WO2009150632A2

WO2009150632A2 - Apparatus for preparing controlled amounts of liquid for cytometry

Info

Publication number: WO2009150632A2
Application number: PCT/IB2009/052518
Authority: WO
Inventors: Giovanni Marchini
Original assignee: Hta S.R.L.
Priority date: 2008-06-13
Filing date: 2009-06-12
Publication date: 2009-12-17
Also published as: SM200800035A; SM200800035B; WO2009150632A3

Abstract

An apparatus for preparing controlled amounts of liquid for cytometry, which comprises a sampler (2) having: motion imparting means (3) for moving a main tray (4) with one or more tubes thereon, each containing a controlled amount of liquid; a support and moving unit (6) for supporting and moving a syringe (7) that transfers preparation liquids into the tubes (5); a motion imparting mechanism (8) for moving the piston of the syringe (7). The apparatus (1) comprises a centrifuge (10) located beside the sampler (2) and having an access opening (11) for receiving the tubes (5), for selective removal of residues to be discarded from the controlled amounts of liquid. Furthermore, the sampler (2) comprises a motorized gripping mechanism (12) disposed in the support and moving unit (6) and coaxial with the syringe (7), the support and moving unit (6) being movable to allow the tubes (5) to be carried from the main tray (4) to the centrifuge (10) and vice versa.

Description

APPARATUS FOR PREPARING CONTROLLED AMOUNTS OF LIQUID FOR CYTOMETRY

Field of the invention

The present invention finds application in the field of clinical-diagnostic and/or biological assays and relates to an apparatus for simplifying a special type of analysis, i.e. cytometry.

Particularly, the present invention discloses an automatic preparation apparatus, particularly adapted to prepare controlled amounts of liquid to be submitted to cytometric examination.

State of the art

The term "cytometry" is intended to identify a type of analysis aimed at finding and counting microorganisms and/or cells in a sample of liquid, generally organic material to be analyzed.

For instance, in certain environments, disinfection efficiency is evaluated in terms of reduction of the microbial count, which is measured by determining the total number of bacteria, total and fecal conforms. In fact, traditional examinations do not always provide adequate results.

Cytometry is an effective response to many problems concerning microorganism counting in the environment, and allows many assays to be performed in a very short time, with considerably simpler procedures and at lower costs than bacterial culture methods, which always take at least forty- eight hours.

As anticipated above, cytometry is also used in the field of clinical-diagnostic assays and is generally divided into at least two application classes, hematology and immunology.

In any case, it allows qualitative and quantitative cell examination and is based on optical reading of relevant parameters. In greater detail, according to a classical concept, cytometry is a method of analysis that allows cell measurement, i.e. determination of cell counts, as well as certain physical properties, such as the size and internal structural complexity of cells. More recently, cytometry has been defined as a method of analysis that allows qualitative and quantitative evaluation of the molecules expressed on the surface of the cells or inside them, for accurate cell type identification.

Immunological cytometry is used for cell classification in patients affected by immune system-related diseases, i.e. immunodeficiency both in adult and pediatric patients, and autoimmune conditions such as disreactive collagen diseases and immune-based cytopenias.

In the field of hematology, possible applications of cytometry include the diagnosis of acute leukemia and lymphoma, prognosis assessment, minimal residual disease detection in hematopoietic malignancies, and stem cell count and characterization in cases of autologous and allogeneic transplant.

Thus, an important application of cytometry is clearly white blood cell analysis and count.

A basic step for the above assays is the preparation of the sample to be analyzed.

Such step is generally carried out in a sampler and consists in removing foreign bodies from the sample for proper reading.

Considering, for instance, the case of white blood cell counting, the sample preparation process in the sampler includes a first step of red blood cell breakdown, i.e. lysis, and a subsequent step, i.e. a washing step, which allows selective removal of residues.

Previously prepared tubes are obviously used, which contain aliquoted blood (0.1 ml). Then, lysis is carried out by adding a lysing reagent into the tube. The whole is stirred and allowed to rest at ambient temperature for a predetermined time to allow inclubation.

