CN111829863A

CN111829863A - Whole blood separation device and separation method thereof

Info

Publication number: CN111829863A
Application number: CN202010756507.5A
Authority: CN
Inventors: 曹志刚; 徐怀海; 张峰; 陆春; 张平; 马金玉; 刘开中
Original assignee: Shenzhen Uno Biotechnology Corp
Current assignee: Shenzhen Uno Biotechnology Corp
Priority date: 2020-07-31
Filing date: 2020-07-31
Publication date: 2020-10-27

Abstract

The invention discloses a whole blood separation device which comprises a sample containing cavity, wherein an isolation structure is arranged in the sample containing cavity and divides the sample containing cavity into a whole blood area and a plasma area, the isolation structure comprises an isolation piece and a filtering structure arranged on the isolation piece, and the filtering structure is used for separating the whole blood to obtain the plasma. When in separation, the whole blood sample is added into the whole blood area, the plasma component in the whole blood sample is filtered to the plasma area through the filtering structure, and the plasma sample is taken from the plasma area for reaction, so that the quick separation of the whole blood and the plasma is realized. The whole blood separation device has the advantages of simple structure, convenient operation and excellent performance, and can directly carry out rapid in-vitro immunodiagnosis on the whole blood.

Description

Whole blood separation device and separation method thereof

Technical Field

The invention relates to the technical field of in-vitro diagnosis and detection, in particular to a whole blood separation device and a separation method thereof.

Background

The in vitro diagnostic reagent refers to a reagent for in vitro detection of human body samples in the processes of prevention, diagnosis, treatment monitoring, prognosis observation, health state evaluation and prediction of hereditary diseases, in particular to a reagent for detecting various body fluids, cells and tissue samples of human bodies, which is closely related to human health, for example, the in vitro diagnostic reagent is required for detecting pathogens, antibodies, inquiring blood types, genes and hereditary diseases when people go to physical examination. The in vitro diagnostic reagent comprises blood, biochemistry, immunity, molecular biology, bacteria, POCT and the like. The first three are the in vitro diagnostic reagent market, which is subdivided according to diagnostic methods, immunodiagnosis, clinical biochemical diagnosis and molecular diagnosis from the global in vitro diagnostic reagent market, the clinical biochemical diagnosis and immunodiagnosis market in developed countries has been approaching to maturity, and POCT and molecular diagnosis are the main growth points of the diagnostic market. The immunodiagnosis reagent of China, clinical biochemical diagnostic reagent, is the two major external diagnostic reagent markets.

The immunodiagnostic reagent is developed through radioactive immunoassay, enzyme-linked immunosorbent assay (ELISA), fluorescence immunoassay, colloidal gold immunochromatography, chemiluminescence immunoassay, etc.

In the field of in vitro diagnosis, the type of a commonly used blood sample is serum or plasma, and because interfering substances such as red blood cells do not exist, the detection result is accurate and reliable. However, plasma or serum samples generally require whole blood samples to be subjected to blood processing steps, often requiring waiting for a long time or requiring cumbersome processing steps. The serum sample is collected by adopting a non-anticoagulation blood collection tube, and the blood coagulation time of at least 30 minutes is required to obtain the serum sample. Although the plasma sample does not need to wait for coagulation, the plasma sample also needs to stand still or be centrifuged by a centrifuge for 5-10 minutes, the plasma sample cannot be used for detection immediately after blood collection, and the centrifugal force can also damage red blood cells to cause hemolysis. But at present, the clinical requirement on rapid detection is higher and higher, and particularly, in emergency departments, ICU departments and other departments, a detection report can be issued within 20 minutes after blood collection. Therefore, there is a definite need for clinical applications to perform assays using whole blood that is not processed.

Patent document CN1243251A discloses a whole blood filtration method in which red blood cells are collected by a hemagglutinin and then filtered. However, this method requires additional anti-erythrocyte antibodies or lectins, is less stable and increases reagent costs. Patent document CN2485656Y discloses a chromatographic kit, in which blood is separated from blood cells by horizontal migration through the lamination of multiple layers of filter membranes, and the patent mainly relates to chromatographic products, which cannot be applied to liquid phase reaction reagents, and has low product detection sensitivity and repeatability. Patent document CN109925884A discloses whole blood filtration including a mode in which a hemagglutinin membrane and a hydrophilic filter membrane are stacked one on top of the other, and this patent also has a problem of using hemagglutinin and is inferior in stability. There are also manufacturers' reagents that dilute and mix a whole blood sample with a sample diluent containing a surfactant, and then take the diluted sample for immunoassay, but this method still reacts the whole blood sample with subsequent reagents such as magnetic beads and enzyme marker reagents, and interference substances such as red blood cells in the whole blood sample are likely to cause false positive or false negative results.

