RELATED APPLICATIONS

This application is related to international patent application PCT/IB2018/055278 filed on Jul. 17, 2018 and titled Device and Method for the Quantification of Cellular and Non-Cellular Blood Components, the entire content(s) of which is/are incorporated herein by reference.

TECHNICAL FIELD

The present invention relates to a device and relative method for the quantification of corpusculated and non-corpusculated components present in a solution containing a blood sample, by means of the concentration and magnetophoretic separation of the components of interest from the rest of the sample and the impedentiometric detection of the quantity of these components.

DESCRIPTION OF THE RELATED ART

For the purposes of the present description “corpusculated components” refer to the cell components of the blood such as red blood cells, white blood cells and platelets.

“Non-corpusculated components” refer instead to those non-cell components, in any case with a volume smaller than or comparable to that of the cells, yet greater than that of the single molecules. These non-corpusculated components can be, for example, crystals of some substances which develop in particular pathological conditions, such as for example the crystals of haemozoin produced by the plasmodium of malaria.

More particularly, the present invention relates to a device and relative method which allows and provides for the isolating and concentrating spatially of one or more corpusculated haemocomponents, as well as one or more substances present in the plasma, exploiting the differences between the magnetic properties of said haemocomponents and/or said substances and the magnetic properties of the other haemocomponents or substances not of interest.

Once separation and concentration have taken place, the device and the relative method of the present invention provide, therefore, for the quantification, of these corpusculated haemocomponents and/or of these substances present in the plasma to take place by means of the measurement of the variation in impedance between two or more electrodes placed in proximity of the zones of concentration.

The field of application of the present invention relates therefore to the diagnosis of all those pathologies that cause an alteration of the magnetic properties of one or more types of blood corpuscles and/or give rise to the formation of substances with different magnetic properties from plasma, said substances being absent or in a different concentration in physiological conditions.

More particularly, pathologies are known that cause the alteration of the magnetic properties of the erythrocytes, or red blood cells, such as malaria, as well as pathologies which cause the alteration of the magnetic properties of the white blood cells. For example, in the case of malaria, it is known how the plasmodium, during the malaria pathogenesis, produces a particular substance mentioned above, which takes the name of haemozoin and is a paramagnetic substance. More particularly, the haemozoin is produced in the form of crystals which are accumulated in the infected erythrocytes, making them paramagnetic. Moreover, in the non-early phases of malaria, the membrane of the infected red blood cells breaks, giving rise to the release of the crystals of haemozoin in the plasma, which is, instead, diamagnetic.

Pathologies are also known in which it is not the magnetic properties of corpuscles of the blood that vary but instead their density. An example of this type comes from sickle cell anaemia, where, while the diamagnetism of the red blood cells remains unchanged, their density changes. In this case, by adding a strongly paramagnetic substance to the plasma, such as for example gadolinium, it can be thought to exploit the magnetic difference between the red blood cells and the gadolinium solution added to the plasma together with the difference in density between the sick red blood cells and healthy red blood cells in order to obtain the separation and, therefore, perform the count of the pathological erythrocytes.

At the state of the art, techniques are known of separation of corpusculated components of the blood, based on the different magnetic behaviour assumed by these components in physiological and pathological conditions.

More particularly, in the patent application U.S. Pat. No. 5,985,153A a device is described for the separation of cells or other magneto-sensitive biological entities comprising: a substrate, a generator of an external magnetic field and a microfluidic system for the loading and unloading of the blood.

In document US0127222A a generic system is instead described for the immobilisation of cells previously marked with magnetic particles, in such a way that they can be attracted by ferromagnetic structures formed on a chip and placed in an external magnetic field.

In the application WO2010091874 a particular ferromagnetic structure is described, composed of magnetic conduits, capable of attracting magnetic particles in particular points in which magnetic domain walls are located. In all the prior art documents mentioned above, as well as in a part of the scientific literature listed in the bibliography [S. Bhakdi et al.,Optimized high gradient magnetic separation for isolation of Plasmodium-infected red blood cells, Malaria Journal2010, 9:38]; [J. Nam et al.,Magnetic Separation of Malaria-Infected Red Blood Cells in Various Developmental Stages, Anal. Chem.,85, 7316-7323 (2013)]; [Ki-Ho Han and A. Bruno Frazier,Paramagnetic capture mode magnetophoretic microseparator for high efficiency blood cell separations, Lab Chip,6, 265-273 (2006)], only the magnetophoretic separation of the components of interest from the rest of the blood sample is described, and no mention is made of the detection of the number of these components.

