WO2009123447A1

WO2009123447A1 - Microcentrifuge device for collecting and separating blood

Info

Publication number: WO2009123447A1
Application number: PCT/NL2009/050154
Authority: WO
Inventors: Jan Hessels
Original assignee: Jan Hessels
Priority date: 2008-04-02
Filing date: 2009-03-27
Publication date: 2009-10-08
Also published as: NL1035244C2

Abstract

A microcentrifuge device for collecting blood, separating blood into plasma/serum and blood cells and stably storing plasma/ serum, comprising a rotor body (1) provided with connecting means for connecting the rotor to a rotary drive unit, and a holder (2) on at least two sides of the rotary shaft for attachment of the upper side of a centrifuge tube (3), such that the centrifuge tubes extend radially from said rotary shaft.

Description

MICROCENTRIFUGE DEVICE FOR COLLECTING AND SEPARATING BLOOD

The invention relates to a microcentrifuge device and a method for collecting blood, separating blood mio plasma/serum and blood cells and stably storing plasma/serum.

Blood is composed of blood fluid, also called plasma, white blood cells, red blood cells and blood platelets. When blood starts to coagulate after being drawn, and fibrinogen is converted into fibrin, the blood fluid xs called teium. When blood is decoagulated with usual anticoagulants after being drawn, the blood fluid is called plasma. The separation of blood cells from plasma or serum is crucial for clinical chemical and biochemical examination. In most cases, blood examination takes place in plasma or serum. It is very important that this separation into plasma/serum and blood cells takes place quickly, completely and efficiently, without lysis of blood cells occurring. Tn particular lysis of red blood cells has a dramatic effect, on the one hand, because of the intensely red colour, on various analyses in plasma or serum, , and on the other hand, because of an entirely different composition from plasma, on the concentration of various components in plasma, such as, for example, potassium, phosphate and LDH. Furthermore, the metabolic process continue m whole blood also after the blood has been drawn, and this affects the composition of the plasma; thus, a decrease of glucose, pyruvate and alanine and an increase of lactate, 3-hydryoxy butyric acid and ammonia take place within one hour after blood has been drawn. This underscores the importance of separating blood cells and plasma/serum as quickly as possible, preferably within half an hour. The method most used in laboratories for separating blood cells and plasma/serum is cer.trifugatior.. To that end, blood contained in a tube or cup is vigorously centrifuged in a centrifuge (1600 - 2500 G), after which the cells will be located in che lower part of the tube and the plasma/serum will be located in the upper part of the tube. To improve this separation process and prevent the cells from mixing in part with plasma/serum again, it is usual to use centrifuge tubes containing a plasma/serum separator gel based on, for example, polydimethyl siloxane, patented as long ago as 1981 by L. Prandi (U.S. Patent No. 4,387,031; filed July 1981) or, more recently, on the basis of cyciopentadiene oligomer by H. Anraku (U.S. Patent No. 7,090,970; filed December 2002) . This plasma/serum separator has a specific rass of 1.03 - 1.07 g/ml precisely between that of plasma/serum (1.02 g/ml) and that of blood cells (1.08 g/ml) and has thixotropic properties, so that it will form a "solid mass" separation between blood cells and plasma/serum after centrifugation.

The classic way of drawing blood for collecting plasma/serum is a by means of a venepuncture in a vacuum tube containing the above-described plasma/serum gel separator. To obtain plasma, the tube also contains an anticoagulant, preferably heparin, EDTA, citrate or oxalate, whilst usually a coagulation activator is used for obtaining serum. The volume of said classical blood drawing tubes is about 4 - 10 ml, but there are also microcups having a blood volume of 250 - 500 μl . However, the current chemical analysers for clinical chemical examination in plasma/serum usually require about 2 - 10 μl of plasma/serum for an analysis, and a minimum cuvette volume of 50 - 100 μl . The blood volume to be drawn can be strongly reduced, therefore. This reduction can be carried through even further by using diluted plasma/serum for analysis. A large number of tests exhibit sufficient sensitivity and linearity to make it possible to carry out measurements in diluted plasma with a sufficient degree of accuracy. Starting from blood having a hematocrit of 0.45 and 3x dilution, a blood volume of 40 - 80 μl, corresponding to one to two drops, and a corresponding plasma volume of 22 - 44 μl and a diluted plasπa volume of 66 - 132 μl, are amply sufficient for about 4 - 10 clinical chemical analyses.

In lanoratories, large and heavy centrifuges capable of processing several tubes at a time are _^sed for centrifuging blood. For microcups, Smaller, table-top model centrifuges capable of processing several microcαps at a time. Turnaville (U.S. Patent No. 5,924,972) descriDes a portable lightweight DC centrifuge for two microtubes havirg a 12 V power supply from a vehicle's cigarette lighter receptacle. Said centrifuge requires precise balancing, however, which is realised by two diametrically opposed tubes having the same weight, one tjbe containing blood and the other tube containing the same volume of a random fluid as counterweight. The entire centrifuge needs to set level prior to use. Because of its dimension and weight, it can be used in a general practitioner's surgery or vehicle, for example, but on account of the complexity of the balancing and levelling operation it is not suitable for home use by (non-expert) patients or clients.