Accordingly, the present invention has been made in an effort to develop a whole blood separation apparatus capable of separating whole blood in a short time without requiring a whole blood processing process.

Disclosure of Invention

The invention aims to overcome the defects of the prior art and provide a device capable of quickly separating whole blood, which has the advantages of simple structure, convenience in operation and excellent performance.

In order to achieve the purpose, the invention provides a whole blood separation device, which adopts the following technical scheme:

the whole blood separation device is characterized by comprising a sample accommodating cavity, wherein an isolation structure is arranged in the sample accommodating cavity and used for separating the sample accommodating cavity to form a whole blood area and a plasma area, the isolation structure comprises an isolation piece and a filtering structure arranged on the isolation piece, and the filtering structure is used for separating the whole blood to obtain the plasma.

In one embodiment, the filtration structure is a filtration microwell or membrane.

In one embodiment, the filtration pores have a pore size of 0.5 μm to 5 μm.

In one embodiment thereof, the filter membrane is selected from at least one of a cellulose acetate membrane, a polyamide composite membrane, a cellulose acetate membrane, a polysulfone membrane, a polyethersulfone membrane, a polyamide membrane, a polyacrylonitrile membrane, a nitrocellulose membrane, a composite membrane, an acetate-nitrocellulose composite membrane, a polycarbonate membrane, a polyamide membrane, a glass fiber membrane, and filter paper.

In one embodiment, the filter membrane is treated with a surfactant to increase filtration rate, the surfactant comprising an anionic, cationic, zwitterionic or nonionic surfactant.

In one embodiment thereof, the non-ionic surfactant is tween or triton.

In one embodiment, the angle between the partition and the vertical direction is 0-60 degrees.

In one embodiment, the isolation structure comprises an isolation piece and a transverse plate connected with the middle area or the lower area of the isolation piece, the filtering structure is arranged on the transverse plate, the included angle between the isolation piece and the vertical direction is 0-60 degrees, and the included angle between the transverse plate and the isolation piece is 15-180 degrees.

In one embodiment, the sample-containing chamber is cylindrical, and the bottom of the sample-containing chamber is a spherical surface, a conical surface, two spherical surfaces, or two conical surfaces.

In one embodiment, the whole blood separation device is a stand-alone structure or the whole blood separation device is embedded within an in vitro diagnostic reagent strip.

In one embodiment, the whole blood separation device further comprises a pressure device, wherein the pressure device is connected with the whole blood area so as to form positive pressure on the whole blood area to accelerate the filtration speed;

or the pressure device is connected with the plasma area, so that negative pressure is formed on the plasma area to accelerate the filtration speed.

The invention also provides a separation method of the whole blood separation device, which is characterized in that a whole blood sample is added into the whole blood area of the sample containing cavity, plasma components in the whole blood sample are filtered to the plasma area through the filtering structure, and a plasma sample to be detected is obtained in the plasma area.

Compared with the prior art, the invention has the following beneficial effects:

(1) the whole blood sample is loaded for detection, and a sample processing process is not needed. In immunodiagnostic reagents, the common sample type is serum or plasma. Waiting for 0.5-1 hour after blood sampling of a serum sample to enable blood to be coagulated to separate out a serum type for detection, or using a serum procoagulant tube, but carrying out centrifugal operation on the procoagulant tube for 5-10 minutes; after blood sampling of the plasma sample, a centrifuge with a specific rotating speed is needed to be used for centrifuging for about 10 minutes so as to obtain the plasma sample. The sample types have the disadvantages of complicated operation steps and long time consumption (nearly half an hour in the whole operation process). The whole blood separation device provided by the invention has the advantages that after anticoagulation is collected, no treatment process is needed, a whole blood sample is directly added, and a plasma sample is obtained after whole blood is filtered.

