METHOD FOR DETECTING SICKLE-CELL DISEASE AND KIT FOR IMPLEMENTING SAME

Abstract

A method for detecting sickle-cell disease in an individual. A blood sample from an individual is brought into contact with an agent for inducing the sickling of red blood cells suitable for placing the red blood cells in a hypoxic condition. The blood sample is filtered through a porous membrane of which the pore size is determined to retain the sickled red blood cells, and allow the non-sickled red blood cells to pass through. The possible presence of a residue on the membrane is detected, during and/or after the filtering step. The presence indicating that the individual is suffering from sickle-cell disease.

Claims

1-16. (canceled)

17. A method of testing for sickle cell disease in an individual, comprising the following successive steps, steps a) and b) being carried out successively or simultaneously: a) bringing a blood sample from the individual into contact with an agent for inducing sickling of sickle red blood cells capable of placing red blood cells contained in the blood sample in a hypoxic condition; b) filtering said blood sample containing the red blood cells through a porous membrane having a pore size that retains the red blood cells which have undergone said sickling, and allows the red blood cells which have not undergone said sickling to pass through; and c) detecting a presence of a residue on the porous membrane, during or at an end of the filtering step, the presence of the residue indicating that the individual has a sickle cell disease characteristic.

18. The method as claimed in claim 17, wherein the agent for inducing sickling is a metabisulfite salt.

19. The method as claimed in claim 17, wherein a porosity of the membrane is between 2 and 8 μm.

20. The method as claimed in claim 19, wherein the porosity of the membrane is between 3 and 6 μm.

21. The method of claim 19, wherein the porosity of the membrane is between 6 and 7 μm.

22. The method as claimed in claim 17, wherein the bringing step comprises a step of mixing said blood sample with a buffer solution having a pH of between 6.8 and 7.4, containing the agent for inducing sickling and free of a cell lysis agent.

23. The method as claimed in claim 22, wherein the buffer solution contains between 1% and 10% by weight of the agent for inducing sickling, relative to a volume of the buffer solution.

24. The method as claimed in claim 23, wherein the buffer solution contains between 2% and 5% by weight of the agent for inducing sickling, relative to the volume of the buffer solution.

25. The method as claimed in claim 17, wherein the bringing step is carried out for a period of greater than or equal to 30 seconds.

26. The method as claimed in claim 25, wherein the bringing step is carried out for a period of greater than or equal to 1 minute.

27. The method as claimed in claim 17, wherein the bringing step is carried out, prior to the filtering step, by depositing said blood sample on a porous filter with a pore size that allows the red blood cells which have not undergone sickling and the red blood cells which have undergone said sickling to pass through, the porous filter is impregnated with the agent for inducing sickling and the filter is placed on the porous membrane.

28. The method as claimed in claim 17, wherein a volume of said blood sample is between 10 and 50 μl.

29. The method as claimed in claim 17, wherein the detecting step comprises a step of detecting a red coloration of the porous membrane.

30. The method as claimed in claim 17, wherein the porous membrane is placed on a member made of absorbent material; and wherein the detecting step comprises a step of detecting a change in coloration of the member made of absorbent material, an absence of said change in coloration is representative of the presence of the residue on the porous membrane.

31. A kit to perform the steps of the method as claimed in claim 17, comprising the agent for inducing sickling of sickle red blood cells capable of placing the red blood cells contained in said blood sample in the hypoxic condition; the porous membrane having the pore size that retains the red blood cells which have undergone said sickling and allows the red blood cells which have not undergone sickling to pass through; and wherein the kit is free of a cell lysis agent.

32. The kit as claimed in claim 31, further comprising a member made of absorbent material.

33. The kit as claimed in claim 31, wherein the agent for inducing sickling of sickle red blood cells is contained in a buffer solution having a pH of between 6.8 and 7.4 and free of cell lysis agent.

