CN113005177B - Blood sample obtaining method, blood sample and application - Google Patents

Blood sample obtaining method, blood sample and application Download PDF

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CN113005177B
CN113005177B CN202110314977.0A CN202110314977A CN113005177B CN 113005177 B CN113005177 B CN 113005177B CN 202110314977 A CN202110314977 A CN 202110314977A CN 113005177 B CN113005177 B CN 113005177B
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collection tube
separation gel
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文中伟
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Hangzhou Biquan Health Technology Co ltd
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Hangzhou Beiqiang Medical Technology Co ltd
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Abstract

The embodiment of the invention relates to the field of biotechnology, and particularly discloses a blood sample obtaining method, a blood sample and application.

Description

Blood sample obtaining method, blood sample and application
Technical Field
The embodiment of the invention relates to the technical field of biology, in particular to a blood sample obtaining method, a blood sample and application.
Background
Blood samples (including human blood samples and animal blood samples) are the most common samples in biomedical research, and are also the most common samples in clinical examination, and blood samples are usually obtained by blood drawing.
At present, in most medical institutions, a vacuum blood collection tube, a procoagulant serum blood collection tube, an EDTA (ethylene diamine tetraacetic acid) anticoagulated whole blood collection tube, a heparin anticoagulation blood collection tube, a sodium citrate anticoagulation blood collection tube and the like are used for blood collection. In general, EDTA anticoagulation blood collection tubes are used in only a few items such as hematology and glycated hemoglobin tests, while procoagulant serum collection tubes are used in a large number of clinical tests such as biochemistry, tumor markers, various proteins, electrolytes and the like.
With the popularization of automatic inspection instruments, the procoagulant serum blood collection tubes used in medical institutions at present have fixed specifications, are hollow cylindrical tubes, have the blood collection amount of 4mL, almost contain separation gel in the blood collection tubes, and separate blood cells from liquid components in blood through the separation gel. However, in the case of a blood collection tube containing a separation gel, after separation is completed, the separation gel covers the blood cells, so that the blood cells below are difficult to reuse. Therefore, at present, almost all serum blood collection tubes containing separation gel in medical institutions can not effectively obtain blood samples for extracting blood cell nucleic acid, and most of the blood samples are directly discarded, thereby causing resource waste.
Disclosure of Invention
An object of the present invention is to provide a method for obtaining a blood sample, so as to solve the problem that the prior serum blood collection tube containing a separation gel cannot effectively remove the separation gel to obtain the blood sample. According to the embodiment of the invention, the blood sample is obtained from the blood collection tube containing the separation gel through researching the blood sample obtaining method, and then the blood sample is used for extracting blood cell nucleic acid from the blood collection tube from which blood cells and liquid are separated by the separation gel, so that the resource utilization rate is effectively improved, and the method has an important significance for further expanding the application field of the coagulation-promoting serum blood collection tube.
In order to achieve the above purpose, the embodiments of the present invention provide the following technical solutions:
a method of blood sample acquisition comprising the steps of:
1) removing upper layer liquid from the blood collection tube containing the separation gel after blood collection, wherein the upper layer liquid is the separation liquid above the separation gel in the blood collection tube;
2) placing the blood sampling tube with the upper layer liquid removed into an adhesion tube for adhering separation gel, and injecting cell lysate into the blood sampling tube before or after placing the blood sampling tube into the adhesion tube;
3) and centrifuging the blood collection tube injected with the cell lysate, and then removing the adhesion tube to take out the separation gel and the insoluble blood clot together with the adhesion tube to obtain a separated blood sample.
As a further scheme of the embodiment of the invention: the separation liquid is serum and/or plasma.
As a further scheme of the embodiment of the invention: the heparin tube that contains the separating gel is including the procoagulant heparin tube that contains the separating gel, contains the anticoagulation heparin tube of separating gel and contain any one in the anticoagulation heparin tube that separating gel, and is specific, the heparin anticoagulation heparin tube, the anticoagulation heparin tube etc. of blood collection tube for having accomplished separation gel blood cell and liquid separation and all heparin tube that anticoagulation and not the anticoagulation are not untied, for example procoagulant serum heparin tube, the anticoagulation whole blood tube of EDTA to and other special heparin tube that use the separating gel clinically, specifically select according to the demand, do not limit here.
As a further scheme of the embodiment of the invention: the cell lysate is salt ion solution which can crack cells, and the density of the salt ion solution is at least higher than that of the separation gel.
It is another object of embodiments of the present invention to provide a blood sample prepared by the above blood sample obtaining method.
Another object of the embodiments of the present invention is to provide a use of the above blood sample obtaining method in nucleic acid extraction.
