Method for pathomorphological determination of prescription of myocardial infarction

FIELD: medicine.

SUBSTANCE: pathomorphological determination of the prescription of myocardial infarction is ensured by fixing a tissue sample and placing it into paraffin. Sections are prepared, de-waxed, heated, washed in a buffer solution, incubated in a moisture chamber and processed with a developing agent, dehydrated and enclosed by a medium. The reagent is presented by matrix metalloprotease 9 antibodies in dilution 1:100-1:250. The sections are incubated with the reagent at a temperature of 25°C and a relative humidity of 100% for 60 minutes. If the microscopy detects the bright-coloured neutrophils in peri-infarction vessels and within the infarction zone, the prescription is stated to be 2 hours to 1 day. If observing the neutrophil degranulation and the bright colour of an extracellular matrix within the infarction, the prescription is 1 to 2 days. If the coloured fibroblasts are found in the infarction border, the prescription is stated to be within 3 to 30 days.

EFFECT: method enables differentiating the prescription of myocardial infarction within 2 hours to 30 days.

1 ex, 3 dwg

 

The present invention relates to medicine, namely to pathological anatomy.

It is known that the exact determination of the period of limitation of myocardial infarction is of great importance for the pathologist.

The known method of macroscopic diagnosis of early stages of myocardial infarction based on the interaction of tellurite potassium dehydrogenase tissues. Outside of myocardial telluric potassium colors myocardium in grey or black color, and the area of necrosis remains unpainted, due to the destruction of these enzymes in the zone of myocardial infarction (Fingers M.A., Anichkov NM Pathological anatomy. 2 so, Vol.2, Part 1. - M.: Medicine, 2001. - S).

The disadvantage of this method is the lack of differential timing of the onset of myocardial infarction. There is a method allows to detect only necrotic stage of myocardial infarction (the time from 2 hours up to 3 days), because after the onset of the stage of organization of the activity of dehydrogenases in the tissue is gradually restored.

In addition, the use of this method makes it difficult to obtain high-quality samples for histological and immunohistological studies and reduces the quality of the documentation of the morphological changes in the myocardium due to the fact that the dyes steadfastly dyed fabric in grey color. When [daln] Isham mandatory study material this coloration prevents staining of standard dyes.

The closest in technical essence to the present invention is a method for the diagnosis of ischemic necrosis of the heart muscle (U.S. Pat. 2094806 Russian Federation, IPC G01N 33/68, G01N 33/50, a method for the diagnosis of necrosis in histological sections / Kozlov D.V.; applicant and patentee, Novokuznetsk state Institute of advanced training of physicians, No. 94005367/14; Appl. 14.02.1994; publ. 27.10.1997).

The known method is as follows. Fragments of the cardiac muscle is fixed with 15% aqueous neutral formalin solution for days at room temperature. Subsequently the material is subjected to paraffin filling, then prepare histological sections, which deparaffinized processing in three portions of o-xylene and subsequent heating in a thermostat at 37°C for 3 hours. Dehydration slices reach the 3 minute treatment in each of the four portions of ethanol (absolute, 96% 70% and 56%). Next, the slices for 5 minutes, transferred to 0.05 M buffered saline with a pH of 7.6. Blocking of endogenous peroxidase carry out a fresh solution of 3% hydrogen peroxide for 10 minutes, washed with buffered saline for 5 minutes. Further cuts stand under the drop of a 5%solution of horse serum albumin. After that, the excess albumin shake and gently wipe the glass around the slices. the and last put a drop afinno-purified rabbit primary antibodies against associated with pregnancy Alphas-glycoprotein at a concentration of 8 µg/ml Incubation of slices is carried out for 30 minutes at 37°C in a humid chamber, and then washed twice buffered saline solution. Incubated histological sections in the secondary biotinylated antibodies in a dilution of 10 µg in 1 ml of buffered saline solution for 30 minutes, then washed buffered saline for 5 minutes. Then treated slices complex avidin-peroxidase for 30 minutes and washed buffered saline solution. Immunological manifestation exercise 0.03% solution of the substrate on the basis of diaminobenzidine (DAB Chromogen) for 10 minutes the Reaction is stopped by rinsing the sections in two portions of distilled water. Cell nuclei Domracheva with hematoxylin for 3 minutes Then cuts to carry through the fresh spirits ascending concentrations (56% 70% 96%absolute ethanol) and three servings of o-xylene, and then sign in balsam.

