RU2531086C1 - Method for prediction of clinical course of reparative regeneration following surgical management of mechanical injury of liver - Google Patents

Abstract

FIELD: medicine.

SUBSTANCE: invention refers to medicine, namely to clinical and experimental abdominal surgery, and can be used to assess the clinical course of reparative regeneration following surgical management of a mechanical injury of the liver. Substance of the method: on the 3^rd and 7^th postoperative day, blood serum is examined for alpha-fetoprotein, and a regeneration coefficient (RC) is calculated by formula: $$RC=\frac{AF{P}_7}{AF{P}_3},$$

wherein: RC is the regeneration coefficient in standard units; AFP₇ is blood serum alpha-fetoprotein (ng/ml) on the 3^rd postoperative day; AFP₃ is blood serum alpha-fetoprotein (ng/ml) on the 7^th postoperative day; if the RC value is 1.0 and more standard units, a slow clinical course of reparative regeneration is predicted.

EFFECT: invention provides higher accuracy and simplified procedure of the prediction of the clinical course of reparative regeneration following surgical management of the mechanical injury of the liver.

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Description

The invention relates to medicine, namely to clinical and experimental abdominal surgery, and can be used to predict the nature of the course of reparative regeneration after surgical treatment of mechanical liver injury.

Currently, comparative morphological, morphometric, immunological, biochemical and microcirculatory aspects of operations to hide the wounds of the liver, spleen and kidney by the desired autograft (Musatov O.V., Zurnadzhan S.A., Khairulin Yu.Kh. Dynamics of tissue IgG after wound gastroplasty are actively being investigated liver, spleen and kidney in the experiment // News. VolSMU. - 2009. - No. 4. - P.72-75; Musatov OV, Zurnadzhan SA Dynamics of the total titer of heterologous antibodies to Shigella Flexner in blood serum after gastroplasty of wounds of the liver, spleen and kidneys in the experiment // Surgery News. - 2011. - T. 19. - No. 4. - P.10-15; Musatov OV, Trizno M.N. Comparative assessment of microcirculation of gastric autografts (experimental work) // Bulletin surgical gastroenterology. - 2012. - No. 2. - P.44-49.).

To confirm the effectiveness of the use of this surgical method in experimental and clinical surgical hepatology, the search for prognostic criteria for the healing of parenchymal organs, especially the liver, becomes an urgent task.

It is known that to study the functional state of the liver in clinical laboratory diagnostics, a lot of biochemical tests and markers are used (A. Khazanov. Functional diagnosis of liver diseases. - M .: Medicine, 1988. - 304 p.).

However, the abundance of indicators of individual liver functions to date has not solved the problems of adequate laboratory parameters, reflecting the regenerative potential of the operated parenchymatous organ.

Existing methods for predicting the nature of the course of reparative liver regeneration after surgery are based on non-invasive instrumental or invasive laboratory methods.

The most common clinical methods for non-invasive studies of destructive and reparative processes in the liver at the moment are ultrasound (ultrasound) and computed tomography (CT), which allow you to establish a destructive process in the liver with a probability of 94% (Zagainov V.E., Gorokhov G.G. ., Zarechnova N.V., Belsky V.A. A method for predicting the development of liver failure in the postoperative period in patients undergoing hemihepatectomy // RF Patent No. 2409317 of 08/11/2009).

CT refers to an expensive and accompanied by a fairly high radiation exposure method (Munoz-Bongrand N., Panis Y., Soyer P. et al. Serial computed tomography is rarely necessary in patients with acute pancreatitis: a prospective study in 102 patients // J Am. Coll. Surg. - 2001. - No. 193 (2). - S.146-152.).

The indicators of the effectiveness of regeneration during plastic surgery in parenchymal organs performed in experimental surgery are the positive results of histological, immunological, biochemical tests.

Known morphological methods for assessing and predicting the regenerative processes of a damaged liver (Antopolskaya E.V., Shveinov I.A., Konoplya A.I., and Ushkalov A.V. Method for evaluating regenerative processes of the liver // RF Patent No. 2303031 from 12.28.2005 )

The disadvantages of morphological methods are:

1) the predominant element of subjectivity, based on a grading scale known for its conventionality;

2) the invasiveness of the method, which consists in obtaining biopsy material for histological sections;

3) the method is applicable mainly for the issuance of pathological and anatomical findings, but not in vivo studies.

