/ml On the basis set within the above interval (150 PCG/ml and higher) values of TNF-&x003B1; in the serum of the patient is predicted favorable for rheumatoid uveitis. Subsidence of the inflammatory process, the disappearance of pericorneal injection and partial resorption of precipitates on the endothelium of the cornea, were noted on day 12 after the onset of the disease, which confirmed the expected us favorable prognosis.

Example 6. Patient M., 48 years old. Diagnosis at admission: acute rheumatoid uveitis. The content of TNF-α in the serum of the patient on the 7th day from the onset of the disease was equal 1173,7 PCG/ml, which assumed favorable for RU. Indeed, already on day 10 of disease during treatment there was a positive trend: no pain, decreased hyperemia of the conjunctiva of the sclera, the cornea is partially resorbed precipitates, synechia exploded. The improvement of clinical picture of the disease testified about the correct prognosis of PN.

Thus, the proposed method of prognosis of rheumatoid uveitis allows to evaluate the clinical course of this disease in the acute stage (between 7 and 10 day) on the contents of TNF-α in the serum of patients. Prognosis of RU is favorable when the level of TNF-α in the serum of patients 150 PCG/ml and above from 7 to 10 days from the beginning of the disease and adverse - when its concentration in the range from 50 to 100 PCs who/ml in the same period.

Method of prognosis of rheumatoid uveitis by determining the concentration of tumor necrosis factor α in the acute period of the disease in the serum, wherein the tumor necrosis factor α determine serum from 7 to 10 days from the beginning of the disease and its contents to 150 PCG/ml and above for rheumatoid uveitis assessed as favourable, and at a concentration in the range from 50 to 100 PCG/ml as negative.



 

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