Method for verification of phase of suppurative septic complications accompanying acute desctructive pancreatitis

FIELD: medicine.

SUBSTANCE: ultrasound-assisted transcutaneous targeted needle liver-biopsy is used in the patients with acute destructive pancreatitis; that is followed by the immunohistological analysis of the biopsy micropreparations in parallel sections 5 mcm thick marked with monoclonal antibodies: CD 79a for identifying B-Lm and CD 68 for identifying MF. That is followed by optical microscopy at magnification x400 with morphometry of B-Lm periportal regions and MF lobe parenchyma; the results are further processed by automated image analysis systems. A relative area taken by the analysed cell elements in the micropreparation is determined by formula (A). If the value A for the B-Lm periportal regions falls within the range of 0.4 to 1.14, while the value A for the MF lobe parenchyma falls within the range of 0.89 to 2.95, a pre-infectious phase of acute destructive pancreatitis is stated. If the value A for the B-Lm periportal regions falls within the range of 1.41 to 4.98, while the value A for the MF lobe parenchyma falls within the range of 4.34 to 9.52, a phase of suppurative septic complications of acute destructive pancreatitis is stated.

EFFECT: using the method provides the well-timed correction of the therapeutic actions; the method is attended by a lower risk of the needle biopsy; it possess high accuracy and simplicity of implementation.

2 tbl, 2 ex, 6 dwg

 

The invention relates to medicine, in particular to histology, morphometry, abdominal surgery.

Tactics of treatment and diagnostic procedures for acute destructive pancreatitis (CCT) is determined by the phase of the disease (confeccionar phase and the phase of purulent-septic complications).

To verify the infection of necrosis of the pancreas and retroperitoneal tissue in patients with CCT determined using biochemical tests, serological markers, such as C-reactive protein, procalcitonin, calcium, IL-10, IL-18, glucose, phospholipase A2, carboxypeptidase, trypsinogen and others; this requires a dynamic analysis of these indicators (J. Iovainna. "Pancreatology. From bench to bedside". Marseille: Springer, 2009).

But with the help of these criteria can only indirect assessment of the pancreas at a specific moment, laboratory methods of diagnostics in pancreatic infection have a lower sensitivity and specificity (B.C. Saveliev, M.I. Filimonov, BYR Gelfand, SZ Burnewicz. "Infected pancreatic necrosis". URL: http://epidept.spb.ru/Content/SSI/Topic/cont/7/b_vi.htm used indicators require dynamic control, which also makes them the disadvantage of the high cost of research.

To verify the infection of necrotic tissue lesions of the pancreas and retroperitoneal cellular and at CCT typically use data bacteriological analysis of biomaterial, obtained by fine-needle puncture of the retroperitoneum under the control of computed tomography (CT) or ultrasound guidance (ultrasound). When there are positive results of bacteriological tests (smear and culture examination aspiration of material) is judged on the onset of purulent-septic complications, with proven infection with necrotic foci is an indication for surgical intervention (Saveliev B.C. Standards of diagnostics and treatment of destructive pancreatitis. Moscow; Russian state medical University based on data from questionnaires surgical clinics of the Russian Federation. URL: http://doctormedcin.narod.ru/TEXT/pancreatit.htm; Büchler M.W., Gloor C., Müller S., Friess H., Seiler S.A. and Uhl W. "Acute necrotizing pancreatitis: treatment strategy according to the status of infection". // Ann. Surg. 2000; 232:619-626).

Disadvantages fine-needle puncture retroperitoneal tissue, in addition to possible complications (damage to the vascular and nervous structures and so on), is a certain risk of a secondary infection (Werner J., Feuerbach, S., Uhl W. and Büchler M.W. "Management of acute pancreatitis: from surgery to intensive care interventional". Gut. 2005; 54:426-436; J. Iovanna. "Pancreatology. From bench to bedside". Marseille: Springer, 2009; 84). In addition, this method requires expensive equipment in terms of operating and qualified professionals, using the method in widespread clinical practice, t is the train in some cases limited by the lack of liquid formations in the presence of a nidus of infection or "echo box" for targeted puncture structures focus of necrosis and small experience in clinical research (B.C. Saveliev, M.I. Filimonov, BYR Gelfand, SZ Burnewicz. "Infected pancreatic necrosis". URL: http://epidept.spb.ru/Content/SSI/Topic/cont/7/b_vi.htm).

