CN1198531A - Agglutination reaction and separation vessel - Google Patents

Abstract

A container for performing a hemagglutination assay is disclosed. A barrier retains the reagents in the upper chamber during incubation and then, in response to a force, allows the reagents to enter the lower chamber containing the matrix for separation of agglutinates.

Description

Agglutination reactions and separation vessels

This application is a continuation-in-part of U.S. Patent Application No. 08/093,106, filed July 16, 1993, which is U.S. Patent Application No. 08/092,157, filed July 15, 1993 A follow-up application to a now-abandoned U.S. patent application.

The present invention relates to the field of agglutination assays, in particular to containers for performing agglutination assays and isolating agglutinates.

Blood group serology requires the determination of hemocompatibility between blood donor and recipient prior to transfusion to a patient or organ transplantation. Cell compatibility is determined by demonstrating the absence of an immunological reaction between antibodies contained in the patient's serum and antigens in blood cells from the donor.

Many different blood group antigens are found on the surface of each individual's red blood cells. Blood typing is usually the process of testing red blood cells to determine the presence of antigens. This is usually done using antibodies of known specificity.

To test for antibodies in a patient's serum or plasma, a reagent containing blood cells with known antigens is mixed with the serum sample. The reaction is incubated for a period of time sufficient to cause agglutination of the red blood cells when antibodies to those antigens are present. The mixture is then centrifuged and, if agglutinated red blood cells are present, these agglutinates are clearly visible at the bottom of the reaction vessel, indicating the presence of antibodies directed against known antigens on the red blood cells in the sample. If no antibodies directed against known antigens on red blood cells are present in the sample, agglutination will not occur, indicating that there are no agglutinated red blood cells after centrifugation.

More recently, systems have been developed in which the agglutination reaction is carried out in one part of the vessel while the separation is performed in another part of the same vessel using a matrix that separates the agglutinated cells from the other components of the reagent/sample mixture. Aggregated red blood cells. Such a system is disclosed and described in co-pending U.S. Patent Application Nos. 08/407,747 and 08/112,402, which are successor applications to (now abandoned) U.S. Patent Application No. 08/023,500 , these applications are generally owned by the occupants of this application. The content of each application is hereby incorporated by reference. The agglutination reaction and separation vessel according to the present invention is also useful in the invention disclosed in the aforementioned application text and is manufactured and sold by Ortho Diagnostic Systems Inc., Raritan, New Jersey, under the trademark BIOVUE ^(TM) . Such a reaction vessel is cylindrical with an upper chamber and a lower chamber, wherein the upper chamber has a larger diameter than the lower chamber. The lower chamber contains the matrix used to separate agglutinated cells from non-aggregated cells. The diameter of the lower chamber is narrow enough that when reagents and samples are added to the upper chamber (typically with a pipette), if no additional force is applied, the reagents and samples appear to remain in the upper chamber without Enter the lower room.

The indirect antiglobulin test (called the Coombs test) is a blood test used to determine whether a patient's serum has IgG antibodies directed against a specific antigen on the surface of red blood cells. In the Coombs test, serum is incubated in the presence of a red blood cell reagent to allow antibodies to bind to antigens on the surface of red blood cells. These IgG antibodies themselves hardly agglutinate erythrocytes, or only slightly agglutinate them, insufficient for visual inspection by conventional techniques. It is often necessary to add a secondary antibody against human IgG in order to easily obtain observable agglutination.

In erythrocyte typing, a blood test used to determine the presence or absence of specific antigens on the surface of red blood cells, the red blood cells to be analyzed are added to the upper chamber and a force, such as centrifugal force, is applied to cause the red blood cells to enter cells containing antibodies against the specific red blood cell antigen and separate substratum. If the red blood cells have antigens on their surface that bind to specific antibodies in the lower chamber, agglutinates form and are separated with a matrix.

In other types of blood assays (such as reverse typing), to measure directly agglutinated antibodies against erythrocyte antigens in the patient's serum, the patient's serum and a reagent of erythrocytes with known antigens on their surface are added to the upper chamber, And a force (such as centrifugal force) is applied to cause the reactants to enter the lower chamber containing the liquid medium and separation matrix but no antibody. In this assay, directly agglutinated antibodies present in patient serum produce agglutinates that are separated using a matrix.

