US 3452924 A
Description (OCR text may contain errors)
July l 1969 v c. A. scHLUTz 3,452,924
SYSTEM AND METHOD FOR WASHING BLOOD AND THE LIKE Filed Feb. s, 1965v sheet of 2 INVENTOR. C24/wis Alfa/U72 ATTORNE V SYSTEM AND METHOD FOR WASHII-IG BLOOD AND THE LIKE Fild Feb. s, 19e-5 C. A. SCHLUTZ Julyvl, 1969 Sheet INVENTOR. (f4/mw Es A62-#1. z/rz United States Patent O U.S. Cl. 233-14 6 Claims ABSTRACT OF THE DISCLOSURE Method and apparatus for continuously washing blood or other biological cells in suspension by introducing said suspension at a plurality of radially spaced apart regions within separate tubes that are readily arrayed and move centrifugally around a common axis.
This invention relates to a method and apparatus for continuously washing blood or other biological cells in suspension by passing a saline or other Washing solution through said suspension under centrifugation to remove contaminating proteins or other materials attached to the cells.
The apparatus herein may also be used for washing other types of both organic and inorganic particles in various suspending media, as may be required or necessary.
Heretofore, the washing of blood has been accomplished by a batch process in which a vial of sterile blood has added to it a first volume of saline washing solution, after which `the resultant mixture is vigorously shaken manually to effect mixing, and then inserted into a centrifuge. After the vial has been centrifuged, the saline solution together with some plasma contaminants are withdrawn, and a second fresh quantity of saline solution is added. The foregoing process is repeated at least four times to achieve the desired purification. This entire processing takes as much as 'four hours or more. One undesirable byproduct of this batch process is that the long time of processing causes the blood cells to exude a number of mucoid substances which contaminate the sera to which these cells might be added for absorptive purposes.
The present invention overcomes these drawbacks of the batch process by teaching a system of balanced hydrodynamic centrifugal fluid columns in which the saline washing fluid component is continuously interjected into a plurality of centrifugal acceleration regions, so that a counterow of blood cells and washing solution simulate the previous shaking operation, thereby combining the mixing, washing, and centrifugal separation processes, and reducing the processing time to the order of one-half hour so as to obviate the exudation of mucoids, and to minimize t-he technician-hours spent in processing the blood.
It has been determined by an exhaustive research program using a centrifugal column of glass beads and packed human erythrocytes with the supernate solution dyed by a neutral Acriflavin dye, that the optimum washing of a 50 ml. volume centrifuge tube takes place when the Washing solution is introduced simultaneously on at least several spaced apart regions of centrifugal acceleration. Subsequent tests with the disclosed gradient chamber apparatus have Verified the effectiveness of the invention in rapidly removing the unwanted residual plasma proteins.
An additional feature of the invention provides sterile means to transfer the cleansed blood cells from the centrifugal gradient chambers into a sterile storage container.
3,452,924 Patented July 1, 1969 lCe It is an object of this invention to provide a method of blood washing which 'will continuously and rapidly under sterile conditions pass a large volume of Washing solution in counterflow relation to the blood or other biological cells, and which will thereafter permit the sterile transfer of the cleansed cells to a storage container.
Another object of the invention is to provide a design of centrifugal gradient chamber which will permit the Iintroduction at a plurality of acceleration regions of a mixture of blood and washing solution while continuously withdrawing the effluent.
Still another object of the invention is to provide a hydraulic circuit in which a sterile blood bag, a sterile washing solution reservoir, `a positive displacement pump, a balanced column rotating joint continuous flow centrifuge, a centrifugal gradient washing chamber, and an exhaust duct can be interconnected to effect the efficient Washing of the blood cells or other biological cells. In such an arrangement, disposable sterile blood bags may be utilized and the complete system may be maintained in a sterile condition from -the input end to the output end.
