Cell washing centrifuge

BACKGROUND OF THE INVENTION ₂5 (1) Field of the invention

The present invention relates to centrifuges and, more particularly, to a. multispeed apparatus for centrifuging biological materials which are subjected, successively, to θθ centrifuging and decanting operations.

(2) Description of the prior art

As far as applicants are aware, there is no single apparatus known to them that performs successive func- 35 tions of centrifugation and decanting. In previous apparatus, as disclosed in U.S. Patent No. 2,739,750, biological materials have been subjected to centrifugation in swinging test tubes after which the test tubes had to be removed from the centrifuge for decantation and returned to the ⁴⁰centrifuge for further processing with fresh washing liquids or the like. This not only was excessively time-consuming but also involved the handling of apparatus which might be deleterious to the contents of the test tubes. Applicants have solved these problems by providing for biological ⁴⁵materials to remain in the test tubes between successive centrifugation and decanting steps without the necessity for disassembling the centrifuge and transporting test tubes containing the biological materials from one place to another. Applicants’ apparatus provides for faster, more ⁵⁰convenient, and more efficient washing of biological materials by means of the apparatus disclosed herein whereby all of the processing takes place within a single instrument until the work is done.

SUMMARY OF THE INVENTION

The present invention comprises a centrifuge apparatus having a plurality of swinging test tube holders which pivotally support respective test tubes in which biological _6Qmaterials are treated by centrifugation. The apparatus also comprises means for operating the centrifuge at different speeds; first, at high speed, for subjecting the biological materials to centrifugation in the outwardly pivoted test tubes and then, optionally, at a lower speed ₆₅of rotation of the centrifuge during which the test tubes are held parallel or at a small negative or small positive angle relative to the vertical axis of the centrifuge rotor whereby supernatant washing liquid is decanted automatically from said test tubes. Further means are provided for ₇θ suspending pivoting or swinging test tube holders on a ‘ removable carrier mounted on the rotating shaft of the centrifuge, whereby the test tubes may be initially filled at a location separate from the centrifuge and thereafter mounted on the centrifuge. After processing is completed, the carrier with the test tubes thereon is removed from the centrifuge and can be set upon a laboratory table or other suitable support element for subsequent analysis or testing of the centrifugal materials. Alternatively and optionally, the test tubes may be filled while they are located in the centrifuge apparatus.

BRIEF DESCRIPTION OF THE DRAWINGS

FIG. 1 is an elevation of the apparatus incorporating the present invention;

. FIG. 2 is a greatly enlarged vertical central section view of the upper portion of FIG. 1, showing details of the centrifuging apparatus, some parts being shown in elevation and with the centrifuging locations of the test tubes being shown in dotted outline;

FIG. 3 is a fragmentary view of a portion of the apparatus shown in FIG. 2 wherein the test tubes are retained in a negative angle decantation position;

FIG. 4 is a fragmentary view taken on line 4—4 of FIG. 3;

FIG. 5 is an enlarged fragmentary view of the lower end of a test tube holder as engaged by the locking ring for the decanting operation;

FIG. 6 is an exploded view, in three sections, showing the manner in which the test tube carrier is removed from the centrifuge shaft, and the manner in which said carrier may be positioned on a laboratory table or stand, some parts being shown in section, some in elevation, and some in dotted outline;

FIG. 7 is a view taken approximately on line 7—7 of FIG. 6;

FIG. 8 is a view taken on line 8—8 of FIG. 6;

FIG. 9 is similar to FIG. 2 showing the test tubes retained in a decanting position at a small positive angle relative to the axis of the centrifuge rotor;

FIGS. 10 and 11 are similar to FIG. 3 and comprise a composite fragmentary view of a further modification of the invention showing a test tube retaining element capable of holding the tubes in either a small negative angle or small positive angle position; and

FIG. 12 is a fragmentary view of a portion of the apparatus showing an additional component for automatically locating the test tubes in a positive angle decanting position.

DESCRIPTION OF THE PREFERRED EMBODIMENTS

While several embodiments of the present invention are illustrated in separate figures of the drawings and described herein, it will be understood that similar parts will be designated throughout all the figures of the drawings by similar reference numerals.

Referring now to the drawings in detail, the apparatus of the present invention is shown mounted on a base or stand, generally designated 11, comprising a platform 12, having three radially and downwardly disposed legs 13, two of which are shown in FIG. 1. Connected to the underside of platform 12 is a flexibly mounted electric motor M which is connected by a suitable line cord 14 to a source of electric power, not shown. Connected to the motor M is a tachometer 16 which regulates the speed of motor M.

