KR100503257B1 - Agglutination reaction and separation vessel - Google Patents

Abstract

The present invention discloses a vessel for performing hemagglutination assay. During incubation, the barrier retains the reactants in the upper chamber and, according to the force, causes the reactants to enter the lower chamber containing the matrix for flocculation separation.

Description

Agglutination reaction and separation vessel

FIELD OF THE INVENTION The present invention relates to the field of aggregation analysis, and more particularly to vessels useful for performing aggregation analysis and separating aggregates.

Blood group serology requires the determination of blood cell compatibility between the blood donor and the patient recipient prior to transfusion or organ transplantation. The affinity of the blood cells is confirmed by the lack of an immune response between the antibody contained in the patient's serum and the antigen present in the blood cells of the blood donor.

Many different blood group antigens are found on the surface of red blood cells of every individual. Blood grouping is generally the process of testing red blood cells to determine which antigens are present and which antigens are not present. This is generally accomplished by using antibodies of known specificity.

In order to detect the serum of a patient or the antibody of a plasma, the reagent containing the blood cell which has a well-known antigen is mixed with a serum sample. The reactants are incubated for a time sufficient to allow aggregation of red blood cells that occur when antibodies are present against the antigen. The mixture is then centrifuged and the aggregated blood cells are clearly visible at the bottom of the reaction vessel if there are aggregated blood cells, thus indicating the presence of antibodies in the sample against known antigens on the red blood cells. If no antibody is present against the known antigen of the erythrocyte cells in the sample, no aggregation occurs, indicating that there is no aggregated erythrocytes after centrifugation.

Recently, aggregation reactions are carried out at one point in the vessel, and the separation of aggregated red blood cells is done at different points in the same vessel using a matrix that separates aggregated cells from other components in the reagent / sample mixture. Has been developed. Such a system is described in US patent applications 08 / 407,747 and 08 / 112,402, which are now serial applications of US patent application Ser. No. 08 / 023,500, which are now abandoned, and these applications are owned by the applicant. The contents of each of these applications are described by reference in this description. Agglomeration and separation vessels according to the invention, which are also useful in the invention described in the above-mentioned applications, are manufactured and sold under the name BIOVUE ^™ by Otto Diagnostic Systems, Ltd., of Lattan New Jersey ^. do. This reaction vessel is in the form of a column having an upper chamber and a lower chamber, the upper chamber having a larger diameter than the lower chamber. The lower chamber includes a matrix for separating the aggregated cells from the non-aggregated cells. The diameter of the lower chamber is typically such that when reagents and samples are added to the upper chamber using a pipette, the reagents and samples remain in the upper chamber and do not enter the lower chamber unless additional force is applied. Narrow enough

Known indirect antiglobulin tests, such as the coombs test, are blood tests used to confirm the presence of IgG antibodies in the serum of a patient for specific antigens on the surface of red blood cells. In the Cooms test, serum is incubated in the presence of red blood cell reagents so that the antibody binds to an antigen on the surface of the red blood cells. Such IgG antibodies generally do not agglomerate red blood cells by themselves, or are insufficient to aggregate visually by one general technique. The addition of a second antibody indicating human IgG is usually necessary to facilitate visually visible aggregation.

In erythrocyte typing, a blood test is used to determine what antigens are present on the surface of red blood cells, and the analyzed red blood cells, for example, into a lower chamber containing, for example, antibodies against the red blood cell antigens and a separation matrix. It is added to the upper chamber following the application of forces such as centrifugal forces to move them. If red blood cells have antigen (s) on their surface to bind specific antibodies in the lower chamber, aggregates will form and will be separated by the matrix.

In other forms of blood analysis, such as reverse typing, in which direct aggregation is analyzed for red blood cell antigens in the serum of the patient, the patient's serum and red blood cell reagents with known antigens on the surface are added to the upper chamber, for example For example, centrifugal forces act to move the reactants into the lower chamber, including the liquid medium and separation matrix, but without the antibody. In this assay, analysis for the presence of antibodies aggregated directly in the patient's serum will produce aggregates separated by the matrix.

In another form of blood assay, antibody reagents with known specificities for erythrocyte antigens are placed into the upper chamber with the erythrocytes of the patient. If the antibody reagent is an agglutinated antibody, for example, a force such as centrifugal force acting without prior consideration is applied and the contents are forced into the lower chamber containing the separation matrix in the aqueous solution. The aggregates are then separated by the matrix. Alternatively, following incubation, the patient's red blood cells are placed in an upper chamber and an antibody reagent IgG with known specificity is added to attach the antibody to an unknown antigen on the surface of the red blood cells. After incubation, a force such as, for example, centrifugal force, acts to move the reaction mass into the lower chamber containing the specific anti-IgG antibody for the IgG antibody reagent used to culture the red blood cells in the separation matrix and in the upper chamber. If the antibody reagent is present on the patient's cell surface, the anti-IgG antibody in the lower chamber facilitates the formation of aggregates separated by the matrix.

