Patent Application
20220317013
This invention relates to a method of separating particles having different sedimentation velocities in a fluid sample through centrifugal sedimentation. The invention also relates to an apparatus for performing the method.
When curing blood poisoning, for example, the pathogenic bacteria have to be detected among blood cells. The bacteria then usually have to be cultivated from a blood draw. Occasionally, the cultivation process may however take such a long time that the patient risks dying of blood poisoning before the bacteria are identified.
An object of this invention is to develop a method where particles such as bacteria, in a fluid sample such as blood, can be separated from other particles such as blood cells in a new and time saving centrifugal manner.
In an aspect of the invention the method comprises:
As the sample is thereby subjected to rotation about two different axes, a particle will sometimes be between the primary axis and the centre axis of the cylinder and sometimes be located radially outside both axes. This causes the particle to alternately move from and towards the axis of the cylinder. Thereby, the sedimentation of particles of a certain size will at some point be counteracted by the rotation of the secondary axis so that the particle lies still relative to the primary axis. Each particle size has its equilibrium position when settled in the sample. Calculations show that the particle will search for the equilibrium position regardless of where in the sample it starts. The equilibrium position depends, among other things, on the particle's sedimentation velocity, which in turn is affected by the size. The larger the particle, the farther from the secondary axis it ends up, and supposedly always on a straight line from the secondary axis.
While not necessarily required for performing the method, the first and second axes are conveniently parallel to each other.
An insight that underlies the invention is that there should be possible to separate particles such as bacteria from blood cells provided that there is a small difference in sedimentation rate. That is, it should be possible to fractionate particles of different sizes. A particle of a certain size will then search for equilibrium and it is possible to calculate how long it takes.
Another application is to produce monodisperse particles, i.e. particles or particle dusters where all particles have the same size. Such particles are used inter alia to calibrate particle analyzers that analyze the frequency of particles having a certain size. Still other applications are imaginable.
In another aspect of the invention the equilibrium or end position of particles as calculated or obtained by previous operations of the method may then be used to more easily find or spot the particles of interest to be further analysed and eventually identified. The method may then further comprise:
Alternatively, the method may comprise:
For ease of identification, steps e and f may then comprise tabulated data for sets of known particles in areas of interest.
The step of measuring the distance is here considered inherently equivalent to determining the sedimentation rate/velocity under the given conditions.
If the separator is stopped a flow will be initiated in the cylindrical container. This flow will mix the fractionated particles. To avoid this, spotting or finding of the particles in the settled fractions may be facilitated by blocking different regions of the sample from mixing with each other.
Specifically, the blocking may be obtained by inserting a framework or grid of axially open compartments into the sample having the settled fractions, for dividing the sample into said regions defined by the compartments. A radially succession of compartments may then function as a multistage filter that facilitates identification of particles found therein. Particles found in a certain compartment may then be more easily identified by being in a close range of sedimentation rates or end locations for known particles of interest.
In the bacteria application the purpose is to concentrate the bacteria level and lessen the blood cell level that may interfere with subsequent analysis. After the separation process the type of bacteria is analyzed by other methods.
It would also be possible to sample particles from different regions in separate samples.
The second rotational speed is higher than the first rotational speed. In contrast, the relative rotational speed, ω, of the sample should be very small in relation to the main rotational speed, Ω, i.e. first rotational speed. As further explained later, ω may however not be too small not to prolong the separation process.
The sample may be a blood sample. In that application bacteria can be separated from blood cell as mentioned above.
An apparatus for performing the method of the invention comprises a cylindrical container for enclosing the sample, a first rotator for rotating the container about the primary axis, and a second rotator for rotating the container about the secondary axis.
The first rotator may comprise a disk supported for rotation about the primary axis and a first electric motor for rotating the disk, and the second rotator may comprise a second electric motor for rotating the cylindrical container supported at the disk for rotation about the secondary axis.
In an alternative embodiment the first rotator also comprises a disk supported for rotation about the primary axis and an electric motor for rotating the disk, and the second rotator comprises a stationary gear concentric with the primary axis and in gear engagement with a gear for rotating the cylindrical container supported at the disk for rotation about the secondary axis. Importantly, such arrangement will make certain that the first and second rotational speeds are synchronized. Other arrangements, e.g. belt arrangements, may be equally practicable.
The apparatus may also comprise a plurality of said container peripherally distributed around the primary axis. Thereby it will be possible to process larger volumes of samples in a single separation process.
Other features and advantages of the invention may be apparent in the following detailed description and the appended claims.
The fractionation apparatus shown in
Other arrangements are likewise applicable, such as various gear or belt transmissions to rotate disk 10 and container 20 synchronously at different speeds by a single electric motor 14.
