• 专利附录
  • 专利说明
  • 权利要求
  • 类似技术
  • 同族专利
  • 引用文献
  • 法律状态
  • 优先权
    • 专利摘要:

    公开号:NL2009403A
    申请号:NL2009403
    申请日:2012-09-03
    公开日:2013-04-03
    发明作者:Sylwia Makulska；Slawomir Jakiela；Piotr Garstecki
    申请人:Inst Chemii Fizycznej Polskiej Akademii Nauk；
    IPC主号:
    专利说明:

    Method for determination of blood group and system for the same.
    The subject matter of the invention is a method for determination of blood group comprising preparation of the sample by mixing the tested material with the reference material and detection of agglutination in the sample so prepared. The invention comprises also a system for determination of blood group allowing to make use of the method.
    Due to a small droplet volume, and consequently low consumption of reagents needed for analyses, microfluidic systems find more and more applications, not only in the research as labs-on-chip, but also biomedical applications as point-of-care devices.
    Typically, droplet-based microsystems possess a multitude of microfluidic channels, with their inlets and outlets that can join and divide inside the system, where droplets of solutions are surrounded by continuous phase immiscible therewith. Further, the droplets inside the systems can be merged, transported along the channels while their contents are being mixed, stored under specific or varying conditions, and finally sorted or split in channel junctions and recovered from the system. The use of microlaboratories to perform chemical and biochemical reactions inside microdroplets offers the following advantages [H. Song, D. L. Chen, and R. F. Ismagilov, Ang. Chem. Int. Ed., 2006, 45, 7336-7356]: i) no dispersion of time of residence for fluid elements in a channel, ii) rapid and efficient mixing, iii) reaction kinetics can be easily controlled, iv) multiple reactions can be performed in parallel, v) low consumption of reagents, and vi) fast detection of the outcome of reactions (due to a small droplet volume the reaction products reach faster measurable concentrations).
    These characteristics make the microdroplet-based microsystems a valuable tool for analytical chemistry, synthetic chemistry, biochemistry, microbiology, medical diagnostics, or molecular diagnostics.
    In the state of the art the determination of blood group relies on testing red blood cells isolated from the patient blood in contact with reference antibodies. To confirm the result, patient’s serum is tested in contact with reference blood cells containing specific antigens. Such assays are carried out in test tubes or on glass plates, by mixing individual samples with each other and monitoring for appearance of agglutination. If a sample contains antigens and antibodies that are specific for these antigens, agglutination, i.e., clumping of red blood cells takes place and they form agglomerates of various size. The phenomenon is detected with naked eye by a laboratory assistant.
    The blood group is determined based on the presence of specific antigens on tested red blood cells. Such an analysis takes about 10-15 minutes, and the amount of blood taken from the patient is 5-10 ml.
    In serological compatibility testing of the donor and the recipient prior to a blood transfusion (cross-matching), blood groups of both patients are first determined as described above. Then, using the same method, tests are performed to check for the presence of the most frequently occurring antigens. After obtaining the full blood profile of the donor and the recipient, the actual cross-matching is performed. The cross-match involves testing recipient’s serum in contact with donor’s red blood cells to check if it does not contain any other antibodies directed against donor’s red blood cells. No agglutination indicates serological compatibility. The full cross-match takes about 60 minutes and requires taking additional blood samples from patients [about 10 ml from each].
    In the state of the art there are already solutions aiming at reduced consumption of biological material and shortened duration of the test [Anal. Bioanal. Chem., 2011, 399, 1869-1875; Anal. Chem., 2010, 82, 4158^164; Lab Chip, 2006, 6, 794-802], they rely, however, on a naked eye detection which in the case of poorer agglutinating antigens can result in an unintentional misinterpretation of the result by a person performing the test. Moreover, such solutions do not prove correct in the case of cross-matching that requires performing over a dozen or even a few tens of similar tests.
    In the state of the art there are also solutions involving complex detection systems to eliminate human error [Anal. Chem., 2008, 80, 6190-6197; Biomed. Microdevices, 2009, 11, 217-229], they still, however, require the person performing the test to be involved in the interpretation of the result.
    All methods of blood group determination known in the state of the art require distinguishing agglutinated samples from non-agglutinated ones based on a light scattering measurement. The measurement is performed either subjectively by a laboratory assistant (with naked eye, or using optical instruments), or by automated devices distinguishing intensity of light transmitted through the sample or reflected from the sample.
