RU2484464C1 - Method for evaluating clinical effectiveness in vitiligo - Google Patents

Abstract

FIELD: medicine.

SUBSTANCE: exudation cells of a skin window are counted. The neutrophil percentage and the macrophage percentage are assessed, and a coefficient K is determined by formula: $$К=\frac{N\left(\%\right)}{М\left(\%\right)},$$

wherein N is a relative neutrophil count in the skin window exudation (%), M is a relative macrophage count (%). A positive response to treatment is stated if observing 10 ≥ K ≥ 1.5.

EFFECT: use of this method enables evaluating the correct therapeutic approach and monitoring the clinical effectiveness before the re-pigmentation signs occur.

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Description

The invention relates to medicine, namely to dermatology, and relates to a method for evaluating the effectiveness of treatment of vitiligo.

Vitiligo is an acquired chronic skin disease from the group of dyschromia, characterized by the appearance on the skin of depigmented spots of white color, prone to peripheral growth, fusion due to the absence or decrease in the content of melanin in the skin. According to the WHO, vitiligo affects from 0.2 to 8% of the total population (about 30 million people) in the world. The urgency of the problem of vitiligo is largely associated with its adverse effect on the psychosocial status of the patient [4].

As you know, the death of melanocytes can cause both exogenous and endogenous effects. Of the exogenous ones, traumatic injuries, constant friction, excessive insolation, chemical, infectious agents, and others should be distinguished. Of the endogenous today, the following pathological processes can be distinguished: neuroendocrine disorders, hereditary predisposition, mental injuries, disorders of the autonomic nervous system, and autoimmune disorders. None of the above factors can fully explain the cause of the disease [3]. The lack of a holistic concept of the pathogenesis of vitiligo complicates the development of effective and reliable methods of treatment, making it impossible to predict the clinical course of this dermatosis [2]. Despite the ease of diagnosis, there is currently absolutely no effective treatment for vitiligo. Regardless of the method used, repigmentation is slow and never complete. Currently, sufficient experience has been accumulated in studying the role of immunological changes in the pathogenesis of vitiligo. However, in most cases, the results of previous immunological studies reflect the state of the whole organism as a whole, therefore it is difficult to talk about the participation of certain cells of the immune system in the development of vitiligo [1].

For an adequate assessment of the situation in the affected area, you need to be well aware of the general principles of its development. Indirect signs indicate that the development of depigmentation may be due to the death of melanocytes on the background of a significant increase in the number and activity of intraepidermal macrophages, activation of proteins of the complement system as a result of pathological changes in the reactivity of the body. At the same time, increased migration of macrophages to the foci of depigmentation may be secondary and is caused by the death of melanin-producing cells under the influence of some other damaging agent. However, no direct confirmation of these hypotheses was obtained [2]. Of greatest interest is the study of the mechanisms of the pathological process in vitiligo directly in the foci of depigmentation.

The known method of the “skin window”, which allows to assess the ability of cells of the phagocytic series to chemotaxis to the site of aseptic inflammation, which is formed by scarification of the epidermis and is traditionally used to assess the state of the protective function of the skin [5]. Disturbances in the macrophage monocyte system are often combined with a weakening of the functions of neutrophils, and it is this mechanism (damage to the neutrophilic link of phagocytosis) that dominates the clinical manifestations of immunological deficiency. Defects in the phagocytic system, as well as immunity in general, often lead to damage to the mucous membranes and skin. Violations of reactivity should be sought not only in circulating cells. They can be stronger in the tissue pool, where the main reactions of phagocytes are realized. At the same time, there are few clinical works characterizing phagocytes from foci of inflammation [6].

Closest to the proposed is a method for evaluating the effectiveness of treatment of vitiligo by determining the state of free radical oxidation and antioxidant systems in the skin lesions in patients with vitiligo. Skin samples are taken, crushed, homogenized, aliquots of the supernatant are separated, the enzyme activity of the antioxidant system is determined spectrophotometrically, the free radical oxidation induction system is selected and the corresponding induction reaction is carried out. The method allows to determine the true ratio of free radical processes and anti-radical protection in damaged skin in vitiligo foci, to assess the severity of the pathological process and select therapy adequate to this skin condition [7].

The disadvantage of this method is the high risk of invasiveness, significant material costs, a large amount of time, the availability of qualified medical personnel. In addition, this method is painful for the patient, has a traumatic effect on the skin and cosmetic defect.

