Method of predicting effect of infliximab therapy in children with crohn's disease
SUBSTANCE: carried out are: determination of the serum concentration of a factor of a tumour necrosis alpha (TNF-α), evaluation of a relative content of Th17-lymphocytes (%CD3+CD4+CD161+ ot CD3+CD4+) and an activity of succinatedehydrogenase in a population of regulatory T-cells (CD3+CD4+CD127low) (SDH-Treg) before and on the following day after infliximab introduction. A prognostic factor of the medicine efficiency K is calculated by formula. If the factor value is K<0.5, a minimal therapeutic effect of infliximab is predicted. When K=0.5-1.5, a moderate therapeutic effect, which demands correction of the medicine introduction plan, is predicted. If K is from over 1.5 to 2.5, a positive effect from introduction of infliximab is predicted.
EFFECT: possibility to predict long-term, not less than 1 year, effect from the infliximab therapy at any stage of therapy.
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The invention relates to medicine, namely immunology, Pediatrics and gastroenterology and relates to a method of predicting the effectiveness of treatment with infliximab in children with Crohn's disease.
Crohn's disease (CD) is a chronic relapsing disease, characterized by transmural granulomatous inflammation with segmental lesions of different parts of digestive tract. The inflammatory process in CD is localized predominantly in the ileum and the colon, but may be affected by any Department of the gastrointestinal tract.
There are various ways of predicting the effectiveness of biological therapy in patients with Crohn's disease (CD), including analysis of clinical, laboratory, endoscopic and pharmacokinetic data. Existing approaches to the development of prognostic criteria are divided into two groups: a retrospective evaluation of possible prognostic parameters and dynamic study of clinical and laboratory findings. Retrospective evaluation of possible prognostic parameters is usually to destination anticytokine therapy and includes a detailed study of clinical and anamnestic data.
There is a method of predicting the effectiveness of infliximab in children with CD, including the assessment of the duration Zab the diseases at the time of initiation of therapy, whereby a shorter duration of the disease is prognostically favorable factor in the effectiveness of the drug (Vermeire s, Louis e, Carbonez, A., Van Assche g, Noman m, Belaiche J. et al. (2002) Demographic and clinical parameters influencing the short-term outcome of anti-tumor necrosis factor (Infliximab) treatment in Crohn''s disease. J Gastroenterol 97:2357-2363).
The method allows only once to predict the effectiveness of therapy without regard to individual response and tolerability.
Known retrospective method for predicting the effectiveness of biological therapy on the basis of the data of the surgical history of the patient and when the patient has surgery on the main disease suggest a lesser therapeutic effect of infliximab compared with patients not operated on for Bq (Orlando A., Colombo E., Kohn, A., L. Biancone, Rizzello f, Viscido A. et al. (2005) Infliximab in the treatment of Crohn''s disease: predictors of response in an Italian multicentric open study. Dig Liver Dis 37:577-583).
The disadvantage of this method is that you cannot use it to evaluate the effectiveness anticytokine therapy in the dynamics.
Known way to predict the effect of infliximab, which includes the determination of the phenotype of CD and when diagnosing the patient stenoderma form of pathology predict a lesser effect of the drug compared to patients with a different disease phenotype (Vermeire s, Louis E. A. Carbonez, Van Assche g, Noman m, Belaiche J. et al. (2002) Demographic and clinical parameters influencing the short-term outcome of anti-tumor necrosis factor (Infliximab) treatment in Crohn''s disease. J Gastroenterol 97:2357-2363).
The disadvantage of this method is the impossibility of its application in patients with mixed phenotype of the disease and the need for confirmation stenoderma form of pathology vysokointensivnymi method colonoscopy.
In predicting the effectiveness of blockers of TNF-A has the value of the localization of the inflammatory process (Arnott I. D., Mcneill g, Satsangi J. "An analysis of factors influencing short-term and sustained response to infliximab treatment for Crohn''s disase" aliment oil displayed pure Pharmacol Ther. 200317:1451-1457). According to studies in patients with isolated colitis occurs most effectiveness of infliximab than in children with different localization of the inflammatory process.
However, the definition of prognostic parameter implies the execution of a colonoscopy, which does not allow to use it as screenimage method to assess the effectiveness of infliximab.
The evaluation of the role of genetic predisposition in the formation of therapeutic response to anticytokine drugs has allowed us to develop a number of prognostic criteria on the basis of genetic analysis.
Known prognostic method of evaluating the effectiveness of infliximab, including the analysis of polymorphism of apoptotic genes (Hiavaty T., Pierik m, Henckaerts L., Ferrante m, Joossens s, Van Schuerbeek N. et al. "Polymorphisms in apoptosis genes predict response to infliximab therapy in luminal and fistulizing Crohn''s disease". Aliment Oil Displayed Pure Pharmacol Ther 2005, 22:613-626). The method includes determining apoptotic pharmacogenetic index API, calculated on the basis of identifying the genotype of a patient from one to three single nucleotide polymorphisms - Fas ligand-843 C/T: Fas a-670G/A; Caapase 93 C/T and when the API is≤1 assume the minimum therapeutic response to infliximab, when API=2 predict a moderate response to anticytokine therapy, when API=3 expect a strong positive effect from the introduction of infliximab.
The disadvantage of this method is a one-time assessment of the prognosis of the effectiveness of therapy that does not take into account changes in the patient's sensitivity to the drug.
In addition, the authors of the method recommend the use of apoptotic pharmacogenetic index in combination with clinical prognostic parameters.
