RU2310191C1 - Method for biopsy of hepatic tissues in neonatal calves - Google Patents

Abstract

FIELD: veterinary medicine.

SUBSTANCE: the present innovation deals with sampling with the help of special device for puncture biopsy being a hollow needle at internal diameter of 2 mm and length of 10 cm, its working end is designed as a saw-tooth trident at tooth length being 2 mm; the angle of sawing between teeth corresponds to 45°C. Moreover, the needle is supplied with a stylet which is 5 mm longer. The innovation enables to control the depth for needle's penetration into hepatic parenchyma and excludes the risk for traumatizing kidneys, pulmonary pleura and lungs.

EFFECT: higher efficiency.

4 dwg, 3 tbl

Description

The present invention relates to veterinary medicine and can be used in normal and pathological histology, clinical diagnosis.

There are various methods of biopsy [RU 2119304 C1, 6 AB 17/34, 09/27/1998; RU 2173091 C1, 7 А61В 10/00, А61В 5/0408, 09/10/2001; RU 2199952 C2, 7 А61В 8/00, 03/10/2003; RU 2230494 C2, A61B 10/00, 06/20/2004], but not one of them can be used for liver biopsy of newborn young cattle.

Closest to the claimed is the method used for biopsy of the liver of cattle (adult animals) [Byrka V.I. On a liver biopsy of cows / V.I. Byrka // Veterinary medicine. - 1969. - No. 3. - S.69-71]. However, it cannot be used for puncture biopsy of the liver in newborn young animals, since it does not completely protect the selection process of punctate. Its essence is that given the length of the needle (21 cm) and the protruding part of the stylet (1 cm), it is difficult to control the depth of immersion, which poses a serious danger to the health of the calf in the form of unjustifiably excessive trauma to both the liver itself and nearby tissues and organs. Our proposed method has a number of design differences that minimize the risk of complications and make possible a liver biopsy of newborn young cattle.

The objective of the invention is to solve the problem of puncture biopsy of the liver of newborn calves for intravital sampling of material for histological examination, which allows for early diagnosis of liver diseases, to study the features of the development of the liver and its structure, to study the ontogenetic basis of liver pathology in cattle, reduce trauma, reduce traumatic the consequences of puncture, simplifying the process of tissue puncture while maintaining the structure of the biopsy ma terial.

The essence of the invention lies in the fact that during the biopsy of the calf is fixed in a standing position. At the puncture site (the last intercostal space on the right, 2 cm below the spinal column), the hair is cut out and the skin is treated with a 5% alcohol solution of iodine. Then, at the injection site, the skin is shifted to the side and a puncture is performed. The skin and peritoneum are pierced with a needle with an inserted stylet, after reaching the capsule of the liver, the stylet is removed and the needle is immersed in the parenchyma to a depth of 2-3 cm, while the needle is directed towards the opposite elbow joint. After immersion, the needle rotates around its own axis. In order for a piece of tissue not to fall out of the lumen of the needle when it is removed, the cannula is closed with a finger and the needle is removed (Fig. 1. Needle for biopsy: 1 - needle tube; 2 - saw-tooth trident; 3 - thickening of the cannula; 4 - cannula; 5 - slot of the locking device; 6 - stylet). The needle has the following characteristics: length - 10 cm, inner diameter - 2 mm, ends with a saw-tooth trident, tooth length - 2 mm, cut angle between them 45 ° and is equipped with an adjacent stylet protruding by 5 mm, which makes it easy to control the depth of penetration of the needle into liver parenchyma, receive punctate up to 50-70 mg by weight, which is quite enough for histological studies. Given its size, the risk of injury to the kidneys, pulmonary pleura and lungs is excluded. Due to the fact that the skin of a newborn is much thinner and more elastic than that of an adult animal, there is no need to use a guiding needle, this allows for an additional reduction in the traumatic effect. The resulting tissue column is pushed out of the needle into the fixative fluid (20% formaldehyde solution). The skin puncture site is again treated with an iodine alcohol solution.