In such case, as indicated in phantom in
As shown in
A fractionation apparatus according to the invention may be operated as follows.
The cylindrical container 20 is filled with a liquid sample or suspension 30 having particles to be detected and identified. The container 20 is closed by a lid 21. Container 20 is then rotated at a speed ξ different from and higher than a speed 0 of the disk 20. Due to the rotation about the primary axis 12, particles, such as bacteria and blood cells in blood, with a different density than the liquid will sediment radially outwards relative to the liquid. By the fact that the liquid does not have the very same rotation as the cylinder has around the primary axis 12, the particle P will sometimes be between the primary and the secondary axes and sometimes be located radially outside both axes. This causes the particle to alternately move from and towards the secondary axis. As previously mentioned, the sedimentation of particles of a certain size will at some point be counteracted by the rotation of the secondary axis so that the particle lies still relative to the primary axis. Each particle size has its equilibrium position when settled in the sample. Calculations have shown that the particle will search for the equilibrium position regardless of where in the sample it starts. The equilibrium position depends, among other things, on the particle's sedimentation rate, which in turn is affected by the size. The larger the particle, the farther from the secondary axis it ends up, and supposedly always on a straight line, L from the secondary axis as illustrated in
When the above described centrifugal operation is completed, the particles may be spotted and identified.
If needed for certain applications, the particles can be spotted during the centrifugation process for example by a camera rotating together with the sample (not shown).
In order to easily classify the particles to be spotted, a sedimentation rate filter 40 is inserted into the settled sample 30 in the container 20. In the embodiment shown, the filter is shaped as a framework 40 defining axially open compartments 42 (
As an alternative to the framework 40, it would also be possible to draw specimens of the settled sample through valves arranged in a pattern substantially corresponding to the compartments 42, for example in the bottom of the container 20, as diagrammatically indicated in
Examples of calculating the behavior of particles subject to operation of a fractionation apparatus according to the invention are described in the following.
Let the difference in rotational speed between the rotation Ω about the primary axis and the rotation ξ of the liquid about the secondary axis, i.e. the axis of the cylinder, be ω. During the time t=π/ω, the centrifugal forces will move the particle away from the cylinder axis and for the same time they will move the particle towards the cylinder axis. In addition, the particle follows the liquid around the cylinder axis. The farther from the centre of the cylinder the centrifugal forces have brought the particle, the faster it is brought back by the rotation of the liquid. This will create a spiralling motion towards the equilibrium position.
An x-y coordinate system may be introduced (not shown) having its centre in the centre of the cylinder and which rotates with the rotational speed Ω of the cylinder around the main centre (located at the distance R far outside the cylinder). In this coordinate system, the centrifugal force is always directed in the y-direction.
Particles will end up at a distance from the cylinder centre which is approximately proportional to the sedimentation velocity. A double radius particle ends up nearly four times further away from the cylinder centre. A different starting position of a particle does not seem to affect the particle's end position.
It is possible to show that
where
α is the particle radius
R is the distance between the two axes
Δρ is the difference in mass density between the particle and the fluid sample
ρ is the mass density of the fluid sample, and
ν is the dynamic viscosity of the fluid.
r is proportional to the square of particle radius. It should therefore be possible to separate particles with almost the same radius a.
It can be shown that the time until the particle has reached its final destination is proportional to 1/ω.
It can also be shown that
determines where in the container 20 the particle ends.
The centrifugal force RΩ2 must be large enough compared to the relative rotation w for the particle to end up far enough from the cylinder centre. At the same time, w must not be too small because then the process takes too long.
With R=0.1 m and
25 turns are required for the particle to reach a relatively stable state at time 150/ω. The main rotation Ω=100 rad/s=955 rpm, results in ω=0.20 rad/s=1.9 rpm. The time is 150/ω=12 minutes. The particle ends 5 mm to the left of the cylinder's central axis. The situation is the same with Ω=400 rad/s=3820 rpm and ω=3.20 rad/s=31 rpm, but the time is only 150/ω=47 seconds.
If it is desired that the particle reaches its final destination without circulating several turns in the liquid, then
should have a high value, but then r becomes rather large.
The foregoing detailed description is given primarily for clearness of understanding and no unnecessary limitations are to be understood therefrom. Modifications will become obvious to those skilled in the art upon reading this disclosure and may be made without departing from the scope of the appended claims.
| Number | Date | Country | Kind |
|---|---|---|---|
| 1950957-9 | Aug 2019 | SE | national |
| Filing Document | Filing Date | Country | Kind |
|---|---|---|---|
| PCT/SE2020/050779 | 8/12/2020 | WO |