After the sample or reagent has been incubated for a time sufficient to allow direct red blood cell aggregation, such as in the case of red blood cell typing, or antigen-antibody reactions, such as in the CUMS test, pressure is applied to the reaction vessel, for example, by centrifugation. The reactants are then released to the bottom of the column and to the separation matrix. As a result of centrifugation, the aggregated cells remain on top of the separation matrix or are distributed into the matrix depending on the extent of aggregation, while the non-aggregated material moves down through the separation matrix. Stronger aggregation reactions result in cells remaining towards the top of the separation matrix, while weaker aggregation reactions result in the distribution of aggregates at various intervals from the top of the matrix.

Retention of the sample and reagents at the top of the column during the culture phase is the result of surface tension across the top edge of the bottom in the column with reduced diameter relative to the top. Two possible errors have been identified in performing the analysis using these columns. First, if reagents and samples are pipetted directly into the center of the reaction chamber with excessive force, the reactants can be placed directly on top of the separation matrix of the lower chamber, but not held in the upper chamber during the culture. Thus, the reactants will begin to enter the separation matrix before the aggregation is complete. Secondly, there is a possibility that diluents or aqueous solutions containing the separation matrix can be introduced into the upper chamber. This can occur, for example, through splashing or other disturbances during transportation or handling of the container. In some cases where a solution or dilution comprising a separation matrix also contains antibodies or other reagents that directly affect the test results, such frying results in cross-contamination of the column with some reagent from another column. do. This can occur when the user inserts a pipette tip into the reaction chamber and the tip is contaminated with the fried reagent, which can then be transferred to another container by the pipette. This can lead to inaccurate results in aggregation analysis.

Accordingly, it is an object of the present invention to provide an improved mechanism for maintaining separation of samples and reagents during the culture state of aggregation assays. It is another object of the present invention to provide a means for preventing displacement of the material contained at the bottom of the column.

The present invention provides an improved vessel for carrying out flocculation reactions and separation of flocculations. This vessel can hold the reactants in the upper chamber holding the reactants, the lower chamber in which the agglomerates separate, separate the chambers and receive the reactants in the upper chamber before the contents of the upper chamber enter the separation matrix, And a barrier means for passing the contents of the upper chamber from the upper chamber to the lower chamber when a large pressure is applied to the barrier. In a preferred embodiment, the barrier comprises a narrow passageway between the upper and lower chambers. Such narrow passages may be made by inserts with narrow holes, by crimps or by inserts having a spiral or other similar shape, which may be shaped or inserted between the upper and lower chambers during manufacture. Can be. Such inserts provide a practical barrier that reduces the size of the hole between the lower and upper chambers, thereby improving the prevention of contamination of the upper chamber by the contents of the lower chamber. Moreover, inserts also improve separation of samples and reagents during incubation. When the insert is helical, the passages, central axis and helix column walls described by the threads are small enough to keep the fluid in the upper chamber under normal gravity and atmospheric pressure. For example, the diameter of the helical insert ranges from about 0.110 to 0.140 inches. The diameter of the helical insert shaft is about 0.030 to 0.090 inch. The helical insert has about 6 to 30 threads per inch.

The narrow passage may also be provided by high frequency welding of the column, which is done after loading reagents into the column. The passageway may also comprise a barrier in the form of a diaphragm. In this embodiment, the diaphragm has a central hole small enough to maintain fluid in the upper chamber under normal gravity and atmospheric pressure. The diaphragm is preferably silicone rubber. The passageway may comprise a barrier in the form of a porous plug. The porous plug has a through passage small enough to maintain fluid in the upper chamber at normal gravity and atmospheric pressure. The porous plug is preferably polypropylene.

In another embodiment, the present invention includes a liner in the form of a conical member having a hole through a narrow vertex. When the sample is placed at the end prior to pipetting into the vessel and extended to the reaction vessel column, such a liner prevents cross-contamination where one diluent or solution from one vessel enters the other vessel, which may cause cross contamination during such pipetting. something to do. The liner is conveniently inserted onto the column by the end user prior to pipetting of the reagents. The liner may have the form of a single unit with six conical members or cells arranged side by side, the overall area and shape of the liner being the same as that of the BIOVUE ™ cassette top.

The liner comprises a body and at least one conical member suspended to the body, the conical member having holes at its narrow vertices and the conical member having sealing means located at a position spaced apart from the narrow vertex. The conical member includes a first end comprising the narrow vertex and a second end adjacent to or spaced from the body, wherein the sealing means is provided at or adjacent the second end. The sealing means comprises an O-ring surrounding the conical member, which may be integral with the conical member. In a preferred embodiment, the liner has six conical members suspended linearly in the body and the liner is sized to fit the reaction vessel of the cassette. The liner is preferably acrylic.

