Patent Publication Number: US-7713708-B2

Title: Immunological assay system and method

Description:
CLAIM OF PRIORITY AND RELATED APPLICATION 
     This application claims priority to and is a continuation of U.S. patent application entitled, “Immunological Assay System and Method,” filed on Jan. 31, 2001 and accorded Ser. No. 09/773,826, which is incorporated herein by reference in its entirety, which claims priority to provisional patent application entitled, “Method for Diagnostic Laboratory Testing using DFS Columns,” filed Jan. 31, 2000 and accorded Ser. No. 60/179,248, which is also incorporated herein by reference in its entirety. 
     This application is related to U.S. patent application entitled “Immunological Assay System and Method” filed on even date herewith and accorded Ser. No. 11/679,581, which is incorporated herein by reference in its entirety. 
    
    
     TECHNICAL FIELD 
     The present invention is generally related to an immunologic assay system and, more particularly, is related to a system and method for separating components of immunological and immunohematological samples. 
     BACKGROUND OF THE INVENTION 
     Immunologic assays are designed to detect reactions between antibodies and antigens. These assays commonly employ cells, such as red blood cells (RBCs) or beads as antigen “carriers.” In the appropriate assay configuration, antibodies can cross-link the antigen carriers, generating a large three-dimensional antigen-antibody aggregate from what were initially individual antigen carriers and antibodies. In other configurations, antibodies bind to the antigen carriers without cross-linking them. 
     Immunohematology testing in the blood bank setting uses RBCs and antibodies to determine compatibility between transfusion donor and recipient prior to transfusion. For example, the donor and recipient are incompatible if antibodies from the recipient cross-link (agglutinate) RBCs from the donor, resulting in the formation of large RBC aggregates. Current commercially available testing reagents are designed to distinguish these aggregates from individual, non-agglutinated RBCs. For example, in standard “tube testing,” RBCs are mixed with antibodies, centrifuged at approximately 1000×g for a brief period, approximately 30 seconds, to enhance the formation of antigen-antibody complexes, and then gently resuspended by hand in order to be able to distinguish agglutinated from non-agglutinated RBCs. Tube testing is labor-intensive, not amenable to automation, and the results are difficult to standardize from lab to lab since they depend on the skill of the individual operator. 
     An alternative approach used to identify agglutinated RBCs is spin column technology, which is based on standard chromatographic principles. With this methodology, tubes filled with a homogeneous matrix material, e.g., beads, gel, or polyacrylamide, are used to separate aggregated from individual RBCs. The matrix material is designed with holes or pores of a specified size such that under carefully controlled centrifugal forces large (“4+”) aggregates barely enter the matrix. However, successively smaller aggregates (“3+” through “1+”) do enter the matrix to increasing degrees, and non-agglutinated RBCs not only enter the matrix, but sediment completely to the bottom of the tube. In order for a single homogeneous chromatographic matrix to effectively separate individual RBCs from RBC aggregates of various sizes, a relatively long centrifugation run, approximately 10 minutes, must be carried out under carefully controlled low-speed centrifugation conditions of 80×g. Deviations from optimal centrifugation conditions, e.g., higher centrifugation speeds in an attempt to shorten the assay run, lead to poor separation of RBCs, compromising the assay ability to determine compatibility between blood donor and recipient. This methodology is to some extent amenable to automation, and less dependent on operator skill. 
     Spin column technology is significantly more expensive than tube testing, due to costs of producing the columns. The matrix material is in solution, and carefully controlled packaging, shipping, and storage conditions are typically necessary. In addition, testing is slower than with tube testing because of the prolonged centrifugation step, approximately 10 minutes, versus approximately 30 seconds with tube testing. Interpretation of assay results also requires operator training, since the readout is on an “analog” scale, i.e., the distance of RBC migration through the matrix must typically be estimated. 
     Thus, a heretofore unaddressed need exists in the industry to address the aforementioned deficiencies and inadequacies. 