The washing step may be carried out with a variety of methods. The most common method is manual, and includes high speed centrifugation, for agglomeration of the bodies to be analyzed into a solid element, known as pellet, which is formed on the bottom of the tube. Then, the residual liquid, known as supernatant, is eliminated by controlled transfer.

Now, the solution is reconstructed by the addition of a compatible biological liquid (PBS) and vigorous stirring to cause the pellet to break and the solution to be resuspended. For effective separation, such operation is preferably repeated various times.

Other methods include the use of hollow-fiber filtering systems, and a selection method including vortexing of the tube and suction of the central part.

Nevertheless, these methods are never as effective as the above described manual method, which requires, on the other hand, a centrifuge.

For faster sample preparation, automatic samplers are known, which carry out most of the above mentioned steps. These samplers are associated with centrifuges and with the cytometry instrument proper.

Nevertheless, the presence of an operator is always required to check the sampler operation and displace the sample tubes from the sampler to the centrifuge and from the centrifuge to the cytometry instrument. Also, in certain cases, the operator is required to manually carry out certain tasks on the sampler, such as adding lysing liquids, supematants and resuspension liquids using a syringe that has to be washed by the operator in between samples.

This obviously involves a waste of analysis time and human resources and forces operators to supervise and follow the whole process.

Furthermore, since certain operations are required to be carried out by an operator, prior art methods and apparatus have a certain degree of unsafety in terms of process procedure and organization. Namely, the tubes are manually handled during certain steps of the process, which involves the possibility of human errors, i.e. tube mix-up.

Also, the amounts and types of preparation liquids to be poured into the tubes may not be correct, and the syringe may be washed with inadequate accuracy.

Disclosure of the invention

A general object of the present invention is to overcome the above drawbacks by providing an apparatus for preparing controlled amounts of liquid for cytometry.

A particular object within the above main object is to provide a preparation apparatus that can autonomously carry out the whole sample preparation process, without requiring the presence of an operator during the process.

Yet another object of the present invention is to provide a preparation apparatus that almost totally eliminates the possibility of sample preparation errors, i.e. tube mix-up, wrong amounts of preparation liquids or inaccurate washing of the syringes used in the preparation process.

These and other objects, as more clearly explained hereafter, are fulfilled by an apparatus for preparing controlled amounts of liquid for cytometry as defined in the main claim.

Particularly, the preparation apparatus comprises at least one sampler having:

- motion imparting means for moving a main tray with one or more tubes thereon, each containing a controlled amount of liquid;

- at least one support and moving unit for supporting and moving at least one syringe that transfers preparation liquids into said tubes;

- at least one motion imparting mechanism, for moving the piston of said syringe, wherein said apparatus comprises at least one centrifuge located beside said sampler and having an access opening for receiving said tubes, for selective removal of residues to be discarded from the controlled amounts of liquid, and wherein said sampler comprises at least one motorized gripping mechanism disposed in said support and moving unit and coaxial with said syringe, said support and moving unit being movable to allow said tubes to be carried from said main tray to said centrifuge and vice versa.

Therefore, the present preparation apparatus obviously combines automatic control of the syringe that is used to transfer preparation liquids from and to the individual tubes and automatic transport of such tubes from the sampler to the centrifuge and back.

This allows the apparatus to prepare controlled amounts of the liquid to be analyzed in a wholly autonomous manner, without any interaction with an operator, except at the very beginning, for setting up the apparatus and at the end of the preparation process, to withdraw the treated liquids and introduce them into the appropriate cytometry instrument. In one aspect of the invention, in order to safely prevent tube mix-up errors while tubes are handled by the apparatus of the invention, both the sampler and the centrifuge are equipped with absolute position sensors which allow to determine the angular position of the tubes and ensure that they move back to the exact starting position at the end of the preparation process.

The apparatus obviously also has an automatic syringe washing unit, which avoids any operator intervention for syringe washing.

According to another aspect of the invention, electronic means are also provided for controlling and monitoring at least the sampler and the centrifuge. These generally include a microprocessor board which is adapted to execute an IT product for controlling all the parts of the apparatus to carry out the desired liquid preparation steps.