Due to the above limitations, it is common practice that the blood must at all times be centrifuged at a αecentralized location, for example at a laboratory or possibly at a general practitioner's surgery. Because of the transport to a decentralized laboratory, the time that passes between the moment blood is drawn and the moment centrifugation takes place is usually more than two hours, or iorger. Consequently, additives are used in metaoolite analysis for retarding metabolic processes, thas, sodium fluoride is used for glucose analysis. This is satisfactory only in part, and has tne additional drawback that the plasma m question can only De used for glucose analysis any more. For coagulation factor analysis, the blood is usually decoaσulated with citrate, but coagulation activation will nevertheless occur if the citrate plasma is not separated from the blood cells within an hour. The most ideal solution is therefore to use a microcentrifuge which is also suitable for home use by (non-expert) patients or clients, and which preferably has dimensions such that it can be sent by post.

There have been published a multitude of patents which describe techniques for "micro blood collection" and separation of plasma linked to an analytic device for measuring cholesterol or glucose, for example, the so-called "point of care tests" or "home tests". Practical experience has taught that the correctness and precision of these tests are generally insufficient for diagnostic use and that the development of new tests is complex and costly. Only a lirrited number of tests are commercially available in the form of "point of care tests" or "home tests". Furthermore, a new type of meter is generally needed for each type of analysis. The majority of these devices use various types of filters for separating plasma/serum and blood cells (U.S. Patent Nos. 5,064,641; 5,266,219; 5,423,989; 5,262,067). Such filters usually consist of a glass fibre material having a pore size selected so that blood cells remain behind in the filter and plasma can be collected in an absorption filter. The drawback of this method is that only part of the plasma can be easily collected, and that the rest remains behind in the filter. Various techniques are used for increasing the separation efficiency and the plasma recovery: the addition of an agglutinant, such as acids, lectins, polylysin or red blood cell antibodies, described by, for example, Jeng { U.S. Patent No. 5,064,541) , Sand ( U.S. Patent No. 5,118,428) and WiIk ( U.S. Patent No. 5,262, 067) , in which tne retention of red blood cells in the separation filter is increased; an agglutinant conjugated to plastic particles, in which larger agglutinates provide an increased retention of the red biood cells (U.S. Patent No. 5,652,148); an agglutinant conjugated to metal particles, in which a magnetic field captures the agglutinated particles (Murto, WO/2002/029406) . In addition to that, Zander (U.S. Patent No. 6,194,138) describes the addition of gelatine and other additives for increasing the plasma flow through the filter. Generally, however, all this still does not lead to a satisfactory plasma yield. In many cases, however, said yield does suffice for devices in which an integrated analytic system is coupled to the separation filter, for example in the case of a direct glucose measurement on the filter strip. If the plasma needs to be collected for diagnostic tests to be carried out at a decentralised location, however, even higher demands are made of the quality, speed and efficiency of the plasma separation. Recently De Rooij ( U.S. Patent No. 6,245,244; NL Patent No. 1002296) described a combination and techniques and additives for collecting about 80% plasma: the use of an agglutinant, plasma dilution and a sub-atmospheric pressure m the tube behind the filter for sucking plasma therefrom. In the case of a number of analyses, however, diluted blood or diluted plasma cannot be used. Very high analytic standards are set for clinical chemical analyses for components which are regulated within close tolerances in the blood, i.e. which exhibit little biological variation, such as sodium, calcium, albumin and magnesium.

Dilution of plasma while blood is being drawn may in that case lead to an inadmissible increase of the inaccuracy factor. Furthermore, diluted plasma/serum cannot be used in the case of a number of analyses because of the change m free and bound fraction of the substance to be measured, such as free thyroxin, free tri-iode thyronine, testosterone and Cortisol. Dilution may also lead to an inadmissible loss of sensitivity, for example in the case of serologic tests for HIV and hepatitis. For coagulation analysis, only a mimipun αiiation of 1 : 9 with citrate is allowed, which is difficult to realise, if at all, in filter separation techniques.

In those cases in which it is actually possible to use diluted plasma, the dilution factor of the blood must be determined. Said dilution factor can be determined m two ways: a. By mixing an exactly known blood volume with an exactly known ouffer volume. In practice use may for that purpose be made of calibrated "er.d-to-end" capillaries for the Diood volume and of a previously pipetted buffer volume in the device. However, this nethod requires a careful and expert use of "pnd-to-end" capillaries and is susceptible to dilution errors, therefore. A complicating factor in thxs regard is furthermore the fact that the dilution factor depends on the hematocrit, ^hich must be known, therefore, b. By mixing a random blood volume with an exactly known amount of volume calibrator. In practice use can be made of a stable substance which is easy to measure in plasma for a volume calibrator, which substance only spreads in the plasma and not in the intracellular compartment. Another method for obtaining a precise amount of a small volume of plasma is described by Jeng (U.S. Patent No. 5,064,541), in which a reproducible amount of plasma per surface area unit is collected m a collection filter. Because of desiccation, said plasma will not remain stable for a prolonged period and is unsuitable for transport and analysis at a decentralized location. The same principle is used in the collection of whole blood in the so-called "dried blood spot" filter, currently used for neonatal screening, in which the blood is actually dried and does remain stable for a pro_onged period. A punch from the dried blooα spot having a precisely determined surface area determines the olood volume in the punch. After extraction of blood from the filter, said blood can be subjected to clinical cnemcal analyses. However, the "dried blood spot" method has a r.urtber of drawbacks: 1. a chromatographic effect occurs when the blood spreads through the filter, so that the concentration of components to be measured is not exactly the same throughout filter, 2. the method can be used only for whole blood/hemolysate measurements, 3. the concentration of components to be measured is influenced by hematocrit of the blood. Because of the above drawbacks, the "dried blood spot" method is unsuitable for clinical chemical analyses in which high standards are set for correctness and precision.