An example of a specific implementation. The calf is black-motley breed, belongs to the BelSA farm “Centralnoe”, date of birth 04/28/2000, stationary number 40, body weight at birth 26 kg, is contained in a typical individual cage. At the time of the procedure (05/03/2000) - age 5 days, clinical status: temperature - 38.6 ° C, pulse - 140 beats per minute, breathing - 36 respiratory movements per minute, general condition is satisfactory, coat, skin and visible mucous membranes without changes or disorders, the cardiovascular and respiratory systems within the generally accepted norms, heart sounds are clear, distinct, the cardiac impulse is localized, breathing without extraneous noise, even. Percussion borders of the liver are not changed, painless. Results of a general clinical blood test: ESR (erythrocyte sedimentation rate) - 0.5 mm / h, erythrocytes - 5.92 million / ml, hemoglobin - 122 g / l, SGE (average hemoglobin in erythrocytes) - 20.6 pg ( picograms), CP (color index) - 1.28, leukocytes - 8.64 thousand / ml, cell coefficient - 685, leukocyte formula (%): B (basophils) - 0, E (eosinophils) - 0, Yu (young neutrophils) - 0, P (stab neutrophils) - 7, C (segmented neutrophils) - 37, Lim (lymphocytes) - 52, Mon (monocytes) - 4. There are no contraindications for the puncture biopsy. The method is as follows. During a biopsy, the calf is fixed in a standing position. At the puncture site (the last intercostal space on the right, 2 cm below the spinal column), the hair is cut out and the skin is treated with a 5% alcohol solution of iodine. Then, at the injection site, the skin is shifted to the side and a puncture is performed. The skin and peritoneum are punctured by a needle with an inserted stylet, after reaching the capsule of the liver, the stylet is removed and the needle is immersed in the parenchyma to a depth of 2-3 cm, while the needle is directed towards the opposite elbow joint. After immersion, the needle rotates around its own axis. In order to prevent a piece of tissue from falling out of the lumen of the needle when it is removed, the cannula is closed with a finger and the needle is removed. The resulting tissue column is pushed from the needle into a container with a fixing liquid (20% formaldehyde solution). The skin puncture site is again treated with an iodine alcohol solution. Upon further observation of the calf, the negative effects of the intervention by both clinical and laboratory research methods were not detected.

Example. The method was used on 47 newborn calves of black-motley breed for the purpose of intravital diagnosis of morphological changes in the liver. During biopsies, calves were fixed in a standing position near individual cells. At the puncture site (the last intercostal space on the right, 2 cm below the spinal column), the hair was cut out and the skin was treated with 5% alcohol solution of iodine. Then, at the injection site, the skin was shifted to the side and a puncture was performed. The skin and peritoneum were punctured with a needle with an inserted stylet, after reaching the capsule of the liver, the stylet was removed and the needle was immersed in the parenchyma to a depth of 2-3 cm, while the needle was directed towards the opposite elbow joint. After immersion with a needle, rotation around its own axis was performed. In order to prevent a piece of tissue from falling out of the lumen of the needle when it was removed, the cannula was closed with a finger and the needle was removed. The resulting tissue column was pushed from the needle into the fixative fluid (20% formaldehyde solution). The puncture site was again treated with an iodine alcohol solution. After the puncture, no complications were revealed, the five-day survival rate was 100% (this indicator is used in assessing most minimally invasive research methods, and it characterizes the safety of a particular study with a high degree of objectivity).

A study of histological preparations found that in animals that had a symptom complex of hypotrophy and biochemical changes characteristic of a functionally inferior liver (see tables 1-3), changes in the architectonics of liver tissue were also noted. Basically, these changes were reduced to the lack of formation of hepatic beams, a sharp decrease (or disappearance) of the number of binuclear hepatocytes and the presence of fields of destructively altered cells with the phenomena of karyolysis and macrophage reaction. Also noted: nuclear polymorphism, the presence of hyperchromic nuclei in hepatocytes and Kupffer cells, foci of micro- and macro-necrosis of the parenchyma of the liver (Fig. 2. Necrosis focus. Calf liver. Coloring hematoxylin and eosin. Lens 40, eyepiece 7). One of the calves showed changes characteristic of the initial stage of cirrhotic degeneration of the organ (Fig. 3. Extensive necrosis in the parenchyma, phenomena of hyalinization of necrotic masses, lymphoid infiltration of the outbreak, cytolysis and karyolysis in hepatocytes, phenomena of karyopiknosis. Calf liver. Coloring hematoxylin and hematoxylin. Lens 40, eyepiece 7). The histological picture of the liver of clinically healthy calves was as follows: the beam structure is relatively well expressed, the sinusoids are moderately dilated, the hepatocytes are polygonal in shape, binuclear cells are often found (Fig. 4. Calf liver. Coloring hematoxylin and eosin. Lens 20, eyepiece 7). Tests of the method revealed the following advantages: it is technically simple and easy to use, atraumatic, it can be reused in this animal, taking material with its help for histological examination does not affect its results under various functional conditions of the organ.

