| Start of section
Production, amateur Radio amateurs Aircraft model, rocket-model Useful, entertaining |
Stealth Master
Electronics Physics Technologies Inventions |
Secrets of the cosmos
Secrets of the Earth Secrets of the Ocean Tricks Map of section |
  |
| Use of the site materials is allowed subject to the link (for websites - hyperlinks) | |||
Navigation: => |
Home / Patent catalog / Catalog section / Back / |
|
INVENTION
Patent of the Russian Federation RU2202796
METHOD FOR DETERMINING THE ABILITY OF MAN HUMAN ERYTHROCYTES
SORBY PREDNISOLONE
The name of the inventor: Kirdey EG; Dmitrieva LA; Belokhvostikova Т.S.
The name of the patent holder: State institution Institute of Traumatology and Orthopedics NTSRVH VSNTS SB RAMS
Address for correspondence: 664003, Irkutsk, ul. Wrestlers of the Revolution, 1, Institute of Traumatology and Orthopedics, National Research Center of the Siberian Branch of the Russian Academy of Medical Sciences, Patent Group, RN Kharlamova
Date of commencement of the patent: 2000.01.10
The invention relates to the field of medicine, in particular to immunology. The method provides for increasing the effectiveness of treatment by determining the individual sorption activity of the patient's erythrocytes to prednisolone before the use of immunopharmacotherapy in the complex of therapeutic measures. The reaction of cellular immunosorption is performed and the supernatant is recovered, and the isolated erythromass is incubated with a solution of prednisolone at a concentration of 1 μg / ml in equal volumes for 60 minutes, centrifuged and the supernatant is obtained, then 0.05 ml of 199, The second - 0.05 ml of the prednisolone solution at the initial concentration and the third - 0.05 ml of the supernatant obtained after the incubation of prednisolone with erythromass, then 0.05 ml of the nitrous tetrazolium solution and 0.1 ml of the whole patient's blood are added to all the samples , After which they incubate, centrifuge, remove the supernatant, prepare the smears from the cell pellet, fix it with methanol and stain with neutral red, count the percentage of neutrophils in the cytoplasm of which contain the granules of diplazan, then evaluate the biological activity of prednisolone in the first test sample by the percentage reduction of the NTS- Activity of neutrophils in comparison with the control sample without prednisolone and determine the sorption activity of erythrocytes from the difference in the indices in the sample with the supernatant and in the sample with the solution of prednisolone.
DESCRIPTION OF THE INVENTION
The present invention relates to the field of medicine, in particular to immunology, and can be used in immunopharmacotherapy using auto-erythrocytes, for example, loaded with prednisolone in a complex of therapeutic measures for various chronic inflammatory diseases accompanied by signs of hyperactivation of the immune system.
The method closest to the proposed method is the determination of the sorption activity of the receptor apparatus of human erythrocytes. The essence of the known method consists in the following: the patient's blood is taken in an amount of 4-5 ml in a tube containing 0.1 ml of 2.5 thousand units of heparin, the erythrocytes are washed three times by centrifugation for 5-7 minutes at 1500 rpm in phosphate-buffered saline . The washed red blood cells are diluted with phosphate buffered saline so that a 3-5% suspension is obtained and 1.5 ml of the resulting suspension is incubated at room temperature for 40-45 minutes with 1.5 ml of diluted murine serum. At the end of the incubation, the mixture is centrifuged and the optical density of the supernatant is measured against the control sample on a spectrophotometer. The control sample is 1.5 ml of the mouse serum of the dilution taken, to which 1.5 ml of phosphate buffered saline is added and treated and, as an experimental test. The obtained value is compared with the average donor control and the difference reflects the sorption activity of the receptor apparatus of human erythrocytes (1).
However, the known method has certain drawbacks, namely:
- The known method of determining the sorption activity makes it possible to judge the functional state of the human erythrocyte receptor apparatus and the prognosis for recovery, while the proposed method can be used to evaluate the sorption activity of red blood cells with respect to a particular preparation and the possibility of carrying out immuno-pharmacotherapy with auto-erythrocytes , Loaded with prednisolone for various diseases, accompanied by hyperactivation of the immune system.
Proceeding from the known level of technologies for determining the sorption activity of erythrocytes, the task was set: to increase the effectiveness of treatment by determining the individual sorption capacity of the patient's erythrocytes to prednisolone before the use of immunopharmacotherapy in the complex of therapeutic measures.
The problem is solved as follows.