Compared with the prior art, the embodiment of the invention has the beneficial effects that:
according to the blood sample obtaining method provided by the embodiment of the invention, the separation gel can be simply, quickly, cleanly and low-cost completely removed from the blood collection tube, and the nucleic acid is released by dissolving the blood cells in the blood collection tube, so that the nucleic acid extraction is facilitated, the problem that the separation gel cannot be effectively removed to obtain the blood sample in the existing blood collection tube containing the separation gel is solved, and the wide market prospect is achieved.
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Fig. 1 is a schematic flow chart of a blood sample obtaining method according to an embodiment of the present invention.
Detailed Description
The invention is described in further detail below with reference to the figures and specific examples. The following examples will assist those skilled in the art in further understanding the embodiments of the present invention, but are not intended to limit the embodiments of the present invention in any way. It should be noted that various changes and modifications can be made by those skilled in the art without departing from the spirit of the embodiments of the present invention. These are all within the scope of the embodiments of the present invention. The embodiment of the invention is only a brand-new blood sample obtaining method, part of the content adopts standard equipment or the prior art, and the original problem does not exist, namely the part which is not related in the embodiment of the invention can be realized by adopting the prior art, and the description is not repeated.
The embodiment of the invention provides a method for obtaining a blood sample, in particular to a method for obtaining a blood sample from a blood collection tube containing separation gel, which comprises the following steps:
1) removing upper layer liquid from the blood collection tube containing the separation gel after blood collection, wherein the upper layer liquid is the separation liquid above the separation gel in the blood collection tube;
2) placing an adhesion tube for adhering the separation gel into the blood sampling tube from which the upper layer liquid is removed, and injecting cell lysate into the blood sampling tube before or after the blood sampling tube is placed into the adhesion tube;
3) and centrifuging the blood collection tube injected with the cell lysate, and then removing the adhesion tube to take out the separation gel and the insoluble blood clot together with the adhesion tube to obtain a separated blood sample.
As another preferred embodiment of the present invention, the blood sample is a blood cell lysate containing at least nucleic acid, and can be directly used for nucleic acid extraction.
As another preferred embodiment of the present invention, the separation liquid is serum and/or plasma, and specifically, the separation liquid may be serum, such as in procoagulant separation gel serum collection tubes; or plasma, such as in a sodium citrate anticoagulant gel blood collection tube.
As another preferred embodiment of the present invention, the blood collection tube containing a separation gel is any one of a procoagulant blood collection tube containing a separation gel, an anticoagulated blood collection tube containing a separation gel, and an anticoagulated blood collection tube containing a separation gel, and specifically, the blood collection tube containing a separation gel is all of procoagulant, anticoagulated, and anticoagulated blood collection tubes that have completed separation of cells from a liquid, such as procoagulant serum blood collection tubes, EDTA anticoagulated whole blood collection tubes, heparin anticoagulated blood collection tubes, sodium citrate anticoagulated blood collection tubes, and the like, and other special blood collection tubes that use a separation gel clinically, and is specifically selected according to the needs, and is not limited herein.
As another preferred embodiment of the present invention, the method for removing the supernatant includes pouring and/or sucking, and the main function of removing the supernatant is to make room for injecting the cell lysate, specifically, the liquid can be poured by directly pouring the blood collection tube, and the liquid can also be sucked by using a pipette tip. The removal or the suction is not necessarily complete, and a small amount of the remaining liquid does not affect the treatment effect, but the removal is preferably performed as much as possible. After the upper layer of liquid is removed, the separation gel can be seen directly, covering the blood cell layer. At this moment, the separation glue is firmly combined with the wall of the blood collection tube, and the internal structure of the blood collection tube cannot be damaged when the blood collection tube is overturned and shaken.
As another preferred embodiment of the present invention, the adhesion tube is a tube with two open ends, and the shape of the adhesion tube is adapted to the blood sampling tube, the outer diameter of the adhesion tube is close to but slightly smaller than the inner diameter of the blood sampling tube, the difference between the two is not more than 4mm, and preferably smaller than the inner diameter of the blood sampling tube by 1-2 mm.
As another preferred embodiment of the present invention, the length of the adhesion tube should be longer than the length of the blood collection tube, preferably more than 1-2 cm.
As another preferable embodiment of the present invention, the wall thickness of the adhesion tube is not greater than 1/3 of the inner diameter of the blood collection tube, preferably about 0.5 mm.
As another preferred embodiment of the present invention, the strength of the material of the adhesion tube is determined by that the adhesion tube can support the centrifugal force of 2000-3000 g in the blood lysate liquid environment without deformation or collapse.