Mounted on the top of platform 12 and raised therefrom on a plurality of posts 17 is a bowl-shaped enclosure 18 of circular shape. Enclosure 18 has a central circular opening at the bottom thereof through which drive shaft 19 extends vertically, said drive shaft being connected to and rotated by motor M.

3,420,437 ³

Mounted on the open top of enclosure 18 is a removable circular cover lid 21, said cover lid having an annular resilient gasket 22 whose annular recess 23 coacts with the upper edge of eclosure 18 to form an air-tight and liquid-tight seal therewith. See FIG. 2. Connected by suitable means to the bottom surface of cover lid 21 is a circular plate 24 which has a downwardly and outwardly extending annular baffle or deflector flange 26 whose function will be described hereinafter. Also connected to the bottom surface of cover lid 21 by suitable means is the circular horizontal flange 27 of a circular collection trough 28 comprising a vertical annular portion 29 and an inwardly and upwardly extending circular lip 31, the inner circular edge of said lip having a somewhat smaller diameter than the outer peripheral edge of baffle flange 26 whereby said lip and said baffle flange overlap each other radially. The bottom portion of collection trough 28 is inclined at a slight angle and at the lower portion thereof there is formed an outlet tube 32 that extends outwardly of said collection trough. Connected to the outer end of tube 32 is a drain tube 33 made of rubber or the like which extends through a suitable aperture in enclosure 18.

In some embodiments, cover lid 21 may be connected to enclosure 18 by means of a suitable hinge, not shown, whereby said lid may remain secured to said enclosure while in the open position.

Drive shaft 19 extends upwardly into enclosure 18, and to the lower portion of said shaft within said enclosure there is connected, by means of set screw 34, a drive hub 36 which rotates in unison with said shaft. Connected by suitable means to drive hub 36 is a rotor bowl 37 which rotates with said hub within the chamber formed by the enclosure 18.

Removably mounted on the upper portion of shaft 19 and fitting telescopically therewith is an elongated tubular or cylindrical hub 41. Connected to the interior of the upper end of hub 41 is a stub 42, the bottom of which is spaced apart from the top of shaft 19. Stub 42 extends upwardly and terminates in a grip knob 43 which enables the manual removal and replacement of hub 41 and its associated parts relative to said shaft. Connected centrally to the upper end of hub 41 is a horizontal carrier plate 44 extending outwardly from said hub. Arrayed in a circle coaxial with shaft 19 near the outer portion of said carrier is a plurality of evenly spaced apart slots 46 which are each pivotally engaged by the curved hinge 47 of a skeleton tube carrier, generally designated 48, integrally formed with said hinge. See FIG. 8. Each tube carrier 48 comprises an elongated spine 49 made of sheet metal or the like, said spine having at least one or more spaced apart circular tube bands or rings 51. The bottom portion of each spine 49 terminates in an outwardly curved support flange 52, a portion of said flange being split to form a hook or tine 53 which extends downwardly from said flange and, together with said flange portion, forms a forked element whose function will be described hereinafter.

Removably insertable through rings 51 of each of the skeleton tube carriers is a test tube 54 made of glass, aluminum, stainless steel, or other suitable material, the bottom of said test tube resting upon and being supported by flange 52.

Connected to the bottom of hub 41 is a circular bottom plate 56 which has an integrally formed downwardly extending collar 57 which terminates in an outwardly extending flange 58. Bottom plate 56 has a pair of diametrically spaced apart apertures 59 through which extend the upper ends of respective drive pins 61 mounted in hub 36. The sides of drive pins 61 are tapered inwardly toward their bottom portions whereby said tapered sides bear against the side apertures 59 to urge bottom plate 56 downwardly during rotation of hub 36. The inner portion of bottom plate 56 rests upon the top of drive hub 36.

Although rotation of hub 41 and support plate 44 has been described as being provided by the action of drive pins 61 upon bottom plate 56, it is understood, in some embodiments, that rotation of said hub and support plate may otherwise be provided by a friction fit between hub 41 and shaft 19, with said hub being readily removable from said shaft notwithstanding said friction fit.

