Method of evaluating immunosuppressive response rate in children with chronic glomerulonephritis

FIELD: medicine.

SUBSTANCE: invention describes a method of evaluating an immunosuppressive response rate in children with chronic glomerulonephritis characterised by determining the peripheral blood cell kinetics by flow DNA-cytometry; evaluating a cell fraction in a proliferation phases not less than 0.74% enables to consider the immunosuppressive therapy to be prescribed, and observing a decreased cell level in proliferation phases in 2-3 weeks after the beginning of administration at least in 2 times, the therapy is considered to be effective; the absence of positive dynamics requires dosage change or preparation replacement.

EFFECT: higher accuracy and reduced number of research injures, derived clinical laboratory criteria of immunosuppressive prescription and prediction of a therapeutic effect.

4 dwg, 1 tbl, 3 ex

 

The invention relates to medicine, namely to immunology and Nephrology, can be used to assess the activity of the process and the effectiveness of immunosuppressive therapy in children with chronic glomerulonephritis (CG).

Among the acquired kidney diseases in children, HGH is the second most common disease after infection of the urinary tract. Over the last 10-15 years has increased the frequency of latent and chronic forms of glomerulonephritis occurring early in impaired renal function. Still a high percentage of patients with poor clinical outcomes. Glomerulonephritis is one of the main reasons for the development of chronic renal failure, disability, children, youth and young age.

Actual problem of modern Nephrology remains the defining markers of activity of the process with chronic hepatitis, clinical and laboratory criteria for the appointment of immunosuppressants, evaluation of the effectiveness of therapy and prediction of treatment outcome.

It is known that the basis of HCG is immune glomerulopathy, in which there is dysfunction of the various elements of the immune system with a predominance of processes activate it (Msipath, Agurbash "IMMUNOSUPRESSIVE therapy for nephrotic syndrome in children", Moscow, 2003, p.40-43).

Proto is ipom of the invention is a method of evaluating the activity of the process with HGH consisting of characteristic histomorphological changes in the kidneys on the basis of microscopic studies of kidney biopsies. This method is also used for determining the nephrotoxicity of drugs ("Microscopic technique", a manual for physicians and laboratory technicians, edited Dsirqeumay and Ulanova, Moscow, "Medicine", 1996, s-519). Direct indications for biopsy are relapsing course of HGH hematuria and/or hypertension, hormonereplacement (Msipath, Agurbash "IMMUNOSUPRESSIVE therapy for nephrotic syndrome in children", Moscow, 2003, p.24). Disadvantages of the method are invasive, the risk of complications after biopsy, a large list of absolute and relative contraindications, negative attitudes of patients and parents. In addition, on the basis of morphobiological characteristics it is difficult to assess the degree of activity of the process, the effectiveness of therapy, the need for urgent connection cytostatics. Re-inspection biopsy of the kidney is possible only in 1.5-2 years, which limits the use of biopsy to monitor the process.

Thus, to resolve the question of the appropriateness and effectiveness of immunosuppressive therapy with HCG promising is the development and use of the latest low is Vasiunyk technologies.

It is known that increased proliferative activity of immune cells may be a marker for aggressiveness of the process. In this regard, the immunosuppression drugs directly influencing the phase of cell proliferation is pathogenetically justified. Determination of the level of proliferation of blood cells can be used as a marker of activity of the process, and be one of the criteria for the appointment of immunosuppressants. To assess the effectiveness of therapy requires monitoring of this indicator.

Currently, to characterize the proliferative activity is used to define the level of cells in the phase of DNA synthesis. One of the methods to estimate the parameters of the kinetics of the cell cycle is running DNA cytometry of peripheral blood cells (Daskalov, Geekazine "Laboratory and clinical significance of flow-cytometrical blood analysis", Moscow, 2004, p.8).

Object of the present invention to provide an efficient and easy to use method of evaluating the degree of activity of the process and effectiveness of cytostatic therapy.

The technical result in the use of the invention is to simplify and reduce the time for determining the activity of the process, improve the accuracy and reduce the morbidity of study is of, obtaining clinical and laboratory criteria for the appointment of immunosuppressants and prediction of treatment outcome.

The proposed method is as follows. A survey conducted in patients with hormoneresistant and hormone-dependent variants and mixed nephrotic syndromes, and in patients with gematrical form of HCG, a disease which is characterized by high activity and resistance to ongoing basic therapy. Collect peripheral blood with heparin in an amount of 5 ml in the morning on an empty stomach. By flow cytometry to determine the kinetics of peripheral blood cells. When determining the proportion of cells in the phases of proliferation (G2 and S) not less 0,74% consider is shown holding immunosuppressive therapy. Control tests carried out 2-3 weeks after you start taking the drug. By reducing the level of cells in the phases of proliferation of not less than 2 times therapy is considered effective. In the absence of positive dynamics consider shows the change of tactics of treatment: dose adjustment or replacement immunosupressant. Subsequent monitoring of this indicator is carried out with an interval of 1-2 months. The best would be reduced proliferative activity to normal levels. The proposed method is a safe, minimally invasive, technically sushestvam.