Preferably, the adhesion tube body can be a thin-wall plastic tube, generally a disposable tube (a hollow cylindrical disposable plastic tube with openings at the upper and lower ends), the appearance of the adhesion tube body is adapted to the inner part of the blood sampling tube, the adhesion tube body is a circular straight tube, the upper and lower ends of the adhesion tube body are provided with openings at the broken ends, the outer diameter of the adhesion tube body is slightly smaller than the inner diameter of the blood sampling tube, and the outer diameter of the adhesion tube body is preferably smaller than the inner diameter of the blood sampling tube by 1-2 mm. The total length of the thin-wall plastic tube is larger than that of the blood collection tube, preferably more than 1-2 cm. The wall thickness of the thin-wall plastic tube is not more than 1/3 of the inner diameter of the blood collecting tube, preferably about 0.5mm, and the strength can support the centrifugal force of 2000-3000 g without deformation or collapse. The shape, thickness and strength of the thin-wall plastic pipe are similar to those of a plastic straw which is generally sold on the market and used for coffee milk tea. The operation is carried out by directly placing the plastic tube into the plastic tube, and the lower opening of the plastic tube is contacted with the separating glue. The effect is not influenced when the thin-wall plastic pipe is slightly inserted into the separating glue or is contacted with the separating glue part. If meet the condition that the pipe diameter of heparin tube the latter half is less than heparin tube upper end opening part pipe diameter, can cut open the latter half of thin-walled plastic tube, and the first half keeps the original state, can avoid appearing can't insert the bottom or fill up the too tight condition after the centrifugation like this.
It should be noted that the thin-walled plastic tube is inserted as a disposable material to which the release glue is adhered. The order of the steps of placing the adhesion tube and injecting the cell lysate can be interchanged, but it is preferable to place the adhesion tube first.
As another preferred embodiment of the present invention, the cell lysis solution is a salt ion solution that can lyse cells, and the density of the salt ion solution is at least higher than that of the separation gel.
As another preferred embodiment of the present invention, the injected cell lysate contains a conventional lysate containing guanidine thiocyanate, guanidine isothiocyanate, sodium dodecyl sulfate, proteinase K, etc. (refer to the description of nucleic acid extraction method in 2011, volume 28, page 12, pages 75-78 of the publication of animal quarantine, china, and the description of similar scientific and technical papers), and the density of the cell lysate is increased by increasing the concentration of original salt ions or adding other salt ion compounds, so that the density of the cell lysate is greater than that of the separation gel.
As another preferred embodiment of the present invention, the density of the salt ion solution is at least 1% higher than the density of the separation gel, and specifically, the lower limit of the amount of the salt ion solution added is that the density of the salt ion solution is at least 1% higher than the density of the separation gel, and the upper limit is that the salt ion is saturated in the salt ion solution.
As another preferred embodiment of the present invention, the injection amount of the cell lysate may be the same volume as the amount of blood in the blood collection tube, but should not be less than 1mL, and too little cell lysate will reduce the amount of lysed cells, weaken the action of lysing some cells in the thrombus, and reduce the yield of nucleic acid extraction. The maximum volume of added cell lysate should not exceed the volume of the blood collection tube to avoid overflow. After the cell lysis solution is added, the visible liquid is positioned in the thin-wall plastic tube and above the separation gel.
As another preferred embodiment of the present invention, the injection amount of the cell lysate is 1-3mL, and the formulation of the cell lysate is a salt ion solution that is conventionally used for cell lysis, such as guanidine thiocyanate, guanidine isothiocyanate, SDS (sodium dodecyl sulfate), proteinase K, etc., and also includes other high concentration salt ion solutions having cell lysis effect. The components of the cell lysate used can be referred to published data and are not described in detail herein, except that the cell lysate described herein should have an increased salt ion concentration in order to achieve a higher density than the gel and clot, or at least above the gel.
The specific gravity of the separation gel to pure water is generally 1.04 to 1.05. The cell lysate is typically prepared by adding one or more water-soluble compounds to pure water to form a salt ion solution. When the amount of the water-soluble compound added per 100 parts by mass of the pure water is more than 5 parts by mass, the specific gravity of the cell lysate will start to be larger than that of the separation gel. To ensure that the specific gravity of the cell lysate is greater than that of the separation gel, the mass of the added water-soluble compound can be increased, for example, by more than 10 parts. There are two methods for increasing the concentration of salt ions in a cell lysate, one is to increase the mass content ratio of the components of the cell lysate in the lysate. The other is adding other water soluble compounds which do not affect nucleic acid extraction, when the concentration of salt ions in the conventional cell lysis solution is not high enough, the added compounds can be guanidine hydrochloride, guanidine thiocyanate, guanidine isothiocyanate, sodium chloride, ammonium chloride and the like by adding other salt ions which do not affect nucleic acid extraction. The specific salt ion species can be adjusted according to the different subsequent extraction reagents. The lower limit of the adding amount is that the solution density is at least 1% higher than the separating gel, and the upper limit is that the salt ions reach saturation in the solution.
