RU2157994C1 - Method and device for carrying out clinical and biochemical analysis of biological fluids - Google Patents

Abstract

FIELD: medicine. SUBSTANCE: method involves taking and storing samples and placing a sample to be studied on reagent zone of diagnostic strip having immobilized reagents for performing particular kind of analysis. The reagent zone color intensity is measured at the place the sample is positioned and quantity or activity of the analyzed substance in the sample is calculated. The device has unit for taking and storing the samples under study, unit for their positioning on the diagnostic strip reagent zone and unit for making sample analysis. The unit for taking and storing the samples under study has plate with drop-shaped cells. The unit for positioning the samples has a comb with pins which number corresponds to the number of cells on the plate. Another version of the unit for positioning the samples is designed as holder having pins with adjustable distance between their ends. The samples are arranged in the diagnostic strip reagent zone as an ordered matrix. EFFECT: wide range of functional applications. 4 cl, 4 dwg

Description

 The invention relates to biology, medicine, veterinary medicine and is intended, in particular, for laboratories conducting clinical and biochemical analyzes of biological fluids.

 The invention can be used for medical and veterinary diagnostics, clinical biochemistry, in particular, to increase the efficiency of the method of conducting clinical and biochemical analyzes of biological fluids, namely, blood, serum, urine and lymphatic fluid.

 A known method for conducting clinical and biochemical analysis of biological fluids, including taking the analyzed sample, applying it to the reagent zone of the diagnostic strip, holding for the reaction, placing the diagnostic strip in the image analyzer and reading the obtained analysis results. According to this method, pre-treated surfaces are used for analysis on which analysis is performed (Catalog N of Jonson & Jonson, 1998). This method is called - dry chemistry. The surface of the diagnostic strip contains a so-called reagent zone measuring several square millimeters in size, which is attached to a plastic or paper strip (diagnostic strips from Roche, Kodak, Johnson & Johnson, NDP, etc.). Reagents necessary for conducting clinical or biochemical analysis of a particular biological fluid are immobilized in the reagent zone. Reagents interact with the analyzed sample to form a colored compound. For analysis using a diagnostic strip (e.g., Johnson & Johnson stripes), the reagent zone is coated with an assay sample applied to the surface as a drop. When the sample interacts with reagents immobilized in the reagent zone, the color of this zone changes. The amount, concentration or activity of the analytes in the sample is determined either visually by changing the color of the reagent zone, or measured using an appropriate recording device. Diagnostic strips are available for analysis of the content or activity of dozens of components of biological fluids. An individual diagnostic strip is used for each analysis. Analysis of the samples is carried out sequentially with the application of the sample on each strip. For storage of the analyzed samples, sample “storages” are used, which are standard or specialized multichannel plates (for example, 96 well immunological plates); conventional micropipettes are used to collect the analyzed sample from the “drive”.

The disadvantages of this method are as follows:
The high cost of one analysis, determined by the cost of the strip, where the most expensive part is the reagent zone. Each analysis is carried out on an individual diagnostic strip with an individual reagent zone, which interacts with the sample. Another parameter or other sample is analyzed using a new diagnostic strip.

 The need for consistent analysis of samples, which increases the time and complexity of analyzing a series of samples and can lead to the appearance of systematic or random errors due to variations in the conditions in which the samples are located in successive measurements.

 The relatively large volume of the sample required for one analysis prevents the possibility of using capillary blood obtained from the finger for several clinical blood tests of one patient. Usually, each analysis requires 50-100 μl of the sample, therefore, for several tests it is necessary to resort to blood sampling from a vein instead of a capillary.

 Measurement error arising due to uneven coverage of the reagent zone with a sample. This error occurs quite often due to the fact that the sample does not cover the entire reagent zone; in particular, in the analysis of capillary blood, due to the large surface of the reagent zone and the difficulty of uniformly covering it with a small volume of blood.

