RU2519030C1 - Device for preparation, storage and transportation of dry samples of liquid objects to be subject to laboratory analysis - Google Patents

Abstract

FIELD: medicine.

SUBSTANCE: device for preparation, storage and transportation of dry objects of liquids comprises a dry container with an adsorption element whereon a liquid aliquot containing analysed components can be applied and dried. An adsorption element is made of a moisture absorbing porous material having the adsorption capacity not less than 40 mg per cm² and an ability for reversible desorption of dry components in an immersible sample solution of at least a working area of the adsorption element with the dry sample in the sample solution with an effectiveness of 30% at the end of no more than 300 sec after the contact of the dry sample with the sample solution.

EFFECT: invention enables obtaining the reproducible results of the analysis of biological liquids, facilitates the structure of the sorbent element.

10 cl, 4 tbl, 3 ex

Description

The invention relates to the field of medicine and veterinary medicine, in particular, to means for taking, storing and transporting samples of biological fluids (blood, serum, urine, lymph, sweat, milk, tissue extracts, etc.) with the aim of subsequent analysis of the material on the content of biologically active substances by ELISA, PCR, etc.

Known (SU, copyright 1017343) container for storing blood samples. The known container contains a container with a lid on which a rod for attaching absorbent paper is mounted. A desiccant is placed in the lower part of the tank with a gap with respect to the rod and a means for fixing it is installed, containing a nylon mesh and a spring. A known container is used as follows. The lid of the container is opened and a drop of blood moistens the surface of the absorbent paper, a desiccant is placed on the bottom of the container and fixed by the action of a nylon mesh and a spring. After applying a drop of blood to absorbent paper, the lid of the container is closed, thereby placing absorbent paper with a drop of blood inside the container above the desiccant. Under the action of a desiccant, a blood sample dries on absorbent paper.

A disadvantage of the known container should be recognized as the complexity of its design. In addition, the characteristics of the sample absorber — absorbent paper — are not specified, which does not allow taking into account the volume of the blood sample during the analysis, while the accuracy of the analysis is reduced.

A widely known approach (Edelbroek PM, van der Heijden J and Stolk LM. Dried blood spot methods in therapeutic drug monitoring: methods, assays, and pitfalls. Therapeutic Drug Monitoring 2009; 31 (3): 327-336), based on the use of porous cellulose paper for applying blood stains in the form of drops to several places previously indicated by circles, drying the resulting paper card and storing or sending the samples to a specialized laboratory, where they subsequently cut out circles of paper with sorbed blood samples using a hole punch and subsequent desorption of sample into the solution for the purpose of further analysis for the content in the original sample of various biologically active substances.

In the application of Cohen et al. (US Patent Application Nr. 2012/0045792 Al) shows the use in this embodiment as a membrane material instead of porous cellulose (paper) of a fiberglass membrane with circles located on it to indicate the places where blood drops were applied.

The disadvantages of these approaches should be recognized as the need to apply blood drops strictly in the center of the circles marked on paper, the impossibility of accurately dosing the amount of applied sample and the need to use special holes with a fixed diameter of the cut paper hole for subsequent cutting of the membrane circles with the sample for their subsequent desorption. In addition, this method of obtaining dry blood stains does not allow the separation of cellular elements of blood for subsequent use in the analysis of only plasma or serum.

To improve the consumer properties of the device, it is proposed to use (US Patent 5,516,487) membranes in the form of a small sheet on which circles are pre-punched in certain places on which the analyzed blood sample is applied. This solution does not require the use of a hole punch, but its disadvantage is the determinate surface area of the membrane with the sample being determined, which does not allow changing the volume of the analyzed blood sample, the need to apply blood drops strictly in the center of the perforated circles, which often leads to difficulties in obtaining samples under non-laboratory conditions, and consequently, significant errors in the analysis. Perforated circles can spontaneously separate from the membrane before the start of the analytical procedure.

The specified technical solution was used as the closest analogue of the developed device.

The technical result obtained as a result of the implementation of the developed technical solution consists in simplifying the design of the sorbing element while expanding the scope by expanding the possible objects of analysis.

To achieve the specified technical result, it is proposed to use the developed device for receiving, storing and transporting dry samples of liquid objects intended for subsequent laboratory analysis. The developed device contains a protective container in which at least one adsorption element is placed, made with the possibility of applying an aliquot of a liquid object containing the analyzed components on it, and then drying it, and the adsorption element to obtain a dry sample is made of absorbent porous material with an adsorption capacity of at least 40 mg of water per cm ² capable of reversible desorption of the dry components of a liquid object into a solvent for the sample upon immersion, in m at least the working area of the adsorption element with the dried sample in the sample solvent with an efficiency of at least 30% after not more than 300 s after the contact of the dried sample with the sample solvent.