As another preferred embodiment of the present invention, the centrifugation is to sink the cell lysate into the underlying blood cell layer by gravity to perform the cell lysis function, and simultaneously, the thin-walled plastic tube is cut into the separation gel regularly and cleanly around the circumference. The separation gel floats on the uppermost layer under the action of gravity, the upper surface and the lower surface are flat, and blood clots float in the blood cell layer and adhere to the lower end of the separation gel. Wherein the centrifugal force and the duration of centrifugation are such that the cell lysate sinks through the separation gel as a starting point, and the centrifugal force and the duration of centrifugation are increased as to maximize the limit of not causing rupture of the blood collection tube.
As another preferred embodiment of the present invention, the centrifugation is performed using a centrifuge suitable for centrifugation of blood collection tubes, and the whole blood collection tube (containing thin-walled plastic tube and added solution) is placed in the centrifuge for centrifugation. Specifically, the fixed-inclination centrifuge and the vertical barrel centrifuge may be used, but the fixed-inclination centrifuge is preferred, and the centrifugal force of the centrifuge should be more than 500 g, so that the centrifuged separation glue adheres to the inner wall of the adhesion tube (thin-walled plastic tube), and the cell lysate is completely immersed in the blood cell layer of the originally collected blood. The magnitude of the minimum centrifugal force required is related to the cell lysate density, with smaller densities requiring greater centrifugal forces and greater densities allowing the centrifugal force to be reduced. Generally, about 2500 g is preferred, and the centrifugal force is larger, so that the centrifugal time is shorter, but the centrifugal force should not exceed the bearing force of the blood collection tube, so as to prevent the tube from breaking. Under 2500 g centrifugal force, the centrifugation time generally needs 1-3 minutes, but the subsequent operation can not be influenced by increasing the centrifugation time to 5-10 minutes. After centrifugation, the thin-wall plastic tube is inserted into the bottom of the blood collection tube tightly attached to the inner wall of the blood collection tube, and the separation glue is adhered to the inner wall of the thin-wall plastic tube. The cell lysate completely sinks into the layer of primary blood cells. The blood clot acted by the cell lysate floats upwards in the blood cell layer and can be generally adhered to the lower end of the separation gel, but the blood clot cannot be adhered due to lower centrifugal force.
The conversion of the number of revolutions of the centrifuge and the centrifugal force is G =1.11 × 10 -5 ×R×(rpm) 2 Calculating, wherein R is the distance between the center of the rotating shaft of the centrifugal machine and the inner wall of the bottom of the centrifugal sleeve; rpm (revolution per minute) is the number of revolutions per minute of the centrifuge; g is the relative centrifugal force (in grams) expressed as the gravitational force, i.e. the multiple of the gravitational acceleration.
As another preferred embodiment of the present invention, the method for obtaining a blood sample further comprises a step of performing a standing process before removing the adhesion tube, wherein the standing blood collection tube is used to lyse the blood cells as much as possible from the cell lysate below the separation gel, lyse the blood cells to release nucleic acids, and shrink blood clots. The standing time is generally 3 to 5 minutes, and extension or reduction of the time may increase or decrease the amount of the finally extracted nucleic acid, but the effect is small. After standing, the head end of the thin-wall plastic tube can be directly grasped and pulled out, at the moment, the separation gel and the blood clot which cannot be dissolved are brought out together with the thin-wall plastic tube, and the separation gel and the blood clot are all abandoned into the special medical garbage bag. At the moment, the blood sampling tube has no separation gel and blood clot, and blood cell lysate containing nucleic acid is remained. The extraction of the blood cell nucleic acid can be continued by conventional procedures.
As another preferred embodiment of the present invention, the above steps can be used manually or the program can be designed to be automatically operated by a machine.
The blood sample obtaining method provided by the embodiment of the invention has the advantages that the liquid above the separation gel in the blood collection tube is removed, the thin-wall plastic tube is placed in the blood collection tube, the cell lysate is injected into the thin-wall plastic tube, the whole blood collection tube and the placed object are centrifuged in the centrifuge and stood for a moment, then the thin-wall plastic tube is pulled out, the obtained liquid in the blood collection tube can be directly used for extracting nucleic acid as a blood sample, the originally waste material can be effectively used by a simple and low-cost method, compared with the conventional method for sucking or digging out the separation gel, the contact time with blood is short, the table top is not easily polluted, the separation gel is thoroughly and cleanly removed, the treated blood sample can still be stored for a long time, the nucleic acid can be sampled and extracted at any time, the cost is low, the operation is simple and rapid, and the total amount of the subsequently extracted nucleic acid is close to that of a whole blood sample.