 A device for conducting clinical and biochemical analysis of biological fluids is known, including a unit for collecting and storing analyzed samples, a sample distribution unit on the reagent zone of the diagnostic strip and an image analyzer unit (Catalog N of Jonson & Jonson, 1998). According to this device, the analyzed samples are collected in a multi-channel unit for collecting and storing samples, from which samples are subsequently taken using the distributor unit and applied to the reagent zone of the diagnostic strip in turn. After keeping the samples on the reagent zone (the holding time after application is determined by the type of analysis), the color intensity of the reagent zone is determined using an analyzer instrument, and the amount of analyte is calculated using predetermined formulas. Currently, there are many devices for densely applying biological samples to a solid substrate (see Genome Research Review, 7, 943-946), including single-channel and multi-channel mechanical and piezoelectric pipettes, single-pin and multi-pin sample dispensers from immunological dies, etc. As a rule , the price of such devices is several thousand dollars (in some cases, tens of thousands of dollars), and they are not very suitable for applying tens of samples on a surface several tens of square millimeters in size trov.

 The technical result of the invention, both in part of the method and in the device, is to reduce the cost of analyzes, increase the speed of analysis and reduce the volume of the analyte, which allows the use of capillary blood instead of venous blood for analysis and simplifies the analysis.

To achieve this result, in the proposed method for conducting clinical and biochemical analysis of biological fluids, including taking the analyzed sample, applying it to the reagent zone of the diagnostic strip, holding for the reaction, placing the diagnostic strip in the device - image analyzer and reading the obtained analysis results, take more than one of the analyzed sample and applying them simultaneously or sequentially to the reagent zone of the diagnostic strip in in the form of an ordered matrix so that at least two drops are placed on 1 cm ^{2 of} its surface and the results are read separately for each sample.

To achieve the above result in the proposed device for clinical and biochemical analysis of biological fluids, which includes a unit for collecting and storing the analyzed samples, a unit for distributing samples on the reagent zone of the diagnostic strip and an image analyzer unit, a unit for collecting and storing samples is a plate with holes made on it having a drop-shaped shape and arranged so that the wide parts of the holes are at a greater distance from one another than the narrow part, the sample distribution block is a comb with pins, the number of which corresponds to the number of holes on the plate, and the distance between them is equal to the distance between the narrow parts of the holes, and the analyzed samples are applied to the reagent zone of the diagnostic strip in the form of an ordered matrix so that at least two drops were placed on 1 cm ^{2 of} its surface and the results were read separately for each sample.

To achieve the above result, according to a variant of the proposed device for clinical and biochemical analysis of biological fluids, which includes a unit for collecting and storing the analyzed samples, a unit for distributing samples on the reagent zone of the diagnostic strip and an image analyzer unit, the unit for collecting and storing samples is a plate with holes, and the sample distribution block is made in the form of a holder with pins mounted on it with the possibility of changing the distance between their ends, contact test samples with the reagent zone of the diagnostic strip, while the analyzed samples are applied to the reagent zone of the diagnostic strip in the form of an ordered matrix so that at least two drops are placed on 1 cm ^{2 of} its surface and the results are read separately for each sample.

 The proposed method for conducting clinical, biochemical analysis of biological fluids is as follows.

From the block for collecting and storing the analyzed samples (multi-hole dies), the sample distribution block produces parallel or sequential sampling of the analyzed samples (the sample volume is 1 μl) and apply them to the reagent zone of the diagnostic strip in separate drops arranged in the form of an ordered matrix, thus so that at least two drops are placed on 1 cm ^{2 of} its surface.

 The diagnostic strip with the applied samples is maintained for the time required for the interaction of the sample with reagents immobilized in the reagent zone (the holding time after application is 1 - 1.5 minutes and is determined by the type of analysis).

 Using the block of the image analyzer, the analysis results are read at the place of application of the sample separately for each sample.

 It is advisable to apply at least one drop containing a reference sample and serving as an internal control to the reagent zone. This improves the accuracy of the analysis.

 Example.

 The block for collecting and storing the analyzed samples is a plate containing 12 wells, in the wells are individual samples of capillary blood, in which it is necessary to determine the glucose content. The shape of the radially arranged drop-shaped holes with a wide and shallow outer end and a narrow and deep central one allows the samples to drain into the middle of the die. Thus, the analyzed samples are poured into the wells using standard devices from the wide parts of the holes, the centers of which are located at a distance of about 7 mm from each other, after which the samples are collected in the narrow parts of the holes, where the distance between the centers of the holes is 1.5 mm. The sample distribution block is a comb with individual pins, the distance between which is 1.5 mm. The pins of the sample distribution block are immersed in the wells of the plate and, when they are removed from the wells, a drop of the corresponding sample with a volume of 1 μl remains on each pin (the size of the drop depends on the diameter of the pin). The pins are brought into contact with the reagent zone of the diagnostic strip, as a result of which drops of samples pass from the pins to the corresponding places in the reagent zone. In this case, an ordered matrix is formed on the reagent zone with an element spacing period of 1.5 mm. Several dozen sample samples can be placed on the reagent zone of a standard diagnostic strip. Samples interacting with the reagent zone cause the formation of a colored product at the application site. The diagnostic strip is placed in the image analyzer unit and after 1 minute the glucose values in each analyzed sample appear on the computer screen. The total analysis time for all samples is almost equal to the analysis time for one sample in existing systems. In the analysis of blood glucose in 12 patients, the difference between the readings obtained using the described device and the readings obtained using the standard method did not exceed ± 8%.