Specification of the sorbing / desorbing characteristics of the adsorption element allows to obtain reproducible results in the analysis of biological fluids, since comparable amounts of adsorbed, dried and desorbed samples of the biological fluid will be used for the analysis.

As moisture-absorbing porous material of the adsorption element, cellulose and its derivatives, as well as glass or polyester microfiber, can be used. In addition, the adsorption element may be made of polysulfone. The choice of the specific material from which the adsorption element is made depends on the conditions of sampling, the conditions and duration of storage of the sample, as well as on the type of biological fluid to be analyzed.

In some embodiments, the adsorption element is made in the form of a strip with a width of 0.2 to 2 cm and a length of 1 to 10 cm, and at the end of the strip with the working area at the same distance in increments of 0.2-1.0 cm from each other identification lines are drawn in the transverse direction, restricting areas of equal size. The use of an adsorption element in this embodiment allows uniform application of the analyzed sample on the membrane by contacting the end of the membrane with the sample for a certain time, sufficient to sorb the liquid on the membrane with a certain surface area. The application of identification lines on a strip with a membrane allows, by cutting the membrane with ordinary scissors, different in area of its sections, to obtain, with good accuracy, dried samples of the analyzed liquid with different fixed contents of the analyzed material.

The adsorption element may further comprise a porous membrane capable of retaining the cellular elements of the blood as it passes through its blood structure and located in contact with the working area of the strip.

In some embodiments, the protective container may be made of a dense material in the form of a folded envelope that provides external protection of the adsorption material on both sides.

Also, the protective container may be a standard polymer tip for an automatic or semi-automatic dispenser. In this embodiment, a membrane material capable of effectively absorbing blood or other biological fluid of a strictly defined size (area or volume) is inserted into the tip, a fixed volume of biological fluid (smaller than the volume of fluid absorbed by the membrane) is introduced into the tip using an automatic dispenser, after which the membrane with the liquid is dried (for example, using a hairdryer or in the presence of an effective desiccant desiccant). Storage or transfer of the obtained samples is carried out in polymer bags that prevent the membrane from absorbing moisture from the environment. For analysis, a tip with a membrane and a dried sample is attached to the dispenser and the volume of the buffer solution equivalent to the initial sample is taken out by the dispenser, incubated for several minutes to desorb the determined components into the solution, the solution is transferred to the analyzer cuvette for analysis using appropriate reagent kits.

An implementation option is possible when the protective container is made of dense material in the form of a lockable case containing a desiccant that provides external protection for the adsorption material.

In any embodiment of the protective envelope, it must isolate the adsorption element with the breakdown of the biological fluid on it from the external environment.

The device is configured to analyze biological fluids (blood, serum, urine, lymph, sweat, milk, tissue extracts, etc.) of living things, as well as water and water-containing samples.

Below are examples of the use of the developed device.

Example 1. A device for obtaining dry blood samples for laboratory enzyme-linked immunosorbent assay of antibodies to infectious bursal disease (IBD) pathogens in the blood of immunized chickens and chickens.

Using the commercial BioChek enzyme-linked immunosorbent reagent kit (England), antibody titers were determined in the blood of immunized chicken chickens against infectious bursal disease (IBD) and infectious encephalomyelitis (AE) pathogens in native and membrane-dried samples. To obtain dry blood stains, a device was used in the form of three separate paper strips 0.5 cm wide and 7 cm long, located in a cardboard envelope, while one of the ends of each of the strips was fixed in an envelope that provided external protection on both sides of the strips, and the second end was in a free state. The strips were made of Whatman 903 porous filter paper, on which thin lines were drawn in the transverse direction at an equal distance of 0.5 cm from each other. The sorption capacity of the sample used paper was 90 μl of water per 1 cm ² the surface area of the membrane. The free end of the strip was immersed in a sample of the blood for 2-3 s (the time during which the blood under the action of capillary forces passed over a distance of more than two designated divisions). The strip was removed from the liquid and dried in air for 2 hours. The dried samples were stored at room temperature for 2 days before analysis. During the analysis using scissors, a piece of the membrane corresponding to the two applied divisions was carefully cut from the free end of the strip (the area of the membrane with the dried serum sample was 1 cm ² ). A cut-off piece of paper with a dry sample was placed in a test tube into which 270 μl of 0.01 M phosphate buffer solution containing 0.14 M NaCl was added, which was three times the initial sorbed volume of the blood sample. The tube was shaken for 3 minutes. For analysis, an aliquot of the solution was taken from a test tube in a volume of 20 μl, which was then diluted 167 times so that the final dilution required by the kit instruction was 1: 500. The results for determining the titer of antibodies to IBD and AE using the proposed device and native blood samples are shown in tables 1 and 2, respectively.

