RU2146367C1 - Method for predicting wound development process course - Google Patents

Abstract

FIELD: medicine. SUBSTANCE: method involves checking peripheral blood characteristics describing macro-organism functional state in patients having pyo-inflammatory diseases. The peripheral blood characteristics are determined concurrently with those determined on the blood taken from wound. The blood characteristics correlation analysis is carried out. The inverse correlation being detected, torpid or complicated course is predicted to take place. Direct correlation being discovered, wound cohesion process without complications is to be predicted. EFFECT: enabled early diagnosis of pyoseptic complications. 5 dwg, 6 tbl

Description

 The present invention relates to medicine and can be used in purulent-septic surgery to predict the course of the wound process and early diagnosis of emerging septic complications.

 It is known that neutrophilic leukocytes are one of the first cellular factors of the natural nonspecific resistance of the body in contact with infectious agents, and further dynamics of the development of the inflammatory process in conditions of antigenic overload depends on their functional and morphological fullness. Their study is of particular interest due to the fact that the main complex of transformations characteristic of the human body as a whole occurs in leukocytes, and thus, a change in the metabolic activity of leukocytes reflects to a certain extent the metabolism of the whole organism.

 When analyzing various cytochemical parameters, it is also necessary to take into account where reactive neutrophil changes occur - at the periphery or in the process of granulopoiesis, since defects in its enzyme systems can be acquired at the level of mature cells. They can occur when neutrophils come into contact with the products of bacterial activity, with excessive production of oxidants due to a lack of their inactivators, etc. On the other hand, damaging environmental factors can stimulate the biological activity of neutrophils. Their activation is a reparative phenomenon. It arose in evolution as an adaptation mechanism. The essence of this mechanism is that in a situation of a sharp change in the conditions of the existence of a cell that threatens her death, she tries to adapt her metabolism to them. At the same time, an emergency status is triggered by a signal from the cell surface, and a massive genome depression occurs. The entire (or key) set of transcripts is urgently expressed. This way the genome offers the cell a choice. If there is a metabolic need for new transcripts, the expression of this part of the program is epigenically fixed, and its implementation begins to be supported by the transcriptional-translational conveyor. If these measures allow the cell to adapt to the situation and establish homeostasis, the activation signal stops, and transcription of unnecessary genes, which is not supported by the metabolic method, fades. Reprogramming is complete.

 Externally, signs of neutrophil activation can manifest itself in a change in the physicochemical properties of the chromatin of their nuclei, a change in chemotaxis activity, complement-dependent and complement-independent phagocytosis, the degree of permeability of cell membranes, the intensity of expenditure of protective cytoplasmic substrates in the focus of inflammation. It should be borne in mind that the leading and relatively easily identifiable sign of neutrophil activation is a change in the physicochemical properties of the chromatin of their nuclei, which should precede all other activation phenomena.

 Thus, signs of altered reactivity of neutrophilic leukocytes must be sought not only in cells circulating in peripheral blood. They can be more pronounced in the tissue pool, directly in the wound barrier, that is, directly where the main reactions of neutrophils take place. This approach to assessing the functional state of the body (periphery-focus) is universal in nature and applies not only to the cytochemical study of neutrophilic leukocytes, but also to other research methods - immunological, biochemical, etc.

 Currently, there are known attempts at local assessment of the purulent foci - pH-metry, the method of wound prints, biochemical examination of purulent discharge and a number of other methods. (Fenchin K.M. Wound healing.- Kiev, 1979.- 168 p.). However, these methods are not widely used due to their low information content and large errors in research results. There are many reasons for this - the heterogeneous composition of pus, a change in its properties depending on the type of pathogen, the number of dead neutrophils in the discharge, etc.