There is a method of predicting the effectiveness anticytokine therapy, including determination of serum content antineutrophilic cytoplasmic antibodies (LIKE) by the method of indirect immunofluorescence and in identifying the patient's atypical variant glow of LIKE predicting the minimum therapeutic response to treatment with infliximab (Taylor K. D., Plevy S. E., Yang, H., Landers, C. J., Barry, M. J., Rolter J. I., S. R. Targan, ANCA pattern and LTA haplotype relationship to clinical responses to anti-TNF antibody reatment in Crohn''s disease. Gastroenterology.2001, 120(6): 1347-55).
The method does not allow to determine dynamic changes forecast the effectiveness of infliximab in a short time due to the long circulation of antibodies in systemic blood flow.
There is a method of monitoring and predicting the effectiveness of infliximab, including the evaluation of pharmacokinetic parameters, namely the residual concentrations of infliximab in serum. According to this method in patients with BC, with the value of the residual concentrations of infliximab over 1,40 g/ml, not earlier than after 6 injections of the drug predict a strong positive effect of infliximab (Maser, E. A., Villela R., Silverberg, M. S., Greenberg, G. R. "Association of Trough Serum Infliximab to Clinical Outcome After Scheduled Maintenance Treatment for Crohn''s Disease, Clinical Gastroenterology and Hepatology Volume 4, Issue 10, Pages 1248-1254, October 2006).
The disadvantage of this method is the inability to predict the effect from the first introduction of the drug for the timely correction therapy.
Closest to the claimed is a method for predicting the effect anticytokine therapy in adults with Crohn's disease, based on the determination of the initial serum concentration of IL-17A, IL-23, IL-12 and with a significant increase in the content of these cytokines before therapy is predicted unstable effect of the introduction of infliximab (Kotaro Ogawa' Takayuki Matsumoto, Motohiro Esaki, Takehiro Torisu, Mitsuo Iida - Proiles of circulating cytokines in patients with Crohn''s disease under maintenance therapy with infliximab Volume 6, Issue 5, June 2012, Pages 529-535).
The disadvantage of this method is that it does not include the study of changes in the concentration of cytokines that play an important pathogenetic role in the development of diseases, in particular tumor necrosis factor alpha (TNF - α), and does not allow to evaluate individual response to the drug.
The method selected as the prototype.
The present invention is to develop a diagnostic method with high accuracy to predict the long-term effect of treatment with infliximab in children with Crohn's disease.
The technical effect of the implementation of the tasks is to improve the accuracy of predicting the effectiveness of treatment with infliximab in children with Crohn's disease. Calculating a prognostic factor efficiency allows adjustment of the scheme is the introduction of infliximab.
The task is carried out through a comprehensive diagnostic assessment of the functional state of the immune system with subsequent calculation of predictive efficiency ratio To
K=[0,564+0,0076×(SDH Treg)to-0,0458×(Th17)to+0,0031×(TNF-α)to]+[0,564+0,0076×(SDH Treg)after-0,0458×(Th17)after+0,0031×(TNF-α)after]/2,
where (SDH Treg)before/afterthe activity of succinate dehydrogenase in regulatory T-lymphocytes (CD3+CD4+CD127low) to / on the trail of the matter the day after the infusion of the drug, srvc.ed.;
(Th17)before/after- the relative amount of Th17-cells (%of CD3+CD4+CD161+ CD3+CD4+) to / on the next day after infusion of the drug,%;
(TNF-α),before/after- serum concentration of TNF-α to/ on the next day after infusion of the drug, PG/ml,
and when the value of K<0.5 to predict the minimum therapeutic effect of infliximab For K=0,5-1,5 predict a moderate therapeutic effect requiring correction scheme the introduction of the drug when from more than 1.5 to 2.5 predict a strong positive effect from the introduction of infliximab.
The method is implemented as follows. In children with Crohn's disease with clinical and endoscopic indications for anticytokine therapy or already receiving infliximab spend the serum concentrations of TNF-α, an assessment of the relative content of Th17-cells (%of CD3+CD4+CD161+ CD3+CD4+) and activity of the mitochondrial enzyme succinate dehydrogenase (SDH) in the population of regulatory T-cells (CD3+CD4+CD127low) before and on the next day after administration of infliximab.
Determination of serum concentrations of TNF-α perform multiplex quantitative method using sets FlowCytomix human Th1/Th2 11 plex kits (Bender MedSystems, Austria) and flow cytometer Beckman Coulter FC 500 (USA). The evaluation of the relative number of Th17-cells (% of CD3+CD4+), make use of fluo eccentric monoclonal antibodies to cell surface markers CD3, CD4, CD161, using flow cytofluorimetry (flow cytometer Beckman Coulter FC 500, USA). Measurement of dehydrogenase activity of peripheral blood lymphocytes carried out immunocytochemical method (Patent RF №2302635). Method of immunocytochemistry is based on the formation of insoluble granules formazan - product of the enzymatic reaction in permeabilizing cells during their incubation with the specific substrate - amber-acidic sodium - and dye - p-nitrotetrazolium purple. The enzyme activity is assessed by the change of the coefficient of lateral light scattering after carrying out enzymatic reactions, which increase in proportion to the number and density of the formed granules. Immunocytochemical method allows to evaluate the functional activity of mitochondria in different populations of lymphocytes.
