GB2202469A

GB2202469A - A centrifuging chamber arrangement for cytological examination of cell suspensions

Info

Publication number: GB2202469A
Application number: GB08805230A
Authority: GB
Inventors: Gunter Eberle
Original assignee: Andreas Hettich GmbH and Co KG
Current assignee: Andreas Hettich GmbH and Co KG
Priority date: 1987-03-05
Filing date: 1988-03-04
Publication date: 1988-09-28
Anticipated expiration: 2008-03-04
Also published as: DE3706998A1; FR2611903A1; GB2202469B; FR2611903B1; DE3706998C2; GB8805230D0

Abstract

A centrifuging chamber arrangement comprises a test tube (1) into which is inserted a sample chamber which divides the test tube into upper and lower reception spaces (34, 35). The sample chamber includes an upper part (4, 40) and a base part (2, 42) which are fitted together with an object carrier (3) trapped therebetween. Means are provided which normally seals the upper space (34) from egress of liquid, but which allows flow to the lower space (35) to occur at high rotational speeds. In one embodiment (Fig. 1), an annular surface (41) at the lower end of the upper part (4) normally sealingly engages the upper surface of the object carrier (3). When the cell suspension in the upper space (34) is subjected to high centrifugal forces during centrifuging, the cell suspension liquid is forced out radially between the annular surface (41) and the object carrier (3), through openings defined by slots (16) in the surrounding side wall of the base part (2), and via slots (17) in the underside of the base part into the lower space (35). In another embodiment (Fig. 9) sealing is by a resilient collar (45) which deforms outwards at high speed rotation. Also shown is a tinting insert (5) which may be filled with an appropriate tinting etc. fluid. <IMAGE>

Description

A CENTRIFUGING CHAMBER APPANGEMENT FOR CYTOLOGICAL EXAMINATION OF CELL SUSPENSIONS The invention relates to a centrifuging chamber arrangement for cytological examination of cell suspensions.

More particularly, the invention relates to a centrifuging chamber arrangement of the type comprising a sample chamber for receding a cell suspension, and having a base surface on which an object carrier is arranged. Openings are prcvided in the sample chamber adjacent the object carrier, through which the cell suspension, or the liquid in which the cells are suspended, is drawn off intc a reception chamber or space during a centrifuging operation.

Centrifuging chamber arrangements of this type have already been proposed by the present applicant.

It is an object of such arrange:t.=nts that a portion of the sample suspension (namely -ne proximal part) from the centrifuging chamber is introduced into an adjacent chamber during the centrifing operation, without stopping the centrifuge. The introduction of the sample is effected by means c compressed air, which increases the complexity of the arrangement.

It is an object of the invention to provide a centrifuging chamber arrangement of the type referred to, in which transfer of at least a portion of a sample fluid may be accomplisheA successfully during a centrifuging operation, with a ess complex structure.

According to the invention, the centrifuging chamber arrangement comprises a test or reaction tube or other tubular receptacle into which the sample chamber is inserted, thereby to form upper and lower reception spaces in the test tube, the sample chamber comprising a base element, the base surface of which has the object carrier arranged on it or is formed by the object carrier, and cross-section or other means for blocking the flow of the cell suspension or the fluid thereof towards the lower reception space are situated adjacent the surface of the object carrier, which allow the fluid to flow from the upper reception space to the lower reception space via the object carrier, only at higher rotational speeds of the centrifuge.

The test tube is divided into an upper reception space and a lower reception space by insertion of the sample chamber. To this end, the sample chamber is situated in the upper reception space and is filled with the cell suspension which is to be examined. The centrifuging chamber assembled in this manner is then inserted into a centrifuge and centrifuged. Under the action of centrifugal force, the heavy particles of the sample suspension travel towards the object carrier which rests on or forms the base surface of the sample chamber. This causes sedimentation of the cells which are to be examined, on the object carrier.