In another type of blood assay, antibody reagents with known specificities for red blood cell antigens are deposited with the patient's red blood cells in the upper chamber. If the antibody reagent is a direct agglutinated antibody, a force (eg, centrifugal force) is applied without prior incubation and the contents are forced into the lower chamber, which contains the separation matrix in aqueous solution. The agglutinates are then separated with a matrix. Alternatively, the patient's erythrocytes can be pelleted in the upper chamber and an IgG antibody reagent of known specificity added followed by incubation to allow the antibody to bind to a predetermined antigen on the surface of the erythrocytes. Following incubation, a force (eg, centrifugal force) is applied to draw the reactants into the lower chamber, which contains the separation matrix and anti-IgG antibodies specific for the IgG antibody reagent used to incubate the erythrocytes in the upper chamber. If antibody reagents are present on the patient's cell surface, anti-IgG antibodies in the lower chamber tend to form agglutinates that are separated by the matrix.

After incubating the sample and reagents for a sufficient time to allow direct agglutination (as in cell typing assays) or antibody-antigen reactions (as in Coombs assays) to occur, the reaction vessel is pressurized by centrifugation so that The reactants are extruded to the lower part of the column and onto the separation matrix. As a result of the centrifugation, non-agglutinated material migrates down through the separation matrix, while agglomerated cells remain on top of the separation matrix or are distributed within the matrix depending on the degree of agglutination. Stronger agglutination reactions result in cell retention in an upper position close to the separation matrix, whereas weaker agglutination reactions result in the distribution of agglutinates at various distances above the matrix.

Sample and reagent retention in the upper portion of the column during incubation is the result of surface tension across the tip of the lower portion of the column, where the diameter of the lower portion of the column is reduced relative to the upper portion. Two potential sources of error in assays performed with this column have been pointed out. First, if reagents and samples are dropped directly into the center of the reaction chamber with excessive force, the reactants can settle directly on top of the separation matrix in the lower chamber and not remain in the upper chamber during incubation. In this way, reactants begin to enter the separation matrix before agglutination is complete. Second, diluents or solutions containing the separation matrix have the potential to enter the upper chamber. Such errors can arise, for example, through spills or other disturbances when transporting or handling the container. In the case of solutions or diluents containing the separation matrix as well as antibodies or other reagents that directly affect the test results, such splashing can lead to cross-contamination of the column with certain reagents from other columns. This phenomenon occurs when a user inserts a pipette tip into a reaction chamber, and spilled reagents contaminate the tip, which is then transferred by the pipette to another container. This can lead to erroneous results in agglutination assays.

It is therefore an object of the present invention to provide an improved mechanism for keeping the sample and reagents separated during incubation in an agglutination assay. Yet another object of the present invention is to provide means for preventing displacement of substances contained in the lower part of the column.

The present invention provides an improved vessel for performing agglutination reactions and separating agglutinates. The vessel includes an upper chamber for containing reactants, a lower chamber for separating agglutinates, and a separation chamber barrier means that allows the reactants to remain in the chamber prior to introducing the contents of the upper chamber onto the separation matrix. In the upper chamber, and when a force (ie, greater than atmospheric pressure) is applied to the barrier, the contents of the upper chamber can be caused to migrate from the upper chamber to the lower chamber. In a preferred embodiment, the barrier includes a narrow passage between the upper and lower chambers. The narrow passage can be formed during manufacture by casting or inserting an insert with a narrow hole, a crimped or helical or other similar geometry, between the upper and lower chambers. This insert provides a physical barrier that reduces the pore size between the lower and lower chambers, thereby preventing the contents of the lower chamber from contaminating the upper chamber. Furthermore, the insert allows the separation of sample and reagents during incubation. When the insert is helical, the passageway defined by the screw of the helix, the central shaft and the wall of the column is small enough for liquid to remain in the upper chamber under normal gravity and atmospheric pressure. For example, the diameter of the helical insert is in the range of approximately 0.110-0.140 inches. The diameter of the helical insert shaft is approximately 0.030-0.090 inches. Spiral inserts have approximately 6-30 threads per inch.

Narrow pathways can also be formed by ultrasonic welding of the column followed by loading the column with reagent. The passageway may also contain a bulkhead-shaped barrier. In this embodiment, the partition has a central hole small enough to retain fluid in the upper chamber under normal gravity and atmospheric pressure. The separator is preferably silicone rubber. The passageway may also comprise a barrier in the form of a porous plug. The porous plug has passages therethrough that are small enough to retain fluid in the upper chamber under normal gravity and atmospheric pressure. The porous plug is preferably polyethylene.

