WO1996037301A1

WO1996037301A1 - Improvements in filtration

Info

Publication number: WO1996037301A1
Application number: PCT/GB1996/001140
Authority: WO
Inventors: Gary James Clements; Richard Michael Ireland
Original assignee: Haemocell Plc
Priority date: 1995-05-23
Filing date: 1996-05-14
Publication date: 1996-11-28
Also published as: GB9510395D0

Abstract

A method and device for concentrating cellular material for subsequent analysis involves a tubular body containing a porous disc covered with a filter membrane. A dispersion of the cellular material is passed through the body so that the cellular material is collected on the membrane. The disc with the membrane attached is subsequently releasable so that it falls out of the tubular body into a collection vessel for analysis of the cellular material.

Description

IMPROVEMENTS IN FILTRATION

In many industries it is necessary to check a product, such as beer, shampoo or detergent for microbiological contamination. Traditional microbiological quality assurance techniques can take anything between two to seven days to produce results. During this time, manufacturers are faced with the choice of storing their product for positive release or releasing the product without test results, running the risk of costly and damaging recall. The length of time involved in such testing is primarily because the microbial material may be present in very small quantities and cultures have to be grown to obtain the minimum quantity necessary for conventional analytical techniques, such as the use of ATP (Adenosine Triphosphate) bioluminescence, a sensitive enzyme based system, which is indicative of live material, by optical radiation. Typically, in the preparation of a sample for assay, a supposed dispersion of cellular material in, say, 500 ml of a liquid medium is passed through a filter membrane so that any cellular material present is retained by the membrane. The membrane is then placed in a Petri dish, nutrient is added, and the dish is incubated for a period of time to produce a culture of the material. A lysing solution is added and thereafter a sample of say 0.2 ml of the lysate is extracted from the dish by means of a pipette and subjected to analysis, for example by being deposited into a reagent in a luminometer cuvette. This technique suffers the disadvantages that there are many stages during which the material is exposed to the atmosphere and may be contaminated by exogenous ATP, and in that the final sample used for analysis is only an aliquot of the available material.

The present invention provides a filtration technique for use in the preparation of a sample of concentrated cellular material in conjunction with rapid micro¬ biological quality assurance, or with non-microbiological applications, such as in mammalian or insect cell tissue culture, or in the analysis of certain clinical samples.

According to a first aspect of the present invention, a filtration device comprises a tubular body having at one end an inlet which is arranged to be sealed to the outlet of a source of a dispersion of the cellular material which can be put under pressure, at its other end an outlet, and between the inlet and the outlet a seating; a disc which is perforated between its opposed major faces and which is provided over one of its faces with a filter membrane, the disc being held on the seating with the membrane both facing the one end and closing the passage through the body between the inlet and the outlet so as, in use, to filter out and retain thereon cellular material in a dispersion flowing through the tubular body from the inlet; and means for releasing the disc whereby in use, the disc with the retained cellular material on the membrane is free to pass through the outlet into a collection vessel for subsequent analysis. According to a further aspect of the invention, a method of evaluating a cellular material comprises forcing a dispersion of a cellular material from a source through a tubular body the passage through which is closed by a filter membrane carried by a supporting disc which is held in the body whereby the cellular material is retained on the membrane; and releasing the disc so that the disc and membrane pass from the body into a collection vessel, and subsequently carrying out an appropriate evaluation.

The source of the dispersion of the cellular material to be forced through the tubular body of the new filtration device may be a conventional syringe. The syringe outlet may be conventionally tapered and either held, or secured as an interference fit, in the inlet of the tubular body, but is preferably connectable to it positively by means, e.g., of a Luer lock. The use of a syringe is particularly appropriate if the filtration is to follow primary filtration in a system in which the recovered concentrated cellular material is collected in a vessel formed by a syringe. The same syringe containing the same concentration can then be removed from the primary filtration system and directly fitted to the tubular body of the secondary filtration device with minimum atmospheric exposure.

The vessel which is used to collect the disc, filter membrane and retained material, following the secondary filtration in the new device, may be a reaction vessel for analytical equipment, such as an impedance device; or an assembly for microscopic analysis. However the vessel will be a tube, such as a luminometer cuvette, in the typical case in which the evaluation is to be carried out by bioluminescence or epifluorescence. In that case, the shape and dimensions of the disc, and the internal dimensions of the cuvette or other tube are preferably such that the disc cannot turn over as it falls into the tube so that the membrane-covered surface of the disc remains uppermost, i.e. facing the open end of the tube. Most simply the peripheral surface of the disc is cylindrical with a diameter sufficiently smaller than the internal diameter of the tube to provide substantially only the minimum clearance necessary to allow the disc to fall freely into the tube. This minimum clearance will accommodate only a minimum amount of liquid reagent subsequently to be added to the tube, so that a highly concentrated dispersion of the cellular material may be formed in the tube immediately above the membrane-covered surface of the disc. A typical luminometer cuvette has an internal diameter of the order of 9 mm whereas a conventional syringe may have a typical internal volume of up to 20 ml. Consequently, the concentration of the cellular material in the tube above the disc may be a factor of 20 or even 200 times greater than that in the syringe prior to the filtration. Furthermore, if the sample thus prepared is to be evaluated by means of a luminometer, the great majority of the dispersion formed in the tube will be above the disc which will consequently provide little interference to optical assay through the wall of the tube.