    Microfluidic modules described in earlier patent applications of Prof. Piotr Garstecki’s research team (e.g., Polish patent applications no. P-390250, P-390251, P- 393619, and an international patent application no. PCT/PL2011/050002) could be used for fast and reliable blood group determination and entirely automatic full cross-matching, without any involvement of a third party. Earlier attempts to use microfluidic systems for that purpose encountered obstacles because of problems (described above) with the detection of possible agglutination in the samples (droplets) of very low volumes - on the order of one microliter or even less.
    It is known that physical properties of a droplet, and in particular its viscosity, may have an effect on the speed of a droplet moving inside a microchannel [S. Jakiela, S. Makulska, P. M. Korczyk, P. Garstecki, Lab Chip, 2011, 11, 3603-3608], In particular, the speed of droplets in a channel is a complicated and non-monotonic function of the flow rate of the continuous phase, the droplet size, the droplet shape, the surface tension between the droplet and the continuous liquid, the viscosity of the droplet forming liquid, and the viscosity of the continuous liquid.
    Unexpectedly, the Inventors of the present invention observed, as the first, that clumping of red blood cells contained in a droplet into aggregates under the influence of specific antibodies (agglutination) has a noticeable and measurable effect on the speed of droplet movement in a microchannel, and that the effect allows for developing of a simple method for blood group determination: since a droplet containing agglutination products changes its physical properties, then it is possible to construct a system for fast recognition of agglutination and distinguishing in this system agglutinated droplets from the non-agglutinated ones based on the change in droplet viscosity, by measuring the time a tested droplet in microchannel needs to pass between the detectors measuring the change in light intensity.
    Additionally, the Inventors of the present invention noticed unexpectedly that in the droplet where the agglutination reaction has occurred, the agglomerates of clumped red blood cells separate and are displaced to the back of the droplet in the direction opposite to that one the droplets are moving in a microchannel. As a result of the phenomenon observed, the outset of the droplet becomes transparent, as opposed to the back of the droplet, which is opaque.
    Therefore, using a detector measuring the intensity of the incident light, one can distinguish a droplet, where the agglutination reaction took place from a droplet, where the reaction did not take place, thanks to the fact that the droplet outset is transparent for an agglutinated droplet, and opaque for a non-agglutinated droplet.
    Therefore, the purpose of the present invention is to propose a new method for determination of blood group, wherein the detection of a possible agglutination in the sample relies on the unexpected properties mentioned above. More particularly, the determination of blood group in the method according to the invention is performed by: i) the measurement of the time of flow of a droplet containing samples of specific blood groups exposed to specific antibodies - performed for instance by measuring the time of flow in a microchannel between two detectors sensitive to change in light intensity, or by ii) monitoring changes in light intensity initiated by a droplet flowing in front of the detector, where the agglutination reaction did (or did not) take place - performed for instance by the analysis of light transmitted through a tested droplet in a detector sensitive to change in light intensity.
    According to the invention, the method for determination of blood group comprising preparation of the sample by mixing the tested material with the reference material and detection of agglutination in the sample so prepared is characterised in that the said mixing of the tested material with the reference material is performed in a channel of a microfluidic system.
    Preferably, the said detection of agglutination is performed by measuring the time of flow of the sample in a microfluidic channel between two detectors.
    Alternatively preferably, the said detection of agglutination is performed by measuring the intensity of light transmitted through the sample as a function of the sample length in a microfluidic channel.
    Preferably, red blood cells isolated from patient’s blood are used as the tested material, and reference antibodies are used as the reference material.
    Alternatively preferably, serum isolated from patient’s blood is used as the tested material, and reference red blood cells are used as the reference material.
    In a preferred example of embodiment of the invention, the tested material and the reference material have a form of droplets suspended in an oily continuous phase, preferably in oil from the group of simple or functionalised hydrocarbons, mineral oil, silicone oil, or fluorinated oil.
    The invention comprises also a system for determination of blood group comprising the first microfluidic channel and the second microfluidic channel, connecting with each other at a junction, and the third microfluidic channel, running from this junction to the first detector, characterised in that the said first detector is a detector sensitive to change in light intensity.
    Preferably, the system according to the invention comprises additionally the second detector sensitive to change in light intensity, located behind the first detector, looking down the said third microfluidic channel.
    Preferably, the said third microfluidic channel comprises a meandering section, located between the said junction and the first detector.