 To check the accuracy of the determination using the described method, the same blood sample was placed in 12 holes of the block for storing samples. The measurements showed that the accuracy of the analysis, including the spread between the pins of the 12-channel sample distribution block, and the spread of measurements, as well as possible heterogeneity of the reagent zone, was no more than ± 5%.

 To increase the accuracy of the measurements, ten blood samples for analysis and two control samples containing known amounts of glucose were placed in the sample storage unit, and all 12 samples were applied to the reagent zone of the diagnostic strip with a 12-channel sample distribution unit. The amount of glucose in each sample was determined using the image analyzer block, and to calculate the glucose values in the analyzed samples, the obtained values for the control samples were taken into account. This procedure is very important for checking the quality of diagnostic strips and in those cases when they are used in extreme conditions of humidity or temperature.

 The proposed method has the following advantages.

 One diagnostic strip with one reagent zone can be used for several identical analyzes of different samples, which reduces the cost of one analysis. In particular, using one standard diagnostic strip from Jonson & Jonson to determine glucose in human serum at a cost of $ 0.5, the proposed method can be used to determine the glucose content in the sera of several dozen patients with a corresponding reduction in the cost of one analysis.

 The method dramatically increases speed when conducting a series of homogeneous analyzes of different patients. Currently, the analysis of each patient is carried out sequentially, on an individual strip and analysis of 200 patients takes several hours. In the proposed method, with the parallel application of samples on a diagnostic strip, the color intensity of all samples is determined simultaneously 1-1.5 minutes after application of the samples.

 The proposed method allows a much more economical use of the analyzed sample. Typically, 50-150 μl of the sample is applied to one diagnostic strip, while in the proposed method the sample consumption for one analysis is 1 μl. This is very significant, since when conducting different blood tests of one patient, you can restrict yourself to capillary blood obtained from a finger, rather than taking blood from a vein, as is currently being done.

 The proposed method allows to avoid errors caused by incomplete coverage of the reagent zone with the analyzed sample, since it determines the intensity of the developing color specifically at the place of application of the sample in the form of a microdrop.

 The use of comparison samples with a known analyte content as an internal control can increase the accuracy of the analysis, which is especially important when conducting analyzes in the field or in rooms with poorly controlled temperature and humidity.

 To implement this method of clinical and biochemical analysis of biological fluids, a device is proposed that includes a unit for collecting and storing the analyzed samples, a sample distribution unit on the reagent zone of the diagnostic strip and an image analyzer unit and a variant of this device.

 The invention is illustrated by the drawing, in which in FIG. 1, a schematic representation of the device, FIG. 2 - the same - option, in FIG. 3 - a plate with teardrop-shaped wells, in FIG. 4 - sample distributor used in the embodiment of the device.

 A device for conducting clinical and biochemical analysis of biological fluids contains a block for storing samples (Fig. 1, pos. 1) a distribution block of samples (Fig. 1, pos. 2), which allows you to take samples from the block for storage and apply them to the reagent zone a diagnostic strip in the form of an ordered matrix (Fig. 1, pos. 3), and an image analyzer block (Fig. 1, pos. 4).

 The storage unit is a multi-hole dice (Fig. 1, item 1). The holes of the plate (Fig. 3, pos. 5) have a drop-shaped shape, which allows them to be positioned relative to each other so that the centers of the “wide” line of the wells (Fig. 3, pos. 6) are at a greater distance from each other than the centers of the elongated "narrow" parts of the holes (Fig.3, pos.7). This shape of the holes having a "wide" part on the one hand allows you to fill them using standard micropipettes.