Accordingly, it is an object of the invention to provide a small, efficient, inexpensive and/or lightweight microcentrifuge, preferably with an inbuilt balancing mechanism, wherein the collection of blood, the separation of plasma/serum from blood cells and/or the (prolonged) storage for transport take place in one and the same device. In addition to that it is an object to realise prolonged stabilisation of the obtained plasma/serum, so that it is still possible, even after transportation, to measure all the components in plasma/serum while maintaining high standards for correctness and precision.

According to the invention, the microcentrifuge device for collecting blood, separating blood into plasma/serum and blood cells and stably storing plasma/serum to that end comprises a rotor body provided with connecting means for connecting the rotor to a rotary drive unit, and a holder on at least two sides of the rotary shaft for attachment of the upper side of a centrifuge tube, such that the centrifuge tubes extend radially from said rotary shaft.

In a preferred embodiment of a microcentrifuge device according to the invention, said holders comprise a tubular projecting part for receiving the centrifuge tube over part of the tube's length, with the centrifuge tube surrounding said tubular projecting part.

Ir. another preferred embodiment of a microcentrifuge device according to the invention, said holders further comprise at least one cup provided with an opening and a passage to rhe ends of the holders for receiving a finger tip with a drop of blood.

In another preferred embodiment of a microcentrifuge device according to the invention, said passages comprises a capillary tube .

In another preferred embodiment of a microcentrifuge device according to the invention, the means of attachment further comprises a closable vent hole, which vent hole is designed to be open upon receiving said finger tip and passing the blood from the cup to the centrifuge tube.

In another preferred embodiment of a microcentrifuge device according to the invention, the vent hole can be closed by depressing the cup.

In another preferred embodiment of a ipicrocentrifuge a device according to the invention, che holders further comprise a fluid reservoir for containing a balancing fluid, which fluid reservoir is provided with a passage to the ends of the holders.

In another preferred embodiment of a microcentrifuge device according to the invention, the rotor body comprises recesses or protrusions for radially fixing the holders. In another preferred embodiment of a microcentrifuge device according to the invention, the rotor body comprises second recesses or protrusions for radially fixing the centrifuge tube.

In another preferred embodiment of a microcentrifuge device according to the invention, the connecting means for connecting the rotor to a rotary drive unit comprise a bore for receiving a drive shaft.

Another preferred embodiment of a microcentrifuge a device according to the invention further comprises said rotary drive unit .

In another preferred embodiment of a microcentrifuge device according to the invention, said rotary drive unit is a mini motor or a high-speed rotary tool.

In another preferred embodiment of a microcentrifuge device according to the invention, the holders comprise a sleeve-iike body having an external diameter of 13 mm and a length of about 25 mm, which is provided with a fluid reservoir for the balancing fluid and plasma/serum, which fluid reservoir has a cylindrical sidewall with a diameter of about 11 mm and a substantially conical bottom, and an opening provided with a sealing cap on the side remote from the bottom, such that the holders are suitable for being placed in a standard clinical chemical micro analyser.

In another preferred embodiment of a microcentrifuge device according to the invention, the holders are provided with a tubular projecting part at the bottom side of the sleeve-like body for receiving the centrifuge tube over part of the latter* s length, with tne centrifuge tube surrounding said tubular projecting part.

In another preferred embodiment of a microcentrifuge device according to the invention, the holders comprise an annular body provided with a snap edge for attaching the centrifuge tube.

In another preferred embodiment of a microcentrifuge device according to the invention, the holders are provided with an externally operated valve for opening and closing the conical bottom part of the fluid reservoir.

The invention also relates to holders for use in the microcentrifuge device according to any one of the above embodiments.

The invention also relates to a method for collecting blood, separating the blood into plasma/serum and blood cells and stably storing the plasma/serum thus formed, wherein a balancing fluid is introduced into the fluid reservoir, a drop of blood is deposited in the opening of the holders or the centrifuge tube, a centrifuge tube is attached to the holders, the holders are placed m the rotor body, and the rotor body is connected to the shaft of the rotary drive unit and the rotor body is rotated at a high speed.

