System and method for the continuous extraction of a liquid phase of microsamples, and automated installation for taking them, for carrying out the extraction and taking measurements

Abstract

The present invention relates in particular to a continuous automated extraction system and method for physically extracting at least one liquid phase of a series of liquid microsamples which are taken beforehand in discrete packets, both in space and in time, and are in stored. An extraction system ( 10 ) according to the invention comprises a centrifuge ( 13 ) provided with a plurality of microholders ( 12 ), at least one of which is filled with the corresponding microsample and includes a filling upper portion that is extended by a separating lower portion of smaller cross section than that of the upper portion. According to the invention, the or each holder thus filled has a mass which is more than ten times the mass of this microsample, in such a way that it is possible to extract, by centrifugation, at a given instant, only a single microsample contained in a single microholder, or several microsamples contained in some or all of these microholders, progressively as the latter are filled.

1 . A continuous automated extraction system for physically extracting at least one liquid phase of a series of liquid microsamples, comprising a centrifuge provided with a plurality of microholders, at least one of which is filled with the corresponding microsample and includes a filling upper portion that is extended by a separating lower portion of smaller cross section than that of the upper portion, wherein said or each holder thus filled has a mass which is more than ten times the mass of this microsample, in such a way that it is possible to extract, by centrifugation, at a given instant, only a single microsample contained in a single microholder, or several microsamples contained in some or all of these microholders, progressively as the latter are filled. 
     
     
         2 . The continuous automated extraction system as claimed in  claim 1 , wherein said or each microholder thus filled has a mass that is more than 100 times the mass of this microsample. 
     
     
         3 . The continuous automated extraction system as claimed in  claim 1 , wherein said portions of each microholder have substantially identical volumes, each volume being intended to contain in practice one microsample. 
     
     
         4 . The continuous automated extraction system as claimed in  claim 1 , wherein each microholder further contains, before it is filled with the corresponding microsample, at least one extraction agent which is capable of extracting said phase from the microsample and the density of which is chosen to be intermediate between that of said phase and that of the remainder of the microsample in such a way that, during centrifugation, this agent migrates to the interface between said phase and the remainder of the microsample, forming a physical barrier thereat. 
     
     
         5 . The continuous automated extraction system as claimed in  claim 4 , wherein said extraction agent comprises a MAGIC (Methacrylic and Ascorbic acid in Gelatin Initiated by Copper) polymer gel. 
     
     
         6 . The continuous automated extraction system as claimed in  claim 4 , which further includes a micropipette which is designed to transfer a specified amount of said extraction agent into each microholder and is mounted so as to move on an articulated arm. 
     
     
         7 . The continuous automated extraction system as claimed in  claim 1 , which further includes transfer means which are mounted so as to move on an actuating member and are intended to transfer, via a metering pump, a specified volume of each microsample from a storage container to at least one of the microholders waiting to be filled. 
     
     
         8 . The continuous automated extraction system as claimed in  claim 7 , wherein said metering pump is a peristaltic pump. 
     
     
         9 . The continuous automated extraction system as claimed in  claim 6 , wherein these transfer means comprise a micropipette and an articulated arm for actuating it, which are identical to or different from said micropipette and said arm that are intended to transfer said extraction agent. 
     
     
         10 . The continuous automated extraction system as claimed in  claim 7 , which further includes a computer-aided control device which is capable of controlling said transfer means and their actuating member, the operation of said centrifuge and means for ejecting the microholders. 
     
     
         11 . The continuous automated extraction system as claimed in  claim 1 , wherein each microsample contained in the corresponding microholder is a whole blood microsample from a mammal having a volume of between 1 μl and 100 μl, for example about 8 μl in the case of a mouse blood microsample or equal to 30 μl in the case of a rat blood microsample. 
     
     
         12 . The continuous automated extraction system as claimed in  claim 11 , wherein said phase to be extracted from each microsample is blood plasma that said system separates from cells of the blood, such as red corpuscles. 
     
     
         13 . A continuous automated extraction method for physically extracting at least one liquid phase of a series of liquid microsamples, such as whole blood microsamples from a mammal, which are taken beforehand in discrete packets, both in space and in time, and are stored, wherein this method comprises the use of at least one microholder filled with a microsample and having a mass that is more than 10 times and preferably more than 100 times the mass of this microsample, and the control, by a computer-aided control device, of the centrifuge, a metering pump placed upstream of this centrifuge and means for inserting and ejecting the microholders, in such a way that it is possible, at a given instant, to carry out the extraction by centrifuging all or some of the microholders, progressively as the latter are filled. 
     
     
         14 . The continuous automated physical extraction method as claimed in  claim 13 , which comprises, before each microholder is filled, the incorporation into the latter of an extraction agent which is capable of extracting said phase from the microsample and the density of which is chosen to be intermediate between that of said phase and that of the remainder of the microsample in such a way that, during the centrifugation, this agent being initially located in said upper portion and then, by centrifugation, in said lower portion of each microholder in order to migrate to the interface between said phase and the remainder of the microsample, forming a physical barrier thereat, said extraction agent preferably being a MAGIC (Methacrylic and Ascorbic acid in Gelatin Initiated by Copper) polymer gel. 
     
