Method and device for fractionated distribution of a blood sample

A method for fractionated distribution of a blood sample, includes the steps of: PA1 a) drawing up a blood sample into a needle; PA1 b) distributing via the needle an aliquot corresponding to a specific fraction of the blood sample drawn up, into a flow of a reagent, to thereby produce a mixture of the aliquot and of the reagent; PA1 c) collecting the mixture in a mixing and/or measuring receptacle; and PA1 d) repeating steps b) and c) at least once to distribute a further aliquot corresponding to a further specific fraction of the blood sample, into a further flow of reagent.

BACKGROUND OF THE INVENTION
 The invention relates to the field of haematological analysis, and more
 specifically to the fractionated distribution of a blood sample.
 The expression "blood sample" in this case denotes a volume of blood
 obtained from a blood sample taken from a patient, and contained in an
 analysis container, which for example can be an open tube, or a tube
 closed by a stopper. In general the blood in question is whole or full
 blood, taken with an anticoagulant.
 In haematological analysis, it is often necessary to obtain several
 fractions of the blood of a patient, which are still known as "aliquots",
 and are destined to be diluted with different reagents in order to obtain
 different analysis results.
 This is the case in particular for globule counters, which count and
 differentiate white globules.
 For practical reasons, it is preferable to take only a single sample of the
 patient's blood in a tube, then to fractionate this sample into different
 aliquots, each of which will then be added to a suitable reagent.
 For this purpose, fractionating of the blood sample by means of a sample
 valve is known, according to the teaching, for example, of publication FR
 2 622 692 in the name of the applicant.
 These sampling valves have the advantage that they operate at high speeds,
 which makes them particularly advantageous for specific types of
 analysers.
 However, their main disadvantages are that they require volumes of blood
 which are far greater than the theoretical volumes necessary for the
 analysis, they have a complex and costly structure, and they additionally
 need frequent cleaning, which is difficult to carry out.
 In the field of biochemistry, it is also known to collect all of a blood
 serum in a needle, and then to discharge various successive volumes of
 this sample, for the purpose of different dilutions.
 This specific technique is used for a blood serum, in other words blood
 from which the globules have been removed, and which thus has lower
 viscosity than whole or full blood.
 SUMMARY OF THE INVENTION
 The object of the invention is in particular to eliminate the
 aforementioned disadvantages, and to permit fractionated distribution of a
 blood sample, in highly efficient conditions.
 In particular, an object of the invention is to provide fractionated
 distribution, using means which are simple, reliable, and easy to
 maintain.
 A further object of the invention is to permit distribution of this type on
 the basis of a minimal blood sample volume, without any risk of
 contamination of the reagents with one another, and in each case ensuring
 that an aliquot of blood is well mixed with the reagent concerned.
 For this purpose, the invention provides a method for fractionated
 distribution of a blood sample, comprising the following operations:
 a) drawing up a blood sample into a needle;
 b) distributing via the needle an aliquot of blood, corresponding to a
 specific fraction of the sample drawn up, into a flow of a reagent, in
 order to produce a mixture of the aliquot and the reagent;
 c) collecting the mixture in a mixing and/or measuring receptacle; and
 d) repeating operations b) and c) at least once on another aliquot of the
 same sample, and another flow of reagent.
 Thus, the method according to the invention makes it possible to
 fractionate a blood sample contained in a needle, into at least two
 aliquots which are each mixed by dilution with an appropriate reagent, in
 each case in a mixing and/or measuring receptacle.
 As a result, each aliquot, which corresponds to a specific fraction of the
 sample, is forced into the flow of reagent, thus permitting thorough
 mixing of the blood and reagent to be analysed.
 The aliquots have selected volumes which can be identical or different,
 thus providing the method according to the invention with considerable
 flexibility of use.
 The above-described operation a) advantageously comprises drawing up the
 blood sample into the needle which already contains a fluid, a bubble of
 gas, for example of air, being formed on the interface between the fluid
 and the blood sample.
 By this means, there is no risk of mixing taking place between the blood
 sample and the fluid, since the latter simply acts as an intermediary for
 drawing up the sample, and then for gradual ejection of this sample in
 various aliquots, according to the analyses to be carried out.
