RU2632694C1 - Device for determination of complement functional activity - Google Patents

Abstract

FIELD: medicine.

SUBSTANCE: device includes a housing inside of which a measuring unit is located containing a module for test samples, light sources for sample illumination and a camera with micro-lenses for sample state recording, a power supply unit and a microcontroller connected to a personal computer. The module for the test samples is made in the form of a strip with cells and is equipped with a shaking unit. Video cameras with micro-lenses are combined into a block so that each cell of the strip has a video camera with a micro-lens placed above it. The measuring unit is equipped with a thermostating unit made in the form of a heat pump based on the Peltier element and a temperature sensor. At that, the light sources for sample illumination are placed under each strip cell and form a lighting unit of dark-field microscopy.

EFFECT: increased reproducibility of analyzes, reduced complexity of their conduct, reduced time of sample preparation, increased reliability of the device and reduced size.

2 cl, 4 dwg

Description

The invention relates to the field of medical and veterinary immunology, and in particular to devices for determining the functional activity of complement in human blood in the diagnosis of a number of diseases and is intended to determine the functional activity of complement by its toxic effect on free-floating infusoria.

The complement system refers to the basic systems of innate immunity, the function of which is to distinguish between “own” and “not own”.

The immunity created by anatomical, physiological, cellular and molecular factors, which are the natural constituent elements of the body, is otherwise called innate.

Thanks to the presence of the immune system, the body is protected from most pathogens (viruses, bacteria, fungi, protozoa, helminths) and toxic products of their vital activity. Immunity also protects the body from exposure to various substances with foreign properties (for example, plant and animal poisons), from the development of tumor cells.

Complement is a complex enzymatic system of many proteins found in blood serum. Under normal conditions, they are inactive; upon activation, they participate in protective reactions. The system is activated spontaneously by certain pathogens or the antigen: antibody complex. Activated proteins either directly destroy the pathogen (killer action), or provide their best absorption by phagocytes (opsonizing effect); or perform the function of chemotactic factors, attracting inflammatory cells into the pathogen penetration zone.

It is known that complement has a nonspecific toxic effect on biological test objects, in particular ciliates [Sinclair I.J.B. The role of complement in the immune reactions of Paramecium aurelia and Tetrahymena pyriformis. Immunology 1958. V.1. P. 291-299].

There is a method of determining complement activity at Tetrahymena pyriformis ciliates, based on the registration of kinetic survival curves of this type of ciliates under the influence of different doses of the test object's serum. A suspension of Tetrahymena pyriformis cells and test blood serum are added to the measuring cells of the device for counting the number of living ciliates, followed by determination of the number of living cells per minute. The coincidence of the dynamics of changes in the number of living cells over time for the test serum and the control, representing a pool of 10 sera of healthy donors, suggests the equality of the activities of serum complement in these sera [RF patent No. 2518739, 2013].

A device is known for assessing the quality of products of animate and inanimate nature (RF patent No. 2122025, 1997), the principle of operation of which is based on counting the number of living ciliates in the tested samples located in the cells of a moving tablet that are being monitored. Product quality is determined on the basis of 2 measurements of the number of living cells - at the beginning of the experiment and at the end of it.

The disadvantages of the known device are the low reliability of the results, their low reproducibility, due to both the specifics of the Tetrahymena pyriformis infusoria and the design features of the described device, which does not involve conducting studies at a constant temperature, the inability to reliably determine the starting time of the study, the complexity of the preparatory stage of the experiment .

As the authors previously discovered, with the action of complement on the Tetrahymena pyriformis ciliates, after a certain period of time, depending on the amount of active complement, the process of immobilization of ciliates begins, proceeding at a speed that also depends on the amount of active complement.

Both measured values: the incubation time required to achieve immobilization of half of the microorganisms, as well as the rate of transition of the moving ciliates to the stationary ones, can be used to calculate the number of functionally active complement.

The method of determination involves introducing into the measuring cells of the device a suspension of ciliates in a buffer solution, adding blood serum in the required amount as a complement source, and automatic cyclic counting of the remaining mobile cells in time.

The calculation methods are based either on determining the incubation time of the ciliates with complement necessary for the death of half of the ciliates, or on determining the cell death rate constant, which can be conveniently calculated from the slope of the logarithm of the number of mobile cells versus time during the period of the observed drop in the number of living cells. Both values, depending on the amount of active complement, can be compared with complement activity in a pool of at least 10 human sera, which is chosen as the standard. Thereby, the determination of the functional activity of complement is achieved by a method suitable for standardization.

Closest to the proposed one is a device for determining the functional activity of a blood complement, including a housing inside which there is a measuring unit containing a module for placing test samples, light sources for illuminating samples and video cameras with micro lenses for fixing the state of samples, a power supply unit and a microcontroller connected to personal computer [RF patent 2361913, 2006]

Work with this device necessarily includes the preparatory phase, which includes turning on the device, starting up the computer, filling and installing the tablet with samples on the tablet table. The main stage of work begins with the launch of the control program.