Study of diagnostic data parameters was performed in 70 children with Bq in age from 10 to 18 years undergoing treatment with infliximab. All children before starting biological therapy underwent a comprehensive examination, including assessment of clinical, laboratory parameters (hemoglobin, hematocrit, leukocytes, platelets, ESR, CRP) and data of endoscopic studies. The criteria for the appointment of infliximab was a high degree of activity, Bq, inefficiency of the Russian Academy of Sciences the e therapy, the presence of hormonal dependence or resistance. Measurement of selected laboratory parameters: serum concentrations of TNF-α, the relative content of Th17-cells (%of CD3+CD4+CD161+ CD3+CD4+) and SDH activity in a population of regulatory T-cells (CD3+CD4+CD 127low) was performed in all patients immediately before infusion and on the next day after administration of infliximab. The number of analyzed at baseline for each patient ranged from 2 to 23, 50 children the evaluation of the selected parameters was performed with the first infusion of the drug.
The efficacy of infliximab were assessed in a year anticytokine therapy, or sooner in cases of involuntary termination of treatment. Therapeutic response was considered minimal in the event high disease activity in clinical laboratory data: pediatric index of Crohn's disease (PCDIA) - more than 10 points and/or endoscopic evidence of active inflammatory process after a year of therapy with infliximab in the standard scheme, and also with a high probability of involuntary cessation of therapy due to security threat to the life of the patient, such as acute allergic reactions, infectious complications and other
Moderate therapeutic effect stated in achieving clinical and laboratory remission (PCDIA<10) and the absence of endoscopic is Reznikov active inflammation after 1 year of therapy after correction schemes the introduction of infliximab - increase doses and/or shorter intervals between infusions of the drug.
Persistent positive effect from the introduction of infliximab corresponded to the cases of endoscopic and clinical and laboratory remission of the disease (PCDIA<10) not less than 1 year after initiation of therapy with infliximab in the standard scheme of administration of the drug. In accordance with the effect of infliximab all the children were divided into 3 groups of patients with minimal, moderate and strong positive therapeutic response to the drug. Average values cytochemical and immunological parameters of patients in the 3 groups are presented in Table 1.
Based on cytochemical and immunologicheskikh indicators a step-by-step method of multiple regression was obtained mathematical equation to calculate a prognostic factor of the effectiveness of infliximab:
K=[0,564+0,0076×(SDH Treg)to- 0,0458×(Th17)to+0,0031×(TNF-α)to]+[0,564+0,0076×(SDH Treg)after- 0,0458×(Th17)after+0,0031×(TNF-α)after]/2, where (SDH Treg)to/afterthe activity of succinate dehydrogenase in regulatory T-lymphocytes (CD3+CD4+CD127low) to / on the next day after infusion of the drug (Th17)to/after- the relative amount of Th17-cells (%of CD3+CD4+CD161+ CD3+CD4+) to / on the next day after infusion of the drug (TNF-α)
Regression coefficients and confidence level for the prognostic equations for calculating the K - factor
On the basis of the prognostic factor of the effectiveness of infliximab determine the estimated effect of anticytokine therapy.
When the values Of<0.5 to predict the minimum therapeutic response: a high activity of disease on clinical laboratory data (pediatric index of Crohn's disease (PCDIA) - more than 10 points) and/or endoscopic evidence of active inflammatory process after a year of therapy with infliximab in the standard scheme is administered, and a high probability of forced termination of therapy due to security threat to the life of the patient (acute allergic reactions, infectious complications, and others).
At values of K=0,5-1,5 suggest a moderate therapeutic effect, namely the achievement of clinical and laboratory remission (PCDIA<10) and no endoscopic evidence of active inflammation after 1 year of therapy in the correction schemes infliximab is a - increase doses and/or shorter intervals between infusions of the drug.
When the values from more than 1.5 to 2.5 predict a strong positive effect from the introduction of infliximab - endoscopic and clinical and laboratory remission (PCDIA<10) not less than 1 year after initiation of therapy with infliximab in the standard scheme of administration of the drug.
The separation of patients on therapeutic effect of infliximab on the basis of calculated and empirically identified, the efficiency of product: 0 - minimal therapeutic effect of infliximab; 1 - moderate therapeutic effect of infliximab requiring correction schemes, and 2 - persistent positive effect of infliximab, are presented in Fig.1.
Examples of the clinical implementation of the method.
Example 1. The patient Was 12 years. Crohn's disease, sick since November 2006.
2008 was observed in the gastroenterology Department with a diagnosis of Crohn's disease, terminal REIT, right-sided colitis, high degree of activity, the stage of exacerbation, pediatric index of the activity of Crohn's disease (PCDIA) was 30 points. Given the continuing high level of activity on the background of anti-inflammatory and immunosuppressive therapy, the formation of the hormone dependence was launched anticytokine therapy chimeric mono is analnyj antibodies to tumor necrosis factor (TNF-α) - infliximab at a dose of 5 mg/day (200 mg) according to the scheme 0-2-6-8 week. After the start anticytokine therapy the child has noted a significant improvement in well-being and appetite, weight gain, lack of abdominal pain and normalization of stool. Objectively, Hematology tests, was observed to stabilize normal blood counts. According to the colonoscopy one year after the beginning anticytokine therapy was visualized only scar biogenic, and histological examination of the intestinal mucosa testified to moderately severe chronic nonspecific inflammation. Based on these results it is possible to speak about the formation of remission of Crohn's disease after a year anticytokine therapy.
Predictive factors of efficacy of infliximab was calculated for this patient at the first (K1), the second (K2), fourth (K4the infusion of the drug and was
Data values (>1.5) was consistent with the prediction of persistent positive therapeutic response to infliximab, which was confirmed to improve clinical and endoscopic parameters.