Research method

To characterize the ploidy and proliferative activity of cells using a method based on the binding of DNA with fluorescent probe propidium-iodide, which allows to determine the ploidy and the proportion of cells in different phases of the mitotic cycle: G0/G1, S and G2+M

In a test tube with 100 µl of blood successively added tripeny buffer 250 ál trypsin inhibitor with RNA Asai 200 μl, and the solution propidium-iodide 200 ál with intermediate mixing and 10-minute incubations, the sample was then filtered through a nylon cloth with cells 50 μm. Samples within 3 hours after addition of PI (Vindelov L.,1989) (not less than 20,000 cells) analyze on flow cytometer FACSCalibur equipped with a module of discrimination doublets in the program ModFit.

A comprehensive survey of 72 children with chronic hepatitis, including, along with traditional laboratory methods, DNA-cytometry and?. Is a direct correlation between clinical and laboratory indicators of activity immunopathological process, the results of the biopsy of the kidney and the level of proliferative activity of the cells. The absence of effects from the basic treatment was the basis for inclusion in the complex of therapeutic measures immunosuppressive drugs. According to DNA cytometry in children with HCG to held what I immunosuppressive therapy was observed a high level of proliferative activity of the cells, that was Me=1,12% (0,74-2,04%). When studying the kinetics of the cell cycle in healthy children, it was found that the proportion of peripheral blood cells in the phase of DNA synthesis (G2 and S) is IU=0,12% (0,09-0,15%) (Nghikembua "Immunological reactivity, ploidy and cell cycle kinetics of peripheral blood cells in various pathological conditions in children, the dissertation on competition of a scientific degree of candidate of medical Sciences, Ufa, 2009). Thus, the proliferation rate in the group of children with HCG was higher standards not less than 4,93 times (0,74:0,15=4,93). The analysis of the results of DNA-cytometry allowed us to identify the features of the kinetics of the cell cycle on the background of immunosuppression. With the re-examination after 2-3 weeks of immunosuppressive drugs was a decrease rate of not less than 2 times. Reducing the proliferative activity of the cells on the background of immunosuppression was accompanied by positive dynamics of clinical and laboratory data and testified about the effectiveness and adequacy of therapy. Subsequent monitoring of the indicator, as a rule, noted a strong tendency towards the decrease in the proportion of cells in the phase of DNA synthesis. The lack of dynamics or increased levels of cell proliferation in the G2 and S phases were used as the basis for dose adjustment or replacement immunosupressant.

Listed who's particular DNA cytometry of peripheral blood cells reflect the body's response to basic therapy. An integrated approach that combines clinical picture of the disease, histomorphologically diagnosis, immunological parameters, the kinetics of the cell cycle, allows you to comprehensively evaluate the activity of the process and the effectiveness of immunosuppressive therapy.

Based on the analysis of research results, including the observation of different stages of disease, we recommend that monitoring of the kinetics of the cell cycle as a marker of activity of the process and the effectiveness of immunosuppressive therapy with HCG.

The invention is illustrated by the following drawings: figure 1 shows the DNA histogram of the peripheral blood cells of the patient K. (before immunosuppressive therapy); figure 2 - DNA-histogram cells of peripheral blood of the patient K. (hormonal immunosuppression); figure 3 - DNA histogram cells of peripheral blood of the patient K. (on the background of immunosuppression-CellCept); figure 4 - DNA-histogram cells of peripheral blood of the patient K. (2 weeks immunosuppression with Myfortic).

We present examples of clinical use of the proposed method.

Example 1.

Baby K., age 15, No. 5186. Clinical diagnosis of primary chronic glomerulonephritis, mixed shape, harmonistically option. Complicated is e: Chronic renal failure, stage of compensation. Iron deficiency anemia. Renal osteodystrophy. Systemic osteoporosis. Secondary immunodeficiency. Angioretinopathia on the background of toxic affection of the Central nervous system, hormonal therapy. Companion: Chronic gastroduodenitis, the period of exacerbation. Encephalopathy complex Genesis with hypertensive syndrome.

Anamnesis morbi: ill since April 2, 2009, when the background of SARS appeared eyelid swelling, decreased urine output. Was treated at home by furosemide, broth bearberry within 4 days. After cooling the swelling increased swelling in the legs. For medical assistance at the place of residence 08.04.2009, Patient K. was admitted to the Nephrology Department of the RCCH 09.04.09, with complaints of swelling, weakness, lethargy, decreased amount of urine. Diagnosis at admission: acute glomerulonephritis, nephrotic form. With 09.04.09, 23.06.09, was treated at the Nephrology Department of our hospital with a diagnosis of acute glomerulonephritis with nephrotic syndrome, harmonisierte form.