     SUMMARY OF THE INVENTION 
     The present invention provides a system and method for immunological and immunohematological assaying. Briefly described, the assay system includes a filter vessel capable of containing an assay sample, an incubator into which the filter vessel may be placed, a sample separation system in close proximity to the incubator, an image acquisition system in close proximity to the sample separation system, and a robotic pipettor including a robotic arm within reaching distance of the filter vessel, the incubator, the sample separation system and the image acquisition system. The filter vessel includes a filter that may be made of an inert material that includes a plurality of pores. 
     The present invention can also be viewed as providing a method for immunological and immunohematological assaying. The immunological method identifies interactions between a sample and a testing reagent where one contains an antigen carrier (RBC or bead) and the other contains an antibody. In this regard, the method can be broadly summarized by the following steps: placing an immunological assay sample into a filter vessel including a filter, adding a testing reagent to the filter vessel, incubating the sample and reagent mixture in the filter vessel, separating the sample and reagent mixture in the filter vessel into components above and below the filter, and analyzing the filter vessel to determine the presence of interactions between the sample and reagent. 
     In an alternative embodiment, if the step of analyzing the filter vessel produces unclear results, the method may also include the steps of placing the sample in the filter vessel in a washer, separating the antigen carrier from liquid components of the sample by capturing the antigen carrier above the filter in the filter vessel, washing the antigen carrier in the filter vessel under vacuum pressure or centrifugation with a physiological salt solution to remove excess antibody not bound to the antigen carrier, adding antibody reagents to the washed antigen carrier in the filter vessel, incubating the mixture in the filter vessel, separating the antigen carrier from liquid components of the mixture by capturing the antigen carrier above the filter in the filter vessel, and again analyzing the filter vessel to determine the presence of interactions between the sample and the reagent. 
     Other methods, features, and advantages of the present invention will be or become apparent to one with skill in the art upon examination of the following drawings and detailed description. It is intended that all such additional methods, features, and advantages be included within this description, be within the scope of the present invention, and be protected by the accompanying claims. 
    
    
     
       BRIEF DESCRIPTION OF THE DRAWINGS 
       The invention can be better understood with reference to the following drawings. The components in the drawings are not necessarily to scale, emphasis instead being placed upon clearly illustrating the principles of the present invention. Moreover, in the drawings, like reference numerals designate corresponding parts throughout the several views. 
         FIG. 1  is a diagram illustrating an immunological system of the present invention. 
         FIG. 2  is a diagram illustrating a filter vessel system, one example of the type of filter vessel that serves as a component of the immunological system of  FIG. 1 . 
         FIG. 3  is a diagram illustrating a vacuum, one example of a sample separation system component of the immunological system of  FIG. 1 . 
         FIG. 4  is a diagram illustrating a centrifuge, another example of a sample separation system component of the immunological system of  FIG. 1 . 
         FIG. 5  is a flowchart of an immunological assay method of the present invention, which uses the immunological system of  FIG. 1 . 
     
    
    
     DETAILED DESCRIPTION OF THE PREFERRED EMBODIMENT 
     In general, the present invention pertains to a system and method for separating and analyzing components of immunological and immunohematological samples. In this regard, an immunological assay system overcomes the drawbacks of current tube testing and spin column technology, while simultaneously rendering the technology of immunological assay more amenable to automation. In a preferred embodiment, the immunological assay system is an instrument that includes a filter vessel system including one or more filters that have discrete molecular weight and size cutoffs due to the presence of a plurality of holes or pores of specified sizes in the filter. An immunological sample is mixed with a reagent and placed above the filter(s). After vacuum or centrifugation or some other method of inducing the sample through the filter is applied, the components of the sample are separated from one another according to their size by the various filters. 
     The immunological system of the present invention can be used to measure interactions between antibodies and cells, or in some cases between antibodies and synthetic beads that can be modified and/or configured to act as antigen carriers. The immunological system can be used in at least two different ways. In one method, “cellular components” of patient samples, e.g., red blood cells (RBCs), white blood cells (WBCs), or platelets, are mixed with “reagent antibodies.” The components of the mixture are separated, and then analyzed to determine the presence of interaction between the cellular components and the reagent antibodies. In another method, the immunological system may be used in an assay method that mixes patient antibody-containing samples, e.g., plasma or serum samples, with antigen carriers that may be synthetic beads or reagent cells, e.g., RBCs, WBCs, or platelets. This mixture is then separated, and the components are analyzed to determine the presence of interactions between the antibody samples and the reagent cells or synthetic beads. 