The sections or other means which block the flow of the fluid of the sample suspension are situated between the upper and lower reception spaces of the test tube. These flow-blocking means preferably form a hermetic seal up to a centrifugal force of, for example, 50g (g=acceleration due to gravity), so that no fluid may escape from the upper reception space into the lower -reception space in this acceleration range of the centrifuge, whilst the cells of the cell suspension are precipitated on to the object carrier.

The hermeticity of the flow-blocking section is thus a direct function of the centrifugal force. To this end, it is preferred that the flow-blocking section should comprise a simple annular base flange of the upper part of the sample chamber, which rests on the surface of the object carrier, with the full area of the flange, without interrution, contacting the object carrier. What is important is that the contact pressure of the annular 'oase flange is a direct function of the centrifugal force, i.e. the base flange is pressed onto the surface of the object carrier with greater or lesser force as a function of the centrifugal force.In the case Or low levels of centrifugal force, within the range of, for example 50g, the fluid pressure of the cell suspension in the upper reception space is insufficit to uncovee an appropriate flow path between the annular base flange of the upper part of the sample chamber and the associated surface of the object carrier.

However, the fluid pressure of the cell suspension in the upper reception space is increased so greatly by raising the centrifugal force, for example to 1500g, that the fluid seeps generally radially outwards under the annular base flange tithe edge of the object carrier, from where it flows into the lower reception space of the test tube via fluid passages (for example slots and grooves).

At the end of the centrifuging operation, most of the sample fluid is consequently withdrawn from the upper reception space and is transferred to the lower reception space, whilst the object carrier is coated with the cells which are to be examined.

The object carrier may then be immediately removed and moved onwards to another examination stage. Alternatively, the centrifuge may be stopped following this sedimentation operation, and a colouring or staining container or insert containing an appropriate colouring or staining fluid may be fitted into the previously drained upper reception space. The centrifuge is then switched on again, and the colouring or staining fluid runs out of the colouring container onto object carrier, and tints the cell structure precipitated thereon.

The present invention embraces two different forms of centrifuging chamber arrangement, namely an arrangement for a so-called dry centrifuging operat ion", and for a "wet centrifuging operation".

In a "dry centrifuging operation", the fluid component of the cell suspension situated in the upper reception space and which is to be examined, flows -entirely, and without leaving fluid residues, into the lower reception space in the test tube, thereby obtaining a dry object carrier after completion of the centrifuging operation, on to which the cell structure to be examined is precipitated.

In a "wet centrifuging operation", it is necessary to ensure that a specific layer of fluid of, for example a few tenths of a millimetre, remains on the object carrier, and cannot flow out. Thus, a damp coating of the cell structure which is to be examined, is obtained on the object carrier.

The application of the colouring, staining or tinting inserts previously described is possible in both the dry and wet centrifuging operations.

Furthermore, in addition to or as an alternative to the tinting inserts previously described, fixing inserts or the like may be utilised in ar. analogous manner.

For an uncomplicated installation of the sample chamber, which in essence comprises an upper part and a lower or base part, the upper part may comprise a cup- or beaker-like hollow cylindrical part, which is provided at its upper extremity with an annular flange of enlarged diameter intended to rest on t.e top edge of the test tube. The lower portion of the upper part is formed as a collar of reduced-diameter, so that the base part may be pushed over the reduce & diameter collar from below, an encircling surface formed by a sleeve of the base part being pushed over te reduceddiameter collar of the upper part.

So that this push-on action may occur against frictional resistance and with a close fit, the sleeve of the base part is provided with axially extending slots which extend through the wall of the sleeve, so that the sleeve can undergo a degree of olametrical variation when pushed onto the lower portico, i.e. the collar, of the upper part of the chamber.

To secure a dry object carrier sface, the surface of the object carrier should Le situated above, for example, 0.2 mm. above, an associated edge of the upper portion of the base part, so that the fluid may flow into the lower reception space without obstruction via slots and grooves situated in the base part, under centrifugal forces exceeding, for example, 1500g.

