The present invention relates to a device and a system for the separation of blood plasma or blood serum from other blood components by centrifugation of blood, and particularly to the separation of small blood volumes for analytical purposes.

Separation of blood samples in a conventional centrifuge, where a plurality of tubes containing blood samples are placed in a rotor which is then rotated at high speed so that the heavier blood cells are collected at the bottom of the tubes, has several disadvantages. Among other things, a number of manual operations are normally required. Thus, for balancing the rotor, it is necessary to accurately determine the amount of sample in the different tubes or to apply counterweights in tubes placed opposite to each other. If the blood from the donor has been collected in tubes which cannot be placed directly in such a centrifugal separator, the samples must first be transferred to tubes designed for the centrifuge that is being used. Moreover, in many analytical contexts it is desired that one blood sample at a time is centrifuged and analysed, and a conventional centrifuge will then be unnecessarily complicated to use and cannot be integrated into a more or less automated system.

Some of the above disadvantages can be eliminated by another type of centrifugal separators, viz. so-called axial centrifugal separators which are characterised by the fact that the centrifugal tube is brought to spin about its longitudinal axis. This results in the heavier phase of the sample (the blood cells) collecting at the walls, while the lighter phase (the blood plasma) collects nearer the central axis of the tube.

US-A-4,828,716 discloses an axial centrifugal separator for batchwise treatment of a liquid, such as blood. This separator is placed in an axial rotor and brought to spin about its longitudinal axis. The centrifugal tube has a phase-separating element provided

with one-way valves and disposed within the tube to be movable therein. At the beginning of the centrifugation, the separating element is at the top of the tube above the blood volume to be separated. The tube is spun at sufficient speed to separate the blood concentrically in the tube so that the blood cells are in a layer at the tube wall and the blood plasma is in a layer inside the former layer. The separating element is then brought downwards within the tube, while the tube is still rotating, by means of a probe inserted into the tube and rotating therewith, and a non-rotating rod, the compressive force of which is transmitted to the probe. By virtue of the centrally arranged one-way valve system, the blood plasma is pressed through the separating element when the latter is pushed downwards within the tube. The blood plasma collects above the separating element, whereas the blood cells collect below the same. The pushing of the separating element downwards within the tube is stopped when all the blood plasma is above the separating element. With the blood thus separated, the plasma can easily be taken out through an inserted cannula or the like. It is readily understood that the arrangement of a movable separating element actuated via a push rod/rotating probe during the rotation of the tube requires a relatively complex construction with high demands on the precision thereof.

Another type of axial centrifugal separator is described in US-A-3,465,957. In this separator the centrifugal tube has a removable receptacle at its open end. This receptacle has a greater diameter than the tube. At its open end the tube has a portion of reduced diameter, such as a flange, causing the heavier phase (e.g. blood cells) , which during centrifugation is close to the tube wall, to be retained in the tube, while the lighter phase (e.g. blood plasma) , which is nearer the centre of the tube, may flow over to the receptacle at the top of the tube and thereby be separated from the heavier phase. A clear disadvantage of this device is the necessary variation of the rotor speed during centrifugation. Thus,

the speed must initially be limited to prevent the sample from flowing over to the receptacle before the phases have separated and the heavier phase forms a layer on the tube wall. Only then may the rotor speed be increased so that the lighter phase may flow over to the top receptacle.

The object of the present invention is to provide a blood separation receptacle and blood separation system, respectively, which are based upon axial centrifugation but which are devoid of the drawbacks of the centrifugal separators described above, which in a short time will give an acceptable separation, will permit closed handling of the blood (to eliminate the risk of contamination by blood viruses) , and can readily be integrated into various analytical systems. While the invention is primarily _ intended to be used for the treatment of small blood quantities for analytical purposes, it may, of course, be applied in other situations where blood is to be separated.

The above and further objects and advantages of the invention, which will become clear from the following description, are achieved with a device which has the features given in claim 1. Preferred embodiments are given in the subclaims.

Hereinafter, embodiments of a blood separation tube and a blood separation system, respectively, according to the invention will be described in more detail with reference to the accompanying drawings.