       FIG. 1  depicts the immunological system  100  of the present invention. The immunological system  100  is an instrument that includes a filter vessel  105  capable of containing an assay sample, an optional incubator  110  into which the filter vessel may be placed, a sample separation system  115  disposed in close proximity to the incubator  110  or disposed therein, an optional image acquisition system  130  in close proximity to the sample separation system  115 , and an optional robotic pipettor  135  that includes a robotic arm within reaching distance to the filter vessel  105 , the incubator  110 , the sample separation system  115  and/or the image acquisition system  130 . The immunological system  100  may also include an optional washer  140  disposed therein, an optional turntable system  145  which has disposed therein sample holders  146  for holding the assay sample. Further included in the immunological system  100  may optionally be tubes with the assay sample  147  and/or tubes with reagent  148 . 
     The optional incubator  110  disposed within the immunological system  100  is of a shape and size that allows a filter vessel  105  to be disposed therein. While many sizes and shapes of an incubator may be used, in a preferred embodiment, the incubator  110  is of a shape and size so as to allow a plurality of filter vessels  105  or a plate of filter vessels  106  to be disposed therein. The incubator  110  may further include an optional heating element capable of heating the filter vessels when they are disposed in the incubator  110 . 
     The sample separation system  115  is also of a shape and size so as to allow a filter vessel  105  to be disposed therein. While many sizes and shapes of a sample separation system may be used, in the preferred embodiment, a plurality of filter vessels  105  and/or a plate of filter vessels  106  may be disposed therein. The sample separation system  115  may be, for example, but is not limited to, a vacuum  120 , a centrifuge  125 , and/or an applied electric field. The sample separation system  115  is of a type that when the filter vessel  105  is placed within the sample separation system  115 , an assay sample  147  disposed within the filter vessel  105  is drawn through a filter  150 , thereby separating out the assay sample into various components based on size. 
     The optional image acquisition system  130  may be, for example, but is not limited to, a camera, a flow cytometer, a special lens such as a microscope, or even a human eye. Usually, an assay sample is analyzed by the image acquisition system  130  after it has been removed from the sample separation system  115 . The image acquisition system  130  allows analysis of the filter vessel  105  in order to determine the presence or absence of material above the filter  150  disposed within the filter vessel  105 . The image acquisition system  130 , particularly when it takes the form of a flow cytometer, may also be used to determine the size of the material above the filter  150 , for example whether the material is in the form of individual antigen carriers or aggregates of antigen carriers, as well as to determine whether antibodies are bound to the antigen carriers that are present above the filter. 
     The optional robotic pipettor  135  used within the system is of the type commonly known and used by those skilled in the art. For example, but not limited to, the robotic pipettor system that is manufactured by and commercially available from Tomtec, Inc. (Hamden, Conn., U.S.A.) or CRS Robotics Corporation (Burlington, Ontario, Canada) may be used in accordance with one embodiment of the present invention. 
     The optional washer  140  is disposed within reaching distance of a robotic arm of the robotic pipettor  135  from the image acquisition system  130 . If analysis of the material above the filter  150  in the filter vessel  105  by the image acquisition system  130  produces unclear results, the assay sample may be as disposed within the filter vessel  105  may be placed within the washer  140 . The washer  140  is of a size and shape so as to allow the filter vessel  105 , a plurality of filter vessels  105 , and/or a plate of filter vessels  106  to be disposed therein. The washer  140  is designed so as to wash all reagents from the antigen carriers present in the assay mixture, and through the filter  150  of the filter vessel  105 . While there may be many configurations of the washer  140 , in the preferred embodiment, the washer  140  is the same system as the vacuum  120 . 
