The method for determining the activity of leprosula process

 

The invention relates to medicine, namely to laboratory research methods. The essence of the method: assess the degree of accumulation of lipids in peripheral blood leukocytes of the patient, which calculate an average cytochemical coefficient and its value is 2.6 units, and below determine the presence of active leprosula process. The method is simple to perform, does not require costly maintenance can be carried out both in laboratory and in field conditions, while providing an accurate evaluation of the activity leprosula process. 1 table, 3 Il.

The invention relates to medicine, namely to laboratory methods, and can be, in particular, used to determine the activity leprosula process.

From the practice of medicine known method for determining the activity of leprosula process laboratory methods of research status of T - and b - systems of immunity, consisting of quantitative and functional assessment of cells of hematopoietic origin T - and b-lymphocytes (Seleznev S. P. the Relationship of the characteristics of the clinic and immune disorders in patients with leprosy: a dissertation on the need to parallel execution 7 immunological laboratory methods, that requires the researcher to certain special skills training, the use of peripheral (both venous and capillary) blood of the patient as a biological material and the impossibility of long-term storage, the presence of a large number of expensive and not readily available reagents, and the long duration of the complete method, which does not allow to obtain a specific technical result, simplification of the way.

There is also known a method of determining the activity of leprosula process laboratory investigation, namely, that determine the dependent nature of the pathologic process nonspecific functional activity of cells of hematopoietic origin of peripheral blood phagocytes (Pervukhin Y. C., Nazarov, K. I., Evstratova C. A., Loginov C. K., Kurochkin B. I. Functional activity of phagocytes in patients with leprosy. // Scientific notes of the Institute for the study of leprosy. Astrakhan, 1972, No. 7(12), S. 141-144). The disadvantages of this method are: the impossibility of long-term storage of biological material, which requires the implementation of the method immediately after the blood of the patient, a certain ratio of the number is used for the study of sediment nonspecific test microbe constant concentration, multiple preparation of blood smears after incubation at a constant temperature with a suspension of non-specific test microbe, and consequently, the need for a stationary thermostat, the possible impact on the estimated rate of comorbidity of both communicable infectious nature that makes it necessary to conduct additional, more sophisticated laboratory methods. All of the above ultimately not possible to obtain a specific technical result, simplification of the way.

Closest to the present invention is a method for determining the activity of leprosula process laboratory research technique lies in the fact that in cells of hematopoietic origin, tissue macrophages leprosy granuloma determine the degree of accumulation of lipids (D. S. Ridley. Skin Biopsy in Leprosy. Histological Interpretation and Clinical Application. - Basel, 1977, 57 p.). The similarity of this method to offer is that they both relate to laboratory research methods based on the determination of the degree of accumulation of lipids in cells of hematopoietic origin.

The disadvantages of this method are:

is a multistage process consisting of a fence Mat is awakening alcohols of different concentrations of preparation of multiple histological sections on a microtome and staining for lipids with additional color on acid-fast bacteria, microscopic examination at high magnification, which ultimately determines the large complexity and technical complexity in the implementation of the method even for the researcher possessing certain skills and vocational training;

most traumatic way as a result of repeated physical and psychological trauma patient due to the sampling of the material by the method of minor surgery - biopsy of the area of the skin with specific leprosie changes within apparently unaltered skin that requires prior anesthesia, observance of the rules of asepsis and the optimal choice of location, depth and size byobserving part of the skin that affects the outcome of the study;

- the need to use a large number (at least 22 items) expensive, scarce and toxic chemicals;

- long duration of the method is not less than 110 hours;

- expensive technical maintenance of way (cutting instruments, microtome, microscope, providing biomorphological laboratory.

Thus, these shortcomings do not allow to obtain a specific technical result, simplification of the way.

The present invention solves the main task is to facilitate the method.

The essence of the invention is expressed by a set of essential features that are sufficient to provide the technical invention of the result.

The problem is solved in the invention by the fact that determine the degree of accumulation of lipids in peripheral blood leukocytes and on average cytochemical coefficient (CCS) 2,6 conventional units and below judge activity leprosula process.

The simplified method gives the possibility to exclude the four stages of the implementation of the prototype method, namely the extraction of the material from the patient using biopsy transactions alcohols of different concentrations of the preparation of microcuts and their staining acid-fast bacteria and microscopic examination under high magnification, which allows for a shorter period of time (approximately 50 times smaller in comparison with the method of the prototype) to carry out an individual approach to the choice of dose and duration of chemotherapy, assess its effectiveness and the possibility of transfer of the patient to outpatient treatment; implementation of the stationary equipment and a large number of expensive, toxic agents and characterized required to work in the field of low invasiveness of sampling of biological material and the possibility of long-term storage.

The problem of determining the activity of leprosula process remains one of the most important in modern leprology. While its most important aspect is the timely detection of signs of active disease, which, on the one hand, promotes adequate individual assignment leprosy drugs and reduces the risk of dozozawisimy side effects sulfones, and on the other hand, allows you to assess the effectiveness of the treatment and the possibility of transfer of the patient to outpatient treatment.