Fig. 1 is a sectional view of an embodiment of a blood separation tube.

Fig. 2 is a sectional view of the blood separation tube in Fig. 1 provided with adapters and arranged for filling of blood.

Fig. 3A is a sectional view of the syringe adapter and Fig. 3B is a corresponding sectional view of the cannula adapter in Fig. 2. Fig. 4 is a sectional view of a filled separation tube placed in a centrifugal apparatus.

Fig. 5 is a corresponding view to Fig. 1 of only the blood separation tube in Fig. 4 filled with whole blood.

Fig. 6 is a corresponding view to Fig. 5 of the blood separation tube after the spinning of the tube has begun.

Fig. 7 is a corresponding view to Fig. 5 of the blood separation tube after the blood has been separated. Fig. 8 is a corresponding view to Fig. 7 after completed centrifugation and with a suction needle for plasma introduced into the separation tube.

Fig. 9 is a sectional view of another embodiment of the blood separation tube. Fig. 10 is a schematic illustration, partially broken up and as an exploded view, of a blood separation tube, together with a centrifugal apparatus with a vacuum device, during the sucking up of blood from a patient.

The blood separation tube illustrated in the figures (particularly Fig. 1) , generally designated by reference numeral 1, is designed for axial centrifugation in an axial rotor, as will be described in more detail below. The tube has a doubly cone-shaped inner chamber 2 for receiving a blood sample, normally whole blood. The chamber 2 consists of a downwards tapering lower part 3 and an upwards tapering upper part 4. At the transition between the lower part 3 and the upper part 4 there is a narrow radial slit 5, opening into the top portion of a concentrically arranged outer chamber 6. The latter is designed to completely hold the blood fraction of red blood cells. This fraction is forced into the outer chamber via the slit 5 during the centrifugation of the blood separation tube, as will be described more precisely below. While it is preferred that the slit 5 runs around the whole circumference of the chamber 2, the slit may also, without affecting the centrifugation process too much, be divided into two or more slits separated by small connecting portions between the opposed chamber parts.

In the case shown, the inner chamber 2 has two openings, a lower opening 7 and an upper opening 8. Both openings 7, 8 are closed by pierceable septa, such as rubber stoppers, 9 and 10, respectively. The lower septum 9 is provided in a recess 11 in the bottom part of the

separation tube 1, and the upper septum 10 is provided in a corresponding recess 12 in the top part of the separation tube.

Depending on the method used for filling the separation tube with blood, it may, however, be sufficient with a single opening, preferably the upper one. This will be discussed in more detail below. Furthermore, it is not necessary for one or both openings 7, 8 to be closed by pierceable septa, but other _^ means of closure through which blood can be introduced and air evacuated, respectively, such as various forms of valves and filters, are, of course, also conceivable. This will also be discussed further below.

The proportions between the inner chamber 2 and the outer chamber 6 are adapted such that the outer chamber can hold the whole blood cell fraction of a sample, which preferably substantially fills up the inner chamber. Since the blood cell content of the blood varies between men and women as well as between different individuals, the outer chamber 6 should be dimensioned according to the top level of this range. Thus, as an example of suitable proportions between the inner and outer chambers may be mentioned an inner chamber volume of about 2 millilitres and an outer chamber volume of about 1.2 millilitres. When the blood separation tube is rotated about its longitudinal axis, the inner wall inclinations of the chamber portions 3, 4, i.e. the conicity of the two chamber parts, will, as is readily understood, create a force component which forces the blood upwards and downwards, respectively, towards the transition between the upper and lower chamber portions where the inner chamber is widest and the slit 5 is situated. A suitable inclination in each particular case is easily determined by the skilled person. An inclination which is too small gives too small a vertical force component or, alternatively, requires too high a rotation speed and/or too long a centrifugation time, while an inclination which is too great inter alia gives a cumbersome separation tube.