The present invention is contemplated as a liner comprising a foil sealed cassette comprising six reaction vessels disposed linearly therein, and a liner comprising a body and six conical members suspended linearly to the body, wherein the conical member is attached to the foil sealing body. A narrow vertex suitable for drilling is included, and when inserted, the liner is frictionally coupled to the cassette to seal the connection between the cassette and the liner. The means for sealing this connection is an O-ring surrounding each conical member and is preferably integrated with each conical member.

According to the present invention, a vessel for carrying out the flocculation reaction and separating the flocculate will be described by various embodiments. Certain embodiments of the present invention are clearly understood through the description of the prepared agglomeration reaction and separation vessel manufactured and sold in cassette form by Ortho Da Agagnostic Systems, Inc., LA, Titanium, under the trade name BIOVUE. Can be.

The container of the present invention, like glass or various plastics, can be made of any suitable material that shows results and does not interfere with the aggregation reaction or separation. In a preferred embodiment, the container is made of polyflopropylene.

The upper chamber of the vessel may be of any shape and specification useful for supporting reagents and specimens while the culture is running. Typically, the upper chamber is cylindrical at the top. The barrier between the upper and lower chambers generally defines the upper boundary of the lower chamber and the lower boundary of the upper chamber. In a preferred embodiment, the barrier forming the bottom of the upper chamber is conical and has a vertex extending into or towards the lower chamber, as shown in any of the first, 5, 6, 7 degrees. One portion of the barrier is configured to allow fluid to flow from the first chamber to the second chamber when a force such as increased pressure or centrifugal force is applied, while reagents and specimens are maintained during incubation under normal gravity and atmospheric pressure. This can be accomplished by various means such as small holes, membranes, plugs, inserts or screens having any one of several geometries, and combinations thereof. In a preferred embodiment, the barrier comprises a hole having a diameter small enough to allow fluid to flow under increased pressure while preventing fluid flow from the first chamber to the second chamber under normal gravity or atmospheric pressure. As shown in FIG. 1, 5 or 7 the hole 1 is at the apex of the conical part of the upper chamber in the insert 2 or in the conical part of the upper chamber integrally formed in the upper chamber as shown in FIG. Is located at the vertex of.

The aperture can be any diameter that allows surface tension to be overcome while the surface tension of the fluid in the upper chamber under normal gravity or atmospheric pressure is small enough to prevent flow from the upper chamber to the lower chamber, and thus the upper under increased pressure or gravity It facilitates the passage of contents from the chamber to the lower chamber. The hole diameter can be changed depending on the magnitude of the force used, for example a smaller diameter when a larger force is applied, and a larger diameter when a smaller force is applied. The diameter can be varied to accommodate particles of different sizes in the reagents. In a preferred embodiment, the diameter of the holes ranges from about 0.010 to 0.050 inches. In a particularly preferred embodiment, the diameter of the hole is 0.020 inches.

In another embodiment, the barrier means for separating the upper and lower chambers comprise a helical insert; Such helical inserts may be of threaded construction. Spiral inserts are preferably circular or cylindrical in diameter, although the elliptical structure is described. Like the holes described above, passages represented by shafts, helical threads and walls of the chamber allow fluid flow under increased force or pressure, while under normal gravity or atmospheric pressure, fluid flows from the upper chamber to the lower chamber. It has a diameter small enough to prevent passage of the furnace. Another function of the insert is to reduce the likelihood of splashing of the diluent or solution from the lower chamber to the upper chamber to contaminate the upper chamber. Such frying may occur, for example, during transportation and handling. The spiral body may be located within the upper chamber at the bottom of the upper chamber 3, as shown in FIG. 11, or may be integrally molded in position with the reaction vessel.

If the helical insert is inserted separately into the upper chamber, the insert may be molded of a suitable material that does not interfere with the agglomeration reaction or separation, for example, such as glass or plastic. The material is preferably, for example, polypropylene, polyamides such as nylon, acetal resins such as Delrin ™ or Delrin P®, crosslinked polystyrene / Rexolite® Plastics such as divinylbenzene, polycarbonate or polyethylene.

Helicals allow surface tension to be overcome to facilitate passage of the contents from the upper chamber to the lower chamber under increased pressure or gravity, while under normal gravity or atmospheric pressure the surface tension of the fluid in the upper chamber from the upper chamber to the lower chamber It can be of any geometry to prevent fluid flow. For example, the pitch of the helix thread prevents contamination of the upper chamber with the matrix or fluid from the lower chamber while fluid (eg, contains blood cells) under increased pressure from the upper chamber to the lower chamber. It can be at any angle that allows the passage of. The pitch may change depending on the magnitude of the force used, i.e. the smaller space represented by the helix axis, the thread and the chamber wall when the larger force is applied, and the larger space when the smaller force is applied. have. The helix structure can also be modified to accommodate particles of different sizes in the reagents. The insert also improves frying prevention of the column contents (solution or diluent comprising the separation matrix) into the upper chamber.