       FIG. 2  depicts the filter vessel  105  component of the immunological system  100  of  FIG. 1 . “Filter vessel”  105  means a vessel capable of containing an assay sample and including one or more filters disposed therein.  FIG. 2  represents the filter vessel  105  (a) before being placed in the sample separation system  115 , and (b) after removal from the sample separation system  115 . As seen in  FIG. 2 , disposed within the filter vessel  105  is/are one or more filters  150 . The filter  150  comprises an inert material that includes a plurality of pores. The pore size of the filter  150  may be varied, according to the various embodiments of the invention. For example, pores of the filter  150  may be of a size ranging from approximately 0.01 microns to approximately 50 microns. The size of the pores of the filter  150  will depend on the application of the filter vessel  105 . 
     If it is desired that the filter  150  be used to retain, for example, RBC aggregates, while allowing individual red blood cells to pass through the pores of the filter  150 , in one embodiment of this application, the range of pore sizes is between approximately 3 microns to approximately 40 microns. While other pore sizes may be used, in the preferred embodiment, the pore size ranges from approximately 3 microns to approximately 5 microns. If, however, the filter vessel  105  is used to filter fluid away from the antigen carriers (RBCs, WBCs, platelets, or synthetic beads) where the filter  150  is used to retain the antigen carriers, but allow fluid containing antibodies to pass therethrough, the range of pore sizes in the preferred embodiment is approximately 0.1 to approximately 3 microns. The optimal pore size for this methodology is 0.2 microns. 
     The thickness of the filter  150  may also vary in the different embodiments of the filter vessel  105 , depending upon the application of the filter  150 . For example, the thickness of the filter  150  may range from approximately 3 microns to approximately 5 mm. In the preferred embodiment the filter  150  is between approximately 3 microns to approximately 100 microns. Optimally, the thickness of the filter  150  is between approximately 10 microns and approximately 75 microns. 
     The material used for the filter  150  may be any material in the various embodiments of the filter vessel  105 , preferably an inert material, that includes a plurality of pores. The material of the filter  150  may be varied, depending on the application of the filter  150 . If the function of the filter  150  is to retain RBC aggregates, while allowing individual RBCs to pass therethrough, then the filter material may be, for example, but not limited to, a polyester mesh, a nylon mesh, or a polycarbonate track-etched membrane. A filter  150  having material of this type is manufactured by and commercially available from Sefar, Inc. in Kansas City, Mo., U.S.A. If the application of the filter  150  is to retain all RBCs, but allowing other fluids containing antibodies to pass therethrough, in the preferred embodiment the filter material used is a 0.2 micron polyvinylidene difluoride filter membrane manufactured by and commercially available from Corning Life Sciences, Inc. in Acton, Mass., U.S.A. Alternatively, a supported cellulose acetate membrane, for example, Acetate Plus™ membrane manufactured by and commercially available from Osmonics, Inc. (Minneapolis, Minn., U.S.A.), may also be used for this application. 
     As can be seen in  FIG. 2 , the filter vessel  105  may contain a plurality of filters  150 . If a plurality of filters  150  are used, the filters  150 ′ disposed lower or below the first filter  150 , are usually, in the preferred embodiment, of a smaller pore size, thereby breaking out an assay sample into its various components by size. As can be seen in  FIG. 2 , an assay sample  147  (not necessarily part of the invention) is placed within the filter vessel  105  above the first filter  150 . In (b), after undergoing sample separation, the largest-sized aggregates of antigen carriers  155  of the sample  147  remain above the first filter  150 . Non-agglutinated or smaller aggregates  156  of the sample  147  remain above or pass through the second filter  150 ′. 
       FIG. 3  depicts the vacuum system  120 , which is one type of sample separation system  115  component of the immunological system  100 . The vacuum  120  is shown in  FIG. 3  with a plurality of filter vessels  105  or a plate of filter vessels  106  disposed therein. It should be understood that included within the scope of the invention is a vacuum with one or more filter vessels included therein. Connected to the individual filter vessels  105  is vacuum tubing  160 , which passes through a vacuum controller  165 , which is connected to a vacuum pump  170 . 