Using the proposed method allows to obtain tissue samples sufficient for morphological assessment of the state of the organ. The absence of registered complications (five-day patient survival was 100%) significantly reduces the number of contraindications for the procedure. Thus, the proposed biopsy method significantly expands the possibilities of early diagnosis of morphofunctional changes in the liver of newborns, and therefore, facilitates the work of clinicians in identifying young animals with liver diseases and resolving the feasibility of further use of the animal, and can also be used in research on the ontogenetic basis of pathology liver in cattle, eliminates the risk of injury to the kidneys, pulmonary pleura and lungs.

Table 1 Biochemical blood parameters of newborn calves (2-4 days) Indicator Hypotrophics Normotrophics Total protein, g / l 49.7 ± 1.1 61.4 ± 1.3 Albumin, g / l 31.0 ± 0.8 26.3 ± 0.9 Alpha Globulins, g / l 5.9 ± 0.8 9.5 ± 0.95 Beta Globulins, g / l 6.9 ± 1.3 7.4 ± 0.6 Gamma globulins, g / l 5.5 ± 0.8 18.5 ± 1.3 Albumin: Globulins 1.98 ± 0.2 0.79 ± 0.05 Sample Veltman, ml of 0.5% solution of CaCl ₂ 0.75 ± 0.03 0.56 ± 0.03 AlAt (alanine transaminase), mcmol / ml / hour 0.26 ± 0.21 0.12 ± 0.08 AcAt (aspartic transaminase), mcmol / ml / hour 0.36 ± 0.1 0.50 ± 0.09

table 2 Biochemical blood parameters of newborn calves (6-8 days) Indicator Hypotrophics Normotrophics Total protein, g / l 52.2 ± ± 1.7 61.4 ± 1.0 Albumin, g / l 34.5 ± 0.6 30.8 ± 1.2 Alpha Globulins, g / l 6.3 ± 0.9 9.5 ± 0.6 Beta Globulins, g / l 4.9 ± 1.1 6.1 ± 0.7 Gamma globulins, g / l 6.7 ± 1.5 15.5 ± 1.2 Albumin: Globulins 2.16 ± 0.22 1.05 ± 0.07 Sample Veltman, ml of 0.5% solution of CaCl ₂ 0.66 ± 0.05 0.5 ± 0.03 AlAt (alanine transaminase), mcmol / ml / hour 0.23 ± 0.15 0.1 ± 0.04 AcAt (aspartic transaminase), mcmol / ml / hour 0.26 ± 0.12 0.3 ± 0.11

Table 3 Biochemical blood parameters of newborn calves (10-12 days) Indicator Hypotrophics Normotrophics Total protein, g / l 52.2 ± 1.2 61.0 ± 1.5 Albumin, g / l 35.3 ± 1.1 32.9 ± 1.3 Alpha Globulins, g / l 7.2 ± 0.5 10.4 ± 2.1 Beta Globulins, g / l 3.9 ± 0.8 3.9 ± 0.6 Gamma globulins, g / l 5.6 ± 0.8 14.2 ± 1.6 Albumin: Globulins 2.26 ± 0.22 1.24 ± 0.12 Globulin index 2.39 ± 0.35 1.34 ± 0.34 Sample Veltman, ml of 0.5% solution of CaCl ₂ 0.56 ± 0.03 0.45 ± 0.03 AlAt (alanine transaminase), mcmol / ml / hour 0.06 ± 0.03 0.1 ± 0.07 AcAt (aspartic transaminase), mcmol / ml / hour 0.23 ± 0.09 0.36 ± 0.1

Claims (1)

A method for biopsy of liver tissue in newborn calves, including sampling using a puncture biopsy device, characterized in that the puncture biopsy device is a hollow needle with an inner diameter of 2 mm and a length of 10 cm, the working end of which is made in the form of a saw-tooth trident with a length teeth 2 mm, and the cut angle between the teeth is 45 ° C, while the needle is equipped with a stylet, which is 5 mm longer than it.

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