In another embodiment, the invention includes a bushing in the form of a tapered member having a bore through a narrow tip. This liner is placed on the tip and extends into the column of the reaction vessel before pipetting the sample into the vessel, thus avoiding cross-contamination of diluent or solution from one vessel into another, which would otherwise occur during pipetting Another container may be cross-contaminated during the process. The end user conveniently inserts the liner onto the top of the column prior to pipetting reagents. The liner may be in the form of a unit having six conical sections or pools arranged side by side. The total area and shape of the liner is the same as the top side of the BIOVUE ^™ box.

The bushing includes a main body and at least one tapered member extending from the main body, wherein the tapered member has a hole at a narrow tip thereof, and the tapered member is at a position spaced from the narrowed tip There is a sealing device. The tapered member includes a first end having said narrow tip and a second end spaced from a location proximate to the body, wherein said sealing means is located on or near the second end. The sealing means comprises an O-ring surrounding said conical part and the O-ring is integrally integral with the conical part. In a preferred embodiment, the liner has six tapered parts projecting linearly from the body and is sized to match the reaction vessels of the cartridge. The bushing is preferably acrylic.

The present invention further contemplates utilizing a foil-sealed box comprising six reaction vessels linearly arranged therein, and a liner comprising a main body and six conical parts projecting linearly therefrom, wherein the cones The shaped part includes a narrow tip adapted to pass through the foil seal. And this bushing which is now inserted frictionally engages in said box so as to seal the junction between the box and the bushing. The means for sealing the joint is an O-ring surrounding and preferably integral with each cone.

Figure 1 shows a reaction and separation vessel with an insert having a narrow perforation in the upper reaction chamber.

Figure 2 is a top view of a cassette with six reaction vessels, where the reservoir has no insert, one vessel (second from the left) contains an insert as shown in Figure 1, and one vessel (third from the left) contains the reaction agent.

Fig. 3 is a cross-sectional view of the cartridge with reaction vessels taken along line 3-3 of Fig. 2 .

Figure 4 is a side view of a cartridge with reaction vessels.

Figure 5 is a sectional view taken along line 5-5 of Figure 2 showing an insert having a narrow perforation in the upper chamber of the reaction vessel.

Figure 6 shows the upper chamber of a reaction vessel with a narrow perforation.

Figure 7 shows the insert with an extension having a perforation in the lower chamber.

Fig. 8 is a sectional view along line 8-8 of Fig. 3 .

Figure 9 shows the reaction and separation vessel that has been crimped beneath the upper chamber.

Fig. 10 is a sectional view taken along line 10-10 of Fig. 9 .

Figure 11 is a side view of a reaction and separation vessel with a helical insert 1 in the upper chamber 2, the lower part 3 of which has been modified to accommodate said insert.

12 is a side view of the construction of a helical insert; (A) shows an insert with an overall diameter 1 of 0.120 inches and an inner shaft diameter 2 of 0.060 inches; (B) shows an overall diameter 1 of 0.120 inches with an inner shaft 0.08 inch diameter insert.

Figure 13 is a side view of a helical insert with an overall diameter 1 of 0.120 inches and an inner shaft diameter of 0.06 inches.

Figure 14 shows a pool of liners fitted into the upper chamber of the reaction vessel column. Each cone or pool has an O-ring 1 around the pool below the bushing body.

Figure 14A is a plan view of a cell designed to fit into a liner in the upper chamber of a reaction vessel column. Each pool has an O-ring 1 located around the pool under the body of the bushing. This figure shows the angle of the narrow pointed top of the liner pool, designed to pierce the seal of the basin.

Figure 14B is a top view of one of the reservoirs of the liner with the innermost circle further illustrating the perforations therethrough.

Figure 14C is a plan view of a well of the liner showing the angle of the top end of the liner so designed to pierce the sealed cartridge.

Figure 15 shows a liner comprising six conical members or pools with pointed tips that fit into the upper chamber of a cassette with six reactor columns. Each part has an O-ring 1 around the part under the bushing body. Each part also has a tip 2 for piercing the foil seal covering the reaction vessel column of the cassette.

Figure 15A is a top view of a liner comprising six conical members or pools.

Figure 15B is a plan view of a single liner cell showing the angle of the top end of the liner designed to pass through the sealed box.

Figure 15C is a plan view of a liner comprising six conical members or wells with pointed tips that fit into the upper chamber of a cartridge with six columns of reaction vessels by piercing the seal of the cartridge.