In the study of prepared microslides mark a clear staining foci of ischemic necrosis of the heart muscle in the area of myocardial infarction. The focus of necrosis has a brown color, and other fabric - yellow-gray color.

The disadvantage of this method, as similar, is the lack of a precise definition of the period of limitation of myocardial infarction, because the prototype method call which allows to identify only necrotic stage.

The disadvantages of this method include that the half-life of complexes associated with pregnancy A2-glycoprotein with proteinase does not exceed 2 minutes. Therefore, in half an hour, virtually all complexes of enzymes associated with pregnancy A2-glycoprotein reach target organs, particularly the liver, where and are metabolized. Outside of the blood vessels this distribution has different characteristics, as high molecular weight complexes prevents them from entering the blood vessels (Zorin N.A. Associated with pregnancy-ALPHA-2-GLYCOPROTEIN. NEWS "Vector-best", N 9, September, 1998. CIT. The link http://www.vector-best.ru/nvb/st9_5.htm 29.04.12 year). Therefore, identifying complexes in the border zone of myocardial infarction is limited to a very short period; identification of the complexes is only possible in the zone of necrosis, i.e. outside the zones intact blood supply.

The task of the invention is to develop a method for determining the period of limitation of myocardial infarction with pathomorphological studies.

The technical result of the proposed method is the determination of the period of limitation of myocardial infarction from 2 hours to 30 days.

The technical result is achieved in that the method pathological determining the limitation of the occurrence of a heart attack is ICARDA includes the fixation of the tissue sample, place it on the wax, making slices, dewaxing and warming up, laundering in the buffer solution, incubation in a humid chamber with the reagent and processing developing agent, dehydration and conclude on Wednesday.

The main distinctive technique proposed method is that as the reagent is used antibodies to the matrix metalloprotease 9 in a dilution of 1:100-1:250.

Distinctive reception of the proposed method is that the incubation with the reagent is carried out at a temperature of +25°C and a relative humidity of 100%. Incubation duration is 60 minutes.

Differences between the proposed method lies in the fact that microscopic detection in drug brightly colored neutrophils in the blood vessels periinfarct zone and brightly colored neutrophils in the zone of infarction establish a period of limitation of infarct from 2 hours to 1 day, in identifying the degranulation of neutrophils and bright colors of the extracellular matrix in the zone of the infarct - from 1 to 2 days, and when the detection of stained cells fibroblast number in the border zone of the infarct - from 3 to 30 days.

Comparative analysis of the proposed technical solutions to the prototype allows to make a conclusion on the conformity of the proposed technical solution the criteria of the invention of "novelty."

The inventive method provides achievement is the perceived by the applicant of the technical result, namely the ability to determine when pathomorphological studies of the period of limitation of myocardial infarction from 2 hours to 30 days while reducing the complexity and duration of the study.

Thus, the authors of the proposed method is established that the used primary antibodies bind with substances synthesized by neutrophils, fibroblastoma, as well as substances in the extracellular matrix in a strictly defined time intervals after myocardial infarction. Therefore, the use of immunohistochemical staining of histological preparations, as the primary antibody to the matrix metalloprotease 9, allowed the authors of the proposed method to differentiate the expiration of the onset of myocardial infarction with pathomorphological study.

The advantage of the proposed method is that it allows you to identify not only the necrotic stage of myocardial infarction, but also the stage of the organization that determines its strength in comparison with the prototype.

The above can conclude that the technical solutions according to the criterion of "inventive step".

The method constituting the invention, intended for use in medicine. The possibility of its fulfillment is confirmed as described in the application techniques and the medium is you. This gives grounds to believe that the proposed solution meets the criteria of the invention "industrial applicability".

The essence of the proposed method is illustrated by a specific example. The following example serves to illustrate, but not limit the invention.

Example

The object of the study was postmortem material 30 patients who died from acute myocardial infarction. In all cases diagnosed with primary acute transmural myocardial infarction. The average age was 63.7 years. Male 18, female 12. The timing of the onset of heart attack patients were distributed as follows: 16 patients died in the period up to 3 days; 14 patients - in time from 3 days to 1 month.

Tissue samples from the zone of myocardial infarction and edge to her area recorded a 10% solution of neutral formalin and embedded in paraffin blocks according to the standard technique (Merkulov, G. A. Course histopathological techniques. - L.: Medicine, 1969. - S-15, 52-59).

