RU2216740C2 - Method for predicting development of retinopathy in premature babies - Google Patents

Abstract

FIELD: medicine, ophthalmology. SUBSTANCE: one should carry out immunological testing of blood serum. The presence of IgG and IgM antibodies to retinal S-antigen is detected due to immuno-enzymatic assay (IEA). At values of relative density being above 0.1 for each of them at terms of 29-40-wk-long gestation it is possible to predict the development of retinopathy in premature babies. EFFECT: higher efficiency of prediction. 2 ex

Description

 The present invention relates to ophthalmology and is intended to predict the risk of developing retinopathy of premature infants. Retinopathy of premature infants (PH) is a serious disease of premature infants, often leading to visual impairment from an early age. The frequency of pH varies in different countries, reaching 24.7 per 100,000 live births. About 5,000 premature babies survive annually in Moscow, and more than 100,000 in the Russian Federation. The incidence of pH among surviving premature babies is 27%, and among deeply premature babies with birth weight less than 1000 g it reaches 84% (Khvatova A.V. et al., 2000; Katargina L.A., Skripets P.P., 2000 ) Moreover, the data of various authors on the frequency of pH at the same body mass vary widely.

 According to the international classification, active pH is subdivided into the stages of the process, its localization and extent.

 Stage 1 - the appearance of the demarcation line at the border of the vascular and avascular retina.

 Stage 2 - the appearance of the shaft (or ridge) in place of the demarcation line.

 Stage 3 - characterized by the appearance of extraretinal fibrovascular proliferation in the shaft.

 Stage 4 - partial retinal detachment - is divided into 4a (without involving the macular zone) and 4c (with retinal detachment in the macula).

 Stage 5 - complete or total retinal detachment.

 Despite many years of clinical and experimental studies, the pathogenesis of pH is not fully understood. The most difficult task is to predict the development and nature of the course of the disease.

 Clinical methods for predicting the development of pH include the identification of children at risk depending on body weight at birth, gestational age at the time of birth, duration of oxygen therapy and mechanical ventilation, concomitant somatic pathology, etc. (Khvatova A.V. et al., 1999). They have low predictive value, because very indirectly associated with the pathogenesis of the disease.

 The proposed method for the study of blood serum on the content of antibodies in it - IgG and IgM to the retinal S-antigen, allows you to determine the risk of developing pH in premature babies.

 The closest analogue is a method for predicting the risk of pH development by the presence of periods of significant fluctuations in gas levels (pCO2 from 30 to 100 mm Hg and pO2 from 33 and below to 75 mm Hg) in the blood during the period of nursing of a premature baby (Kogoleva L.V., 2001).

 The technical justification of the invention is an earlier and more reliable prediction of the risk of developing PH in premature babies.

 The technical result is achieved by identifying elevated levels of IgG and M to the serum S antigen in premature babies during incomplete retinal formation (the presence of avascular zones without signs of retinopathy) at the age of 29-40 weeks of gestation due to activation or development of organ-specific immunity of the child to retinal structures (S-antigen).

 The method is as follows.

Blood from a child’s vein in an amount of 3 ml is kept in an thermostat at 37 ^o C for 30-45 min, then cooled in the refrigerator for 15-20 min to 0 ^o C and placed in a centrifuge for 15 min at a speed of 1000 rpm. After that, the obtained blood serum without any shaped elements was frozen at -18 ^o C. The density level of IgG and M to the retinal S-antigen was determined using enzyme-linked immunosorbent assay (ELISA) using the solid phase method using horseradish peroxidase as an indicator enzyme.

The studied and control serum samples were added to a well of an ELISA polystyrene plate, on which retinal S-antigen isolated from bovine eyes (mm 48 kDa) was immobilized first, then mouse monoclonal antibodies against human IgG or IgM conjugated to enzyme label - horseradish peroxidase (company "Sigma"). After adding an enzyme-specific substrate solution (CRF) and a stop reagent (1N H ₂ SO ₄ ), the reaction is recorded on a vertical scanning spectrophotometer (Multiscan, Finland) at a wavelength of 492 nm. As a reference serum (positive control) in ELISA, lyophilized serum is used, which is a mixed pool of sera obtained from patients with various forms of retinal diseases (diabetic retinopathy, uveoretinitis, ideopathic retinal detachment, etc.).

 As a negative control, a phosphate-buffered saline (PBS, pH 7.2) containing bovine serum albumin at a concentration of 1 mg / ml (protein) is used. At the same time, we considered it necessary and sufficient to use the FBI, which is used to dilute test serum samples with the addition of a minimum amount of non-specific protein preparation, given that in normal newborn babies the total content of immunoglobulin proteins is low.

 It should be emphasized that there is a significant difference between the “negative control” used in the study of children with pH and the negative reference sera commonly used in ELISA studies of blood serum of adult patients and older children, when the standard samples are serum obtained from a blood pool from practically healthy ones ( ophthalmologically and somatically) people of different age groups.

 Evaluation of the reaction is carried out by the difference in the optical density (AOP) values obtained by spectrophotometric measurement of the test and standard, normal test serum obtained from a pool of blood serum of healthy people. For this, the average OD value was calculated for each analyzed and control (standard-normal) sample. Then, the average OD of the normal control serum was subtracted from the average OD value of the test serum. If the resulting difference (ΔOP) was greater than or equal to 0.20, the result of the study was considered positive.

 To control the reaction, a difference was found between the average OD value of the positive control serum (obtained from the pool of blood serum of patients with severe retinal pathology and used as reference serum) and normal control serum. This difference should be at least 0.20.

 Example 1. Patient Sh., Born on November 14, 1998 at 28 weeks of gestation with a body weight of 1250 g at 4 weeks of life (32 weeks of gestation), when examining the fundus, avascular areas of the retina without signs of active pH were revealed. To determine the risk of developing PH in this child, a blood test was performed for the presence of antibodies to the retinal S-antigen in the manner described above. As a result, high relative densities of IgG (0.57) and IgM (0.32) were revealed, indicating a risk of pH development. During dynamic observation, the development of active stage 1 pH at the 35th week of gestation (7th week of life) was revealed in the child. Inspections were carried out once a week. Stage 2 PH was diagnosed at 36 weeks of gestation (8 weeks of life) and progressed to 3 threshold stages (at 38 weeks of gestation, 10 weeks of life). The child underwent prophylactic cryocoagulation of the avascular zones of the retina, after which the pH passed to the cicatricial stage within 2 weeks.

 Example 2. Patient B., born 05.11.98, at 28 weeks of gestation with a body weight of 1340 g at 4 weeks of life (32 weeks of gestation), examination of the fundus revealed avascular areas of the retina without signs of active pH. To determine the risk of developing PH in this child, a blood test was also performed for the presence of antibodies to the retinal S-antigen in the same way. As a result, low relative densities of IgG (0.07) and IgM (0.02) were revealed, which indicated that there was no risk of developing pH. During the dynamic observation, the child did not show signs of pH, and the growth of the retinal vessels ended in the formation of a normal vasculature, which indicates that at low relative densities of IgG and IgM, the risk of developing pH is minimal.

 From the above examples it is seen that the proposed method allows to predict the development of retinopathy of premature infants in risk groups with an accuracy exceeding the previous methods for predicting this condition.

Claims (1)

 A method for predicting the development of retinopathy of premature infants, including an immunological study of blood serum, characterized in that the presence of antibodies of the IgG and IgM class to the retinal S antigen is determined by enzyme-linked immunosorbent assay and with relative density indices> 0.1 of each of them within 29-40 weeks of gestation predict the development of retinopathy of premature infants.