Diagnostic technique for membrane toxicity

FIELD: medicine.

SUBSTANCE: method involves taking blood at 1 ml per a sample into the Vakutainer evacuated test tube with sprayed ethylene diaminetetraacetic acid 2.2 mg; a reference sample 1.0 ml and a sample 1.0 ml with weighted xenobiotics are taken from the total volume into the Eppendorf tubes; 30-minute incubation is followed by using a uniform procedure to prepare blood smears by applying on a dry slide to prepare a smear to be stained according to Leishman; blood corpuscles are analysed by the Zeiss light microscope at a magnification 1500. A microscope field showing echinocytes with styloid processes of the membrane not found in the reference sample is a sign of the xenobiotic membrane toxicity.

EFFECT: invention enables an instant assessment of the membrane toxicity of the substance.

 

The invention relates to medicine, namely membranology, and can be used to test embryotoxicity xenobiotics used in therapeutic and orthopedic dentistry as restorative and reconstructive materials, as well as in toxicology and pharmacology along with traditional methods of evaluating the toxicity of substances, medicines.

There is a method of determining the potential cytotoxicity of biomaterials on cell cultures of mouse fibroblasts (L-929), based on the determination of qualitative and quantitative changes in cell cultures after incubation with a filling material.

The disadvantage of this method is that it is time-consuming, costly and not sufficiently specific.

Known "Method of determining embryotoxic properties of xenobiotics" (Patent RF №2073244, date of publication - 10.02.1997) using the reaction spontaneous rosethorne lymphocytes before and after administration of the test substances in experimental animals, which is close to the estimated effect and the achieved result.

The disadvantage of this taken as a prototype of this method is the length, complexity, the multiple steps involved, the need for the use of experimental animals with subsequent treatment of the tissue, receiving only delayed is the result.

The aim of the invention is to develop a method of assessing embryotoxicity xenobiotics, which allows a rapid assessment of the substance.

The technical result is achieved by the fact that in a vacuum tube Vakutainer with powder 2.2 mg ethylenediaminetetraacetic acid blood is taken at the rate of 1 ml per sample, then the total volume taken by 1.0 ml tube (Eppendorf, control and contains a sample of xenobiotics, after 30-minute incubation on a standardized method to prepare blood smears on dry glass slide put a drop of blood, get a PAP smear and paint it according to the method of Leishman and conduct the study of the formed elements of the blood with the help of light microscope "Zeiss" with increasing 1500. Indicator of embryotoxicity of the xenobiotic is the emergence of the field of view of echinocytes - erythrocytes with subulate appendages membrane absent in the control.

The technical result, which directed the establishment of this invention is the development of rapid, specific, cost-efficient method of determining embryotoxicity xenobiotics.

The method is as follows: in the vacuum tube Vakutainer with powder 2.2 mg ethylenediaminetetraacetic acid took 4 ml of blood was then collected in 1.0 ml of 4 Eppendorf tubes: control (1)and containing 25 mg of hanging the cured dental composites and adhesive Filtek Ultimate (2), Filtek Bulk Fill (3) and Single Bond Universal (4). After 30-minute incubation on a standardized methodology was preparing blood smears on dry glass slide was put a drop of the studied blood and distributed it using pure grinding glass, holding at an angle of 45°. Blood smears were located at a distance of 1.0-1.5 cm from the edges of the glass, occupying almost the whole of its length, and ended the "whisk". Received drugs (1-4) was dried in the air, marked, stained by the method of Leishman, washed with distilled water, dried in the air. Criteria of a good color was pink erythrocytes, violet staining granularity of neutrophils on a pink background, gentle Ashrafinia grain monocytes. Morphological examination of blood cells was performed using a light microscope "Zeiss" with increasing 1500. Indicator of embryotoxicity of the xenobiotic is the emergence of the field of view of echinocytes - erythrocytes with subulate appendages membrane absent in the control. In the sample 2 with Filtek Ultimate, their number amounted to 1%, in the sample 3 with Filtek Bulk Fill is 3%, and the sample 4 with a Single Bond Universal number of echinocytes was 12%. Thus, due to the research of the adhesive has a sufficiently strong embryotoxicity properties that require hermetic isolation from contact with the tissues Polo the tees mouth.

The method can be used for rapid assessment of embryotoxicity various xenobiotics.

Literature

1. GOST RISO 10993-5-2009.

2. "Method for determining embryotoxic properties of xenobiotics" (Patent RF №2073244, date of publication - 10.02.1997).

The way the rapid assessment of embryotoxicity of xenobiotics, including the study of membrane structures of reactive cells under the influence of the analyte, characterized in that the vacuum tube Vakutainer with powder 2.2 mg ethylenediaminetetraacetic acid blood is taken at the rate of 1 ml per sample, then the total volume taken by 1.0 ml tube (Eppendorf, control and contains a sample of xenobiotics, after 30-minute incubation on a standardized method to prepare blood smears on dry glass slide put a drop of blood, get a PAP smear, paint his method Leishman and conduct the study of the formed elements of the blood with the help of light microscope "Zeiss" with increasing 1500, the emergence of the field of view of echinocytes - erythrocytes with subulate appendages membrane absent in the control, is indicative of embryotoxicity of the xenobiotic.



 

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