GB1590120A

GB1590120A - Preparation of cell spreads

Info

Publication number: GB1590120A
Application number: GB3481276A
Authority: GB
Original assignee: National Research Development Corp UK; National Research Development Corp of India
Current assignee: National Research Development Corp UK; National Research Development Corp of India
Priority date: 1977-08-03
Filing date: 1977-08-03
Publication date: 1981-05-28

Description

CELL IMPROVEMENTS IN THE PREPARATION OF CELL SPREADS (71) We, NATIONAL RESEARCH DEVELOP MENT CORPORATION, a British Corporation established by Statute, of Kingsgate House, 66-74 Victoria Street, London S.W.l, do hereby declare the invention for which we pray that a patent may be granted to us, and the method by which it is to be performed, to be particularly described in and by the following statement:- The present invention relates to the field of cell-biology and in particular to the preparation of cell spreads from liquid cell suspensions.

A prerequisite of many techniques e.g.

immunofluorescence, autoradiography and quantative hi stochemistry, customarily used for cell investigation is the preparation on a suitable surface such as a microscope slide of a closely spaced spread of cells which are sufficiently flattened to display their cytoplasmic and nuclear structure.

Historically cell spreads were prepared by tedious and cell damaging smear techniques, but in recent years a centrifugal technique has been proposed (bore and Balfour, Immunology, Volume 9 No. 4, October 1965, pages 403-405) for preparation of cell spreads from liquid cell suspension and a commercially available cell-centrifuge apparatus has been used extensively in clinical and other laboratories. In this apparatus cells are centrifuged from a liquid suspension on to an area of microscope slide surrounded by a ring of filter paper which absorbs the fluid, and thus exerts a lateral pull on the cells which thereby stretches and flattens them on the surface of the slide.

This method, however, suffers from the drawback that cells are drawn into the filter paper with the fluid making it impossible to accurately estimate the proportion of a particular type of cell initially present in the cell population. A modified cell centrifuge is now proposed which overcomes this drawback and is also generally applicable to the preparation of cell spreads.

Accordingly the present invention provides an improved cell centrifuge for the preparation on microscope slides of cell spreads from liquid cell suspensions, in the form of a centrifugal rotor comprising an assembly of centrifugal passageways radiating from the axis of the rotor, the outer ends of the passageways provided separately with fixing means to hold microscope slides in place across the passageway outlets, the outlets being constructed in an appropriate form and/or material to provide a seal with the microscope slides and the passageways provided with a common means for introduction of cell fixative thereto whilst the centrifuge is rotating.

The invention also includes a method for the preparation on microscope slides of cell spreads from liquid cell suspensions, in which cells are centrifuged from a cell suspension on to a microscope slide and cell fixative is introduced to the cells and fixes them on to the microscope slide whilst they are held on the microscope slide by centrifugal force.

The centrifuge and method of the present invention may be used widely for the preparation of cell spreads in general and may include cell spread preparations previously carried out using prior art apparatus andlor techniques. Thus the present invention may be applied to the preparation of cell spreads from unicellular arganisms such as bacteria, though more usually the invention is applied to the preparation of cell spreads derived from the organs and tissues of higher organisms. More especially the invention finds particularly advantageous application for the preparation of cell spreads from cell populations which contain a low proportion of a distinguishable e.g. aberrant, cell which it is desired to monitor. Generally the type of cell suspensions which are used and their methods of preparation from organs and tissues are similar to those which are well known in the art.

The centrifuge of the present invention comprises a rotor typically regular in crosssection e.g. circular, square or hexagonal, and usually mounted, with cross-section horizontal, symmetrically on a vertical rotary shaft coupled to a suitable drive means such as an electric motor. The rotor as a whole may be housed within a suitable enclosure, for instance to shield the user against injury from the rotating machinery and to guard against release of cells e.g. pathogenic cells, into the surrounding environment.

The centrifugal passageways may be provided within the main body of the rotor itself, for instance as passageways symmetrically disposed about the axis of rotation of the rotor and radiating therefrom, the inner ends of the passageways accessible usually through the top surface of the rotor and their outer ends terminating in openings conveniently in a face or faces provided around the circumference of the rotor.