A new technical task is to develop a method for evaluating the effectiveness of treatment of vitiligo, which has accuracy and informativeness, which helps the practitioner to choose the right treatment for this disease.

To solve the problem in a method of evaluating the effectiveness of treatment of vitiligo by examining the characteristics of the patient’s skin cells in the depigmentation focus, determine the cellular composition of the exudate of the “skin window” before and after treatment, after which the coefficient K is calculated by the formula:

$$\mathrm{TO}=\frac{N(\%)}{M(\%)}$$

where H is the relative content of neutrophils in the exudate of the skin window (%), M is the relative content of macrophages (%), and at values of 10≥K≥1.5, the treatment is evaluated as effective.

New in the method is that they determine the presence and relative content of immunocompetent cells - mononuclear phagocytes and polymorphonuclear neutrophils on smears-fingerprints in the repigmentation site, determine their ratio in the exudate of the "skin window" before and after treatment and calculate the coefficient K by the formula:

$$\mathrm{TO}=\frac{N(\%)}{M(\%)}$$

where H is the relative content of neutrophils in the exudate of the skin window, M is the relative content of macrophages. At values of 10≥K≥1.5 after the course of therapy, treatment is assessed as effective.

In the absence of a clinical effect in the form of repigmentation, one can judge the positive or negative effect of the treatment by the dynamics of changes in the cellular composition of the “skin window” and the numerical value of the coefficient. If the values of 10≥K≥1.5 after treatment fall into the above interval, a delayed positive effect is predicted (repigmentation for the next 6 months).

With values of K <1.5, a lack of therapeutic effect from a set of measures selected for a given patient is noted and new therapeutic approaches are planned.

The proposed solution is new, because in the patent and medical literature analyzed by the authors no similar methods for assessing the effectiveness of the treatment of this disease were found.

The proposed method meets the criteria of the invention "inventive step", since this set of distinctive features does not follow explicitly for specialists from the prior art.

The proposed method for evaluating the effectiveness of treatment of vitiligo meets the criteria of the invention “industrially applicable”, as it was tested at the Department of Dermatovenereology SBEI HPE SibGMU of the Ministry of Health and Social Development of Russia.

The method is as follows.

1. The focus of depigmentation is disinfected with alcohol. Using a sterile disposable scalpel, remove the upper layer of the epidermis to a shiny layer without damaging the vessels with an area of 1 × 1 cm.

2. A sterile glass slide is placed on the scarified area and fixed with a band-aid.

3. After 6 hours, the glass is removed.

4. Imprint smears are fixed in ethyl alcohol for 10 minutes, stained with azure-zosin according to the Romanovsky-Giemsa method.

5. The smears are microscopic under a 90 × 10 immersion lens.

6. The results are expressed as a percentage of each cell type in inflammatory exudate. Keep track of the percentage of neutrophils and mononuclear phagocytes per 100 cells.

7. Calculate the coefficient K, reflecting the ratio of the percentage of neutrophils to the percentage of macrophages:

$$\mathrm{TO}=\frac{N(\%)}{M(\%)}$$

where H is the relative content of neutrophils in the exudate of the skin window, M is the relative content of macrophages. At values of 10≥K≥1.5, treatment is assessed as effective.

The proposed criteria of the method are based on the analysis of clinical observations.

It is known that inflammation is characterized by the active emigration of inflammatory cells from the blood to the area of damage. Neutrophils dominate in the early stage, then phagocytic cells of the macrophage system appear in the focus of inflammation. However, normally neutrophils remain the predominant type of cells in the lesion area for several days.

A survey was conducted of 34 patients with various forms of Vitiligo disease. As a control group, 13 healthy volunteers were examined.

The table shows the results of a survey of patients with vitiligo and a group of healthy volunteers.

When assessing the number of immunocompetent cells in healthy volunteers, the predominance of neutrophils in the early phase was revealed, which indicates a normal cellular response to damage. An analysis of “skin window” preparations in patients with vitiligo prior to treatment indicates the predominance of mononuclear phagocytes and a decrease in the yield of neutrophils in the focus of vitiligo. When analyzing the exudate of the “skin window” after treatment, a decrease in the number of macrophages in the composition of cell exudate and an increase in the coefficient K with successful treatment in 100% of cases were found.

An example of a specific implementation of the method

Example 1

Patient P., 20 years old. Medical history No. 1120

Diagnosis: Vitiligo, generalized form.

The diagnosis was confirmed by the presence of characteristic signs - spots of milky white color, with clear boundaries, rounded in shape were located on the extensor surface of the elbow joints, the inner surface of the wrists, in the axillary areas, the skin pattern was preserved in the foci, there were no signs of atrophy.