The disc, assembled with its membrane, may be secured within the tubular body by a variety of means, provided that it may be released by remote manipulation into the collection vessel. In one construction, claws extend radially inwardly through apertures in the wall of the tubular body to urge the disc assembly axially onto the seating. The claws may be manipulated externally of the tubular body to cause them to move radially outwardly to release the disc.

The disc assembly must close the passageway through the tubular body and the necessary seal may be provided by direct engagement of the peripheral portion of the membrane with a seating in the form of an annular shoulder in the body.

The tubular body may extend a sufficient distance downstream of the seating for the disc assembly to provide a guard which inhibits contact, and potential contamination, of the disc assembly. This guard, will necessarily have an internal cross-section large enough for the disc to fall through it into the collection vessel. However, its internal cross-sectional dimension may exceed the peripheral dimension of the disc sufficiently to accommodate the rim at the open end of a cuvette or other tubular connection vessel. In this case the tubular collection vessel will be inserted into the outlet end of the tubular body up to a position adjacent to the disc, before the disc is released, whereby the disc passes directly into the collection vessel.

The advantages of the new filtration technique are that all the available cellular material is collected and is subsequently available for analysis as a very highly concentrated dispersion in the minimum quantity of liquid medium. The collection and concentration of the cellular material is carried out with the minimum opportunity for external contamination of the sample from the environment. The technique therefore enhances the detection of low level contaminating micro organisms in the liquid product, with minimum danger of the introduction of agents such as ATP, which may interfere with the relevant analytical technique. An example of a filtration system utilising the present invention is illustrated in the accompanying drawings in which:-

Figure 1 is an exploded diagrammatic perspective view; Figure 2 is an enlarged view of part of Figure 1; and, Figure 3 is an axial cross-section through part of Figure 2 in greater detail than Figure 2.

As shown, the heart of the system is a device comprising a tubular body 4, having an inlet end 5 provided with a Luer-lock connector 6 and an outlet end 7.

Approximately the downstream third of the tubular body 4 is stepped radially outwardly and forms a guard section 8. Internally, where the body 4 is stepped outwardly, there is formed an annular seating 9 facing in the downstream direction. The guard section 8 is formed with two diametrically opposed apertures 10. The body 4 is a plastics moulding and is formed integrally with a pair of resilient claws 11 which extend radially inwardly through respective ones of the apertures 10, are connected to the tubular portion of the body 4 by respective bridges 12, and continue into respective fingerpieces 13 which overlie the outside of the body 4 with a clearance. The arrangement is such that the claws 11 can be moved radially outwardly through the apertures 10 by squeezing the fingerpieces 13 together.

Located on the seating 9 is a disc assembly D which, as shown in Figure 3 , consists of a skirt 14, a web 15 perforated by a number of holes 16, and circular wafer of a filter membrane 17 which is secured within a shallow recessed end of the disc. One or more circular wafers 22 of loosely woven or other perforate compressible material may be interposed between the membrane wafer 17 and the base of the recess. In the initial position in which the device is supplied, the claws 11 overlie the skirt 14 and hold the disc assembly on the seating 9 with the peripheral portion of membrane 17 in sealing engagement with the seating. Conformity of the membrane to the seating is improved by the compressibility of the wafer(s) of compressible material. Over the larger amount of its axial length, the radially outer peripheral surface 18 of the skirt 14 is cylindrical. In use, a syringe 19 having a neck 20 with a complementary Luer lock connector, is coupled to the inlet end of the body 4. The syringe contains, for example, a primary filtered or other dispersion of cellular material in a liquid medium and when the syringe plunger is depressed, this is forced into the tubular body 4, and filtered by the membrane 17, the filtrate passing through the holes 16 and out through the outlet end 7 to waste. The cellular material will simultaneously be retained by the membrane. In order to provide an even spread over the membrane, the operation will normally be carried out with the axis of the tubular body 4 substantially vertical with its inlet 5 uppermost. This provides an intensive concentration of the cellular material on the membrane 17. In order to prepare this for evaluation, the open end of a tubular collection vessel, such as a luminometer cuvette 21, is inserted upwardly through the outlet end 7 of the body 4, within the guard 8 until it is adjacent to the claws 11. This is possible because, as shown in Figure 3, the guard 8 is stepped internally between a smaller diameter part 8A, which closely surrounds the disc D, and a larger diameter part 8B, which will accept the cuvette. The finger pieces 13 are then squeezed towards one another, causing the claws 11 to be displaced radially outwardly, and releasing the disc assembly so that it falls into the cuvette 21, the cylindrical peripheral surface 18 of the disc skirt maintaining the disc at the same orientation within the cuvette, with the membrane facing upwardly. An intensive concentrated dispersion of the cellular material for the appropriate analysis, such as bioluminescence, is then created by introducing into the cuvette just sufficient reagent to immerse the cellular material on the membrane, and to provide a sufficient depth of the dispersion within the cuvette above the membrane-covered surface of the disc. The disc is a close fit within the cuvette so that there is very little wasted dispersion in the annular clearance between the periphery of the disc and the wall of the cuvette. A typical aliquot of reagent for bioluminescence would comprise 100 μl of lysing agent followed by 100 μl of enzymatic agent.

