RU2524627C1 - Diagnostic technique for senile amyloidosis in experimental laboratory animals - Google Patents

Abstract

FIELD: medicine.

SUBSTANCE: diagnostic technique for AL-amyloidosis consists in the fact that a biological fluid drop with a crystal inductor is subject to wedge dehydration and then a prepared fascia is analysed under the microscope with using qualitative criteria.

EFFECT: technique is accessible, easy to implement, enables detecting systemic amyloidosis over a short period of time.

Description

The invention relates to medicine, namely to the laboratory diagnosis of amyloidosis.

Currently, there are a number of methods for diagnosing amylodosis. This is a histological examination of a biopsy of the affected organ in combination with histochemical stains and microcopy under a polarizing microscope [1]. This method is accurate, but has several disadvantages. Firstly, it is the difficulty of obtaining a sample of the studied tissue. Secondly, these are complications inherent in the biopsy method. The third, and key flaw, is the study time, which takes several days.

Another way to diagnose amyloidosis is a functional test with Congo red or methylene blue. The essence of the test is that the dye introduced intravenously should be fixed with a pathological protein (amyloid) and not excreted in the urine [1, 2]. But dyes have a tropism for many proteins, and this may be the reason for the frequent false results. It is also possible to develop toxic and allergic reactions in a patient, since these dyes are not completely inert to living tissues. Of great negative importance is the psychological factor, since this procedure is not always perceived positively by the patient.

Another diagnostic method is the determination of the amino acid sequence of an amyloid protein by enzyme immunoassay [3]. The disadvantage of this method is the variability of the composition of amino acids in various subspecies of amyloid proteins, and at the same time, varieties of pathological proteins of different chemical composition constantly appear, which interferes with the diagnostic search. Significant disadvantages include the need to collect a large number of serums to fill the strips during the analysis, which makes it economically impractical to conduct single analyzes. Relative disadvantages include the high cost of antibody kits, flushing fluids, and the large time required for reactions.

As a result of the patent information search, the closest analogue was not identified.

The objective of the invention is to provide a method for the diagnosis of amyloidosis, simple to implement, not requiring large material costs and which is carried out in a short time.

The essence of the invention lies in the collection of blood serum in animals, mixing it in a volume of 100 μl with 50 μl of an isotonic NaCl solution and applying to a previously defatted glass slide in a volume of 20 μl, then placing the glass slide in a Petri dish for 1 hour at a temperature of 22 ° C and humidity 55-75%, after which facies are analyzed under a microscope at a magnification of 100 times using quantitative parameters as criteria, such as the number of crystallization centers in the quadrant, the number of secondary rays and the value of the peripheral protein roller.

The method was developed on 30 outbred white rats - males. These rats were divided into 2 groups: experimental with primary systemic amyloidosis AL and intact. The conditions of detention, the diet is identical. To obtain a model of systemic amyloidosis, we used a model with the introduction of Freund's incomplete adjuvant. The adjuvant in combination with native egg albumin was administered intraperitoneally for 7 days every other day, 1 ml per 300 g of body weight. After that, on day 14, all rats were withdrawn from the experiment with the collection of blood serum and organs (liver, kidneys, spleen, lungs, heart) )

For the wedge-shaped dehydration method, 100 μl of blood serum and 50 μl of a 0.9% NaCl solution were taken, then the two solutions were mixed, and the resulting substrate in a volume of 20 μl was applied to a previously defatted glass slide. Glass was placed in a Petri dish for one hour at a temperature of 22 ° C and an air humidity of 55-75%. After that, the facies obtained by wedge-shaped dehydration was analyzed under a microscope with a 100-fold increase.

The crystallization temperature was chosen due to two phenomena: first, it was at this temperature that we obtained the most accurate and detailed images of crystallization; a temperature change of 4 degrees Celsius above or below 22 degrees showed much worse results; secondly, this temperature is equal to room temperature, and for its reconstruction does not require any special devices.

