CN119756979A - A system, method and application for preserving human body fluids and excrement - Google Patents
A system, method and application for preserving human body fluids and excrement Download PDFInfo
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Abstract
The invention provides a preservation system, a preservation method and application of human body fluid and excrement, wherein the preservation system comprises a sample box body, a sample carrier for carrying a sample is arranged in the sample box body, the sample carrier separates the inner space of the sample box body to form a sample area and a drying area, a first drying agent is filled in the drying area, the first drying agent is in non-contact with the sample carrier, the sample carrier has water absorption and air permeability, and the sample box body has a sealing structure. The invention expands the application range through a separation type drying mode, and can store both solid samples and liquid samples.
Description
Technical Field
The invention relates to the technical field of biology and disease diagnosis, in particular to a preservation system, a preservation method and application of human body fluid and excrement.
Background
Human excreta contains a large amount of biological information including lesion bleeding of organs, abnormal reproduction of microorganisms, abnormal increase or decrease of metabolites, and the like. The information is closely related to the health of the person, and the biomarker is detected and monitored, so that the health condition and disease development of the person can be known in time. At present, the excrement routine and urine routine of hospitals and physical examination centers should be low in cost and high in efficiency, and are welcomed by vast doctors and patients. However, the detection often requires the patient to go to the hospital or the physical examination center for sampling, so that the time cost is high, and particularly, in some countries and regions with lack of medical resources, a large number of ordinary people cannot enjoy the cheap and efficient physical examination service.
With the vigorous development of the express industry, the detection of a sample delivery inspection center after home sampling becomes possible. At present, DNA stabilizers of human feces and urine have made a technological breakthrough, wherein a large number of intestinal cancer home sampling detection products are marketed. Protein stabilizers are much more difficult than DNA stabilizers. Taking the fecal sample as an example, a strong denaturant can solve all the problems in order to inhibit the activity of DNase and the proliferation of bacteria, and the denaturant itself does not affect the biological activity of DNA and can be used as a DNA stabilizer. However, the biological activity of the denaturing agent on the protein is fatal, and the stability of the protein is far from that of DNA, so that the stability of protein components in feces and urine is ensured, but the inhibition of bacterial growth and the activity of various proteases is still a technical problem.
There are many products on the market for human fecal sample stabilizers, such as the precision scientific product "Cologuard" which contains a DNA stabilizer and a separate protein stabilizer. The protein stabilizer can ensure that the hemoglobin in the fecal sample is stable in the process of transportation for 3 days at normal temperature, but the time of more than 3 days cannot be ensured, so that the sample is required to be mailed to a laboratory within 3 days, the OC-sensor developed in Japan comprises a fecal sample sampling bottle, and the stabilizer can ensure that the hemoglobin in the fecal sample is stable for about 2 weeks at normal temperature, but the stability is poor at 37 ℃ and the degradation is serious along with the time. The method is more suitable for community sampling and centralized detection, and if the time for mailing the sample is longer and the temperature condition is not fixed, hidden danger exists in the products. In addition, the temperature difference is large in different areas and seasons, and particularly in summer, so that the temperature difference is a huge test on the products. Therefore, developing a stabilizer with high temperature resistance and good stability is a key for realizing a household sampling service mode.
The Chinese patent literature discloses a fecal sample treatment method and application thereof, the publication number of which is CN104931314A, and the patent discloses a simple and effective fecal sample stabilization method which is low in cost and convenient to use, can meet the requirements of qualitative hemoglobin detection, and is widely applied to the market of intestinal cancer screening at present. However, the method can only meet the requirement of qualitative detection, and quantitative FIT requires more stable sample and more reliable data, so that the method needs to be greatly optimized and improved. According to the method reported in this patent, the stool sample is stable well after drying, but during the time not yet dried, the stability is poor and the protein degradation is severe, as to how quickly a dried stool sample is obtained, the patent fails to find a solution. On the other hand, the dryness of the sample directly affects the stability of the sample, however, the method recorded in the patent only ensures that the water content of the sample is less than 10%, and the drying is not completely complete, which is one of reasons for unsatisfactory stability of the method. Therefore, how to quickly and thoroughly dry the sample is a problem that still needs to be solved at present. Furthermore, this patent has a technical problem of limited application range by forming a mixture of faeces and a desiccant by adding the desiccant into the container, which is not suitable for the stabilizing treatment of liquid samples. Human body fluid, particularly urine, is a biological sample with abundant information, has important clinical detection significance, but lacks an effective preservation means.