In the spiral insert embodiment of the present invention, the number of threads per thread inch and the thread depth are controlled (at the bottom of the range) by the effect of this resulting helix to prevent contamination of the upper chamber by the fluid from the lower chamber and under increased pressure. It will only be limited (at the top of the range) by the ability of the sample to pass the helix down, for example red blood cells. In order to facilitate the above-mentioned reasons and other aggregates and red blood cell passage through the barrier means, it is desirable for the tissue and finish to be relatively smooth for threads, central axes and column walls representing passages. The walls of the threaded portion of the helical insert facing the column wall can be sharp or flat.

In a preferred embodiment, in order to use the current BIOVUE ™ cassette structure comprising six reaction vessels, the helix has about 6 to about 30 threads per inch, more preferably about 12 to about 20 threads per inch. It will have a thread, and the pitch measured in inches from the top of one thread to the top of the next thread is from about 0.033 to about 0.166 inches, more preferably from about 0.05 to about 0.083 inches.

The overall diameter of the helical insert is in the range of about 0.110 to about 0.140 inches, more preferably about 0.120 to 0.130 inches.

The axis of the insert of the helix is in the range of about 0.030 to about 0.090 inches, more preferably about 0.060 to about 0.080 inches. Reference is made to FIGS. 11, 12 and 13 which illustrate examples of the relative structure of insert elements. Referring to FIG. 13, suitable geometries (in inches) may be as follows.

Also provided herein is an elliptical helix that may have a similar pitch structure as an alternative to a round diameter helical insert positioned to the modified lower portion of the upper chamber as shown in FIG. As already described for the round diameter helical insert, the elliptical barrier means may take the form of a helical tightening molded into the form of an insert or a unit with a chamber.

As another alternative, a narrow passage between the upper and lower chambers may be provided by high frequency welding of the column after the reagents have been loaded. Alternatively, the barrier may comprise a disk or plug of porous material located on top of the column or lower chamber. The plug is cylindrical in size to fit between the upper and lower chambers, suitable for delaying splashing of the lower chamber contents (eg, reagent and separation matrix) into the upper chamber. Porous plugs prevent the sample from passing through immature conditions (eg, prior to centrifugation), but are suitable for allowing the sample to pass through at pressures greater than atmospheric pressure (eg, centrifuged). Porous plugs are not involved in agglomeration reactions or separations, or are involved in non-specific binding or showing the results of any component of, for example, glass and more particularly, scintered glass, or plastic, for example polypropylene. It does not consist of any material.

Other suitable barriers may take the form of flanges fixed in a spiral, stepped cascade around the inner wall of the chamber or other similar warped path designs that function as described above to prevent contamination of the upper chamber contents. The barrier may also take the form of a circular valve or diaphragm located in the column. Such a diaphragm may have a central hole or a through hole, for example, and may be marked radially from the column wall to the central hole. When a force is applied, the diaphragm will allow the sample contents to pass from the upper chamber to the lower chamber. The diaphragm will consist of any suitable material that does not show results or interfere with the flocculation reaction or separation. The diaphragm is made of any suitable flexible material such as, for example, silicone rubber.

When the vessel of the present invention is used to carry out flocculation reactions and separations, reagents and samples are added to the upper chamber for incubation. The barrier retains samples and reagents in the upper chamber while incubation takes place. In a preferred embodiment, when the barrier has a hole or is in the form of a spiral insert, the structure of the helix or the diameter of the hole, respectively, is small enough that the surface tension of the specimen and the solution crosses the hole, or normal gravity and atmospheric pressure. Under the helix the body will retain the contents in the upper chamber. After sufficient incubation time, the force is exerted by any of a variety of means, such as centrifugation, pressure or suction, against the barrier in a direction substantially along the axis from the upper chamber to the lower chamber. The force must be sufficient to allow the contents of the upper chamber to pass through to the lower chamber and overcome the barrier. In a preferred embodiment, the surface tension is controlled by force if the barrier comprises a hole or the barrier means comprises a helical insert. Overcoming, the contents flow from the upper chamber to the lower chamber to the separation matrix.

Barriers are also important in providing means to reduce contamination of the upper chamber due to diluents or solutions in the lower chamber, as may be caused by frying or other disturbances during transportation and handling.

