RU2241994C1 - Method for preparing bone samples to morphological diagnostic analysis - Google Patents

Abstract

FIELD: medicine.

SUBSTANCE: method involves decalcinating bone samples during 7-8 days in 5% nitric acid solution on the basis of 5% formalin solution, after having fixed them in formalin. Then, the bone samples are washed with flowing water during 2 h and immersed into 5% lithium sulfate solution for 12 h and next to it they are washed with flowing water once more during 20-30 min and dehydrated in alcohols of growing concentrations.

EFFECT: accuracy treatment process; produced high quality histological slices with all bone tissue structure being retained.

Description

The invention relates to medicine, namely to morphology, can be used for morphological diagnosis of bone tissue pathology.

Two main groups of decalcification methods are known - acid-free and acidic [4, 5, 7, 8, 9]. The acid method of decalcification [2, 3] consists in preparing the material for decalcification and decalcification. Preparation for decalcification consists of 1) fixation of bone samples in a 15-20% formalin solution for no more than 48 hours; 2) in washing bone samples with running tap water for 24 hours; 3) in their dehydration in alcohol solutions of 50%, 70%, 96%, 70%, 50%, during the day in an alcohol solution of each concentration.

Decalcification:

1) following the rules of decalcification, bone samples are placed in a 5-7.5% solution of nitric acid based on distilled water for 10-12 days; 2) then the bone samples are placed in a 5% solution of sodium sulfate or lithium for 24 hours to eliminate swelling; 3) bone samples are washed in running tap water for 1-2 days; 4) pouring into celloidin or paraffin or cutting on a freezing microtome.

The disadvantages of this method of decalcification are: the duration of decalcification (10-12 days). the duration of the washing of bone samples in running water (2-3 days), which contributes to their saturation with liquid, reduces the quality of the color of the sections and the clarity of the manifestation of bone tissue structures.

The objective of the proposed invention is to reduce the time of decalcification, increasing the plasticity of bone samples and maintaining the structure of bone tissue.

The task is carried out due to the fact that the decalcification of bone samples is carried out for 7-8 days in a 5% solution of nitric acid based on a 5% solution of formalin, after which they are washed for 2 hours with running tap water and for 12 hours immersed in a 5% solution of lithium sulfuric acid, then again washed with running water for 20-30 minutes and subjected to dehydration in alcohols of increasing concentrations.

The method is as follows.

Bone samples measuring 1.0 × 1.0 cm (cadaveric material) are fixed in a 7-8% formalin solution for 2 days. Then they are washed with running tap water for 20-30 minutes and immersed in a 5% solution of nitric acid based on a 5% formalin solution [1, 6].

The formalin-based acid solution is changed daily. Decalcification lasts 7-8 days, to determine its completion using the puncturing of the samples with a dissecting needle, a test incision and their compression. Then the bone samples are washed with running tap water for 2 hours and immersed for 12 hours in a 5% solution of lithium sulfate to eliminate their swelling.

Then, after washing with running water for 20-30 minutes, the bone samples are subjected to dehydration in alcohols of increasing concentrations (50%, 70%, 90%, 96%, absolute alcohol, alcohol - ether mixture). In each solution, the samples are incubated for a day. Then follows their pouring into celloidin.

A complete decalcification is achieved by a nitric acid solution prepared on the basis of a 5% formalin solution, which has the effect of demineralization. An increase in the plasticity of bone samples is facilitated by a reduction in their residence time in lithium sulfuric acid solution, their dehydration only before pouring, and pouring into celloidin, which in its properties (plasticity) is close to decalcified bone samples. To evaluate the completeness of decalcification and plasticity of bone samples is possible only when preparing sections. There is no crunch and complete, even sections are obtained. The preservation of the structure of bone tissue can be assessed only after staining of the sections (according to Schmorl) and histomorphometry. On the studied drugs, bone layers, osteon structures, and cells are determined.

The proposed method allows to reduce the duration of decalcification by 5-7 days, to achieve complete decalcification, to increase the plasticity of bone samples, to preserve the structure of bone tissue.

List of references

1. Valenson B.A. The method of accelerated decalcification in acid-formalin salt solution. Archive patol. 1950, vol. XII, N.5, p. 93. 2. Volkova OV, Yeletsky Yu.K. Basics of histology with a histological technique. - M.: Medicine, 1971, p.271. 3. Eliseev V.G. Fundamentals of histological technique. - M.: Medicine, 1967, p. 209. 4. Klevezal G.A. The recording structures of mammals in zoological studies. - M .: Nauka, 1988, p. 285. 5. Lilly R. Pathological technology and practical histochemistry. - M.: Mir, 1969, p.645. 6. Merkulov G.A. The course of histological technology. - L .: Medicine, 1969, p. 424. 7. Minkov S.A. To the question of decalcification of teeth and jaw fragments. Bull. East Siberian. scientific Center SB RAMS. - Irkutsk, 1993, issue 3-4, p.150-154. 8. Nikonorov S.G., Kozlova T.A. Decalcification of bone tissue in increasing concentrations of Trilon B. salt. Archive anat., Histol. and embryol. 1986, vol. XCL, N.1, pp. 78-80. 9. Sarkisov D.S., Perov Yu.L. Microscopic technique. A guide for doctors and laboratory assistants. - M.: Medicine.

Claims (1)

A method of preparing bone samples for morphological diagnosis, including fixing bone samples in a formalin solution, washing them with water, decalcifying with a 5% solution of nitric acid, maintaining in a 5% solution of sodium sulfuric acid followed by washing with water, characterized in that decalcification carried out for 7-8 days in a 5% solution of nitric acid based on a 5% solution of formalin, after which for 2 hours the samples are washed with running tap water and immersed for 5 hours in a 5% solution of lithium sulfuric acid then in t chenie 20-30 min of again washed with running water and dehydrated in alcohols of increasing concentrations.