Disclosure of Invention
In view of the above-mentioned drawbacks of the prior art, the present invention aims to provide a preservation system, a preservation method and an application of human body fluid and excrement, which are used for solving the problems that the existing fecal sample treatment method has poor sample stability under preservation conditions of more than 37 ℃, different areas, different post times, different seasons and different transportation conditions and is not suitable for preserving liquid samples.
To achieve the above and other objects, the present invention provides a method for improving the preservation stability of human body fluid and excrement, wherein a sample of human body fluid and/or excrement is rapidly dehydrated and then thoroughly dehydrated, so as to achieve the stable preservation of the sample of human body fluid and/or excrement.
The invention also provides a preservation system for human body fluid and excrement, which comprises a sample box body, wherein a sample carrier for carrying a sample is arranged in the sample box body, the sample carrier separates the inner space of the sample box body to form a sample area and a drying area, a first drying agent is filled in the drying area, the first drying agent is in non-contact with the sample carrier, the sample carrier has water absorption and air permeability, and the sample box body has a sealing structure.
The sample preservation device in the prior art can only preserve solid samples, such as solid fecal samples, and the preservation system for human body fluid and excrement expands the application range through a separation type drying mode, so that the solid samples can be preserved, and liquid samples, such as human urine samples, can be preserved. The preservation system is a completely closed container, when the preservation system is applied to preservation of a liquid sample, the liquid sample can be rapidly dispersed on a sample carrier with water absorption and air permeability, the sample carrier is a compact, porous and air-permeable material, the liquid sample can be rapidly dispersed under the action of capillary action when being dripped on the carrier and uniformly attached to the carrier, the sample can reach a relatively dry state in the process, instant preliminary drying is realized, the sample on the sample carrier is subjected to gas exchange with a drying agent in a sample box body, and the non-contact water absorption effect of the drying agent is further combined, so that the sample on the sample carrier is thoroughly, rapidly and thoroughly dehydrated, long-term stability of the sample is realized, and protein components in the sample preserved by adopting the preservation system of the application are not denatured and deactivated and do not influence subsequent detection because of the complete dehydration state.
The sample carrier has two functions of rapidly dispersing samples in the early stage and achieving a primary drying effect instantaneously, and is breathable and porous in the later stage, so that gas exchange is facilitated, and the samples can be rapidly and thoroughly dehydrated by a drying agent. The preservation system can realize absolute stability of samples under different conditions, eliminates the differences among the samples caused by different areas, different post times, different seasons and different transportation conditions, ensures that the reliability of clinical detection meets the stability of the samples under different areas and season conditions of community service, and meets the stability of the samples in post process of the samples in uncertain environments for a long time in a long distance after home sampling.
More preferably, the first desiccant occupies 2/3 of the volume of the drying zone, thereby avoiding overfilling of the first desiccant resulting in contact with the sample carrier.
Preferably, a first ventilation filter screen is arranged below the sample carrier, and the first ventilation filter screen is fixed on the inner wall of the sample box body. The first air-permeable screen can ensure that the first desiccant and the sample are not in contact, but that the gas exchange is good.
Preferably, the inner wall of the sample box body further comprises a carrier supporting net arranged above the first ventilation filter net, the sample carrier is fixed at the center of the carrier supporting net, and the sample carrier is in non-contact with the inner wall of the sample box body, so that air in the sample box body has good circulation, and drying efficiency is improved.
Preferably, the sample box body is provided with a sealing cover, a filling pipe with a hollow hole-shaped structure is arranged at the bottom of the sealing cover, a second drying agent is filled in the filling pipe, and the second drying agent is in non-contact with the sample carrier.
The sealed lid can guarantee that inside and the outside air of sample box body are isolated, fills the drying effect that the second drier can further improve the sample in sealed lid.
Preferably, a second air-permeable screen is arranged between the filling tube and the sample carrier. The second air-permeable screen ensures that the second desiccant is in contact with the sample, but the gas exchange is good.
Preferably, at least one of the first and second desiccants irreversibly reacts with water molecules.
Preferably, the first desiccant and the second desiccant are selected from one or both of a chemical desiccant and a physical desiccant, and include at least a chemical desiccant.