Another embodiment of the present invention is a liner that can be inserted into the top of the reaction vessel just prior to adding the specimen. Such a liner comprises a body and one or preferably a plurality of members or cells suspended from the body, which cells have a conical or funnel shape. When inserted into the cassette, the narrow apex of the cell containing the aperture is directed towards the inside of the container as shown in FIGS. 14 and 15. The liner may be used in the container with or without barrier means such as crimps or inserts. In a preferred embodiment, the liner is (1) until the vessel is forced (e.g., if the incubation time is not prolonged by the insulation effect of any air gap between the vertex and the separation matrix in the column). By means of centrifugation) a cell having a diameter small enough to contain a specimen delivered to the container; (2) If the test system is able to withstand early contact with reagent splashes, include a cell with a diameter large enough to allow the specimen to easily pass into the reaction vessel chamber. The cells of the liner preferably have sealing means located at a position spaced apart from the narrow vertex, for example at an outer end just under the liner body. Reference is made to FIGS. 14 and 15 in this regard. The sealing means is preferably a protruding ring integral with the liner. The liner cell fits into the reaction vessel upper chamber so that it is not easily removed during normal handling. Raised rings or O-rings will be placed in the margin around the container on the upper surface of the cassette, thus sealing the joint between the liner and the cassette. Protruding rings or O-rings prevent capillary action of any fried column contents from one column to another.

As described above, in the absence of an insert provided in the container, a solution or diluent comprising the separation matrix may enter the upper chamber during shipping and handling. In this case, and where the separation matrix may also contain antibodies or other reagents that directly affect the test results, such frying may result in cross contamination of the columns with reagents from other columns. This can occur when the user inserts the pipette tip into the reaction vessel upper chamber, which contaminates the tip with the splashed reagent, which can then be transferred to another vessel on the pipette. The latter can lead to misleading results in aggregation analysis.

The purpose of the liner is to prevent cross contamination of the reagents from one vessel to the next during pipetting, and the reagents may be splashed into the upper chamber during shipping and handling.

The liner may take the form of a single conical member or cell so that it is placed individually on top of each reaction vessel. However, in a preferred embodiment, and with reference to FIG. 15, the liner takes the form of a single unit with six conical cells aligned side by side, integral with the liner body. Such a structure allows a single liner unit comprising six individual cells to rest on top of six reaction vessel BIOVUE cassettes.

Referring to Figures 14 and 15, each conical cell of the liner has narrow sharp edges with through holes. The sharp vertices of the liner under artificial pressure will penetrate the foil sealing body over all six columns because the BIOVUE container is provided as a cassette comprising six columns and is sealed across its top by a foil strip. Specimen samples can then be delivered directly to the cells via a pipette. Thus, clean liner cells are free from any contamination reagents or separation matrices that can come into contact with the specimen and be handed over to other columns.

The liner is conveniently inserted into the column by artificially using an end user or a pronged tool. In order to insert six cell liners into the BIOVUE cassette, the cassette foil is drilled as liner tips and fully inserted by the shaking action of the liner. The use of a fork tool aids in the alignment of the liner and cassette during insertion. The liner may remain intact during processing of the cassette since the liner body is typically the same size and shape as the top surface of the cassette. Using the liner in this manner does not interfere with analytical performance and results (eg, centrifugation, free migration of unaggregated red blood cells through the separation matrix and inflow of aggregated red blood cells into the column). However, columns without O-rings do not prevent column reagent cross contamination during use. See Examples 19 and 11 for a contrast functional test of liners with and without O-rings. The results indicate that the O-ring prevents cross contamination of column reagents that may result from reagent "Wicking" between cassette foils as a resultant flow of reagents across the cassette top.

The liner and cell may be made of any suitable material that does not interfere with the flocculation reaction or separation, for example glass or plastic. The material should be suitable for drilling the foil sealing body on the cassette. For effective sample delivery from the liner cell to the upper chamber, the liner cell wall is preferably relatively soft in tissue and finish. The material is preferably a plastic such as, for example, polyester, acetal, acrylic, acrylonitrile butadiene styrene (ABS), nylon, polycarbonate, polyamide or polypropylene. In a preferred embodiment, the material is acrylic .

In the Biovue ^™ system, 10 μl of red blood cell reagent incubation in 40 μl of patient serum with 40 μl of low ion concentration solution takes place in the upper chamber for 10 minutes to allow the putative patient's IgG antibody to bind to the red blood cell surface antigen (s). These assay components are added separately and they can be mixed so that it is important that the components remain in the upper chamber to provide the serum with a percentage of low ion concentration solution for red blood cells each time the assay. The barrier makes it easy under normal gravity and pressure. This works to reduce the chance that certain analytical components are added to the lower chamber while adding the sample. The barrier may also allow the analyte to remain in the upper chamber throughout the incubation period.

Barriers are also important to prevent premature binding of anti-human IgG antibodies to putative anti-erythrocyte antibodies in patient serum before the antibodies bind to erythrocytes, and reduce the chance of final occurrence in the lower chamber. After incubation, the contents of the upper chamber are passed through the barrier into a lower chamber containing anti-human IgG that binds the patient's IgG at the surface of the reagent erythrocytes forming an aggregate so that centrifugal force acts and does not pass through the matrix to the bottom of the lower chamber. Move it.