In the patent application US20120003687A and in the scientific publications [E. Du, et al.,Electric Impedance Microflow Cytometry for Characterization of Cell Disease States, Lab Chip.2013 Oct. 7; 13(19): 3903-3909] e [M. Ibrahim, J. Claude, D. Kourtiche and M. Nadi,Geometric parameters optimization of planar interdigitated electrodes for bioimpedance spectroscopy, J Electr Bioimp, vol.4, pp. 13-22, 2013] techniques of impedentiometric quantification of corpusculated components are described.

These techniques have not however ever been used in association with magnetophoretic separation and concentration. Impedentiometric detection requires that the volumetric fraction of the corpuscles in proximity of the electrodes is sufficiently high, in order to obtain a signal-to-noise ratio in the output signal which is sufficient in order to guarantee a correct quantification of the separated components.

This concentration is usually obtained with microfluidics techniques which considerably increase the degree of complexity of the system and make it poorly suited to a use by a non-specialised user, for example the actual patient.

The device proposed intends to overcome these difficulties by replacing the microfluidics part with a system of magnetic separation and concentration of the components of interest on zones of the substrate in which the detection electrodes are located. In order to perform the measurement the non-specialised user has to dispense on the support a drop of newly sampled blood and then place it in contact with the substrate on which the concentrator elements and the electrodes are housed, in turn placed face downwards within an external magnetic field.

For a volume of the drop of blood sampled of the order of around ten microlitres and supposing that the capture of the components of interest takes place at most at a distance from the concentrators comprised between 20 and 200 micrometres, the dimensions of the active area for the capture on the substrate must be of the order of a few cm2and, in particular, comprised between 0.5 and 5 cm2. The support must also have approximately the same dimensions.

On these values of active area a high concentration of components of interest is necessary in order to ensure an adequate signal-to-noise ratio. As will be explained in greater detail here below, this concentration can be quantified by means of a so-called concentration factor Fe which comes from the ratio between the active area of the substrate within which the drop containing the components which are to be quantified is confined and the area defined by the detection electrodes. In order to have an adequate signal-to-noise ratio in the output signal the concentration factor Fe must preferably be at least around 100.

The object of the present invention is therefore that of providing a device and relative method which are able to quantify the haematic components of interest starting from a quantity of blood such as that which can be extracted by means of pricking with a needle the finger of a patient (5-10 microL) and produce a signal in output with a signal-to-noise ratio such as to allow the detection of corpusculated and non-corpusculated components of the blood with lower limit of concentration up to 10 components per microlitre.

This object is achieved by the present invention with a measurement of the impedentiometric type performed by means of appropriate detection electrodes. The device of the present invention comprises, in fact:at least one pair of detection electrodes, said at least one pair of detection electrodes comprising at least one first electrode connected with a first input apt to receive a first signal in input (V+) and a second electrode;at least one pair of reference electrodes; said at least one pair of reference electrodes comprising a first electrode connected with a second input configured to receive a second signal in input (V+) of opposite polarity to the first input signal (V+) and a second electrode connected to the second electrode of said at least one pair of detection electrodes, in a common point wherefrom an output signal (Out) is picked up;at least one concentrator of ferromagnetic material, configured to co-operate with a magnetic field external to the device, in such a way as to cause the concentration of said components on said at least one pair of detection electrodes;a substrate configured for the housing of: said at least one pair of detection electrodes; said at least one pair of reference electrodes and said at least one concentrator;a support configured to receive a sample of blood or of solution containing blood; andat least one spacer element, configured to confine in the plane of the substrate the blood sample and to distance said substrate from said support.

Said at least one concentrator can be a cylinder or a parallelepiped or an element of another shape placed on the substrate, placed at the detection electrodes and is constituted by ferromagnetic material. The concentrator, attracting towards itself the components to be quantified, ensures that the latter are not distributed everywhere in the area covered by the substrate but concentrate, instead, in proximity of said concentrator, and therefore in proximity of the detection electrodes.