 According to another characteristic of the invention, the blood sample is
 drawn up into the needle from a tube of an open or closed type, containing
 a volume of blood to be analysed.
 The blood sample which is drawn up into the needle advantageously has a
 volume which is greater than the sum of the respective volumes of the
 aliquots to be distributed, such as to provide an optional excess of
 volume, in order to obtain an initial volume of blood in the needle on
 completion of the operation a), and/or a residual volume of blood in the
 needle on completion of the operation d), thus making it possible to keep
 the aliquots intact.
 The initial volume of blood can be discharged before the fractionated
 distribution takes place.
 The residual volume of blood alone is affected if there is residual fluid
 present before sampling of the blood takes place. This residual volume can
 be discharged after the distribution, for example into a rinsing vessel,
 which allows the needle to be used to collect a further blood sample.
 According to a further characteristic of the invention, the operation b)
 comprises injection of the aliquot of blood and injection of the flow of
 reagent under controlled conditions, and substantially simultaneously.
 According to a preferred embodiment of the invention, this operation b)
 comprises in succession:
 b1) injection of an initial part only of the flow of reagent;
 b2) simultaneous injection of the aliquot of blood and of an intermediate
 part of the flow of reagent; and
 b3) injection of a final part only of the flow of reagent.
 According to a further characteristic of the invention, in operation b),
 the flow of reagent is injected such that this flow reaches the end of the
 needle by means of which the aliquot of blood is injected.
 The aliquot of blood is preferably injected in a substantially vertical
 descending direction, whereas the flow of reagent is injected in a
 direction which is horizontal or inclined relative to the horizontal, such
 as to assist rotary motion or eddying, thus permitting thorough mixing of
 the blood and the reagent.
 The method according to the invention can comprise at least one additional
 operation, consisting of depositing an aliquot of blood in a fluid medium
 or on a solid support, for example on a glass plate.
 This makes it possible to combine operations of mixing aliquots and
 reagents, with operations of a different kind.
 According to another aspect, the invention relates to a device for
 implementation of the above-described method, comprising:
 a needle which can draw up a blood sample;
 a first volumetric distributor, which is connected to the needle, in order
 to draw up and eject selected volumes of blood;
 at least one reagent injection nozzle, which can be disposed in the
 vicinity of the end of the needle;
 a second volumetric distributor, which is connected to the nozzle, in order
 to eject selected volumes of a flow of reagent;
 a control unit which is connected to the first volumetric distributor and
 to the second volumetric distributor (s), in order to actuate the latter
 in a coordinated manner; and
 at least one mixing and/or measuring receptacle, in order to collect the
 mixture formed from the blood and the reagent, which are distributed
 respectively by the needle and the injection nozzle.
 The injection nozzle is preferably oriented towards the open end of the
 needle, via which the blood is ejected.
 The needle is advantageously substantially vertical, with its open end
 facing downwards, whereas the injection nozzle extends in a direction
 which is horizontal or inclined relative to the horizontal.
 The injection nozzle is advantageously integral with a mixing and/or
 measuring receptacle.
 This receptacle can be a vessel which is delimited by a peripheral wall,
 the injection nozzle opening into the vessel in the vicinity of the
 peripheral wall.
 According to a variant embodiment, the receptacle is a T-shaped connection
 with a first, a second and a third branch, wherein the free end of the
 needle opens into the first branch, the second branch acts as an injection
 nozzle for the reagent, and the third branch constitutes an outlet for the
 mixture.
 According to a preferred embodiment of the invention, the device comprises
 motor means, which can displace the needle, and bring it in succession
 towards the receptacles, under the control of the control unit.
 It will be appreciated that the needle can be fixed, and the receptacles
 can be brought in succession close to the needle.
 The first volumetric distributor and the second volumetric distributor(s)
 each advantageously comprise at least one syringe piloted by a motor,
 which is for example of the step type.
 The following description, which is provided by way of example, refers to
 the attached drawings.

DESCRIPTION OF THE PREFERRED EMBODIMENTS
 The device in FIG. 1 comprises a needle 1, which can contain a blood sample
 E, this needle extending in a substantially vertical direction and having
 a lower end 2 which is open, and an upper end 3 which is connected by
 means of a duct 4 to a first volumetric distributor 5, consisting of a
 syringe 6 actuated by a step motor 7. A control unit 8 comprising a
 microprocessor 9 is provided in order to pilot the step motor 7, and thus
 allow the needle to draw up the blood sample E, and then to eject the
 fractions or aliquots of this sample under controlled conditions, as will
 be described hereinafter.
