Method for predicting chronic pyelonephritis in children

FIELD: medicine, pediatrics, immunology.

SUBSTANCE: in patients' peripheral blood one should detect the expression level of CD95 lymphocytes markers due to indirect immunofluorescence technique and at the content of CD95 markers being above 21.31% one should detect chronic flow of pyelonephritis. The method is simple and of high information value. It enables to evaluate quantitative marker of affected mechanisms of apoptosis at chronic pyelonephritis quickly and objectively for 2.5 h after blood sampling, moreover, it is of high information value for differential diagnostics of chronic pyelonephritis and provides rational complex of therapeutic procedures in due time.

EFFECT: higher efficiency of diagnostics.

1 ex, 1 tbl

 

The invention relates to medicine, in particular to Pediatrics, immunology. It can be used in the clinic for the diagnosis of chronic pyelonephritis (CP) in children.

A known method for the diagnosis of CP on clinical grounds according to which CP differentiate from cystitis, glomerulonephritis, hereditary nephritis and other diseases (Vozianov A.F., maidannyk VG, a Prominent VG, Bagdasarova IV Basics of Nephrology children. - K.: Book plus, 2002. - S-91).

A known method for the diagnosis of pyelonephritis to determine the level of cytokines, which is selected as the closest analogue. This method comprises detecting increased levels of cytokines (tumor necrosis factor-alpha, transforming growth factor-beta 1) in plasma by ELISA (Khvorostov, I.N., Smirnov IE, Zorkin, S.N., Kucherenko A.G. cytokines with obstructive uropathy in children // Pediatrics. - Annex 1: proceedings of the X Congress of pediatricians of Russia "Ways of increasing the effectiveness of health care for children". - 2005. - V.4. - S). The disadvantage of this method is its high cost.

The aim of the invention is to improve the accuracy, efficiency and objectification of the differential diagnosis of chronic pyelonephritis, which will ensure the speed of diagnosis, economical, and timely the military and rational realization of a complex of therapeutic measures.

The essence of the proposed diagnostic method is that along with clinical data and urinalysis determine the level markers CD95 lymphocytes of peripheral blood of a child by indirect immunofluorescence using monoclonal antibodies and content markers CD95 more 21,31% of diagnosed chronic pyelonephritis.

CD95 (weight fasr) refers to the receptor protein family factors tumor necrosis (TNF - a. The main function of the system Fas-receptor-Fas-ligand (FasL) - induced apoptosis in the cells. When the binding of the ligand to the receptor occurs oligomerization of the cytoplasmic proteins, resulting activated apoptosisinducing protease (caspase-8) and develop characteristic for apoptosis processes. In addition to genetically determined mediators of apoptosis, the cell may be exposed to compounds that regulate this process, such as cytokines (interleukins 2, 4, 10)circulating in the blood or associated with biomembranes. In infectious processes development of apoptosis depends on specific factors secreted by microorganisms (superantigen-exotoxins often act as inductors activation of apoptosis). The destruction of the cells of the tubular epithelium, along with necrosis, can occur by apoptosis. In the final stages of inflammation, adopt the zu has an important role, because in this period the elimination of activated cells of the immune system, performed their functions. Possible negative role of apoptosis in the development of secondary immunodeficiency in patients with chronic pyelonephritis.

The method is as follows: samples (4-5 ml) of heparinized blood taken on an empty stomach in children, isolated lymphocytes on the gradient ficoll-urografin, then 1-0,1 million cells in a volume of 50 μl contribute to the wells of 96-well round-bottom of the tablet. To the cells, add 5 µl of the tested monoclonal antibodies (CD95) and incubated for 30-45 min at +4°C. Then add 150 μl of Hanks solution and centrifuged 5 min at 200 g. After removal of the supernatant contribute 50 μl of Hanks solution, the cells are suspended, add 150 μl of Hanks solution and centrifuged 5 min at 200 g. To the precipitate washed cells add 50 μl of F(ab˜)2fragments of sheep antibody to mouse Ig labeled with FITZ and diluted 1:100 (for cultivation using the Hanks solution containing 0.5% gelatin and 0.1% sodium azide). Cells are suspended and incubated for 30 min at +4°C, the cells are then washed 2 times as described above. Stained cells analyzed by fluorescence microscope. Cells are suspended, transferred to a glass slide, covered with a cover glass, and looking at the drug under immersion with increasing (#x000D7; 3; lens ×90). The number antigenpositive cells is defined as the percent of fluorescent cells while browsing 200 lymphocytes minus % fluorescent cells observed in the preparation of the negative control. As a negative control, using drugs, prepared similarly, but without the processing of monoclonal antibodies. When the expression of markers CD95 more 21,31% lymphocytes establish chronic pyelonephritis.