In this way, dimensioning appropriately both the concentrator and the detection electrodes, the concentration factor can increase up to the value necessary for obtaining an adequate signal-to-noise ratio.

The device described above co-operates with means for the generation of a static magnetic field, with which it forms an apparatus. These means for the generation of said field are permanent magnets configured so as to generate a magnetic field, optionally, characterised also by a macroscopic gradient.

Said field is able to attract uniformly towards the substrate the components sought, when they are at a great distance from the concentrators, and of magnetising the aforementioned concentrators to create therefore an intense local magnetic field gradient, which completes the separation of the components to be quantified from the rest of the solution and produces their accumulation on the concentrators.

This separation takes place thanks to the competition between the gravitational force which aims towards the ground and the magnetic attraction force in the direction opposite to that of the gravitational force. The detection electrodes are placed in proximity of said concentrator elements, while the reference ones are placed in areas without said concentrators.

In this way the separated components accumulate selectively on the detection electrodes but not on the reference ones, causing a specific variation of the impedance between the detection electrodes with respect to the spurious one possibly recorded between the reference electrodes. The output signal of the impedentiometric quantification system is therefore proportional to the difference between the impedance variation recorded between the detection electrodes and the one between the reference electrodes. The number of components of interest of this output signal can then be estimated through comparison with an appropriate calibration curve, performed by means of a processor.

As mentioned above, the device and relative method of the present invention can be applied to the diagnosis of any pathology which is the cause of a variation of the magnetic properties of one more haematic components, as well as to the diagnosis of pathologies which cause a variation in the density of one or more components, in this case providing for the addition in the sample of blood to be analysed of a solution of different magnetic properties with respect to the plasma.

Among the various pathologies for the diagnosis of which it is possible to use the device of the present invention, malaria is however of particular interest, in that the diagnostic devices for this type of pathology, today present on the market, have some limitations which make them not always easy to use in particularly disadvantaged contexts, such as the typical ones of endemic zones, often located in developing countries. The most sensitive method currently available for the diagnosis of malaria is based in fact on gene recognition of the various strains of plasmodium by means of PCR (polymerase chain reaction).

This type of method is particularly complex and delicate and, therefore, difficult to apply in contexts that are not technologically advanced. Moreover PCR is not a pan-plasmodium method but is targeted at specific strains and subject, therefore, to the problems arising from the continuous mutations of the plasmodium.

The method, instead, of the “thin smear and/or thick drop” which consists in counting under the optical microscope the red blood cells infected by the plasmodium in a drop of blood, while not requiring complex instrumentation, needs highly expert staff, entails a certain variability in the interpretation of the results and long analysis times.

The rapid tests (RDT) based on the antibody-antigen interaction are, instead, characterised by such low sensitivity as to prevent use thereof for early diagnosis. Moreover, due to the latent presence of the antigen in the body of patients in an endemic zone, the methods based on the antibody-antigen interaction give rise to a high number of false positives.

A second object of the present invention is therefore that of providing a device and relative method which also allow the early diagnosis of malaria, is pan-plasmodium, has adequate sensitivity and is of such simplicity and economical nature as to be able to be used also in those zones where the economic means available do not allow the use of complex instruments and specialist personnel.

This object is achieved by the device and method of the present invention, in that the latter is able to perform the magnetic separation and the quantification both of the infected erythrocytes and the magnetic separation and direct detection of the free haemozoin crystals in the plasma.

The quantification of the infected erythrocytes allows a direct valuation of the parasitemia to be obtained, which is normally quantified by calculating the ratio between infected erythrocytes and healthy erythrocytes, optionally also in the early phase of the disease, before the completion of the first cycle of reproduction of the plasmodium (48-72 hours).

The direct detection of the crystals of haemozoin is, instead, particularly useful, in the non-initial phases of the disease, such as for example concurrent with the first fever attack, since, in these phases, the erythrocytes have already undergone the breakage of the membrane, and the only thing which can effectively be quantified in circulation is the free haemozoin.

These and further objects of the present invention will be made clearer by the reading of the following detailed description of some preferred embodiments of the present invention, to be understood by way of a non-limiting example of the more general concepts claimed, and from the examples relating to experimental tests performed on the present invention.

