RU2616239C2 - Method for erosive and ulcerative skin lesions treatment - Google Patents

Abstract

FIELD: pharmacy.

SUBSTANCE: method for autologous platelet-rich plasma manufacture includes blood withdrawal from patient's peripheral vein into vacuum tubes, blood mixing with sodium citrate by tubes revolution, centrifugation, platelet-rich autologous plasma upper layer exctration into a separate tube, platelet-rich autologous plasma exctracted upper layer activation by calcium ions by calcium gluconate addition into the tube, the obtained liquid is stirred by tube revolution under certain conditions. The method for erosive and ulcerative skin lesions treatment by means of autologous platelet-rich plasma.

EFFECT: method allows to obtain autologous platelet-rich plasma, with intense reparative having activity against erosive and ulcerative skin lesions, the resulting effect is persistent and characterized by absence of recurrence.

9 cl, 1 ex

Description

The invention relates to medicine, namely to dermatovenerology, and can be used to treat erosive and ulcerative skin lesions resulting from various causes, for example, with chronic venous insufficiency, as well as for the treatment of certain skin diseases accompanied by an inflammatory reaction: acne, rosacea , seborrheic dermatitis, etc.

Erosive-ulcerative skin lesions in the presence of chronic venous insufficiency (CVI), according to domestic and foreign researchers, are found in 2% of the population. Against the background of diabetes mellitus (DM), they occur in 8-10% of patients and present a serious interdisciplinary medical problem, in the solution of which dermatovenerologists are largely involved. About 67% of patients suffering from erosive and ulcerative lesions of the lower extremities become disabled, and in 81% of patients the quality of life decreases, which is reflected in disability. In this regard, despite the variety of treatment methods and a wide arsenal of drugs, the problem of treating erosive and ulcerative skin lesions is still relevant.

More than 200 conservative and surgical methods are known, and up to 1000 various systemic and external preparations are used both independently and in combination for the treatment of erosive and ulcerative skin lesions.

Along with pathogenetic and etiotropic systemic therapy in the treatment complex, the most important place belongs to local therapy of erosive and ulcerative skin defects.

Means for local treatment of skin lesions are conditionally divided into several groups: antiseptic solutions; proteolytic enzymes; ointments and creams on a water-soluble basis; wound coverings; biological products, etc.

A known method of conservative treatment of erosive and ulcerative skin defects (The effectiveness of the drug Ebermin in the treatment of trophic ulcers of venous etiology, LI Bogdanets, VN Lobanov, AI Kiriyenko. Journal "Phlebology". 2009, No. 4, p. 9-14), including the combined use of ointments and creams: the ebermin preparation, which is a hydrophilic ointment, 100 g of which contains 1 mg of recombinant human epidermal growth factor (EHF) and 1 g of silver sulfadiazine (SDS), which are applied directly to skin defect surface. Dressings are performed daily or every other day, depending on the clinical situation. Treatment is carried out for 2 months with follow-up visits 1 time per week.

The disadvantage of this method is its low efficiency. The healing of a ulcerative defect occurs only in 47% of cases, frequent complications in the form of allergic dermatitis and eczema, the development of secondary infection and, as a result, the chronicity of the course of the pathological process.

Currently, wound coverings and biological products that are applied directly to the defect area are increasingly used for conservative treatment, which, due to the biologically active substances contained in them, leads to its healing.

There is also known a method of treating erosive and ulcerative skin lesions (The use of the living skin equivalent in the complex treatment of patients with trophic ulcers of the lower extremities of venous etiology / Ivashkin A.N., Fominykh E.M., Maksimenko V.N., Gasanov I.K., Smirnov A.V., Fedorov D.N., Dashinimaev E.B., Kiseleva E.V. Military Medical Journal, 2009, No. 11, p. 51-52), including the use of wound dressing, which is a living skin equivalent. It is applied to the area of skin damage pretreated with an isotonic sodium chloride solution by simultaneous displacement at the rate of 1 cm2 of graft per 1 cm2 of ulcer surface. A wound cover is placed on top of the graft so that it is captured at least 1-2 cm from the edges of the ulcer. Then a dry gauze napkin is applied from above without pressure and bandaging is performed. The first ligation is carried out on the 4th-5th day after transplantation.