In General, the analysis of blood: the lake. 18,4·109/l, eritr. 5,77·1012/l, HB 162 g/l, a blood clot. 346·109/l, hematocrit of 49.5; p/I - 2%, limp. - 29%, mon. 10%, neutral. 59%, ESR 35 mm/h

In urinalysis: protein 5.0 g/l, erythrocytes and leukocytes 0-1 in the field of view, beats. weight 1010, pH 7.0.

Biochemical blood analysis: total protein - to 42.2 g/l, albumin of 18.3 g/l, cholesterol - was 10.82 mmol/l, urea - to 4.81 mmol/l, creatinine - 86 µmol/l, total bilirubin. - 3,9 KMOL/l, ALT 11TH/l, 4,1, CA ion. 1,041.

The results of the ELISA blood test for antibodies to hepatitis b virus, cytomegalovirus, chlamydia, Toxoplasma the negative.

A/body to DNA, CRP - negative.

The results of immunological tests:

Ig A to 1.37 g/l, Ig M 1.07 g/l, IgG - 5.0 g/l, the complement of 1:32, CEC 26 USD CD3+- 57%, CD8+- 28%, CD4+- 31%, CD4+/CD8+- 1,11, CD19+- 40%, CD16+- 7%, HLA-DR+- 8%, CD25+- 28%.

The results of DNA-cytometry (before pulse-therapy Solumedrol) G1 - 98,27%, G2 - a 0.02%, S - 1,71%, CV - 2,92 (figure 1).

Conclusion: a marked In-lymphocytosis, high levels of the activation markers HLA-DR+and CD 25+characterize recuperability autoimmune process. The tendency to the formation of T-cell deficiency.

The high level of proliferative activity in S-phase confirms the results of immunophenotyping.

Conducted treatment: hormonal therapy for 8 weeks (prednisolone 60 mg daily for 4 weeks, then medrol 48 mg - 4 weeks), antibiotic therapy, electrophoresis with heparin 8000 UNITS in region of kidneys.; /drip aq albumin 10% - 100,0 (No. 2), reopoliglyukin 200,0.

On the background of basic therapy a slight improvement in the form of disappearance of edema syndrome. In the biochemical analysis of blood total protein increase is ILSA to 50 g/l, albumin - 25.4 g/l, cholesterol 15,74 mmol/l, urea of 4.95 mmol/l, creatinine - 48 µmol/L.

Discharged 23.06.2009, outpatient treatment for 5 days with recommendations to continue therapy medroom scheme.

29.06.2009, the child was hospitalized again at the Nephrology Department of our hospital with worsening conditions after hypothermia with complaints of swelling, abdominal pain, changes in the urine in the form of proteinuria, lethargy, weakness, loss of appetite. Objectively the child expressed swelling on the face, torso, limbs, multiple stretch marks all over my body.

In General, the analysis of blood from 30.06.09: lake. 26,8·109/l, eritr. 5.2 x 1012/l, HB 164 g/l, a blood clot. 317·109/l, Miele. 10%, u - 6%, p/I - 2%, neutral. 59%, limp. - 19%, mon. - 4%, ESR 60 mm/h

In the General analysis of urine from 30.06.09: protein 5.0 g/l, erythrocytes 0-1 in the field of view, beats. weight 1015, pH 6,0.

Biochemical analysis of blood from 30.06.09: total protein of 40.3 g/l, albumin of 16.7 g/l, cholesterol - 15,91 mmol/l, urea - 5,71 mmol/l, creatinine - 84 µmol/l, total bilirubin. - 3,9 KMOL/l, ALT 15 U/l

In gemostaziogramma from 30.06.09: activated time rekaltsifikatsii - 37 sec, thrombin time of 17.7 seconds, PETIT - 106%, the concentration of fibrinogen to 7.75 g/l

According to the biopsy revealed membrane-proliferative glomerulonephritis with severe tubular and moderately strong vascular component, the active phase.

Results immunologics the CSO survey on 21.07.09 (until the appointment of cytostatic therapy): Ig A 0,83 g/l, Ig M 2.5 g/l, IgG - 0,94 g/l; CD3+- 78%, CD8+- 32%, CD4+- 52%, CD4+/CD8+- 1,62, CD19+- 19%, CD16+- 5%, HLA-DR+- 8%, CD25+- 30%.

DNA-cytometry hormonal immunosuppression (figure 2): G1 - 99,2%, G2 - 0,06%, S - 0,74%, CV - 2,99.

Conclusion: a High degree of activity of the autoimmune process involving high proliferative activity of the cells. The severity of the autoimmune component is a marker of the inefficiency of the ongoing immunosuppression. Extremely low levels of IgG.

Given the resistance to the ongoing hormonal therapy with prednisolone at a dose of 2 mg/kg per os for 10 weeks and pulse-therapy salt-medrala, rapidly progressing disease, the results of the biopsy in the basic complex of therapeutic measures 5.08.09 included cytostatic CellCept in a dose of 1 g per os. With 11.08.09 dose of cytostatic increased to therapeutic (2 g per day).