Then cooked slices with a thickness of 5 μm, which were mounted on glass coated with poly-L-lysine. Was deparaffinization drugs beginning in toluene - 5 minutes, and then in alcohols 100°, 96°, 90°, 70° and 50° for 1 minute. Then the preparations were kept in distilled water for 10 minutes.

For enabling IRQ-unmasking antigen preparations were placed in CIT is atny buffer (rn) and heated for 8 minutes in the microwave power 800W at 100% power, and then 10 minutes at 60% power. Then samples were cooled to 25°C in citrate buffer (rn) and washed with distilled water 2 times for 5 minutes. The tissue sample was fortified with a hydrophobic pencil Dako Pen (Dako, Code S2002).

For inactivation of endogenous peroxidase for each drug was applied in 50 μl of 3%hydrogen peroxide and kept for 5 minutes. Then laundered Tris-buffer (rn) twice for 5 minutes. Then put 50 ál of 0.4% solution of casein, and incubated for 5 minutes, washed Tris-buffer (rn) 2 times, 5 minutes each.

Next, for each drug was applied primary antibodies - antibodies to MMR rabbit monoclonal IgG antibody (Epitomics, Clone ID: EP1254, Cat. N2551-1, Lot YG 11300 IP) working dilution of 1:100-1:250 and incubated in a moist chamber at 25°C and 100% humidity for 1 hour. Then laundered Tris-buffer (rn) 2 times, 5 minutes each. Then the preparations were applied in 50 μl of a solution containing 10% serum in Tris-buffer (rn), incubated in a humid chamber at a temperature of +25°C for 30 minutes. Washed Tris-buffer (rn) 2 times, 5 minutes each. Then put the secondary antibodies Novolink Polymer (Novocastra, REF=7112, Lot 711236)labeled with peroxidase, 50 ál, incubated in a humid chamber at a temperature of +25°To 30 minutes. Washed Tris-buffer (rn) 2 times, 5 minutes each. Added 50 μl of the substrate mixture containing 50 μl of 1.74% 3'3'-diaminobenzidine in 1 ml of 0.05% hydrogen peroxide Novocastra, REF=RE7105, Lot 710550), kept for 5 minutes. Washed with water. The slices they finished painting areas 0.02% solution of hematoxylin for 30 seconds. Washed with water. Dehydrational consistently alcohols 70°, 90°, 96°, 100° for 1 minute. Then in toluene for 5 minutes. Placed in the enclosing environment Permanent Slide Mounting Medium (Novocastra, REF=7137, Lot 713708) under a glass cover. Microscopic examination was performed using a light microscope Nikon 80L

Established that the deaths that have developed within a few hours after myocardial infarction, recorded brightly coloured neutrophils in the blood vessels periinfarct zone (Figure 1, item A), as well as brightly coloured neutrophils in the area of infarction (Figure 1, item B).

In the period of limitation of infarct from 1 to 2 days recorded the degranulation of neutrophils, loss of staining of the cytoplasm of neutrophils. In parallel with this was fixed coloration of the extracellular matrix in the area of the heart that reflects the allocation of MMP-9 from neutrophils in the tissue (Figure 2, item C). In the case of deaths at a later date (3-30 days) was noted colouring cells fibroblast number in the border zone. The maximum intensity of staining noted at 7-14 day (Figure 3, item D).

The authors note a good reproducibility of the proposed method is staining of serial sections in different parties reached an identical re ulitity staining.

Thus, the proposed method allow to clearly differentiate the limitation period of the onset of myocardial infarction with pathomorphological study. This method can be used in medicine, namely in pathological anatomy.

How pathological determining the limitation of the onset of myocardial infarction, including the fixation of the tissue sample and placing it in paraffin, making slices, dewaxing and warming up, laundering in the buffer solution, incubation in a humid chamber with the reagent and processing developing agent, dehydration and concluding on Wednesday, characterized in that the reagent is used antibodies to the matrix metalloprotease 9 in a dilution of 1:100-1:250, duration of incubation with the reagent at a temperature of 25°C and 100% relative humidity is 60 minutes, and microscopic identification of the preparation of brightly coloured neutrophils in the blood vessels periinfarct zone and brightly colored neutrophils in the zone of infarction establish a period of limitation of infarct from 2 hours to 1 day, in identifying the degranulation of neutrophils and bright colors of the extracellular matrix in the infarction zone from 1 to 2 days, and when the detection of stained cells fibroblast number in the border zone of the infarct - from 3 to 30 days.



 

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1 ex

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2 ex, 3 tbl

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