It should be noted that the separation gel is a viscous soft gel with a specific density, and functions to separate blood cells from liquid components in blood. When the blood collection tube containing the separation gel is centrifuged, blood cells pass through the separation gel and are concentrated below the separation gel due to the density of the blood cells being higher than that of the separation gel, while the liquid part passes through the separation gel and is concentrated above the separation gel due to the density being lower than that of the separation gel, and the separation effect of the separation gel is generated due to the difference of the densities. Besides procoagulant serum blood collection tubes, other blood collection tubes are clinically used with separation gel, such as sodium citrate anticoagulated blood collection tubes and other special blood collection tubes. For blood collection tubes containing separation gel, when separation is completed, the separation gel covers the blood cells, making the underlying blood cells difficult to reuse. For extracting nucleic acid substances from blood cells under the separation gel, the separation gel must be removed first. The separating gel is soft and has large viscosity, the separating gel is difficult to completely remove by adopting methods such as suction, digging and the like, and as long as a small piece of separating gel is not removed, the suction head is easy to block when blood is sucked, so that a blood sample cannot be obtained. Moreover, the operation of removing the separation gel in the prior art is very easy to pollute the experiment operation table. Therefore, at present, serum blood collection tubes containing separation gel are hardly used for extracting blood cell nucleic acid in medical institutions, and the number of the serum blood collection tubes containing separation gel in clinical examination is far more than that of EDTA (ethylene diamine tetraacetic acid) anticoagulant blood collection tubes, and the blood collection tubes are largely discarded every day, which is a waste of resources. The embodiment of the invention, as a new method, can completely remove the separating gel from the blood collection tube containing the separating gel which is completely separated by blood cells and liquid easily and cleanly, better utilizes resources, has simple operation and low cost, and comprises the steps which are more favorable for extracting the blood cell nucleic acid from the coagulated blood, thereby overcoming the problems that the existing method has a plurality of obstacles to effectively utilizing the blood collection tube containing the separating gel to extract the nucleic acid and researchers are reluctant to use the blood collection tube containing the separating gel to extract the blood cell nucleic acid.
The embodiment of the invention also provides a blood sample prepared by the blood sample obtaining method, wherein the blood sample is blood cell lysate at least containing nucleic acid, and the extraction of blood cell nucleic acid can be continuously carried out according to the conventional steps.
The embodiment of the invention also provides application of the blood sample obtaining method in nucleic acid extraction.
As another preferred embodiment of the present invention, the blood sample obtaining method is a method for obtaining a blood sample from a blood collection tube containing a separation gel, and is used for rapidly and cleanly obtaining a blood sample for nucleic acid extraction, and is particularly suitable for extracting blood cell nucleic acid from a blood collection tube from which blood cells and liquid have been separated by a separation gel, and can be completely applied to extraction of blood cell DNA (deoxyribonucleic acid) and various types of RNA (ribonucleic acid). By taking the extraction of DNA as an example, by using the method, after the serum and the separation gel are removed from a conventional 4mL procoagulant serum blood collection tube containing the separation gel, DNA with the total amount of 5-10 micrograms can be averagely extracted, and almost all DNA detection researches can be met.
The technical effects of the blood sample collection method according to the embodiment of the present invention will be further described below with reference to specific examples.
Example 1
A method of obtaining a blood sample, as illustrated with reference to fig. 1, comprising the steps of:
the first step is as follows: serum (02) in the blood collection tube (01) was aspirated by a pipette. Specifically, as shown in figure 1, the treated blood collection tube (01) is greiner bio-one brand, 4mL standard, contains Z Serum Sep clock Activator coagulation factor as separation gel, namely, is a Serum coagulation blood collection tube containing separation gel, has collected about 3mL of blood, and the blood collection time is 3 days away from the treatment time. The blood collection tube (01) is separated after blood collection, the content in the blood collection tube (01) is divided into three layers, the upper layer is about 1mL of serum (02), the middle layer is separation gel (03), and the lower layer is a blood cell layer (04) containing blood clots, which is about 2 mL.
The second step is that: a disposable thin-walled plastic tube is inserted into the blood collection tube (01) as an adhesive tube (05).
The third step: 2mL of cell lysate (06) is injected from the upper opening of the disposable thin-wall plastic tube by a pipette tip, and the components of the cell lysate (06) are 10 percent of sodium dodecyl sulfate, 10 mu g/mL of protease K and 2mol/L of sodium chloride.