Prognostic factor efficiency more than a year anticytokine therapy (ninth infusion of the drug) was equal To9=[0,564+0,0076×184,53-0,0458×9,5+0,0031×27,37]+[0,564+0,007×179,36-0,0458×8,5+0,0031×41,71]/2=1,63, testified to the continuing forecast of a stable positive effect of the drug. The dynamics of the prognostic factor of the effectiveness of infliximab in the patient with persistent positive effect is presented in Figure 2.
Example 2. Patient E., age 14. Crohn's disease of the colon and small intestine, sick since autumn 2009, was hospitalized at the ncla RAMP in April 2011 for admission to the Department of state was regarded as difficult due to cachexia, metabolic disorders, symptoms of intoxication, expressed depressive syndrome. For diagnostic purposes was performed colonoscopy and diagnosed with ulcerative REIT and proctitis. PCDIA was more than 40 points. Given the high inflammatory activity of the disease, the formation of sterilizability was launched anticytokine therapy with infliximab at a dose of 5 mg/kg according to the scheme 0-2-6-8 weeks. On the background of anti-inflammatory drug therapy 5-ASA at a dose of 50 mg/kg, cytostatic therapy azatioprina - 1 mg/kg on a background of biological therapy the child's condition has improved significantly: stopped abdominal pain, normalized stool, decreased complaints of weakness, the girl gained weight (+6 kg), gone are the symptoms of depression, decreased laboratory disease activity (CRP levels decreased to 2,93 mg/l). PCDIA decreased to 10 points. The next is colonoscopy examined by the departments of colon and terminal ileum consistent with endoscopic variant rules. However, at 6-7 infusion in a child there was a decrease of sensitivity to infliximab: deterioration of health, weight loss, increase clinical and laboratory signs of inflammation. Given the high degree of activity, Bq, no endoscopic remission dose infliximab was increased to 10 mg/kg with the same interval between infusions. On the background correction therapy showed improvement status and reduced activity of the inflammatory process.
Predictive factors of efficacy of infliximab was calculated for this patient at the first (K1), the second (K2), fifth infusion (K5) drug and was
Values (K=0,5-1,5) corresponded to the forecast of moderate therapeutic response requiring correction scheme the introduction of the drug. Increasing the dose of infliximab was conducted on clinical and endoscopic indications when the seventh administration of the drug. Prognostic efficiency ratio for the year anticytokine therapy (eighth infusion of the drug) was equal To8=[0,564+0,0076×209,64-0,0458×24,5+0,0031×38,77]+[0,564+0,0076×154,61-0,0458×20,30+0,0031×0,00]/2=0,98, which corresponded to the continued forecast of moderate efficacy of the drug.
The dynamics of the prognostic factor of the effectiveness of infliximab in this patient, with a moderate effect is tons of infliximab, requiring correction dose are presented in Figure 3.
Example 3. Patient C., for 15 years. Crohn's disease, a high degree of activity, pacolet, active form): fistula anorectal region, rectovaginal fistula, continuously relapsing course. The girl has been sick since 2008, when the survey was diagnosed with Crohn's Disease. Within 4 years the child treatment is not received. Were observed at the place of residence. In March 2011 due to the sharp deterioration girl was referred to the gastroenterology Department of Hepatology group of the fgbi "the ncla RAMS. On admission, the child's condition was regarded as serious due to intestinal syndrome and symptoms of intoxication. Inspection of the perineum revealed swelling, redness over the entire surface with the transition to large labia, there is deformation, melting, multiple vegetation, on the back of the perineum - the skin defect going to the coccyx. According to colonoscopy diagnosed erosive-ulcerative pancholi. Given the high risk of developing septic process, the child was performed surgery: the formation of terminal and an ileostomy, appendectomy. Given the high inflammatory activity, the fistulous Crohn's disease, biological therapy monoclonal antibodies to TNF-α - inflict the MAB at a dose of 5 mg/day (150 mg) according to the scheme 0-2-6-8 week. Anticytokine therapy was carried out in combination with an anti-inflammatory drug 5-aminosalicylic acid (5-ASA) - pentasa dose of 50 mg/kg and cytostatic azathioprine 1.5 mg/kg On the background conducted induction rate was observed moderate positive trend in the decrease of laboratory disease activity, normalization of stool, improve well-being and appetite, weight gain. However, according to the results of endoscopic examination, the child remained erosive-ulcerative pacolet high degree of activity. Due to lack of efficacy of combined anti-inflammatory, immunosuppressive and anticytokines of drug therapy chimeric monoclonal antibody to TNF-α infliximab at a dose of 5 mg/kg dose was increased to 10 mg/kg (350 mg) under the old scheme (every 8 weeks). Against the backdrop of ongoing biological therapy were positive dynamics, the relief of inflammatory manifestations in the anus, lower laboratory disease activity, however, complete remission Bq was not observed. When the control hospitalization 10 months after the start anticytokine therapy in girls was observed deterioration: fever, weakness, repeated vomiting, liquid stool. Upon examination, the child was observed laboratory and endoscopic activity is aesni Crown. Given the ineffectiveness of conservative therapy, the girl had surgery: laparoscopic associated total colectomy.