Since different process steps are provided according to the type of cytometric analysis to be carried out, the apparatus comprises at least one user interface having at least one keyboard and one display to allow an operator to select the desired preparation type in the preliminary preparation step.

Brief description of the drawings

Further features and advantages of the invention will be more readily understood from the detailed description of a few preferred non exclusive embodiments of an apparatus for preparing controlled amounts of liquid for cytometry according to the invention, which is shown as a non limiting example with the help of the annexed drawings, in which:

FIG. 1 is a perspective view of a preparation apparatus of the invention; FIG. 2 is a front view of the preparation apparatus of Fig. 1 ;

FIGS. 3 to 5 show detains of the preparation apparatus of Fig. 1. Detailed description of a preferred embodiment

Referring to Fig. 1 , there is shown an apparatus 1 for preparing controlled amounts of liquid for cytometry according to the invention. It is found to first include at least one sampler 2 having motion imparting means 3 for moving a main tray 4 with one or more tubes 5 thereon, each containing a controlled amount of liquid.

As shown in Fig. 2, the sampler 2 also comprises a support and moving unit 6 for supporting and moving a syringe 7 to transfer preparation liquids, such as lysing liquids, supernatant liquids and/or resuspension liquids into the tubes 5.

The embodiment as described herein shall be only intended as an example, without limitation to the invention, and different embodiments, e.g. including multiple samplers, multiple main trays, multiple support units and/or multiple syringes, shall not be excluded from the scope of the invention.

In one aspect of the invention, the support and motion and imparting unit 6 has a motion imparting mechanism 8 for moving the piston of the syringe 7 for autonomous suction and ejection of said liquids, without any action by an operator.

In this respect, the sampler 2 has one or more suitable containers, not shown, for the preparation liquids, which may be withdrawn by the syringe 7, i.e. via nozzles. In another aspect of the invention, empty container sensors may be provided, for checking the availability of each preparation liquid.

Since the preparation liquids are transferred in an automatic manner, the sampler 2 is generally equipped with a washing unit for washing the syringe

7, which may be of the type comprising a pump for causing the washing liquid to flow into and out of the syringe needle 9. The washing operation may be carried out each time that the syringe 7 is filled and emptied to ensure that the following cytometric assays are correct.

A peculiar feature of the invention is that the apparatus comprises at least one centrifuge 10, located beside the sampler 2 and having an access opening 11 for receiving the tubes 5. This centrifuge 10 allows selective removal of residues to be discarded from the controlled amounts of liquid.

Furthermore, it will be appreciated from the detail of Fig. 3 that the sampler 2 comprises at least one motorized gripping mechanism 12 coaxial with the syringe 7 in the support and moving unit 6. The latter is movable and particularly of the rotary type, to transfer the tubes 5 from the main tray 4 to the centrifuge 10 and vice versa.

As anticipated and understandable from the above, the present apparatus 1 allows preparation of controlled amounts of liquid to be analyzed in a wholly autonomous manner, without any interaction with an operator, except, as better explained below, at the very beginning, for setting up the apparatus and at the end of the preparation process, to withdraw the main tray 4 with the treated liquids and introduce them into the appropriate cytometry instrument.

This is ensured both by the automation of this particular embodiment of the sampler 2, in which the syringe 7 is automatically operated to perform all the tasks assigned to the sampler 2, and by the provision of the centrifuge 10 beside the sampler 2 and the presence of rotating support and motion imparting means 6 equipped with the gripping mechanism 12 which allows automatic transfer of the tubes 5 from the main tray 4 to the centrifuge 10 and vice versa.

The automatic operation of the apparatus is ensured by electronic means 15 for controlling and monitoring at least the sampler 2 and the centrifuge 10. The latter generally include a microprocessor board and at least one user interface which includes, in the most general embodiment, at least one keyboard 16 and one display 17. These allow the operator to set up the apparatus 1 at the start of the process, by suitably programming the steps for preparing the controlled amounts of liquid according to the type of analysis to be performed.