Subtle immunochemical methods for determining various protein markers can inform the state of the regenerative potential to the greatest extent using immunosensor technology, enzyme-linked immunosorbent assay (ELISA) and immunochromatographic analysis (IHA) based on the methods of dry chemistry (Menshikov VV Portable analytical devices in operational laboratory support medical care // Clin.Lab. Diagnostics. - 2008. - No. 9. - P.31-32).

The use of specific immunochemical regeneration markers in abdominal surgery is especially promising, having a number of advantages over standard laboratory analysis methods. Oncofetal proteins, tumor markers, primarily claim the role of such markers.

It was shown that the production of these proteins in the adult organism is not a specific reaction to malignant transformation, but a general biological pattern characteristic of changes in the proliferative regime of tissues and immunological changes that occur during “proliferation diseases” (Miroshnikov V.M., Restorative reactions of the body and stage-specific antigens. - Saratov, 1992 .-- 160 p.).

The first immunochemical indicator of proliferative processes in the liver (Tatarinov Yu.S. Detection of embryospecific protein in primary liver cancer // Abstracts of the I All-Union Biochemical Congress. M. - L .: AN USSR, 1963. - T.2. - P.274) is protein alpha-fetoprotein (AFP). Alpha-fetoprotein (AFP) is a serum protein of a developing mammalian embryo. It almost completely disappears from the blood shortly after the birth of an animal or human, but appears in the blood of adults with the development of liver cancer and some tumors of the reproductive system. In these cases, AFP is used as a specific indicator (marker) of these tumors for their detection and monitoring the effectiveness of treatment (Abelev G.I. Alpha-fetoprotein: a look into the biology of development and the nature of tumors // Soros Educational Journal. - 1998. - No. 9. - S.8-13).

Commercial AFP diagnostic kits are widely used in medicine for the diagnosis of hepatocellular carcinoma and teratoblastoma (Tatarinov Yu.S. The past and future of oncofetal proteins // Lecture on the Act Day of the Institute. - M. - 1988. - P.23-25). Prospects for its use as a marker of recovery processes in traumatology, combustiology and surgery have been found (Miroshnikov V.M. Stadiospecific antigens and recovery processes. Saratov, SSU, 1986. - 176 pp.), As well as in pediatrics (Silishcheva N.N. Clinical significance of the determination of alpha-fetoprotein in the postnatal period and in certain diseases in children: Abstract of thesis of a doctor of medical sciences - Astrakhan., 1978. - 45 p.) And neonatology to assess the degree of maturity of the liver in premature infants ( Poretskova G.Yu., Zakharova LI Method of evaluating f functional maturation of the liver in premature infants // RF Patent No. 2312355 of 11/23/2004).

It is known about the relationship between the level of AFP in the bloodstream and the degree of liver damage in viral (Tatarinov Yu.S. Past and Future Oncofetal Proteins // Lecture on the Act Day of the Institute. - M. - 1988. - P.23-25) and toxic hepatitis (Konyaeva A.G., Vishnevetsky F.E. On the effect of glucocorticoids on the synthesis of alpha-fetoprotein and the liver structure of mice in acute carbon tetrachloride poisoning // Bull.experimental biol. And medical.- 1977. - T.83. - No. 2. - S.151-153).

In addition, there are a large number of biochemical markers of the functional state of the liver based on the determination of various proteins and their combinations in blood serum: for example, (Romensky R.V., Romenskaya N.V., Kapustin R.F. Method for assessing the functional state of the liver // RF patent No. 2305844 C1 dated 12/20/2005), (Parshikov V.V., Nemov V.V. Method for assessing the degree of liver damage // RF Patent No. 2132071 dated 04/21/1998), (Tony E. Hugli, Craig M. Jeckson Diagnostic markers of liver dysfunction // US patent No. 20030087316 A1 of 05/08/2003).