(2) The whole blood sample has a fast filtration speed. After addition of 150uL of the whole blood sample, the whole blood filtration process was completed in about 1 minute. Or the whole blood area is connected with a pressure device to apply certain pressure to the whole blood area, or the plasma area is connected with the pressure device to form certain negative pressure in the plasma area, so that the filtering speed is accelerated, the whole blood filtering process can be completed within 10 seconds, and the plasma sample is immediately taken from the other side for detection. The whole blood separation device is particularly suitable for occasions with higher requirements on detection speed in departments such as emergency departments and ICUs of hospitals.

(3) The whole blood separation device has excellent testing performance. The plasma sample obtained after the whole blood filtration is reacted with a conventional immune reagent for testing, and the testing performance of the plasma sample can be comparable to any conventional immune diagnostic reagent.

Drawings

In order to more clearly illustrate the embodiments of the present application or technical solutions in the prior art, the drawings needed to be used in the embodiments will be briefly described below, and it is obvious that the drawings in the following description are only some embodiments described in the present invention, and other drawings can be obtained by those skilled in the art according to the drawings.

FIG. 1 is a schematic structural view of a whole blood separation device according to an embodiment of the present invention;

FIG. 2 is a schematic diagram of a whole blood separation device embedded in an in vitro diagnostic reagent strip according to an embodiment of the present invention;

FIG. 3 is a cross-sectional view of a spacer with filter pores in a filter structure according to an embodiment of the invention;

FIG. 4 is a cross-sectional view of a separator with a filter membrane as a filtration structure according to one embodiment of the present invention;

FIG. 5 is a cross-sectional view of a sample-receiving chamber according to one embodiment of the present invention;

FIG. 6 is a cross-sectional view of a sample-receiving chamber according to another embodiment of the present invention;

FIG. 7 is a cross-sectional view of a sample site with a whole blood compartment connected to a pressure device according to one embodiment of the present invention;

FIG. 8 is a cross-sectional view of a sample site with a plasma region coupled to a pressure device in accordance with one embodiment of the present invention;

FIG. 9 is a graph showing the correlation between the measured value of a clinical sample obtained by inserting the whole blood separating device into the in vitro diagnostic reagent strip according to the embodiment of the present invention and the inlet Beckmann reagent.

Detailed Description

In order to make the technical solutions of the present invention better understood, those skilled in the art will now describe the present invention in further detail with reference to the accompanying drawings. It is to be understood that the described embodiments are merely exemplary of the invention, and not restrictive of the full scope of the invention. All other embodiments, which can be derived by a person skilled in the art from the embodiments given herein without making any creative effort, shall fall within the protection scope of the present invention. The experimental procedures in the following examples are conventional unless otherwise specified. The test materials used in the following examples were purchased from a conventional biochemical reagent store unless otherwise specified.

Example 1

As shown in FIG. 1, the whole blood separation device 100 comprises a sample containing cavity, an isolation structure 120 is arranged in the sample containing cavity, the isolation structure 120 separates the sample containing cavity into a whole blood zone 130 and a plasma zone 140, the isolation structure 120 comprises an isolation member and a filter structure 150 arranged on the isolation member, and the filter structure 150 is used for separating the whole blood into plasma.

In this embodiment, the sample accommodating cavity is cylindrical, the axis of the cylindrical sample accommodating cavity is parallel to the vertical direction, and the bottom of the sample accommodating cavity is spherical, but in other embodiments, the bottom of the sample accommodating cavity may be one spherical surface, one conical surface, two spherical surfaces, or two conical surfaces.

As shown in fig. 3, in the present embodiment, the filter structure 150 is disposed in the lower region of the separator, the filter structure 150 is the filter pores 160, and the pore size of the filter pores 160 is 0.5 μm to 5 μm, preferably 5 μm. Further, the included angle between the isolating piece and the vertical direction is 0-60 degrees, and the included angle can be adjusted according to the actual condition, and is preferably 0 degrees.

In use, a whole blood sample is applied to the whole blood zone 130, the plasma component of the whole blood sample is filtered through the filter pores 160, and the filtered plasma sample is obtained in the plasma zone 140. Typically 150 μ L of whole blood sample can be filtered out in about 1 minute.

