Method for early diagnostics of perinatal hypoxic cns lesions in mature neonatals born in gestosis-suffering mothers

FIELD: medicine, neonatology.

SUBSTANCE: in umbilical cord blood at the moment of a child's birth one should detect the ratio of the parameters for relative content of CD3+HLA-DR+ against CD3+ lymphocytes and at its value being equal to 8.1% or above it one should diagnose perinatal hypoxic CNS lesion at accuracy of 89.28%. Application of the present method enables to diagnose perinatal hypoxic CNS lesion in mature neonatals in gestosis-suffering women at earlier terms.

EFFECT: higher accuracy of diagnostics.

3 ex, 1 tbl

 

The invention relates to medicine, namely to neonatology, and can be used for early diagnosis of perinatal hypoxic CNS lesions in full-term newborns from women with preeclampsia.

The urgency of development of the method is determined by the increase in the frequency of perinatal hypoxic lesions of the Central nervous system, high rates of mortality and disability by them [1]. Diagnosis of perinatal CNS lesions in newborn children is often difficult because it is not always clear picture of neurological symptoms [2]. One of the main reasons for the development of perinatal CNS lesions in newborns is preeclampsia in the mother. The incidence of this pathology in children born to women with preeclampsia, according to literature data, reaches a 49.9%, which is 4.8 times higher than in uncomplicated pregnancies [3]. Clinical manifestations of CNS pathology in newborn not always reflect the true severity of the condition, the degree of CNS damage and future forecast of development of the child [4]. Therefore, development of a method for early diagnosis of perinatal hypoxic lesions of the Central nervous system of the newborn is important. The method will allow you to diagnose perinatal hypoxic lesions of the Central nervous system in the first day of life, to assign adequate therapy of cerebral disorders and prevent the debilitating consequences.

WPI the walls of the method for the diagnosis of perinatal CNS lesions in newborn by holding neurosonography brain. In the presence of neurological symptoms in newborns pathological changes by ultrasound examination of the brain was detected in 83% of children [Sidorov, I.S., Asnis N.P., Makarov ACTING Ultrasound diagnosis of perinatal damage of the Central nervous system in the fetus and newborn. // Russian journal of Perinatology and Pediatrics. - 1995. No. 3. - P.15-18].

The disadvantages of the method:

1) when carrying out this method reveals predominantly rough damage of brain tissue: intraventricular hemorrhage, the periventricular leucomalacia and remain inaccessible hypoxic-ischemic injuries that occur at the cellular level [4];

2) there is a risk of incorrect interpretation of data neurosonography [4];

3) conclusion on neurosonography correctly implemented not earlier than the 3rd day of a child's life [Finger A.B., Shabalov, I.E. Hypoxic-ischemic encephalopathy in newborns. St. Petersburg: Piter, 2001. - 224 S.];

4) for the implementation of the method requires special equipment.

A known method for the diagnosis of severe perinatal hypoxic lesions of the Central nervous system in newborns by research in peripheral blood serum concentrations of protein S-100 in the dynamics on the 1st, 3rd, 7th, 14th and 21st day of life. With increasing content of protein S-100 based on the reference level, but exceeding the mu diagnosed with severe perinatal CNS damage [Voice G.S., Petrukhin A.S., Markevich K.A., Trifonov per unit change in the level of protein S-100 in neonates with perinatal hypoxic lesions of the Central nervous system. // Pediatrics. - 2004. No. 1. - P.10-15].

The disadvantages of the method:

1) the level of protein S-100 significantly increased only in severe forms of perinatal lesions of the Central nervous system that cannot be used for the diagnosis of perinatal CNS lesions (mild or moderate severity;

2) for the implementation of the method requires five times the blood, causing a painful injury to the newborn and may adversely affect the course of the underlying disease;

3) high economic value method (the need to purchase expensive special sets of reagents for determining the level of protein S-100 by indirect enzyme-linked immunosorbent assay).

The closest technical solution is a method of early diagnostics of disorders of blood and CSF circulations of hypoxic Genesis in newborns by examining the level of production by monocytes cord blood interleukin 1β (IL-1β) and tumor necrosis factor α (TNF-α). Disorders of blood and CSF circulations of hypoxic Genesis monocytes cord blood was spontaneously produced more IL-1β (0,746 ng/ml against 0,259 ng/ml) and significantly more F. the O-α (1,301 ng/ml against 0,128 ng/ml) [Tabolin V.A., Volodin N.N., Degtyarev M.V., DMITRY Degtyarev. et al. Current issues in perinatal immunology. // International Journal on Immunorehabilitation. - 1997. No. 6. - P.112-122].

The disadvantages of the method:

1) IL-1β and TNF-α are not specific indicators, because their contents can be modified in various pathological conditions, particularly in sepsis [Kovalchuk L.V., Gankovsky L.V., Khoreva M.V. Sokolova, E.V. System cytokines, complement and modern methods of immunological analysis. M., 2001. - 158 S.];

2) for carrying out this method requires more time (3 hours)than for the proposed method;

3) do not specify the diagnostic accuracy of this method.