The disadvantages of the known methods for predicting the functional state of the liver, based on the use of immunochemical techniques for the determination of various marker proteins and their combinations in blood serum are:

1) tracking only the current state of the liver at the time of the examination, which does not allow to predict the nature of the course of reparative regeneration, therefore, cannot be used as prognostic tests;

2) monitoring the functional state of the liver includes not only the state of its regenerative potential, but also other liver functions (protein-synthesizing, detoxifying and bile-forming)

Closest to the proposed one is a method involving the collection of blood samples from a patient before and after surgery in the dynamics of the 1st, 7th and 14th day and determining in it the levels of total protein, medium molecules and the activity of alanine aminotransferase, then calculating the ratio of the level of total protein to the level of average molecules and the ratio of the activity of alanine aminotransferase to the level of average molecules and with a value of the first indicator of 255.2-284.2, the second of 2.1-3.15 predict a favorable course, and with a value of the first indicator of 150.5-196.8 the second 5.53-7.2 - unfavorable the course of the postoperative period (Alperovich B.I .; Arefieva E.S .; Tskhai V.F .; Fedorova T.S. A method for predicting the severity of the course of the postoperative period in patients with focal liver lesions // RF Patent No. 2040797 of 07.25.1995 )

The disadvantages of the prototype are:

- a large number of points of blood sampling (four points);

- a large number of heterogeneous indicators of blood samples (three) requiring different laboratory equipment;

- lengthening the analysis procedure;

- the complexity of the methods used for practical use;

- A large number of different coefficients (at least nine) that can give conflicting forecasts.

The invention is aimed at improving the accuracy and simplifying the procedure for predicting the nature of the course of reparative regeneration after surgical treatment of mechanical injury to the liver.

The specified technical result is achieved by the fact that on the 3rd and 7th day after surgery on the liver in the blood serum, the level of alpha-fetoprotein is examined, the regeneration coefficient (CR) is determined by the formula:

, $$\mathrm{TO}R\frac{\mathrm{BUT}\mathrm{<figure-callout\; id="7"\; label="F\; P"\; filenames="00000005.png"\; state="\{\{state\}\}">F\; </figure-callout>}{P}_{}{}_7}{\mathrm{BUT}\mathrm{<figure-callout\; id="3"\; label="F\; P"\; filenames="00000005.png"\; state="\{\{state\}\}">F\; </figure-callout>}{P}_{}{}_3}$$

,

where KR is the regeneration coefficient in arbitrary units;

AFP ₇ - serum alpha-fetoprotein level (ng / ml) on the 7th day after surgery on the liver;

AFP ₃ - serum alpha-fetoprotein level (ng / ml) on the 3rd day after surgery on the liver,

and with a value of CR equal to 1.0 or more conventional units, the sluggish nature of reparative regeneration is predicted.

The proposed method is based on the phenomenon of quantitative differentiated biosynthesis and the release of embryospecific alpha-fetoprotein protein into the blood after autoplasty of liver wounds with various autografts and hepatraphy in the experiment (see Fig. 1 and Table 1).

Preliminary experiments showed that after autoplasty of the liver wound with a serous-muscular flap of a sick stomach curvature on the vascular pedicle, there was a productive type of inflammatory-reparative process with adequate regeneration and development of a gentle connective tissue scar at the wound site under the flap in the long-term period. After the usual suturing of the liver wound, a chronicization of the inflammatory-reparative process with a pronounced proliferation of connective tissue at the site of its modeling and the formation of a rough fibrous scar was revealed.

The proposed method was successfully tested at the Department of Surgery with topographic anatomy of the Astrakhan State Medical Academy during 2011-2012.

Below are the results of testing.

Example 1. The study was performed on 45 Chinchilla rabbits. Under anesthesia, a laparotomy was performed, a sero-muscular flap was cut out from the large curvature of the stomach on the vascular pedicle, which covered the simulated lacerated wounds of the liver (hepatoplasty). Duration of observation - 1st, 3rd, 5th, 7th, 14th, 21st, 30th, 60th, 90th, 120th, 150th, 180th 360th day after surgery. Determination of AFP titers in the blood of operated rabbits was carried out by the method of enzyme-linked immunosorbent assay with a conjugate of antibodies against rabbit AFP from the bank of immunochemical test systems of the Department of Biochemistry AGMA with horseradish peroxidase (Serva) (sensitivity 4 ng / ml).

Starting from the third day (Table 1.), the level of AFP increased 3-4 times in all rabbits after hepatoplasty in blood compared to the control intact group (6.1 ± 1.82 ng / ml, n = 21). In this group, after hepataphia, the mean AFP values on the 3rd day (AFP ₃ ) were 35.9 ± 2.40 ng / ml, and on the 7th day (AFG ₇ ) - 27.0 ± 1.95 ng / ml , Raman regeneration coefficient = 0.75 (the dispersion of individual Raman scores is from 57 to 0.89; the median is 0.75).