     The preferred configuration of the vacuum  120  as shown in  FIG. 3  uses a 96-filter vessel plate format. While other configurations are possible in other embodiments, in the preferred embodiment the vacuum system vacuum  120  is one that independently contacts each of the filter vessels  105  and applies pressure to each filter  105  independently of the other filter vessels  105 . In this embodiment, each filter vessel  105  of the filter vessel plate  106  ( FIG. 1 ) is contacted underneath by an individual gasket, which is in turn is attached to an individual piece of tubing  160  that connects the filter vessels  105  to the vacuum pump  170 . Thus, there are  96  segments of tubing from the pump  170  to the plate of filter vessels  106 , and each segment of tubing  160  could independently have vacuum pressure switched on or off. However, it should be understood that the vacuum  120  may also apply the vacuum to a subset of filter vessels  105  at any one time, or could also apply vacuum to the entire plurality of filter vessels  105  or filter vessel plate  106  simultaneously. 
     The pressure that may be applied by the vacuum system may range from approximately −0.1 to approximately −100 inches mercury (in. Hg). In the preferred embodiment, the range of pressure is between approximately −0.1 to approximately −10 in. Hg. Optimally, the pressure maintained by the vacuum  120  is approximately −0.1 to approximately −3 in. Hg. 
       FIG. 4  depicts the centrifuge  125 , which is another type of the sample separation system  115 , a component of the immunological system  100 . It should be understood that any type of centrifuge system known and used by those skilled in the art may be used as the centrifuge  125 . For example, a typical centrifuge manufactured by and commercially available from Beckman Coulter, Inc (Fullerton, Calif. U.S.A.) may be used in accordance with one embodiment of the present invention so long as the centrifuge is modified to hold the filter vessels  105 . The centrifuge  125  shown in  FIG. 4  shows the angle of centrifugation used in the preferred embodiment. While many angles could work, in a preferred embodiment the filter vessel  105  is placed at a 45° angle to the axis of rotation  175 . 
     In one embodiment of the immunological system  100  of the present invention, the orientation of the filter vessel  105 , the sample, and the filter  150  is such that the sample separation system  115  can cause the sample to contact the filter  150  and allow components of the sample that are smaller than the nominal pore size of the filter  150  to pass through the filter  150  into a capture reservoir below the filter  150 , or the next filter  150 ′, and thus be separated from the components of the sample that are too large to fit through the filter pores and that remain in the filter vessel  105  above the filter  150 , and possibly filter  150 ′. Also included within the present invention is an immunological assay method. The immunological assay method  180  is depicted in the flowchart of  FIG. 5 . The immunological assay method  180  includes the optional step of, as can be seen block  185 , placing an immunologic assay sample in the filter vessel  105 . In another embodiment of the present invention, the immunological assay sample may also be placed in a standard test tube or microcentrifuge tube. Other vessels may be used for holding the assay sample in other embodiments of the method  180 . Block  190  shows the next optional step of adding assay reagents to the filter vessel  105 . The next step, shown in block  195 , is mixing the sample with the reagent to form a sample mixture  200 . Block  205  shows the optional step of incubating the sample mixture  200 . In the incubation step of block  205 , the sample mixture  200  may be incubated at a temperature ranging from approximately 4° C. to approximately 37° C. In a preferred embodiment, the sample mixture  200  is incubated at a temperature range between approximately room temperature (20-25° C.) and approximately 37° C. The incubation time of the sample mixture  200  can range from no incubation to approximately 30 minutes. In a preferred embodiment, the incubation time is from approximately 2 to approximately 5 minutes. 
     After the optional incubation step, the next optional step as depicted in block  210  is to separate the sample mixture  200  into its various components. This step is usually accomplished by placing the sample mixture  200  in the sample separation system  115 . If the sample separation system  115  used in the separating step of block  210  is the centrifuge  125 , the centrifuge speed is usually between approximately 10 to 10,000×g, although other speeds may be used. In the preferred embodiment, the speed of the centrifuge is between approximately 1000 to approximately 5000×g. Optimally, the speed of the centrifuge is 3000×g. The centrifuge time may range from approximately 5 seconds to approximately 5 minutes. Although other times may be used, in the preferred embodiment, the centrifuge time is between approximately 10 to approximately 30 seconds. Optimally, the centrifuge time is between approximately 15 to approximately 20 seconds. 