According to the present invention, a vessel for performing an agglutination reaction and separating agglutinates will be described in various embodiments. Certain embodiments of the present invention will be best understood from the description of the agglutination reaction and the cartridge-shaped separation vessel manufactured and sold by Ortho Diagnostic Systems Inc., Raritan, New Jersey, under thd trademark BIOVUE ^(TM ).

The container of the present invention can be made of any suitable material (such as glass or various plastics) that does not interfere with the agglutination reaction or separation and visual results. In a preferred embodiment, the container is made of polypropylene.

The upper chamber of the container can be of any shape and size for containing reagents and samples while the incubation is taking place. Typically, the upper chamber is cylindrical over most of its upper portion. The barrier between the upper and lower chambers generally defines the lower interface of the upper chamber and the upper interface of the lower chamber. In a preferred embodiment, the barrier forming the lower portion of the upper chamber is tapered and its tip projects towards or into the lower chamber, as shown in FIGS. 1 , 5 , 6 and 7 . A portion of the barrier serves to retain reagents and samples in the upper chamber under normal conditions of gravity and atmospheric pressure during incubation, while allowing fluid to flow from the first chamber when a force (such as increased pressure or centrifugal force) is applied to the second room. This can be accomplished by various means such as small holes, membranes, pistons, stoppers, inserts of any geometry, or screens, and combinations of any of these. In a preferred embodiment, the barrier comprises an aperture of sufficiently small diameter to prevent fluid flow from the first chamber to the second chamber under normal gravity or atmospheric pressure, but to allow fluid flow therethrough under increased pressure. The hole 1 is located at the top of the conical part of the upper chamber, which can be included in the insert 2 (as shown in Figure 1, 5 or 7) and also in the upper chamber (as shown in Figure 6).

The diameter of the orifice is small enough that the surface tension of the fluid in the upper chamber prevents the fluid from flowing from the upper chamber to the lower chamber under normal gravity or atmospheric pressure, and under increased pressure or gravity, the surface tension can be overcome, so, The contents flow from the upper chamber to the lower chamber. The diameter of the hole is changed according to the amount of force applied, ie the hole diameter is smaller when a higher force is applied and larger when a lower force is applied. The diameter can also be varied to accommodate different sized particles in the reagent. In a preferred embodiment, the diameter of the holes is in the range of approximately 0.010-0.050 inches. In a particularly preferred embodiment, the diameter of the holes is 0.020 inches.

In another embodiment, the barrier means separating the upper and lower chambers comprises a helical insert; the helix may be of screw-shaped configuration. The helix is preferably circular or cylindrical in diameter, although elliptical configurations may also be used. Similar to the orifice described above, the passageway defined by the shaft of the helix, the threads, and the walls of the chamber is sufficiently small in diameter to prevent fluid flow from the upper chamber to the lower chamber under normal gravity or atmospheric pressure, while under increased force Or the fluid can flow under pressure. Yet another function of the insert is to reduce the possibility of diluent or solution being spilled from the lower chamber into the upper chamber, thereby contaminating the upper chamber. Such splashing can occur, for example, during shipping and handling. The helix is located in the upper chamber on the bottom 3 of the upper chamber as shown in FIG. 11 , or is cast in one piece with the reaction vessel in this position.

Where the helical inserts are individually inserted into the upper chamber, the inserts may be molded of any suitable material such as glass or plastic that does not interfere with the agglutination reaction or separation or visual results. The material is preferably a plastic such as polypropylene, polyamide (e.g. nylon), acetate (e.g. Delrin ^™ or DelrinP ^™ ), cross-linked polyethylene/divinylbenzene (e.g. Rexolit-e ^™ ), polycarbonate or polycarbonate vinyl. In a preferred embodiment the material is polypropylene.

The helix can be any geometry such that the surface tension of the fluid in the upper chamber prevents the flow of fluid from the upper chamber to the lower chamber under normal gravity or atmospheric pressure, and overcomes the surface tension under increased pressure or gravity, Thus, the contents flow from the upper chamber to the lower chamber. For example, the pitch angle of the helical flight can be any angle that allows fluid (e.g., containing blood cells) to flow from the upper chamber to the lower chamber under increased pressure while avoiding contamination of the upper chamber with fluid or substrate from the lower chamber . The pitch angle can be varied according to the amount of force applied, ie the area defined by the screw shaft and threads and chamber walls is smaller when higher force is applied and larger when lower force is applied. The geometry of the helix can also be varied to accommodate different sized particles in the reagent. Furthermore, the insert enhances the possibility of avoiding the contents of the column (containing the separation matrix and diluent or solution) from splashing into the upper chamber.