Example 2. The study was performed on 17 chinchilla rabbits. Under anesthesia, a laparotomy was performed, a lacerated wound of the liver was modeled and its suturing (hepatraphy) was performed. The condition of the animals immediately after the operation is satisfactory. The terms of observation and determination of AFP titers are the same as in example 1 - from one day to a year. Starting from the third day (Table 1), the level of AFP increased by 3-4 times in all rabbits in the blood compared to the control intact group (6.1 ± 1.82 ng / ml, n = 21), in which, after hepatraphy, the average AFP values on the 3rd day (AFP ₃ ) were 35.1 ± 4.47 ng / ml, and on the 7th day (AFP ₇ ) - 33.8 ± 4.41 ng / ml, Raman regeneration coefficient = 0 97 (scatter of individual Raman scores from 0.75 to 1.20; median 1.00). A retrospective analysis based on morphological research methods revealed that the group is heterogeneous: 7 rabbits with signs of sluggish regeneration. The Raman regeneration coefficients calculated in the first week were 1.20 in these animals; 1.14; 1.11; 1.07 and thrice 1.00, and 6 rabbits have less than one.

From figure 1 it follows that the most prognostically significant differences in the levels of AFP and, accordingly, the maximum regeneration coefficient - (CR) are observed when determining AFP titers on the 3rd and 7th day, and not on the 5th or 14th not to mention the follow-up dates for determining AFP levels.

The results showed that the dynamics of serum AFP after the investigated operations depends on the type of surgical intervention and on the plastic properties of the used autograft. The use of serous-muscular flap of the stomach on the vascular pedicle contributes to a more intensive release of AFP into the bloodstream in the period of 5-7 days due to the activation of an immune system trauma. The use of a serous-muscular flap of the stomach on the vascular pedicle contributes to a less stressed state of reparative regeneration in the process of healing of liver wounds compared to hepatraphy due to a more productive course of the inflammatory-reparative process. With safe operations, serum AFP is restored in the shortest possible time of the postoperative period.

The proposed method improves accuracy and simplifies the procedure for predicting the nature of the course of reparative regeneration after surgical treatment of mechanical injury to the liver.

Thus, the proposed method allows to identify complications developing in the postoperative period in the form of sluggish liver regeneration.

Table 1 Levels of alpha-fetoprotein (ng / ml) in rabbit serum at various times after various options for surgical treatment of mechanical liver injury №№ Duration, day Hepatoplasty 45 animals Liver wound closure (hepatraphia) 17 animals one one 9 ± 0.81 11 ± 1,03 2 3 36 ± 2,40 35 ± 3.47 3 5 33 ± 2.94 36 ± 3.18 four 7 27 ± 1.95 34 ± 2.41 5 fourteen 15 ± 1.32 18 ± 1.62 6 21 14 ± 1.21 16 ± 1,50 7 thirty 12 ± 0.81 14 ± 1.55 8 60 10 ± 0.93 13 ± 1.06 9 90 7 ± 0.64 10 ± 1.19 10 120 5 ± 0.47 7 ± 1,06 eleven 150 7 ± 0.55 7 ± 1.53 12 180 0 8 ± 0.44 13 360 0 8 ± 0.71

Claims (1)

A method for predicting the nature of the course of reparative regeneration after surgical treatment of a mechanical injury to the liver, consisting in a biochemical blood test, characterized in that on the 3rd and 7th day after surgery on the liver, serum alpha-fetoprotein is examined, and the regeneration coefficient is determined ( KP) according to the formula
$$\mathrm{TO}R=\frac{\mathrm{BUT}F{P}_7}{\mathrm{BUT}F{P}_3}$$

,
where KR is the regeneration coefficient in arbitrary units;
AFP ₇ - serum alpha-fetoprotein level (ng / ml) on the 7th day after surgery on the liver;
AFP ₃ - serum alpha-fetoprotein level (ng / ml) on the 3rd day after surgery on the liver,
and with a value of CR equal to 1.0 or more conventional units, the sluggish nature of reparative regeneration is predicted.

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