The results of immunological examination from 19.08.09 (2 weeks immunosuppression-CellCept): Ig And - 0,62 g/l, Ig M - 1.22 g/l, IgG - 1 g/l; CD3+77%, CD8+- 32%, CD4+- 46%, CD4+/CD8+- 1,44, CD19+- 24%, CD16+- 3%, CD25+- 33%.

DNA-cytometry (figure 3): G1 - 97,6%, G2 - 0%, S - 2,4%, CV of 3.6.

Conclusion: No significant dynamics of immunological parameters. Remains extremely low levels of IgG. On the background of immunosuppressive treatment is AI CellCept in a week marked increase in proliferative activity of cells. Recommended immunological monitoring after 2 weeks to assess the effectiveness of immunosuppressive therapy after switching to another drug on Myfortic.

Due to severe gastrointestinal complications (nausea, vomiting, liquefied chair), lack of positive dynamics of clinical, laboratory, immunological parameters 19.08.09, CellCept replaced by Myfortic dose of 1080 mg / day per os with further increase of the dose up to 1440 mg per day.

The child on the background of immunosuppression with Myfortic had positive clinical dynamics in the form of improved health, no nausea, normalization of appetite.

The results of immunological examination from 31.08.09 (2 weeks immunosuppression with Myfortic): Ig And - 1,14 g/l, Ig M - 1,95 g/l, IgG - 1.86 g/l; CD3+- 74%, CD8+- 33%, CD4+- 44%, CD4+/CD8+- 1,33, CD19+- 17%, CD16+- 6%, CD25+- 30%.

DNA-cytometry (figure 4): G1 - 98,78%, G2 - 0,01%, S - 1,21%, CV - 2,65.

Conclusion: On the background of positive clinical dynamics there is a trend towards normalization of proliferative activity by DNA-cytometry. Recommended to continue therapy Myfortic.

At discharge, the General analysis of blood lake. 7,4·109/l, eritr. 4,27·1012/l, HB 115 g/l, a blood clot. 335·109/l, limp. - 36%, mon. - 7%, neutral. 57%, ESR 8 mm/h

In urinalysis: protein 5.0 g/l, beats. weight 1015, pH 6,0.

Biokhimicheski the first blood analysis: Gen. protein to 40.4 g/l, albumin - to 18.9 g/l, cholesterol - 10.1 mmol/l, urea - 6,37 mmol/l, creatinine - 63 µmol/l, ALT 6 E/L.

5.10.09, the child was discharged to outpatient treatment with the following recommendations: continue immunosuppressive therapy medroom and Myfortic.

Example 2.

Patient L., No. 16249, 6 years old, was admitted in the Nephrology Department of the RCCH 21.12.2007 with a preliminary diagnosis of haemolytic uraemic syndrome?".

Complaints on admission: cough, abdominal pain, vomiting, weakness, lethargy, rash on body, increase body temperature.

Objectively: a serious condition, skin pale, whole body hemorrhagic rash.

Data from laboratory studies:

General blood analysis: the hands of Eritrean. is 4.7×1012/l; ESR 25 mm/h; Hb - 107 g/l; lake. and 12.4×109/l; PAL./the cores. - 13%; segm./the cores. - 64%; limp. - 18%; Mont. - 5%.

Urinalysis: color - C/W; turbidity - clear; reaction - neutral; Udis - 1001; protein - 1,33 g/l; lake. - 1-0-1-0 in the p/SP.; salt - single. in the p/SP.

The results of immunological tests: Ig And - 1,82 g/l; Ig M - 1,03 g/l; Ig G and 10.8 g/l; CEC - $ 29; CD3+- 38%; CD4+/CD8+- 1,10%; CD19+- 23%; CD4+- 22%; CD8+- 20%; CD16+- 5%, CD25+- 36%.

DNA cytometry: G1 - 98,09%, G2 - 0,28%, S - 1,63%, CV - 3,38.

As a result of the survey revealed an active inflammatory process: accelerated erythrocyte sedimentation rate, leukocytosis, deep T-cell is a deficit, activation of b - lymphocytes and a high level of cells with markers of early activation (CD25+). According to DNA cytometry of peripheral blood cells 1,91% of the cells were in the phases of proliferation, 98,09% in G0/1 phase.

Conducted antibacterial and immunosuppressive (glucocorticoids) therapy. After stabilization of spent fine-needle biopsy of the kidneys, the results of which are diagnosed with chronic glomerulonephritis, matangi-proliferative with tubulointerstitial component, a mixed form.

The child was conducted clinical and immunological monitoring, the results of which are presented below.

General analysis of peripheral blood from 11.01.2008: the hands of Eritrean. - 2,78×1012/l; ESR - 5 mm/h; Hb - 85 g/l; lake. - 4,8×109/l; EOS. - 4%; Basov. - 13%; segm./the cores. - 42%; limp. - 44%; Mont. - 8%.