More preferably, the chemical desiccant is calcium oxide.
More preferably, the physical desiccant is activated alumina or anhydrous calcium chloride.
The two types of desiccants, namely the chemical desiccant and the physical desiccant, can be matched for use, wherein the chemical desiccant combines all moisture in the sample box body through chemical reaction, so that the sample on the sample carrier is thoroughly dehydrated, and the moisture in the container is thoroughly absorbed. The physical drying agent is made of porous and large-surface-area materials, and is assisted in rapid water absorption. Sometimes the desired effect can be achieved by using calcium oxide alone.
Preferably, the material of the sample carrier is cellulose or animal hair.
More preferably, the sample carrier is made of industrial wool felt.
The invention also provides a preservation method of the human body fluid and the excrement, and the preservation system of the human body fluid and the excrement is adopted to place a human body fluid or an excrement sample on the surface of a sample carrier and seal the sample box body.
Preferably, when the stored environmental temperature is higher than 37 ℃, the method further comprises a sample pretreatment process, a buffer solution is added into the human body liquid or excrement sample, the buffer solution contains a protein protective agent, the stability of the sample before thorough dehydration can be improved by adopting the buffer solution, and an auxiliary stabilizing effect is achieved by matching with a storage system.
The prior art can only achieve the stability of a sample at 37 ℃, and the stability of the sample cannot be guaranteed at a higher temperature. In the process of mailing samples, in some cases, such as highway transportation in summer, the temperature in the carrier may reach 60 ℃ and above in a short time, and the sample preservation in the prior art will fail, resulting in unknown detection error results. The preservation system of the invention can still ensure the stability of 70% of the sample at 65 ℃ by combining with the preservation method.
More preferably, the protein protectant is selected from one or more of bovine serum albumin, trehalose, heme and protease inhibitors.
The invention also provides application of the preservation system for human body fluid and excrement in medical detection.
As described above, the present invention has the following advantageous effects:
(1) The application range is widened through a separation type drying mode, so that not only can solid samples be stored, but also liquid samples such as human urine samples can be stored;
(2) The preservation system is a completely closed container, when the preservation system is applied to preservation of a liquid sample, the liquid sample can be rapidly dispersed on a sample carrier with water absorption and air permeability, the sample carrier is a compact, porous and air-permeable material, the liquid sample can be rapidly dispersed under the action of capillary action when being dripped on the carrier and is uniformly attached to the carrier, the sample can reach a relatively dry state in the process, instant preliminary drying is realized, the sample on the sample carrier exchanges gas with a drying agent in a sample box body, and the non-contact water absorption effect of the drying agent is further combined, so that the sample on the sample carrier is thoroughly, rapidly and thoroughly dehydrated, and long-term stability of the sample is realized;
(3) The biological components in the sample preserved by the preservation system are in a complete dehydration state, so that the biological components are not denatured and inactivated, the subsequent detection is not influenced, and the biological components have wide application prospects in the field of medical detection;
(4) When the stored environmental temperature is higher than 37 ℃, the buffer solution containing the protein protective agent is added into the human body liquid or excrement sample through the pretreatment procedure, the buffer solution can improve the stability of the sample before thorough dehydration, and the auxiliary stabilizing effect is achieved by matching with a storage system;
(5) The liquid preservation methods commonly used in the prior art begin to denature and inactivate proteins above 50 ℃ and most species above 60 ℃ can be completely denatured, which can lead to subsequent detection failures or errors. In the method, protein components in the sample are in a completely dehydrated state, so that the protein components cannot be denatured and deactivated, and the subsequent detection is not influenced.
Drawings
Fig. 1 is a schematic diagram showing the structure of a preservation system for human body fluid and excrement of example 1.
Fig. 2 is a schematic diagram showing the structure of the preservation system of human body fluid and excrement of example 2.
FIG. 3 shows a line graph simulating the hemoglobin content of a stool sample over time at different temperatures.
FIG. 4 shows a line graph simulating the hemoglobin content of a urine sample over time at various temperatures.
The reference numerals illustrate the cartridge body 1, the sample area 11, the drying area 12, the first desiccant 121, the sealing cover 13, the filling tube 14, the second desiccant 141, the sample carrier 2, the first air-permeable screen 3, and the carrier support net 4.