 The prototype is a method for determining the myeloperoxidase of neutrophilic leukocytes according to A.Sato (1927), (Kaplow LS Simplified myeloperoxidase Stain using benzidine dihydrochloride // Blood.- 1966. - Vol. 26. - N 3. - P. 215), based on oxidation benzidine system hydrogen peroxide - peroxidase with the formation of a colored reaction product. However, the blood sampling for cytochemical studies in the classical version is carried out from the patient’s finger or cubital vein. The study of blood taken directly from the wound barrier was not carried out, which significantly reduces the information content of the studies, since changes in a number of indicators in the peripheral blood are already observed with a fairly pronounced pathological process, when there is already a systemic response of the macroorganism in response to the spread of the infectious agent. In cases where the purulent process is local in nature, there may not be any obvious data suggesting functional depletion of neutrophilic leukocytes in the peripheral blood. While in the purulent focus in these periods there are already signs of developing decompensation.

 The purpose of the invention: providing early diagnosis of purulent-septic complications and predicting the course of the wound process.

 Objective: to increase the information content of existing methods for assessing the course of the wound process.

 The essence of the invention: the simultaneous study of indicators characterizing the functional state of the macroorganism both in the peripheral blood and directly in the purulent focus, with the subsequent determination of their correlation relationship.

 The method is as follows. In peripheral blood, studies are carried out according to generally accepted methods. Simultaneously with the blood sampling from the periphery, blood is taken from the purulent focus using a sterile capillary by scarification of the wound wall with a sterile instrument. The blood thus obtained is then studied also by generally accepted methods. Upon receipt of the results, the obtained data are compared in the peripheral blood and in the purulent focus.

 For example, cytochemical methods for determining the NBT test and myeloperoxidase of neutrophilic leukocytes, the physicochemical state of chromatin of the nuclei of leukocytes, and also the determination of the effective concentration of albumin in patients with purulent-inflammatory soft tissue diseases can be cited.

 The collection of material for the study (peripheral blood and blood from the wound barrier) was carried out from 9-00 to 10-00 daily during dressings using sterile heparinized capillaries. The study of enzyme systems was carried out according to standard methods, followed by the calculation of the SCK according to LSKaplow and the construction of histograms. (Kaplow LS Histochemical procedure for localizing and evaluating leukocyte alkaline phosphatase activity in smears of blood marrow // Blood. - 1955. - Vol. 10. - P 1023-1029).

 The results of the research.

 1. HCT test. The reaction for the determination of the HCT test was carried out according to the standard method of I.V. Nesterova (Nesterova I.V. Intra-leukocyte microbicidal system of neutrophilic granulocytes in healthy children with purulent-septic diseases: Abstract of thesis. Candidate of Medical Sciences. - M., 1980. - 20 p.). When studying the HCT test in the peripheral blood, a spontaneous HCT test was performed, in the wound, taking into account the contact of neutrophils with bacterial antigens, the HCT test was considered as stimulated. In this case, the HCT test in peripheral blood (HCT-p) characterized the degree of activation of neutrophils for certain disorders of homeostasis, that is, the severity of the inflammatory process, while the HCT test in the wound (HCT-p) characterized the functional reserve of neutrophils, revealing them potential ability to complete phagocytosis. Localized forms of purulent-inflammatory process with a favorable clinical course were characterized by a high degree of neutrophil activation in the HCT-p-test (2.19 ± 0.02) with low HCT-p-test values (1.31 ± 0.03), while in the torpid course of wound process, with a tendency to purulent-septic complications, there was a decrease in formazan-positive cells in the HCT-p test with a high degree of irritation of neutrophils in peripheral blood. It should be noted that the most informative were the cytochemical parameters in the wound, which directly correlated with the dynamics of the wound process and responded to changes within 4-6 hours (a repeated decrease in HCT-p together with MP-p with an increase in HCT-p, indicating the pathology of oxygen-dependent bactericidal mechanisms of neutrophils and (or) the humoral link of the phagocytic system), while in peripheral blood statistically significant differences in the dynamics of the indicators were observed only in severe purulent-septic complications phenomena and at a later date (table. 1, Fig. 1).