Alternatively the centrifugal passageways may be provided by passageway modules which are separable from the main body of the rotor, in which case the rotor is normally provided with suitable means for locating the passageway modules in a radiating arrangement. For example, the rotor may have an annular wall around its circumference provided with suitable seatings for location of passageway modules, such as the commercially available Shandon Cytospin apparatus. (Shandon is a registered Trade Mark.) Generally the centrifugal passageways are constructed with a view to minimising hindrance to the passage of cells from suspension to the surface element and are usually smoothly contoured, for instance as parallel sided tubes.

The passageways terminate at their outer ends in openings which are provided separately with means for making a seal with the surface element, e.g. microscope slide, on to which the cells are being centrifuged.

There is provision of clips or other fixing means to hold microscope slides in place across the passageway outlets and typically the outlets are constructed in an appropriate form and/or material to provide the required seal with the microscope slides. In a particularly preferred embodiment the passageway outlet is provided by an inset of a suitable resilient material e.g. silicone rubber, preferably with the mouth of the outlet constructed in such a way that there is no significant distortion of the mouth when the microscope slide is held in sealing engagement against it. For example, a double-lip outlet arrangement, in which the mouth of the outlet is provided at the centre of a disc shaped surface surrounded by two raised concentric ridges has been found to be highly satisfactory.

Characteristically the passageways are provided with a common means for introduction of cell fixative thereto whilst the rotor is spinning. It will be apparent that there are many ways in which this may be achieved and all of these are included within the scope of the invention. Generally, however, the introduction means takes the form of a common inlet means connected to all of the passageways, usually towards their inner ends, and typically symmetrically located with regard to the axis of rotation of the rotor. For instance, the introduction means may take the form of an inlet means e.g. well, located at the axis of rotation of the rotor and which interconnects with the radiating passageways.Alternatively in a preferred embodiment the introduction means may comprise an annular groove in the top surface of the rotor at a fixed radius from the axis of rotation, the groove interconnecting with the inner ends of the centrifugal passageways.

Also in systems in which the passageways are separable from the rotor, such as the Cytospin apparatus, the means for introduction of fixative may be supplied by a suitable separable adapter comprising a common inlet means to the passageways which is located symmetrically with respect to the axis of rotation. For instance, the adapter may take the form of a disc having an annular groove in its top surface located at a fixed radius from the axis of rotation, the groove having outlet passageways which coincide with the centrifugal passageways distributed around the circumference of the rotor. Such adapters are included within the scope of the present invention.

Generally the cell fixatives which may be used in the present invention are already well known in the art and include ethyl alcohol and formaldehyde.

In use the outer ends of the centrifugal passageways are sealed with microscope slides, cell suspensions are delivered to the passageways as desired and the rotor is set in motion. The cell concentration of the suspensions may previously be adjusted, for instance to the level required to give a monolayer of cells covering the area of microscope slide corresponding to the outer end of the centrifugal passageway. Centrifugal force due to rotation of the rotor causes sedimentation of the cells from the suspensions on to the microscope slide, and subsequent addition of cell fixative to the passageways fixes the cells to the microscope slides whilst they are held in position by the centrifugal force in an advantageously flattened state. The rotor is then stopped and the supernatant fluid is discarded as the microscope slides are removed from the centrifuge. The cell spreads so prepared may be stained or otherwise treated and examined as desired. In some cases, in particular when the supernatant is of significantly greater density than the fixative, the layer of supernatant remaining after sedimentation of cells may hinder the passage of fixative to the cells. Thus, in a modification, the centrifuge may be provided with suitable outlet means through which super natant may be displaced from the passageways consequent upon the introduction of fixative, thereby diminishing hindrance to the passage of fixative to the cells. For example, in one arrangement, each passageway has a suitably shaped tubular outlet e.g.

a curved hypodermic needle, extending along the axis of the passageway from the direction of the axis of rotation towards the passageway outlet, such that introduction of fixative to the rotating centrifuge causes supernatant to be displaced from the passageways through the tubular outlets. In addition, the supernatant outlet may advantageously provide a conveninet inlet for introduction of cell suspension to the passageways prior to centrifuging. This modification does not appear to be an essential feature as satisfactory results are readily obtained using apparatus unmodified in this respect.