    The method presented in the present patent application allows for fast identification of blood group on the basis of a definitely simpler optical measurement requiring only to ascertain the presence of the droplet in a given point at a given moment in time. This solution definitely reduces the requirements put on optical measurements and may allow for i) simplifying the design of the apparatus for determination of blood groups, and ii) development of portable devices for determination of blood groups.
    Detailed description of the invention
    The invention is now explained more in detail in preferred embodiments, with reference to the accompanying figures, wherein:
    Fig. 1 shows a general scheme of construction of a typical microfluidic system for determination of blood groups according to the invention,
    Fig. 2 shows a schematic diagram of microchannels in a system designed for distinguishing of droplets containing samples of different blood groups, according to example 1 and 2,
    Fig. 3 shows times of flow for droplets containing samples of each blood group, according to example 1, and
    Fig. 4 shows variations of voltage measured during flow of an agglutinated droplet (dashed line) and an agglutinate-free droplet (solid line), according to example 2.
    In a preferred embodiment of the invention, the speed of droplets containing mixtures of red blood cells and serum is determined in a microfluidic cartridge containing microfluidic channels (Fig. 1). In a preferred embodiment, the network of microfluidic channels contains channels 1 and 2, used to introduce a serum droplet 8, and a droplet of suspension of red blood cells 9, respectively, said red blood cells suspended in a continuous fluid 10 immiscible therewith. In preferred examples of embodiments, the continuous fluid 10 is any fluid immiscible with aqueous solutions, for instance oil from the group of simple or functionalised hydrocarbons, mineral oil, fluorinated oil, or other. Channels 1 and 2 preferably join each other into one channel at the junction point 3. At the junction point 3, serum droplets 8 and droplets of red blood cells suspension 9 may be merged into a droplet containing a mixture of serum and red blood cells suspension 11. Preferably, the microfluidic cartridge contains a meandering channel section 4 providing for easy mixing of the content of a droplet flowing through it. Preferably, after mixing, the droplet flows into a channel section 5 furnished with two sensors of droplet presence 6 and 7 that are installed at a known distance from each other. The sensors 6, 7 may be optical or electrical sensors, or any other sensors allowing for detection of presence of a droplet 11. Comparison of the time of flow of droplets 11 containing different mixtures of red blood cells and serum between sensors 6, 7 allows for determining in which droplets 11 the red blood cells have agglutinated, and based thereon for determination of blood group.
    Preferred examples of embodiment of the invention
    Example 1
    In a preferred embodiment (Fig. 2), two sorts of reagents are being introduced through T-junctions into a channel of a microfluidic system: the reagents containing antigens (tested red blood cells or reference red blood cells with specific blood group) -this are junctions in the area A, and the reagents containing antibodies (tested serum or reference reagents containing specific antibodies) - this are junctions in the area B. The reagents are being introduced through the longest arm of the junction, used also as a storage space, preventing the contact of biological material with the electromagnetic valve. The continuous phase in a form of oil (hexadecane) is introduced through the inlets of remaining junctions, both in the area A and in the area B.
    In a preferred example of embodiment, using electromagnetic valves controlled by the computer software and installed at the inlets of T-junctions (area A and B), two droplets are released - one coming from the area A, from any T-junction, and the other one coming from the area B, also from any junction. In a preferred example of embodiment both droplets flow through arms 1 and 2 to the reaction chamber (area C), where electrocoalescence (merging of droplets stimulated by an external electromagnetic field) is induced using high voltage (500-3000 V) of a frequency 100-59300 Hz supplied to the system by the electrodes. A droplet so produced contains two sorts of reagents (antigens and antibodies) and is directed from the chamber 3 to a mixing channel 4, i.e., a microchannel comprising meandering sections, and used for droplet mixing (area D). The first detector 6, measuring the intensity of the incident light, is installed between the reaction chamber and the mixing channel, whereas the second detector 7, measuring the intensity of the incident light, is installed behind the mixing channel and before the outlet channel 5.
    In a preferred example of embodiment, the time of flow of a droplet between the first detector 6, and the second detector 7, is measured. Based on the differences in speeds of droplets it is possible to distinguish droplets, where the agglutination took place, from those where the process did not occur. In a preferred embodiment this observation may be used to characterise the blood group based on the analysis of sets of antigens and antibodies, and the speeds of flow of droplets containing different combinations of these reagents.