Detailed Description
Other advantages and effects of the present invention will become apparent to those skilled in the art from the following disclosure, which describes the embodiments of the present invention with reference to specific examples. The invention may be practiced or carried out in other embodiments that depart from the specific details, and the details of the present description may be modified or varied from the spirit and scope of the present invention.
It should be understood that the process equipment or devices not specifically identified in the examples below are all conventional in the art.
It is to be further understood that the use of one or more method steps in the present invention does not exclude the presence of other method steps before or after the combination step or the insertion of other method steps between the explicitly mentioned steps, unless otherwise indicated, and that the use of a combined connection between one or more devices/means in the present invention does not exclude the presence of other devices/means before or after the combination device/means or the insertion of other devices/means between the explicitly mentioned two devices/means, unless otherwise indicated. Moreover, unless otherwise indicated, the numbering of the method steps is merely a convenient tool for identifying the method steps and is not intended to limit the order of arrangement of the method steps or to limit the scope of the invention in which the invention may be practiced, as such changes or modifications in their relative relationships may be regarded as within the scope of the invention without substantial modification to the technical matter.
Example 1
As shown in fig. 1, embodiment 1 of the present application provides a preservation system for human body fluid and excrement, comprising a sample box body 1, a sample carrier 2 and a sealing cover 13, wherein the sample carrier 2 is arranged in the sample box body and is used for carrying a sample, the inner wall of the sample box body is provided with a carrier support net 4, the sample carrier is fixed at the center of the carrier support net, so that the sample carrier and the inner wall of the sample box body are in non-contact to ensure good air permeability, the sample carrier separates the inner space of the sample box body to form a sample area 11 and a drying area 12, a first air-permeable filter screen 3 is arranged below the sample carrier, the first air-permeable filter screen is fixed on the inner wall of the sample box body and is positioned in the drying area, and the carrier support net is arranged above the first air-permeable filter screen. The drying area is filled with a first drying agent 121, the first drying agent is in non-contact with the sample carrier, the sample carrier has water absorption and air permeability, and the sample box body and the sealing cover form a sealing structure after being sealed and fixed. The first desiccant was a chemical desiccant of calcium oxide and the sample carrier was an industrial wool felt with a thickness of 2 mm.
Example 2
As shown in fig. 2, embodiment 1 of the present application provides a preservation system for human body fluid and excrement, comprising a sample cartridge body 1, a sample carrier 2 for carrying a sample provided in the sample cartridge body, and a sealing cover 13, wherein a filling tube 14 having a hollow hole-like structure is provided at the bottom of the sealing cover, and a second desiccant 141 is filled in the filling tube, and the second desiccant is in non-contact with the sample carrier. The inner wall of the sample box body is provided with a carrier support net 4, the sample carrier is fixed at the center of the carrier support net, so that the sample carrier and the inner wall of the sample box body are in non-contact, good air permeability is guaranteed, the sample carrier separates the inner space of the sample box body to form a sample area 11 and a drying area 12, a first air-permeable filter screen 3 is arranged below the sample carrier, the first air-permeable filter screen is fixed on the inner wall of the sample box body and is positioned in the drying area, and the carrier support net is arranged above the first air-permeable filter screen. The drying area is filled with a first drying agent 121, the first drying agent is in non-contact with the sample carrier, the sample carrier has water absorption and air permeability, and the sample box body and the sealing cover form a sealing structure after being sealed and fixed. The first desiccant is a mixture of chemical desiccant calcium oxide and the second desiccant is a mixture of chemical desiccant calcium oxide and physical desiccant activated alumina. The sample carrier is made of industrial wool felt with the thickness of 2mm, and is cut into 8 mm multiplied by 8 mm squares when in use.
Example 3
Example 3 differs from example 1 in that the first desiccant is of a different type, specifically using a physical oxidant activated alumina.
Comparative examples 1 to 5
Comparative examples 1 to 5 are different from example 1 in that the sample carriers are different, as shown in table 1.
Comparative examples 6 to 10
Comparative examples 6-10 differ from example 1 in that the sample carrier used a physical oxidizer, activated alumina, was different in the type of the first desiccant, and the sample carrier is shown in table 1.