Advantageously the apparatus and method of the present invention avoid loss of cells into the filter paper as with prior art cell-centrifuge apparatus, substantially all the cells from the suspension being retained on the microscope slide. Conveniently also, it is not generally necessary to fix the cells subsequent to removal of the slides from the centrifuge and prior to staining and other treatment, as would be required with prior art cell centrifuge apparatus and methods.

Without prejudice to the scope of the present invention, the apparatus and method are further illustrated in the following specific description which refers to the accompanying diagrams in which: Figure 1 represents a plan view from above of a centrifugal rotor according to the invention; Figure 2 represents a side elevation of the same rotor; Figure 3 represents a side view of a vertical section taken through the line A-A of Figure 1; Figure 3a represents the same view as Figure 3 but including an additional supernatant outlet modification; Figure 4 represents a plan view from above of an alternative centrifuge design having an adaptor to provide means for introduction of cell fixative to centrifugal passageway modules located around the circumference of the rotor and separable therefrom; and Figure 5 represents a side view of a ver tical section through the line B-B of Figure 4.

Figures 1, 2 and 3 show a cell centrifuge according to the present invention in the form of a solid Perspex rotor 1 of hexagonal cross-section having a tapering bore hole 2 extending through it along its axis of rotation to accommodate a centrifuge spindle coupled to an electric motor drive, neither of the latter being shown. (Perspex is a registered Trade Mark.) The upper opening of the bore hole 2 is closed with a clamping knob 3 having a threaded recess 4 in its base for locking engagement with the threaded end of the centrifuge spindle (not shown) and a further recess 5 in the middle of its upper surface to accommodate a tachometer spindle (not shown). The clamping knob 3 is held in position by a retainer plate 6 fixed to the upper surface of the rotor 1 by three countersunk screws 7.

Each of the six vertical faces 9 around the circumference of the rotor 1 has two holes 10 drilled perpendicular to its surface, each lined with a tubular resilient silicone rubber inset 11 providing six pairs of parallel sided tubular centrifuge passageways 12 radiating from the axis of rotation, each passageway 12 being perpendicular to the face 9 in which it is located. The insets 11 have flanges 13 at their outer ends which fit drilled recesses in the faces 9 with the outermost ends of the inset 11 projecting slightly. The outer end faces of the insets 11 each carry two concentric annular ridges 14 and 15, one adjacent the passageway outlet and the other adjacent the circumference of the flange 13, which provide a fluid-type seal when the surface of a microscope slide (not shown) is held in pressure against them.At the inner end of each passageway 12 the inset 11 has a smoothly moulded chamfer 16 leading into the periphery of an outwardly slanting annular groove 17 machined at a fixed radius from the axis of rotation into the top surface of the rotor 1. This annular groove 17 constitutes a means of delivering cell fixative to the passageways 12 whilst the rotor 1 is rotating.

Each face 9 of the rotor 1 has a slide retainer mechanism to hold a microscope slide against the outer ends of the insets 11, which consists of a rectangular brass base plate 18 attached to the bottom surface of the rotor 1 and a retainer spring 19 attached to its upper surface adjustable by means of an adjuster knob 20. The brass plate 18 consists of an oblong brass strip having two transverse slots 21 opening through one elongate side, the other elongate side turned up along itsedges to form an elongate lip 22. The base plate 18 is attached to the bottom surface of the rotor 1 by two screws 23 fitted with shake proof washers 24 pass ing through the two slots 21, and the up turned elongate lip 22 projects beyond the edge of the face 9 providing an elongate channel 25 to engage the lower edge of a microscope slide (not shown).The slide retainer spring 19 consists of a flexible rectangular bronze sheet fastened at its inner edge to the upper surface of the rotor 1 by two round-headed screws 26. The bronze sheet 19 is turned down along its outer edge to form an elongate lip 27 which engages the upper edge of a microscope slide (not shown). The spring 19 is set with an angle of elevation of about 5" in the radial direction and is adjustable by means of an adjuster knob 20 connected to a threaded shaft 28 which passes through the spring 19 and engages with a screw threaded recess 29 provided within the rotor 1.