Determination of the ability of human erythrocytes to sorb prednisolone is performed by setting up a cellular immunosorption reaction and obtaining a supernatant. New in the proposed method is that the isolated erythromass is incubated with a solution of prednisolone at a concentration of 1 μg / ml in equal volumes for 60 minutes, centrifuged and a supernatant is obtained. Then 0,05 ml of medium 199 are introduced into the first test, 0.05 ml of prednisolone solution at the initial concentration and into the third sample of 0.05 ml of supernatant obtained after incubation of prednisolone with erythromass. Then, 0.05 ml of a solution of nitrogens tetrazolium and 0.1 ml of whole blood of the patient are added to all samples. After that, incubate, centrifuge and remove the supernatant. From the cell sediment, smears are prepared, fixed with methanol and stained with neutral red, the percentage of neutrophils counted, in the cytoplasm of which the granules of diformazan are contained. Then, the biological activity of prednisolone in the first test sample is estimated by the percentage reduction of the neutrophil NST activity in comparison with the control sample without prednisolone and the sorption activity of erythrocytes is determined by the difference in the indices in the sample with the prednisolone solution in the initial concentration and in the sample with the supernatant and when the difference between the These indicators more than 5% consider it expedient to perform extracorporeal immunopharmacotherapy using autoerithrocytes treated with prednisolone solution.
We explain the essentiality of the distinctive features of the proposed method of immunopharmacotherapy.
When erythromass is incubated with a solution of prednisolone, erythrocytes have the ability to nonspecifically sorb and concentrate on its surface prednisolone and, when administered, have a regulatory effect on the lymphoproliferation processes that underlie the immune response.
Obtained after incubation and centrifugation of erythromass with prednisolone solution in the initial concentration, the supernatant is used to establish the HCT test to evaluate the sorption capacity of erythrocytes, determined by the degree of decrease in the prednisolone biological activity after its incubation with erythrocytes.
The first sample, with the addition of 0.05 ml of medium 199, was used as a control for subsequent comparison with the test samples.
A second sample, with 0.05 ml of prednisolone at a concentration of 1 μg / ml, was used to evaluate the prednisolone biological activity compared to the control sample.
A third sample with the addition of 0.05 ml of supernatant obtained after the incubation of prednisolone in the initial concentration with erythromassa is used to evaluate the sorption capacity of erythrocytes in terms of the degree of decrease in biological activity of the preparation in comparison with the second sample.
The addition of 0.05 ml of the soluble nitrosine tetrazolium dye and 0.1 ml of the whole patient's blood to all samples followed by incubation in a thermostat at 37 ° C and centrifugation is necessary for the preparation of smears and the determination of the percentage of neutrophils in the cytoplasm of which the insoluble diamorphine granules are contained.
The biological activity of prednisolone is assessed by the percentage of decrease in the neutrophil NST activity in the sample with the addition of the prednisolone solution at the initial concentration as compared to the control sample without prednisolone.
The presence and magnitude of the erythrocyte sorption activity is calculated from the difference in the sample values with the supernatant and the sample with the prednisolone solution at the initial concentration.
The 5 percent difference eliminates the error in the counting of HCT-positive cells and suggests obtaining reliable results when deciding whether it is expedient to conduct extracorporeal immunopharmacotherapy in a particular patient using auto-erythrocytes treated with prednisolone in a set of therapeutic measures.
The conducted patent studies on the subclass G 01 N 33/53, A 61 K 37/02 and the analysis of scientific and medical information reflecting the existing level of methods for determining the sorption capacity of peripheral blood erythrocytes, did not reveal technologies identical to the one proposed. Thus, the proposed method of determining human erythrocytes to sorb prednisolone is novel.
The interrelation and interaction of the essential techniques of the proposed method for determining the ability of human erythrocytes to sorb prednisolone provides a new medical result in the solution of the task, namely: to increase the effectiveness of treatment by determining the individual sorption capacity of the patient's erythrocytes to prednisolone.
The proposed method can be widely applied in practical public health, since it can be reproduced repeatedly and does not require the use of expensive equipment and reagents.
The essence of the proposed method for determining the ability of human erythrocytes to sorb prednisolone is as follows.
The patient's blood is withdrawn from the ulnar vein in sterile conditions on heparin at a rate of 25 units per 1 ml of blood, part of which is used to obtain erythromass by triple washing in an isotonic sodium chloride solution. Equal volumes of washed erythromass and prednisolone solution at a concentration of 1 μg / ml are incubated in a thermostat at 37 ° C for 1 hour. After incubation, the slurry is centrifuged at 1000 rpm for 10 minutes, the supernatant is removed, which is used in further studies. The NST-test is performed in plates for immunological studies. In the first sample, 0.05 ml of medium 199 are introduced, the second - 0.05 ml of prednisolone solution at a concentration of 1 μg / ml, in the third - 0.05 ml of the supernatant recovered after erythrosorption. A solution of nitrous tetrazolium in a volume of 0.05 ml and 0.01 ml of whole blood of the patient is introduced into all samples. The samples are incubated in a thermostat at 37 ° C. and for 30 minutes, centrifuged for 10 minutes at 1000 rpm. Cells are prepared from the cell pellet, fixed with methanol, stained with neutral red, and the percentage of neutrophils containing the granules of diphenazan (HCT-positive cells) in the cytoplasm is determined.