34. The kit as claimed in claim 31, further comprising a porous filter with a pore size that allows the red blood cells which have not undergone sickling and the red blood cells which have undergone said sickling to pass through, the porous filter is impregnated with the agent for inducing sickling.

35. The kit as claimed in claim 31, further comprising a filtering module.

36. A device to perform the steps of the method as claimed in claim 17, comprising: a receptacle, containing the agent for inducing sickling of the sickle red blood cells, collects and brings said blood sample from the individual into contact with the agent for inducing sickling of sickle red blood cells; the porous membrane to filter said blood sample containing the red blood cells directly after said blood sample in the receptacle comes into contact with the agent for inducing sickling of sickle red blood cells; an optical detector to detect the presence of the residue on the porous membrane; a conveyor to convey the receptacle containing said blood sample to and from the porous membrane and the optical detector; and a controller to automatically control operations of the receptacle, the porous membrane, the optical detector and the conveyor to perform the bringing step, the filtering step and the detecting step of the method.

Description

[0084] The characteristics and advantages of the invention will emerge more clearly on reading the examples hereinafter, given simply by way of illustration and which are in no way limiting of the invention, with the support of FIGS. 1 to 4, in which:

[0085] FIG. 1 represents diagrammatically the constituents of a kit for testing for sickle cell disease in accordance with the present invention, and illustrates the various steps of a method of testing for sickle cell disease in accordance with the present invention, carried out by means of this kit;

[0086] FIG. 2 shows a photograph of the membranes obtained after carrying out a method according to the invention, using a blood sample, (A) from a normal individual, (B) from a sickle cell individual, for dilution rates of the blood sample in a solution containing an agent for inducing sickling respectively of 100, 50 and 10 μl of blood sample in a total volume of 8.6 ml of this solution;

[0087] FIG. 3 shows images obtained by observation under a microscope, with a magnification of 200 times, of the membranes of FIG. 2, corresponding to the volume of 10 μl of blood sample, (A) for the normal individual, (B) for the sickle cell individual; and

[0088] FIG. 4 illustrates diagrammatically the steps of a method of testing for sickle cell disease according to a variant of the invention.

[0089] A kit for testing for sickle cell disease according to the invention is represented diagrammatically in FIG. 1. The relative dimensions of the various constituents of this kit are not representative of reality.

[0090] This kit comprises a first tube 10, termed sampling tube, comprising a body 11 and a stopper 12 comprising a closure member 13 which can be pierced with a needle, known as a septum. An anticoagulant, in particular EDTA, is contained in the body 11 of the tube 10.

[0091] The kit also comprises a filtering module 20. This filtering module can in particular be as described in patent documents FR 2952069A or FR 2926090A in the name of the present applicant.

[0092] Diagrammatically, the filtering module 20 comprises the following elements. For reasons of clarity, some of these elements are represented in FIG. 1 separated from one another, whereas they are in reality initially assembled together.

[0093] The filtering module 20 thus comprises a reservoir 21 for receiving the sample to be filtered, comprising, at a first end 211, an opening for feeding with sample.

[0094] At a second end 212, in particular opposite said first end 211, the reservoir 21 is secured to a member 25 for receiving a filtering membrane 24. This filtering membrane 24 is mounted for this purpose in a rigid membrane support 241.

[0095] The membrane 24 is for example made of polycarbonate. It has for example a random pore distribution, a pore diameter of 3.5 μm, and a pore density of 100 000 pores/cm.sup.2.

[0096] The filtering module also comprises a hollow end piece 22, reversibly attached around the member 25 for receiving the membrane, and comprising a support 23 for a tip for piercing a septum, pierced with a channel 231 which opens out at its end, for discharging the filtrate and applying the suction.

[0097] The filtering module also comprises a tube 26 with a reduced internal pressure, of VACUTAINER® type, termed collecting tube, comprising a body 27 and a stopper 28 with a septum 29. The internal pressure in the collecting tube 26 is for example initially between 76 and 200 mmHg.