The disadvantages of the known device are as follows:

- the design of the known device does not allow to achieve high reproducibility of the results of the analyzes (by reproducibility of the results we mean obtaining close results of two tests obtained by the same method, under identical conditions, in different laboratories) due to:

- the lack of a temperature control unit, which does not allow research at a constant temperature. Since complement is a protein enzymatic system, its activity is significantly dependent on temperature. Therefore, the measurement of complement activity should be carried out at a constant temperature;

- specifics of the ciliates Tetrahymena pyriformis itself. Tetrachimena is a highly mobile infusoria. Therefore, it is initially quickly and evenly distributed throughout the field of view of the camcorder. However, this type of ciliates has a tendency to concentrate in the parietal zone, as a result of which, during kinetic studies, the number of living ciliates in the field of view is underestimated;

- due to the fact that kinetic studies require reliable determination of the starting point in time. In the case of analysis, where many samples are involved at once, this becomes very relevant;

- the known device uses non-standard wells, which eliminates the use of multi-channel automatic pipettes. This in turn increases the complexity of the analyzes and reduces their productivity;

- the known device contains electromechanical moving parts, which reduces its reliability and increases the size.

The technical problem solved by the invention is the creation of a reliable, simple and easy to use device that allows with maximum efficiency and reliability to obtain reliable results of studies of the functional activity of complement and to ensure their reproducibility.

The technical result of the invention is the provision of the possibility of obtaining reliable research results, increasing their reproducibility, by increasing the reliability of the device, reducing the complexity of research, reducing the time for sample preparation, while ensuring a reduction in the dimensions of the device and cheaper design.

To achieve a technical result, in a device for determining the functional activity of a blood complement, including a case, inside which there is a measuring unit containing a module for placing test samples, light sources for illuminating samples and video cameras with micro lenses for fixing the state of samples, a power supply unit and a microcontroller connected to personal computer

the module for placing test samples is made in the form of a strip with cells and is equipped with a shaking unit, video cameras with micro lenses are combined in a block so that each cell of the strip has a video camera with a micro lens placed above it, and the measuring unit is equipped with a temperature control unit made in the form of a heat pump on based on a Peltier element and a temperature sensor, while light sources for illuminating the samples are placed under each strip cell and form a lighting unit of dark-field microscopy.

In this case, it is preferable that the lighting unit of dark-field microscopy is made in the form of a diaphragm and a board with concentrically placed LEDs on it and a light-absorbing plate under each strip cell.

The implementation of the module for placing the tested samples in the form of a strip with cells allows, unlike the known devices, to work with standard consumable (disposable and sterile) material, which makes it possible to work, for example, with automatic pipettes, dispensers, readers, etc.) . At the same time, multichannel automatic pipettes make it possible to introduce samples into the strip cells simultaneously, and not sequentially, as in the known devices. This accurately identifies the beginning of the kinetic process of the death of ciliates, which, in turn, increases the reliability of studies, the reproducibility of registration of kinetic curves, and hence the reproducibility of studies in general. In addition, labor costs are reduced and research time is saved.

Supply of a module for placement of test samples with a shaking unit solves the problem of uneven distribution of ciliates in the sample volume. At rest, ciliates prefer to concentrate in the parietal zone of the cell, which complicates video analysis and masks (underestimates) the number of living cells. The shaking unit briefly excites (agitates) the cells and prevents them from sticking to the cell walls. This eliminates the bias and improves the reliability of cell counting, as well as the reproducibility of the analysis as a whole. The shaking unit can be made by any known method, for example, based on a miniature vibromotor.

The combination of a video camera with micro lenses in a block so that each cell of the strip has a video camera with a micro lens placed above it allows microscopic analysis of objects simultaneously in the entire strip. This makes it possible to take more detailed and statistically significant kinetic curves of cell survival than with electromechanical scanning, as in known devices. This, in turn, increases the reliability of the registration of kinetic curves and the reproducibility of the analysis as a whole. The combination of a video camera with micro lenses in a block can be carried out by one of the methods known and applicable in this case, for example, using a bar. The placement of light sources to illuminate the samples under each cell of the strip provides individual illumination of each cell. This eliminates the need for electromechanical scanning of the strip, which ensures the absence of moving parts in the device and thus increases the reliability of the device, and also reduces its dimensions.

Providing the measuring unit with a temperature control unit, made in the form of a heat pump based on a Peltier element and a temperature sensor, allows measurements at a fixed temperature, while the temperature sensor is a feedback element that controls the actual temperature in the measuring unit. The need for a temperature control unit is due to the fact that the activity of the complement critically depends on temperature. Therefore, the shape of the kinetic curves of cell survival depends on temperature. Thus, temperature stabilization in the measuring unit increases the reproducibility of the registration of kinetic curves, and hence the reproducibility of the analysis as a whole. The Peltier element is a thermoelectric converter, the principle of which is based on the Peltier effect - the occurrence of a temperature difference during the flow of electric current. The advantage of the Peltier element is its small size, the absence of any moving parts, as well as gases and liquids. When reversing the direction of the current, both cooling and heating are possible - this makes it possible to thermostat at an ambient temperature both above and below the thermostat temperature. Another advantage is the absence of mechanical parts and the absence of noise. All of the above explains its use in the temperature control unit.