In the embodiment of the helical insert of the present invention, only this helix is used to prevent the effect of fluid from the lower chamber contaminating the upper chamber (low end of the value range) and passage of the sample (e.g. red blood cells) under increased pressure. The power of the screw (the high end of the scale) defines the number of threads per inch and the depth of the threads. For the foregoing reasons, in order to facilitate the passage of red blood cells and other agglutinates through the barrier device, it is desirable that the threads, central shaft and post walls defining the passage be relatively smooth in texture and finish. The wall of the threaded portion of the helical insert facing the wall of the column may be pointed or flat.

In a preferred embodiment, the helix has about 6-30 threads per inch, more preferably about 12-20 threads per inch, for use in a BIOVUE ^™ cassette of present configuration including six reaction vessels , the pitch measured from the top of one thread to the top of the lower thread below is in the range of about 0.033 to about 0.166 inches, more preferably about 0.050 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 about 0.130 inches.

The helical insert shaft is in the range of about 0.030 to about 0.090 inches, more preferably about 0.060 to about 0.080 inches. Turning to Figures 11, 12 and 13, these figures illustrate the relative geometry of the insert components. Referring to Figure 13, the approximate geometry (in inches) is shown as follows:

Thread/inch DI M3 DI M6

12 0.083 0.020

14 0.070 0.020

16 0.060 0.015

As an alternative to the modified lower circular diameter helical insert placed in the upper chamber as shown in Figure 11, an elliptical helix with a pitch of similar geometry could also be used here. As previously described for the circular diameter helical insert, the elliptical barrier means can be either in the form of an insert or a helical obstruction cast as a unit with the chamber.

As yet another alternative, the constricted passage between the upper and lower chambers can be formed by ultrasonically welding the column after loading the column with reagents. Alternatively, the barrier may comprise a disc or piston of porous material positioned at the top of the lower chamber or column. The piston is cylindrical and sized to fit between the upper and lower chambers, adapted to prevent the contents of the lower chamber (eg reagents and separation matrix) from splashing into the upper chamber. The porous piston is also adapted to prevent premature passage of the sample (eg before centrifugation), but instead the sample passes through the piston at a pressure greater than atmospheric pressure (eg under centrifugation). The porous piston can be made of any material that does not interfere with agglutination or separation, or any visual outcome, or that does not bind specifically to any of its components, examples of which are glass, especially scintillation glass, and plastics (e.g. polypropylene).

Other suitable barriers are flanges built into the helix, stepped grilles around the chamber wall, or other similar tortuous paths, all of which have the effect of preventing contamination of the upper chamber contents as previously described. The barrier can also be an annular valve or baffle placed inside the column. Such a partition can have, for example, a central hole or perforation and the partition is scored radially from the column wall to the central perforation. Upon application of force, the diaphragm opens, allowing sample contents to flow from the upper chamber to the lower chamber. The separator can be made of any suitable material that does not interfere with agglutination or separation, or any visual results. The spacer can be made of any suitable flexible, moldable material such as silicone rubber.

When the container of the present invention is used for agglutination reactions and separations, reagents and samples are added to the upper chamber for incubation. Samples and reagents are kept in the upper chamber while the barrier is incubated. In a preferred embodiment, the barrier has a hole, or the barrier is in the form of a helical insert, the diameter of the hole or the geometry of the helix, respectively, being small enough to allow liquid and sample to pass through the hole or the surface tension of the helix under normal gravity and atmospheric conditions. The contents are retained in the upper chamber under pressure. After a sufficient incubation time, a force is applied to the barrier substantially in the axial direction from the upper chamber to the lower chamber by any of various means such as centrifugation, pressure or absorption. This force must be sufficient to overcome the barrier and allow the contents of the upper chamber to pass into the lower chamber. In a preferred embodiment, the barrier comprises a hole, or the barrier means comprises a helical insert, the surface tension is overcome by force and the contents flow from the upper chamber into the lower chamber into the separation matrix.

The barrier is also important in that it reduces the possibility of contamination of the upper chamber by diluent or solution in the lower chamber that would otherwise occur, for example, due to spills or other disturbances during shipping and handling.