The following examples are provided for the purpose of illustrating the invention and are not intended to limit the scope of the invention.

Example 1

The Bioov ( ^TM ) column with inserts was compared to the column without inserts to determine the efficacy of the efficiency of each shape to maintain the air layer separating the reactants from the separation matrix during the incubation period. Inserts with holes of 0.04 inches were used. 40 μl of buffer was added to each of the 840 test columns. Manual pipettes were used which were maintained at an angle of about 45 degrees from the vertical axis of the column to deliver 40 μl. The column was then observed to determine if the air space below the reaction chamber was maintained. The number of "break-throughs" columns is listed in Table 1.

Table 1

Example 2

Reagents were also added to the columns (with and without inserts) and incubated at 37 ° C. for 10 minutes. To each of the 480 test columns was added 40 μL buffer, 40 μL serum and 10 μL red blood cell suspension. A pipette maintained at an angle of about 45 degrees was used to deliver the reactants. After the incubation period, the column was examined to determine if the air layer below the reaction chamber was maintained. The frequency of "break-throughs" is listed in Table 2.

TABLE 2

Example 3

40 μL of buffer was charged to the column using an automatic pipette maintaining an angle of about 45 degrees. Automatic pipettes typically deliver more force than models manually operated. Observation after filling was performed to determine whether the air layer below the reaction chamber was maintained. The measured results on the column with and without the insert are shown in Table 3.

TABLE 3

Example 4

40 μL of buffer was packed into 240 columns using a single pipette that was held vertically. By keeping the pipette vertical, the fluid will exert greater force on the aperture, thus better destroying the air layer separating the reaction chamber from the separation chamber. The results of this experiment are listed in Table 4.

Table 4

Example 5

The 240 column reaction chamber was also filled with 40 μl of buffer using a vertically maintained automatic pipette that would more likely destroy the underlying air layer than when the automatic pipette was held at an angle. The results of this test using columns with inserts and columns without inserts are shown in Table 5.

Table 5

Example 6

In addition to maintaining an air layer between the reaction chamber and the separation matrix during the culturing phase of the test, the present invention also provides splashing that can occur during transport and handling where a portion of the contents of the lower separation chamber may spring up into the upper reaction chamber. Acts as a means of preventing. Cassettes with inserts and cassettes without inserts were transported from New Jersey to California and returned to test anti-frying efficacy. Transportation used by air and land transport included shipping, unloading and delivery to laboratories. The method used was common to this product line. After being conveyed, the cassette was inspected for the presence of splashed liquid in the reaction chamber. The results are given in Table 6.

Table 6

Example 7

Additional transport tests were performed to test frying reduction with inserts having reduced size holes. Openings between the reaction chamber and the separation matrix were 0.025, 0.020 and 0.015 inches in diameter. Each of these inserts was mounted in 600 columns. No insert was used as a control. Cassettes were packed and an internal surrogate transport test was performed to drop the box ten times from three feet high. The angle of the box was adjusted such that the vessel fell towards one edge and three edges as well as all six flat surfaces. This standardized test represents the worst case in transportation and handling. The results given in Table 7 show the inverse relationship between hole size and frying reduction.

TABLE 7

Example 8

Another means that can reduce the orifice between the reaction chamber and the bottom of the separation chamber is to "crimp" the cassette. This can be done by an compression extrusion that compresses the neck of the cassette directly below the reaction chamber. The force and duration of compression determine the extent to which the opening is reduced. The shape of the compression tool determines the shape of the opening. Various shapes are possible. The crimping process can take place in the production line after the column is filled with reagents and glass beads.

816 columns in the production line were crimped as described above to narrow the opening between the reaction chamber and the separation matrix. Crimping resulted in the cross sectional form shown in FIG. 10 through the parts shown in parentheses in FIG. They were packaged with 768 uncrimped controls and shipped to California and returned from California as described above. The reduction in frying into the reaction chamber caused by the transport conditions is given in Table 8.

Table 8

Example 9

Spiral insert barrier means can be used to limit the size of the orifice between the reaction (top) chamber and the separation (bottom) chamber. The helical insert was molded from polypropylene and inserted into the neck of the cassette directly below the modified upper chamber. After filling the column with reagents and a separation matrix (eg glass beads), the helical insert is placed in the neck of the cassette during the production process.

Example 10

A biobe column with a liner with an O-ring was compared to a column with a liner without an O-ring to determine if the O-ring prevented cross-column reagent contamination.

The cassettes were simulated and transported to knock or bump the cassette onto a hard working surface so that frying occurred in the upper chamber and on the foil.

Column reagent wicking was observed after inserting 192 cassettes with liners without O-rings into the cassette column. Half (96) of the liner was manually inserted into the cassette and the other half was inserted using the two-fork tool described above.