A tfurther object is to provide a design of auxiliary sterilizable accessory ducts for the inlet and outlet tubes of the Iforegoing acceleration gradient washing chamber which rwill expedite the sterile transfer of the processed blood cells to a storage container.
Still other objects and advantages of the invention will be apparent from the specification.
The features of novelty which are believed to be characteristic of the invention are set forth herein and will be best understood, both as to their fundamental principles and to their particular embodiments, by reference to the specification and accompanying drawings, in which:
rFIGURE 1 is a diagrammatic view of an embodiment of the subject blood or cell washing system, showing the hydraulic circuit;
FIGURE 2 is a side view, partly in section, ont the centrifugal acceleration gradient chamber of the subject invention;
FIGURE 3 is an enlarged, fragmentary view of the lower portion of the inlet gradient injection or distributor tube, a portion of which is shown in FIGURE 2, and which illustrates the spaced apart distributor ports as Well as the restrictive end orifices therein;
FIGURE 4 is a side view showing the installation of the auxiliary intake air filter and exhaust needle with its guard, preparatory to the transfer of washed blood to its storage container; and
FIGURE 5 is a somewhat smaller wiew of the foregoing transfer assembly for gravity transfer of Washed blood cells to its storage bottle.
Referring to the diagram of FIGURE 1, plastic bag 21 contains the sterile blood specimen, which typically might have a volume of from to 400 ml., said bag 21 being suspended at the highest point in the system to generate a hydrostatic head which insures that the blood be deposited into the centrifuge early in the washing cycle. Bag 21 is sterilized and evacuated prior to being lled with the blood sample.
A reservoir bottle 22 for the washing solution is provided With a stopper 23 through which are sealed a short air intake tube 24 and a long deep outlet tube 26. Intake tube 24 communicates with the outside atmosphere through an air filter 27 to maintain the sterility of the system.
The washing solution, which will typically be a hypertonic saline solution having a volume of four to ve times that of the blood sample, is drawn from the bottom of bottle 22 via tubes 26, 28 and 29, at a controlled rate by the action of the positive displacement electric motor driven pump 31. A useful type of pump for this purpose is one which uses a pair of spaced apart rotating rollers on a semi-circular track where they progressively collapse the wall of resilient rubber tube 29 so as positively to displace the fluid without the possibility of contaminating it.
Similarly, the blood from bag 21 is drawn by pump 31 via tube 32 and the tubular junction fitting 33, where the blood and saline washing solution mix, to pass together through pump 31 and tube 29. The mixture then passes through a flow meter 34, the series connecting tubes 36, 37 and 38, thence entering the electrically motor driven rotating-joint continuous iiow centrifuge rotor 39. Such centrifuges, which are well known in the art, are based on the principle of balanced hydrodynamic columns, in the sense that incoming liquid, such as that entering via tube 38, is passed through a rotating seal into a centrifugal chamber or tube 44 having a closed outer end or bottom through a path which carries it into progressively higher centrifugal accelerations.
However, on continuous fiow centrifuges, the outlet column also passes through a separate rotary seal and hydraulically joins the inlet column in the centrifuging chamber. Consequently, except for the difference in density between the incoming and outgoing columns, there is a major balancing of the hydrodynamic columns, thereby permitting the continuous flow into and out of the high centrifugal field forces.
The motivating force which causes fiow of fluid into and out of the centrifugal field is hydraulic head; i.e., a preponderance of fiuid head in the inlet lines over that in the outlet. Thus the incoming fluid is conducted down the rotating axis of the centrifuge to a small central chamber where it is distributed to all the tubes from a point as near the axis of rotation as possible. The outlet tube, on the other hand, feeds to a chamber also concentric with the axis, but whose periphery is radially further away. The difference, in these two resultant radial columns, then, is the hydraulic head which effects continuous iiow through the rotating centrifuge rotor.