General analysis of urine from 11.01.2008: color - brown; turbidity - muddy; the reaction is acidic; beats. weight - 1016; protein - 1,32 g/l; lake. - 2-4-2 in p/SP.; the hands of Eritrean. - 1-3-2 in the p/SP.

The results of immunological examination from 12.01.2008: Ig A - 0,89 g/l; Ig M - 0,97 g/l; Ig - 4,2 g/l; CEC - $ 21; CD3+- 45%; CD4+/CD8+- 0,88%; CD19+- 54%; CD4+- 21%; CD8+- 24%; CD16+- 5%, CD25+- 44%; HLA-DR+- 12%; CD95+- 37%.

The results of DNA-cytometry from 12.01.2008 (3 weeks after treatment with corticosteroids): G1 - 99,9%, G2 - 0,06%, S - 0,04%, CV of 3.1.

As a result of treatment the patient's condition with what was bilizirovali. Hematological parameters (ESR, WBC, neutrophils) had positive dynamics, immunological monitoring showed the formation of secondary immunodeficiency. According to DNA-cytometry the number of proliferating cells decreased more than 2 times and amounted to 0.1%, which is an indicator of the effectiveness of glucocorticoid therapy.

Example 3.

Child A., age 6, No. 732. Clinical diagnosis primary: secondary chronic glomerulonephritis in the background of hemorrhagic vasculitis mixed with renal syndrome.

Anamnesis morbi: ill since April 2007, when amid SARS first appeared rash on the feet. Treated in terms of RSD Neftekamsk with a diagnosis of hemorrhagic vasculitis, cutaneous form. In April 2009 she had had flu, later developed a rash on the feet, legs, buttocks, abdomen. With 17.04.2009, 21.04.2009, was hospitalized in CRH Neftekamsk. In the absence of dynamics, the emergence of abdominal pain, vomiting with blood, was transferred to the cardiological Department of the RCCH Ufa with a diagnosis of hemorrhagic vasculitis, mixed form. The child was transferred to the Nephrology Department of the RCCH 13.05.2009, in connection with the accession renal syndrome, with complaints of fever, discoloration of urine, swelling.

In General, the analysis of blood from 13.05.2009: lake. 13,4·109/l, eritr. ,06·10 12/l, HB 91 g/l, a blood clot. 440·109/l, EO - 3%; p/I - 3%, limp. - 15%, mon. 6%, neutral. 63%, ESR 45 mm/h

In the General analysis of urine from 13.05.2009: protein 5.0 g/l, erythrocytes-250/µ1 and 100 leukocytes/µ1, beats. weight 1020, pH 5.0, color - dark.

Biochemical analysis of blood from 13.05.2009: total protein was 43.6 g/l, cholesterol - 3.63 mmol/l, urea - 8.5 mmol/l, creatinine - 60 µmol/l, total bilirubin. - 3.4 KMOL/l, ALT 8E/l, 4,27.

Conducted infusion, antimicrobial, antiplatelet, hormonal (prednisolone at a minimum dose of 15 mg/day) therapy. In the absence of positive dynamics 10.05.2009, increased dose of prednisone to 40 mg/day. With 20.05.2009. joined acute respiratory viral infection. It was noted the deterioration of health, complaints about low-grade fever, cough, recurrent vomiting with blood, change in urine color.

In General, the analysis of blood from 1.06.2009: lake. 13,6·109/l, eritr. 3,62·1012/l, HB 110 g/l, a blood clot. 350·109/l, EO - 3%; n - 1%, C/I - 3%, limp. - 24%, mon. 6%, neutral. 68%, ESR 35 mm/h

In the General analysis of urine from 1.06.2009: protein 0.75 g/l, erythrocytes-250/µ1 and leukocytes 25/µ1, beats. weight 1010, pH 5.0, color - s/W.

Biochemical analysis of blood from 1.06.2009: total protein of 63.6 g/l, cholesterol - 6,59 mmol/l, urea - 9.3 mmol/l, creatinine - 70.5 mmol/l, total bilirubin. - 6.5 KMOL/l, ALT of 11.2 u/l, 4,27.

The results of immunological examination from 5.06.2009:

Ig A 2,68 g/l, Ig M 0.71 g/l, IgG - 7.0 g/l, complemen is 1:128, CEC $ 19, CD3+- 68%, CD8+- 37%, CD4+- 38%, CD4+/CD8+- 1,11, CD19+- 24%, CD16+- 8%, HLA-DR+- 15%, CD25+- 26%.

The results of DNA-cytometry 10.06.2009, (before cytotoxic therapy) G1 - 97,85%, G2 - 0,98%, S - 1,16%, CV - 3,18.

Conclusion: a marked In-lymphocytosis, high levels of the activation markers HLA-DR+and CD 25+characterize recuperability autoimmune process. Identified immunological features correlate with a high level of cells in the phases of proliferation according to the results of DNA-cytometry. Recommended cytostatic therapy.