The cell centrifuge illustrated in Figures 1, 2 and 3 may be used to prepare cell spreads on microscope slides as follows: Clean microscope slides are attached to the vertical faces 9 around the circumference of the rotor 1, as desired, by means of the slide retainer mechanisms. The adjustors 20 are slackened to raise the outer edges of the slide retainer springs 19 and permit location of the lower edges of the microscope slides in the channels 25 provided between the faces 9 and the upturned lips 22 of the base plates 18. The adjustors 20 are then tightened bring the downward turned lips 27 of the slide retainer springs 19 to bear upon the upper edges of the microscope slides clamping them in place and, in cooperation with the concentric annular ridges 14 and 15 around the ends of the resilient inserts 11, sealing the outer ends of the centrifuge passageways 12.Cell suspensions are then introduced to the passageways 12, for instance by pipette or syringe introduced through the annular groove 17 to the inner ends of the passageways 12. The rotor 1 is set in motion centrifuging cells from the suspensions on to the surfaces of the microscope slides, and whilst the rotor 1 is in motion and cells are held flattened against the slide, cell fixative is introduced to the annular groove 17, flows into the passageways 12 and fixes the cells in position. The rotor 1 is then stopped, the adjustors 20 slackened and the microscope slides removed, the remaining fluid from the suspension being discarded.Substantially all the cells initially present in the suspensions are retained on the surface of the microscope slides, making it possible to accurately estimate the proportion of a particular type of cell initially present in the cell population. The cell spreads so prepared may be strained and/or otherwise treated and examined as desired.

With reference to Figure 3a, the centrifuge of Figures 1, 2 and 3 may be modified by provision of supernatant outlet means.

Each passageway 12 of the centrifuge is provided with a supernatant outlet by means of a curved, square ended hypodermic needle 80 disposed along the axis of each passageway 12. The needle 80 is connected at its external end by way of a collar 84 of resilient material to an enlarged tubular section 81 which is held in a metal clip 82 firmly attached by round-headed screw 83 to the top surface of the perspex rotor 1. The supernatant outlet also provides an inlet means for introducing cell suspensipns to the passageways 12.

In use cell suspensions are introduced into the enlarged tubular sections 82 and drain into the passageways 12. The centrifuge is set in motion and cells sediment on to the microscope slides. Fixative is then added to the annular groove 17, as previously described, and is distributed to the passageways 12. Consequent upon the inflow of fixative, supernatant present in the passageways 12 is displaced through the needles 80, and fixative comes into contact with the cells and fixes them in place on to the microscope slides. Thereafter the centrifuge is stopped and slides removed as previously described.

Figures 4 and 5 show an alternative cell centrifuge arrangement based on prior art cell centrifuge apparatus specially adapted in accordance with the present invention.

The centrifuge rotor is of the type used in the Shandon Cytospin apparatus, comprising a flat circular base plate 50 which has a vertical wall 51 around it periphery. The base plate 50 has an upwardly tapering passageway extending through it along its axis of rotation to accept a centrifuge spindle 52 coupled to an electric motor drive (not shown), the upper portion of the spindle 52 in screw threaded locking engagement with a recess 53 in The base of a central shaft 54. Around the base of the central shaft 54 and in screw-threaded engagement therewith is a clamping plate 55 which when it is tightened down provides the clamping means between the centrifuge spindle 52 and the base plate 50.

The inner face of the vertical wall 51 has twelve recesses at regularly spaced intervals to accommodate sample modules 56, the recesses coinciding with inspection ports 57.