Concerning the centrifuge 10, in the detailed view of Fig. 4, it is shown to include at least one rotating tray 18 with a plurality of tube-holding supports 19 adapted to receive the tubes 5 from the sampler 2. Of course, the centrifuge 10 also includes motorized mechanical means 20 for rotating the rotating tray 18.

Since, as mentioned above, the centrifuge 10 is used to submit the controlled amounts of liquid to centrifugal force for the corpuscles to compact into a pellet at the bottom of the tubes 5, the tube-holding supports 19 swing between a first substantially vertical position, allowing placement, removal, emptying and filling of each of the tubes 6, and at least one second substantially horizontal position which is taken as a result of said centrifugal force generated by the rotation of the rotating tray 18.

Particularly, as shown in the detailed view of Fig. 5, each tube-holding support 19 has a pivot pin 20 which is pivotally associated with the rotating tray 18 to allow free rotation of the tubes 5.

Since the rotating tray 18 is rotated at a particularly high speed, the tubes 5 must be disposed in a balanced arrangement. For this reason, the centrifuge 10 is generally equipped with a balancing tube, to be used when there is an odd number of tubes 5.

According to another aspect of the invention, the centrifuge 10 includes means for closing the access opening 11 , which generally consist of a movable wall having sensors for checking the wall position. This ensures safe operation and prevents liquid leakages into the surrounding environment if a tube 5 breaks during centrifugation.

As mentioned above several times, the basic aspect of the apparatus 1 of the invention consists in the automatic preparation of controlled amounts of liquid for cytometric assays. One of the most apparent risks in this case is a mix-up of tubes 5. To prevent such problem, the apparatus 1 comprises one or more absolute position sensors associated with the motion imparting means 3 and one or more absolute rotation angle detectors associated with the rotating tray 18 of the centrifuge 10. With such sensors and detectors, the apparatus 1 determines at each instant of time the exact position of each tube 5 and can move it back to its original position at the end of the process, to ensure correspondence between the tube 5 and the origin of the liquid to be analyzed contained therein.

In operation, an operator prepares the tubes 5 on the main tray 4 by filling each of them with a controlled amount of liquid to be submitted to cytometry. Then, the operator sets up the operation of the apparatus 1 using the interface means. In case of hematological assay, such as for white blood cell count, the operator selects appropriate liquid preparation steps.

Namely, a lysis step is provided, which includes the addition of a lysing agent into the tubes 5, using the syringe 7. The next stirring and incubation steps are also carried out in the main tray 4.

Obviously, since the syringe 7 is in a fixed position relative to the underlying tube 5, the main tray 4 is rotated by the motion imparting means 3 for the required tasks to be accomplished on all the tubes 5.

This is followed by a washing step, which occurs inside the centrifuge 10. For this reason, the rotating support and motion imparting means 6 pick up each tube 5 from the sampler 2, with the help of the gripping mechanism 12, and place it into the centrifuge 10.

Here, the rotating tray 18 is spun at high speed (about 500 g centrifugal acceleration) so that the tubes 5 can take a substantially horizontal position and the bodies to be analyzed, in this example the white blood cells, can agglomerate into a pellet on the bottom of each tube 5.

Then, the supernatant is sucked off, and the solution is reconstructed by the addition of a compatible biological liquid (PBS) and vigorous stirring to cause the pellet to break and the solution to be resuspended. Now, the tubes 5 are placed back into the main tray 4 of the sampler 2 and the preparation process ends. The tubes 5 are thus in their proper positions, and contain the appropriate liquids, ready for cytometry.

It shall be thus appreciated that the present preparation apparatus fulfills the intended objects and particularly allows preparation of controlled amounts of liquid for cytometry in a wholly autonomous manner, without requiring the presence of an operator during the process.

More particularly, the present invention allows to eradicate any possibility of sample preparation errors, i.e. tube mix-up, wrong amounts of preparation liquids or inaccurate washing of the syringes used in the preparation process.