Another embodiment of the invention is a liner that can be inserted into the top of the reaction vessel prior to adding the sample. The bushing comprises a main body and one or preferably a plurality of members or pools projecting from the main body, the pools being conical or funnel shaped. When the narrow top end of the pool containing a hole is inserted into the box, it is oriented towards the interior of the container, as shown in FIGS. 14 and 15 . Liners are applied to containers with or without barrier devices such as crimps or inserts. In a preferred embodiment, the liner comprises a pool with a perforation, ① the diameter of which is small enough to block the sample to be delivered to the container until the container is subjected to force (for example by centrifugation) and the incubation time will not be extended by the thermal insulation effect of the air gap between the top and the separation matrix in the column; or ② the hole is large enough to allow the sample to be transported to easily flow into the reaction vessel chamber and allow the test system to prematurely contact with the reagent. Contact with splashes. The reservoir of the liner preferably has a seal under the body of the liner at a location spaced from the narrow top end (eg its outer top end). Reference is made to FIGS. 14 and 15 in this regard. The seal is a raised ring which is preferably integral with the bushing. The lined pool fits into the upper chamber of the reaction vessel so that the pool cannot be easily removed during normal operation. A collar or O-ring fits around the rim of the container on the upper surface of the box, thus sealing the junction between the liner and the box. The raised ring or O-ring prevents capillary action of any spilled column contents from one column to the other.

As mentioned above, when the insert is not installed in the container, diluent or solution containing the separation matrix can enter the upper chamber during shipping or handling. In cases where the separation matrix also contains antibodies or other reagents that directly affect the test results, this splatter can cause cross-contamination of the column with reagents from other columns. This cross-contamination occurs when a user inserts a pipette tip into the upper chamber of a reaction vessel, spilled reagent contaminates the tip, and the tip is then transferred with the pipette to another vessel. The latter can lead to erroneous results in agglutination assays.

The purpose of the liner is to prevent cross-contamination of reagents from one container to the next during pipetting; reagents can splash upward into the upper chamber during transport and handling.

The liner can be a conical member or a pool, respectively located at the top of each reaction vessel. However, in a preferred embodiment, referring to Figure 15, the liner is a single unit having six conical pools arranged side by side, integral with the liner body. This configuration allows one liner unit containing six individual wells to sit on top of a six reaction vessel BIOVUE ^™ cassette.

Referring to Figures 14 and 15, each conical pool of the bushing has a narrow tip with a perforation therethrough. When the BIOVUE ^™ container is a box of six columns and the top of each column is sealed with foil tape, the tip of the liner under normal pressure will pierce the foil seals on all six columns. The sample is then transferred to the cell by pipetting. This way, the clean lined cell does not receive any contamination from reagents or separation matrix that would otherwise come into contact with the sample and be carried to another column.

The bushing is conveniently inserted into the post by the end user manually or using a forked tool. To insert the six well liners into the BIOVUE ^™ cassette, the foil of the cassette was pierced with the sleeve tip and fully inserted into the cassette by shaking the liner. Since the body of the bushing is the same area and shape as the top surface of the box, the bushing conveniently remains in place during manipulation of the box. Using the liner in this manner does not interfere with assay performance or results (eg: centrifugation, free passage of unagglutinated RBCs through the separation matrix, agglutination of RBCs into the column) However, columns without O-rings cannot be used during use. Prevent cross-contamination of reagents to the column. See Examples 10 and 11 for comparative performance tests with and without O-rings. The results here demonstrate that O-rings prevent reagent cross-contamination of the column due to reagent "siphoning" between the foil and liner of the cartridge, resulting in reagent flow through the top of the cartridge.

The liner and its reservoir can be made of any suitable material that does not interfere with agglutination or separation, such as glass or plastic. The material must be suitable for piercing the metal derivative seal on the box. In order to efficiently transfer the sample from the cell of the liner to the upper chamber, it is preferred that the cell walls of the liner are relatively smooth in texture and finish. The material is preferably a plastic such as 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 was added to 40 μl of patient serum and incubated with 40 μl of low ionic temperature solution in the upper chamber for 10 minutes to allow binding of predicted patient IgG antibodies to red blood cell surface antigens. The assay components are added separately and, importantly, retained in the upper chamber for mixing so that the ratio of low ionic strength solution to red blood cells and serum remains constant during the assay. Barriers tend to do this under normal gravity and pressure. It also reduces the chance of any assay components being forced into the lower chamber during sample loading. The barrier also allows assay components to remain in the upper chamber throughout the incubation period.