The embodiment of the application also provides a preservation method of human body fluid and excrement by adopting the preservation systems of the embodiments and the comparative examples, wherein the sample box body adopts a 50 mL medical centrifuge tube, the sealing cover adopts a centrifuge tube cover, the types of the sample carrier and the first desiccant are shown in Table 1 in detail, and the preservation method comprises the following steps:
Cutting the sample carrier into square with the size of 8mm multiplied by 8mm by using scissors, fixing the square at the center of a carrier supporting net, adding a first drying agent into a drying area, adding the first drying agent into a 50 mL medical centrifuge tube until the first drying agent is near 35 mL of the centrifuge tube, taking 4 0.5 mL centrifuge tubes, covering a tube cover, vertically inserting the square sample carrier into the first drying agent, and placing the cut square sample carrier into the tube covers of the 4 0.5 mL centrifuge tubes. Tightening a 50 mL centrifugal pipe cover, and attaching a sealing adhesive tape to the pipe orifice;
drying sample carrier, namely carrying out gas exchange on the sample carrier and the first drying agent in a centrifuge tube for 24 hours, and carrying out gas exchange on the sample carrier and the first drying agent
The simulated sample hemoglobin solution was prepared by dissolving human hemoglobin standard in nuclease-free water, and diluting with 10 mg/mL BSA (bovine serum albumin) solution to give a final hemoglobin concentration of 10 ug/mL.
50. Mu.L of the simulated samples were pipetted with 200. Mu.L of each pipette, added to the middle of the sample carriers of the preservation systems of the respective examples and comparative examples, and the seal caps were screwed down to divide the samples into 2 groups, one group stored in a 25℃incubator and the other group stored in a 37℃incubator.
Taking out one sample on days 1,2 and 3 respectively, taking out one sample carrier, transferring to a2 mL centrifuge tube, adding 2 mL nuclease-free water, swirling for 30s, standing for 10min at normal temperature, swirling for 30s again, taking 100 mu L of sample, adding to an immunofluorescence reagent card, and detecting the content of hemoglobin by using a full-automatic immunofluorescence instrument. The original hemoglobin sample was diluted approximately 40-fold to a final concentration of 250ng/mL. Daily test values were recorded in ng/mL and the results are shown in Table 1:
TABLE 1 detection results of the simulated samples at different storage temperatures using the storage systems of examples 1 and 3 and comparative examples 1-10
As can be seen from Table 1, the sample carrier stabilizing effect and elution effect are best industrial wool felt, and the other carriers have poor stability and elution comprehensive properties. The sample carrier is most preferably an industrial wool felt.
The water absorption of the activated alumina as a physical desiccant is reversible, so that a sample cannot be completely and thoroughly dried, and the residual small amount of water causes the sample to be unstable, while the water absorption of the activated alumina as a chemical desiccant is irreversible, so that the water in a sealed container can be completely and thoroughly absorbed in enough time, the sample can be completely and thoroughly dehydrated, and the stable effect is realized. The first desiccant is most preferably calcium oxide;
Comparing the difference of different preservation temperatures of 25 ℃ and 37 ℃, under the condition of 25 ℃, calcium oxide is used as a first drying agent, industrial wool felt is used as a sample carrier, and the stability is good, but under the condition of 37 ℃, the hemoglobin degradation of the combination is 40% on day 1, and the combination is stable on days 2 and 3 relative to day 1, which shows that after the sample is added to the sample carrier, the sample is instantly absorbed and dried, but the drying is not thorough, the stability is poor at high temperature, and the further optimization is needed.
In order to improve the stability of the 37 ℃ sample, the sample was stored in the human body fluid and excrement storage system of example 1, and after the sample was added, the sample was divided into two groups, the test group 1 was placed at normal temperature of 25 ℃ for 24 hours, then the 37 ℃ test was performed, the control group 1 was directly stored at 37 ℃, and then the test combination control group was tested by the same test method as described above, and the results are shown in table 2:
TABLE 2 detection results of different drying modes for different shelf times at 37℃for Experimental group 1 and control group 1
As can be seen from Table 2, the stability of the sample at the subsequent high temperature can be significantly improved by drying the sample under milder conditions (within 25 ℃) for 24 hours after the sample is added.
In order to improve the stability of the sample before complete dehydration in the preservation system of example 1, a sample buffer containing a BSA (bovine serum albumin) solution as a core component was provided as an auxiliary function.