The sample distribution block (Fig. 1, pos. 2) represents a multi-channel dispenser that is a "comb" with pins (Fig. 1, pos. 8) located at a fixed distance from each other. The number of pins corresponds to the number of holes on the die, and the distance between the pins corresponds to the distance of the centers of the elongated "narrow" parts of the holes from which the multichannel dispenser takes samples. When the pins are immersed in the wells of the plate and removed from the wells, a drop of the corresponding sample remains on each pin (Fig. 1, item 9). When the pins come into contact with the reagent zone of the diagnostic strip (Fig. 1, item 3), drops of samples pass from the pins to the corresponding places in the reagent zone. As a result of the transfer, an ordered matrix is formed on the reagent zone, consisting of droplets containing the analyzed samples, and at least two drops are placed on 1 cm ^{2 of} its surface.

 Block - image analyzer is used to determine the content, concentration or activity of the analyte in the samples deposited on the surface of the reagent zone. The determination is made by measuring the color intensity in the appropriate location of the reagent zone. The analyzer contains standard units, namely, a illuminator, a holder of a diagnostic strip, a device for transferring images to the surface of a multi-channel light detector (CCD camera, etc.) and the multi-channel detector itself. The image analyzer unit is connected to a computer (Fig. 1, item 15) containing an image analysis program, it measures the color change of the reagent zone at the points of contact with the samples and, using a specially designed program, calculates the amount or activity of the analyte in the corresponding sample. If control samples are applied to the same reagent zone, the corresponding values are used to correct the measurements and increase the accuracy of the analysis. The analysis of all samples located on the same surface is carried out simultaneously, which avoids errors associated with different processing conditions of the sample on different diagnostic strips (temperature, humidity). The total analysis time for all samples is almost equal to the analysis time for one sample in existing systems.

 A variant of the device for conducting clinical and biochemical analysis of biological fluids also contains a unit for storing samples (Fig. 2, item 11), a sample distribution unit (Fig. 1, item 2), and an image analyzer unit (Fig. 1, item . 4). The block for collecting and storing samples can be a standard 96-well immunological plate, a rack with test tubes, or other devices for collecting and storing the analyzed samples. The sample distribution block is a comb with a handle (Fig. 2, pos. 12) and pins (Fig. 2, pos. 8), moreover, the distance between the pins can be changed using the adjustment handle (Fig. 2, pos. 13) in depending on the stage of the method. In the sampling stage, the distance between adjacent pins (Fig. 2, item 14) provides a parallel sampling from a multi-hole plate or other device for storing samples. At the stage of applying samples to the reagent zone of the diagnostic strip, the pins of the microdoser are shifted so that the distance between the pins provides a dense spatial arrangement of the droplets of the analyzed samples in the form of an ordered matrix on the reagent zone of the diagnostic strip.

 The image analyzer block in the device variant is the same as in the device, and measurements are made in the same way.

Claims (3)

1. A method for conducting clinical and biochemical analysis of biological fluids, which involves taking an analyzed sample, applying it to a reagent zone of a diagnostic strip, holding for a reaction, placing a diagnostic strip in an image analyzer and reading the obtained analysis results, characterized in that when using one diagnostic strip take more than one analyzed sample, and applying them simultaneously or sequentially to the reagent the zone of the diagnostic strip is carried out in the form of an ordered matrix so that at least two drops are placed on 1 cm ^{2 of} its surface and the results are read separately for each sample.  2. A device for conducting clinical and biochemical analysis of biological fluids, including a unit for collecting and storing the analyzed samples, a unit for distributing samples on the reagent zone of the diagnostic strip and an image analyzer unit, characterized in that the unit for collecting and storing samples is a die with her holes, having a drop-shaped shape and arranged so that the wide parts of the holes are at a greater distance from each other than the narrow parts, while the block is distributed of the samples is a comb with pins, and their number corresponds to the number of wells per plate, and the distance between them equal to the distance between the narrow parts of the holes, wherein the samples to be analyzed are applied to the reagent zone of the diagnostic strips in an ordered array.  3. A device for conducting clinical and biochemical analysis of biological fluids, including a block for collecting and storing the analyzed samples, a block for distributing samples on the reagent zone of the diagnostic strip and an image analyzer block, characterized in that the block for collecting and storing samples is a plate with holes, and the sample distribution unit is made in the form of a holder with pins mounted on it with the possibility of changing the distance between their ends in contact with the reagent zone of the diagnostic Oski, wherein the samples to be analyzed are applied to the reagent zone of the diagnostic strips in an ordered array.

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