Method for predicting acute pyelonephritis in children

FIELD: medicine, pediatrics, immunology.

SUBSTANCE: in patients' peripheral blood one should detect expression level of adhesion molecules of CD11β and CD54 lymphocytes due to indirect immunofluorescence technique at applying monoclonal antibodies and at the content of CD11β molecules being above 26.34% and CD54 molecules being above 33.26% one should detect active stage of acute pyelonephritis. The method is simple and of high information value. It enables to evaluate the values of leukocytic adhesion and their transendothelial migration quickly and objectively for 2.5 h after blood sampling, moreover, it is of high information value for proving the activity of microbial-inflammatory process in case of acute pyelonephritis and provides rational complex of therapeutic procedures in due time.

EFFECT: higher accuracy 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 acute pyelonephritis (OP) in children.

A known method for the diagnosis of OP on clinical grounds according to which the OP differentiate from cystitis, glomerulonephritis, interstitial nephritis, hereditary nephritis and other diseases (Vozianov A.F., maidannyk VG, Bidny 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) 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 diagnosis of acute pyelonephritis, which will ensure the speed of diagnosis, efficiency, and Sovrem the TES 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 of adhesion molecules CD11β and CD54 of lymphocytes in the peripheral blood of a child by indirect immunofluorescence using monoclonal antibodies and the content of the molecules CD11β 26,34% and molecules CD54 more of 33.26% diagnose active stage OP.

The output of immune system cells of the vasculature and migrate them into the hearth of the penetration of infectious agents is a key step in the defense system of the body. Interaction CD11β (Mac-1, integrin, a receptor for iC3b) CD54 (ICAM-1, a ligand for LFA-1 and Mac-1) is important in the regulation of a single phase of leukocyte adhesion and transendothelial migration. ICAM-1 and its receptor LFA-1 are additional factors of activated T-lymphocytes. Adhesion molecules CD11β and CD54 induced by inflammatory mediators, such as IL-1, TNFα and IFNγ. This may explain the higher level of expression of adhesion molecules in microbial inflammatory process in the kidneys.

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. Cells DOB is make 5 µl of the tested monoclonal antibodies (CD11β , CD54) 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 drug under immersion with increasing (×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 content of the adhesion molecules CD11β 26,34% and adhesion molecules CD54 more of 33.26% set the active stage of the OP.

EXAMPLE.

Girl Days, 5 years. Sick 2 days. The disease began in the Le of hypothermia with a temperature increase of the body to 39.0° C, appearance of abdominal pain in the lumbar region. When admitted to hospital, the condition of moderate temperature of 39.0°C. pale Skin, swelling and pastoznost tissues no. Vesicular breathing in lungs. NPV 20 in 1 min. heart sounds satisfactory sonority. Pulse 120 in 1 min, BP 100/60 mm Hg Abdomen is soft, painful to palpation in the area of the right flank. The kidneys are not changed, the kidneys are not palpable, the symptom of Pasternacki to the right is positive. Chair decorated. Urination acadeny up to 10 times per day, painless.

In General, the analysis of blood neutrophilic 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 - 15.750 1 ml (neutrophils 80%, lymphocytes 20%), erythrocytes - 1000 in 1 ml Bacteriological examination of urine: 100000 E. coli CFU in 1 ml, sensitive to ampicillin, Ceftriaxone, ceftazidime, gentamicin, nitrofurantoin. 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: heterogeneity Central Ecocomplex due hyperechoic signals, the echo is rsnake inflammatory process right kidney. Consultation of the gynecologist: signs of inflammation of the external genital organs are not detected.

The level of expression of adhesion molecules CD11β peripheral blood lymphocytes 36,00%, molecules CD54 - 46,00%.

Diagnosis. Acute secondary (dysmetabolic) pyelonephritis, active stage. Kidney function is preserved.

Data show that the molecules CD11β and CD54 can serve as a marker of the activity of microbial and inflammatory process in the OP.

Method for the diagnosis of acute pyelonephritis in children according to the level of expression of adhesion molecules CD11β and CD54 by peripheral blood lymphocytes was tested in 14 children with a preliminary diagnosis of acute pyelonephritis set on the basis of clinical signs (main group).

The control group consisted of 30 children with first health group with normal blood and urine tests. All of the respondents had performed blood sampling and determination of the expression level of molecules CD11β and CD54 the 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 was performed pop the RNO using 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% at P<0,05.

In healthy children adhesion molecules CD11β Express no more than MT 18 : 34% of peripheral blood lymphocytes, molecules CD54 - 22,41%.

The data obtained showed that at OP level of expression of adhesion molecules CD11β and CD54 by peripheral blood lymphocytes was significantly increased, therefore, the molecules CD11β and CD54 can be considered as a highly sensitive marker of microbial and inflammatory process in the OP.

Using the proposed diagnostic method has the following advantages:

1) high information to confirm the activity of microbial and inflammatory process in acute pyelonephritis;

2) rapid diagnostics;

Table 1

Method for the DIAGNOSIS of ACUTE PYELONEPHRITIS IN CHILDREN
Acute pyelonephritis (n=14)Healthy (n=30)
M±m (min, max)M±m (mn, max)
CD11β (%)CD54 (%)CD11β (%)CD54 (%)
30,10±1,88*37,00±1,87*18,00±0,17*22,00±0,21*
(26,34-33,86)(of 33.26-40,74)(17,66-MT 18 : 34)(21,79-22,41)
Note: * - p<0,05 - significance of differences between groups when comparing

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

Method for the diagnosis of acute pyelonephritis, including the study of the peripheral blood, characterized in that to determine the level of expression of cell adhesion molecules CD11β and CD54 by indirect immunofluorescence using monoclonal antibodies and the content of the molecules CD11β 26,34% and molecules CD54 more of 33.26% believe the presence of microbial and inflammatory process in acute pyelonephritis confirmed.



 

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2 ex, 3 tbl

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