The connecting slit 5 between the two chambers 2 and 6 is adapted to readily permit a flow of blood cells into the outer chamber 6 from the inner chamber 2 under the action of the centrifugal force in combination with the conical inner walls of the inner chamber portions 3, 4, while simultaneously rendering more difficult the back-flow of blood cells to the inner chamber 2 when the rotation stops. An example of a suitable slit width in connection with the above mentioned chamber volumes is about 0.1 millimetres. The blood separation tube 1 is preferably made of a transparent or opaque material, inter alia for the separation process to be more easily monitored. Suitable blood-compatible materials are, for example, PMMA, polystyrene, or polypropylene. The illustrated blood separation tube may advantageously be made by joining an upper part, comprising the upper chamber part 2 and the external wall 13 of the outer chamber 6, and a lower part, comprising the lower chamber part 3 as well as the inner wall 14 and bottom portion 15 of the outer chamber 6, by means of a welding joint, indicated by reference numeral 16 in Fig. 1.

Hereinafter, the use of the blood separation tube will be described with reference to Figs. 2 to 8.

Fig. 2 shows the filling of the inner chamber 2 of the blood separation tube 1 with whole blood 23 from a whole blood source, such as a patient's blood vessel. To that end the lower part of the blood separation tube 1 is coupled via a first adapter 17, provided with a needle, to a cannula 18 connected to the whole blood source (not shown) , and the upper part of the blood separation tube is coupled via a second adapter 19, provided with a needle, to a syringe 20 for evacuation of air from the inner chamber 2 of the blood separation tube 1.

The cannula adapter 17 and the syringe adapter 19 are - shown in more detail in Figs. 3A and 3B, respectively. . Thus, the cannula adapter 17 (as shown in Fig. 3B) has a protective portion 17a designed to receive the lower part __ of the blood separation tube 1, and a lower connection

portion 17b designed to be operatively coupled to the connection end of the cannula 18, which preferably is of a conventional type. Usually, the connection end of the cannula 18 is conventionally in the form of a Luer female, and the lower part of the adapter 17 is therefore suitably designed as a Luer male. A needle 21 is fixed to the adapter portion 17b and has a sufficient length to be capable of penetrating the lower septum 9 and thereby connecting the cannula 18 to the inner chamber 2 of the blood separation tube. Correspondingly, the syringe adapter 19 (as shown in Fig. 3A) has a protective portion 19a adapted to receive the upper part of the blood separation tube 1, and an upper portion 19b designed to be coupled with the connection part 20a of the syringe 20. The connection part 20a is usually in the form of a Luer male, and the adapter portion is therefore suitably designed as a Luer female. A needle 22 having a sufficient length to penetrate the upper septum 10 and to thereby connect the interior of the syringe with the inner chamber 2 of the blood separation tube, is fixed to the bottom part of the adapter 19. As can be seen in Fig. 2, the needle 22 should only extend into the upper opening channel 8 of the chamber 2 to permit almost complete filling of the chamber 2, i.e. substantially up to the upper end of the cone-shaped portion 4.

By means of the syringe 20, air is evacuated from the interior of the blood separation tube 1. Due to the __ negative pressure created in the inner chamber 2, blood from the whole blood source will effectively flow into the chamber 2 and fill it up. A minor volume of blood may find its way into the outer annular chamber 6.

Preferably, the blood separation tube 1 will already before the filling with blood contain an anti-coagulant, such as citrate, heparin or EDTA, in order to ensure that no coagulation that could cause obstruction of the narrow slit 5 between the inner chamber 2 and the outer chamber 6 will take place.

There are, of course, other ways of filling the blood separation tube 1 than by means of a syringe as described above. Thus, instead of a manual syringe, a vacuum device, which is coupled in the same way as the syringe, may be used for the evacuation of the blood separation tube.

Another alternative is to pre-evacuate the blood separation tube. In this case, a single septum may, of course, be sufficient (i.e. a single opening to the inner chamber 2 of the blood separation tube) , since the evacuation and filling operations are effected on different occasions. In order to prolong the storage life of such a pre-evacuated tube, it may be packed, under a controlled negative pressure, in a gasdiffusion-tight package, e.g. of aluminum foil laminate. A variation of this filling procedure is to use a vacuum device which evacuates the blood separation tube mechanically before use. Still another alternative is to fill the blood separation tube by means of the patient's venous pressure while successively discharging the air from the interior of the tube through a filter that is permeated by air but not by blood, such as a hydrophobic filter. In this last-mentioned case, the top rubber stopper 10 of the previously described blood separation tube is replaced by a hydrophobic filter stopper.