The invention described herein is directed at solving the above-described problems in the current method of drawing blood and separating blood into plasma/serum and blood cells. The object is to develop a device and method which enable a patient or client to collect blood at home himself or herself, separate plasma/serum and blood ceils, stably store plasroa/serum for transport and subsequently send it to a laboratory by post for analysis. In order to accomplish t^Λnis, an automatically balanced microcentrifuge device has been developed for collecting blood, separating plasma/serum and blood cells by centrifugacion as well as stably storing the same for transport in one and the bc-me device. The result is a separation method exhibiting a yield of practically 100% plasma/serum, which remains stable for a prolonged period of time at room temperature. The whole can be sent by regular post to a laboratory, where the analyses can be carried out m plasma/serum with state of the art, advanced and well-calibrated and controlled equipment.

Preferably, the centrifuge tubes contain a thixotropic plasma/serum separator gel for separating plasma/serum and blood cells and keeping the same separated. Said centrifuge tubes may also be commercially available, disposable microcentrifuge tubes, possibly containing an anticoagulant, such as one of the anticoagulants used in the state of the art in usual concentrations, for example heparin or EDTA, for obtaining plasma, or a coagulation factor for obtaining serum. The capillary tube may comprise one of the anticoagulants used in the state of the art in usual concentrations, such as heparin or EDTA, for premature decoagulation of blood. The liquid anticoagulant is for example citrate or oxalate or another usual anticoagulant in usual concentrations. The balancing fluid is for example "pure silicone fluid" (Dow Corning 200 fluids) having a super-low viscosity of 0.65, 1.0, 1.5 or 2 cSt, preferably 1 cSt, with a volatility higher than water and a specific mass lower than water, or another fluid having comparable physical properties that does not mix with water. The aforesaid "pure silicone fluid" may also function as a plasma/serum stabiliser for keeping plasma/serum stable at room temperature for a prolonged period of time without any change in the activity or concentrations of components to be measured. The device is quxte suitable for prolonged stable storage of various fluids, suc~ as plasira/serum, calibration fluid or quality control serum, at room temperature witnout any loss or decrease of components to be rreasured, using the "pare silicone fluid".

Any imbalance as a result of different amounts of blood being introduced into the centrifuge tubes can be corrected by causing an excess of oalanci^g fluid to flow into the centrifuge tube from the fluiu reservoir immediately after centrifugation has starred and subsequently causing the excess balancing fluids to flow out via the upper vent hole, so that the centrifuge tubes will at all times be completely filled with fluid, irrespective of the blood volume being introduced, and are thus optimally balanced.

To further clarify the features of this invention and explain the concomitant advantages and specifics thereof, the microcentrifuge device according to the invention will now be explained in more detail. It will be apparent, however, that tne description below can by no means be interpreted as constituting a limitation of the scope of protection of the present invention as defined m the claims.

The appended figures show the details of a prototype of the microcentrifuge device, m which the numerals between brackets m the text refer to the numerals in the drawings, in which:

Figure 1 shows a perspective top plan view of the microcentrifuge device;

Figure 2 shows a perspective, partially sectional view of the microcentnfuαe device; Figure 3 is a detail drawing, showing a cross-sectional front view and a cross-sectional perspective view of a tαbe holder; Figure 4 shows a perspective top plan view of a rotor with a centrifuge tube; and Figure 5 is a detail drawing, showing a perspective view and a front view of an alternative embodiment of a microcentrifuge device .

A specific aspect of the microcentrifuge device concerns the construction of the rotor I, the tube holder {2} and the centrifuge tube (3). By placing the centrifuge tubes (3) diametrically opposite each other at an angle relative to the shaft (4) of the mini motor, preferably an angle of 90°, the distance between the two centrifuge tubes is greatest and the centrifugal force is maximally utilised, whilst a maximum degree of stability is obtained during centrifugation. This construction may be extended in the same manner with several centrifuge tubes disposed diametrically opposite each other. By placing the microcentrifuge device in a vertical position on a mini motor or a high-speed rotary tool and axially centrifuging it for about 0.5 to 5 minutes, preferably about 1 minute, at a speed of 10,000 to 60,000 rprr, preferably about 20,000 rpn, depending on the spacing between the two centrifuge tubes, an optimum separation between blood cells and plasma/serum is obtained. The thixotropic separator gel (5) in the centrifuge tubes (3) prevents blood cells from flowing back and mixing with plasma/serum after centrifugation. The gel used in the state of the art is a thixotropic gel, which is supplied in commercially available centrifuge tubes by various companies, sucn as Becton Dickinson, Sekisui, Terumo, Kabe or Greiner. The centrifuge tube