Sample carrier preparation the industrial wool felt was sheared with scissors into squares of about 8 mm x8 mm.
The design of a drying agent container, namely adding granular calcium oxide into a 50mL medical centrifuge tube until the granular calcium oxide is near the centrifuge tube 35 mL, taking 4 0.5mL centrifuge tubes, covering a tube cover, vertically inserting the centrifuge tubes into the calcium oxide, and placing the sheared square sample carrier at the tube covers of the 4 0.5mL centrifuge tubes. And (5) screwing the centrifugal tube cover of 50mL, and attaching a sealing adhesive tape to the tube opening.
And (3) drying the sample carrier, namely carrying out gas exchange on the sample carrier and calcium oxide in a centrifuge tube for 24 hours, and carrying out gas exchange on the sample carrier and the calcium oxide.
The hemoglobin solution is prepared by dissolving human hemoglobin standard substance into 2 groups, dissolving experimental group 2 in nuclease-free water, diluting with 50 mg/mL BSA (bovine serum albumin) solution to obtain final hemoglobin concentration of 10ug/mL, and diluting control group 2 with nuclease-free water to obtain final hemoglobin concentration of 10ug/mL
The 2 groups of simulation samples are respectively sucked into 50 mu L of simulation samples by a 200 mu L pipettor, added into the middle of a sample carrier, screwed on a centrifugal tube cover, and the tube mouth is sealed by a sealing adhesive tape. Stored at room temperature 25 ℃.
After 24h, one sample was removed from each group, the sample carrier was removed, transferred to a 2mL centrifuge tube, added with 2mL nuclease free water, vortexed for 30 seconds, allowed to stand at ambient temperature for 10 minutes, and vortexed again for 30 seconds. 100 μl of the sample was taken and added to an immunofluorescent reagent card, and the hemoglobin content was measured using a full-automatic immunofluorescent meter, with the original hemoglobin sample diluted approximately 40-fold to a final concentration of 250ng/mL. The values are recorded.
Samples were divided into 3 groups and stored in 25 ℃,37 ℃ and 65 ℃ incubators, respectively.
One sample was taken from each group on days 1,2,3, the sample carrier was removed, transferred to a2 mL centrifuge tube, 2 mL nuclease free water was added, vortexed for 30 seconds, allowed to stand at ambient temperature for 10 minutes, and vortexed again for 30 seconds. 100 μl of sample is taken and added to an immunofluorescence reagent card, and the hemoglobin content is detected by using a full-automatic immunofluorescence instrument. Daily test values were recorded in ng/mL and the results are shown in Table 3:
TABLE 3 detection results of different shelf lives of Experimental group 2 and control group 2 at different temperatures
From Table 3, it can be seen that the addition of the protective buffer has no significant effect on stability from the comparison of the results at 25℃and 37℃and that the addition of the protective buffer left about 75% of the samples and about 55% of the samples without the addition of the protective buffer compared to the results at 65℃and that the protective buffer did not significantly improve stability under mild conditions (within 37 ℃) but significantly improved stability of the samples under relatively severe conditions.
Application example one simulation of human fecal sample hemoglobin stability study
Sample carrier preparation an industrial wool felt of thickness 2 mm was selected and sheared with scissors into squares of about 8 mm x 8 mm.
The design of a drying agent container, namely adding granular calcium oxide into a 50 mL medical centrifuge tube until the granular calcium oxide is near the centrifuge tube 35 mL, taking 4 0.5 mL centrifuge tubes, covering a tube cover, vertically inserting the centrifuge tubes into the calcium oxide, and placing the sheared square sample carrier at the tube covers of the 4 0.5 mL centrifuge tubes. As an alternative to a gas permeable screen, it can be ensured that the sample carrier and the underlying calcium oxide do not come into contact, but that a sufficient gas exchange is possible. And (5) screwing the centrifugal tube cover of 50 mL, and attaching a sealing adhesive tape to the tube opening.
And drying the sample carrier, namely carrying out gas exchange on the sample carrier and calcium oxide in a centrifuge tube for 24 hours, and carrying out chemical reaction on the residual moisture of the sample carrier and the calcium oxide to thoroughly dehydrate.
The hemoglobin solution was prepared by dissolving human hemoglobin standard in nuclease-free water, and diluting with 50 mg/mL BSA (bovine serum albumin) solution to give a final hemoglobin concentration of 10 ug/mL.