     
         15 . The continuous automated physical extraction method as claimed in  claim 14 , wherein the incorporation of said extraction agent in each microholder to be filled is carried out by displacement of a micropipette actuated by an articulated arm introducing, into each microholder, a specified constant amount of this agent, and then by the rotation of said centrifuge until said agent falls to the bottom of this microholder. 
     
     
         16 . The continuous automated physical extraction method as claimed in  claim 13 , which further includes, before the extraction, a transfer of a specified volume of each microsample from a storage container to at least one of the microholders that has not yet received a microsample, via the displacement of a micropipette mounted on an articulated arm. 
     
     
         17 . The continuous automated physical extraction method as claimed in  claim 15 , wherein the same pipette as that for transferring said agent is used for transferring the microsamples. 
     
     
         18 . The continuous automated physical extraction method as claimed in  claim 13 , wherein, prior to this extraction:
 a) the microsamples are continuously taken, by an automated sampling system, according to a monotonic time function, via the sending at preprogrammed instants by said control device, to this sampling system, of signals for the taking of a microsample of preprogrammed volume; and then   b) these microsamples thus taken follow one another spatially and temporally in a temporary storage line as far as a downstream end of this line where each microsample drops into a receptacle from where it is then continuously transferred, via said metering pump, into the microholders of said centrifuge, the synchronism between this transfer and the progression of the microsamples in this storage line being controlled by this computer-assisted control device.   
     
     
         19 . An automated installation for carrying out, in succession and continuously, the taking of liquid microsamples as discrete packets in space and in time, their storage and the extraction of at least one phase thereof, wherein the installation comprises:
 a continuous automated sampling system for taking microsamples via a metering pump, for sucking up these microsamples in bursts, and for temporarily storing said microsamples;   an extraction system as claimed in  claim 1 , which is placed downstream of this sampling system by means of said metering pump, in which said microholders each have a diameter in their upper portion that is equal to or larger than the diameter of their lower portion, the former diameter being for example twice the latter diameter; and   a computer-aided control device for controlling all these systems and said pump.   
     
     
         20 . The automated installation as claimed in  claim 19 , wherein each microholder has a height equal to or greater than 30 mm and is provided, in its upper portion, with a measurement apparatus for measuring a physical quantity relating to radiation to which the wall of each microholder is transparent, which apparatus, such as a gamma-radiation well counter or a fluorescence counter, is placed on the same axis as this microholder. 
     
     
         21 . The automated installation as claimed in  claim 20 , wherein said counter and a computer-controlled robotic push-rod are placed respectively above and below a location in said centrifuge intended to accommodate each microholder which is to undergo this measurement, the robotic push-rod being capable of momentarily pushing each microholder so as to bring its upper part into a shield for said counter. 
     
     
         22 . The automated installation as claimed in  claim 21 , wherein each liquid microsample obtained from said extraction system and subjected to this measurement consists of whole blood in which the plasma has been extracted from the corpuscles, this measurement apparatus being an absolute gamma-counter of the well-counter type, which is intended to measure the activity of just the plasma or just the corpuscles of each microsample through the corresponding microholder, in order to allow measurement of the activity of just the plasma, the blood corpuscles deposited by centrifugation on the bottom of each microholder remaining outside this shield. 
     
     
         23 . The automated installation as claimed in  claim 19 , wherein said automated sampling system has a succession of lines through which the microsamples pass, the cross-sectional enlargements of said lines all being less than or equal to 20% in terms of area ratios, in such a way that the microsamples in discrete packets both in space and in time that follow one another in this succession of lines, in particular those having a volume of 30 μl or less, do not in practice become mixed together. 
     
     
         24 . The automated installation as claimed in  claim 23 , wherein said automated sampling system comprises a fluid connection device which is intended for transferring a liquid, such as blood microsamples, to be taken, which system is intended to be connected to a first line via a first opening in this device, which includes a second opening through which a second line passes, the second line being intended to communicate with the first line in order to transfer this liquid, the device comprising:
 a female fluid connector that defines said first opening and has an internal fitting surface terminating in a female radial end into which this first line opens; and   a male fluid connector that defines said second opening, which is fitted into the female connector via its external surface and terminates via its male radial end inside the female connector,   this second line being formed from a flexible microtube which is pushed right through the male connector axially beyond said male end, the free end of this second line pressing in a sealed manner against said female end so as to minimize the dead volume between the first line and the male connector.   
     
     
         25 . The automated installation as claimed in  claim 24 , wherein said internal fitting surface of the female connector is a conical surface converging on said female end with the same conicity as said external surface of the male connector, which converges on said male end. 
     
     
         26 . The automated installation as claimed in  claim 25 , wherein said male and female connectors are both connectors of the “Luer” type, as defined by the ISO 59461 standard of 1986, or else of the “Luer-lock” type, as defined by the ISO 594-2 standard of 1998. 
     