The apparatus for preparing controlled amounts of liquid for citometry of the invention is susceptible of a number of changes and variants within the inventive principle disclosed in the annexed claims. All the details thereof may be replaced by other technically equivalent parts, and the materials may vary depending on different needs, without departure from the scope of the invention. While the preparation apparatus has been described with particular reference to the annexed figures, the numerals referred to in the disclosure and claims are only used for the sake of a better intelligibility of the invention and shall not be intended to limit the claimed scope in any manner.

Claims

1. An apparatus for preparing controlled amounts of liquid for cytometry, which comprises at least one sampler (2) having: - moving means (3) for moving a main tray (4) with one or more tubes

(5) thereon, each containing a controlled amount of liquid; at least one support and moving unit (6) for supporting and moving at least one syringe (7) that transfers preparation liquids into said tubes (5); at least one moving mechanism (8), for moving the piston of said syringe (7), wherein said apparatus (1) comprises at least one centrifuge (10) located beside said sampler (2) and having an access opening (11) for receiving said tubes (5)to selectively remove the residues to be discarded from said controlled amounts of liquid, and wherein said sampler (2) comprises at least one motorized gripping mechanism (12) disposed in said support and moving unit (6) and coaxial with said syringe (7), said support and moving unit (6) being movable to allow said tubes (5) to be carried from said main tray (4) to said centrifuge (10) and vice versa.

2. A preparation apparatus as claimed in claim 1 , characterized in that said moving means (3) include at least one absolute position sensor for determining the position of each of said tubes (5).

3. A preparation apparatus as claimed in claim 1 or 2, characterized in that said sampler (2) is equipped with a washing unit for washing said syringe (7), said washing unit being of the type comprising a pump for causing the washing liquid to flow into and out of the needle (9) of said syringe (7).

4. A preparation apparatus as claimed in any preceding claim, characterized in that said sampler (2) comprises at least one nozzle, through which said preparation liquid contained in an appropriate container is withdrawn.

5. A preparation apparatus as claimed in claim 4, characterized in that it has at least one empty container sensor for checking the availability of said preparation liquid.

6. A preparation apparatus as claimed in any preceding claim, characterized in that it has electronic control and monitoring means (15) for controlling and monitoring at least said sampler (2) and said centrifuge (10).

7. A preparation apparatus as claimed in claim 6, characterized in that said electronic control and monitoring means (15) include at least one microprocessor board.

8. A preparation apparatus as claimed in claim 6 or 7, characterized in that said electronic control and monitoring means (15) include at least one user interface, having at least one keyboard (16) and one display (17).

9. A preparation apparatus as claimed in any preceding claim, characterized in that said centrifuge (10) comprises at least one rotating tray (18) having a plurality of tube holding supports (19) suitable for receiving said tubes (5) with controlled amounts of liquid from said sampler (2) through said support and moving unit (6).

10. A preparation apparatus as claimed in claim 9, characterized in that said tube-holding supports (19) swing between a first substantially vertical position, allowing placement, removal, emptying and filling of each of said tubes (5), and at least one second substantially horizontal position resulting from the centrifugal force generated by the rotation of said rotating tray (18) so that the corpuscles under examination may compact into a pellet on the bottom of said tubes (5).

11. A preparation apparatus as claimed in claim 9 or 10, characterized in that said centrifuge (2) comprises motorized mechanical means for rotating said tray.

12. A preparation apparatus as claimed in claim 11 , characterized in that said motorized mechanical means include an absolute rotation angle detector for checking the position of each of said tubes (5).

13. A preparation apparatus as claimed in any claim from 9 to 12, characterized in that said centrifuge (10) is equipped with at least one balancing tube to be used when there is an odd number of said tubes (5) containing controlled amounts of liquid.

14. A preparation apparatus as claimed in any claim from 9 to 13, characterized in that said centrifuge (10) has means for closing said access opening (11).

15. A preparation apparatus as claimed in claim 14, characterized in that said closing means include at least one movable wall having sensors for checking the position of said wall.

16. A preparation apparatus as claimed in any preceding claim, characterized in that said preparation liquids include lysing liquids, supernatant liquids and/or resuspension liquids.