The proposed method is as follows. Prepare the drug - peripheral smear (venous or capillary) of the patient's blood on a glass slide according to the standard technique (in. A. Menshikov, 1999). The product is dried in air at room temperature protected from light. Next drug fix in pairs, 40% formalin for 10 minutes and paint it according to the method of E. Pier (1962) a solution of Sudan black B, which was prepared in advance as follows: dissolve 0.7 g is 100With in a few minutes. Hot dye solution is passed through a four-layer paper filter. The dye solution is cooled to room temperature and filtered through four-layer paper filter one more time. Immediately the color of the drug solution of Sudan black In is as follows: the product is transferred into a vessel with a dye solution for 7 minutes, during which the vessel from time to time shake. Then the product is transferred into a vessel with an 85% solution of ethylene glycol for 3 minutes, during which the vessel from time to time shake. Then the product is transferred into a vessel with a 50% solution of ethylene glycol for 3 minutes, during which the vessel from time to time shake. After this drug carefully washed for one minute in a warm (+30-35C) running water. Dried in the air looking at the drug under the microscope (oil immersion system, the lens h, eyepiece X7 or X10). 100 viewed peripheral blood leukocytes, depending on the intensity of their specific colouring divided into four groups: with slabopolojitelen (1+), positive (2+) and strongly positive (3+) and maximum (4+) specific staining (Fig.1).

Quantity is Regni cytochemical coefficient (CCS) - calculated by the formula

The CCS is a quantitative expression of the determination of the degree of accumulation of lipids in peripheral blood leukocytes in conventional units;

And the number of peripheral blood leukocytes in the group with slabopolojitelen specific staining (1+) in percent to 100 viewed in the product;

B - the number of peripheral blood leukocytes in the group with positive specific staining (2+) in percent to 100 viewed in the product;

In the number of peripheral blood leukocytes in the group with strongly positive specific staining (3+) in percent to 100 viewed in the product;

The number of peripheral blood leukocytes in the group with the maximum specific color (4+) in percent to 100 viewed in the product;

1, 2, 3, 4 - the number of pluses corresponding to each of the four groups, viewed in the product 100 peripheral blood leukocytes;

100 - the total number reviewed in the preparation of peripheral blood leukocytes in percent.

All used reagents were valid for laboratory research category of quality. The work was carried out using a microscope Patriotic about is whether the MICROSOFT EXCEL spreadsheets with calculation the following statistical parameters: arithmetic mean (M), the mean-square deviation (S) and student criterion.

Summary of the invention illustrated by table and Fig.1-3.

The table shows that the average value (M) medium cytochemical coefficient (CCS) in patients with leprosy with signs of activity leprosula process (the first group) was 2.0 conventional units. Taking into account the mean-square deviation (S) upper bound of the allowable range (Mą2S) was equal to 2.6 conventional units. She was adopted as a criterion, if the values are equal and below which patients with leprosy were identified active leprosy process. In the group of patients who have leprosy process was characterized by the absence of signs of activity and was at the stage regression (the second group), the average value of the cytochemical ratio was 3.1 conventional units. Thus the lower bound of the confidence interval with accounting standard deviation corresponded to 2.7 standard units. The examined group of patients significantly (P <0.001) and differed in the degree of accumulation of lipids in peripheral blood leukocytes. Thus, in patients with leprosy with signs of activity leprosula process is marked camping this indicator as a laboratory test for determining the activity of leprosula process in patients with leprosy.

Fig.1 - the group of peripheral blood leukocytes with different intensity of specific staining:

1+ - kernel does not contrast or low contrast; staining of the cytoplasm at the periphery of the cells in the form of a thin pale blue bezel;

2+ - core has blurred the contours on the background of diffuse painted in pale blue cytoplasm;

3+ - kernel clearly contractarian on the background of intensely colored blue cytoplasm;

4+ - core has sharp contours; diffuse staining of the cells in blue color with the maximum location of the dye on the periphery of the cytoplasm; the deposition of dye in the form of individual granules.

Fig.2 - unit with slabopolojitelen specific color of the peripheral blood leukocytes of a patient with leprosy signs of activity leprosula process, indicating a low degree of accumulation of lipids in them;

Fig.3 - group of peripheral blood leukocytes of a patient with leprosy with no signs of activity leprosula process that have slabopolojitelen, positive and maximum specific colour, indicating a relatively higher degree of accumulation of lipids in comparison with patients with leprosy, with signs aktivnosti on 87 patients with leprosy during 2000-2002. From all examined patients fourteen patients at the time of the study had clinical manifestations activity leprosula process, which were confirmed by laboratory methods. The remaining 73 people the disease was characterized by the absence of clinical and laboratory signs of activity leprosula process. All patients were divided into two groups. The first group included patients with signs of activity leprosula process (13). The second group included 73 patients with leprosy, who had been at the time of the study of signs of activity leprosula process.