(3) is clamped over the tubular projecting part (6) of the tube holder (2) . The tube holder (2) with the centrifuge tube (3) is noved into the space (7), which is to that end available in the rotor (1), from above. The lower part of the tube holder (8) is clamped against an upright inner edge (9) of the rotor (1) , whilst the upper edge (10) of the centrifuge tuoe fits exactly in the notch (11) in the rotor (1). A recess (12) at the upper side of the outer edge of the rotor (1} ensures that the centrifuge tαbe (3) is clamped from above and cannot move upwards at high rotational speeds. In practice this construction of the microcentrifuge device has been found to lead to a high degree of stability during centrifugation by means of a mini motor or a high speed rotary tool, with a vibration-free rotation being observed in all posbible positions, whilst it is not oven necessary to keep the microcentrifuge upright. This is a major advantage over the current blood centrifuges, which need to be manually balanced and must be positioned upright and level. This has several advantages in the case of (non-expert) home use by the patient or client. Furthermore, this microcentrifuge and this method of plasma/serum separation solve many problems that occur in filter separation: full recovery of plasma/serum; prolonged stabilisation of plasma/serum that has been formed (see below) ; it can be used both for diluted and for undiluted plasma/serum; it is suitable for every metabolic examination without using metabolic inhibitors; a chromatographic effect does not take place and the method does not depend on the hematocrit, this in contrast to the use of whole blood in "dried blood spot" filters. The microcentrifuge device is for example made of polycarbonate or polyethylene terephthaiate (PET) or any other suitable synthetic or lightweight material. The rotor (1) is placed on the shaft of a mini motor or a high-speed rotary tool via a connecting piece or adapter and can be disconnected therefrom after centrifugation, possibly after transport. The mini motor or high-speed rotary tool and the rotor (1) with the box and the safety cover can be reused, whilst the tube holders

(2) and the centrifuge tubes (3) are disposable. For the present application, use has been nade of commercially available centrifuge tubes from Kabe Labortechnik (Germany) , type GK 150, and Dickinson (USA) , type Micro container, both with various anticoagulants and separator gel. The wide part (13) of said centrifuge tube (3) is filled by the tubular projecting part (6) of the tube holder (2), so that the remaining space of the narrow part (14) of the centrifuge tube (3) will be about 250 μl. The mini motor is a DC mini motor which is fed from the mams via an adapter or from a (rechargeable) battery. The DC irini motor may be a mini motor having a height of about 1 - 3 cm, preferably about 2 cir., which is commercially available from companies such as, for example, Faulhaber, Maxon or Danaher. Alternatively, a commercially available high-speed rotary tool may be used, such as a Dremel© from Bosch. The microcentrifuge device comprises an on-off switch with a variable or fixed centrifuging time and is protected by a protective cover, which may automatically activate the mini motor after being closed. The overall microcentrifuge device in the box with the protective cover and the mini motor has small dimensions, preferably about 15 x 15 x 3 cm (1 x w x h) at most, in any case such that it can be used at any location, for example at home or m a general practitioner's surgery, for plasma/serum preparation and be sent to a laboratory by regular post. In the laboratory, the collected and posted plasma/serum can be analysed with the most advanced state of the art, correctly caiiorated and verified equipment. The correctness and precision of said equipment is in many cases better than those of the current "point of care" meters and home meters, such as glucose meters, PSA meters, TSH meters, etc. It is furthermore a major advantage that several determinations, to be selected from a large and diverse arount of a few hundred tests, can be made from the same drop(s) of plasma/serum with the current laboratory equiprr.er.t . The microcentrifuge device described herein is a universal device, which is suitable for preparing and collecting serum as well as all kinds of plasma, both in diluted and in undilu^ted form.