Table 1 Antibodies to viruses Antibody titer in native blood samples (n-14) Interpretation of the result Membrane The average titer of antibodies Interpretation of the result % to titer of antibodies in the blood 1: 15408 + 1: 13940 + 81.9 1: 15429 + 1: 10455 + 67.8 1: 9158 + 1: 7472 + 81.6 1: 10725 + 1: 8169 + 76,2 1: 14226 + 1: 12071 + 84.9 1: 9603 + 1: 972 1 + 101.2 1: 6491 + 1: 9574 + 147.4 IBB 1: 9433 + 1: 6911 + 73.3 1: 8285 + 1: 11365 + 137.2 1: 10745 + 1: 11287 + 105.0 1: 11074 + 1: 1931 + 107.7 1: 14184 + 1: 16372 + 115.4 1: 10405 + 1: 13124 + 126.1 1: 10576 + 1: 11406 + 107.8 Average value 101.0

Tab. 2 Antibodies to viruses Antibody titer in native blood samples (n = 14) Interpretation of the result Membrane The average titer of antibodies Interpretation of the result % to titer of antibodies in the blood 1: 9606 1: 8197 + 85.3 1: 5622 + 1: 3999 + 71.1 1: 4800 + 1: 3364 + 70.1 1: 6984 1: 4935 + 70.7 1: 6372 1: 5822 + 91.4 1: 8712 - 1: 9591 + 100.1 1: 7328 1: 8835 + 120.5 1: 7112 1: 7043 + 99.0 AE 1: 2504 - 1: 3566 + 142.4 1: 6402 1: 7463 + 116.6 1: 4 Neg. 1:79 Neg. Neg. 1:45 Neg. 1:76 Neg. Neg. 1:28 Neg. 1:94 Neg. Neg. 1:13 Neg. 1:68 Neg. Neg. Average value 96.7

For all the studied sera, the data obtained using dry spots correlate well with the results of determining the titer of antibodies in native sera, which indicates the possibility of using a device for producing dry serum stains for subsequent serological studies.

Example 2. A device for obtaining a dry serum sample for laboratory analysis of chicken serum for the presence of antibodies to Newcastle disease (NB) pathogens.

A comparative determination of antibodies to Newcastle disease pathogens was determined in native sera containing antibodies in different titers using the hemagglutination inhibition test (RTGA) in samples of native sera and samples obtained after drying on membranes and subsequent elution. To obtain dry serum stains, a device was used in the form of three separate paper strips 0.5 cm wide and 7 cm long located in a cardboard envelope, with one of the ends of each of the strips being fixed in an envelope that provided external protection on both sides of the strips, and the second end was in a free state. The strips were made of porous filter paper from Munktell, on which thin lines were drawn in the transverse direction at an equal distance of 0.5 cm from each other. The sorption capacity of the sample used paper was 70 μl of water per 1 cm ² the surface area of the membrane. The free end of the strip was immersed in a sample of the studied blood for 2-3 seconds (the time during which the blood under the action of capillary forces passed at a distance of more than two designated divisions). The strip was removed from the liquid and dried in air for 2 hours. The dried samples were stored at room temperature for 2 days before analysis. During the analysis using scissors, a piece of the membrane corresponding to the two applied divisions was carefully cut from the free end of the strip (the area of the membrane with the dried blood sample was 1 cm ² ). A cut-off piece of paper with a dry sample was placed in a test tube into which 210 μl of 0.01 M phosphate buffer solution containing 0.14 M NaCl was added, which was three times the initial sorbed volume of the serum sample. The tube was shaken for 3 minutes. An aliquot of the solution in a volume of 20 μl was taken from the tube for analysis. The results for determining the titer of antibodies using the proposed device and native sera are shown in table 3.

Table 3 Serum No. The antibody titer in rtga (log2) % to the titer of antibodies in native
serum Native serum After elution from the membrane one four 5 125 2 5 5 one hundred 3 5 four 80 four 5 four 80 5 2 2 one hundred 6 3 3 one hundred 7 3 3 one hundred 8 3 3 one hundred 9 3 four 133 10 3 3 one hundred eleven four four one hundred 12 four four one hundred 13 one one one hundred fourteen four 5 125 fifteen four 5 125 16 5 5 one hundred 17 7 7 one hundred eighteen 6 6 one hundred 19 8 8 one hundred twenty 9 9 one hundred 21 8 7 88 22 8 8 one hundred 23 6 6 one hundred 24 eleven 10 91 Average
value 102

As can be seen from the table, the antibody titer determined in eluents using dry serum samples obtained using the proposed device corresponded to the original 88-125%, which indicated the suitability of these membranes for the preparation and storage of bird serum samples for further serological studies.

Example 3. A device for obtaining dry samples of cow's milk for laboratory analysis of milk to determine the content of progesterone in order to determine the pregnancy of cows.