The high level of proliferative activity in the phases of proliferation, correlating with clinical and laboratory markers of underlying disease (makrogematuriya, proteinuria, hypoproteinemia), indicates the need for more inclusion in the basic course of remedial measures cytostatics.

With 16.06.2009 in the basic course of remedial measures included cytostatic therapy with azathioprine 50 mg/day 25.06.2010 prednisolone replaced by medrol 32 mg/day for 4 weeks, then the dose was gradually reduced to 20 mg/day. In addition, it was held antimicrobial, antiplatelet therapy.

On the face of the base combined immunosuppressive therapy significant improvement in the form of disappearance of the rash, edema syndrome. the biochemical analysis of blood, 1.07.2009 protein total increased to 70 g/l, albumin was 42.1 g/l, cholesterol 5,52 mmol/l, urea of 5.6 mmol/l, creatinine - 49,5 µmol/L.

In the General analysis of urine from 1.07.2009: protein 0.33 g/l, erythrocytes-10 in field of view, beats. weight 1010, pH 6,0.

The results of immunological examination from 1.07.09: Ig A 1,62 g/l, Ig M 1,25 g/l, IgG - 8.8 g/l, CEC $ 39, the titer of the complement is 1:32.

The results of DNA-cytometry 1.07.2009, (after 2 weeks of combination therapy medroom and azathioprine) G1 - 99,84%, G2 - 0,08%, S - 0,09%, CV is 2.51.

Was discharged for outpatient treatment with recommendations to continue the combined immunosuppressive therapy medroom and azathioprine under the scheme.

Since may 2009, regularly observed in the Nephrology Department of the RCCH (held control tests and inpatient treatment in August, October, December 2009 and January 2010). Since January 2010, there has been diagnosed with secondary chronic glomerulonephritis in the background of hemorrhagic vasculitis mixed with renal syndrome. Since June 2009 and is currently receiving combination immunosuppressive therapy medroom and azathioprine. Along with omegamassagechair, immunologicheski methods were conducted regularly monitoring the proliferative activity of the cells according to the DNA-cytometry, the results of which are shown in the table.

As can be seen from the table, the indicators DN is-cytometry was characterized by consistently low proliferative activity of the cells.

The course of the disease, since June 2009, was characterized by recurrent makrogematuriya, proteinuria. Clinical manifestations of the underlying disease in the form of swelling and rash was not. Other laboratory parameters (according to the General and biochemical blood tests) was characterized by relatively stable normal level. Long combined immunosuppression was accompanied by the accession of opportunistic infections. In November 2009 witnessed a deterioration in the urine in the form of appearance of proteinuria up to 1.5 g/l, macrohematuria. When examined by ELISA detected antibodies in classes M and G to the virus Epstein-Barr, which is characteristic of acute infection. In January 2010 noted the accession of acute cytomegalovirus infection (current cytomegalovirus infections), identified according to the ELISA blood test with determination of specific antibodies of classes M and g On the background of the current cytomegalovirus infections has been declining in the urine in the appearance of microproteinuria, macrohematuria. According to immunological examination, it was found persistent overproduction of Ig And characteristic of the underlying disease. According to DNA-cytometry level of proliferative activity of the cells exceeded the norm by no more than 2 times. Along with basic, was conducted antiviral therapy against which noted the positive dynamics in the form of smart the decision of proteinuria and erythrocyturia.

Thus, despite the emergence of laboratory markers of activity of the process, the proportion of cells in the phases of proliferation remained consistently low, indicating adequate immunosuppressive therapy, which does not require dose adjustment of drugs.

Indicators DNA-cytometry patient A. in example 3.
Dates DNA-cytometryG1 (%)G2 (%)S (%)CV (%)
1.10.2009.99,820,020,162,92
20.11.2009.99,740,180,082,75
15.12.2009.99,820,110,073,44
15.01.2010.99.89 per0,030,072,68

A method of evaluating the effectiveness of immunosuppressive therapy in children with chronic glomer what lonerism, characterized by the fact that determine the kinetics of peripheral blood cells by the method of running DNA-cytometry, by identifying the proportion of cells in the phases of proliferation of not less than 0,74% believe shows the assignment of immunosupressant and by decreasing the level of cells in the phases of proliferation 2-3 weeks after start of treatment not less than 2 times therapy is considered effective, in the absence of positive dynamics consider shows a change in dose or replacement of the drug.



 

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1 tbl, 3 ex

FIELD: medicine.

SUBSTANCE: method of quantitative determination of human chitinase-like protein YKL-40 (another name - human cartilaginous glycoprotein-39, or HcGP-39) is offered. When implementing the method, colloidal chitin or heparin is sorbed in microplate wells, then solutions containing a certain amount of chitinase-like protein, and analysed samples are introduced in the wells. The amount of YKL-40 is analysed by the amount of an enzymatic reaction product formed by interaction of an enzymatic conjugate with YKL-40 antibodies against with a substratum of this enzyme.

EFFECT: method allows well reproducible and high sensitive YKL-40 analysis in various human biopsy materials and excretes.