 2. Myeloperoxidase. On the first day of the postoperative period, all patients showed a significant decrease in the level of myeloperoxidase in the cells, accompanied by the release of enzyme crystals beyond the neutrophil (MP-p from 2.38 ± 0.02 decreased to 1.56 ± 0.06, MP-p from 2.46 ± 0.02 to 2.01 ± 0.02, respectively) , which was the result of the bactericidal and antitoxic effects of neutrophil in response to surgical trauma, and these changes were most pronounced in the wound (MP-p). In the next day of the postoperative period with uncomplicated course, a steady increase in the level of MP in the cell occurred (on the 7th day, MP-p 2.58 ± 0.01, MP-p 2.55 ± 0.02), which was inversely related to the level of bacterial contamination of the wounds.

 In the torpid course of the wound healing process, in baroactive conditions, or in the event of purulent complications, equalization of the MP-p index was not observed for a long period of time (Table 2, Fig. 2).

 3. The effective concentration of albumin. Albumin plays a large role in binding various toxic low- and medium-molecular weight organic substances that enter the bloodstream, thereby acting as an important link in the body's detoxification system. This ability is realized due to the presence in the albumin molecule of several active centers designed to bind various toxic substances. When toxic metabolites are combined with albumin, the number of free active centers in the molecule decreases, leading to a decrease in the effective concentration of albumin.

 Thus, this indicator can be used to assess the general intoxication of the body.

 The fluorescence measurement can be performed on any fluometer having an excitation wavelength in the region of 410-450 nm and a fluorescence detection wavelength in the region of 510-550 nm.

 In our studies, the AKL-01 "ZOND-3" analyzer was used. The analyzer is calibrated using the fluorescent calibrators included with the instrument, and the measurement result is presented on the instrument’s digital indicator in units of the effective concentration of albumin (ECA, g / l).

 As a result of the studies we obtained the following results (table. 3, Fig. 3). During the first three days after the operation, the total concentration in the blood and in the purulent focus of active albumin centers in all patients was approximately at the same level: ECA-p 60.0 ± 3.52 g / l; ECA-p - 20.1 ± 3.43 g / l (P <0.001). Subsequently, depending on the course of the wound healing process, the dynamics of ECA parameters in the peripheral blood, and especially in the wound, differed significantly. In the uncomplicated course of the wound healing process, the ECA-p index fluctuations during the entire observation period were insignificant, gradually increasing from 60 to 64.7 g / l by the 9th day of observation (P> 0.05), with the transition of the degenerative-necrotic and degenerative-inflammatory phases into regenerative in patients, there was a rather sharp increase in the ECA-p indicator, which towards the end of the observation period was close to the ECA in the cubital vein (61.3 ± 3.11 g / l). The most obvious was the dynamics of the ECA indicator in the torpid and complicated course of the wound process, and the most informative were these indicators in the wound barrier, which responded to purulent complications earlier and to a greater extent, while a significant decrease in ECA was observed in the peripheral blood only if the patient has severe purulent intoxication.

 In peripheral blood in such cases, ECA-p was 40.3 ± 3.18 g / l on the first day, reaching 43.5 ± 3.22 g / l on the 9th day, while in the wound, ECA-p progressively decreased from 1 to 5 day 7 with the occurrence of non-sanitized septic ulcers, gradually increasing to 14 ± 3.31 g / l on the 9th day after opening.

 A direct correlation relationship (r = 0.87) was observed for the dynamics of the indicators of effective albumin concentration with cytochemical indicators of neutrophilic leukocytes characterizing their bactericidal antitoxic potential (HCT-p-test, myeloperoxidase), the inverse correlation with HCT-p-test (g = 0.91) .

