WO1982004201A1

WO1982004201A1 - Test-tube for the centrifuging and collecting of a liquid

Info

Publication number: WO1982004201A1
Application number: PCT/CH1982/000073
Authority: WO
Inventors: Memorial Inst Battelle
Original assignee: Ringrose Anthony
Priority date: 1981-05-29
Filing date: 1982-05-25
Publication date: 1982-12-09
Also published as: EP0069053A1

Abstract

The test-tube comprises a cylindrical portion (1$i(a)) having a large cross-section and of which the bottom is closed by a piston (2), and is intended to receive a liquid to be centrifuged. This cylindrical portion (1$i(a)) is connected to a cylindrical portion (1$i(b)) of a smaller cross-section by a linking portion (1$i(d)). When the liquid is centrifuged, an actuating rod (3) pushes the piston (2) which brings the separate constituants into the portion (1$i(b)) thereby increasing the height of the different layers proportionally to the square of the ratio between the radii between the cross-sections of the portions (1$i(a)) and (1$i(b)). The constituants may be collected as the piston (2) moves at the outlet of the portion (1$i(b)).

Description

TEST FOR CENTRIFUGATION AND COLLECTION OF A LIQUID

The present invention relates to a test tube for separation by centrifugation of a physiological liquid and collection of at least one of the constituents of given density.

Centrifugation is a well known method for the separation and purification of cells, viruses and cellular particles. Generally this separation takes place in a centrifugation medium capable of generating a continuous or discontinuous density gradient when it is subjected to centrifugal force. Such substances are manufactured in particular by Pharmacia Fine Chemicals AB in Uppsala in Sweden and sold under the trade names "Per coll" and "Ficoll-Paque".

In practice, the centrifugation tenps necessary for the separation of the particles, in a medium of given viscosity, is a function of the height of the column of liquid in the analysis tube. It is common for the particles to be separated and extracted to form, after centrifugation, a very thin layer, of the order of a millimeter or less. It follows that the extraction of these particles is a delicate operation which requires great meticulousness and skill. This operation is carried out manually and its automation obviously poses problems in terms of the sensitivity of the means necessary to detect such a thin layer, as well as that of the means capable of exclusively but totally sucking up these particles.

As we have seen above, the reduction in the diameter of the test piece would lead to a proportional lengthening of the centrifugation tenps necessary to obtain the separation. However, an increase in the thickness of the particle layer should be very sensitive to allow the separation and automatic removal of such particles using simple detection and sampling means.

In addition to the lengthening of the centrifugation tenps, the reduction in the diameter of the test tube gives a less good separation which results in contamination of the red blood cells by lymphocytes as well as in an increase in the number of dead cells.

It has already been proposed in US Pat. No. 3,513,976 to form a test piece with an intermediate part of appreciably smaller section, the volume and position of which relative to the other parts of the test piece is chosen to correspond roughly to the portion of the volume of leukocytes contained in a sample corresponding to the volume of blood which the test tube can contain as well as to the position of these leukocytes after centrifugation of the test tube, the longitudinal axis of which is oriented radially with respect to the axis of rotation . It is also planned to finely adjust the level of the leukocytes by adding mercury to the bottom of the test tube. The centrifuge in such a test tube requires a transfer of the components of different densities through the throttle of the intermediate part. If this constriction is significant, separation by centrifugation may prove to be practically impractical or, at the very least, require a very considerable lengthening of the centrifugation period.

It was moreover, described by Harry Cutts on page 132 of his book "Cell separation methods in Bsmatology" published by Académie Press New York - London an installation including a test tube in which the blood is centrifuged. This test piece is placed inside a support, the bottom of which is provided with a neoprene seal. A hollow needle is inserted through the bottom of the support, the neoprene seal and the wall of the test piece. This needle is connected to a brcπobenzene tank itself connected to a water tank by a peristaltic pump. By pumping water into the brcαobenzene reservoir, it is expelled into the test tube through the hollow needle. A conical plug was fixed to the sonnet of the test tube, the center of which is crossed by a tube of small diameter, which serves to recover the various liquid phases, as they are brought out of this pe tube tit diameter by brαnobenzene.

