Chemistry: analytical and immunological testing – Including sample preparation – Liberation or purification of sample or separation of...
Reexamination Certificate
1998-07-17
2001-05-22
Soderquist, Arlen (Department: 1651)
Chemistry: analytical and immunological testing
Including sample preparation
Liberation or purification of sample or separation of...
C436S174000, C436S178000, C436S180000, C436S183000, C494S037000
Reexamination Certificate
active
06235537
ABSTRACT:
FIELD OF THE INVENTION
This invention relates to a method and apparatus for washing biological cells that is compatible with automated equipment.
BACKGROUND OF THE INVENTION
Many procedures involving the preparation of biological cells for analysis require that unreacted reagents and cellular debris be separated from the cells of interest. Traditionally batch centrifugation has been the method of choice to perform this separation. However, batch centrifugation is not readily adaptable to automated sample preparation systems.
Most automated sample preparation equipment utilize circular or rectangular arrays of disposable test tubes. The test tubes are transported to appropriate positions so various operations can be performed sequentially and discretely on each test tube. In many sample preparation procedures, the time between discrete operations must be carefully controlled to obtain reliable and reproducible results. However, the operation of cell washing by centrifugation, as it is practiced in the art, is a batch operation. This batch operation cannot be performed sequentially and discretely on each test tube. The presence of a batch operation by definition disrupts the timing of the discrete operations which precede and follow the batch operation.
Batch centrifugations require substantially equal volumes of liquid in each tube to balance the centrifuge rotor, which may not always be desirable in an automated sample preparation system.
Batch centrifugations require rotational alignment of the centrifuge rotor with the loading/unloading system which introduces additional complexity into the centrifuge drive. Samples also require positive sample identification after centrifugation to verify rotational alignment was achieved.
Lastly, the centrifuge rotor and loading/unloading apparatus increases the size, weight and complexity of the system.
In spite of these limitations, automated sample handling systems that utilize batch centrifugation do exist. The ASHS system, marketed by Automed, automates the loading and unloading of conventional centrifuges using robotics; however such systems are large and costly, and only suitable for high volume laboratories.
Another category of instruments has been developed for blood washing and processing, as described in U.S. Pat. Nos. 5,405,308, 4,983,158, 4,668,214, 4,300,717, and 4,086,924. Generally these instruments consist of a bowl assembly with a central feed tube to introduce blood or wash solution to the bowl, feed tube and seal assembly which provide an input feed line to the bowl and an output line from the bowl, and a core assembly that imparts angular velocity to the incoming fluid. These instruments are not suitable for cell washing in automated sample preparation equipment because they require specialized bowl/core structures to enhance the processing of large quantities (ca 500 ml) of undiluted blood.
A number of companies market automatic cell washers. One such system, the Centra-W Automatic Cell Washer marketed by IEC, automate the aspiration of supernatant and addition of dilutent, but still require manual loading and unloading of the sample containers into and from the centrifuge rotor. Consequently, these types of instruments are not compatible with automated sample preparation equipment.
It would be desirable to have a method and apparatus to wash cells that are compatible with automated sample preparation equipment. Such a method and apparatus should operate on each sample individually so it can be synchronized with the other discrete operations performed on the samples.
It would also be desirable to have an apparatus that could be implemented as a discrete processing station on the periphery of a carousel or linear track sample preparation system.
Furthermore, it would be desirable to perform the wash step in the same disposable test tubes commonly used in automated sample preparation and analysis equipment.
It would also be desirable for the cell washer to effectively wash out the undesired cellular debris and unreacted reagents, and to concentrate the desired cells.
Lastly, it would be desirable to have the cell washer apparatus operate without an elaborate system to transport the sample containers to and from the cell washing apparatus.
SUMMARY OF THE INVENTION
The invention described herein provides an improved apparatus and method for removing debris and unbound reagents from cellular suspensions contained in disposable test tubes. The cell washer invention can be implemented into a variety of sample processing systems by a variety of suitable handling system embodiments familiar to those skilled in the art.
A disposable test tube containing the cell suspension to be washed is rotated about its longitudinal center line at speeds sufficient to force the cell suspension up the inner wall of the test tube. This film, typically less than a millimeter thick, is retained by an o-ring near the end of a spindle assembly concentric with the test tube. The o-ring also transfers torque from the spindle to the test tube to rotate it.
After a few seconds of rotation, the larger, more dense cells will migrate radially to the inner wall of the test tube under the action of centrifugal forces. At this time, a cell compatible washing fluid is delivered to the bottom of the test tube from an external reservoir. This wash fluid displaces the fluid containing smaller and less dense cells, cellular debris, and unbound reagent upwards, through radial passageways in the spindle, and out through suitable passages to an external waste reservoir. Wash fluid thus displaces and removes the unwanted supernatant cell suspension fluid and thus effects a washing of the cells. This process continues until the desired degree of cell washing is achieved. The wash fluid and waste fluids may be moved by suitable pumps which may produce a vacuum inside of the test tube. In the preferred embodiment described a pump producing a vacuum is used because it is a simple system and allows easy introduction and removal of air and washing fluid. The annular outflow passage in the spindle assembly has an outer diameter which in conjunction with the test tube inner diameter establishes the radial thickness of the cell suspension fluid. The final suspension volume may be controlled by the final rotational speed of the spindle. If the rotational speed is not decreased at the end of the wash cycle, the final suspension volume is represented by the annular volume between the outer diameter of the spindle. If the rotational speed of the spindle is reduced while continuing to introduce wash fluid, the reduction in rotational speed causes the wash fluid to assume a parabolic shape, thus increasing the final suspension volume. In this manner, the final suspension volume of the sample may be controlled.
The sedimentation velocity for spherical shapes is related to the difference in density of the particle and the surrounding fluid, the particle diameter, the fluid viscosity, and the particle acceleration by Stokes Equation. For irregular shapes, the density, shape and size of the particle and the viscosity of the surrounding fluid and the centrifugal forces determines the sedimentation velocity.
After the cells are adequately washed, and the resuspension volume has been established, the wash fluid flow is stopped. Then the drive motor is rapidly stopped by braking, either mechanically or electrically. Dynamic braking of a permanent magnet field direct current motor is used in the preferred embodiment because it is simple, convenient, and does not add parts which can wear out. The rapid stopping of the test tube rotation causes the fluid inside the test tube to continue rotating which washes over the cells at the test tube inner wall, which in turn resuspends the cells. Not all cells may be resuspended by a single rapid stopping of the test tube rotation, so the test tube may be rotated and stopped several times to increase cell recoveries. Also, the use of a cell compatible surfactant may be used in the wash fluid to inhibit cell sticking to the test tube.
At this point, c
Chase Eric
North Howard L.
Schulte Harvey
Cytek Corporation
Schneck David M.
Schneck Thomas
Soderquist Arlen
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