     As shown in block  215 , after the optional separation step  210 , the filter vessel  105  may optionally be analyzed to determine the presence or absence of interactions between the assay sample and reagent that remain above the filter  150 . The sample is analyzed by placing the filter vessel  105  in the image acquisition system  130 . If interactions between assay sample and reagent are detected in the material above the filter  150  by the acquisition system  130  in the analyzing step of block  215 , the immunological assay method  180  is completed. Material will be detected above the filter if there have been interactions, for example, between cellular components in the assay sample and antibody reagents. The interaction will evidence itself in the form of agglutination, or clumping together, of the cellular components by the antibodies. This agglutination may be detected by the image acquisition system  130 . Likewise, the absence of evidence of agglutination indicates that there were no interactions between the cellular components and the antibody reagents. Similarly, the assay method may be used to detect interactions between antibody components in the assay sample and cellular reagents by detecting presence or absence of agglutination of cellular reagents by the antibody components by the image acquisition system  130 . 
     If the results of the analyzing step of block  215  are not clear, and no interactions are detected in the material above the filter  150  of the filter vessel  105 , the immunological assay method  180  may optionally continue to the step  220  of washing the sample mixture  200  in the washer  140 . As noted previously, in the preferred embodiment the washer  140  is also the vacuum  120 . While vacuum may be applied for varying lengths of time according to various embodiments, in a preferred embodiment, a vacuum is applied until all fluid has been vacuumed through the filter  150  of the filter vessel system  105 . The length of time the vacuum is applied is dependent on the vacuum pressure, but is often approximately 5 seconds to approximately 2 minutes, or until such time the liquid component of the sample mixture  200  has been largely or completely drawn through the filter  150 . 
     In the optional washing step of block  220 , the sample mixture  200  may be washed with a physiological salt solution. The salt solution may be for example, but not limited to, saline, e.g., 0.9% sodium chloride (NaCl) solution, a phosphate-buffered saline, or any physiological salt solution that preserves the viability of cellular components during the assay method. The washing step may comprise the steps of providing the physiological salt solution, adding approximately 10 microliters to approximately 5 milliliters of the physiological salt solution to the sample mixture  200 , separating the antigen carriers, either cellular components or synthetic beads, that remain above the filter  150  from the liquid components that pass through and remain below the filter  150 , and repeating these adding and separating steps from one to approximately ten times, until the sample mixture  200  is washed sufficiently for the application. 
     After the optional washing step of block  220 , the immunological assay method  180  begins again at the optional step  190  where assay reagents are added the washed antigen carriers that remain above the filter  150  in the filter vessel  105 . Following the optional mixing step  195  the resulting sample mixture  200  proceeds to the optional incubation step of block  205 , wherein the sample mixture  200  is again placed in the incubator  110 . Upon incubation, the sample mixture  200  is optionally placed in the sample separation system  130  as shown in block  210 , and then optionally analyzed again, as shown in block  215 . 
     In the embodiment of the present invention in which the assay sample is first placed in a standard test tube or microcentrifuge tube, the samples are assayed first with the image acquisition system  130 . Then, provided the testing results of the image acquisition system  130  are unclear, the sample mixture  200  is then placed in the filter vessel  105 , where it proceeds to the washing step of block  220 , followed by the addition of testing reagents in block  190 , the mixing of sample and reagents in block  195 , the incubation step of block  205 , the sample separation step of block  210 , and the analysis step of block  215 . 
     It should be emphasized that the above-described embodiments of the present invention, particularly, any “preferred” embodiments, are merely possible examples of implementations, merely set forth for a clear understanding of the principles of the invention. Many variations and modifications may be made to the above-described embodiment(s) of the invention without departing substantially from the spirit and principles of the invention. All such modifications and variations are intended to be included herein within the scope of this disclosure and the present invention and protected by the following claims.