When the inner chamber of the blood separation tube 1 has been substantially filled with whole blood, the tube is centrifuged in a centrifugal device which is schematically illustrated in Fig. 4. To that end, the filled blood separation tube 1 is placed in a specially designed recess 24 in the axial rotor, or chuck, 25 of the centrifugal device. The chuck is driven by an electric motor 26 via a flexible axial coupling 27 and an axial bearing 28. In the illustrated case, both the electric motor 26 and the axial bearing 28 are supported by a chassis 29 which is vertically adjustable on a column 30. The centrifuging position of the chassis 29 is shown in solid lines. Above the chuck 25, the centrifugal device has a sucking needle 31 fixed in a protective cover 32, which in turn is supported by a horizontal frame member 33.

The centrifuging process will now be briefly described with reference to Figs. 5 to 8, which for the sake of simplicity only show the blood separation tube 1.

Fig. 5 shows the filled blood separation tube 1 in a stationary position. When the chuck 25, and thereby the blood separation tube 1, start to rotate, the blood 23 is pressed towards the sides of the inner chamber 2 by the action of the centrifugal force, thereby also filling up the outer chamber 6 via the slit 5, as is shown in Fig. 6. The heavier blood cells are affected by a greater force than the lighter plasma and will therefore be collected in the outer chamber 6. Plasma which has initially come into the outer chamber will successively be forced out as the centrifugation continues. By virtue of the doubly conical chamber sides, the blood in the lower chamber part 3 is forced upwards/outwards towards the slit 5 during the centrifugation, and the blood in the upper chamber part 4 is forced outwards/downwards towards the slit 5. This ensures that the blood, and primarily the blood cells, will be effectively pressed into the outer chamber 6 through the very narrow slit 5.

In Fig. 7, the blood is shown separated into plasma 34 and blood cells 35. As already mentioned, the outer chamber 6 is dimensioned to hold the whole blood cell fraction, and it is seen in Fig. 7 that this is the case, the innermost layer 34b in the outer chamber 6 consisting of plasma.

A blood separation tube having the above given exemplary dimensions has in centrifugation at more than 10,000 to 20,000 rp for about 30 to 60 seconds proved to give a fully satisfactory separation for most analytical purposes, as will be apparent from a working example presented below.

Fig. 8 shows the blood separation tube after the rotation has stopped and the tube is stationary again. Due to the narrow slit 5, re-entry of blood cells from the outer chamber 6 to the inner chamber 2 when the rotary motion is stopped is substantially prevented. With reference now also to Fig. 4, the plasma suction needle 31

is at this stage forced through the upper rubber stopper 10 and down to the bottom of the lower chamber part 3 by bringing the chassis 29 upwards to the position indicated in dash-dotted lines, so that the plasma can be efficiently sucked from the blood separation tube and transferred to a desired receptacle for further processing or storage. Exchange of blood separation tubes after the suction procedure is completed may then be effected by lowering the chassis 29 to the lower position, also indicated in dash- dotted lines.

The suction arrangement is sufficient for sucking at least small amounts of plasma from the inner chamber 2. For greater plasma amounts, however, access of air to the inner chamber during the suction procedure may be necessary. This may, ^" for example, be accomplished by providing the suction needle 31 with a jacket, so that air may be supplied via the slit between the needle and the jacket, or by designing the top part (attachment part) of the needle with a wedge or chisel portion or the like, which will widen the rubber stopper 10 in the penetration to permit- air to pass through (not shown) .