It is obvious that such a solution does not make it possible to envisage the automatic treatment of series of samples, as this must be done in bio-medical laboratories in hospitals and blood transfusion centers, as the necessary operations are delicate . In addition, such a solution requires at least the emptying and periodic recharging of the brcmobenzene reservoir and the change of the hollow needle intended to pass through the bottom of the test piece, insofar as the latter comes into contact with the lcn sample to analyze. It should also be noted that the conical plug, added in the opening of the test piece, has sharp edges which are liable to create eddies during the flow of the liquid to be analyzed and therefore to mix the different phases separated by the centrifugation. The object of the present invention is to remedy at least in part the above-mentioned drawbacks so as to facilitate the removal of the particles separated by centrifugation without increasing the time necessary for the separation or affecting the quality of this separation, by a solution which is particularly suitable. well at the tαnatisation of this sample.

To this end, the present invention relates to a test tube for separation by centrifugation of a physiological liquid and for the collection of at least one of the constituents of given density, having two parts of different circular sections connected by a connecting part whose section progressively passes from one to the other of said sections. This test piece is characterized in that it is open at its two ends, the sections of these openings corresponding to said respective sections, the opening having the largest of these sections forming the end of a centrifugation cylinder which receives a piston tightly fitted and the other of these openings, having the smallest section, constituting the opening through which said separate constituents in said cylinder can be collected, following the movement of said piston in the direction of this opening.

The advantage of the test tube which is the subject of the invention essentially resides in itself autonomy, since it comprises the elements which allow the constituents to be separated by centrifugation and those which then allow these constituents to be brought to the level of the reduced section opening with a view to collecting them, this reduced section opening considerably increasing the thickness of the different layers and reducing their interfaces in function of the square of the ratio of the rays between the cylindrical body of the test piece and the reduced section opening where these constituents are collected. It therefore lends itself well to an automatic installation which would include a centrifugation station and a collection station for the constituents after which the test piece can be discarded, given the very low cost that it represents.

The single figure of the attached drawing illustrates, schematically and by way of example, an embodiment of a device for implementing the method which is the subject of the invention. This device comprises a transparent test piece 1 having a portion 1a of diameter greater than a portion 1b, connected by a portion 1d in which the section varies gradually. The bottom of the part la is constituted by a piston 2 retained by a flange lc. For the sampling operation, the piston 2 is arranged coaxially with a rack drive rod 3 engaged with a pinion 4 secured to the output shaft of an electric motor 5 connected to a control station 6. This control station is connected to a photoelectric cell 7 placed opposite a light source 8, these two elements being on either side of part 1b of the test piece 1 near its outlet and used to detect the level of the various products resulting from the separation of the physiological liquid introduced into the test tube. Two pipette tips 9 and 10 are introduced into the open end of the test piece 1 and are connected to the control station 6. One of these pipettes is intended to take the parts of liquid situated above the layer of particles to be sampled while the other pipette will collect these particles.

Since the constituents of the centrifuged liquid are intended to be collected as they flow through the exit opening of the test piece, the length of the part lb is immaterial and the exit opening can therefore lie directly at the level where part ld has its weakest section corn. Furthermore, the rim 1 intended to retain the piston 2 during centrifugation is not essential insofar as, during centrifugation, the test piece can be placed in a cylindrical housing oriented radially to the centrifugation axis and the bottom of which, facing outwards, is used to hold the piston 2.

For example, the device described can be used to separate and collect lymphocytes from a patient's blood sample in order to carry out a macrophage electrophoretic mobility test based on the detection of sensitivity. lymphocytes vis-à-vis an antigen from a tumor.

The ratio between the thickness or the height of the layer of lymphocytes and its section is an inverse function of the fourth power of the diameter of the tube. Indeed, if h = height of the layer, s = cross section of this layer, v = volume of the layer, ra = h / s, c = constant, f = function.

v = h x s = c

ra

In practice, if, for example, the diameter of the tube decreases from 20 mm to 5 mm (a factor of 4), the height of the layer of lymphocytes increases by a factor of 16, that is to say of the order of 2 mm. 32 mm and the h / s ratio increases by a factor of 256 or goes from 0.006 to 1.54.

To carry out this separation and this removal of lymphocytes according to the invention, the procedure is first of all carried out according to the usual technique, namely, the patient's blood sample is diluted, for example, with a solution of Bank salts (Hank's BSS), 5 ml of Ficoll-Paque (registered trademark), which is a so- aqueous luticn with a density of 1.077 + 0.001 g / cm ³ containing 5.7 g of Ficoll 400 which is a synthetic polymer of high molecular weight (M _w 400,000) of sucrose and epychlorohydrin easily soluble in water and 9 g of an anti-bacterial agent, sodium diatrizoate, per 100 ml, in a test tube 1 of 20 ml. 10 ml of diluted blood are then added to the test tube 1. This test tube is centrifuged with its inclined or horizontal axis and the piston 2 directed outwards at 400 g for 30 to 40 min.