The fourth step: the whole blood collection tube (01) was centrifuged at a fixed inclination of 40 degrees for 3 minutes at 2500 g.
The fifth step: after completion of centrifugation, the blood collection tube (01) was taken out and left standing for 3 minutes. The disposable thin-wall plastic tube is pinched to expose the part of the blood collection tube (01), and the disposable thin-wall plastic tube is pulled out and discarded into a special medical waste garbage bag. At the moment, the separating gel is completely adhered in the disposable thin-wall plastic tube, and the blood clot (07) which can not be completely dissolved is adhered at the lower end. The hemocyte lysate left in the blood collection tube can be freely sucked, and no separation gel and blood clot residues exist. Subsequently, the hemolysate was used as a Blood sample (08), and the total amount of nucleic acid was 7.5. mu.g by extracting nucleic acid using QIAmp Blood Mini Kit (Qiagen).
Example 2
A method of obtaining a blood sample, as illustrated with reference to fig. 1, comprising the steps of:
the first step is as follows: the blood plasma in the blood collection tube (01) is directly removed. Specifically, as shown in fig. 1, the blood collection tube (01) is a domestic three-strength sodium citrate separation gel anticoagulated blood collection tube, which contains sodium citrate separation gel and collects about 8mL of blood. The blood collection tube (01) is separated after blood collection, the content in the blood collection tube (01) is divided into three layers, the upper layer is about 3mL of blood plasma, the middle layer is separation gel (03), and the lower layer is blood cell layer (04), which is about 2 mL.
The second step is that: a disposable thin-walled plastic tube is inserted into the blood collection tube (01) as an adhesive tube (05).
The third step: 4mL of cell lysate (06) is injected from the upper opening of the thin-wall plastic tube by a pipette tip, and the components of the cell lysate (06) are 4mol/L of guanidine isocyanide and 10 mu g/mL of proteinase K.
The fourth step: the whole blood collection tube (01) is centrifuged on a vertical barrel type blood vessel special centrifuge, the centrifugal force is 2500 g, and the centrifugation time is 3 minutes.
The fifth step: after completion of centrifugation, the blood collection tube (01) was taken out and left standing for 1 minute. The disposable thin-wall plastic tube is pinched to expose the part of the blood collection tube (01), and the blood collection tube is pulled out and discarded into a special medical waste garbage bag. At this time, the separating glue is completely adhered in the disposable thin-wall plastic tube and is completely removed along with the disposable thin-wall plastic tube. The hemolysate left in the blood collection tube (01) can be freely sucked. The hemocyte lysate is subsequently used as a blood sample, and extracted by using a blood genome DNA extraction kit of Beijing Tiangen Biochemical company to obtain the total amount of 13 micrograms of nucleic acid.
Example 3
A method for obtaining blood sample from blood collection tube containing separation gel, as shown in figure 1, comprises the treatment of blood collection tube (01) whose content has been separated into blood cells and liquid by separation gel, and the operation process comprises the following five steps:
step one, removing separation liquid (02) above a separation gel in a blood collection tube (01); wherein, the removing mode is that the blood sampling tube (01) is directly poured for removing, and then the residual separation liquid (02) is sucked by a pipette tip;
step two, putting a thin-wall plastic tube into the blood collection tube (01) as an adhesive tube (05); the adhesive tube (05) is a tube with openings at two ends, the shape of the adhesive tube is matched with that of the blood collection tube (01), the outer diameter of the adhesive tube (05) is close to but slightly smaller than the inner diameter of the blood collection tube (01), and the difference between the outer diameter and the inner diameter is 1 mm; the length of the adhesion tube (05) is 1cm longer than that of the blood collection tube (01); the thickness of the adhesion tube (05) is 1/3 of the inner diameter of the blood collection tube (01);
step three, injecting cell lysis solution (06) into the thin-wall plastic tube;
step four, centrifuging the whole blood collection tube (01) and the placed object in a centrifuge;
and step five, pulling out the thin-wall plastic tube after standing for a moment, and taking the residual liquid in the blood collection tube as a blood sample (08) which can be directly used for nucleic acid extraction.
Example 4
The procedure was as in example 3 except that the order of step three and step two was changed as compared with example 3.
Example 5
The procedure was the same as in example 3 except that the separation liquid (02) in the blood collection tube (01) above the separation gel was a mixture of serum and plasma, as compared with example 3.
Example 6
The same procedure as in example 1 was repeated, except that the blood collection tube (01) was an anticoagulated blood collection tube containing a separation gel, as compared with example 1.