An alternative embodiment of the blood separation tube is shown in Fig. 9. Like the blood separation tube 1 in Fig. 1, this blood separation tube, generally designated by the reference numeral 1', has a doubly cone-shaped inner chamber 2 ' , formed by a lower part 3 ' and an upper part 4 ' , and a concentrically arranged outer chamber 6' connected to the inner chamber 2' via a radial slit 5'. In contrast to the blood separation tube 1 in Fig. 1, the blood separation tube l ¹ has, however, only a single opening 8' placed at the top of the upper chamber part 4 ' and sealed by a septum 10', such as a rubber stopper, which is placed in a recess 12' connecting directly to the opening 8' of the chamber part 4' (i.e. without any channel-shaped extension of the opening 8' as in Fig. 1). Further, a protrusion 6'a is provided in the outer chamber 6' in front of the slit 5', so that the entrance to the slit from the chamber 6' will be labyrinth-shaped (simultaneously as the slit 5' will be

longer) in order to render the back-flow of blood cells to the inner chamber 2' more difficult when the rotation of the tube stops at the end of the centrifugation.

Fig. 10 schematically shows the use of the above described blood separation tube 1 (alternatively the blood separation tube 1') in a centrifugal device integral with a vacuum device. The apparatus illustrated here has, inside a casing 36, a centrifugal device with an axial rotor, or chuck, indicated by the reference numeral 37, at the projecting part of a blood separation tube 1, the lower part of which is received in the chuck. The vacuum device is indicated by a vacuum pump 38 visible through the partially broken up casing side and connected to a cannula 39 in a socket 40 for the blood separation tube 1. At 41 is indicated a micro-switch to be actuated by the blood separation tube for activating the vacuum pump 38 when the blood separation tube 1 is inserted.

In use, air is first evacuated from the blood separation tube 1 by inserting it into the vacuum socket 40, the cannula 39 penetrating one of the rubber stoppers of the blood separation tube 1 (or the one rubber stopper in the case of the blood separation tube 1' , which only has a single septum-sealed opening) and the vacuum pump being activated. Then, the evacuated blood separation tube 1 is inserted into a cannula adapter 43 provided with a guide sleeve 42 and connected to a cannula 44, which has previously been introduced into a patient's bend of the arm 45. Hereby the upper cannula end penetrates through the rubber stopper that seals the chamber opening in question and into the interior of the blood separation tube, and blood is sucked into the evacuated inner chamber of the blood separation tube. The filled blood separation tube is then put into the centrifugal chuck 37, here surrounded by a protective tube 46, and thereafter separation of the blood is effected in a similar manner to that described above.

A centrifugal device like that described with reference to Fig. 4, or an integral centrifuge and vacuum

system like that described with reference to Fig. 10, may advantageously be integrated into various types of analytical systems. As an example of such a blood analysis system may be mentioned the system for determining cardiac infarction markers that is disclosed in our pending international application PCT/SE92/00386 (publication number WO 92/21973) .

EXAMPLE For separating whole blood by centrifugation a blood separation tube described in connection with Fig. 1 above was used, as well as a conventional blood tube as a means of comparison. The volume of the inner chamber 2 of the blood separation tube 1 according to Fig. 1 was about 2 ml and the volume of the outer chamber 6 was about 1.2 ml. About 2 ml of anti-coagulated whole blood were introduced into the inner chamber 2 of the blood separation tube in Fig. 1 as described above by means of a vacuum produced by a syringe. The whole blood had a red blood cell concentration of about ,5 x 10 ⁶ per μl and a white blood cell concentration of about 5 x 10 ³ per μl. After centrifugation for about 1 minute at about 14,000 rpm, essentially all blood cells had collected in the outer chamber. The plasma (about 0.8 ml) was sucked from the inner chamber 2 and exhibited a residual content of red blood cells of about 10,000 per μl and of white blood cells of about 200 per μl. Usual centrifugation in a so-called swing-out centrifuge at 4,000 rpm for 10 minutes of about 10 ml of the same whole blood in the conventional blood tube gave a residual content of about 1,000 red blood cells per μl and about 20 white blood cells per μl.

The invention is, of course, not restricted to the embodiment specifically described above and shown in the drawings, but many changes and modifications may be made within the scope of the general inventive concept as stated in the following claims.