EXAMPLE.

Girl Days, 5 years. A year ago she was hospitalized for acute pyelonephritis; noted the symptoms of intoxication, abdominal pain syndrome, bladder syndrome, is represented by leukocyturia, bacteriuria. Two days ago after a short hypothermia increased body temperature up to 38,50°C, pains in the stomach, in the lumbar region. When admitted to hospital, the condition of moderate temperature of 38.5°, pale skin, periorbital cyanosis. Swelling and pastoznost tissues no. Vesicular breathing in lungs. NPV 20 in 1 min. heart sounds satisfactory sonority. Pulse 122 1 min, BP 100/60 mm Hg Abdomen is soft, painful to palpation in the area of the right flank. Symptom of Pasternacki to the right is positive. Urination acadeny up to 10 times per day, painless. In General, the analysis of blood stoichiometric who think leukocytosis, increased ESR. Biochemical blood analysis: total protein, urea, creatinine within normal limits, C-reactive protein - negative. In the General analysis of urine - leukocyturia, oxaluria. Urine analysis according to Nechyporenko: leukocytes - 25.500 1 ml (neutrophils 82%, lymphocytes 18%), erythrocytes - 1000 in 1 ml Bacteriological examination of urine: 100000 E. coli CFU in 1 ml sensitive to Ceftriaxone, ceftazidime, gentamicin, norfloxacin. Reduced antichristianity the ability of urine to the oxalate of calcium. According to the results of research on clearance of endogenous creatinine, urine analysis zimnitsky - kidney function is preserved. Ultrasound of the kidneys and bladder: the heterogeneity of the Central echo complex due hyperechoic signals, broken relationship parenchyma and Cup-pelvis system right, echo signs of inflammation of the right kidney. Excretory urography: allocation of the contrast agent is not broken, there is a smoothness of Vernikov right kidney.

The level of expression of markers CD95 the peripheral blood lymphocytes of 25.00%.

Diagnosis. Chronic secondary (dysmetabolic) pyelonephritis, relapsing course, active stage. Kidney function is preserved.

Data show that the level markers CD95 of peripheral blood lymphocytes may serve as an additional criterion in the differential diagnosis of CP

A method for the diagnosis of chronic pyelonephritis in children according to the level of expression of markers CD95 by peripheral blood lymphocytes was tested on 38 children with a prior diagnosis of chronic pyelonephritis set on the basis of clinical signs (main group). The comparison group consisted of 14 children with a diagnosis of acute pyelonephritis, the control group of 30 children from the first group of health with normal blood and urine tests. All of the respondents had performed blood sampling and determination of the level of expression of markers CD95 by peripheral blood lymphocytes listed above.

The results of the study are presented in the table. For processing the received data, and statistical analysis in the studied groups was used for the statistical processing of information "STATGRAPHICS PLUS". To calculate the average and the average error (M±m). Comparison of study groups conducted in pairs, using the parametric methods because the study sample could be approximated by a normal distribution. The comparison was performed using t-test (t) (comparison means) and Fisher (comparison of mean square deviations). The confidence level was assessed value R. Differences when the comparison was considered significant with a probability of more than 95% if < 0,05.

Table

A method for the DIAGNOSIS of CHRONIC PYELONEPHRITIS IN CHILDREN
CD95 (%)

M±m (min, max)
Chronic pyelonephritis25,45±2,07*
(n=38)(21,31-29,59)
Acute pyelonephritis14,20±1,38*
(n=14)(11,44-16,96)
Healthy8,00±0,32*
(n=30)(of 7.36-8,64)
Note: * - p<0,05 - significance of differences in the groups in the comparison.

In healthy children markers CD95 Express no more than 8,64% of peripheral blood lymphocytes in children with acute pyelonephritis - no more 16,96%.

The obtained data showed that in CP level markers CD95 of peripheral blood lymphocytes was significantly increased compared with levels in children with SOS and control group, therefore, the level of expression of CD95 molecules by peripheral blood lymphocytes can be considered as a highly sensitive marker in CP.

Using the proposed diagnostic method has the following advantages:

1) high information content for the differential diagnosis of CP;

2) is informative method of quantification brand is and violations of apoptosis mechanisms in CP dynamics;

3) rapid diagnostics;

4) ensure timely and efficient implementation of the complex of therapeutic measures.

A method for the diagnosis of chronic pyelonephritis, including the study of the peripheral blood, characterized in that to determine the level of expression of lymphocyte markers CD95 by indirect immunofluorescence using monoclonal antibodies and when the content in the peripheral blood markers CD95 more 21,31% believe the presence of chronic pyelonephritis confirmed.



 

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