Predictive factors of efficacy of infliximab was calculated for this patient at the first (K1), the second (K2), third (K3the infusion of the drug and was
Value (<0.5) is consistent with the prediction of the minimum therapeutic response from the introduction of infliximab, which was confirmed persistent high clinical, laboratory and endoscopic activity of the inflammatory process. Increasing the dose of infliximab was conducted on clinical and endoscopic evidence on the sixth administration of the drug, however, the improvement in clinical status and reduced inflammatory activity was not observed. Prognostic factor efficiency in the sixth introduction of infliximab was To6=[0,564+0,0076×210,09-0,0458×47,5+0,0031×0,00]+[0,564+0,0076×159,01-0,0458×44,4+0,0031×0,00]/2=-0,15, that was in line with expectations minimum efficacy of the drug. After 10 months anticytokine therapy (seventh infusion of the drug) is a prognostic factor of the effectiveness of infliximab amounted To7=[0,564+0,0076×153,46-0,0458×43,7+0,0031×0,00]+[0,564+0,0076×181,19-0,0458×50,3+0,0031×0,00]/2=-0,33, that was in line with expectations minimum therapeutic resp is the introduction of infliximab. The child was observed clinical, laboratory and endoscopic signs of pronounced inflammatory process, which was a testimony to the scheduled surgical interventions for the underlying disease. The dynamics of the prognostic factor of the effectiveness of infliximab in this patient with minimal therapeutic response from the introduction of infliximab, are not amenable to correction when schema changes are the introduction of the drug, is presented in Figure 4.
The method of predicting the effectiveness of infliximab in children with Bq allows us to predict the long-term, not less than 1 year, the effect of therapy with infliximab at any stage of treatment takes into account individual patient response to the drug and is particularly relevant in cases of inadequate performance anticytokine therapy requiring correction schemes the introduction of infliximab.
A method for predicting therapeutic effect of infliximab in children with Crohn's disease, including the identification of immunological and cytochemical indices, wherein spend the serum concentration of tumor necrosis factor alpha (TNF-α), assessment of the relative content of Th17-cells (%of CD3+CD4+CD161+ CD3+CD4+) and dehydrogenase activity in a population of regulatory T-cells (CD3+CD4+CD127low) (SDH-Treg) prior to and following su the key after the introduction of infliximab and calculate prognostic factor efficacy -
To the formula:
K=[0,564 + 0,0076 × (SDH Treg)to- 0,0458 × (Th17)to+ 0,0031 × (TNF-α)to] + [0,564 + 0,0076 × (SDH Treg)after- 0,0458 × (Th17)after+ 0,0031 × (TNF-α)after] / 2,
where (SDH Treg)to/afterthe activity of SDH-Treg to / on the next day after infusion of the drug, the river.ed.; (Th17)to/after- the relative amount of Th17-cells (%of CD3+CD4+CD161+CD3+CD4+) to / on the next day after infusion of the drug, %; (TNF-α)to/after- serum concentration of TNF-α to/ on the next day after infusion of the drug, PG/ml,
and when the value of K<0.5 to predict the minimum therapeutic effect of infliximab For K=0,5-1,5 predict a moderate therapeutic effect requiring correction scheme the introduction of the drug when from more than 1.5 to 2.5 predict a strong positive effect from the introduction of infliximab.
SUBSTANCE: invention relates to analytical chemistry, in particular to aspects, related to methods of the nonenzymatic determination of the presence or a quantity of carbohydrates in a sample, and describes a method, device and set for the sample analysis to determine the presence or a quantity of an analysed substance, in particular a carbohydrate, in particular, sugar, in the sample with the application of fabric. The method includes the application of the sample on a synthetic fabric; chemical modification of the said carbohydrate, present in the sample, with an oxidising agent with a sufficient oxidising potential in order to cleave the carbohydrate between two hydroxyl groups; inactivation of the said oxidising agent, which would prevent the identification of the chemically modified carbohydrate; identification of the presence or the quantity of the said chemically modified carbohydrate by means of a copper-containing compound to obtain the visible change of colour.
EFFECT: invention can be used for the determination of the presence of a carbohydrate on the surface to indicate the surface contamination, in particular contamination with a substance, promoting the microbial growth.
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SUBSTANCE: invention relates to medicine, namely to method of determining expression of inflammatory process in case of osteoarthrosis. Essence of method consists in the following: carried out is luminol-dependent iron-induced chemi-luminescence of model system, which has the following composition: 2.72 g of KH2PO4, 7.82 g of KCl, 1.5 g of sodium citrate C6H8O7Na3*5,5H2O per 1 liter of distilled water, pH 7.45 with 0.2 ml of 10-5 M luminol solution, after that, intensity of model system luminescence is determined in presence of synovial fluid before and after addition of synovial fluid. Degree of suppression of intensity of model system chemi-luminescence is calculated by formula. If its value is from 1.71 to 6.48%, high activity of inflammatory process is determined, if its value is from 6.49 to 21.55%, medium activity is determined, from 21.56 to 55.46 - small activity.
EFFECT: application of the method makes it possible to reduce time of determination and increases accuracy of assessment of inflammatory process degree in case of osteoarthrosis.
1 tbl, 1 dwg, 3 ex
SUBSTANCE: diagnostic test element (110) for the detection of an analyte in a sample (126) of a body fluid, in particular whole blood with the volume not less than 2 microlitres, contains a test field (116) with a reagent-indicator, where the reagent-indicator is capable of feeling a detected change, in particular an optic change, in case of the analyte presence. The test field (116) includes a detector layer (118), containing the reagent-indicator, where the detector layer contains particles (137). 90% of all particles (137) of the indicator layer (118) have the actual size smaller than 10 microns. The diagnostic test element (110) contains a bearing element (112), which has a transparent region (114), where the test field (116) with its detection side (120) is partially applied on the transparent region (114).
EFFECT: detected change is an optically detectable change, where an optic detector with spatial resolution is applied for the detection of the detected change.
