Liquid purification or separation – Magnetic
Reexamination Certificate
2000-09-11
2001-10-16
Reifsnyder, David A. (Department: 1723)
Liquid purification or separation
Magnetic
C210S695000, C356S038000, C356S246000
Reexamination Certificate
active
06303030
ABSTRACT:
BACKGROUND OF THE INVENTION
Ferrography is a method of separating suspended particles displaying magnetic properties from a liquid by passing the liquid through a magnetic field. The interaction between the magnetic field through which the particles move and the magnetic dipole moments of the particles causes the particles to deposit onto a substrate in the region of strongest field gradient. This approach, applied to removing wear particles from used lubricating oil, was first described in U.S. Pat. No. 4,047,814, herein incorporated by reference.
Ferrographic techniques have also been applied to cell sorting methods that exploit differences in surface protein compositions of biological cells, such as find wide application in research and in diagnostic procedures relating to cancer and immunodeficiency diseases, as well as in other biomedical applications. This type of analysis is based on the ability of magnetic tags conjugated to appropriate antibodies to attach to specific surface protein compositions. For example, after magnetic tagging, lymphocytes may be readily sorted ferrographically. This approach to cell sorting efficiently concentrates the cells of interest and thus does not require sophisticated fluidic or optical systems for subsequent study or counting of the cells.
U.S. Pat. No. 5,714,059, herein incorporated by reference, discloses an analytical ferrograph, also called an analytical magnetic cytometer, suitable for magnetically driven cell deposition. Its magnet has pole members defining an interpolar gap therebetween with a relatively high magnetic flux density. For ferrographic analysis of biological cells suspended in a sample fluid, an aliquot of the sample fluid is passed through a liquid-tight flow pathway, including a flow chamber which is part of a larger flow unit, disposed in the fringing magnetic field. In the flow chamber, this flow pathway is defined by opposing parallel plates, one of which, the substrate, is mounted against the pole members and over the gap. Particles that are magnetically susceptible (either naturally or due to prior preparation by techniques well known to those skilled in the art) are separated from the balance of the sample fluid, and are deposited onto a deposition surface, i.e., the interior surface of the substrate, facing away from the pole gap. After the entire aliquot has passed through the flow chamber, the flow unit is disassembled and the deposit is analyzed.
A flow chamber in the flow unit is defined by a hole through a spacer sheet, or gasket, of elastomeric material disposed between the two plates, one of which is the substrate and the other of which is referred to herein as the platen. Pressure applied perpendicularly to the substrate surface maintains the integrity of the seal around each flow chamber. Or, electrostatic or frictional forces can hold the plates against the spacer sheet, parallel to one another. Free ends of the sheet extending beyond the edges of the substrate and platen facilitate disassembly of the unit without disruption of the substrate or deposit.
A flow unit including several parallel flow chambers, each defined by a hole through the gasket, is used to permit simultaneous processing of several aliquots under identical flow and magnetic field conditions. Collection of the resulting deposits in compact form on a single deposition surface facilitates comparison of different deposits derived from the same fluid source.
Several features of the flow unit promote optimal performance of such a system. Typically, the substrate onto which the cells are deposited is a thin slide of common borosilicate glass. The optical transparency, mechanical rigidity, smooth surface, and low chemical reactivity of glass facilitate analysis of the deposit after removal of the flow chamber from the apparatus. A very thin substrate—preferably on the order of 100 micrometers—allows the deposition surface to be as close as possible to the interpolar gap so that tagged cells in the fluid flowing adjacent the gap encounter strong field gradients which efficiently draw the cells to the surface, where they are deposited in compact, well-defined regions. Finally, the flow unit is economically disposable after a single use to satisfy hygienic requirements.
Although known ferrographic systems effectively separate biological cells, several characteristics of past fluid pathway implementations have limited their serviceability. For example, to form a fluid pathway, thin tubing is typically joined to the flow unit at two orifices in the platen which serve as ports for a single flow chamber. As the sample fluid passes through such a pathway, surface irregularities—such as discontinuities, crevices or diameter changes—along the length of tubing and especially at the joints, induce suspended material, including magnetically tagged bodies of interest, to be caught and remain at these unintended deposition sites. Specimen matter is lost in greater quantity with an increase in the tubing length or the number and/or abruptness of diameter changes experienced by the sample fluid before it reaches the desired deposition site on the substrate. Processing protocols entailing moving the sample fluid through the flow chamber in one direction and then reversing the flow direction to pass the same fluid through the flow chamber a second time also are predisposed to loss of specimen matter.
Another difficulty with known systems is related to the processing of several fluids. The aliquot of sample fluid is typically followed through the fluid pathway by one or more additional liquids in order to interact with material deposited on the substrate. For example, one such liquid may stain deposited cells to facilitate their visual examination. A rinse fluid may then be applied to remove extraneous stain material or, even if no stain is applied in situ, to reduce the number of nonmagnetic elements entrained in the deposit. Such a multi-fluid sequence is often implemented by consecutively disposing the several liquids in one of the lengths of tubing and then moving the liquids through the flow chamber. Gas bubbles at the interfaces between adjacent fluids collect in the flow chamber, interfering with deposition of specimen material. The total amount of sample fluid that can be conveniently processed is limited by the tubing volume. The surface irregularities and extended distance which the interface between adjacent fluids traverses dispose the fluids to premature and uncontrolled mixing which, in the case of a staining liquid following the sample fluid, impairs control of the staining operation.
DESCRIPTION OF THE INVENTION
OBJECTS OF THE INVENTION
It is, accordingly, an object of the invention to provide a ferrographic apparatus and method that allows processing of a sample fluid without introducing bubbles into the fluid pathway.
It is another object of the invention to provide such an apparatus and method capable of processing arbitrarily large sample volumes.
It is yet another object of the invention is to provide such an apparatus and method that reduces the loss of specimen material by unintended deposition of suspended material before it passes through the magnetic field.
It is yet another object of the invention to provide such an apparatus and method capable of capturing cells present in a sample fluid at very low concentrations.
It is yet another object of the invention to provide such an apparatus and method capable of consecutively processing several fluids without introducing gas bubbles into the fluid pathway, and with minimal mixing therebetween.
Yet another object of the invention is to provide such an apparatus and method that affords improved exposure control of specimen material displaying magnetic properties to staining agents.
Another object of the invention is to provide such an apparatus and method that minimizes subjection of the specimen materials to destructive conditions, such as passage through a peristaltic pump, before they pass through the flow chamber.
It is another object of the invention to provide such an apparatus a
Desjardins John B.
Seifert William W.
Wenstrup Robert S.
Westcott Vernon C.
Guilfoyle, Inc.
Reifsnyder David A.
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