     
         27 . The automated installation as claimed in  claim 26 , wherein said second line extends beyond said male end by an axial length at least equal to the minimum distance (dl) separating said respective ends of the two connectors when the male connector is pushed right into the connection position in the female connector in accordance with one or other of said standards. 
     
     
         28 . The automated installation as claimed in  claim 24 , wherein said second line is provided, around its cylindrical wall and near its free end, with a stiffening means capable of stiffening it inside said female connector. 
     
     
         29 . The automated installation as claimed in  claim 28 , wherein said stiffening means is formed from a ring made of a material that has a stiffness at least equal to and preferably greater than that of said second line and is capable of being fastened thereto, this ring being for example based on a polymeric resin and being mounted so as to bear between said second line and said conical internal fitting surface of said female connector. 
     
     
         30 . The automated installation as claimed in  claim 24 , wherein said second line is made of a material based on a low-density polyethylene (LDPE), for example in order to minimize the absorption of beta-radiation for the purpose of particle counting carried out downstream of this device. 
     
     
         31 . The automated installation as claimed in  claim 24 , wherein said connection device is equipped with said first line, this being a flexible microtube suitable for taking said liquid, such as a flexible catheter to be implanted into the caudal vein of a small mammal for the purpose of taking blood microsamples. 
     
     
         32 . The automated installation as claimed in  claim 23 , wherein said succession of lines has an approximately constant cross section, so that the microsamples following one another each flow over an axial length that is greater than at least five times the largest internal transverse dimension of these lines. 
     
     
         33 . The automated installation as claimed in  claim 19 , which further includes a counting device for counting, with improved sensitivity, elementary particles emitted by the microsamples, this counting device including a line for transferring these microsamples and, placed to the outside of said line, means for detecting these particles, said particles being attenuated by a wall of said line and/or by these microsamples, this counting device comprising at least a counting portion of oblong cross section that joins together two adjacent portions of this line, having a larger flow section and which has an [internal height (h)/internal width (l)] ratio of 20% or less, in which the internal height and internal width represent the smallest and largest transverse dimensions, respectively, of this portion, this being measured along two approximately perpendicular directions, said detection means extending transversely to this portion facing its entire width and on either side thereof. 
     
     
         34 . The automated installation as claimed in  claim 33 , wherein this counting portion has an [internal height (h)/internal width (l)] ratio of between 5% and 10%. 
     
     
         35 . The automated installation as claimed in  claim 33 , wherein the ratio of the flow section of the portion to that of each adjacent portion is equal to 35% or less. 
     
     
         36 . The automated installation as claimed in  claim 35 , wherein said internal height (h) of the counting portion is less than 20% of the inside diameter of each of the adjacent cylindrical portions and wherein said internal width (l) of this portion is 1.3 times greater than this inside diameter. 
     
     
         37 . The automated installation as claimed in  claim 33 , wherein this counting portion has an approximately rectangular cross section, the long sides and/or the short sides of which are curved with mutually symmetrical curvatures, so that this portion has at least in part an approximately convex or concave external face. 
     
     
         38 . The automated installation as claimed in  claim 33 , wherein said line is suitable for the flow of blood microsamples, said internal height (h) of the counting portion being between 100 μm and 250 μm and said internal width (l) of this portion being greater than 1.3 mm, whereas the or each adjacent cylindrical portion has a diameter of between 0.8 and 1.2 mm. 
     
     
         39 . The automated installation as claimed in  claim 38 , wherein the area of the flow section of said counting portion is between 0.15 mm 2  and 0.25 mm 2  and wherein this portion has a length of between 30 mm and 40 mm so as to be able to contain a microsample of about 8 μl facing said detection means. 
     
     
         40 . The automated installation as claimed in  claim 33 , wherein said counting portion has a wall thickness e, expressed in μm, and a density d, expressed in g/cm 3 , the product e×d of which is less than 100, in such a way that the attenuation by this portion of these particles to be counted is minimized when these particles are electrons or positrons from the beta-radioactive radiation emitted by said microsamples. 
     
     
         41 . The automated installation as claimed in  claim 40 , wherein this product e×d is less than 50. 
     
     
         42 . The automated installation as claimed in  claim 40 , wherein said counting portion is based on a thermoformed polymer having a density of 1.5 g/cm 3  or less, and wherein this portion has a wall thickness of less than 50 μm and preferably less than 30 μm. 
     
     
         43 . The automated installation as claimed in  claim 33 , wherein said detection means comprise two sets of detectors placed respectively against or in the immediate vicinity of two approximately planar large faces of said counting portion, which faces are separated from each other by said height (h) and are joined together by two small faces of this portion, these sets of detectors extending beyond said small faces in the direction of said width (l). 
     
     
         44 . The automated installation as claimed in  claim 19 , which is used to measure entry functions of mammals for nuclear imaging, in particular for quantitative imaging of tracers using TEP (positron emission tomography), each liquid microsample being blood from this mammal, such as a rat or a mouse.