The implementation of light sources for illuminating samples in the form of a lighting unit of dark-field microscopy is due to the following circumstances.

Since the ciliates are slightly colored and translucent, microscopic analysis is performed in a dark field (dark-field microscopy). The analysis is based on the illumination of an object by oblique rays of light. The rays do not fall into the lens of the camera, so the field of view looks completely black. If the cell contains live ciliates, then bright luminous objects are visible on the black field. This approach speeds up and improves the reliability of subsequent video analysis. The result of the video analysis is the number of living cells in the field of view of the video camera. As a result, the use of dark-field microscopy increases the reliability of the registration of kinetic curves, and therefore the reliability and reproducibility of the analysis as a whole. To perform the lighting assembly of dark-field microscopy in the form of a diaphragm and a circuit board with LEDs and light-absorbing plates placed on it under each strip cell, printed circuit technology and micro-miniature SMD components were used, which in general leads to a reduction in size and the cost of the entire structure.

The proposed device is shown in the drawings, where in FIG. 1 shows a block diagram of a device; FIG. 2 and 3 - schematically, the lighting unit of dark-field microscopy, in FIG. 4 - typical survival curves of ciliates.

A device for determining the functional activity of the blood complement contains a housing 1, inside which a measuring unit 2 with a module 3 is placed for placement of the tested samples, light sources 4 for illuminating the samples, a video camera 5 with micro lenses 6 for fixing the state of the samples, a power supply 7, a microcontroller 8, connected to a personal computer 9.

Module 3 for placing the test samples is made in the form of a strip 10 with cells 11 and is equipped with a shaking unit of the tested samples 12.

Video cameras 5 with micro-lenses 6 are combined into a block by means of a rod 13. so that above each cell 11 of the strip 10 there is a video camera 5 with a micro-lens 6.

The measuring unit 2 of the device is equipped with a temperature control unit 14, made in the form of a heat pump 15 based on a Peltier element and a temperature sensor 16.

The illumination node of dark-field microscopy is a board 17 with sequentially placed light sources for illuminating samples 18. On the board 17, under each cell 11 of the strip 10, SMD LEDs 19 are concentrated, the light-absorbing plate 20 and the diaphragm 21 bounding and forming the light cone. The axial line of the light cone coincides with the axial line of the cell 11 located above it (Fig. 2, 3).

In FIG. Figure 4 shows kinetic curves for the survival of ciliates at different doses of blood plasma. So, 10 μl (curve A), 15 μl (curve B) and 20 μl (curve C) of human blood serum were added to 100 μl of a suspension of Tetrahymena periformis infusoria. The complement contained in serum is a non-specific toxicant for this species. It is clearly seen that with an increase in dose, the time causing the death of 50% of ciliates is reduced. These curves are the starting material for various statistical processing of toxicological data.

The device operates as follows.

After it is connected to a household network and a personal computer 9, the operator launches a specialized application on computer 9 that monitors the operation of the device in an interactive mode. After the temperature of the measuring unit 2 is stabilized, the device is ready for analysis. For analysis, a sterile strip 10 is placed in the measuring unit 2. Using an automatic pipette, 100 μl of the optimal concentration concentration of ciliates are placed in all cells 11 of the strip 10. The optimal concentration of cells gives the least error in determining their number. With a low concentration of cells, the standard error will increase, while with a high concentration, a systematic error in cell counting will appear. After 5-minute thermal stabilization of the strip 10 using the temperature control unit 14, samples of diluted blood serum are introduced into it using an 8-channel automatic pipette. The lid (not shown) of the measuring unit 2 is closed, which serves as a signal for fixing the time reference when constructing kinetic curves of the survival of ciliates. From this moment, the light sources are automatically turned on to illuminate cells 11 and the analysis of video data begins. With a frequency of 1 min, strip 10 is shaken using the shaking unit of the test samples 12 for 1 s, while the current video data is ignored. The frequency and duration of shaking strip 10 can be adjusted depending on the type of ciliates used. Further, in accordance with a predetermined data processing protocol, a complement titer characterizing its activity is calculated, or other statistical processing is performed.

Claims (2)

1. A device for determining the functional activity of a blood complement, including a housing inside which a measuring unit is located, containing a module for placing test samples, light sources for illuminating samples and a video camera with micro lenses for fixing the state of samples, a power supply unit and a microcontroller connected to a personal computer, characterized in that the module for placing the tested samples is made in the form of a strip with cells and is equipped with a shaking unit, a video camera with micro lenses about are combined into a block in such a way that each strip cell has a video camera with a micro lens placed above it, and the measuring block is equipped with a temperature control unit made in the form of a heat pump based on a Peltier element and a temperature sensor, while light sources for illuminating the samples are placed under each strip cell and form a lighting unit of dark-field microscopy. 2. The device according to claim 1, characterized in that the lighting unit of dark-field microscopy is made in the form of a diaphragm and a board with LEDs concentrically placed on it and a light-absorbing plate under each strip cell.

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