[0098] The kit also comprises a buffer solution at pH 7.2, termed reducing solution, containing, for 1 liter of solution, 8.41 g of phosphate buffered saline (PBS), 1.8 g of EDTA and distilled water in a sufficient amount to obtain a total volume of 1000 ml. The pH thereof is adjusted to the desired value with sodium hydroxide (NaOH). The reducing agent, sodium metabisulfate, is added to this solution at a concentration of 5% w/v.

[0099] A method according to one particular embodiment of the invention is carried out in the following way, by means of this kit.

[0100] For an individual to be tested, a blood sample is taken and collected in the sampling tube 10, according to the standard protocol for taking a blood sample dedicated to the instructions for a complete blood count (CBC). Schematically, after having placed a tourniquet on the arm, the radial or cubital vein is punctured using a vacuum device, for example by means of a beveled needle connected to the sampling tube 10. A volume of blood 111, for example 5 ml, is collected in the sampling tube 10, and immediately brought into contact with EDTA.

[0101] The blood samples are preferably used within 24 h after they have been taken.

[0102] The desired volume of the blood sample is then taken from the sampling tube 10, and diluted in the reducing solution, so as to achieve the desired total volume. The sickle red blood cells possibly contained in the blood sample then undergo the sickling phenomenon.

[0103] After 1 to 2 min, the final solution thus obtained is placed in the reservoir 21 of the filtering module 20, as indicated at 31 in FIG. 1. The tip attached to the tip support 23 of the end piece 22 is then inserted through the septum 29 of the collecting tube 26, as indicated at 32 in FIG. 1.

[0104] Owing to the reduced pressure in the collecting tube 26, the content of the reservoir 21 is suctioned through the membrane 24. The filtrate 33 passing through this membrane 24 and collected in the collecting tube 26 comprises the blood containing the normal red blood cells and the leucocytes. The sickle red blood cells which are rigid and sickle-shaped are for their part retained by the membrane 24.

[0105] At the end of the filtration, the filtering module 20 is disassembled, as indicated at 34 in FIG. 1, so as to collect the membrane 24, as indicated at 35.

[0106] This membrane 24 is observed visually, so as to detect thereon any possible red coloration and/or to detect the possible presence of an overthickness on its surface. Such a possible coloration and/or such a possible overthickness are indicative of an individual suffering from sickle cell disease.

[0107] Such a method according to the invention was carried out in the following way, for 16 patients suffering from sickle cell disease and for 16 normal patients.

[0108] The biological diagnosis of sickle cell disease had been established beforehand either by electrophoresis in the presence of ampholines (diagnosis referred to as focusing of the hemoglobin), or by HPLC.

[0109] Volumes respectively of 100, 50 and 10 μl of blood from each of the individuals were diluted in the reducing solution, so as to achieve a total volume of 8.6 ml.

[0110] For each individual and each dilution, the diluted sample thus obtained was placed in the reservoir 21 of a filtering module 20, and subjected to filtration through the membrane 24.

[0111] The filtration time was between 1 and 2 min.

[0112] At the end of the filtering step, each membrane was recovered and visually observed.

[0113] For the three dilution levels, an example of the membranes obtained for a normal individual (A) and for a sickle cell individual (B) are shown in FIG. 2. These results are representative of each category of individuals tested.

[0114] A coloration of the membranes from the sickle cell individual is clearly observed, this being for all the initial volumes of blood sample, including for the volume of 10 μl. This coloration is dark red in reality. No coloration is observed for the membranes from the healthy individual.

[0115] An observation under the microscope of the membranes obtained for an initial volume of blood sample of 10 μl was carried out. The images obtained are shown in FIG. 3. It is observed therein that the membrane used to filter the normal blood sample comprises no red blood cell on its surface, whereas the membrane used to filter the sickle blood sample comprises red blood cell aggregates on its surface.