In one embodiment, the whole blood separation device is a stand-alone structure or the whole blood separation device is embedded within an in vitro diagnostic reagent strip. As shown in FIG. 2, the in-vitro diagnostic reagent strip further comprises reagent sites, including a labeled reagent site 210, a first wash fluid level 310, a second wash fluid level 410, a third wash fluid level 510, a solid phase reagent site 710, a sample dilution fluid level 810, a substrate fluid level 910, a detection site 930, and the like. The reagent sites contain reagents pre-dispensed therein, including the corresponding immunodiagnostic reagents. In one embodiment, the labeled reagent site 210 contains an enzyme-labeled reagent, such as an antibody to be detected labeled with horseradish peroxidase or alkaline phosphatase, the solid-phase reagent site 710 contains magnetic beads, the particle size of the magnetic beads is 100 nm-10 μm, and the substrate solution is luminol or AMPPD and the like.

The whole blood separation method includes the steps of feeding a whole blood sample into the whole blood zone 130, filtering plasma components in the whole blood sample through the filter pores 160, and filtering the filtered plasma sample in the plasma zone 140. Further, the detection method comprises the steps of sucking 50 mu L of filtered plasma sample from the plasma area 140, sucking 50 mu L of sample diluent in the sample diluent level 810, diluting and uniformly mixing the sample diluent to the empty hole position 920, sucking 50 mu L of the diluted and uniformly mixed sample, sequentially sucking 50 mu L of enzyme-labeled reagent, adding the enzyme-labeled reagent to the solid phase reagent position 710, and carrying out immunoreaction for 5 minutes after uniform mixing. After the reaction is finished, the reactant product is magnetically separated, sequentially passes through the first cleaning liquid level 310, the second cleaning liquid level 410 and the third cleaning liquid level 510, is washed for 3 times by the cleaning liquid, and then the substrate liquid in the substrate liquid level 910 is sucked to the detection position 930 for photoelectric detection. The instrument automatically calculates the content of the component to be detected according to the detected photoelectric value. The whole detection process takes about 15 minutes, and can meet the clinical requirement of rapid diagnosis.

Example 2

As shown in FIG. 4, the sample-receiving chamber is divided into a whole blood region 130 and a plasma region 140 by the isolation structure 120. The isolation structure 120 includes a spacer and a filtering structure 150 disposed on the spacer, and the filtering structure 150 is a filtering membrane 170. The filter membrane 170 is at least one selected from a cellulose acetate membrane, a polyamide composite membrane, a cellulose acetate membrane, a polysulfone membrane or a polyethersulfone membrane, a polyamide membrane, a polyacrylonitrile membrane, a nitrocellulose membrane, a composite membrane, an acetate-nitrocellulose composite membrane, a polycarbonate membrane, a polyamide membrane, a glass fiber membrane, and filter paper, and preferably is a polyethersulfone membrane, a composite membrane, a glass fiber membrane, and the like.

Further, the filter membrane is treated by a surfactant to accelerate the filtration speed, and the surfactant comprises an anionic surfactant, a cationic surfactant, a zwitterionic surfactant or a nonionic surfactant, and preferably the nonionic surfactant is selected from Tween, Triton and the like.

Example 3

As shown in FIG. 5, as an optimized design, the sample accommodating cavity can increase the hole depth properly to form a column shape with a certain depth, and the depth is at least larger than the diameter of the sample accommodating cavity opening. The material of the whole blood separation device is selected from at least one of polyethylene, polypropylene, polyvinyl chloride, polystyrene and acrylonitrile-butadiene-styrene copolymer.

Example 4

As shown in fig. 6, the separating structure 120 includes a separating member and a horizontal plate 122 connected to a middle region or a lower region of the separating member, the horizontal plate 122 is disposed in the plasma region 140, and the filtering structure 150 is disposed on the horizontal plate 122, wherein the filtering structure 150 is a filtering micro-hole or a filtering membrane. In this embodiment, the spacer is a spacer plate, and the lower portion of the spacer plate is spaced from the bottom surface of the sample-receiving chamber by a gap 121 through which the whole blood sample can flow to the underside of the filter structure 150. The included angle of the partition and the vertical direction is 0-60 degrees, preferably 0 degree, and the included angle of the transverse plate 122 and the partition is 15-180 degrees, preferably 90 degrees. This kind of structural design can make full use of the principle of linker, utilizes the effect of gravity, makes the whole blood sample pass through the filtration on the diaphragm 122, obtains the plasma sample in plasma district 140 diaphragm 122 top, and this structure can accelerate whole blood filter speed, improves whole blood filtration efficiency. Of course, other preferable structural designs should fall within the protection scope of the present invention.