A 0.9% NaCl solution was used as a crystallizer, since it is isotonic to biological media of the body and does not cause structural and quantitative changes in them, while other crystallizers, which are often used in other works (CuSO ₄ , glycine), induce changes in crystallogenic fluid properties and distort the true crystallogram. In addition, the advantage of isotonic NaCl solution is its low cost and availability.

Based on a series of experiments, we established the optimal crystallization time, which is equal to one hour. If an object crystallizes less than a given time, then crystallogenesis does not have time to complete and there are crystalline zones on the studied facies. An increase in time over one hour does not change the crystallogram, since it is precisely this time that is sufficient to completely stop crystallogenesis.

Of primary importance are qualitative indicators, such as the shape of the crystals, their branching and spatial relationship. In the experimental group, we obtained large cross-shaped crystals that decreased in size and thickened closer to the center of the dehydrated drop, while the number of branches was minimal. In the intact group, the crystals have characteristic "gentle lines" that differ significantly from the resulting crystals in the first group. A number of quantitative parameters have also been developed that are used as diagnostic criteria:

1. The number of crystallization centers in the square of the study. The dimensions of the latter were obtained experimentally - we obtained the area of the entire drop, and then divided it into 16 quadrants with an area equal to 500 conventional units. In the experimental group, the number of crystallization centers is 2 ± 1 (p = 0.05). In the intact group, this indicator is 5 ± 1 (p = 0.05).

2. The number of secondary rays. In the group of animals with AL-induced amyloidosis, this parameter is equal to 7 ± 2 (p = 0.05). In potentially healthy laboratory animals from the intact group, the number of secondary rays is 13 ± 2 (p = 0.05).

3. The striation of the peripheral protein roller. In the group of experimental animals, this parameter is 9 ± 2 (p = 0.05). It should be noted that these cracks have the wrong shape and direction. In the group of intact animals, the number of lines on the protein roller is 12 ± 2 (p = 0.05), and these lines have a strictly ordered parallel course, which also serves as a diagnostic criterion in contrast to facies with AL amyloidosis.

All the obtained and interpreted results of the analysis of the obtained facies were compared with a histological examination of autopsy organs (liver, kidneys, spleen, lungs, heart) to confirm the correlation of parameters and the presence of pathology. Based on this study, we obtained 100% confirmation of amyloidosis using the developed method.

The technical result of using the method:

1. The minimum amount of time.

2. Reliability, nativeness and physiology of the study.

3. The absence of allergic and toxic effects on the part of the patient.

4. Reducing the cost of diagnosing AL-amyloidosis.

5. The availability of equipment for research.

Literature

1. Warsaw, V.A., Proskurneva, E.P. Importance and methods of morphological diagnosis of amyloidosis in modern medicine. Practical Nephrology. - 1998. - No. 2, - p.16-23.

2. Houdner, P., Rausing, A., Steen, K., Torp A. Diagnosis of cardiac amyloidosis by Myocardial Biopsy. // Acta med. Scand - 1975 - No. 6, p. 525-528.

3. Skinner M. Amyloidosis Current Therapy in Allergy, Immunology, and Rheumatology. // Mosby-Year Book. - 1996, p.235-240.

Claims (1)

A method for the diagnosis of systemic amyloidosis in experimental laboratory rats, which consists in collecting blood serum, mixing it in a volume of 100 μl with 50 μl of an isotonic NaCl solution and applying to a previously defatted glass slide in a volume of 20 μl, then placing the glass slide in a Petri dish for 1 hour at at a temperature of 22 ° C and an air humidity of 55-75%, after which the analysis of the facies under a microscope is carried out at a magnification of 100 times using quantitative parameters as criteria, such as the number of crista centers lysis in the quadrant, which is the area of the entire drop, divided into 16 quadrants, the number of secondary rays and the striation of the peripheral protein cushion, and the diagnosis of systemic amyloidosis is considered established if the number of crystallization centers in the study quadrant is 2 ± 1 (p = 0, 05), the number of secondary rays is 7 ± 2 (p = 0.05) and the striation of the peripheral protein roller is 9 ± 2 (p = 0.05), while the cracks have the wrong shape and direction.