Another aspect of the microcentrifuge device concerns the 5 construction for collecting blood. The upper side of blood passage capillary tube (15) is provided with a flexible cup (16) provided with a hollow space in which a fingertip with the drop of blood precisely fits. Said hollow space has a diameter of 3 - 9 mm, preferably 5 - 7 mm, and a depth of 2 - 8 mm, preferablyC 4 - 6 mm. Because of this construction, the drop of blood on the finger is led into the capillary in a simple and hygienic manner. To prevent an overpressure situation in the centrifuge tube (3) while the tube is being filled with blood, a small notch (17) of about 0.1 - 0.4 mm, preferably 0.2 mm, is formed at the upper5 side on the two tubular projecting parts (6) of the tube holder (2), which function as vent holes (17). This is conducive to a quick and spontaneous flow of blood in the blood passage capillary tube to the centrifuge tubes (3) . 0 A specific aspect of the microcentrifuge device concerns the construction of the fluid reservoir (18) with a capillary tube (19) between the centrifuge tube (3) and the fluid reservoir (18) and a vent hole (20) opening into the upper side of the tube holder (2) on the one hand and into the fluid reservoir (18) on the other5 hand. The fluid reservoir (18) is filled beforehand with a balancing fluid (see below) via the vent hole (20) . This construction makes it possible to store the balancing fluid for a prolonged period of time in the fluid reservoir (18) in the closed condition of the vent holes (17 and 20) and the blood0 passage capillary tube (15), without the balancing fluid coming into contact with the separator gel (5) in the centrifuge tubes 3. After the upper vent hole (17) and the blood passage capillary tube (15) have been opened, one or more drops of blood can be : 7 introduced without ary balancirg fluid flowing out of the fljid reservoir (18), irrespective of the position of the microcentrifuge device. Directly after centrifugation has started, the oalancmg fluid and any other fluids will flow from the fluid reservoir (18) into tne centr.fuge tube (3) under the influence of the centrifugal force. Because of the slightly larger volume of tne fluid reservoir (18) m comparison with the volume of the centrifuge tube (14) tnat is available for fluid, the excess of balancing fluid will flow out via tne upper vent hole (1^"?) . Because of the low specific mass, said oalancmg fluiα is located m front of the plasma/serun layer during centrifugation and thus prevents the outflow of blood and/or plasma/serum via the upper vent hole (17) . The centrifuge tubes (3) are thus completely filled at all times during centrifugation. This guarantees a vibration-free rotation up to a rotational speed of at most 60.000 rpm. One of the unique aspects of this invention is the fact that the microcentrifuge device is automatically balanced in a very simple manner without any specific operations being required, which is a major advantage over most current centrifuges. An optimum balance leads to an enhanced separation efficiency, a longer life of the mini motor and a lower noise production. After centrifugation, the plasma/serum that has been formed can flow back into the fluid reservoir (18) togetner with the balancing fluid. After the vent holes (17 and 20) and the blood passage capillary tube (15) have been closed, the plasma/serum and the balancing fluid can be stored (for a prolonged period of time) m the fluid reservoir (18) , with the Dalancing fluid also functioning as a plasma/serurr stabiliser (see below for more deta_ls) . The opening and closing of the vent holes (17 and 20) and the blood passage papillary (15) can take place by depressing the flexible cup (16) with the sealing edge (21) or Dy means of a stop, plug, shut-off valve or any otner mechanism. Tne advantages of this construction are: a. the fluids in the fiuiα reservoir (18) do not come into (prolonged) contact with the thixotropic gel (5) in the centrifuge tube (3) and cannot interact with said gel during storage; b. the fluids m the flαid reservoir (18) are sealed airtight, so that there can De no evaporation, making it possible, for example, to stably store a buffer with a volume calibrator or anticoagulants therein; c. fluids present in the centrifuge tube cannot obstruct the upper vent hole (17) while filling with blood takes place and prevent the passage of blood; d. no fluids can leak out via the vent hole (17) while filling with blood takes place. An alternative construction, in which a blood passage capillary tube is vertically disposed, for example, and directly opens into a larger fluid reservoir, has the advantage that the blood will flow out more quickly, but a disadvantage is the fact that fluids can leak from the fluid reservoir while blood is being drawn. After this, the entire microcentrifuge device can be sent to a laboratory for analysis. Tn the laboratory, the tube holders (2) with the centrifuge tubes (3) can be simply moved off the rotor (1) and subsequently the tube holder (2) can be removed from the centrifuge tube (3} and be sealed with a cap until analysis takes place. The plasma/serum may also be pipetted from the fluid reservoir (18) via the vent hole (20) . To simplify this procedure, a recess (22) has been formed at the bottom side of the fluid reservoir (18).

Another specific aspect of the microcentrifuge device concerns the use of a specific balancing fluid in the fluid reservoir (18) and a method for automatically balancing the microcentrifuge device. The balancing fluid is an a-polar fluid which does not mix with plasma/serum and which has the following physical characteristics: it does not absorb water and does not mix with plasma/serum; it does not absoro any components from plasma/serum; it is volatile; it has a low specific mass and floats on the plasma/serum layer, sealing in airtight, In the present application, we use the volatile "pure silicone fluid" (Dow Corning 200 fluids) having a super-low viscosity of, for example, 0.65, 1.0, 1.5, 2 or 3 cSt, preferably 1 cSt, and a low specific mass of < 0.90 g/ml for this purpose. The total volume of the fluid reservoir (18) with "pure silicone fluid" is at least 10 - 100 μl, preferably about 20 μl, more than the volume of the free space in the centrifuge tube (14). In this way, both centrifuge tubes containing one or a few drops of blood are filled up with the "pure silicone fluid" directly after centrifugation has started. Because of the low specific mass, said "pure silicone fluid" is present on top of the aqueous fluids as a front layer during centrifugation. As a result, four different layers are successively formed; blood cells, thixotropic gel (5) , serum/plasma, possibly mixed with buffer or other aqueous fluids, and finally "pure silicone fluid". As a result of this construction, only the excess "pare silicone fluid" will disappear via the upper vent hole (17) immediately after centrifugation has started. An additional advantage of the above-described "pure silicone fluid" is the fact that it floats on the plasma/serum, thereby providing an airtight seal and completely preventing evaporation. Evaporation is a major problem when very small volumes need to be stored for a prolonged period of time. Table 1 shows that a small volume of heparin plasma of 100 μl in an open cuvette covered with 200 μl "pure silicone fluid" will retain a stable volume at room temperature for 9 days, derived from stable concentrations of various plasma components during this period. In an unsealed microtube, an increase in the concentration or activity of about 35% of all the components due to evaporation could be observed already after 1 day (Table 1} . Without 'cjre sillcone I fluid 'pure si Iicone fluid¹

DAY 0 DAY 1 DAY 2 DAY 3 !DAY 0 DAY 3 DAY 7 DAY 9

PROTEINS rotisl 56 ηη ICO Cb. d. 56 57 57 58

Albumin 29 39 50 c.b.d. 29 30 31 31

AF 104 Kl 188 c.b. d. 104 99 100 99

ALAT 88 120 159 c.D. α. 88 86 81 79

7.4 9.8 12.3 c.b.d. 7.4 ~^> .6 7.7 7.7

TSH 3.2 4.4 5.2 c.b.d. 3.2 3.2 3.0 3.0

METABOLITES

Bilirubir* - 3 1 c.b.d. 7 2 1

Cholesterol 3.5 4.6 6.0 c.b.d. 3.5 3.6 3.7 3.6 iLactaie 3.9 5.2 6.7 c.b.d. 3.9 4.1 4.3 4.3