[0116] These results are representative of all the results obtained, for each of the two categories of individuals tested. For all of the samples taken, a deposit of red blood cells was observed at the surface of the membranes when the blood came from affected patients. This deposit was never observed when the blood came from healthy individuals. The sickling induced by sodium metabisulfite thus made the sickle red blood cells rigid and undeformable, incapable of penetrating, by deformability, through the pores of the membrane, as the red blood cells from the normal individuals did.

[0117] This clearly demonstrates the reliability of the method in accordance with the invention for testing for sickle cell disease. Even using a blood sample with a volume of 10 μl, this method makes it possible to distinguish between individuals suffering from the disease and healthy individuals.

[0118] FIG. 4 illustrates a particularly advantageous embodiment of the invention, using a kit in accordance with the invention, comprising a stratified filtering assembly 40 comprising the following successive constituent layers: a porous filter 41, a membrane 24 and a member made of absorbent material 42. In this figure, these various layers are represented spaced out from one another for reasons of clarity. They are in reality in contact with one another. Moreover, the relative thicknesses of these various layers are not in any way representative of reality.

[0119] The membrane 24 corresponds to the characteristics described above.

[0120] The filter 41 and the member made of absorbent material 42 can be made of the same material, or of different materials.

[0121] The filter 41 has a pore size that is sufficient to allow red blood cells which have not undergone sickling and red blood cells having undergone sickling to pass through.

[0122] For example, the filter 41 and the member made of absorbent material 42 are both WHATMAN® No. 40 filtration papers, based on cotton fibers, having a thickness of 22 μm, a grammage of 95 g/m.sup.2 and a retention of 8 μm.

[0123] This filter 41 and this member made of absorbent material 42 are for example in the form of disks, in particular having a diameter of between 8 mm and 2 cm, for example of approximately 9 mm.

[0124] The filter 41 was pre-impregnated by soaking in a solution containing the agent for inducing sickling, for example sodium metabisulfite, at a concentration of 25% w/v, then dried.

[0125] A method of testing for sickle cell disease can be carried out in the following way, by means of such a filtering assembly 40.

[0126] In a first step, illustrated at 43 in FIG. 4, a blood sample 44 is deposited on the upper surface of the filter 41. The volume of this blood sample 44 is for example approximately 15 μl, that is to say approximately the equivalent of a drop of blood.

[0127] This sample passes through the filter 41, by capillary action. During this phase, it is in contact with the agent for inducing sickling. Upon this contact, the sickle red blood cells possibly contained in the blood sample 44 undergo the sickling phenomenon.

[0128] Depending on whether the blood sample comes from an individual suffering from sickle cell disease or from an individual who is healthy with respect to this disease, the following events then occur.

[0129] If the individual is healthy with respect to sickle cell disease, as indicated at 45 in FIG. 4, the blood sample 44, which does not contain red blood cells having undergone sickling, passes through the membrane 24, still by capillary action, and is absorbed by the member made of absorbent material 42. The latter changes color, to a red color. The filter 41 for its part retains its initial coloration. The testing for an absence of sickle cell disease characteristic is thus carried out by observation of the filter 41 and/or of the member made of absorbent material 42, the first having kept its initial coloration and the second having become red in color.

[0130] If the individual is suffering from sickle cell disease, as indicated at 46 in FIG. 4, the red blood cells 47, contained in the blood sample 44 and having undergone sickling, are blocked by the membrane 24. Observation of the member made of absorbent material 42 after a few seconds shows that the latter has kept its initial coloration, since the red blood cells never came as far as said member. The filter 41, for its part, is still in contact with the sickle-shaped red blood cells 47, some of which, contrary to what is represented in FIG. 4, still remain trapped in its pores. It consequently has a red coloration. Thus, the testing for having a sickle cell disease characteristic is also carried out by observation of the filter 41 and/or of the member made of absorbent material 42, the first having becoming red in color and the second having kept its initial coloration.

[0131] This testing was advantageously very simple and rapid to carry out, and what is more using a very small volume of blood.