Preparation of a simulated sample, namely simulating the process of sampling and preparing a fecal sample before conventional fecal occult blood detection. A small amount of human fecal sample, about 0.2 gram of fecal sample, was added to the prepared hemoglobin solution of about 10mL and mixed by shaking.
50. Mu.L of the simulated sample was pipetted with a 200. Mu.L pipette, added to the middle of the sample carrier, screwed down the centrifuge tube lid and left to stand at room temperature. A total of 20 identical samples were prepared.
After 1 hour, after the sample on the carrier is completely dried, one of the sample carriers is taken out, transferred to a 2mL centrifuge tube, added with 2mL nuclease-free water, vortexed for 30 seconds, allowed to stand at normal temperature for 10 minutes, and vortexed again for 30 seconds. 100 μl of sample was taken and added to an immunofluorescent reagent card, hemoglobin content was measured using a full-automatic immunofluorescent meter, and the value was recorded as control day, value T.
The rest sample centrifugation tube openings are sealed by sealing adhesive tapes and stored at room temperature.
After 24 hours, one of the samples was removed, the sample carrier was removed, transferred to a 2 mL centrifuge tube, added with 2 mL nuclease free water, vortexed for 30 seconds, allowed to stand at room temperature for 10 minutes, and vortexed again for 30 seconds. 100 μl of the sample was taken and added to an immunofluorescent reagent card, and the hemoglobin content was measured using a fully automatic immunofluorescent apparatus, and the value was recorded as day 0, value T0, and the result is shown in table 4.
The remaining 18 samples were divided into 3 groups, 1 group in a 25 degree incubator, 2 group in a 37 degree incubator, and 3 group in a 65 degree incubator.
One sample was taken on each 1,4,6,8,11,13 th day and the hemoglobin concentration was measured according to the procedure of step 9, with the values recorded as 25T1,25T4,25T6. Cndot. T followed by the numbers representing the days of sample storage, and 37. Cndot. Was recorded as 37T1, 37T4,37T6. Cndot. T followed by the numbers representing the days of sample storage.
And dividing the hemoglobin content of other dates by T by taking the control day value T as a standard to obtain the residual hemoglobin percentage. A line graph was made from the percentage amounts and the sample stability was analyzed and the results are shown in fig. 3.
TABLE 4 test results of stability test for simulated fecal samples
As can be seen from Table 4 and FIG. 3, excluding the error of immunofluorescence itself, T0 to T is about one hundred percent, indicating good sample stability during room temperature drying, and excluding the error of immunofluorescence itself, 25C and 37C samples, hemoglobin concentrations within 13 days are nearly one hundred percent compared to control day T, indicating that hemoglobin in fecal samples is stable according to the methods of the present invention. At 65 ℃, about 70% remains after the first day of hemoglobin degradation, but can remain stable later. It shows that under relatively extreme conditions, trace residual moisture still has an effect on sample stability, but after the subsequent dehydration is complete, a stabilizing effect can be achieved.
Application example II, experiment for simulating hemoglobin stability of human urine sample
Sample carrier preparation an industrial wool felt of thickness 2 mm was selected and sheared with scissors into squares of about 8 mm x 8 mm.
The desiccant container was designed to add granular calcium oxide to a 50 mL medical centrifuge tube to around centrifuge tube 25 mL and to continue adding activated alumina to around centrifuge tube 25 mL. Taking 4 0.5 mL centrifuge tubes, covering a tube cover, vertically inserting the tube covers into activated alumina, and placing the sheared square sample carriers at the tube covers of the 4 0.5 mL centrifuge tubes. As an alternative to a gas permeable screen, it can be ensured that the sample carrier and the underlying activated alumina do not come into contact, but that gas exchange is sufficient. And (5) screwing the centrifugal tube cover of 50 mL, and attaching a sealing adhesive tape to the tube opening.
And (3) drying the sample carrier, namely carrying out gas exchange for 24 hours on the sample carrier and two desiccants in a centrifuge tube, and carrying out chemical reaction on the residual water of the sample carrier and calcium oxide to thoroughly dehydrate, wherein the surface area of the activated alumina is larger, so that the gas exchange can be quickened.