Example 7
The same procedure as in example 1 was repeated, except that the blood collection tube (01) was an anticoagulated blood collection tube containing a separation gel, as compared with example 1.
Example 8
The same procedure as in example 1 was repeated, except that the blood collection tube (01) was a whole blood collection tube anticoagulated with EDTA, as compared with example 1.
Example 9
The same procedure as in example 1 was repeated, except that the blood collection tube (01) was a heparin anticoagulant blood collection tube, as compared with example 1.
Example 10
Compared with example 3, except that the adhesion tube (05) should be close to but slightly smaller than the inner diameter of the blood collection tube (01), the difference between the two is 4 mm; the length of the adhesion tube (05) is 2cm longer than that of the blood collection tube (01); the thickness of the adhesion tube (05) was the same as that of example 3 except that the inner diameter of the blood collection tube (01) was 1/15.
Example 11
Compared with example 3, except that the outer diameter of the adhesion tube (05) should be close to but slightly smaller than the inner diameter of the blood collection tube (01), the difference between the two is 2 mm; the length of the adhesion tube (05) is 1.5cm longer than that of the blood collection tube (01); the thickness of the adhesion tube (05) was the same as that of example 3 except that the inner diameter of the blood collection tube (01) was 1/10.
Example 12
Compared with example 3, except that the outer diameter of the adhesion tube (05) should be close to but slightly smaller than the inner diameter of the blood collection tube (01), the difference between the two is 3 mm; the length of the adhesion tube (05) is 4cm longer than that of the blood collection tube (01); the thickness of the adhesion tube (05) was 0.3mm, and the procedure was otherwise the same as in example 3.
Example 13
Compared with example 3, except that the outer diameter of the adhesion tube (05) should be close to but slightly smaller than the inner diameter of the blood collection tube (01), the difference between the two is 3.5 mm; the length of the adhesion tube (05) is 5cm longer than that of the blood collection tube (01); the thickness of the adhesion tube (05) was 0.5mm, and the procedure was otherwise the same as in example 3.
Example 14
The same as example 1 except that the centrifugal force was 600 g as compared with example 1.
Example 15
The same as example 1 except that the centrifugal force was 1100 g as compared with example 1.
Example 16
The same as example 1 except that the centrifugal force was 2000 g as compared with example 1.
Example 17
The same as example 1 except that the centrifugal force was 3500 g as compared with example 1.
Example 18
The same as example 1 except that the centrifugal force was 1500 g as compared with example 1.
The principle that the blood sample obtaining method provided by the embodiment of the invention can produce corresponding effect is that the cell lysate is generally high-concentration salt ion solution, or the density is improved by adding salt ions which do not influence nucleic acid extraction, so that the density of the cell lysate is higher than that of separation gel and blood clots. When the centrifugation is carried out, the high-density cell lysate can penetrate through the separation gel to enter blood cells below the separation gel to directly lyse the blood cells. The thin-wall plastic tube is tightly attached to the inner wall of the blood collection tube under the action of centrifugal force and inserted to the bottom of the blood collection tube, the inner wall is tightly attached to the separation glue, the process can occur before the cell lysate sinks, when the cell lysate sinks subsequently, the separation glue and the blood clot are pushed upwards by the lysate, the separation glue is attached to the blood clot and tightly attached to the inner wall of the thin-wall plastic tube, and when the thin-wall plastic tube is pulled out, the separation glue and the blood clot which cannot be dissolved are completely removed. The method has the beneficial effect that the originally abandoned materials can be effectively used by a simple and low-cost method. The embodiment of the invention obtains the blood sample by removing the separation gel, is simple, does not have the effect of subsequent nucleic acid extraction, and the nucleic acid extraction is influenced by different kits.
In the above embodiments of the present invention, after removing the liquid above the separation gel in the blood collection tube, placing the adhesion tube and injecting the cell lysis solution, then centrifuging the blood collection tube in a centrifuge, standing the blood collection tube for a moment, and then removing the adhesion tube, so as to obtain the liquid remaining in the blood collection tube as a blood sample, which can remove the separation gel from the blood collection tube simply, quickly, cleanly and at low cost, and promote the blood cells in the blood collection tube to dissolve and release nucleic acid, thereby facilitating the extraction of nucleic acid. After the treatment by the method, the obtained blood sample can be directly used for nucleic acid extraction.
It should be noted that the blood sample obtaining method provided by the embodiment of the present invention has the following main advantages:
the time for contacting blood is obviously reduced, and the table top is not easy to be polluted. The existing method uses a digging or sucking method to remove the separation glue, blood needs to be exposed in the air for a long time in the operation process, a human face needs to face the exposed blood for a long time, and the blood is easy to spill on the operation table top by actions such as digging, digging and the like. By adopting the method, only the steps of adding liquid, centrifuging, pulling out the tube and discarding are needed, blood can be seen only at the moment when the thin-wall plastic tube is discarded, the blood exposure time is extremely short, and the table top is not easy to be polluted.