The principal disadvantages of this method using modern wound dressings are: high cost, laboriousness and complexity of execution, the need for preliminary cleaning of the wound surface, the rapid absorption of the active substance in the dressing and the impossibility of creating the necessary concentration of the drug that directly affects the pathological process.

Similar disadvantages have the majority of modern methods of treatment of erosive and ulcerative skin defects based on biological products.

As the closest analogue, a method has been adopted for treating patients with erosive and ulcerative skin lesions, in which the treatment efficiency is improved by using growth factors in the accompanying therapy complex (Autologous Platelet Rich Plasma in Chronic Venous Ulcers: Study of 17 Cases // J. Cutan Aesthet Surg . 2013; 6 (2): 97-99).

From the patient's peripheral vein, 20 ml of blood is sampled into tubes containing citric acid, sodium citrate and dextrose in a ratio of 9: 1 (blood: anticoagulant), centrifuged at a speed of 5000 rpm for 15 minutes to separate red blood cells from plasma with platelets. Plasma with platelets and white blood cells are centrifuged again at a speed of 2000 rpm for 5-10 minutes. Then take the lower layer of the obtained plasma - about 1.5 ml, add 10% calcium chloride in the ratio: 0.3 ml per 1 ml of plasma.

A coating impregnated with autologous platelet-rich plasma is applied to the erosive-ulcerative skin defect and dressing is done once a week until the ulcer is completely epithelized. The procedure is repeated for 6 weeks.

However, the known method does not allow to obtain a pronounced reparative effect in the treatment of erosive-ulcerative skin lesions, since in the process of preparing an autologous platelet-rich plasma at such a high speed of rotation of the centrifuge due to mechanical and chemical effects, growth factors are destroyed. In this regard, the application of liquid plasma with preserved growth factors is insufficient to effectively affect the tissue affected by the ulcer process.

In addition, due to the need for double centrifugation, the method is extremely time-consuming, which complicates its use.

The objective of the invention is to provide a simpler highly effective method of treating erosive and ulcerative skin lesions, which allows to obtain a pronounced and persistent clinical effect in a short time.

The essence of the invention lies in the fact that in a method of treating erosive and ulcerative skin lesions, including taking blood from a patient's peripheral vein, mixing it with an anticoagulant, centrifuging and subsequent exposure to a skin defect of an obtained autologous platelet-rich plasma, the blood is centrifuged for 5 minutes with centrifuge speed of 800 rpm, the extracted top layer of platelet-rich autologous plasma in a separate tube is activated with calcium gluconate at a rate of 50 μl per 1 ml of plasma, ready-to-use plasma is used for 5-10 minutes, while at least a third of the plasma obtained in liquid form is collected in 1 ml insulin syringes and an ulcer-erosive defect is punctured around the periphery intradermally at the rate of 0.1- 0.5 ml for each injection, the remaining plasma is placed in a glass Petri dish and placed in a thermostat for 20 minutes at 37 ° C until a transparent thin film of pale yellow color is formed, which is applied to the ulcer defect in the form of an application, closed with a dry cloth and bandage for 24 hours CA, the procedure is carried out once a week until the defect is completely healed.

Using the invention allows to obtain the following technical result.

The method provides a pronounced clinical effect, which is manifested in an intensive reparative process and provides fast (within 2-5 weeks) and complete healing of the defect.

The resulting effect is persistent and is characterized by the absence of relapse.

The method is simple and quick to execute. To prepare autologous platelet-rich plasma, only a single, slow, short-term (within 5 minutes) centrifugation is required.

The method is also cost-effective, as it allows the use of conventional laboratory equipment with low cost.

The method has wide functionality. It can be used to treat damage to the skin, has no age restrictions and contraindications due to the fact that the plasma is autologous, can be used in medical institutions of any type and can be used for treatment in both inpatient and outpatient settings.

Since the effect is pronounced and quick, the social adaptation of patients improves and their quality of life improves.

The technical result is achieved due to the new original technology developed by the authors for the preparation and combined use of autologous platelet-rich plasma, while the technique developed by the authors provides for a significant simplification of the process of obtaining autologous plasma and allows to preserve the integrity of platelets containing growth factors, to ensure their delivery and preservation in the outbreak due to the combined sequential effect on the affected area, during the implementation of which first, plasma is injected intradermally in liquid form by cleaving the defect, and then after the film is formed from the remaining liquid form in the thermostat, the latter is applied to the affected area as an application.