The modules 56 are in the form of perspex blocks each having a passageway 58 through it, the outer end being horizontal but extending upwards towards its inner end at an oblique angle to an exit perpendicular to an oblique face 59 machined in the upper inner edge of each block 56. The outer orifice of each passageway 58 has a resilient silicone rubber inset 60 to provide a seal with the surface of a microscope slide 61 held in place between one of the blocks 56 and the inner face of the vertical wall 51 by a leaf spring 62 located at its inner end in the clamping plate 55 and with its outer end bearing upon an abutment 63 on the inner face of the block 56, The inner ends of the passageways 58 also have resilient silicone rubber insets 64 to provide seals with an angled face 65 around the lower periphery of an annular Perspex adapter 66.Machined into the upper surface of the adapter 66 is an outwardly directed annular groove 67 which extends at its outer extremities into twelve radial passageways 68 regularly spaced around the periphery of the adapter 66. These passageways 68 correspond with the inner ends of the centrifuge passageways 68 and are maintained in register therewith by a locating peg 69 keying into a recess in the adapter 66 through a hole in the vertical wall 51. The adapter 66 is mounted axially on the vertical central shaft 54 and a pressure plate 70 recessed into its upper surface in co-operation with a locking knob 71, which is in screw threaded engagement with the top ends of the central shaft 54, holds the adapter 66 in pressure against the sample blocks 56.The underside of the adapter 66 is machined around its periphery giving a shallow step 72 to provide clearance between the sample blocks 56 and the undersurface of the adapter 66.

The specially adapted apparatus of Figures 4 and 5 may be used to prepare cell spreads. Clean microscope slides 61 and sample blocks 56 are located in the recesses around the inner face of the vertical wall 51 as desired, and held in place by the leaf springs 62. Cell suspensions are then introduced into the passageways 58. The adapter 66 is fitted over the central shaft 54, the pressure plate 70 located in the recess in its upper surface and the locking knob 71 screwed in place on the ends of the central shaft 54 to hold the adapter 66 in pressure against the sample blocks 56. The centrifuge is then set in motion and cells centrifuge on to the surface of the microscope slides 61.Whilst the centrifuge is still in motion and the cells are held flattened against the slides 61, cell fixative is introduced to the annular groove 67 and flows via the passageways 68 into the centrifuge passageways 58 fixing the cells to the surfaces of the slide 61. The centrifuge is then stopped, the locking knob 71 unscrewed and the adapter 66 and pressure plate released from the central shaft 54. The sample blocks 56 and microscope slides 61 may then be removed from the recesses around the inner face of the vertical wall 51, the pressure of the leaf springs 62 being released from the abutments 63. The excess fluid from the cell suspension is discarded. The cell spread preparations on the surfaces of the microscope slides 61 may then undergo subsequent treatment as desired.

WHAT WE CLAIM IS: 1. A method for the preparation on microscope slides of cell spreads from liquid cell suspensions, in which cells are centrifuged from a cell suspension onto a microscope slide and cell fixative is introduced to the cells and fixes them onto the microscope slide whilst they are held on the microscope slide by centrifugal force.

2. A cell centrifuge for the preparation on microscope slides of cell spreads from liquid cell suspensions, in the form of a centrifugal rotor comprising an assembly of centrifugal passageways radiating from the axis of the rotor, the outer ends of the passageways being provided separately mAth fixing means to hold microscope Aides in place across the passageway outlets, the outlets being constructed in an appropriate form and/or material to provide a seal with the microscope slides and the passageways being provided with a common means for introduction of cell fixative thereto whilst the rotor is rotating.

3. A centrifuge according to Claim 2, in which the rotor is housed within a suitable enclosure.

4. A centrifuge according to Claim 2 or 3, in which the centrifugal passageways are provided within the main body of the rotor.

5. A centrifuge according to Claim 2 or 3, in which the centrifugal passageways are provided by passageway modules which are separable from the main body of the rotor, the rotor being provided with suitable means for locating the passageway modules in a radiating arrangement.

6. A centrifuge according to any of Claims 2-5, in which the passageway outlets are provided by insets of a suitable resilient material.

7. A centrifuge according to Claim 6, in which the mouth of the passageway outlet is constructed in such a way that there is no significant distortion of the mouth when the microscope slide is held in sealed engagement against it.

8. A centrifuge according to Claim 7, in which the mouth of the passageway is provided at the centre of a disc-shaped surface and the outlet is surrounded by two raised concentric rings.

9. A centrifuge according to any of Claims 2-8, in which the means for introduction of cell fixative to the passageways comprises a well located at the axis of rotation of the rotor and which interconnects with the radiating passageways.

10. A centrifuge according to any of Claims 2-8, in which the means for introduction of cell fixative to the passageways comprises an annular groove in the top surface of the rotor, at a fixed radius from the axis of rotation, the groove interconnecting with the inner ends of the centrifuge passageways.