Example 5

As shown in FIG. 7, the whole blood separation device further comprises a pressure device connected to the whole blood region to create a positive pressure on the whole blood region to increase the filtration rate. Alternatively, as shown in fig. 8, a pressure device is connected to the plasma region to create a negative pressure on the plasma region to increase filtration rate. This design completes the whole blood filtration process within 10 seconds and immediately takes a plasma sample from the plasma region 140 for testing. The whole blood separation device is particularly suitable for occasions with higher requirements on detection speed in departments such as emergency departments and ICUs of hospitals.

Example 6

In one embodiment, the step of treating the filter membrane with the surfactant comprises preparing the surfactant into a solution with a suitable concentration by using purified water, such as a surfactant solution with a concentration of 1% prepared by tween-20 or triton X-100, soaking the filter membrane, such as a polyethersulfone membrane, a composite membrane, a glass fiber membrane and the like, for 0.5-1 h by using the prepared surfactant solution, drying at a temperature of 35-38 ℃, cutting the filter membrane into a suitable size, and assembling the filter membrane on the separator.

The method screening tests were performed on different filtration membranes treated with different surfactants, and the filtration time, plasma yield and hemolysis were measured, respectively, with the results shown in table 1:

TABLE 1

The data in the table 1 can be used for obtaining that the polyether sulfone membrane is selected to be treated by TW-40 or TW-80, the filtration time is within 1 minute, the plasma yield is high, and no hemolytic reaction exists; selecting composite membrane to process with TW-80, filtering time is about 1 minute, plasma yield is higher, and no hemolytic reaction; selecting glass fiber membrane to be treated by TW-80, filtering time is within 1 minute, plasma yield is high, and no hemolytic reaction exists.

Example 7

And (3) performance testing: the whole blood separation devices of examples 1-6 were used to insert them into troponin-i (ctni) chemiluminescent reagent strips containing troponin-i (ctni) alkaline phosphatase enzymatic chemiluminescent reagents and the reagents were tested for their detection limits, linearity range, reproducibility, and clinical sample correlation.

Detection limit test: after the plasma sample obtained by filtering in the sample accommodating cavity is diluted by the sample diluent, the detection is carried out according to the detection method in the embodiment 1, three batches of whole blood detection devices are adopted, each batch is repeatedly measured for 20 times, the RLU value (relative luminous value) of the result is obtained for 20 times, the average value (X) and the Standard Deviation (SD) are calculated, X +2SD is obtained, two-point linear regression fitting is carried out according to the result of the concentration between the sample diluent and the RLU value of the adjacent calibration product to obtain a linear equation, and the RLU value of the X +2SD is substituted into the equation to obtain the corresponding concentration value, namely the blank limit. The results are shown in table 2:

TABLE 2

As can be seen from the data in Table 2, the whole blood separation device of the present invention is embedded in the reagent strip to detect troponin-I (cTnI), the detection limit is not more than 0.008ng/mL, and the sensitivity is high.

And (3) linear verification: high concentration cTnI samples were diluted with negative plasma sample gradients to 50ng/mL, 25ng/mL, 5ng/mL, 1ng/mL, 0.2ng/mL, 0.01 ng/mL. The test was repeated 3 times for each concentration of sample using the whole blood separating device embedded reagent strip in the example of the present invention, the average value thereof was calculated, the resultant average value and the dilution ratio were subjected to straight line fitting by the least square method, and the linear correlation coefficient (R) was calculated. The results are shown in Table 3:

TABLE 3

From the data in table 3, the correlation coefficient R was calculated to be 1.0, i.e., the linear range of 0.01 to 50ng/mL for troponin-i (ctni) detected when the whole blood separation device of the present invention was inserted into the test strip, with a wide linear range.

And (3) repeatability test: using the same lot of whole blood separation apparatus as in example 1, clinical samples with acute myocardial infarction critical value + -50% level were repeatedly tested 10 times, and the average of 10 measurements was calculated

And the Standard Deviation (SD), and the Coefficient of Variation (CV) is obtained according to the formula (1). The coefficient of variation CV value of the repetitive samples is required to be not more than 10.0%. The results are shown in Table 4.