The biological activity of prednisolone in the sample with its initial concentration and in the sample with the supernatant obtained after the incubation of the prednisalone solution with erythromassa is calculated as a percentage of the decrease in the neutrophil HCT activity in comparison with the control sample without prednisolone. The sorption activity of human erythrocytes to prednisolone is calculated from the difference in the indices in the sample with the supernatant and in the sample with the prednisolone solution and, if the difference is found, more than 5 percent consider it appropriate to perform extracorporeal immunopharmacotherapy using autoerithrocytes treated with prednisolone solution.
The essence of the proposed method is explained by clinical examples.
Example 1. Patient U., 52 years old. Diagnosis: Diabetic fracture of the right tibia, chronic osteomyelitis, IDS of hypo-suppressor type with signs of hyperactivation of phagocytes.
The sorption activity of erythrocytes (SAE) was evaluated by the ability of prednisolone at a concentration of 1 μg / ml to suppress neutrophil activity before and after its incubation with erythrocytes, incubation was carried out in a thermostat at 37 ° C for 1 hour after mixing equal volumes of erythromass with prednisolone solution. EPS was calculated as a percentage of the decrease in the biological activity of prednisolone after incubation with erythrocytes.
In the control sample without prednisolone, the percentage of NSA activity was 62%. In the first test sample (with the addition of a solution of prednisolone in the initial concentration), the neutrophil NST activity was 46%. In the second test run (with the addition of supernatant after the incubation of erythrocytes with prednisolone), the neutrophil NST activity is 57%. The SAE index was 11% (57% - 46% = 11%).
When determining the ability of the patient's erythrocytes to sorb prednisolone, the index of HCT activity of nitrophils in the sample with the supernatant obtained after incubation of the patient's erythromass with the prednisolone solution in the initial concentration exceeded the indicator of HCT activity in the sample with the addition of prednisolone. Thus, this patient is advisable to perform extracorporeal immunopharmacotherapy with the use of autoerithrocytes, treated with a solution of prednisolone in a complex of therapeutic measures in order to reduce the hyperactivation of the immune system.
Example 2. Patient D., 19 years old. Diagnosis: primary-open infection with a double fracture of the middle third of the right tibia with displacement, necrosis of soft tissues in the middle third of the right tibia, infected wound of the right foot, VAT of a hypersupressive type of pronounced character with signs of hyperactivation of phagocytic leukocytes.
In the control sample (without prednisolone), the percentage of neutrophil NST activity was 77%. In the first test sample (with the addition of a solution of prednisolone at the initial concentration), the NST activity was 58%. In the 2nd test run (with the addition of a supernatant after the incubation of erythromass with a prednisolone solution), the neutrophil HCT activity is 54%. The indicator of EPS was 5% (58 - 54% = 4%).
Therefore, when determining the ability of erythrocytes of a given patient to sorb prednisolone, the neutrophil count of neutrophil activity in the sample with supernatant obtained after incubation of erythromass with prednisolone solution exceeds the index of HST activity in the sample with a prednisolone solution in the initial concentration by no more than 5%. Carrying out extracorporeal immunopharmacotherapy with the use of autoerithrocytes treated with prednisolone is inadvisable, in view of the low sorption activity of erythrocytes.
Thus, the proposed "Method for determining the ability of human erythrocytes to sorb prednisolone" allows, in comparison with known technologies, to increase the effectiveness of treatment by determining the individual sorption capacity of the patient's erythrocytes to prednisolone before the use of immunopharmacotherapy in the complex of therapeutic measures.
THE SOURCE OF INFORMATION
1. A.S. USSR N1778707, BI 44, G 01 N 33/531.
CLAIM
A method for determining the ability of human erythrocytes to adsorb prednisolone by setting up a cellular immunosorption reaction and obtaining a supernatant, characterized in that the isolated erythromass is incubated with a solution of prednisolone at a concentration of 1 μg / ml in equal volumes for 60 minutes, centrifuged and the supernatant obtained, Sample 0.05 ml of medium 199, the second - 0.05 ml of prednisolone solution at the initial concentration and in the third 0.05 ml of the supernatant obtained after incubation of prednisolone with erythromassa, then 0.05 ml of a solution of nitrogens tetrazolium And 0,1 ml of whole blood of the patient, after which they are incubated, centrifuged, the supernatant is removed, smears are made from the cell pellet, fixed with methanol and stained with neutral red, the percentage of neutrophils counted in the cytoplasm of which contains the granules of diplazane, then the biological activity of prednisolone in The first test sample for the percentage of decrease in the neutrophil NST activity in comparison with the control sample without prednisolone and determine the sorption activity of erythrocytes according to the difference in the indices in the sample with the supernatant and in the sample with the prednisolone solution.
print version
Date of publication 02.04.2007gg
Comments
When commenting on, remember that the content and tone of your message can hurt the feelings of real people, show respect and tolerance to your interlocutors even if you do not share their opinion, your behavior in the conditions of freedom of expression and anonymity provided by the Internet, changes Not only virtual, but also the real world. All comments are hidden from the index, spam is controlled.