11. A centrifuge according to any of Claims 2-10, comprising outlet means for

**WARNING** end of DESC field may overlap start of CLMS **.

Claims

**WARNING** start of CLMS field may overlap end of DESC **. seals with an angled face 65 around the lower periphery of an annular Perspex adapter 66. Machined into the upper surface of the adapter 66 is an outwardly directed annular groove 67 which extends at its outer extremities into twelve radial passageways 68 regularly spaced around the periphery of the adapter 66. These passageways 68 correspond with the inner ends of the centrifuge passageways 68 and are maintained in register therewith by a locating peg 69 keying into a recess in the adapter 66 through a hole in the vertical wall 51.The adapter 66 is mounted axially on the vertical central shaft 54 and a pressure plate 70 recessed into its upper surface in co-operation with a locking knob 71, which is in screw threaded engagement with the top ends of the central shaft 54, holds the adapter 66 in pressure against the sample blocks 56. The underside of the adapter 66 is machined around its periphery giving a shallow step 72 to provide clearance between the sample blocks 56 and the undersurface of the adapter 66. The specially adapted apparatus of Figures 4 and 5 may be used to prepare cell spreads. Clean microscope slides 61 and sample blocks 56 are located in the recesses around the inner face of the vertical wall 51 as desired, and held in place by the leaf springs 62. Cell suspensions are then introduced into the passageways 58. The adapter 66 is fitted over the central shaft 54, the pressure plate 70 located in the recess in its upper surface and the locking knob 71 screwed in place on the ends of the central shaft 54 to hold the adapter 66 in pressure against the sample blocks 56. The centrifuge is then set in motion and cells centrifuge on to the surface of the microscope slides 61.Whilst the centrifuge is still in motion and the cells are held flattened against the slides 61, cell fixative is introduced to the annular groove 67 and flows via the passageways 68 into the centrifuge passageways 58 fixing the cells to the surfaces of the slide 61. The centrifuge is then stopped, the locking knob 71 unscrewed and the adapter 66 and pressure plate released from the central shaft 54. The sample blocks 56 and microscope slides 61 may then be removed from the recesses around the inner face of the vertical wall 51, the pressure of the leaf springs 62 being released from the abutments 63. The excess fluid from the cell suspension is discarded. The cell spread preparations on the surfaces of the microscope slides 61 may then undergo subsequent treatment as desired. WHAT WE CLAIM IS:

1. A method for the preparation on microscope slides of cell spreads from liquid cell suspensions, in which cells are centrifuged from a cell suspension onto a microscope slide and cell fixative is introduced to the cells and fixes them onto the microscope slide whilst they are held on the microscope slide by centrifugal force.

11. A centrifuge according to any of Claims 2-10, comprising outlet means for

displacement of supernatant from the passageways consequent upon the introduction of fixative thereto.

12. A centrifuge according to Claim 11, in which each centrifugal passageway has a suitably shaped tubular outlet extending along the axis of the passageway from the direction of the axis of rotation of the rotor towards the passageway outlet, such that introduction of fixative to the rotating centrifuge causes supernatant to be displaced through the tubular outlets.

13. A cell centrifuge according to Claim 2, substantially as hereinbefore described and illustrated with particular reference to Figures 1, 2, 3 and 3a.

14. A centrifuge according to Claim 5 in which the means for introduction of fix ative is supplied by a separable adapter comprising a common inlet means for introducing cell fixative to the centrifugal passageways, said inlet means being adapted to communicate with the passageways and being located symmetrically with respect to the axis of rotation of the centrifuge.

15. A centrifuge according to Claim 14, in which the adapter is in the form of a disc having an annular groove in its top surface located at a fixed radius from the axis of rotation thereof, the groove having outlet passageways which are adapted to coincide with the centrifugal passageways distributed around the circumference of the rotor.

16. A cell centrifuge according to Claim 15, substantially as hereinbefore described and illustrated with particular reference to Figures 4 and 5.

17. A method for the preparation of cell spreads from liquid cell suspensions according to Claim 1, substantially as hereinbefore specifically described.