 A fluid L which fills the syringe 6, the duct 4 and partially the needle 1,
 acts as an intermediary in order to control the steps of drawing up into,
 and ejection from the needle 1.
 The control unit 8 can also displace the needle 1 by suitable motor means
 (not shown).
 In the position in FIG. 1, the needle 1 is in the vicinity of an injection
 nozzle 10, which can convey a flow F of a reagent R in the direction of
 the open lower end 2 of the needle, in order to permit thorough mixing of
 an aliquot of blood A (ejected by the needle 1), and of the flow F of the
 reagent R.
 The nozzle 10 is connected by means of a duct 11 to a second-volumetric
 distributor 12, comprising a syringe 13 activated by a step motor 14,
 which is also controlled by the control unit 8.
 The injection nozzle 10 is supported by a mixing and/or measuring
 receptacle, in this case consisting of a vessel 15, which can receive the
 mixture formed from the aliquot of blood A and the flow F of reagent R.
 As can be seen more particularly in FIGS. 2 and 3, the vessel 15 is
 delimited mainly by a peripheral wall 16, which has a generally
 cylindrical shape and a vertical axis. This wall 16 has an open upper part
 17, and is extended in its lower part by a frusto-conical wall 18, which
 leads to an outlet duct 19 for discharge of the mixture M.
 The injection nozzle 10 has an axis XX (FIG. 2) which is slightly inclined
 relative to the horizontal, and intersects the axis YY of the needle 1.
 The latter is disposed in the vicinity of the wall 16 of the vessel 15,
 such that the aliquot of blood A is forced by the flow F of reagent to a
 point close to the wall 16, thus assisting thorough mixing of the blood
 and the reagent by means of a rotary motion which creates a type of eddy.
 The mixture M thus created is then measured and/or ejected through the
 outlet duct 19 of the vessel 15.
 FIG. 4 shows schematically a device which is similar to that in FIG. 1, and
 comprises three mixing and/or analysis vessels 15-1, 15-2 and 15-3, which
 are similar to the vessel 15 previously described, and support
 respectively three injection nozzles 10-1, 10-2 and 10-3, which are
 similar to the injection nozzle 10 previously described.
 These injection nozzles are connected respectively to three volumetric
 distributors 12-1, 12-2 and 12-3 (shown schematically), which are similar
 to the volumetric distributor 12 previously described.
 On each occasion, the distributors 12-1, 12-2 and 12-3 can convey a
 specific volume of reagent into the corresponding vessel. The volumetric
 distributors 12-1. 12-2 and 12-3 are piloted by the control unit 8.
 In the embodiment in FIG. 4, the device additionally comprises a rinsing
 vessel 20, with which there is associated an injection nozzle 21, which is
 connected to a volumetric distributor 22, in order to permit distribution
 of a rinsing fluid. The volumetric distributor 22 is also piloted by the
 control unit 8.
 The latter also pilots motor means 23, which can displace the needle 1, and
 bring it in succession towards the rinsing vessel 20 and the vessels 15-1,
 15-2 and 15-3, according to a sequence of operations programmed by the
 microprocessor 9.
 Thus, the needle 1 can be introduced into an analysis tube 24 which
 contains the blood S of a patient, this tube being in the form of an open
 tube in the present case.
 The motor means 23 can displace the needle in vertical or horizontal
 translation, as shown by the double arrows.
 The functioning of the device in FIG. 4 is now described, by way of
 example.
 The needle 1 is displaced by the motor means 23, under the control of the
 control unit 8, such as to penetrate in the interior of the analysis tube
 24. The volumetric distributor 5 is then actuated by the control unit 8,
 in order to draw up a specific volume of blood, which constitutes a sample
 E. This sample E is contained in the needle 1, and is separated from the
 fluid L by an air bubble A (FIG. 5) which forms an interface, and thus
 prevents mixing between the blood and the fluid. The latter acts as an
 intermediary in order to control the drawing up into the needle 1, and
 ejection from it.