For rapid detection of cow pregnancy, an approach based on determining the concentration of progesterone (PG) in the milk of seeded cows on the 21st day after insemination by the method of solid-phase competitive enzyme-linked immunosorbent assay was used. The concentration of GHG in cow's milk undergoes cyclic changes. The concentration of GHGs less than 2 ng / ml corresponds to the moment of ovulation, and then increases to a maximum (> 10 ng / ml) on the 13-15th day. If pregnancy does not occur, then the level of GHG drops already by 18-20 days. If the cow is pregnant, a high concentration of GHG is maintained. Therefore, by determining the concentration of progesterone on day 21, it is possible to determine the status of the studied animals as pregnant (with high levels of progesterone) or non-pregnant (with low levels of progesterone). A cow with a GH content in milk> 7 ng / ml is considered to be a pregnant.

As the analyzed material, we used both freshly obtained samples of cow’s milk and samples of milk deposited on the porous membranes of the proposed device. Since milk is taken from cows directly on the farm, and the analysis is carried out in specialized laboratories, the processes of transportation and preservation of samples prior to analysis are key. As a rule, to preserve the liquid samples obtained, their freezing is used, which presents additional difficulties. In this case, to test the proposed device for obtaining dry samples of analytical material, we used a variant of the device based on the use of standard tips of automatic dispensers of 200 μl, in which standard Whatman 903 membranes were placed in the form of narrow folded strips with an area of 1 cm ² . Using a semi-automatic single-channel dispenser, 50 μl volumes of milk were taken for each animal, the tips with the samples were placed in a tripod, and the contents were dried using a hairdryer for 1 hour. The obtained samples, as well as the corresponding samples of fresh milk, were transported to the laboratory where the GH content in milk samples was determined using standard commercial reagent kits for enzyme-linked immunosorbent assay of GH produced by Hema-Medica (RF) in order to determine the pregnancy of animals. A total of 101 milk samples from 92 animals were tested. The results are shown in table 4.

Tab. four Linked immunosorbent assay Rectal examination Total samples Liquid milk samples Dry milk samples one hundred% Analysis time (days after insemination) 21 21 65-75 Research result Pregnant Pregnant Pregnant 58 (57%) Pregnant Pregnant Unstable 14 (14%) Unstable Unstable Unstable 29 (29%)

Using the enzyme-linked immunosorbent assay, 29% of the total number of samples (total 29 samples from 27 animals) were diagnosed as belonging to unstable animals, which was subsequently confirmed by rectal examination. The number of animals, defined as pregnant, was fully consistent for samples of liquid milk and dry samples obtained in the proposed device. Thus, there is a complete correspondence between the results of determining GHG in samples of liquid and milk powder, which indicates the applicability of the proposed device for producing dry samples of biological fluid for subsequent analysis.

A comparative analysis of the developed device and the device used as the closest analogue showed an undoubted simplification of the design of the sorbing element while expanding the scope due to the expansion of possible objects of analysis.

Claims (10)

1. A device for receiving, storing and transporting dry samples of liquid objects intended for subsequent laboratory analysis, containing a protective container in which at least one adsorption element is placed, configured to deposit an aliquot of a liquid object containing analyzed components on it , and its subsequent drying, characterized in that the adsorption element for obtaining a dry sample is made of absorbent porous material with adsorption mkostyu at least 40 mg of water per cm ^2, capable of reversible desorption of the dry components of the liquid object in the solvent for the sample when submerged, at least the working area of the adsorption element with the dried sample in the solvent for the sample with an efficiency of at least 30% after no more than 300 s after contact of the dried sample with the sample solvent. 2. The device according to claim 1, characterized in that the protective container is made of dense material in the form of a folded envelope, providing external protection of the adsorption material from two sides. 3. The device according to claim 1, characterized in that the protective container is a standard polymer tip for an automatic or semi-automatic dispenser. 4. The device according to claim 1, characterized in that the protective container is made of dense material in the form of a lockable case, providing external protection of the adsorption material. 5. The device according to claim 1, characterized in that cellulose and its derivatives are used as the moisture-absorbing porous material of the adsorption element. 6. The device according to claim 1, characterized in that glass or polyester microfiber is used as the moisture-absorbing polymer material of the adsorption element. 7. The device according to claim 1, characterized in that the adsorption element is made of polysulfone. 8. The device according to claim 1, characterized in that the adsorption element is made in the form of a strip with a width of 0.2 to 2 cm and a length of 1 to 10 cm, and at the end of the strip with the working area at the same distance in increments of 0.2- 1.0 cm from each other in the transverse direction marked identification lines bounding the same area areas. 9. The device according to claim 8, characterized in that the adsorption element further comprises a porous membrane capable of retaining the cellular elements of the blood when passing through its blood structure and located in contact with the working area of the strip. 10. The device according to claim 1, characterized in that it is arranged to analyze biological fluids of living things, as well as water and water-containing samples.