Following separation by centrifugation, four layers formed in the test piece. the upper layer essentially contains the plasma, then comes the thin layer containing lymphocytes, then the separation medium formed in this example by Ficoll-Paque and finally the red blood cells and other cellular particles. Then, this test piece 1 is placed with respect to the rack drive rod 3.

By pushing the piston 2 upwards, the rack drive rod 3 brings the sormmet of the plasma layer into the constricted part 1b of the test tube 1. When the top of this layer comes towards the detector photoelectric 7, the control station 6 starts one of the pipettes 9 and 10 which is intended to aspirate the plasma as it rises in the part lb of the test piece. As soon as the lymphocyte plasma interface arrives at the photoelectric detector 7, the control station 6 stops the pipette aspirating the plasma and starts the other pipette which then sucks the layer of lymphocytes until the detector photoelectric 7 detects the arrival of the bottom of this layer, marries which the pipette aspirating the lynrohocytes is stopped as well as the motor 5.

The lymphocytes are then washed according to the conventional process which consists in again adding a macrophage electrophoretic medium, in centrifuging at 250 g for 10 minutes, in eliminating the supernatant liquid and in repeating this operation as many times as necessary, for example three time.

Then, the lyirphocytes are counted, incubated with the antigen and the above-mentioned macrophage electrophoretic mobility test is carried out.

Comparative tests were carried out with the blood of a donation male neur using the manual method of sampling with conventional test tube and moreover, also using the manual method of sampling but with the test object of the present invention. A calibration of the components of the blood to be tested was carried out by various microspace counts and by establishing the average of these counts. The average white blood cell count based on six separate counts gave 6650 cells / mnr or 6.65 x 10 ⁶ / ml. The average rate of lyπphocytes was then determined using three counts, which gave 52% of lyizphocytes, ie 6.65 x 52% x 10 ⁶ / ml = 3.46 x 10 ⁶ .

Then, the collection of the lyirphocytes was carried out using the test tube which is the subject of the invention, using a 10 ml sample of blood diluted to 50% in a solution of Hank's salts (Hank's BSS) and treated. prior to heparin centers coagulation. These 10 ml were placed in a test tube whose body is 20.3 mm in diameter and the outlet opening of the lb portion 8 mm by adding 5 ml of Ficoll-Paque (registered trademark). This specimen was placed in a cylindrical support tooth the bottom has an opening of diameter smaller than that of the piston 2 and this sample was centrifuged at 400 g for 35 minutes.

Then, the test piece was placed on a device fitted with a rack drive rod 3, but the collection was carried out manually at the exit opening of the test piece. It has been found that the displacement of the lymphocyte layer is obtained with remarkable stability of its interfaces.

The results obtained are 3.07 x 10 ⁶ / ml of total white blood cells of which 92% of lymphocytes is a yield of 3.07 x 92 / 3.46 = 81.6% compared to the reference count, with less than 2 % of dead cells. The same test carried out with a conventional test tube gave 2.4 × 10 ⁶ / ml of total white blood cells of which 82% of lymphocytes is a yield of 2.4 × 82 / 3.46 = 56.8% and approximately 1% of dead cells.

As an indication, the manufacturer of Ficoll-Paque, Pharmacia Fine Chemicals AB laboratories, indicates a normal yield of 50% + 15%.

It is noted that the test object of the invention makes it possible to obtain hold a very noticeable improvement in these performances.

Of course, the invention is not limited to the separation of lymphocytes but can be used each time it is desired to separate or purify viruses, bacteria, cell particles in a physiological liquid.

Claims

RESELL I CAT I ON

Test tube for separation by centrifugation of a physiological liquid and collection of at least one of the constituents of given density, having two parts of different circular sections connected by a connecting part whose section passes progressively from one to the other of said sections, characterized in that it is open at its two ends, the sections of these openings corresponding to said respective sections, the opening having the largest of these sections forming the end of a centrifuge cylinder which receives a piston tightly adjusted and the other of these openings having the smallest section, constituting the opening through which said separate constituents in said cylinder can be collected, following the movement of said piston in the direction of this opening.