    Depending on whether the analysed droplets contained antigen-specific antibodies or not, they differed in speed. The time of flow for all the droplets not containing reagent-like (in the meaning - inducing agglutination) antigen-antibody pairs was by a few seconds shorter than for the droplets containing agglutinates. In a preferred example of embodiment it is possible to determine blood group if one knows the content of each droplet and the time of flow between the detectors.
    Fig. 3 shows the times of flow for droplets containing samples of each blood group, representing the basis for the distinction thereof.
    Example 2
    In another preferred example of embodiment it is possible to distinguish droplets, where the agglutination took place, from those where it did not. The distinguishing represents the basis for evaluation of the blood group, based on the readouts of variations in the intensity of light transmitted through a tested droplet. Using a detector converting linearly light intensity to voltage one can observe a distinct difference in the recorded amplitude. Fig. 4 shows variations of voltage measured on the detector 7 (Fig.2) during flow of an agglutinated droplet (gray dashed line) and an agglutinate-free droplet (black solid line).
    Fig. 4 shows a preferred situation, wherein agglutination results in the droplet outset being significantly more transparent that the back of the agglutinated droplet (gray dashed line) and in appearance of a distinct maximum in amplitude of the readout from detector. In turn, for a droplet with no agglutination the said maximum does not occur (black solid line). Therefore, the method based on the analysis of the presence of variations in light intensity along a droplet provides a clear answer if the agglutination reaction took place in the tested droplet.
    权利要求:
    Claims (9)
    [1]
    A method for determining a blood group comprising the preparation of the sample (11) by mixing the tested material (8) with the reference material (9) and the detection of agglutination in the thus prepared sample (11) with the characterized in that said mixing of the tested material (8) with the reference material (9) is carried out in a channel (3) of a microfluidic system.
    [2]
    Method according to claim 1, characterized in that said agglutination detection is performed by measuring the time of the flow of the sample (11) between two detectors (6, 7) in a microfluidic channel (5).
    [3]
    Method according to claim 1, characterized in that said agglutination detection is performed by measuring the intensity of the light emitted by the sample (11) as a function of the sample (11) length in a microfluidic channel ( 5).
    [4]
    Method according to one of the preceding claims 1 to 3, characterized in that red blood cells isolated from the blood of the patient are used as the test material (8) and reference antibodies are used as the reference material (9).
    [5]
    Method according to one of the preceding claims 1 to 3, characterized in that serum isolated from the blood of the patient is used as the test material (8) and reference red blood cells are used as the reference material (9).
    [6]
    Method according to one of the preceding claims, characterized in that the tested material (8) and the reference material (9) have a form of droplets incorporated in a continuous oil phase, preferably in oil from the group of single or functionalized hydrocarbons , mineral oil, silicone oil or fluorinated oil.
    [7]
    A system for determining a blood group comprising the first microfluidic channel (1) and the second microfluidic channel (2), which are each connected to each other near a coupling (3) and the third microfluidic channel (5) which from this coupling (3) runs to the first detector (6), characterized in that said first detector (6) is a detector which is sensitive to changes in light intensity.
    [8]
    A system according to claim 7, characterized in that it additionally comprises the second detector (7), which is sensitive to changes in the light intensity and which is placed behind the first detector (6) in the direction of the third microfluidic channel ( 5).
    [9]
    A system according to claim 7 or 8, characterized in that said third microfluidic channel (5) comprises a winding part (4) located between said coupling (3) and the first detector (6).
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    法律状态:
    2017-03-01| HC| Change of name(s) of proprietor(s)|Owner name: SANGO TECHNOLOGIES SPOLKA Z OGRANICZONA ODPOWIEDZI Free format text: DETAILS ASSIGNMENT: CHANGE OF OWNER(S), CHANGE OF OWNER(S) NAME; FORMER OWNER NAME: GEOMOBILE SPOLKA Z OGRANICZONA ODPOWIEDZIALNOSCIA Effective date: 20170215 |
    2020-11-04| PD| Change of ownership|Owner name: FUNDACJA POLSKICH INICJATYW TECHNOLOGICZNYCH; PL Free format text: DETAILS ASSIGNMENT: CHANGE OF OWNER(S), ASSIGNMENT; FORMER OWNER NAME: SANGO TECHNOLOGIES SPOLKA Z OGRANICZONA ODPOWIEDZIALNOSCIA Effective date: 20201015 |
    优先权:
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    PL39649411|2011-09-30|
    PL396494A|PL219803B1|2011-09-30|2011-09-30|Method for determining blood groups and a system for determining blood groups|
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