This method elected us as the closest analogue (prototype). These drawbacks are proposed to be eliminated in the present method.

The technical solution of the proposed method is the early diagnosis of perinatal hypoxic CNS lesions in full-term newborns from women with preeclampsia by defining in umbilical cord blood at birth ratios in the relative content of CD3+HLA-DR+ CD3+ lymphocytes, when the metric value of 8.1 per cent or more, diagnose perinatal hypoxic CNS damage with precision 89,28%.

The novelty of the method

First offered in the early (at birth) diagnostician is the formation of perinatal hypoxic CNS lesions in full-term newborns from women with preeclampsia by determining the ratios of the relative content of CD3+HLA-DR+ CD3+ lymphocytes.

We assume that this parameter has a direct pathogenetic connection with the development of perinatal hypoxic lesions of the Central nervous system, because it reflects the change in the activation of T-lymphocytes, which may be associated with development of autoimmune processes in response to changes in the permeability of the blood-brain barrier, which is formed as a result of chronic intrauterine hypoxia, accompanied by the release in the peripheral circulation automotive antigens.

The increase of the relative content of CD3+ and CD3+HLA-DR+ lymphocytes in the peripheral blood is observed at various infectious diseases, accompanied by activation of the immune response with a predominance of T×1 reactions [5]. The decline in the relative content of CD3+ lymphocytes observed in T-cell immunodeficiency [5], the decrease of the relative content of CD3+HLA-DR+ cells can be observed in atopic dermatitis [6] and obesity in children [7].

Distinctive features of the process

Set diagnostic parameter of the relative content of CD3+HLA-DR+ CD3+ lymphocytes, when the value is equal to 8.1% or more, diagnose CNS damage in full-term newborns from women with preeclampsia.

The method is as follows

1. At the birth of a child produce a fence in a test tube 0.5 ml of cord blood.

2. Allocation l is Mazitov from umbilical cord blood is the standard method - by centrifugation at 1500 Rev/min in a gradient density ficoll-urografin within 30 minutes [8].

3. 1 tube containing 100 ál of lymphocytes in a concentration of 1×106cells/ml, add 20 μl of monoclonal anti-CD3 AT labeled with FITC or PE, in another test tube, add 20 ál monoclonal anti-CD3 AT labeled with FITC or PE, and 20 μl of monoclonal anti-HLA-DR AT labeled with FITC or PE (depending on the first label). Cells incubated with monoclonal antibodies for 30 minutes at room temperature in the dark, then washed 1 time with 1 ml of saline by centrifugation at 1500 rpm for 2 minutes. Cells in test tubes resuspended in 500 μl of physiological solution [9].

4. The reaction accounting is performed on a flow cytometer, in determining lymphocytic gate percentage (relative) content of CD3+ and CD3+HLA-DR+ cells.

5. Calculate the ratio of the relative content of CD3+HLA-DR+ CD3+ cells. When the metric value equal to 8.1% or larger, diagnose perinatal CNS damage with precision 89,28%.

6. The method is carried out for 1 hour 10 minutes.

The essence of the proposed method is illustrated by the following examples.

Example 1. The child Was born from the first pregnancy occurring on the background of obesity II, anemia of I degree, complicated by preeclampsia moderate severity. Timely delivery at 40 weeks. Estimation on Apgar scale 5-6 points. Body weight at birth 3600 g, height 56 see General condition of moderate severity since birth. Locomotor activity was reduced, the loud cry. Marked muscular hypotonia, tremor of extremities, acrocyanosis. Big spring 2×2 see

At birth by the present method in cord blood was determined the content of CD3+, equal to 44.3 percent, and CD3+HLA-DR+ cells, 16.6%, and the ratio of CD3+HLA-DR+ CD3+, which accounted for 37.5%, which is higher than claimed (8,1%) level. Conclusion: diagnosed perinatal CNS damage.

On the third day of life held neurosonography. Based on these data and taking into account the clinical picture was diagnosed with chronic intrauterine hypoxia, perinatal CNS damage, cerebral ischemia II degree of hypoxic-ischemic dysfunction, hypertensive syndrome, neuroelectronic excitability.

Thus, the method of early diagnosis of perinatal hypoxic lesions of the Central nervous system in newborns from women with preeclampsia on the proposed method was confirmed.

Example 2. Baby b was born from VII pregnancies complicated by premature at 12 weeks, ARI 36 weeks, preeclampsia mild severity. Timely II birth at 38 weeks. Estimation on Apgar scale 8-9. Body weight at birth 2800 g, height 48 see Great is th spring 2,5× 2,5 see the Condition of the child at birth is estimated as satisfactory. At birth by the present method in cord blood was determined the content of CD3+, equal to 44.6%, and CD3+HLA-DR+ cells, equal to 3.0%, and the ratio of CD3+HLA-DR+ CD3+, which was 6.7%, which is lower than claimed (8,1%) level. Conclusion: perinatal CNS damage is not diagnosed. During the neonatal period when examining a neonatologist and a neurologist child healthy.