Glucose 5.1 6.9 3.9 c.b.α. 5.1 5.2 5.3 5.2

ELECTROLYTES

Potassium 3.8 5.0 6.5 c.b.d. 3.8 3.8 3.9 3.7

Table 1. Effect of liquid sealing of 100 μl heparin plasma at room temperature in an open micro container with 200 μl "pure silicone fluid" (Dow Corning 200 fluids; viscosity 1 cSt) on the stability of various plasma components.

* It is a well-known phenomenon that bilirubin decreases rapidly under the influence of light, "pure silicone fluid" does not prevent this.

** c.b.d. - cannot be determined, volume too low due to desiccation.

These results furthermore indicate that no absorption of water by the "pure silicone fluid" takes place, and that there is no interference between components from the plasma/serum and the "pure silicone fluid". A second concomitant advantage is that no clot formation could be observed in heparinised plasma, not even after 9 days of storage at room temperature . It is well-known from practice that tne activity of heparin in plasma stored at room temperature decreases and that micro (clots) may form; furthermore, a gelatinous mass nay form after about three days. Even small micro clots may lead to technical problems and major analytical errors when pipetting takes place in automated analysis equipment. The use of the aforesaid "pure silicone fluid" increases the storage life of heparin plasma by at least 9 days and prevents clot formation, and ensures that the plasma will remain fully liquiα. These are important advantages, which make transport at room temperature possible.

According to the broad aspect of the present invention, the microcentrifuge device may comprise a coagulation activator for accelerating coagulation (fibrin formation) m order to obtain berum, or an anticoagulant for preventing fibrin formation in order to obtain plasma. The anticoagulant may be present in the blood passage capillary tube (15) and/or in the centrifuge tube

(3) and/or in the fluid reservoir (18) , in dried form or dissolved in a buffer. The anticoagulant is preferably one of the usual agents in a usual concentration, such as heparin, SDTA, citrate or oxalate. Furthermore, the use of two blood passage capillary tubes (15) to each of the centrifuge tubes (3) provides a possibility to use two different anticoagulants and to collect two different kinds of plasma while drawing blood only once. In this way a universal and widely applicable method for collecting both serum and all kinds of plasma is obtained.

It stands to reason that various changes and modifications to the inventions described and proposed herein will appear to be available to those skilled within this field of the art. Such changes and modifications can be realised within the framework of the above-described inventions described herein, without departing from the spirit and scope of the inventions as described herein and without limiting the concomitant advantages as described. Such changes and modifications are explicitly intended to fall within the scope cf the claims defined herein. Figure 5 shows the derails of an alternative embodiment of the microcentrifuge device, with the numerals between brackets in the text referring to the numerals in the drawing. The dimension and shape of the cube holder (2) have been adapted so that said rube holder can also function as an analyser cup. In this embodiment, use is rnaoe of a commercially available centrifuge tube (3} from Becton Dickinson (microtube) containing separator gel and heparin as an anticoagulant and being provided with a thickened outer edge (10) of about 1 mm at the upper side of the tube. The blood, for example oDtamed by means of a finger prick, is directly collected in the aforesaid centrifuge tube 3. After the blood has been drawn, the centrifuge tube (3) is clamped over the tubular projecting part (6} of the tube holder {2} and at the same time snapped into place behind a snap edge (105) . Said snap edge (105) forms an integral part of the tube holder (2) or is separately screwed tnereon, it consists of a ring provided with three resilient lips (106) with a notch (107) the size of the thickened outer edge (10) of the centrifuge tube (3). When this construction is used, the centrifuge tube will remain snapped in place in case of a centrifugal force of up to at least 3000 G. The tube holder (2) further comprises a fluid reservoir (18) and a two-way cock (109) and is sealed leak-tight by means of a screw cap (100) . The fluid reservoir has the conical shape of a usual micro analyser cup and is closed at the bottom side by the two-way cock (109) . The tube holder (2) with the centrifuge tube (3) snapped in placed thereon is subsequently placed in a rotor (1) and centrifuged, for example in the previously described manner by means of a mini motor (120) or a high-speed rotary tool. The cock (109) must be opened before centrifugation takes place, so that the previously described balancing fluid can flow from the fluid reservoir (18) into the centrifuge tube (3) . The handle of the two-way cock (109) is placed or. the tube holder (2) in such a manner that it only fits in the rotor (1) in the opened position. Zn this way, centrifligation cannot take place if no balancing fluid is present in the centrifuge tube. After centrifugation, the tube holder (2) with the centrifuge tube (3) is removed from the rotor (1) and held vertically for some time, durxng which time plasma/serum and balancing fluid flow down into the fluid reservoir (18) . After the two-way cock (109) has been closed, the plasma/serum and the balancing fluid are separated from the thixotropic gel and the blood ceils, after which it can be stored at room temperature for a prolonged period of time (see Table 1) and be transported. The upper part of the round tube holder (2) has a length of 25 mm and a diameter of 13 mm, whilst the snap edge (105) has an external diameter of about 11 mm and can be fixed therewith in a standard analyser tube (3) having an internal diameter of 11 mm, an external diameter of 30 mm and a length of 75 mm. The overall tube construction (110) now has a dimension of 13 x 100 mm, which can be directly placed into the current clinical chemical analysers. Because the fluid reservoir (18) now functions as a micro analyser cup, it is an additional advantage that the plasma/serum no longer needs to be manually pipetted into a micro analyser cup. Because of the non-polar nature of the balancing fluid, which, as already described before, at least substantially functions as a protection against evaporation, this fluid is not recognised by the pipetting needles of the current clinical chemical analysers, nor does it adhere to the outer side of the needle. An additional major advantage is therefore the fact that the balancing fluid need not be pipetted therefrom, but remains present on the plasma/serum during analysis. Thus, a simple and efficiently usable tube holder is obtained, which, in addition to functioning as a centrifuge tube, also functions as a fluid reservoir and as a microcup analyser.