336 cassettes with liners with O-rings were observed for column reagent wicking as above. Half of the cassette 168 was manually inserted with the liner and 168 were inserted using a two-fork tool.

Column wicking was assessed visually by examining the top of the cassette. Wicking was measured by observation of reagent flow between the top of the cassette foil and the bottom of the liner body.

Table 9 shows the number and percentage of cassettes wicked against the total number of cassettes tested. The liner insertion method did not affect the results with the liner with O-rings, but with the liner without the O-rings. As shown, when the liner was manually inserted (cassette in inverted position), the number of cassettes in which reagent wicking occurred was about twice as long as inserting the liner using a tool (cassette in vertical position).

Table 9

Example 11

Cassettes with liners without O-rings were compared in terms of functional test with cassettes with liners with O-rings.

As described in Example 10, the cassettes were simulated to allow reagent frying to occur in the upper chamber and on the foil.

The liner was inserted into the cassette column manually and using a two-fork tool as described above. In half of the cassette, 10 μl of 4% RBC (in normal saline) was pipetted into each column using a BioOVUE ^™ BioHit (Ortho Diagnostic Systems Inc., Taritan NJ) pipette. In the other half of the cassette, 50 μl of 0.8% RBC (normal saline) with surface antigens as specified in Table 10 below was pipetted into the cassette column. Half of the cassettes used were Ortho BIOVUE ^™ International cassettes; Ortho Diagnostic System Inc., Raritan, New Jersey; Inc., Raritan, New Jersey). As shown in Table 10 below, ABE and RHK cassettes were prepared with antibodies in each column.

Table 10

When the ABE cassette was used, A ₁ rr cells were used as samples. When RHK cassettes were used, R ₁ R ₁ K (−) cells were used as samples. Pipetting was performed from the leftmost cassette column to the right. Cassettes were centrifuged at 794 +/- 16 × g for 2 minutes and then at 1510 +/- 30 × g for 3 minutes in an Ortho Biosystem Centrifuge (Ortho Diagnostic System Inc, Raritan, NJ). Each column with a negative response (non-aggregation of RBCs) was evaluated for the complete absence of red blood cell passage through the entire column. For example, if an anti-A antibody was transferred from column 1 of the ABE cassette to column 2, a false positive reaction would occur and the cells react in column 2.

The results are shown in Table 11. In the column with O-rings, all expected positive reactions were positive. However, all expected negative responses were not negative. There was one false positive result. Although not confirmed, the reason was not reagent wicking. Table 11 shows the number and percentage of cassettes and columns in which the total benefit false positive reaction of the cassettes tested occurred. The liner insertion method did not affect the results with the liner with O-rings, but indirectly with the results with the liner without O-rings, which was found in cassettes where reagent wicking occurred where all false positive results were visible. Because it appeared.

Table 11

1 shows a reaction and separation vessel with an insert having a narrow hole located in an upper reaction chamber,

FIG. 2 shows four vessels without inserts, one vessel with inserts as shown in FIG. 1 (second from left) and one vessel with the reactant (third from left). Top view of cassette of six reaction vessels.

3 is a cross sectional view of the cassette of the reaction vessel taken along line 3-3 of FIG.

4 is a side view of a cassette of a reaction vessel.

FIG. 5 is a cross-sectional view taken along line 5-5 of FIG. 2 showing an insert having a narrow hole inside the upper chamber of the reaction vessel.

6 shows an upper chamber of a reaction vessel configured with a narrow hole.

7 shows an insert with an extended part having a hole disposed in the lower chamber.

FIG. 8 is a sectional view of the line 8-8 of FIG.

9 shows the pleated reaction and separation vessel just below the upper chamber.

10 is a cross-sectional view taken along the line 10-10 of FIG. 9,

11 shows a side view of a reaction and separation vessel with a helical insert (1) located in an upper chamber (2) in which a lower portion (3) has been modified to receive an insert.

12 is a side view of the helical insert structure,

(A) shows an insert with an overall diameter 1 of 0.120 inches and an internal axis with a diameter 2 of 0.060 inches,

(B) shows an insert with an overall diameter 1 of 0.120 inches and an internal axis with a diameter 2 of 0.080 inches.

13 shows a side view of a helical insert having a total diameter (1) of 0.120 inch and an inner shaft (2) having a diameter of 0.060 inch.

14 shows one cell of a liner designed to fit within the upper chamber of the reaction vessel column, each conical member or cell having an O-ring positioned around the cell below the liner body.

FIG. 15 shows a liner comprising six conical members or cells with sharp vertices designed to fit into an upper chamber of a cassette having six reaction vessel columns, each member enclosing a member under the liner body. With a ring (1), each member further has a vertex (2) suitable for penetrating a foil seal covering the reaction vessel column of the cassette.