Thus, the method of early diagnosis of perinatal hypoxic lesions of the Central nervous system in newborns from women with preeclampsia on the proposed method was confirmed.

Example 3. Baby L was born from the third pregnancy, complicated by SARS in 6-7 weeks, the threat of termination 25 and 36 weeks, preeclampsia mild severity. Timely III delivery at 38-39 weeks. Estimation on Apgar scale 7-8 points. Body weight at birth of 3000 g and a height of 50 cm Big spring 1×1 see the first day of life the child's condition is satisfactory.

At birth by the present method in cord blood was determined the content of CD3+, equal to 64.2 per cent, and CD3+HLA-DR+ cells, equal to 5.2%, and the ratio of CD3+HLA-DR+ CD3+, which was 8.1%, which corresponds to the claimed (8,1%) level as a criterion for early diagnosis of perinatal CNS pathology.

On the 2nd day of life the child's condition worsened to moderate, appeared g is personbudget, the tremor of a chin and hands, acrocyanosis. On the basis of clinical and data neurosonography diagnosed with chronic intrauterine hypoxia, perinatal CNS damage, cerebral ischaemia first degree of hypoxic-ischemic dysfunction, hypertensive syndrome, neuroelectronic excitability.

Thus, the method of early diagnosis of perinatal hypoxic lesions of the Central nervous system in newborns from women with preeclampsia on the proposed method was confirmed.

Advantages of the method

1. Early (at birth) diagnosis of perinatal hypoxic CNS lesions in full-term newborns from women with preeclampsia will allow you to assign adequate therapy of cerebral disorders and prevent the debilitating consequences.

2. The inventive method does not require time-consuming and is 1 hour 10 minutes

3. Research can be conducted at any time of the day.

4. The proposed method has high accuracy - 89,28%.

5. Investigated small volume of blood (0.5 ml).

6. The proposed method of retraumatized.

In this way were examined 28 babies born from women with preeclampsia.

The results of the study are presented in table 1.

Table 1
Results studies
The number of the examined28
True-positive results16
True-negative results9
False positive results2
False-negative results1
The accuracy of the method89,28%
The sensitivity of the method94,10%
The specificity of the method81,82%

Literature

1. Starlings I.A., Rani G., Osipenko and other so-CALLED // Journal of neuropathology and psychiatry. - 1990. - T. No. 8. - P.5-10.

2. Beresnev SCI Hypoxic encephalopathy: the hypothesis of the pathogenesis of cerebral disorders and the search for methods of drug therapy. // Russian journal of Perinatology and Pediatrics. - 2002. No. 1. - P.6-13.

3. Afanas'eva N.V., summary of AV Outcomes of pregnancy and childbirth with fetoplacental insufficiency of different severity. // Questions of gynecology, obstetrics and Perinatology. - 2004. - V.3. No. 2. - P.7-13.

4. Bakanov M.I., Altyntsev CENTURIES, Podkopaev NR. New biochemical criteria for the diagnosis and prognosis of perinatal CNS lesions in infants. // Pediatrics. - 1999. No. 6. - Pp.33-37.

5. Beresnev SCI Hypoxic-ischemic encephalopathy the newborn: the contribution of perinatal factors, pathogenetic characteristics and prognosis. // Russian journal of Perinatology and Pediatrics. - 1996. No. 2. - P.29-35.

6. Handbook of therapy for a practitioner. - SPb.: Dialogue, 2002. - 478 S.

7. Bulina O.V., Kalinin N.M. Research of the immune status in children with atopic dermatitis. // Medical immunology. - 2002. - V.4. No. 2. - S.

8. Grishkevich POSTGRADUATE, V.T. Manchuk, Savchenko A.A. Changes in population and subpopulation composition of immune cells in children of different forms of obesity. // Medical immunology. - 2004. - V.6. No. 3-5. - S-284.

9. Boum A. Isolation of mononuclear cells and granulocytes from human blood and bone marrow // Scand. J.Clin. Invest. - 1968. - V.21, No. 97. - P.77.

10. Ohta G., Fujiwara K., Nishi T., Oka H. Normal values of peripheral lymphocyte populatuon and T cell subsets at a fixed time of day: A flow cytometric analysis with monoclonal antibodies 210 in healthy adults // Clin. Exp. Immunol. - 1986. - V.64. - P.146-149.

Method for early diagnosis of perinatal hypoxic CNS lesions in full-term newborns from women with preeclampsia by immunological studies of umbilical cord blood, wherein the blood determines the ratio of the relative content of CD3+HLA-DR+ CD3+ lymphocytes and its value is equal to 8.1% or more, diagnose perinatal hypoxic lesions of the Central nervous system at birth.



 

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