The method of determining sensitivity to pharmaceuticals, dental materials

 

(57) Abstract:

The invention relates to medicine, namely to laboratory diagnosis. The method is based on imposing on the gum sample of the test substance, the count of neutrophils in swabs from the oral cavity, taken before and after contact of the mucosa with substance, centrifugation, determining the level of emigration of neutrophils into the oral cavity and index shift of neutrophils. The neutrophil count is carried out by staining peroxidase-Chromogen solution. The index shift of neutrophils is determined by their optical density according to the formula:

When the value of the shift index of neutrophils in the range from 0.75 to 1.5 body's sensitivity to the subject matter defined as normal. The method allows to reduce terms of research when determining sensitivity to pharmaceuticals and dental materials.

The invention relates to medicine, namely to laboratory diagnosis.

There is a method of determining drug allergies by detecting the level of inhibition of natural emigration of neutrophils into the mouth after mouth rinsing solution of this drug for 1 min (ADO is stvennoi allergies. Methodical recommendations. - M., Institute of Immunology, 1986. - 20 C.). This test is unsafe for the patient, because with the product contacts the mucous membrane of the mouth. In addition, the test is time consuming and requires use of the microscope.

There is a method of determining the sensitivity of the organism to local anesthetics based on the Muco-gingival test (Lebedev, K. A., Maximov Y. M., Kulmagambetov I. R. and others, Muco-gingival test for the determination of hypersensitivity to local anesthetics /Maestro dentistry. - 2003. - №3 (12). - S. 74-78). The method consists in that the carrier of the test drug is ligninases disk with a diameter of 10 mm and thickness of 3 mm, which allows you to create a concentration gradient of the drug and provides long lasting effects on the mucous membrane, which allows to study more deeply the influence of the drug on the cells of the body. Disk impregnated with the solution, is applied to the mucous membrane of the gums and incubated for 50 min, after which cells swabs taken before and after exposure to the test drug, precipitated by centrifugation, determine the number of neutrophils in the swabs. Determine the index of the shift netropa. When values of 0,8-2,0 determine the normal sensitivity of the organism to the drug. This method is safe for patients, because it is based on the use of drug concentrations 10,000 times lower, in addition, the drug not contact the entire surface of the mucous membrane of the mouth, and only a small part of it. However, and in this way the evaluation of the results carried out using a light microscope, which restricts the use of this method by the presence of the laboratory, the evaluation result was 20 min in 1 patient. This method is chosen for the prototype.

The objective of the invention is the possibility of determining sensitivity to any substance, the reduction in terms of determining the sensitivity of the organism. This is due to the fact that the counting of neutrophils carried out by staining peroxidase-Chromogen solution, and the index shift of neutrophils is determined by their optical density according to the formula:

and when the value of the shift index of neutrophils in the range from 0.75 to 1.5 body's sensitivity to the subject matter defined as normal.

In General, the study of 1st patsie is for example 10 people increases, and the research time is 5-6 hours. The time increase due to the evaluation results each time for 20 min scoring under the microscope - 20 min in 1 patient (10 patients - 20 min (10=200 minutes).

In the proposed method study 1 patient takes 1 hour 20 min, and a survey of 10 people takes 2 hours 20 minutes Time is reduced due to the fact that the evaluation of results is carried out by staining peroxidase-Chromogen solution. The proposed method of calculation of results 1 patient - 2 min (10 patients - 2 min+9 min (1=11 min). In addition, research can be carried out in automatic mode (you can explore up to 96 people).

The method is as follows: the patient takes wash from the oral cavity (the patient rinses the mouth with a fixed number of physiological solution, usually 8 ml, for 1 min, then carefully drain it back into the tube). Then to the gum put a sample of the test substance. If the test substance has a liquid consistency, it is possible to apply, for example, ligninases disk with a diameter of 10 mm and thickness of 3 mm, impregnated with a solution of the test drug in a dilution of 10-7from the particular to substances solid consistency, in the form of gels or pastes - a piece of this substance.

After 50 min the sample of the test substance was removed, and the patient takes another wash from the oral cavity when subject to the same conditions as the first flush. Neutrophils from both swabs precipitated by centrifugation. The supernatant liquid is drained and the cells are washed with saline, Prilepa to rain 8 ml of physiological solution and resuspended them (thus remove any traces of saliva, which contains a large number of peroxidase). Cells are again precipitated by centrifugation in the same mode.

Next, the supernatant is poured, and to rain poured an equal amount of distilled water (usually 1 ml), mix thoroughly. After 2 min in vitro cells lyse, the neutrophils released peroxidase. In the wells of flat-bottomed device for conducting immunoassay fill in 0.1 ml from each tube. Poured 0.1 ml of a solution of tetramethylbenzidine (TMB). However after 1-5 min begins to develop a blue color, the intensity of which is proportional to the concentration of peroxidase in the liquid. 10-30 min the reaction is stopped by adding to the wells with 0.1 ml of a 5% solution of sartomer. Determine the index of the shift of neutrophils by their optical density (intensity of staining) according to the formula:

and when the values from 0.75 to 1.5 determine the body's sensitivity to the subject matter as normal.