When the apparatus is normally at rest, skeleton tube carriers 48, together with the test tubes 54 therein, are suspended at a slight negative angle toward their bottom portions relative to the axis of shaft 19 by virtue of the off-center location of hinges 47 relative to the respective axes of tubes 54. Skeleton tube carriers 48 and their respective test tubes 54 are maintained at a substantially uniform negative angle relative to shaft 19 by virtue of the lower portions of spines 49 bearing against collar 57 of bottom plate 56, said angle in some embodiments being in the order of 0° to 5°. The outer periphery of collar 57 is substantially coaxial with the axis of shaft 19.

During the centrifugation step of the processing performed by the apparatus of the present invention, motor M is energized to cause rotation of drive shaft 19 whereby hub 41 and top plate carrier 44 is rotated, whereby test tubes 54 in skeleton tube carriers 48 are caused to swing outwardly on respective hinges 47 under the action of centrifugal force until the bottoms of said test tubes abut the upper inner end portion of the rotor bowl 37 to limit the outward motion of said tubes. During such centrifugation, the heavier mass of materials such as blood cells or the like in test tubes 54 are urged towards the bottom ends of said tubes by the centrifugal forces engendered therein.

After centrifugation takes place, it is then desired to decant the supernatant liquid in test tubes 54. Accordingly, motor M is shut off and the centrifuge bowl 37 decelerates to a stop whereby said tubes pivotally descend to their original slightly negatively inclined position as shown in FIG. 2.

Decantation of the supernatant liquid from test tubes 54 takes place under rotation of the centrifuge rotor while the test tubes are maintained in a parallel or slightly negative angled position of 0° to 5° approximately or more relative to the central axis of the centrifuge depending upon the decantation conditions that are necessary or desired. This is accomplished by providing a vertically movable locking element which comprises a circular horizontal ring plate 63 having a central aperture which surrounds a portion of drive hub 36. Horizontal plate 63 has a peripheral upwardly extending annular flange 64 which terminates in an inwardly extending annular angled locking ring 66. See FIGS. 2, 3 and 5.

During the centrifuging action of the apparatus, plate 63 rests upon the bottom surface of bowl 37 with rim 66 being downwardly spaced apart from the path of the bottoms of tube holders 48, as shown in FIG. 2.

The bottom of bowl 37 has a pair of diametrically spaced apart apertures 67 through which vertical actuator pins 68 freely extend, said pins being connected to horizontal plate 63. The bottom ends of actuator pins 68 rest freely upon a circular actuator ring 69 which surrounds drive hub 36 and which is movable vertically relative thereto.

Mounted on platform 12 is a pivot post 71 to which is pivotally connected a lift fork, generally designated 72, the outer portion of which comprises a manually operated handle 73 and the inner portion of which terminates in a pair of spaced apart tine elements 74 extending to opposite sides of shaft 19. See FIGS. 2, 3, and 7. The ends of tine elements 74 are each formed in the shape of upwardly bowed knuckles 76.

The bottom floor of enclosure 18 has an annualar opening 77 through which knuckles 76 are freely movable vertically. Knuckles 76 are normally in the retracted position, as shown in the solid line representation of FIG. 2. When

3,420,437 ⁵ handle 73 is depressed to raise lift fork 72, knuckles 76 move upwardly through annular opening 77 to bear against and raise actuator ring 69 which, by way of pins 68, lifts ring plate 63 into a position where locking rim 66 is engageable by hook 53 or, optionally, by the outer end 5 of support flange 52 of each tube holder 48.

When bowl 37 is again rotated, the normal outward pivoting action of tubes 54 is prevented by hook 53 or flange 52 engaging locking rim 66 so that said tubes are maintained in a vertical or slightly negative angle position ₁₀relative to the axis of rotor shaft 19. While bowl 37 rotates with the retention of tubes 54 by locking rim 66 against the action of centrifugal force, supernatant liquids discharge from the tops of tubes 54 over onto circular lip 31 and thence into collection trough 28. Said supernatant 15 liquids then flow in trough 38 toward outlet tube 32 through which they are discharged into drain tube 33 for collection or disposal, as may be desired or necessary. Baffle plate 26 prevents splashing of the discharging supernatant liquids which are thereby deflected into trough 28. 20

During decantation operation of the rotor, handle 73 may be continually depressed manually for the comparatively short time that is necessary for such decantation. In some embodiments, however, a bracket 81 mounted on platform 12 may be provided pivotally to support a lever 2a latch 82 which is actuated by a spring 83 to hold handle 73 in the depressed position, as shown in FIG. 4. Handle 73 may be released by manually moving latch 82 out of engagement with handle 73. In other embodiments, solenoid or other electrical means may be provided to operate 30 handle 73 for controlling the position of locking rim 66.