The barrier is also important in that it prevents premature binding of anti-human immunoglobulin (IgG) antibodies to predicted anti-erythrocyte antibodies prior to binding to erythrocytes, thereby reducing the chance of agglutination eventually occurring in the lower chamber. After incubation, centrifugal force is applied to force the contents of the upper chamber through the barrier into the lower chamber, which contains anti-human immunoglobulin (IgG) that binds to patient immunoglobulin (IgG) on the surface of the red blood cell reagent, thereby forming agglutination condensate cannot flow through the matrix to the bottom of the lower chamber.

The following examples are only for understanding the present invention, rather than limiting the scope of the present invention.

example 1

BIOVUE ^(TM) columns with inserts were compared to columns without inserts to determine the effectiveness of each configuration for maintaining a spatial barrier separating the reactants from the separation matrix during incubation. Utilize an insert with a 0.040 inch hole. 40 microliters of buffer was added to each of the 840 columns used in the experiment. The pipette was artificially used at an angle of approximately 45° to the vertical axis of the column in order to deliver the 40 microliters of solution. The column was then observed to determine if an air gap was maintained beneath the reaction chamber. Table 1 gives the number of "leaks out".

Table 1 Number of experiments leaked number leakage percentage Pillars with plugins 840 0 0% Posts without plugins 840 231 27.5%

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 columns used in the test, 40 microliters of buffer, 40 microliters of serum and 10 microliters of erythrocyte suspension were added. Use the pipette at an angle of approximately 45° to pipette the reagents. After a period of incubation, the column is checked to determine whether an air gap remains under the reaction chamber. Table 2 gives the "leakage" frequencies.

Table 2 Number of experiments leaked number leakage percentage Pillars with plugins 480 0 0% Posts without plugins 480 16 3.3%

Example 3

Fill the column with 40 microliters of buffer with an automatic pipette at an angle of approximately 45°. Automatic pipettes typically use higher transfer forces than manual methods. Observations were made after filling to determine whether an air gap remained under the reaction chamber. Table 3 presents the results for columns with and without inserts.

table 3 Number of experiments leaked number leakage percentage Pillars with plugins 240 0 0% Posts without plugins 240 103 43%

Example 4

Fill 240 columns vertically with 40 microliters of buffer using a pipette. Since the pipette is held vertically, the liquid flow presses the perforation with greater pressure, thus possibly breaking through the air gap separating the reaction chamber from the separation chamber. Table 4 presents the results of this test.

Table 4 Number of experiments leaked number leakage percentage Pillars with plugins 240 0 0% Posts without plugins 240 144 60%

Example 5

40 microliters of buffer was also filled into the 240-column reaction chamber using the automatic pipette vertically, which was more likely to disrupt the air space below than holding the automatic pipette at an angle. Table 5 presents the results of these tests using columns with and without inserts.

table 5 Number of experiments leaked number leakage percentage Pillars with plugins 240 0 0% Posts without plugins 240 204 85%

The invention functions in that, in addition to maintaining an air gap between the reaction chamber and the separation matrix during the incubation period of the assay, it also serves as a means of preventing splashing, which can occur during shipping and handling, resulting in the following Part of the contents of the separation chamber splashes upwards into the upper reaction chamber. To test the effectiveness of splash protection, boxes with and without inserts were shipped from New Jersey to California and back. Shipments are made by air and land, and the process includes loading, unloading, and delivery to the laboratory. After shipping back and forth, check the box for spilled liquid in the reaction chamber. The results are shown in Table 6.

Table 6 Number of experiments Number of columns with liquid splash Percentage of columns with spilled liquid Pillars with plugins 816 30 3.7% Posts without plugins 768 571 74.3%

Example 7

Another shipping study was conducted to test that the potential for splashing could be reduced using an insert with a reduced size hole. The opening diameters between the reaction chamber and the separation matrix were 0.025, 0.020, and 0.015 inches. Fill 600 columns with each insert. The control group has the plug-in. Boxes were packed and an indoor surrogate delivery study was performed, ie, boxes were dropped 10 times from a height of 3 meters. Controls the angle of the box so that the container falls on all of its six planes plus a corner and 3 sides. This standardized test represents damage in shipping and handling. The results presented in Table 7 show an inverse relationship between pore size and splash reduction.