 The needle is then withdrawn from the tube 24, and subsequently moved such
 that it is positioned opposite the rinsing vessel 20, and is inserted in
 the latter. The volumetric distributor 5 is activated by the control unit
 8, in order to eject an initial volume VI (see FIG. 5). This initial
 volume is received in the rinsing vessel, and is discharged by means of a
 flow of rinsing fluid L conveyed by the nozzle 21, under the action of the
 volumetric distributor 22 which is piloted by the control unit 8. The
 needle 1 is then extracted from the rinsing vessel 20, moved opposite the
 mixing and/or analysis vessel 15-1, and inserted in the latter, such that
 the end of the needle is positioned opposite the injection nozzle 10-1.
 The volumetric distributor 12-1 is then actuated by the control unit 8, in
 order to eject a fraction of the sample E, which corresponds to a first
 aliquot A1 (FIG. 5). This aliquot A1 is mixed with a flow of a first
 reagent R1 in order to produce a mixture, which is measured and/or
 collected at the outlet of the vessel 15-1.
 The needle 1 is then extracted from the vessel 15-1, and is subsequently
 brought towards the vessels 15-2, 15-3, into which the aliquots A2 and A3
 respectively (FIG. 5) are ejected. These aliquots are mixed with flows of
 reagent R2 and R3, in order to produce mixtures which are measured and/or
 collected at the outlet of the vessels 15-2 and 15-3.
 After these operations, there remains in the needle a residual volume VR of
 blood (FIG. 5), which is still separated from the fluid L by the air
 bubble A. This residual volume VR must be discharged.
 For this purpose, the needle 1 is displaced once more towards the rinsing
 vessel 20, and the residual volume VR is discharged by a flow of the
 rinsing fluid, which at the same time ensures that the lower end 2 of the
 needle 1 is cleaned, so that similar operations can be carried out on
 another sample.
 The control unit 8 makes it possible to pilot the sequence of operations,
 such that drawing up of the blood sample, then distribution of the sample
 in aliquots obtained from fractionating of the sample, are carried out
 under very accurate controlled conditions.
 The various volumetric distributors are each actuated by step motors, which
 consequently make it possible to collect by drawing up, or to distribute
 by ejection, very accurate volumes of blood, reagent or rinsing fluid,
 with flow rates which are likewise accurate.
 In order to ensure that an aliquot of blood and a flow of reagent are mixed
 thoroughly in a mixing vessel, it is advantageous to carry out firstly
 injection of only an initial part of the flow of reagent, then
 simultaneous injection of the aliquot of blood and of an intermediate part
 of the flow of reagent, and to end by injection of only a final part of
 the flow of reagent.
 By way of example, the initial, intermediate and final parts of the reagent
 can correspond respectively to approximately 10%, 80% and 10% of the total
 volume of the flow of reagent.
 It will be appreciated that the above-described operating sequences can be
 subjected to many variants.
 In particular, two successive mixing operations can be carried out in a
 single vessel, and a rinsing operation can be interposed between two
 mixing operations which involve different reagents etc.
 In general, the mixing operations are carried out starting with the least
 pollutant or contaminating reagent, and ending with the most pollutant or
 contaminating reagent.
 In the case in FIG. 6, the needle 1 is associated with a cleaning device 25
 of the type described in publication FR 2 707 760 in the name of the
 applicant. This cleaning device substantially comprises a guiding unit 26,
 which contains an emptying duct 27 and a rinsing duct 28. In addition, the
 device 25 comprises a pre-piercing needle 29, which has a bevelled end 30
 which can pierce the stopper (not shown) of an analysis tube.
 In the embodiment in FIG. 7, to which reference is now made, the mixing
 and/or measuring receptacle no longer consists of a vessel, but of a
 T-shaped connection 31, which has a first, vertical branch 32 in which the
 needle 1 can be introduced, a second, horizontal branch 33, in which the
 reagent is injected, and a third branch 34, which is opposite the branch
 33, and acts as an outlet for the mixture. The branch 33 is disposed such
 that the flow of reagent reaches the open end 2 of the needle.
 It will be appreciated that the invention is not limited to the embodiments
 previously described, and can be subjected to many variants.
 The method and the device according to the invention provide considerable
 flexibility of use, particularly concerning the number and volume of the
 aliquots of blood to be processed.