In the case of a wet centrifuging operation, the bases of the slots are located at a level of 1.2 mm. above the upper surface of the base part, on which the object carrier is situated. Depending on an acceptable thickness of the inserted object carrier, this establishes an outflow dam or wier of a height of 0.1 to 0.3 mm. above the upper surface of the object carrier. This assures that the object carrier is immersed in or forms the base of a sump or reservoir in the case of a wet centrifuging operation.

It is also important that radial slots, for example of a depth of 0.1 mm. and a width of 0.2 mm., be distributed peripherally on the underside of the base part, to prevent too abrupt a sealing off of the sample suspension from the upper reception space in the test tube in a direction towards the lower reception space.

In another embodiment, the flow-blocking section or means consists of a resilient, for example natural or synthetic rubber, annular lip which is peripherally continuous and encircles the collar forming the lower portion of the upper part of the sample chamber. This lip seals off apertures formed by radial slots or openings in the latter collar, for example, situated in the internal periphery or lower edge thereof, which slots or openings form a passage for fluid to flow from the upper to the lower reception space of the sample chamber.

The resilient annular lip thus acts as a flow blocking element, because the cell suspension i the upper reception space and in the slots in the lower collar, up to a certain fluid pressure, presses against the annular lip in radially outward direction but is prevented from flowing out through the sloths by the annular lip. However, when the cell suspension is subjected to high centrifugal forces and is ther~~ore under a higher pressure, this lip is deformed radially outwards by the pressure of the suspension, and tnus opens the apertures formed by the radial slots in a direction towards the lower reception space of the sample chamber.

In order that the invention may be more readily understood, various embodiments will now be described with reference to the accompanying drawings, in which: Figure 1 is a cross-section through a centrIfug- ing chamber arrangement embodying the present in::ent- ion, for carrying out a dry centrifuging operatic; Figure 2 is a partial cross-section throug the component which forms the upper part of the sarple chamber of Figure 1 Figure 3 is a plan view of the components snown in Figure 2; Figure 4 is a side view of the component which forms the base part of the sample chamber of Figure 1; Figure 5 is a plan view of the base part shown in Figure 4; Figure 6 is a cross-section through the base part on the line VI-VI in Figure 5; Figure 7 is a cross-section through the centrif fuzing chamber arrangement of Figure 1, incorporating a tinting insert; Figure 8 is a cross-section similar to Figure 1, trough a further embodiment of centrifuging chamber arrangement for carrying out a wet centrifuging operation;; Figure 9 is a cross-section through a further embodiment of centrifuging chamber incorporating a tinting insert; Figure 10 is a cross-section through the centr ining chamber arrangement of Figure 9, with the tinting insert removed, and illustrating other retails; Figure 11 is a partial cross-section through the component which forms the upper part of the sample chamber ofFigures9 and 10; Figure 12 is a plan view of the component shown i Figure 11; Figure 13 is a side view of the component which forms the base part of the sample chamber of Figures 9 2ne 10; Figure 14 is a plan view of the base part shown in Figure 13; and Figure 15 is a cross-section through the base part on the line XV-XV in Figure 14.

The centrifuging chamber arrangement embodying the invention shown in Figure 1 comprises a tubular reaction container or test tube 1 constructed as an elongate beaker-or cup-like element having an internal step 7, intended to contain and support a two-part sample chamber embodying the invention. The sample chamber comprises a tubular base part 2 which is fitted over an upper part 4 with an object carrier 3 trapped between the two parts.

As shown in Figure 2, the upper part 4 of the sample chamber consists of a beaker- or cup-like hollow cylindrical element which has an upper opening bounded by a peripheral annular flange 12 of enlarged diameter. The upper part 4 rests in the test tube 1 with this annular flange 12 raised above the upper rim of the test tube 1 as shown in Figure 1. The lower portion of the upper part 4. is in the form of a reduced diameter collar or sleeve 13, which opens downwardly as indicated at 36.

Referring to Figures 1, 2 and 3, the upper part 4 is supported on the object carrier 3 via an annular base flange 41 at the lower end of the collar 13. The upper part 4 consequently does not rest on the test tube 1 via the annular flange 12. The base flange 41 is thus capable of defining with the associated supporting surface of the object carrier 3, the cross-section of a passage or gap which allows fluid to pass only at higher speeds of revolution of the centrifuge.