The hemoglobin solution is prepared by dissolving human hemoglobin standard substance in nuclease-free water, and the final concentration of hemoglobin is 200 ug/mL.
Preparation of a simulated sample, namely taking 18 mL fresh urine, adding 1 mL hemoglobin mother solution, wherein the final concentration of hemoglobin is 10 ug/mL, and 1 g BSA (bovine serum albumin), and shaking and mixing uniformly. The final hemoglobin concentration was about 10 ug/mL and BSA (bovine serum albumin) concentration was about 50 mg/mL.
50. Mu.L of the simulated sample was pipetted with a 200. Mu.L pipette, added to the middle of the sample carrier, screwed down the centrifuge tube lid and left to stand at room temperature. A total of 20 identical samples were prepared.
After 1 hour, after the sample on the carrier is completely dried, one of the sample carriers is taken out, transferred to a 2mL centrifuge tube, added with 2mL nuclease-free water, vortexed for 30 seconds, allowed to stand at normal temperature for 10 minutes, and vortexed again for 30 seconds. 100 μl of sample was taken and added to an immunofluorescent reagent card, hemoglobin content was measured using a full-automatic immunofluorescent meter, and the value was recorded as control day, value T.
The rest sample centrifugation tube openings are sealed by sealing adhesive tapes and stored at room temperature.
After 1 day, one of the samples was removed, the sample carrier was removed, transferred to a2 mL centrifuge tube, added with 2 mL nuclease free water, vortexed for 30 seconds, allowed to stand at room temperature for 10 minutes, and vortexed again for 30 seconds. 100 μl of the sample was taken and added to an immunofluorescent reagent card, and the hemoglobin content was measured using a fully automatic immunofluorescent apparatus, and the value was recorded as day 0 of the experiment, value T0, and the result is shown in table 5.
The remaining 18 samples were divided into 2 groups, one group stored in a 25 degree incubator and the other group stored in a 37 degree incubator.
Taking out one sample on 1,4,6,8,11,13 days respectively, detecting the concentration of hemoglobin according to the mode of the step 9, wherein the numerical record is that the samples stored at 25 degrees are respectively recorded as 25T1,25T4,25T6T and the numbers behind the samples represent the days of sample storage; the 37 degree stored samples were recorded as 37T1,37T4,37T6T followed by numbers representing the number of days the sample was stored.
And dividing the hemoglobin content of other dates by T by taking the control day value T as a standard to obtain the residual hemoglobin percentage. A line graph was made from the percentage amounts and the sample stability was analyzed and the results are shown in fig. 4.
TABLE 5 test results of stability test for simulated urine samples
As can be seen from Table 5 and FIG. 4, excluding the error of immunofluorescence itself, T0 to T is about one hundred percent, indicating good sample stability during room temperature drying, and excluding the error of immunofluorescence itself, 25C and 37C samples, hemoglobin concentrations within 13 days are nearly one hundred percent compared to control day T, indicating that hemoglobin in urine samples is stable according to the methods of the present invention. At 65 ℃, about 70% remains after the first day of hemoglobin degradation, but can remain stable later. It shows that under relatively extreme conditions, trace residual moisture still has an effect on sample stability, but after the subsequent dehydration is complete, a stabilizing effect can be achieved.
The stabilization method of the present invention is directed to human body fluid and fecal samples including proteins, carbohydrates, lipids, nucleic acids, human metabolic waste products, and the like, as well as protein and nucleic acid components of microorganisms such as bacteria, fungi, mycoplasma, and the like. Hemoglobin is one of the most well-accepted components of poor stability, and thus is exemplified in the application example. According to the design concept of the invention, all the components lose activity under the condition of complete dehydration, so that protein and nucleic acid cannot be degraded, and all microorganisms stop growing and reproducing, so that metabolic wastes cannot be generated and the content of the original sample cannot be changed. This means that all information of the original sample is truly and accurately saved, and the sample adopted in the subsequent clinical detection can truly reflect the actual situation of the patient.
The above embodiments are merely illustrative of the principles of the present invention and its effectiveness, and are not intended to limit the invention. Modifications and variations may be made to the above-described embodiments by those skilled in the art without departing from the spirit and scope of the invention. Accordingly, it is intended that all equivalent modifications and variations of the invention be covered by the claims, which are within the ordinary skill of the art, be within the spirit and scope of the present disclosure.
Claims (10)
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