Below are the results of testing.

Example 1.

The patient, 1946, R., (history No. 2662). Sick with leprosy since 1957. From October 2001 to the present time there is a relapse of leprosy. The diagnosis of leprosy, lepromatous type, LLS. Active leprosy process. Clinically the disease is characterized by the presence of the following signs of active phase leprosula process: skin - diffuse deep infiltration, leprosy and facies leonina; upper respiratory tract - hoarseness, infiltration of the mucosa of the nasal cavity; peripheral nervous system - the suffering and pain of the elbow and malabaricone stage with the presence of a large number M leprae. For carrying out the method used peripheral blood smear on a glass slide and on average cytochemical coefficient (CCS) activities were determined leprosula process. Preparing the drug - peripheral blood smear of a patient on a glass slide according to the standard technique. The preparation was dried at room temperature in the dark place. The drug was fixed for 10 minutes in pairs, 40% formalin. Were stained with the drug solution of Sudan black In the following way. The drug was transferred into a vessel with a dye solution for 7 minutes, during which the vessel from time to time shook. Then the drug was transferred into a vessel with an 85% solution of ethylene glycol for 3 minutes, during which the vessel from time to time shook. Then the drug was transferred into a vessel with a 50% solution of ethylene glycol for 3 minutes, during which the vessel from time to time shook. After that, the product was carefully washed for one minute in warm running water. Dried in the air, the drug was viewed under the microscope using the oil immersion system.

100 viewed peripheral blood leukocytes, depending on the intensity of the specific color R the tion. Quantitative expression of the determination of the degree of accumulation of lipids in peripheral blood leukocytes - average cytochemical coefficient (cbfv) was calculated by the formula

The value of the CCS was 2.6 conventional units. Thus, the proposed method allowed us to reliably determine the activity leprosula process.

Example 2.

Ill H-a, 1937, R., (history No. 3867). Sick with leprosy for over 15 years. The diagnosis of Leprosy, lepromatous type, LLSactive leprosy process. Clinically - side of the skin, mucous membranes of the upper respiratory tract, peripheral nervous system manifestations of the activity leprosula process. BIN=2,66+. Histology: Infiltration lepromatosis structure containing macrophages in different degrees, the fat vacuolization and homogeneous and granular M. leprae. The determination of the activity of leprosula process was carried out by the method described in example 1. The value of the CCS was 1.5 standard units.

Thus, the proposed method allowed us to reliably determine the activity leprosula process.

Example 3.

A female patient, 1926, R., (history No. 3847). Sick with leprosy since 1977. The diagnosis of Leprosy, lepromatous type, LLS

Thus, the obtained results allowed us to reliably exclude activity leprosula process.

The application of the proposed method compared with the method of the prototype achieved a positive result, which consists in simplifying the determination of activity leprosula process. Simplification of the method is achieved with the exception of the four most time-consuming and lengthy implementation stages of the prototype method, namely the extraction of the material from the patient using biopsy, dehydration of alcohols of different concentrations of the preparation of histological sections, staining for acid-fast bacteria and microscope at high magnification, which makes the method easy to implement even for the researcher, without special training. Excluding stage sampling of material from the patient using biopsy as a result of using the proposed method as a material and does not require for its implementation prior anesthesia, strict compliance with the rules of asepsis, the optimal choice of location, depth and size byobserving skin. To implement the proposed method requires no more than five are available, inexpensive and low-toxic chemicals that 4.5 times less in comparison with the method of the prototype. The proposed method, which takes no more than two hours, quickly run, which is approximately 50 times less in comparison with the method of the prototype. To implement the proposed method does not require the presence of obligatory for the implementation of the prototype method expensive technical support, which enables the proposed method, both in the field and in any laboratory. In addition, the results of clinical trials of the proposed method are evidence of its reliability and ease of implementation, which allows for a shorter period of time to evaluate the effectiveness of the treatment, and therefore, promptly make the necessary adjustments in the prescribed dose and duration of treatment with leprosy drugs, which potentially have a number of dose-dependent side effects. The possibility of predlagaemoj the treatment of patients with signs of activity leprosula process, which in turn will reduce the risk and incidence of severe and debilitating long-term complications of the current leprosula process.

Thus, the authors proposed a new, anyone not previously proposed method for determining the activity of leprosula process.

Further on the basis of the proposed method may be a method of assessment leprosy activity of drugs, as well as the method of determining the effectiveness of leprosy therapy. The proposed method can be recommended for clinical use in leprosy institutions.

Claims

The method for determining the activity of leprosula process, which consists in determining the accumulation of lipids in cells of hematopoietic origin, wherein assess the degree of accumulation of lipids in peripheral blood leukocytes by calculating the average cytochemical coefficient whose values 2,6 conventional units and below show an active leprosum process.

 

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