2 cl, 2 ex, 1 tbl

FIELD: medicine.

SUBSTANCE: once admitted to hospital, a patient suffering acute cholecystitis is examined for the blood serum lactoferrin level to calculate a range score; the serum lactoferrin values 200 ng/ml are taken as 1 point; respectively, the lactoferrin level 8 point and lower enable to observe a destructive form of chronic cholecystitis; the lactoferrin level within 8 to 10 points shows catarrhal cholecystitis, and the level 10 points and higher indicates destructive cholecystitis.

EFFECT: use of the technique allows higher diagnostic accuracy in gallbladder tissue destruction in associated acute cholecystitis.

3 ex

FIELD: medicine.

SUBSTANCE: method of determining individual human drug sensitivity or immunotoxicity is based on individual modelling in vitro of the effect on IFN-alpha and/or IFN-gamma production by patient's whole blood leukocytes/lymphocytes. The method involves evaluating a degree of a stimulating/correcting or inhibitory drug action and/or afteraction on the primary IFN system reactivity values.

EFFECT: method provides individual prescription of adequate drugs and thereby higher clinical efficacy for prevention of potential developing medicinal interferon immunodeficiencies.

3 cl, 4 ex

FIELD: medicine.

SUBSTANCE: for selective recovery of a viable cell population, a biological fluid sample is placed in a microfluid device cell containing a silicon microchannel matrix with through holes of size 3-30 mcm and channel length 50-300 mcm as a filter medium, and passed through the matrix at rate 0.1-4 ml/min. In case of cell size separation, a cell fraction of the size less than that of the through holes in the matrix, are collected after passing of the microchannel matrix, while the larger cells are eluated from the microchannel matrix channels by eluent backflow. In case of receptor-specific cell separation, a matrix surface is pre-modified by specific cell receptor antibodies, while the target cells are eluated from the matrix channels by isotypic antibodies or haptens.

EFFECT: more efficient and faster selective recovery of the viable cell population from biological fluids.

3 cl, 2 dwg, 4 tbl, 4 ex

FIELD: medicine.

SUBSTANCE: for detecting thrombinemia associated with large joint replacement, on the 10th-14th postoperative day, patient's blood plasma is examined for the hemostasis system values: the concentrations of D-dimer, plasminogen and antithrombin III. A discriminator (Z) is calculated by formula: Z=4.3936-0.001×D-0.029×P+0.017×A, where D is the concentration of D-dimer (ng/ml FEU), P is the concentration of plasminogen (percentage of normal concentration), A is the concentration of antithrombin (percentage of normal concentration). If observing the value Z <0, high probability of preserving thrombinemia is diagnosed, and pharmacological antithrombotic prevention is recommended to be continued.

EFFECT: use of the declared method allows high-efficient determination of appropriateness of prolongation of a pharmacological antithrombotic prevention course following the large joint replacement.

2 ex

FIELD: medicine, ophthalmology.

SUBSTANCE: in lacrimal liquid one should detect the content of interleukin 8 (IL-8) and that of interleukin 1 beta (IL-1β) to calculate prognostic coefficient (PC) due to dividing the first value by the second one by the following formula: At PC value being below 10.0 one should predict favorable disease flow, and at PC value being above 10.0 - unfavorable flow.

EFFECT: higher accuracy of prediction.

2 ex

FIELD: medicine, medicinal microbiology.

SUBSTANCE: method involves growing microorganism culture to be studied in solid nutrient medium followed by preparing microbial suspension and its incubation in the presence of lactoferrin. Control sample is prepared in parallel series. Control and experimental samples are incubated, supernatant is removed from bacterial cells and lactoferrin concentration is determined in supernatant of experimental and control sample by immunoenzyme analysis. Then anti-lactoferrin activity is calculated by difference of concentrations of residual lactoferrin in experimental and control samples. This method provides enhancing the sensitivity and precision in carrying out the quantitative evaluation of anti-lactoferrin activity in broad spectrum of microorganisms that is urgent in diagnosis and prognosis of diseases with bacterial etiology. Invention can be used in determination of persistent indices of microorganisms for assay of their etiological significance in pathological processes.

EFFECT: improved assay method.

3 tbl, 3 ex

FIELD: medicine, biology.

SUBSTANCE: invention relates to nutrient medium used for accumulation of cells for the following cytological and/or immunocytochemical analysis carrying out. Invention relates to medium containing salts NaCl, KCl, anhydrous CaCl2, MgSO4 x 6 H2O, MgCl2 x 6 H2O, Na2HPO4 x 2 H2O, KHPO4, NaHCO3, and also glucose and Henx's solution, 10% albumin solution and polyglucin taken in the ratio 1:1:1. Invention provides enhancing the preservation of cells.

EFFECT: improved an valuable properties of nutrient medium.

3 ex

FIELD: medicine, cardiology.