Disease experience: 2 years.

Hospitalized 02.24.11.

According to the proposed method, the next day after hospitalization, the composition of the exudate of the “skin window” was determined before treatment. The results: macrophages - 63%, neutrophils - 37%, indicator K is equal to a value of 0.5, which is significantly different from the control norm.

As a basic therapy, the patient is prescribed: 0.05 g of zinc oxide 3 times a day, 30% sodium thiosulfate solution 10 ml intravenously, intramuscular injections of 1 ml of 6% thiamine bromide solution, 5% vitamin B ₆ solution, 5% ascorbic solution acid, 1% nicotinic acid solution. The course is 15 days. Ultrafonophoresis with hydrocortisone ointment is on the foci of depigmentation.

The day after the end of treatment, a control study of the exudate of the “skin window” was carried out, which showed a decrease in the percentage of macrophages to 28%, the number of neutrophils increased to 78%. The value of K reached 3. In the foci of depigmentation, a clinical result was achieved in the form of perifollicular repigmentation by the end of the first course of treatment. These results indicated a correctly selected comprehensive treatment for this patient.

Example 2

Patient B., 23 years old. Case history No. 1095.

Diagnosis: Vitiligo, generalized form.

Concomitant pathology: Diabetes mellitus, insulin-dependent type.

The diagnosis was confirmed by the presence of spots of milky white color, with clear boundaries, round or oval in shape with localization on the skin of the face, lateral surfaces of the trunk, rear of the hands, feet. In the foci of depigmentation, the skin pattern is preserved, there are no signs of atrophy.

Disease experience: 15 years.

Hospitalized 02.22.11.

According to the proposed method, the next day after hospitalization, the skin window technique was applied, and the composition of the exudate was determined.

The results: macrophages - 88%, neutrophils - 12%, value K = 0.1, which differs significantly from the parameters of the group of healthy volunteers.

As a basic therapy, the patient received 0.05 g of zinc oxide 3 times a day, 10% calcium gluconate solution 10 ml intravenously, intramuscular injections of 1 ml of 6% thiamine bromide solution, 5% vitamin B ₆ solution, 5% ascorbic acid solution , 1% nicotinic acid solution, course 15 days. Ultrafonophoresis with hydrocortisone ointment is on the foci of depigmentation.

The day after the end of the course of therapy, a control study was conducted, where changes in the quantitative composition of the “skin window” were recorded - the number of neutrophils increased to 61%, the number of macrophages decreased to 39%. Level K was 1.5. In this clinical case, a distant clinical result was observed three months later - the foci of depigmentation acquired fuzzy borders on the rear of the hands, the lateral surfaces of the trunk — edge repigmentation, and the absence of fresh spots of depigmentation.

Despite the absence of clinical changes immediately after treatment (in the second example), positive changes in the cellular composition of the “skin window” were recorded, which was confirmed by a delayed positive clinical result.

Example 3

Patient F., 28 years old. Case history No. 5046.

Diagnosis: Vitiligo, generalized form.

Concomitant pathology: Chronic opisthorchiasis.

The diagnosis was made by the presence of spots of finely white color with clear boundaries, localized on the back of the hands, face, forearms, and lateral surfaces of the trunk. Spots of a rounded shape, prone to fusion, have clear contours. There are no signs of atrophy, the skin pattern is saved.

Disease experience: 1 year.

Hospitalized on 08.29.11.

The next day after hospitalization, the composition of the exudate of the “skin window” was determined by the proposed method.

The results: macrophages - 72%, neutrophils - 28%, the value of K = 0.3, which significantly differs from that of the control group.

As an external treatment, a hydrocortisone ointment with the influence of a physiotherapeutic procedure - ultraphonophoresis was prescribed on the foci of depigmentation. In addition to external therapy, the patient received zinc oxide 0.05 g 3 times a day, 30% sodium thiosulfate solution 10 ml intravenously, intramuscular injections of 1 ml 6% thiamine bromide solution, 5% vitamin B ₆ solution, 5% ascorbic acid solution, 1% nicotinic acid solution.

In each clinical case, the full course of treatment is 15 days.

After treatment, a control study was conducted, where the approximation of the quantitative composition of the “skin window” to the control values was not registered - the number of neutrophils 31%, the number of macrophages - 69%. The value of K remains significantly low 0.4.