The objective of the invention is to improve the accuracy of the verification phase of purulent-septic complications, and technical simplification and cheapening of the study methodology.

The essence of the method lies in the fact that patients with CCT under ultrasound carry out target puncture percutaneous liver biopsy, performed immunohistochemical study of specimens biopsies on parallel slices with a thickness of 5 μm labeling monoclonal antibodies: CD79α for the identification of b-lymphocytes (B-Lm) and CD68 to identify macrophages (MF), and then perform light microscopy at magnification ×400 with morphometrics B-Lm periportal areas and Matt parenchyma slices and subsequent processing of results using automated image analysis, thus determine the relative area occupied analyzed the cellular elements in the microscopic slide according to the formula:

A=i=1nAin×S×100%where a is the relative area occupied by analyzer is the subject of cellular elements - in %, Ai is the absolute square of the analyzed cells in the field of view of i - in pixels, n is the number of investigated fields of view in the microscopic slide, S is the total area of the field of view, in pixels, in this case, if the value of the relative area And for B-Lm periportal areas belongs to the interval from 0.4 to 1.14 and the value for MT parenchyma of the liver belongs to the interval from 0.89 to 2.95, determine coinfection phase CCT, if the value for B-Lm periportal areas belongs to the interval from 1,41 to 4.98 and value for MT parenchyma of the liver belongs the interval from 4,34 to 9.52, determine the phase of purulent-septic complications.

For the first time on the basis of objective morphometric studies established the relationship between indicators of relative area B-Lm periportal areas of the liver and the MOF of the liver parenchyma and phases CCT. Increasing values of the relative area MT of the liver in phase purulent-septic complications CCT, presumably caused by increased phagocytic and antigen-presenting activity of these cells in relation to components of microorganisms coming from anatomically and functionally similar massive inflammatory-necrotic lesion after infection. Increasing values of the relative area B-Lm liver in phase purulent-septic complications CCT, feels the part, associated with the activation and proliferation of B-Lm with a massive stimulation by bacterial antigens. Light microscopy at magnification ×400 provides the best visualization and software identification of the analyzed cellular elements, which allows to use the tools of computer systems image analysis mode techniques for automatically determining the area occupied B-Lm and MF, and therefore increases the accuracy and objectivity of research.

The proposed method is as follows.

Patients with CCT after processing the surgical field standard antiseptic solution under ultrasound carry out target puncture percutaneous liver biopsy under local infiltration anesthesia with 0.25% solution of novocaine needles for histological biopsy of soft tissues G16, G18. Biopsy material of the liver is subjected to fixation in 10% neutral buffered formalin, conventional circuits post the material and fill in paraffin, on the microtome receive parallel histological sections with a thickness of 5 μm, the liver specimens subjected to immunohistochemical study by incubation with a "ready to use" CD79a antibodies (rabbit anti-human CD79a monoclonal antibody: clone SP18 firm Spring, USA) and CD68 (anti-human CD68 monoclonal antibody: clone 514H12 company Monosan, the Netherlands) according to the standard procedure then perform light microscopy of histological specimens at magnification ×400 with morphometrics B-Lm periportal areas and Matt parenchyma slices and handling results using an automated system image analysis (for example, "Videotest 4.0"). Then determine the relative area occupied B-Lm periportal areas and Matt parenchyma of slices in the microscopic slide (for example, using MS Excel) by the formula:

A=i=1nAin×S×100%where a is the relative area occupied analyzed the cellular elements in %, Ai is the absolute square of the analyzed cells in the field of view of i - in pixels, n is the number of investigated fields of view in the microscopic slide, S is the total area of the field of view, in pixels. If the value of the relative area And for B-Lm periportal areas belongs to the interval from 0.4 to 1.14 and the value for MT parenchyma of the liver belongs to the interval from 0.89 to 2.95, determine coinfection phase CCT. If the value for B-Lm periportal areas belongs to the interval from 1,41 to 4.98 and value for MT parenchyma of the liver belongs to the interval from 4,34 to 9.52, determine the phase of purulent-septic complications. Presents graphic materials illustrate the changes described above. Figure 1 presents the results of the methods of automatic definition wide-angle is square, occupied B-Lm periportal area micropreparative liver, within the computer program image analysis Videotest 4.0" confecciones phase CCT (immunohistochemistry processing CD79a, light microscopy at magnification 400). Figure 2 presents the periportal B-Lm infiltration of the liver in coinfections phase CCT (marking CD 79α, light microscopy at magnification 400). Figure 3 presents the periportal B-Lm infiltration of the liver in phase purulent-septic complications CCT (marking CD 79α, light microscopy at magnification 400). Figure 4 presents Matt infiltration of the liver parenchyma in coinfections phase CCT (CD 68, light microscopy at magnification 400). Figure 5 presents Matt infiltration of the liver parenchyma in the phase of septic complications CCT (CD 68, light microscopy at magnification 400). Figure 6 presents the method for automatically determining the space MF of the liver parenchyma, in the framework of the computer program "Videotest 4.0" in the phase of septic complications CCT.