The base part 2 shown in Figures 4 to 6 also comprises a beaker- or cup-like cylindrical part defining an upper opening 38 surrounded by an annular sleeve or jacket 39. Axial slots 16 are provided through the sleeve 39, which terminate just above the upper cr internal surface of the base 37 of the base part 2, so that each slot defines a step 22 having an upper edge 23 as viewed in Figure 1. The slots 16 serve the purpose of giving the sleeve 39 a degree of resilience in a radial direction, so that it may be pushed or sprung over the collar 13 of the upper part 4 with a close fit, and also serve the purpose of defining the edges 23 of the steps 22.The base part 2 is provided with a central opening 38 situated above the base 37, and the underside of the base is provided with radial slots 17 extending outwards from, and distributed uniformly around, an opening 18 in the base, te radial slots 17 terminating in the external surface of the sleeve 39.

Ir use, an object carrier 3 in the form of a circular glass plate is initially placed on the internal surface of the base 37 of the base part 2, with the sample chamber disassembled. The sleeve 39 of the base part 2 is then pushed over the reduceddiameter collar 13 of the upper part 4, so that the necked-dwn part 10 at the top edge of the base part 2 is placed in contact with the associated necked-down part c the upper part 4, so that the lower base flange 41 of the collar 13 rests on the upper surface 9 of the object carrier 3 in full and sealing contact.

The sample chamber comprising the base part 2 and the upper part 4 is then inserted into the test tube 1 as shown in Figure 1, thereby placing the lower surface of the base 37 of the base part 2 in contact with the step 7 of the test tube 1.

At this stage, the full areal contact of the base flange 41 on the object carrier 3 prevents undesirable leakage of the cell suspension out of the upper reception volume or space 34 of the test tube 1 into the bottom reception volume or space 35. The centrifuging chamber is then centrifuged at approximately 50g for 1 minute, causing the cells which are to be examined to be deposited on the object carrier 3, without the fluid escaping radially outwards between the base flange 41 and the object carrier 3.

The fluid pressure is increased to a higher level hy increasing the centrifugal force (acting in the direction of the arrows 8) to approximately 1500g.

This causes the cell-depleted supernatant fluid to flqw radially outwards in the direction of the arrows 19, between the base flange 41 and the surface of the object carrier 3, and to flow axially downwardly, deflected in or below the base part 2, into the area of the radial slots 17 in the underside of the base 37, from where the sample fluid then flows into the lower reception space 35 in the direction of the arrows 20.

It is important in this respect, that the plane of the downwardly directed surface of the base flange 41 should be flush with, or above, the top edge 23 of the step 22 of the base part 2, so that the fluid from the sample suspension contained in the upper reception space 34 may drain completely downwards into the lower reception space 35 during the centrifuging operation (at a higher speed of rotation). To this end, the surface of the object carrier 3 may even be 0.2mm.

higher than the edge 23 of the base part 2, to secure complete drainage of the fluid and thus a dry centrifuging operation.

In the case of a wet centrifuging operation the arrangement shown in Figure 8 is employed. In this embodiment, the step 24 is made higher and forms an edge 25 situated above the plane of the upper surface of the base flange 41, so that this step 24 forms an obstacle to fluid flow, i.e. forms a wier or dam,which assures that the sample fluid collecting on the object carrier 3 ces not drain off completely during the centrifuging operation. This means that a sump or reservoir of the sample fluid accumulates on the object carrier 3, the depth of which is established by the distance 26 between the edge 25 and the plane of the base farce 41 or object carrier 3.The surface 9 of the objet carrier 3 thus remains coated with the fluid retainer in this sump.

The centrifuging chamber arrangements shown in Figures 1 any 8 may incorporate a tinting insert 5 as shown in Figures 7 and 10. The insert 5, which has a lid 28, is introduced into the sample chamber, and is filled with an appropriate tinting, staining or colouring fluid. The tinting insert is of hollow cylindrical form and has a conically convergent base 29 leading irto a central outlet opening 30, the downstream end of which is situated in the region of an annular or circular gap 33 disposed just above the object carrier 3. Radially and laterally extending recesses 31 are provided in the base of the tinting insert 5, for the purpose of saving weight and material.