In this clinical example, the treatment effect was assessed as unsatisfactory, lack of repigmentation, as well as the dynamics of the quantitative composition of the exudate of the “skin window”. The patient was prescribed to correct the treatment taking into account the concomitant pathology and repeat the course of therapy.

Thus, the proposed method is highly informative, especially in the absence of signs of repigmentation immediately after the end of the course of treatment.

Studies have shown that the sensitivity of the method is 90%, specificity is 75%. In addition, the method is quite simple for practicing physicians and is applicable in the conditions of a clinical diagnostic laboratory, since it does not require expensive reagents and equipment for its formulation.

Practical application of the method is possible for intermediate monitoring of the effectiveness of treatment until signs of repigmentation appear, which in turn will help in choosing the right method of treatment for this dermatosis.

Information sources

1. Dvoryankova E.V., Tkachenko S.B., Korsunskaya I.M. Features of the immunological status in vitiligo patients / Experimental and clinical dermatocosmetology. Scientific and practical journal. - 2006. - No. 2. - S.9-11.

2. Korsunskaya I.M., Dvoryankova E.V., Efremova E.I. The experience of the use of polyoxidonium in the treatment of vitiligo / Immunology, - 2005. - No. 4. - S.236-238.

3. Koshevenko Yu.N. Vitiligo. Clinic, etiology, pathogenesis, treatment, rehabilitation, prevention. - M.: “Cosmetics and Medicine”, 2002, p. 281-339.

4. Lomonosov K.M. Immunopathogenesis and therapy of vitiligo immunocorrector neovir / Russian Journal of Skin and Sexually Transmitted Diseases. - 2010. - No. 2. - S. 36-39.

5. Mayansky D.N. Lectures on clinical pathology. A guide for doctors. - “GEOTAR - Media”. - 2007. - P.464.

6. Mayansky A.N., Pikuza O.I. Clinical aspects of phagocytosis. - Kazan "Magarif". - 1993 .-- 192 s.

7. Patent RU 2241992 C1, G01N 33/48. A method for determining the state of free radical oxidation and antioxidant systems in areas of skin lesions in patients with vitiligo / I.M. Korsunskaya, E.V.Dvoryankova, E.I. Sazontova, Yu.V. Arkhipenko, E.I. Efremova, A.G. Zhukov. - No. 2003115963/15; Stated May 29, 2003; Publ. 12/10/2004.

Characterization of the exudate "skin window" in patients with vitiligo and healthy volunteers Patient Characterization The composition of the cell exudate TO Macrophages,% Neutrophils,% Before treatment After treatment Before treatment After treatment Before treatment After treatment Patients with a clinical result and positive dynamics of the cellular composition of the “skin window” (n = 25) 56.3 (37.1-77.1) p <0.05 17.0 (13.5-28.0) 39.8 (20.6-57.1) p <0.05 83.0 (72.0-86.5) 0.7 (0.1-0.7) p <0.05 4.0 (1.5-6.0) Patients with a long-term (1-6 months) clinical result and positive dynamics of the cellular composition of the “skin window” (n = 4) 64.7 (53.1-7.08) p <0.05 21.7 (9.0-37.0) 35.2 (22-47) p <0.05 78.2 (63.0-91.0) 0.5 (0.3-0.8) p <0.05 4.8 (1.7-10) Patients who did not achieve treatment results (n = 4) 69.7 (57.0-87.0) p <0.05 66.2 (49.0-75.0) p <0.05 30.2 (13.0-43.0) p <0.05 33.7 (25.0-51.0) p <0.05 0.4 (0.2-0.7) p <0.05 0.5 (0.3-1.0) p <0.05 Group of healthy volunteers (n = 13) 13.5 (10.8-15.3) 83.2 (78.9-86.0) 6.1 (5.1-7.9) Note: p is the level of significance of differences from the corresponding indicator in the group of healthy volunteers, K is the coefficient characterizing the ratio of the relative content of neutrophils and macrophages in the composition of the cell exudate of the “skin window”.

Claims (1)

A method for evaluating the effectiveness of treatment of vitiligo by examining the characteristics of the patient’s skin cells in the depigmentation focus, characterized in that the cellular composition of the exudate of the “skin window” is determined before and after treatment, after which the coefficient K is calculated by the formula:
$$\mathrm{TO}=\frac{N\left(\%\right)}{M\left(\%\right)},$$

where H is the relative content of neutrophils in the exudate of the “skin window” (%), M is the relative content of macrophages (%), and at values of 10≥K≥1.5, the treatment is evaluated as effective.