Claims

1. A microcentrifuge device for collecting blood, separating blood into plasma/serum and blood cells and stably storing

5 plasma /serum, comprising a rotor body (1) provided with connecting means for connecting the rotor to a rotary drive unit, and a holder (2) on at least two sides of the rotary shaft for attachment of the upper side of a centrifuge tube (3), such that the centrifuge tubes extend radially fromC said rotary shaft.

2. A microcentrifuge device according to claim 1, wherein saiα nolders (2) comprise a tubular projecting part (6* for receiving the centrifuge tube (3) over part of the tube's5 length, with the centrifuge tube surrounding said tubular, projecting part.

3. A microcentrifuge device according to claim 1 or 2, wherein said holders (2) further comprise at least one cup (16)0 provided with an opening and a passage (15) to the ends of the holders (6) for receiving a finger tip with a drop of blood.

4. A microcentrifuge device according to claim 3, wherein said5 passages (15) comprise a capillary tube.

5. A microcentrifuge device according to claim 3 or 4, wherein the means of attachment (2) further comprises a ciosabie vent hole (17) , which vent hole is designed to be open upon0 receiving said finger tip and passing the blood from the cup (16) to the centrifuge tube (3). β. A microcentrifuge device according to claim 5, wherein the vent hole (17) can be closed by depressing the cup.

7. A microcentrifuge device according to clam 6, wherein said holders (2) further comprise a fluid reservoir (18) for containing a balancing fluid, which fluid reservoir is provided with a passage (10) to the ends of the holders (2) .

8. A microcentrifuge device according to any one of the preceding claims 1-7, wherein the rotor r>ody (1) comprises recesses (7) or protrusions for radially fixing the holders (2) .

9. A microcentrifuge device according to any one of the preceding claims 1-8, wherein the rotor body (1) comprises second recesses (11) or protrusions for radially fixing the centrifuge tube (3) .

10. A microcentrifuge device according to any one of the preceding claims 1-9, wherein the connecting means for connecting the rotor to a rotary drive unit comprise a bore (4) for receiving a drive shaft.

11. A microcentrifuge device according to any one of the preceding claims 1-10, further comprising a rotary drive unit .

12. A microcentrifuge device according to claim 11, wherein said rotary drive unit is a mini motor (120) or a high-speed rotary tool.

13. A microcentrifuge device according to any one of claims 1-12, wherein the holders (2) comprise a sieeve-like body having an external diameter of 13 mm and a length of about 25 πm, which is provided with a fluid reservoir for the balancing fluid and plasma/serum, which fluid reservoir nas a cylindrical sidewall with a diameter of about 11 mm and a substantially conical bottom, and an opening provided with a sealing cap (100) on the siαe remote from the bottom, such that the holders are suitable for being placed in a standard clinical chemical micro analyser.

14. A microcentrifuge device according to claim 13, wherein the holders {2} are provided with a tjbular projecting part (6) at the bottom side of the sleeve-like body (2) for receiving the centrifuge tube (3) over part of the letter's length, with the centrifuge tube (3) surrounding said tubular pro3ecting part.

15. A microcentrifuge device according to claim 13 or 14, wherein the holders (2) comprise an annular body provided with a snap edge (105) for attaching the centrifuge tube.

16. A microcentrifuge device according to claim 13, 14 or 15, wherein the holders (2) are provided with an externally operated valve (109) for opening and closing the conical bottom part of the fluid reservoir.

17. Holders (2) for use m the microcentrifuge device according to any one of the preceding claims.

18. A method for collecting blood, separating blood into plasma/serum and blood cells and stably storing plasma/serum, wherein a balancing fluid is introduced into the fluid reservoir, a drop of blood is deposited in the opening of the holders or the centrifuge tube, a centrifuge tube is attached to the nolders, the holders are placed m the rotor body, and the rotor body is connected to the shaft of the rotary drive unit and the rotor body is rotated at a high speed.