The tinting insert 5 has an upper encircling annular flange 32 of enlarged diameter which sits on the annular flange 12 of the upper part 4 of the sample chamber, establishing the gap 33, and again establishing a full areal contact between the steps 10 and 11 of the upper part 4 and base part 2.

The sleeve of the base part in Figures 8 and 9 thus has shorter slots 27 machined in it as compared to the previously described slots 16 in the base part of Figures 1 and 7, to form a higher step 24.

Another embodiment of centrifuging chamber arrangement is shown in Figures 9 to 15, in which like parts are designated by the same reference numerals as in the previous embodiments. Whereas Figure 9 shows the tinting insert 5 installed in the sample chamber, the tinting insert 5 is removed in Figure 10. The embodiment of Figures 9 and 10 differs essentially from the previous embodiments in that the flowblocking section or means comprises a resilient annular lip 45 which encircles the lower collar 13 of the upper part 40 of the sample chamber in a peripherally continuous manner.

Figures 10 to 15 show further details of the centrifuging chamber arrangement.

At the pon= of transition between the upper jacket portion of the upper part 40 of the sample chamber and the reduced diameter collar, the upper part 40 has a sep 48 in the region of which are situated downward and radially projecting detent projections 43. These detent projections engage in associated axial slots 46 in the base part 42 and thereby facilitate correct positional assemblage of the upper part 40 and the base part 42.

Instead of n circumferentially continuous base flange 41 as in the earlier described embodiment, a base flange 50 (inure 12) is now provided which is perforated in the peripheral direction by radially directed slots 44. The slots 44 are distributed uniformly around the periphery of the base flange 50 for this purpose.

The annular resilient lip 45 which shuts or valves off the radially external openings of the slots 44, is mounted cr. the outer periphery of the collar 13. As shown in Figure 10, the cell suspension in the upper reception space 34 is also present in the slots 44, and the outflow of the cell suspension into the lower reception space 35 is prevented only by the resilient annular lip 45.

However, under high centrifugal force, the fluid pressure in the upper reception space 34 increases to such a level that the resilient lip 45 is deformed radially outwardly, extending into and being accommodated by an unoccupied annular space 51 (Figure 10).

The lip 45 thereby uncovers the radially outer openings of the slots 44, and the fluid may thus drain out without obstruction via the slots 46, and the grooves 47 (see Figures 13 to 15) in the base of the lower part 42, into the lower reception space 35.

The embodiment shown in Figures 9 and 10 shows a centrifuging chamber arrangement for a dry centrifuging operation, and for this purpose, it will be apparent that the upper surface of the object carrier 3 lies above the edge 23 of the base part 42.

This embodiment may also be modified for wet centrifuging operation by ensuring that the edge 23 of the base part 42 is at a higher level than the upper surface of the object carrier 3, and at the same time by ensuring that the resilient lip 45 may be deformed radially outwards without being impeded by the vertically higher edge 23 of the base part.

A coating operation on an object carrier, and if appropriate a tinting and/or fixing operation on a cell sample suspended on the object carrier, may thus be undertaken with the centrifuging chamber hereinbefore described, in a neat and rapid operation, without any great expense and with a considerable weight reduction.

Claims

1. A centrifuging chamber arrangement, primarily intended for the cytological examination of cell suspensions, comprising a tubular receptacle into which a sample chamber for receiving a cell suspension is inserted to thereby form an upper and a lower reception space in the receptacle, the sample chamber comprising a base part ha'::ing a base surface on which an object carrier is arranged, and having at least one opening adjacent the object carrier through which the cell suspension fluid fffi-cws from the upper reception space into the lower reception space during a centrifuging operation, means being provided to block the flow of the cell suspension fluid towards the lower reception space, situated adjacent the object carrier, which is operable to allow the cell suspension fluid to fle from the upper to the lower reception space only at higher rotational speeds of the centrifuge.