 4. Chromatin anisotropy of leukocyte cell nuclei. The physico-chemical state of the chromatin of leukocyte nuclei is an important criterion for assessing the functional state of the cell as a whole. An increase in the fraction of condensed chromatin indicates a decrease in the functional activity of cells, and its decrease is a sign of reverse processes. The degree of chromatin condensation can be determined by measuring the chromatin anisotropy of cell nuclei that occurs after they are stained with toluidine blue at pH 5.0. In a comparative study of the anisotropy of the nuclei of neutrophilic leukocytes obtained from peripheral blood and wound discharge of surgical patients, it was found that on the first day of the postoperative period, a statistically significant decrease in chromatin anisotropy of the nuclei of segmented neutrophilic leukocytes obtained from the surgical wound was observed compared with similar peripheral blood cells of these the same patients (table. 4, Fig. 4).

 The data obtained indicate that in the examined patients there is an activation of the tissue pool of segmented neutrophils.

 Thus, a comparative analysis of various indicators of homeostasis in the peripheral blood and in the wound can increase the information content of these methods when assessing the functional state of wound barriers and predicting the course of the wound process.

 Example.

 Patient V. - 57 years old (medical history N 32578) was admitted to the hospital on 04.24.96, 10 days after the illness with a post-injection abscess of the right gluteal region. From the anamnesis, it was found that on April 14, 1996, in a clinic 5 days ago, an intramuscular injection of rheopirin was performed for osteochondrosis of the lumbar spine.

The condition at admission is relatively satisfactory. A patient with increased nutrition. Body temperature 38.2 ^o C. Pulse 90 beats per minute satisfactory qualities. HELL = 160/90 mm Hg On examination, slight hyperemia and infiltration in the upper-outer square of the right gluteal region (20 x 10 cm). Blood test: red blood cells - 4.3 t / l, leukocytes - 9.0 g / l, stab - 9%, segmented - 77%, lymphocytes - 12%, monocytes - 2%, ESR - 27 mm / hour, blood sugar - 5.5 mmol / l.

 04.24.96, under intravenous anesthesia (sombrevin), an abscess was opened through the center of the infiltrate up to 20 cm in size - up to 100 ml of pus was obtained (bacteriological culture - Staphylococcus aureus in association with Escherichia coli). During the audit it was found that the abscess cavity measuring 16x18x4 cm has a multi-chamber structure. The treatment was carried out with the treatment of the purulent focus with low-frequency ultrasound, however, a significant improvement in the general well-being of the patient was not observed.

On the 3rd day of the postoperative period, the patient again had an increase in body temperature to 38 ^o C. The studies performed yielded the following results: MP-p - 2.51 cu (initial level - 2.46 cu), MP-p - 1.64 cu. e. (initial level - 2.38 cu); NST-p - 2.16 c.u. and 2.12 cu accordingly, NST-p - 1.67 c.u. and 1.98 cu respectively; ECA-p - 30.43 g / l and 40.35 g / l, respectively; ECA-p - 9.91 g / l and 21.11 g / l, respectively (Fig. 5). Thus, the above research results indicate: in the purulent foci on the 3rd day there is a sharp decrease in ECA-p, MP-p, HCT-p, indicating the presence of a non-sanitized purulent foci and progressive decompensation of the bactericidal-antitoxic potential of neutrophilic, white blood cells directly in the purulent focus, while in the peripheral blood these changes are delayed by a day and cytochemical indices indicate still preserved compensation of the macroorganism and there is no danger of purulent generalization process. Based on the available results, it is possible to choose therapeutic tactics for each specific patient.

 On April 28, 1996, a repeated audit of the purulent focus was performed, and a deep purulent pocket was revealed. Contraperture has been applied and drainage has been installed for suction-flushing drainage.

 On the eighth day from the moment of surgery, the wound defect is closed by secondary intention, the drainage is removed. The patient was discharged on the 10th day from the moment of admission with a good cosmetic effect.

Claims (1)

 A method for predicting the course of the wound process in purulent-inflammatory diseases of soft tissues, including the study of peripheral blood parameters characterizing the functional state of a macroorganism, characterized in that the parameters in peripheral blood and in blood taken from a wound are determined simultaneously, a correlation relationship is established between these indicators and with an inverse correlation relationship, a torpid or complicated course of the wound process is predicted, and with a direct correlation in aimosvyazi - uncomplicated wound healing process.

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