Claims (25)

a) an upper chamber having an opening for receiving a fluid reactant; b) a lower chamber disposed to receive fluid from the upper chamber, the lower chamber including a matrix separating the aggregates; And c) separating the upper chamber from the lower chamber, having means for retaining the fluid in the upper chamber under normal gravity and atmospheric conditions, allowing passage of fluid from the upper chamber to the lower chamber under pressure greater than atmospheric pressure, Agglutination assay vessel comprising a barrier containing. 2. The aggregation assay vessel of claim 1 wherein the helical structure has a passage described by a spiral, central axis and column wall small enough to retain fluid in the upper chamber under atmospheric pressure at normal gravity. The aggregation assay vessel of claim 2, wherein the helical structure comprises a helical insert. a) an upper reaction chamber having an opening containing a fluid reagent and a hole defined by a helical insert having a specification that fluid is retained in the upper reaction chamber for gravity and atmospheric pressure; b) a coagulation assay vessel connected to the top chamber via a helical insert, the coagulation assay vessel including a bottom chamber comprising a separation matrix for separating aggregates. 6. The aggregation assay vessel of claim 5 wherein the helical insert is polypropylene or flexolite. a) a first chamber for receiving and holding fluid samples and reagents; b) a second chamber connected with the first chamber to receive fluid from the first chamber, the second chamber having a separation matrix for separating the aggregates; c) separating the first chamber and the second chamber to allow fluid passage from the first chamber to the second chamber under pressure greater than atmospheric pressure, while at normal or atmospheric pressure the fluid passage from the first chamber to the second chamber Agglomeration analysis vessel that can prevent the, comprising an insert having a spiral barrier. The coagulation assay vessel of claim 6 wherein the helical insert includes a passage of a diameter small enough to retain fluid in the first chamber under normal gravity and atmospheric pressure. 10. The aggregation assay vessel of claim 8 wherein the diameter of the helical insert is in the range of about 0.110 to 0.140 inches. 9. The aggregation assay vessel of claim 8 wherein the diameter of the shaft of the helical insert is between about 0.030 and 0.090 inches. 10. The aggregation assay vessel of claim 9 wherein the helical insert has about 6 to 30 threads per inch. a) an upper chamber having an opening for receiving a fluid reactant; b) a lower chamber disposed to receive fluid from the upper chamber, the lower chamber including a matrix for separating the aggregates; And c) separating the upper chamber from the lower chamber and allowing a passage of fluid from the upper chamber to the lower chamber under pressure greater than atmospheric pressure, while having means for maintaining the fluid in the upper chamber under normal gravity and atmospheric pressure, the diaphragm comprising Wherein the diaphragm has a small central hole sufficient to retain the fluid in the upper chamber under normal gravity and atmospheric pressure. 12. The aggregation assay vessel of claim 11 wherein the diaphragm is silicone rubber. a) an upper chamber having an opening for receiving a fluid reactant; b) a lower chamber disposed to receive fluid from the upper chamber and including a matrix for separating the aggregates; And c) separates the upper chamber from the lower chamber with a means for retaining the fluid in the upper chamber under normal gravity and atmospheric pressure, while allowing passage of fluid from the upper chamber to a lower pressure than atmospheric pressure; Agglomeration assay vessel comprising a barrier comprising a). 14. The aggregation assay vessel of claim 13 wherein the porous plug has a through passage small enough to retain fluid in the upper chamber under normal gravity and atmospheric pressure. 15. The aggregation assay vessel of claim 14 wherein the porous plug is polypropylene. Sealing means including a body and at least one conical member suspended to the body, wherein the conical member has one aperture at its narrow vertex, the conical member being located at a position spaced apart from the narrow vertex. A liner for preventing cross-contamination of reagents or solutions from one column of agglutination assay cassette to another column of the cassette. 17. The liner of claim 16, wherein the conical member comprises a first end comprising a narrow vertex and a second end spaced adjacent to the body, wherein the sealing means is at or adjacent the second end. 18. The liner according to claim 17, wherein the sealing means comprises an O-ring surrounding the conical member. 19. The liner of claim 18, wherein the sealing means comprises an O-ring integral with the conical member. 20. The liner of claim 19 comprising six conical members linearly suspended from said body. 21. The liner of claim 20 having a size to fit within a reaction vessel of a cassette. The liner of claim 21 wherein the liner is acrylic. A main body and six conical members suspended from the main body, each conical member having holes at narrow vertices and spaced from the first end and the first end spaced from the main body and integral with the conical member to surround the conical member. A liner comprising a second end having an O-ring. And six reaction vessels arranged linearly therein and six conical members suspended linearly to the body, wherein the conical members include narrow vertices suitable for drilling the foil sealing body, and the liner is inserted into the cassette when inserted. A foil sealed cassette, frictionally coupled to the cassette to seal the bond between the liners. 25. The foil sealed cassette of claim 24, wherein the means for sealing the joints is an O-ring that is integral with the respective conical members and surrounds each of the conical members.

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