9 cl, 8 dwg, 1 tbl, 2 ex
SUBSTANCE: invention relates to immunology and medical diagnostics and represents method of carrying out immunochromatographic analysis for serodiagnostics. Claimed invention is intended for immunochromatographic determination of antibodies to causative agents of infectious diseases or other antigens, for instance, allergens, in liquid samples. Claimed method of identifying antibodies is characterised by the following: solution for sample dilution contains specific antibodies against test strip of antigen (or antigens) immobilised in analytic zone. Used concentration of antibodies in diluting solution is lower than lower limit of determination of antibodies by said method with application of diluting solution, which does not contain specific antibodies, which results coloration of analytic zone of test strip is not observed if specific antibodies are absent in sample. If sample contains specific antibodies, presence of additional quantity of specific antibodies in diluting solution results in enhancement of intensity of coloration of test strip analytic zone.
EFFECT: application of invention makes it possible to register total concentration of specific antibodies in sample and in diluting solution, which results in reduction of limit of analysis identification due to displacement of working range of concentrations, determined by test, into the area of lower values.
3 dwg, 1 ex
SUBSTANCE: invention refers to medicine and represents a method for accelerated staining of histological specimen for identifying tuberculosis mycobacteria involving the preparation of the histological specimen according to a common technique, the Ziehl-Nelsen staining differing by the fact that the standard preparation of the specimen for staining is followed by a de-waxing procedure by placing them successfully into xylol and alcohols of the decreasing concentration for 5 minutes in a thermostat at 54°C and then placing into water, then in a 1% periodic acid for 2 minutes and washing in flowing water for 10 seconds; thereafter, Ziehl carbol-fuxine is poured on a section coated with a filter paper and heated on an alcohol burner lamp until evaporating for 2 minutes; the stain is left on the section after the heating procedure is completed, for 3-5 minutes; the filter paper is removed, and the sections are washed in water, differentiated in a 1% acid buffer, washed in main water for 1 minute; thereafter Leffler methylene blue is placed on the section and heated on the alcohol burner lamp until evaporating, washed in water, differentiated in a 1% acid buffer and washed with a plenty of water, de-watered in alcohols of the increasing concentration for 1 minute, placed in xylol and balm.
EFFECT: developing the method for the accelerated stain of the histological specimen.
SUBSTANCE: invention refers to medicine and aims at the prediction of postoperative suppurative complications in patients suffering from colorectal cancer by a dynamic blood test. On the 2nd and 5th postoperative day, blood plasma of the patient suffering from colorectal cancer is examined for C-reactive protein (CRP). If the 5th-day CRP level is less than 10.0 mg/dl, a relatively mild postoperative course is predicted. If the CRP level decreases in relation to the second postoperative day, a risk of the complications is considered to be low. If the CRP level increases in relation to the second postoperative day or remains unchanged, a risk of the complications is moderate. If the 5th-day CRP level is more than 10.0 mg/dl, a risk of the postoperative suppurative complications is considered to be high.
EFFECT: invention provides the effective prediction of the suppurative complications in the patients on the second and fifth day following colorectal cancer surgery.
SUBSTANCE: ovarian tumour tissue is examined for a portion of the synthesis cycling-state cell; if the derived value is less than 15.9%, a favourable outcome is predicted, while at the derived value being more than 15.9%, an unfavourable outcome is predicted. The invention can be used more than once in clinics for women and special oncological hospitals for the purpose of predicting recurrent ovarian carcinoma according to the tumour tissue DNA-cytometry findings.
EFFECT: higher prediction accuracy.
SUBSTANCE: technique involves preparing a patient's blood serum sample by drying blood serum, milling the dry deposition and suspending in Vaseline oil. The prepared sample is analysed by IR-spectroscopy by determining a peak height of absorption bands with maxima 1165; 1160; 1150; 1100; 1070; 1050, and 1025 cm-1. And the peak height relations are calculated: the relation of the peak height with maximum 1160 cm-1 to the peak height with maximum 1165 cm-1; the relation of the peak height with maximum 1165 cm-1 to the peak height with maximum 1070 cm-1; the relation of the peak height with maximum 1165 cm-1 to the peak height with maximum 1150 cm-1, the relation of the peak height with maximum 1165 cm-1 to the peak height with maximum 1050 cm-1, the relation of the peak height with maximum 1100 cm-1 to the peak height with maximum 1050 cm-1, the relation of the peak height with maximum 1025 cm-1 to the peak height with maximum 1165 cm-1. The derived values are used to draw a differential diagnostic profile of the patient's blood serum sample to produce a plane polygon formed by 6 beams corresponding to 6 relations and to compare the above polygon to a polygon that is a reference differential diagnostic profile of pulmonary thromboembolism, wherein 6 relations make: 1 (1.85±0.26), 2 (0.28±0.13), 3 (0.39±0.07), 4 (0.25±0.13), 5 (0.58±0.10), 6 (4.47±1.70). If the derived patient's differential diagnostic profile has a similarity with the reference profile, and all 6 relations of the patient's blood serum sample coincide with the reference relations, pulmonary thromboembolism is diagnosed in the patient.
EFFECT: invention possesses high accuracy, easy to implement, and requires no heavy material and time expenses; it enables diagnosing thromboembolism of all pulmonary arteries both in the presence of clinically significant symptoms of the disease, and in the absence thereof.