SUBSTANCE: in peripheral blood one should detect the level of CD95(+) and CD16(+) neutrophilic granulocytes and at combination of increased level of CD95(+) neutrophilic granulocytes by 4 times and more and CD16(+) neutrophilic granulocytes by 0.6 times against the norm with ECG signs of myocardial infarction one should predict lethal result of large-focal myocardial infarction.

EFFECT: higher accuracy of prediction.

FIELD: medicine, parasitology.

SUBSTANCE: one should carry out immunoenzymatic assay to detect diagnostic optic density and that of labeled immune complex in a plot's hole with tested serum measured in conventional units at wave length being 492 nm. One should calculate coefficient of antibodies concentration measured in conventional units by the following formula: CAC = (Odtsh - Odd) x 100, where CAC - coefficient of antibodies concentration, Odtsh - optic density of the hole with tested serum, Odd - diagnostic value of optic density, 100 - coefficient of serumal dilution. By CAC value one should detect the titer of antibodies to Lamblia intestinalis antigens to interpret results of the trial. The method enables to study the dynamics of disease flow.

EFFECT: higher efficiency and accuracy of diagnostics.

1 ex, 1 tbl

FIELD: medicine.

SUBSTANCE: the present innovation deals with studying and treating diseases of inflammatory, autoimmune and degenerative genesis. One should perform sampling of heparinized blood followed by its sedimentation to obtain blood plasma with leukocytes and centrifuging to isolate the latter which are washed against erythrocytic and serumal admixtures, and, also, it deals with calculating the number of cells in samples out of leukocytic suspension after incubation (B) for 1.5 h at 37 C in holes of plastic microplotting board, out of leukocytic suspension one should additionally prepare two samples, one should be applied to calculate total number of leukocytes before incubation (A), the second sample undergoes incubation at the same mode at addition of autoserum to calculate the number of cells remained after incubation (C). One should state upon adhesive properties of leukocytes by the index of spontaneous adhesion (D), where D=(A-B)/B.100%, and effect for enhanced cellular adhesion under the impact of autoserum should be detected by the value of K=(B-C)/C.100% at K ≥ 30%, where B - C - the number of cells undergone additional adhesion after addition of autoserum. The present innovation widens functional possibilities of the suggested method due to obtaining additional values depicting adhesive properties of blood leukocytes.

EFFECT: higher accuracy of detection.

FIELD: medicine, immunology.

SUBSTANCE: one should carry out reaction of blast-transformation, detect proliferation of T-lymphocytes activated with antibodies to CD3 in the presence of interleukin-7 (ACT IL-7) and in the presence of interleukin-7 and dexametazone (ACT IL-7 D), calculate the index for dexametazone action as the ratio of ACT IL-7 to ACT IL-7 D, moreover, the value of dexametazone action index being above 1.2 indicates increased production of cytokins that suppress T-lymphocytes in neonatals. The method enables to detect functional defect of immune system that characterizes neonatal period.

EFFECT: higher efficiency of detection.

2 ex

FIELD: medicine.

SUBSTANCE: method involves measuring forced exhalation volume per 1 s (FEV1) in l, full right ventricle evacuation time (RVE) in ms and angiotensin II value (AII) in ng/l. Discriminant relationship is built as D=0.504·RVE+3.038·FEV1 - 2.0·AII. D being less than 83.88, pulmonary hypertension occurrence is predicted within 1 year. D being equal to or greater than 83.88, no pulmonary hypertension is predicted to occur.

EFFECT: enhanced accuracy of prediction.

FIELD: medicine, medicinal immunology.

SUBSTANCE: method involves determination of heterophilic antibodies in human serum blood by the Paul-Bunnel's method relatively the level of circulating immune complexes, complement-activating properties of heterophilic antibodies by incubation of standardized ram erythrocytes with 0.8% serum for 30 ± 5 min and the following measurement of the erythrocytes lysis degree. The measurement of the effector function coefficient of heterophilic antibodies is carried out by the complement system Keff.f.h.a.-c.s. by the formula: Keff.f.h.a.-c.s. = Y/Tg.a. wherein Y means a lysis degree, %; Tg.a. means a reverse titer of heterophilic antibodies to ram erythrocytes. The damage assay is carried out by comparison of the immune status with the relative level of circulating immune complexes in serum. Method provides detection of preclinic from of immunodeficiency and autoimmune diseases that opens the possibility for their prophylaxis at most early stages of development. Invention can be used for assay of damage in the immune status in human serum blood.

EFFECT: improved method for assay.

5 tbl, 1 ex

FIELD: medicine.

SUBSTANCE: method involves concurrently examining anti-inflammatory IL-4 level in blood serum and lacrimal fluid. The value being within the limits of 60-70 pg/l in blood serum and 5-15 pg/l in lacrimal fluid, disease prognosis is considered to be unfavorable. The IL-4 concentration being within the limits of 90-100 pg/l in blood serum and 20-30 pg/l in lacrimal fluid, disease prognosis is considered to be favorable.

EFFECT: high accuracy of diagnosis.

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