When rotor bowl 37 comes to rest after decantation, the bottom ends of tube holders 48 and tubes 54 move inwardly by pivoting under their own weights on the offcenter hinges 47 until spines 49 bear against collar 57 of 35 bottom plate 56, whereby hooks 53 have become disengaged from locking rim 66. Thereafter, with handle 73 released, locking rim 66, plate 63, and actuator ring 69 descend by gravity, thereby lowering locking rim 66 away from the arcuate path otherwise traversed by hook 53 40 when tubes 54 are subjected to centrifugation. The descent of actuator ring 69 also causes the descent of knuckles 76 when downward pressure is released from handle 73.

When tubes 54 are in the decanting position, the tops thereof are spaced apart from the inner edge of lip 31 of trough 28. Under centrifugal action in this circumstance, supernatant liquid in each tube 54 rises upwardly along the outer portion thereof and when said liquid reaches the top of the tube, the liquid traverses across the gap into trough 28 under centrifugal action without any leakage of ₅θ liquid into rotor bowl 37.

After decantation has taken place, cover lid 21 may be lifted from enclosure 18 so that washing fluid or the like may be added to test tubes 54 for further treatment of the blood cells or other biological materials that are be- 55 ing processed therein. Thereafter, cover lid 21 is closed and successive centrifugation and decantation processing steps may be repeated.

After a sufficient number of cycles of centrifugation and decantation have taken place, cover lid 21 is opened 60 and with locking rim 66 in the disengaged position, knob 43 is grasped and lifted vertically to remove hub 41 from shaft 19 whereby the assembly of top plate 44, bottom plate 66, test tube holders 48 and test tubes 54, is simultaneously removed as a separate, self-contained unit from ⁸⁵the centrifuge, as shown in FIG. 6, with flange 58 serving as a base for location upon a table or platform. It may also be desirable to provide a separate stand for said assembly as shown in another portion of FIG. 6 in the form of a base 86 of suitable shape standing upon a table or bench 87, said base 86 having a vertical dummy shaft 88 which has the same dimensions as shaft 19. Hub 41 is placed upon dummy shaft 88 so that the assembly of the test tubes may be securely positioned in order to facili- 75 tate the handling of said test tubes 'by the laboratory experimenter or technician.

Although deflector 26 and trough 28 are shown in FIG. 2 as being connected to cover lid 21 whereby all of said elements are removable as an assembly from enclosure 18, it is contemplated that suitable modifications can be arranged whereby cover lid 21 and deflector 26 together are removable while trough 28 remains secured to enclosure 18 and is separately removable therefrom for cleaning. In both alternative structures, drain tube 33 may be disengageable from outlet tube 32. Deflector 26 and trough 38 may also be removably attached to cover lid 21 whereby said components may be disassembled for cleaning or replacement and then reassembled.

In some processing operations, it may be desirable to perform the decanting step with the test tubes retained at a small positive angle relative to the axis of the rotor. Accordingly, as shown in FIG. 9, the apparatus is modified by providing that plate 63α has a larger diameter than plate 63 in FIGS. 2, 3 and 5. Plate 63α has an integrally formed vertically extending flange 64α which terminates in an inwardly extending annular angled locking rim 66α, the diameter of locking rim 66α being somewhat larger than the diameter of locking ring 66 in FIGS. 2. 3 and 5.

Thus, when plate 63α is lifted into the hooking position and the rotor is spun, upon outward movement of tubes 54 hooks 53 are engaged and retained in position by locking rim 66α in a location where tubes 54 are secured at a small positive angle relative to the axis of the centrifuge rotor. In this position, when the rotor turns at the lower decantation speed, as described hereinbefore, the extent of decantation can be controlled. Furthermore, it is evident that, because of the small positive angle of tubes 54, which are restrained in that position, a certain controlled amount of residue can be detained in said tubes irrespective of the speed of rotation. By selecting the diameter of plate 63α, it is possible to determine the extent to which a particular desired quantity of material should remain in the tubes after decantation. The positive angle of retention of tubes 54 may vary between 0° to approximately 15° or more relative to the central axis of shaft 19, depending upon the decantation conditions that are necessary or desired.