2 ex, 3 dwg
SUBSTANCE: summary of declared method consists in determining the anticholinesterase effect of solutions of the examined anticholinesterase compounds (AnCEC) of a preparation of cholinesterase - the enzyme propionyl cholinesterase of calamari brain tissue (PCE), namely in estimating a percentage PCE activity inhibition in solutions of low (0.04 U/ml) and high (4 U/ml) concentrations. A toxicity hazard of the examined AnCEC solutions is assessed by a difference of their effect on PCE activity in the solutions of low and high concentrations. If a percentage of PCE activity inhibition in the solution of high concentration goes to zero or does not exceed 5-10%, the examined solution is stated to contain high-toxicity AnCEC of the 1st hazard class. If the inhibitory effect size remains constant or falls by no more than 10%, the presence of low-toxicity AnCEC is stated.
EFFECT: using the declared method enables assessing the toxicity hazard of the anticholinesterase compounds for 30-60 minutes in various situations, where their composition is unknown or they present in the form of solutions of an unknown concentration.
1 tbl, 2 ex
SUBSTANCE: invention relates to monitoring of the environment and biological objects aimed at identification of the content of metal ions in liquid media with the application of photochromic compounds. In the method of spectrophotometric identification of metal ions in liquid media with an application of photochromic compounds of class of chromenes due to formation of complexes between the photoinduced merocyanine form of the said compounds and metal ions as chromenes used are such bischromenes as: 2,2,11,11-tetrakis(4-methoxyphenylphenyl)dioxa(1,12)triphenylene, 2,2,8,8-tetrakis(4-methoxyphenyl)dioxa(1,7)chrysene, 3,3,11,11-tetra-(4-methoxyphenyl)-3,11-dihydro-4,10-dioxadibenzo[a,h]anthracene, 3,3,10,10-tetra-(4-methoxyphenyl)-3,10-dihydro-411-dioxadibenzo[a,h]anthracene.
EFFECT: increased selectivity of identification is achieved.
24 ex, 1 tbl, 6 dwg
FIELD: medicine, analytical biochemistry.
SUBSTANCE: invention relates to laboratory methods of investigations. Method involves sampling specimen from patient to be inspected, extraction of serotonin and histamine from a specimen, chromatography of extract and determination of concentration of serotonin and histamine by the fluorescence intensity value. Saliva is used as biological fluid. Saliva by volume 1 ml is extracted with 4 ml of 1 N hydrochloric acid solution, 2 g of anhydrous potassium carbonate and 5 ml of mixture of butanol and chloroform in the ratio 3:2 are added, extract is shaken up and centrifuged. Organic phase (4 ml) is sucked off from extract and passed through chromatography column (diameter is 3 mm, height is 16 mm) filled with ion-exchange resin KB-4 or KB-4P-2 or Bio Rex-70 in H+-form, size of granules is 0.1 ± 0.02 mm. Histamine is eluted with 4 ml of 0.1 N hydrochloric acid at the rate of eluting solution 0.4 ml/min. Histamine concentration is determined by reaction with ortho-phthalic aldehyde dissolved in ethanol. Serotonin concentration is determined by reaction with ninhydrin in organic passed through column. Method provides assaying the saliva concentration of serotonin and histamine with high precision.
EFFECT: improved assay method.
SUBSTANCE: method involves studying blood samples with venous blood mixed with vital stain like methylene blue. Degree of vital stain absorption by erythrocytes is determined by applying photocolorimetry. The value drop being more than 25%, extracorporal detoxication is to be predicted as ineffective.
EFFECT: simplified method.
FIELD: medicine, infectology.
SUBSTANCE: one should detect the level of terminal stable metabolites of nitrogen oxide (NOx) in whole blood. At its value ranged 39.6-86.0 mcM/l one should evaluate hemorrhagic fever accompanied with renal syndrome (HFRS) of average severity, at NOx value ranged 86.7-141.5 mcM/l - severe form of HFRS and at its values ranged 88.2-128.6 mcM/l at the background of pronounced clinical picture - as complicated disease flow. The method enables to shorten the terms for carrying out the assays.
EFFECT: higher accuracy of evaluation.
3 ex, 1 tbl
FIELD: medicine, biochemistry.
SUBSTANCE: in blood serum one should detect the level of lactoferrin and biliary acids. At their ratio being equal to 5-17 it is necessary to detect chronic hepatitis of high activity.
EFFECT: higher accuracy of detection.
FIELD: medicine, dermatology, clinical laboratory diagnostics.
SUBSTANCE: the present method deals with detecting the focus of neutrophilic phagocytic activity lesion in capillary blood. At the values of cells' capacity to phagocytosis in percentage and phagocytic number on the 10th d of therapy being below 20% and 3.3, correspondingly one should evaluate therapeutic efficiency to be low, if it is above 40% and 4.0, correspondingly - as high.
EFFECT: higher accuracy of evaluation.