The authors first proposed the use of a single slow centrifugation mode with a centrifuge speed of 800 rpm for 5 minutes, which allows to preserve the integrity of platelets containing growth factors that, due to the very high centrifugation speed, can be mechanically injured and / or enter into chemical interactions for due to an increase in temperature, and all this leads to premature platelet degranulation, the destruction of growth factors and, as a result, to the absence of growth ennogo clinical effect.

In addition, due to short-term centrifugation for 5 minutes, platelets do not have time to sink to the bottom of the tube and mix with the layer of leukocytes and red blood cells, but remain suspended in the upper part of the tube, evenly distributed in the plasma, which does not require plasma separation or centrifugation.

Short terms for plasma production and ease of preparation allows the use of this technique in routine medical practice in institutions of any profile.

Due to the combined injection and application of plasma, developed and proposed by the authors, growth factors remain in the lesion for a long time, thus managing to realize their biological effect, attract fibroblasts into the focus, which further synthesize collagen, necessary to restore tissue integrity.

The use of a film from platelet-rich plasma in the form of an application, rather than a plasma in liquid form, also allows you to maximize the positive effect.

Calcium gluconate, used as an activator substance for platelets, stimulates the most complete degranulation of platelet alpha granules with subsequent release of growth factors, without causing side effects when it is introduced into the tissue.

The use of autologous plasma within 5-10 minutes after activation excludes the possibility of platelet precipitation and mixing, and also allows all growth factors to reach the point of application in active form.

At least one third of the obtained autologous plasma is used to chunk the ulcer around the periphery, and a film for application to the ulcer surface is obtained from the remaining two thirds, which makes it possible to evenly distribute the volume of the obtained plasma. When calculating the optimal dose of liquid plasma for one injection - 0.1-0.5 ml, the authors proceeded from taking into account the area of the ulcer and the plasma volume necessary to achieve a clinical effect, pain for patients.

No available sources of information have been found in the available literature, including information on the production and use of platelet-rich plasma by the method proposed by the authors.

The method is as follows.

From the patient's peripheral vein, blood is drawn into vacuum tubes of 3–9 ml in volume (depending on the size of the ulcer).

A 3.8% sodium citrate solution is used as an anticoagulant. To mix blood with citrate, the tubes are inverted 4-5 times. Then the blood is centrifuged for 5 min with a centrifuge speed of 800 rpm. During rotation, the blood in the test tubes is divided into three layers: the lower is red blood cells, the middle is a thin layer of white blood cells, and the upper is plasma enriched with platelets.

Using a laboratory dispenser, the top layer of platelet-rich autologous plasma is removed into a separate tube. After that, it is activated by calcium ions: per 1 ml of plasma - 50 μl of calcium gluconate.

The fluid is mixed by carefully turning the tubes. Ready-to-use autologous plasma is used for 5-10 minutes.

The plasma volume required for the procedure depends on the size of the erosive-ulcerative defect (from 4 ml - small ulcers, up to 16 ml - extensive).

At least a third of the autologous plasma obtained in liquid form is collected in 1 ml insulin syringes. An ulcer is punctured on the periphery intradermally at the rate of 0.1-0.5 ml for each injection.

The remaining 2/3 of the plasma volume is placed in a glass Petri dish and placed in a thermostat with a temperature of 37 ° C for 20 minutes until a transparent thin film of pale yellow is formed, which is applied to the ulcer defect in the form of an application, covered with a dry cloth and bandaged for 24 hours The procedure is carried out once a week until the ulcer defect is completely healed.

The method was applied in clinical conditions in 43 patients (volunteers) who complained of an erosive and ulcerative skin defect of the lower extremities.

The age of the patients ranged from 58 to 95 years, the median age was 72 years, of which 63% were women and 37% were men.

The study found that the number of patients in whom an ulcerative process of the lower extremities occurred on the background of CVI was 2.1 times higher than the number of patients with diabetes (68 and 32%, respectively). Disease duration - up to 6 months, 2.3 times prevailed over that over 6 months (70 and 30%, respectively).