In the composite illustration of FIGS. 10 and 11, an arrangement is provided whereby a single component may be operated to retain the test tubes in either the negative or positive angle decantation positions. Horizontal plate 63 is provided with an integrally formed outwardly extending ring 63b which has an integrally formed upwardly extending annular flange 64Z> which terminates in an inwardly extending annular angled locking rim 66b. Alternatively, hooks 53 may be engaged either by coaxial locking rims 66 or 66b, depending upon whether tubes 54 are to be retained in a negative angled (FIG. 10) or positive angled (FIG. 11) decantation position, respectively, at the choice of the operator of the apparatus. Other suitable equivalent means may be utilized to provide alternative and selective retention of tubes 54 in either the small negative or small positive angled position for decantation purposes.

In order to facilitate the use of the alternatively selectable device shown in FIGS. 10 and 11 for locating the tubes 54 in a positive angle position, a removable ring 91 may be slipped over the shoulder 57 of plate 56 whereby the lower portions of spines 49 will be prevented from moving too far inwardly so that hooks 53 will be engaged only by locking rim 66b.

Although it has been indicated herein that decantation may take place at a lower speed than that of centrifugation, it is to be understood that the particular speed of decantation may be predetermined by the particular angle at which tubes 54 are retained and the acceleration rate of the rotor on which said tubes are mounted. By controlling the negative or positive angle of the tubes relative

3,420,437 to the axis of shaft 19, the acceleration rate of the rotor and the duration of rotation of the rotor, it is possible to decant the supernatant liquid from tubes 54 down to a precise volume. In some embodiments, a suitable variable speed control may be connected to motor M for determining the most advantageous speed of said motor in the circumstances of the particular structure of the apparatus and the gap across which the supernatant fluid is to traverse between the tops of tubes 54 and the inner edge of lip 31 of trough 28. In other embodiments, automatic determination of a specific decantation speed different from the centrifuging speed may be accomplished by providing a microswitch 85 on bracket 81 which is actuated when handle 73 is depressed, said switch being connected to suitable electrical speed control circuitry well known in the art.

It is evident that when tubes 54 are retained at a slight negative angle as shown in FIGS. 3 and 5, the time of rotation of the centrifuge at the lower decantation speed must be limited by the operator to prevent complete evacuation of the contents of said tubes. Accordingly, a definite time limit must be prescribed for decantation at the negative angle either by visual inspection or by predetermined time periods as established by empirical experience so that the desired amount of residue will remain in the test tubes after decantation.

As to the conditions obtaining when decanting from test tubes 54 are retained at a positive angle relative to the rotor axis, the decantation angle will be determined, in addition to the factors of rotor speed and time of rotation, by the dimensions of tubes 54. The retention of tubes 54 at a positive decantation angle will delimit the amount of supernatant liquid that can emerge from the upper and outer edge portion of the mouths of said tubes.

It is evident that the greater the positive angle of decantation retention, the lesser amount of fluid will be discharged by decantation from tubes 54. With positive angle decantation, the inside diameter of lip 31 of trough 28 will be somewhat smaller and would be suitably retracted to provide a small enough gap for the decanting liquid to flow from tubes 54 into trough 28.

The apparatus of the present invention is particularly useful where repeated washing and centrifuging operations are necessary as, for example, in the procedure for testing cells for the D^u factor by the indirect antiglobulin (Coombs) test wherein a saline suspension of red blood cells are successively centrifuged and washed at least three times. It is evident that with centrifuge apparatus that lacks the inherent means for decantation, considerable time and eflort would have to be expended in removing supernatant liquid from a plurality of test tubes for several successive stages of the processing. The present apparatus, however, conserves considerable time and effort whereby the decantation takes place simultaneously from all of the test tubes without the necessity for handling each of them. Refilling of the test tubes with successive quantities of wash solutions in the apparatus described herein is quickly and easily accomplished for the next succeeding centrifuging operation.

The present invention contemplates within its scope the provision of means for removably mounting a plurality of test tubes relative to a centrifuge rotor whereby said test tubes are enabled to swing outwardly during a centrifuging step and are prevented from swinging completely outwardly during a decantation step. Also, simple test tube holders are provided comprising means that not only serve to support the bottoms of the removable test tubes, but also are engageable by a movable locking element associated with the rotor for retaining the holders and the test tubes therein in the decantation position during rotation of the rotor.

Although the present invention has been described with reference to particular embodiments and examples, it will be apparent to those skilled in the art that varia8 tions and modifications can be substituted therefor without departing from the principles and true spirit of the invention.