2 ex, 3 tbl
SUBSTANCE: method involves studying blood serum, processing obtained data and setting disease diagnosis. The study is carried out by preparing dried blood serum sample as suspension in Vaseline oil and doing the infrared spectroscopy analysis in the bandwidth of 120-1000 cm-1 and determining absorption strip peak heights having maximum at 1180; 1165; 1160; 1150; 1130; 1070; 1025 cm-1 and then calculating the following two ratio groups, the first of which is ratio of peak height with maximum at 1165 cm-1 to 1150 cm-1; 1160 cm-1 to 1130 cm-1; 1070 cm-1; 1025 cm-1. The second group has ratio peak having maximum at 1165 cm-1 to 1160 cm-1; 1180 cm-1 to 1130 cm-1; 1065 cm-1; 1070 cm-1. The obtained three-dimensional distribution of the first group is projected to frontal plane for calculating two-dimensional coordinates and comparing to flat reference diagnostic images of hepatic pathologies and to a normal reference diagnostic image represented as flat polygons which boundaries are given by the following values. The norm is represented by X(-2.3;2.0;4.0;4.0) and Y(1.6;0.8;0.8;1.6), respectively. Oncology is represented by X(1.7;1.7;0.0;0.0) and Y(1.9;1.25; 1.25;1.9). Hepatites are represented by X(1.9;2.2;1.8;1.4 and 1.9;1.8;4.0) and Y(1.9;1.9; 0.5;0.5 and 08;0.5;0.8). Cirrhosis is represented by X(1.9;2.6;1.4) and Y(1.6;0.8;0.4). Diseases are differentiated by interpreting point position within particular area. Three-dimensional distribution of the second group is projected to frontal plane and compared to diagnosis images of pathology and norm. Coordinate values of the second group are as follows: norm - X(1.8;2.9;2.5;1.5), Y(2.7;2.0;1.2;1.6); oncological cases - X(0.27;0.67;0.63), Y(0.27;0.67;0.3); hepatitis - X(1.5;2.5;2.4;1.2), Y(1.6;1.2;0.2;0.9); cirrhosis - X(1.1;0.9;0.9). Final diagnosis of pathology is set when particular data values belong to the corresponding pathology zone in both cases.
EFFECT: high accuracy of diagnosis.
SUBSTANCE: method involves studying biological material by applying infrared spectroscopy techniques. The obtained data are processed and diagnosis is set. Blood serum is used as the biological material. The study is carried out by preparing dried blood serum sample as suspension in Vaseline oil and doing the infrared spectroscopy analysis in the bandwidth of 120-1000 cm-1 and determining absorption strip peak heights having maximum at 1170; 1165; 1160; 1150; 1140; 1060; 1050; 1040; 1025 and then calculating the following ratio values like peak height with maximum at 1160 cm-1 to 1140 cm-1; 1165 cm-1 to 1150 cm-1; 1040 cm-1 to 1025 cm-1. The obtained distribution of this group is projected to frontal plane for calculating two-dimensional coordinates and comparing to flat reference diagnostic images of prostate pathologies and to a normal reference diagnostic image represented as flat polygons which boundaries are given by the following values. The norm is represented by X(-1.15;-0.9;0.45;0.0;-0.65) and Y(0.99;4.2;0.9;0.46), respectively. Pathology by X(-1.15;-1.15;0.35;0.0;0.65) and Y(0.99;-0.03; 0.48;0.09;0.46). The norm and pathology are differentiated. Additional mathematical processing is carried out on infrared spectra of blood serum samples of patients belonging to pathology image according to parameter values. First of all, three-dimensional distribution is calculated as peak having maximum at 1160 cm-1 to one having maximum at 1150 cm-1; 1170; 1160 cm-1; 1160 cm-1 to 1025 cm-1. It is projected then to frontal plane and compared to diagnosis images of prostate adenoma and images of prostate carcinoma. The second group relationships the following values are used: oncological cases - X(0.28;0.77;1.24;0.96), Y(0.75;0.46;-0.13;-0.02); adenoma - X(0.28;1.24;2.21;1.24;0.77), Y(0.75;1.24;-0.12;-0.13;0.46). Differential diagnosis of pathologies is set by interpreting point position within particular pathology image.
EFFECT: high accuracy of differential diagnosis.
SUBSTANCE: method involves determining mean cytochemical coefficient of lipid accumulation in peripheral blood leukocytes in conditional units before beginning therapy application (MCC1) and in 2-3 or 5-6, or 10-12, or 20-24 months of therapy application (MCC2). Therapy effectiveness coefficient is calculated in conditional units from formula K= MCC2/MCC1. The value being equal to or greater than 1, leprosy therapy is predicted to be effective.
EFFECT: simplified prognosis method.
1 dwg, 1 tbl
SUBSTANCE: method involves determining infrared radiation absorption coefficient in blood plasma in bandwidth of 1543-1396 cm-1. The infrared radiation absorption coefficient is determined in %. The value being equal to 29.7±1.1%, catarrhal cholecystitis is diagnosed. The value being 26.4±1.4%, phlegmonous cholecystitis is diagnosed. The value being 21.2±1.8%, gangrenous cholecystitis is diagnosed. The value being equal to 18.6±0.5%, gangrenous perforated cholecystitis case is diagnosed. The value in norm is equal to 32.4±0.8%.
EFFECT: high accuracy and specificity of diagnosis.
SUBSTANCE: method involves pouring venous blood treated with heparin into five conic test-tubes in the amount of 0.1 ml. The first three of them contain 0.1 ml of non-colored latex suspension with particle size of 1.5 mcm, the fourth one contains 0.1 ml of medium 199 and 0.1 ml of 0.1% aqueous solution of tetrazole nitro blue, the fifth one contains .1 ml of latex suspension and 0.1 ml of 0.1% aqueous solution of tetrazole nitro blue. The first test-tube is incubated in thermostat for 5 min at37°C, the second one for 30 min, the third one for 1 h, the fourth and the fifth one for 40 min. Smears are prepared from 0.2 ml of incubation mixture on glasses and dried at 37°C, fixed in burner flame, stained with 0.1% aqueous solution of tetrazole nitro blue, repeatedly dried and studied with microscope under immersion with magnification of 90x10. Test results are evaluated from absorption activity in phagocytosis reactions in determining the number of phagocytes, phagocytic number, phagocytic integral index and phagocytosis rate values. Tetrazole nitro blue test response is determined by counting formazan-positive cell number, calculating cytochemical activity index and tetrazole nitro blue test stimulation index.
EFFECT: accelerated test; high accuracy and low cost of examination.
1 dwg, 3 tbl