2. An arrangement according to claim 1, wherein cell suspension fluid flow through the flow-blocking means between the upper and lower reception spaces occurs only at or abc-e a centrifugal acceleration force of the order of 15cho.

3. An arrangement according to claim 1 or 2, wherein the flow-blockir. means comprises a base flange of the upper part of the sample chamber which normally rests in effec -ely full areal contact, and is applied with increasing force when the centrifugal force is increased, o the surface of the object carrier, then upper part and flange being displaceable away from the object carrier to allow cell suspension fluid to flow between the flange and object carrier from the upper to the lower reception space due to the pressure exerted on the upper part by the cell suspension fluid resulting from said higher rotational speeds of the centrifuge.

4. An arrangement according to claim 3, wherein the upper part of the sample chamber comprises a hollow cylindrical element which, at its upper extremity, has an enlarged diameter annular flange intended to be disposed on or adjacent a rim at the upper end of the tubular receptacle, the lower portion of the upper part being formed as a collar of reduced diameter, the lower end of which is annular and forms said base flange.

5. An arrangement according to claim 3 or 4, wherein the base part of the sample chamber comprises a hollow cylindrical element having a surrounding wall which has a first opening at its upper end situated opposite the base surface, the base surface has a second and central opening therein, and generally radial slots are situated at the underside of the base surface which open into the second opening and traverse the surrounding wall of the base part.

6. An arrangement according to claim 5 in combination with claim 4, wherein the surrounding wall of the base part is axially slotted to permit the surrounding wall to be pushed over the collar of the upper part of the sample chamber.

7. An arrangement according to claim 5 or 6, wherein the radial slots situated at the underside of the base surface of the base part have a depth of the order of 0.lmm., an a width of the order of 0.2mm.

8. An arrangement according to claim 1 or 2, wherein the flow-blocking means comprises a resilient annular lip which encircles the upper part of the sample chamber in a peripherally continuous manner, and covers the openings therein through which the cell suspension liquid is draw-,-off from the upper reception space into the lower reception space.

9. An arrangement according to claim 8, wherein the upper part of the sample charter comprises a hollow cylindrical element, the lover portion of which comprises a cylindrical collar of reduced diameter, and wherein the annular lip shuts off openings defined by radial slots in the lower end of the collar which form a fluid passage between the upper and lower reception spaces.

10. An arrangement according to any preceding claim, wherein, in order to obtain a dry sample deposition on the upper surface of the object carrier, the upper surface of the object carrier is located at or above the level of the lower boundaries of the openings in the sample chamber through whIch the cell suspension fluid can flow from the upper to the lower reception space.

11. An arrangement according to any of claims 1 to 9, wherein, in order to chain a moist sample deposition on the upper surface of the object carrier, the upper surface of the object carrier is located below the level of the lower boundaries of the openings in the sample chamber through which the cell suspension fluid can flow from the upper to the lower reception space.

12. An arrangement according to any preceding claim, including an insert provided with a fluid reservoir insertable into the upper part of the sample chamber, the insert having one or more calibrated fluid outlets from the reservoir which, when the insert is operatively inserted, open at a distance above the object carrier and are operative to discharge a tinting, colouring, staining or fixing fluid from the reservoir onto a cell sample deposited on the object carrier.

13. A centrifuging chamber arrangement substantially as hereinbefore described with reference to Figures 1 to 6 of the accompanying drawings.

14. A centrifuging chamber arrangement substantially as hereinbefore described with reference to Figure 7 of the accompanying drawings.

15. A centrifuging chamber arrangement substantially as hereinbefore described with reference to Figure 8 of the accompanying drawings.

16. A centrifuging chamber arrangement substantially as hereinbefore described with reference to Figure 9 of the accompanying drawings.

17. A centrifuging chamber arrangement substantially as hereinbefore described with reference to Figures 10 to 15 of the accompanying drawings.

18. A centrifuge provided with one or more centrifuging chamber arrangements according to any preceding claim.