And secondly, the separation gel is completely and cleanly removed, so that the availability of blood clots is obviously increased. Because the separation glue is sticky, the glue is difficult to remove by methods such as digging, sucking and the like, and only a small blood clot can be dug out from the blood sampling tube for use. After the method is adopted, almost all the separation gel is removed completely, and most of the remaining blood and blood clots can be utilized.
And thirdly, the cost is low, and the operation is simple. Compared with the original methods of digging, sucking and the like, the method only needs to increase the cost of one disposable plastic pipe, and the cost is less than 0.1 yuan. The specific gravity of cell lysate in the commercially available nucleic acid extraction kit is generally higher than that of separation gel, and generally salt ions do not need to be added. Even if salt ions are added, the cost is not higher than 0.1 yuan.
In addition, the processed blood sample can be sampled at any time to extract nucleic acid, and as the method ensures that most blood cell components in the blood sampling tube can be utilized, the total amount of nucleic acid obtained by subsequent extraction is close to that of a whole blood sample.
While the preferred embodiments of the present invention have been described in detail, the embodiments of the present invention are not limited to the above embodiments, and various changes can be made without departing from the spirit of the embodiments of the present invention within the knowledge of those skilled in the art. And are neither required nor exhaustive of all embodiments. And obvious variations or modifications of the embodiments of the present invention are still within the scope of the embodiments of the present invention as thus claimed.

Claims (7)

1. A blood sample obtaining method, characterized by comprising the steps of:
removing upper layer liquid from the blood collection tube containing the separation gel after blood collection, wherein the upper layer liquid is the separation liquid at one end, facing the opening of the blood collection tube, of the separation gel in the blood collection tube;
placing the blood sampling tube with the upper layer liquid removed into an adhesion tube for adhering separation gel, and injecting cell lysate into the blood sampling tube before or after placing the blood sampling tube into the adhesion tube;
centrifuging the blood collection tube injected with the cell lysate, and then removing the adhesion tube to take out the separation gel and the blood clot together with the adhesion tube to obtain a separated blood sample;
wherein the cell lysate is a salt ion solution capable of cracking cells, and the density of the salt ion solution is at least higher than that of the separation gel;
the centrifugal force of the centrifuge is greater than 500 grams.
2. A blood sample acquisition method according to claim 1, wherein in the blood sample acquisition method, the separation liquid is serum and/or plasma.
3. The method of obtaining a blood sample according to claim 1, wherein the blood collection tube containing the separation gel is any one of a procoagulant blood collection tube containing the separation gel, an anticoagulated blood collection tube containing the separation gel, and an anticoagulated blood collection tube containing the separation gel.
4. A method according to claim 1, wherein the method for removing the supernatant liquid comprises pouring and/or aspirating.
5. The method according to claim 1, wherein both ends of the adhesion tube are opened, and the outer diameter of the adhesion tube is smaller than the inner diameter of the blood collection tube, and the difference between the outer diameter of the adhesion tube and the inner diameter of the blood collection tube is not more than 4 mm; the length of the adhesion tube is greater than that of the blood collection tube; the wall thickness of the adhesive tube is not greater than 1/3 of the inner diameter of the blood collection tube.
6. The method for obtaining a blood sample according to claim 1, further comprising a step of standing before removing the adhesion tube.
7. Use of a blood sample acquisition method according to any one of claims 1-6 for nucleic acid extraction.
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CN113751095B (en) * 2015-12-11 2024-01-09 巴布森诊断公司 Sample container and method for separating serum or plasma from whole blood
CN106754871A (en) * 2016-12-01 2017-05-31 厦门大学附属中山医院 A kind of method of the DNA rapid extraction from clot
CN207855702U (en) * 2017-03-06 2018-09-14 湖北伽诺美生物科技有限公司 A kind of convenient disposal vacuum heparin tube of sampling
CN209271495U (en) * 2018-12-20 2019-08-20 张学军 A kind of blood separating mechanism
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CN209449556U (en) * 2019-01-25 2019-10-01 上海益诺思生物技术股份有限公司 A kind of integral type blood sampling cryopreservation tube
CN110029090B (en) * 2019-05-05 2021-10-22 江苏康为世纪生物科技股份有限公司 Preparation method of separation tube for separating tumor cells
CN210545213U (en) * 2019-09-19 2020-05-19 成都瑞琦医疗科技有限责任公司 Blood collection separation tube
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