Example 1.

Patient I., A.N., 44 years (map patient No. 61/11) was treated at at Clinical emergency hospital" gsmarena for acute destructive pancreatitis. Liver biopsy obtained on the 5th day from the start of the clinical PR the phenomena of the disease (No. 21-11): A (B-Lm) amounted to 1.12%, And(MT) amounted to 2.57%, which allowed to conclude confecciones phase CCT. Data General clinical blood analysis (5th day of the disease): Lc 20,8×109/l SW 2%, plasmic order has been revealed 2%, stab neutrophils 37%, segmented 50%, lymphocytes 8%, monocytes 1%. The data of biochemical analysis of blood: creatinine 86 MCOL/l, urea of 6.4 mmol/l, protein 37 g/l, glucose of 7.1 mmol/l At laparoscopy (5th day of the disease) identified a total hemorrhagic pancreatic necrosis, peripancreatic infiltration (aminobutric), aseptic abscess retroperitoneal fat (parapancreatic, parabolically, perirenal areas), enzymatic peritonitis. Bacteriological examination of feces (effusion in the abdominal cavity, stuffing bags) confeccionar phase CCT confirmed. Assigned to comprehensive treatment, including: 1) detoxification, restoration of blood rheological properties, compensation plasmopara; 2) antisecretory therapy, 3) correction of respiratory and hemodynamic disorders; 4) antifermentny therapy.

Example 2.

Patient B. V.E., 82 years (map patient No. 1771/12) was treated in AGBUS "Clinical emergency hospital" gsmarena for acute destructive pancreatitis. Liver biopsy obtained on the 10th day of the disease (No. 600-12): A (B-Lm) stood at 2.52%, And(MOF) was a 4.86%, mo is allowed to set the phase of purulent-septic complications CCT. Data common clinical analysis of a blood (the 10th day of the disease): Lc 10,2×109/l, neutrophils stab 9%, segmented 78%, lymphocytes 8%, monocytes 5%. The data of biochemical analysis of blood: creatinine 75 MCOL/l, urea 16 mmol/l, protein 52 g/l, glucose of 5.4 mmol/l At laparotomy revealed a pancreatic abscess stuffing bags and subhepatic abscess localization, septic foci sanitized, and drained Winternitz tube drainage. The results of the bacteriological examination of feces (the contents of the abscess stuffing bags and abdominal cavity), and intraoperative data confirmed the patient's phase presence of purulent-septic complications CCT. In the postoperative period assigned to comprehensive treatment, including detoxification, restoration of blood rheological properties, compensation plasmopara, correction of respiratory and hemodynamic disorders, antibiotic therapy with broad-spectrum, creates an effective bactericidal concentration in the tissue of the pancreas and peripancreatic tissue for the majority of infectious agents.

Validation of the method was carried out on the material 30 of liver biopsies obtained from patients with CCT, who was undergoing treatment in hospital emergency medical care Smolensk is. The study of the cellular composition In the lymphocytic component infiltrates periportal zones and macrophage infiltration of the parenchyma was performed in parallel slices on the basis of the results of immunohistochemistry by incubation with a "ready to use" monoclonal antibodies for identification of B-Lm CD 79α (clone SP18 firm Spring, USA), MOF-CD 68 (clone N company Monosan, the Netherlands). Depending on the size of the biopsy were analyzed from 3 to 10 fields of view. It was found that in coinfection phase CCT (13 drugs) values of the relative area occupied B-Lm periportal areas ranged from 0.4 percent to 1.14%, in the phase of suppurative complications (16 drugs) values of the relative area occupied B-Lm periportal zones ranged from 1,41% to 4.98%. When assessing the relative area MT of the liver parenchyma in coinfections phase CCT (13 drugs) values obtained from 0.89% to 2.95%. In the phase of septic complications (17 specimens) values of the relative area MT of the liver parenchyma ranged from 4,34% to 9.52%.