To maintain balance, the centrifuge tubes 44 are used in diametrically opposite pairs, and by a double manifold (not shown) the inlet tubes 48 of the several tubes 44 are connected together, and the outlet tubes 49 of these tubes are connected together.
The effluent from the continuous flow centrifuge 39 is passed via serially connected tubes 41, 42 and 43, to a waste discharge. The entire centrifuge 39 together with its motor 46 is housed within a temperature controlled chamber 47 which may also be maintained in sterile condition.
Centrifuge tubes 44 extend radially outwardly and downwardly from the axis of rotation of rotor 39. As said tubes 44 move around said axis of rotation, centrifugal force fields having radial gradients of acceleration are set up in said tubes, the centrifugal forces increasing in different region of said tubes depending upon their distances therein from said axis of rotation.
Referring now to FIGURE 2, which shows the detailed construction of the subject acceleration gradient centrifuge tube 44, there is provided a circular compression element 51 having a central upwardly extending threaded shaft 52 which extends freely through a central aperture in circular cap 53 which extends over and abuts the upper end of centrifuge tube 44. Cap 53 has an annular collar 54 which extends inwardly into the chamber of tube 44. Compression element 51 has an annular recess which accommodates a resilient O-ring 55. When nut 56 is tightened on shaft 52, O-ring 55 is expanded by the increased pressure between compression element 51 and collar 54 to effect a seal between the inner wall of centrifuge tube 44 and the assembly of said compression element and cap.
Both inlet tube 48 and outlet tube 49 are secured and sealed to the compression element 51 and extend freely through suitable apertures, not shown, in cap 53. Outlet tube 49 terminates flush with the inner surface of element 51 while inlet tube 48 continues nearly to the bottom end of the chamber of centrifuge tube 44.
Inlet tube 48 is constricted at its bottom end to form one or two small terminal ports 57 (see FIGURE 3). In addition, tube 48 is provided with groups of spaced apart radial distributor ports 58.
Distributor ports 58 are not only located in spaced apart gradients of centrifugal fields, but their respective locations also facilitate good dispersion and mixing of the wash fluid with the specimen cells. Depending upon the type and size of cells and other particulate materials that are to be washed and upon the nature of the washing medium, the sizes of distributor ports 58 may be selected and determined to provide for optimum efiiciency of the washing process. The size of distributor ports 58 may also be selected and determined to control the rate of dispersion and velocity at which the materials flow through tube 44.
In one embodiment, the dimensions of the holes 58 are approximately drill size 60, but it is contemplated that other drill sizes may be selected for said holes in adapting the apparatus for different types of materials to be processed thereby.
By making ports 57 small enough so that fiow therethrough is comparatively slow, a back pressure is induced throughout inlet tube 48 thereby causing the incoming mixture of blood and washing fluid to flow through all of the ports 58 along the length of said tube into several spaced-apart regions of centrifuge tube 44. By this means, during the centrifuging motion of centrifuge tubes 48, the heavier blood cells will move outwardly and downwardly, while the washing fiuid which is of lesser density than the blood cells will move in a counterfiow direction inwardly and upwardly through centrifuge tube 44 and emerge as an effluent through outlet tube 49 to be transmitted through tubes 41, 42, 43 to a discard location.
The provision of a plurality of regions of introduction of the materials to 'be centrifuged greatly increases the efficiency of the washing action taking place within the centrifuge tube 44 and greatly speeds up the production of washed cells on a continuous basis.
After the washing operation is complete, and before the washing uid in bottle 22 reaches the bottom of tube 26, the centrifuge motor 46 is deenergized to stop the rotor and the centrifuge tubes 44 are removed.
In order to transfer the processed blood cells from tube 44 to a storage bottle 59 (FIGURE 5), an air filter 61 (FIGURE 4) is attached by a sterile tube 62 to tube 48 while a hollow sterile needle 63 with a protective cover 64 is connected to tube 49 by the sterile tube 66.