In the formula:

-an average of the measurements;

SD-Standard deviation;

CV-coefficient of variation.

TABLE 4

According to the data in table 4, the whole blood separation device of the present invention is embedded in a reagent strip to detect troponin-i (ctni), and the repeatability CVs are all within 6%, and the repeatability is high.

Clinical sample correlation test: 40 clinical samples (covering the whole linear range) are selected, wherein the concentration value of 50% of the samples is outside the reference interval, a troponin-I (cTnI) reagent strip embedded in the whole blood separation device is used for being compared with a Beckmann troponin-I chemiluminescence detection system, and the correlation coefficient R of the detection results of the two is calculated. The results are shown in Table 5:

TABLE 5

According to the data in table 5, the results of the measurement using the present invention are plotted on the ordinate and the results of the measurement using the beckmann reagent are plotted on the abscissa for regression analysis, and as shown in fig. 9, the correlation equation is: y is 1.0817X-0.0599, and the correlation coefficient R is 0.998. The results of statistical treatment show that the whole blood separation device is embedded into the reagent strip to detect troponin-I (cTnI) and has good correlation with the measured value of the clinical sample of the inlet Beckmann reagent.

In conclusion, the whole blood separation device of the present invention has very excellent detection performance.

The above embodiments only express a few embodiments of the present invention, and the description thereof is more specific and detailed, but not construed as limiting the scope of the invention. It should be noted that, for a person skilled in the art, several variations and modifications can be made without departing from the inventive concept, which falls within the scope of the present invention. Therefore, the protection scope of the present patent shall be subject to the appended claims.

Claims

1. The whole blood separation device is characterized by comprising a sample accommodating cavity, wherein an isolation structure is arranged in the sample accommodating cavity and used for separating the sample accommodating cavity to form a whole blood area and a plasma area, the isolation structure comprises an isolation piece and a filtering structure arranged on the isolation piece, and the filtering structure is used for separating the whole blood to obtain the plasma.

2. The whole blood separation device according to claim 1, wherein the filter structure is a filter microwell or a filter membrane.

3. The whole blood separation device according to claim 2, wherein the filtration pores have a pore size of 0.5 μm to 5 μm;

the filter membrane is selected from at least one of an acetate fiber membrane, a polyamide composite membrane, a cellulose acetate membrane, a polysulfone membrane, a polyether sulfone membrane, a polyamide membrane, a polyacrylonitrile membrane, a nitrocellulose membrane, a composite membrane, an acetate-nitrocellulose composite membrane, a polycarbonate membrane, a polyamide membrane, a glass fiber membrane and filter paper;

the filter membrane is treated by a surfactant to accelerate the filtration speed, and the surfactant comprises anionic, cationic, zwitterionic or nonionic surfactant.

4. The whole blood separation device according to claim 3, wherein the non-ionic surfactant is Tween or Triton.

5. The whole blood separation device according to any one of claims 1 to 4, wherein the angle between the partition and the vertical direction is 0 ° to 60 °.

6. The whole blood separation device according to any one of claims 1 to 4, wherein the isolation structure further comprises a transverse plate connecting the isolation member with the middle region or the lower region of the isolation member, the filter structure is arranged on the transverse plate, the isolation member has an included angle of 0-60 degrees with the vertical direction, and the transverse plate has an included angle of 15-180 degrees with the isolation member.

7. The whole blood separation device of claim 1, wherein the sample receiving chamber is cylindrical and the bottom of the sample receiving chamber is one spherical surface, one conical surface, two spherical surfaces, or two conical surfaces.

8. The whole blood separation device of claim 1, wherein the whole blood separation device is a stand-alone structure or the whole blood separation device is embedded within an in vitro diagnostic reagent strip.

9. The whole blood separation device of claim 1, further comprising a pressure device connected to the whole blood region to create a positive pressure on the whole blood region to increase filtration rate;

10. The method for separating whole blood as claimed in any one of claims 1 to 9, wherein the whole blood sample is supplied to the whole blood area of the sample-receiving chamber, and the plasma component in the whole blood sample is filtered through the filter structure to the plasma area, where the plasma sample to be tested is obtained.