Evaluation of the results takes 2 min, which is 10 times smaller than the prototype method.

Clinical example 1.

The patient N. (and a/b No. 6014-03), 43 years old, was determined sensitivity to local anesthetics with the aim of selecting the drug for therapeutic dental treatment. On the day determined the sensitivity to 1 drug.

Before the examination the patient had taken control wash: rinse mouth with 8 ml of saline for 1 min Then to the gum was applied ligninases disk with a diameter of 10 mm and thickness of 3 mm, impregnated with a solution of Ultracaine D-S forte in dilution 10-7from therapeutic concentration. The duration of application was 50 minutes Then the patient again took the wash from the oral cavity in the same conditions. Both swabs cells besieged by centrifugation at 200 g for 10 min. the Supernatant was decanted, and the precipitate was added 8 ml of physiological solution, the precipitate resuspendable. This procedure is a response in the same mode. The supernatant was decanted, and the precipitate was added distilled water to a volume of 1 ml were Thoroughly stirred. After 2 min to 0.1 ml of the fluid was poured into the wells of flat-bottomed tablets, there was added 0.1 ml of a standard solution of TMB (production xema-Medica, Russia). After 15 min was added to 0.1 ml of a 5% solution of sulfuric acid. We measured the optical density (OD) using a vertical photometer at a wavelength of 450 nm. OP control flushing was 0,412, after application of the drug - 1,123, i.e. was 2.73 times more (173%). Also can be assessed visually (using a color scale). The conclusion was made about the sensitivity of the patient to the drug and the impossibility of its use for local anesthesia. During questioning of the patient revealed that the previously identified clinical symptoms of Allergy (rash) after application of sulfa drugs (ultracain is a sulfa drug).

In a similar way the following day the patient was determined sensitivity to lidocaine 3% (Russia). OP control flushing was of 0.533, after application of the drug - 0,540, i.e., the excess was 1,013 (1.3%). The conclusion was made about the lack of high sensitive is ntci. The patient successfully underwent therapeutic treatment at the dentist with this drug.

Clinical example 2.

Patient E., b No. 358-03), 39 years old, came to our clinic concerning pain and edema of the mucous membrane in the area 13 of the tooth. History of 6 weeks ago was treated periodontitis of the tooth, the channels are sealed. Rentgenovskii snapshot showed a satisfactory condition obturation of the canals. Suggested that the patient has intolerance material used for fillings (zincoxide paste). Got questions, first, about the confirmation or refutation of this assumption, and secondly, in case of intolerance of this filling material about the selection of a new material for fillings. To conduct Muco-gingival provocative test used the pieces ready filling material in the form in which they filled the channel of the tooth (but not its individual components). Before the examination the patient took control wash: rinse mouth with 8 ml of saline for 1 min Next to the gum put a piece of the test material, the duration of application was swabs besieged by centrifugation at 200 g for 10 minutes Measured OD using a vertical photometer at a wavelength of 450 nm. OP, directly after taking swabs, then was added 8 ml of saline, the cells resuspendable.

The washed cells swabs besieged by centrifugation in the same mode. Then the supernatant liquid was decanted, and the precipitate was added distilled water to a volume of 1 ml, were mixed. After 5 min, 0.1 ml of the fluid was poured into the wells of flat-bottomed plate. Was added on ML ml standard solution of TMB. After 25 min the reaction was stopped by adding one ml of a 5% solution of sulfuric acid. Measured OD using a vertical photometer at a wavelength of 450 nm. OP control flushing was 0,428, rinse after application of the material - 0,830, i.e., the coefficient of stimulation was 1.94 (excess cells was 94%). This is consistent with the presence of the patient reaction of the average force on the material that could give available in her clinical manifestations. In the result, it was decided to make pereplanirovka channel.

For selecting a new filling material, the patient was determined sensitivity to the two filling materials: 1) Silent and 2) incomedependent pasta (COAP) to the t) OP in the control flushing totaled value (0.475), after application of the drug - 0,608 (that is, IP amounted to 1.28. In the study of material 2 (COAP) optical density in the sample washout was 0,377, after application of the drug - 0,622 (i.e. IP was 1.65). For sealing channel was selected material 1 (Silent). After pereplanirovki channel with the use of this material clinical symptoms for which the patient was referred to our clinic, disappeared.

The method of determining sensitivity to pharmaceuticals, dental materials based on application of the gum sample of the test substance, the count of neutrophils in swabs from the oral cavity, taken before and after contact of the mucosa with substance, centrifugation them, determining the level of emigration of neutrophils into the oral cavity, the determination of the index shift of neutrophils, characterized in that the counting of neutrophils carried out by staining peroxidase-Chromogen solution, and the index shift of neutrophils is determined by their optical density according to the formula

and when the value of the shift index of neutrophils in the range from 0.75 to 1.5 body's sensitivity to the subject matter defined as normal.



 

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