Check samples for normality of distribution was carried out using a criterion χ2. Statistical processing was carried out using the nonparametric criterion of Mann-Whitney. The difference between the levels of attributes in the respective samples according to the criterion of Mann-Whitney (data shown in table 1, 2) is su is the natural enemy in the level of statistical significance 1%. In table 1 presents the evaluation of the differences between samples on the relative areas A(B-Lm) periportal areas of the liver specimens of patients with CCT in coinfections phase of the disease (sample 1) and in the phase of septic complications (sample 2) using U-test, Mann-Whitney. Accepted alternative hypothesis, i.e. the difference between the level of variable A(B-Lm) in the samples is substantially at the level of statistical significance 1% (UEMF=0 when Ucrit=51). Table 2 presents the assessment of differences between samples in terms of the relative area(MT) in the liver specimens of patients with CCT in coinfections phase (sample 1) and in the phase of septic complications (sample 2) using U-test, Mann-Whitney. Accepted alternative hypothesis, i.e. the difference between the level of variable A(B-Lm) in the samples is substantially at the level of statistical significance 1% (UEMF=0 when Ucrit=55). The presence of purulent-septic complications of acute destructive pancreatitis corresponded intraoperative data, results of bacteriological analysis of the contents of the abdominal cavity and retroperitoneal space.

Thus, the described changes in morphometric parameters of the liver for B-Lm periportal areas and Matt parenchyma slices can be used as an objective criterion for the verification stage of purulent-septi the definition of complications of CCT, that allows to use this method as a new criterion for the timely correction of remedial measures, including surgical intervention. The described method involves less risk and is characterized by greater simplicity, technical performance biopsy of the liver, rather than puncture retroperitoneal tissue, as well as more profitable (requires CT-control. The cost of immunohistochemistry and morphometric data processing is relatively smaller than when using serological markers of infection of necrosis in the dynamics), the method has high accuracy.

16 29
Table 1
Sample 1, AndRankSample 2, AndRank
1,0281,9318
0,4322,822
1,08113,0124
0,4751,76
0,727is 4.9328
0,443,52,9223
1,0591,7415
0,4411,4114
1,12123,2926
1,14133,5427
1,06103,1825
0,5962,5921
0,443,52,4519
1,8817
to 4.98
2,5220
Amount91344
UEMF=0 (Ucrit=51 at p≤0,01).

Table 2
Sample 1, AndRankSample 2, AndRank
2,95136,8225
2,627,55,6322
2,84105,2419
2,2736,926
2,85115,2720
2,4145,1717
0,8917,1227
2,94124,6715
to 2.5769,5230
2,5258,5329
2,7596,7224
2,627,57,3328
1,425,6121
5,2318
4,3414
5,9123
4,86 16
Amount91374
UEMF=0 (Ucrit=55 at p≤0,01).

Method verification phase of septic complications in acute destructive pancreatitis, including the sampling of biological material and its subsequent analysis, wherein the patient under ultrasound perform percutaneous sighting needle biopsy of the liver and carried out the immunohistochemical investigation of specimens biopsies on parallel sections of labeling monoclonal antibodies CD 79a for the identification of b-lymphocytes (B-Lm) and CD 68 to identify macrophages (MF), then perform light microscopy at magnification ×400 with morphometrics B-Lm periportal areas and Matt parenchyma slices, processing results carried out using automated image analysis determine the relative area occupied analyzed the cellular elements in the microscopic slide by the formula
A=i=1nAinS100%,
where a is the relative area occupied analyzed the cellular elements %; Ai is the absolute square of the analyzed cells in field of view i, the pixels; n - number of studied fields of view in the microscopic slide; S is the total area of the field of view, pixels,
and if the value of the relative area And for B-Lm periportal areas belongs to the interval from 0.4 to 1.14 and the value for MT parenchyma of the liver belongs to the interval from 0.89 to 2.95, determine coinfection phase CCT, if the value for B-Lm periportal areas belongs to the interval from 1,41 to 4.98 and value for MT parenchyma of the liver belongs to the interval from 4,34 to 9.52, determine the phase of purulent-septic complications.



 

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