Tube 44 is then clamped in a support pedestal 67, cover 64 is removed, and the needle 63 is caused to pierce the resilient cap 68 enclosing storage bottle 59. Gravity will then complete the transfer.
In actual experience with the apparatus described herein, sterile conditions have been achieved from the input end to the output end of the system as exemplified by passing twenty liters of thioglycollate media through the system on several occasions with sterile salvage. The efficiency of the wash system has been established as determined by findings of removal of human protein when tested by anti-human precipitant serums.
It is to be noted that the principles of this invention may also be used for the washing of bacterial germ cells in various liquid agents, and in the glycerolization of red cells.
1. The method of washing blood cells or the like in a liquid washing solution comprising the steps of forming a mixture of blood with a liquid washing solution having less density than said blood, moving a radially extending centrifuge tube with a closed outer end around an axis of rotation, introducing said mixture into said moving centrifuge tube at a plurality of radially spaced apart regions therethrough having differing values of centrifugal acceleration whereby the blood cells in said blood move in an outward direction in said centrifuge tube in counterflow to the movement of said washing solution, and withdrawing the effluent washing liquid from said centrifuge tube at a region of least centrifugal acceleration.
2. A method according to claim 1 and further cornprising means for continuously transmitting said mixture into said centrifuge tube as it rotates, and means for continuously removing the efuent washing liquid from said centrifuge tube.
3. A centrifuge structure for washing biological cells such as blood or the like comprising a rotor, a plurality of centrifuge tubes having closed outer ends arrayed radially in said rotor, means for continuously introducing materials to be centrifuged into each of said centrifuge tubes as the latter move around the axis of rotat-ion of said rotor, and outlet tube for continuously removing eluents from said centrifuge tubes during said rotation, said introducing means comprising an inlet tube extending into the interior and substantially throughout the length of each centrifuge tube and a plurality of spaced apart ports on said inlet tube for introducing said materials into the respective centrifuge tube at spaced apart regions therethrough where different degrees of centrifugal acceleration are engendered during rotation of said rotor.
4. A structure according to claim 3 and further comprising a constriction on the outer end of said inlet tube in the interior of said centrifuge tube, said constriction being suficiently small to induce back pressure in said inlet tube to cause the materials to be centrifuged to emerge simultaneously through all of the spaced apart ports therein.
5. A structure according to claim 3 and further comprising a constriction on the end of said inlet tube in the interior of said centrifuge tube, said constriction forming at least one port on the end of said inlet tube, the aperture of said port being sufficiently small to induce back pressure in said inlet tube to cause said materials to emerge simultaneously through all of the spaced apart ports on the remainder of said inlet tube.
6. A continuous Washing system for biological cells in suspension according to claim 3 and further comprising a container for said suspension, a washing solution container', an air inlet duct including an air lter communicating with said washing solution container, a positive displacement pump, first duct means communicating between the inlet of said pump and both said suspension container and said solution container, second duct means communicating between the output of said pump and the said inlet tube of said centrifuge tube, and third duct means communicating between said outlet tube of said centrifuge tube and the exterior of said centrifuge.
References Cited UNITED STATES PATENTS 2,834,541 5/ 1958 Szent-Gyorgy et al. 233-26 3,050,238 8/1962 Doyle et al 233-15 3,133,880 5/1964 Madany 233-15 3,235,173 2/1966 Unger 233-26 XR 2,758,597 8/1956 Elder 12S-214 2,775,240 12/ 1956 Morrissey et al. 128-214 3,064,646 11/ 1962 Earl 128-272 XR4 3,185,154 5/1965 Caccavo et al. 128-272 3,347,454 10/1967 Bellamy et al. 233-26 XR 3,092,106 6/ 1963 Butler 128-214 FOREIGN PATENTS 864,410 4/ 1961 `Great Britain.
HENRY T. KLINKSIEK, Primary Examiner.
U.S. Cl. X.R.