The filter membrane 17 will be selected to have the appropriate pore size for the cellular material to be collected but may typically be a track-etched polycarbonate membrane, with a pore size in the order of 0.2 to 5 micron.

It will be appreciated that the system as illustrated provides the minimum of opportunity for the collected cellular material to be exposed to a contaminant, such as human-carried or environmental ATP. Thus the device consisting of a tubular body 4 with the disc assembly in place will be provided in a sterile and ATP-free packet. Once the syringe 19 has been connected to the tubular body, the cellular material will be isolated from the atmosphere by the membrane 17 and will, after collection of the cellular material, be released into the cuvette 21 without any direct manual contact with the disc assembly.

Claims

1. A filtration device comprising a tubular body (4) having at one end an inlet (5) which is arranged to be sealed to the outlet (20) of a source (19) of a dispersion of cellular material which can be put under pressure, at its other end an outlet (7) , and between the inlet (5) and the outlet (7) a seating (9) ; a disc (D) which is perforated between its opposed major faces and which is provided over one of its faces with a filter membrane (17) , the disc (D) being arranged to be held on the seating (9) with the membrane both facing the one end and closing the passage through the body between the inlet (5) and the outlet (7) so as, in use, to filter out and retain thereon cellular material in a dispersion flowing through the tubular body from the inlet (5) ; and means for releasing the disc (D) whereby in use, the disc with the retained cellular material on the membrane is free to pass through the outlet (7) into a collection vessel (21) for subsequent analysis.

2. A filtration device according to claim 1, wherein the inlet (5) is arranged either to hold, or to secure as an interference fit, a tapered outlet (20) of a source (19) .

3. A filtration device according to claim 1 or claim 2, wherein the inlet (5) is arranged to be connected to the outlet (20) of a source (19) with a Luer lock (6) .

4. A filtration device according to any one of the preceding claims, in conjunction with a syringe as the source.

5. A filtration device according to any one of the preceding claims, wherein the outlet (7) of the tubular body (4) is arranged to accommodate an open end of a collection vessel.

6. A filtration device according to claim 5, in conjunction with a collection vessel.

7. A filtration device according to claim 6, wherein the collection vessel is a tube (21) .

8. A filtration device according to claim 6, wherein the collection vessel is a luminometer cuvette (21) .

9. A filtration device according to any one of claims 6 to 8, wherein the shape and dimensions of the disc (D) , and the internal dimensions of the collection vessel (21) are such that the disc (D) cannot turn over as it passes into the collection vessel (21) so that the membrane-covered surface of the disc remains uppermost, i.e. facing an open end of the collection vessel.

10. A filtration device according to claim 9, wherein the peripheral surface of the disc (D) is cylindrical with a diameter sufficiently smaller than an internal diameter of the collection vessel (21) to provide substantially only the minimum clearance necessary to allow the disc (D) to pass freely into the collection vessel (21) .

11. A filtration device according to any one of the preceding claims, wherein the tubular body (4) extends downstream of the seating (9) for the disc assembly (D) to provide a guard (8) which inhibits contact, and potential contamination, of the disc assembly (D) .

12. A filtration device according to claim 11 when dependent upon any one of claims 5 to 10, wherein the internal cross-sectional dimension of the guard (8) exceeds the peripheral dimension of the disc (D) to accommodate an open end of a collection vessel (21) .

13. A filtration device according to any one of the preceding claims, wherein the disc (D) , assembled with its membrane, when secured within the tubular body (4) , is releasable by remote manipulation into the collection vessel (21) .

14. A filtration device according to claim 13, wherein claws (11) extend radially inwardly through apertures in the wall of the tubular body (4) to urge the disc assembly (D) axially onto the seating (9) , the claws (11) being manipulatable externally of the tubular body (4) to cause them to move radially outwardly to release the disc (D) .

15. A filtration device according to any one of the preceding claims, wherein the disc assembly (D) closes the passageway through the tubular body (4) and the necessary seal is provided by engagement of the peripheral portion of the membrane with the seating (9) in the form of an annular shoulder in the body.

16. A method of evaluating a cellular material, the method comprising forcing a dispersion of a cellular material from a source (19) through a tubular body (4) the passage through which is closed by a filter membrane (17) carried by a supporting disc (D) which is held in the body whereby the cellular material is retained on the membrane; and releasing the disc (D) so that the disc (D) and membrane (17) pass from the body (4) into a collection vessel (21) , and subsequently carrying out an appropriate evaluation.

17. A method according to claim 16, wherein the method is carried out with the axis of the tubular body (4) substantially vertical with an inlet (5) of the tubular body (5) uppermost.