Complete relapse-free epithelization of a ulcer during 2-4 weeks was observed in 33 patients (77%). In 10 patients (23%), ulcer epithelization by more than 50% occurred during this time. The treatment was continued 1 time per week. Complete epithelization was recorded in 8 patients (18%) within 5 weeks, in 2 patients (5%) within 6 weeks. No adverse effects reported. Patients returned to their usual way of life, quickly adapted socially.

Example 1. Patient G., 67 years old. Diagnosis: Ulcer of the left leg. A history of chronic venous insufficiency due to varicose veins. The duration of the ulcer is 6 months.

The patient is treated with the proposed method.

12 ml of blood were taken from peripheral blood (in 4 vacuum tubes of 3 ml each). 3.8% sodium citrate solution was used as an anticoagulant. Then centrifugation was carried out for 5 min with a centrifuge speed of 800 rpm. During rotation, the blood in the test tubes was divided into three layers: the lower — red blood cells, the middle — a thin layer of leukocytes, and the upper — plasma enriched with platelets.

Using a laboratory dispenser, the top layer of autologous plasma was extracted into a separate tube. Then, platelet-rich plasma was activated with calcium ions: 50 μl of calcium gluconate per 1 ml of plasma.

Mixing of the liquid was carried out by carefully turning the tubes. Received 5 ml of platelet-rich plasma.

2 ml of the obtained plasma was collected in 1 ml insulin syringes. An ulcer was injected peripherally intradermally at the rate of 0.1 ml per injection.

The remaining plasma volume was placed in a glass Petri dish and placed in a thermostat for 20 minutes with a temperature of 37 ° C until a transparent thin film of pale yellow color was formed, which was applied to the ulcer defect, covered with a dry cloth and bandaged for 24 hours.

The procedure was carried out once a week for 3 weeks until the ulcer defect was completely healed.

The healing process went smoothly, without complications and side effects. The patient was able to return to normal life. Socially adapted. Relapse of the disease is not observed.

Clinical trials have shown that the proposed method is simple and highly effective for the treatment of erosive and ulcerative skin lesions. It allows you to get a pronounced and persistent clinical effect in a short time.

Claims (9)

1. A method of manufacturing an autologous platelet-rich plasma, characterized in that blood is taken from the patient's peripheral vein into vacuum tubes, the blood is mixed with an anticoagulant, which is used as a solution of sodium citrate, the blood is mixed with the anticoagulant by flipping the tubes, and the blood is centrifuged with the anticoagulant in for 5 min with a centrifuge rotation speed of 800 rpm, the upper layer of platelet-rich autologous plasma is removed into a separate tube, activated treated upper layer of platelet-rich autologous plasma with calcium ions by adding calcium gluconate to the tube, mix the resulting liquid by flipping the tube. 2. A method of manufacturing a plasma according to claim 1, characterized in that the blood from the peripheral vein of the patient is taken into vacuum tubes with a volume of 3-9 ml. 3. A method of manufacturing a plasma according to claim 1, characterized in that a 3.8% sodium citrate solution is used as an anticoagulant. 4. A method of manufacturing a plasma according to claim 1, characterized in that the blood with the anticoagulant is mixed by flipping the tubes 4-5 times. 5. A method of manufacturing a plasma according to claim 1, characterized in that to activate the extracted upper layer of platelet-rich autologous plasma, calcium gluconate is added at the rate of 50 μl per 1 ml of plasma. 6. A method of treating erosive-ulcerative skin lesions using an autologous platelet-rich plasma made by the method according to claim 1, characterized in that within 5-10 minutes from the time of plasma production, part of the plasma in liquid form is collected into syringes and chipped ulceratively erosive defect on the periphery intradermally at the rate of 0.1-0.5 ml per each injection, the remaining plasma is placed in a glass Petri dish and placed in a thermostat at a temperature of 37 ° C until a transparent thin film of pale yellow color is formed, which put on a peptic ulcer in the form of an application, cover with a dry cloth and bandage. 7. The treatment method according to p. 6, characterized in that the plasma is collected in insulin syringes with a volume of 1 ml. 8. The treatment method according to p. 6, characterized in that the glass Petri dish and put in a thermostat for 20 minutes 9. The treatment method according to claim 6, characterized in that the ulcer defect is bandaged for 24 hours, while the procedure is carried out once a week until the erosive-ulcer defect is completely healed.