Table 7 Number of experiments Number of columns with liquid splash Percentage of columns with spilled liquid Column with 0.015 plugin 600 75 13% Column with 0.020 plugin 600 120 20% Column with 0.025 plugin 600 132 twenty two% Column without insert 600 600 100%

Example 8

Another way is to reduce the aperture between the reaction chamber and the separation chamber below by "crimping" the box. This can be achieved by stamping, in which the neck of the box just below the reaction chamber is impacted. The force and duration of the impact determine the extent to which the opening is reduced. The shape of the impact tool determines the shape of the opening. Several configurations are available. The crimping process can be done on the production line after loading the reagents and glass beads into the column.

816 columns from the production line were crimped to define the opening between the reaction chamber and the separation matrix as described above. This crimping results in a cross-sectional shape (as shown in FIG. 10 ) through the area indicated by a parenthesis in FIG. 9 . These columns, along with 768 uncrimped control columns, were packaged and shipped to California and back from there (as previously described). Table 8 presents the results of the reduction in the likelihood of liquid splashing into the reaction chamber due to shipping conditions.

Table 8 Number of experiments Number of columns with liquid splash Percentage of columns with spilled liquid curled pillar 816 548 67% Columns without beading 768 571 74%

Example 9

A helical insert barrier arrangement is used to define the pore size between the reaction (upper) chamber and the separation (lower) chamber. The helical insert was cast from polypropylene and inserted into the neck of the cartridge just below the modified upper chamber. The helical insert is placed into the neck of the cartridge during the manufacturing process after loading the reagents and separation matrix (eg glass beads) into the column.

Example 10

BIOVUE ^™ columns lined with O-rings were compared to columns lined without O-rings to determine whether the O-rings prevent reagent cross-contamination of the column.

By slapping or banging the box against a solid work surface, splashes in the upper chamber and on the foil during transport of the box can be simulated.

The 192 cassettes with no O-rings in the liner were observed to see if the reagent in the column siphoned after the liner was inserted into the column of the cassette. One half (96) of the bushing is manually inserted into the box, while the other half is inserted using the dual prong tool discussed above.

Observe the 336 boxes lined with O-rings for siphoning of reagents in the column as described above. The bushing is manually inserted into one half (168) of the box while the other half (168) is inserted with a double-pronged tool.

The siphonage of the column was determined visually by inspecting the top side of the box. This siphon effect can be determined by observing the reagent fluid between the foil top of the cartridge and the underside of the liner body.

Table 9 shows the number and percentage of boxes with siphons out of the total number of boxes used for each test. The method of bushing insertion does not affect the results with bushings with O-rings, but does affect the results with bushings without O-rings. As shown, when the liner was manually inserted (cassettes in the inverted position), there were approximately double the number of cartridges with siphon reagents than when the liner was inserted with the tool (cassettes in the vertical position).

Table 9 Bushing Insertion Method Bushing without O-ring Bushing with O-ring with tools 39/96 (40.6%) 0/168 artificial 74/96 (77.1%) 0/168

Example 11

A functional test was performed comparing a box lined without an O-ring with a box lined with an O-ring.

Simulate shipping boxes with reagent splashes in the upper chamber and on the metal foil, as described in e.g. 10.

As mentioned above, the bushing is inserted into the post of the cartridge both manually and with the use of a two-pronged tool. In one half of the box, 10 μl of 40% red blood cells (in normal saline) were pipetted into each column using a BIOVUE ^™ BioHit (orthoDiagnostic Systems Inc., Raritan, NJ) pipette. In the other half of the box, 50 μl of 80% erythrocytes (in normal saline) with surface antigens as listed in Table 10 below were pipetted onto the column of the box. In half of the cassettes used for the experiment, the Ortho BIOVUE ^™ ABE International cassette (Ortho Diagnostic Systems Inc., Raritan, New Jersey) was used. In the other half of the box used for the assay, the (Ortho BIOVUE ^™ Bltk box (Ortho Diagnostic Systems Inc., Raritan, New Jersey) was used. The ABE and BHK boxes had antibodies in each column (as shown in Table 10 below)

Table 10 ABE box column Antibody Expected results with A ₁ rr cells 1 Anti-A + 2 Anti-B - 3 anti-AB + 4 Anti-D - 5 anti-CDE - 6 Control (diluent) - RHK box column Antibody Expected results with R ₁ R ₁ K(-) cells 1 Anti-C + 2 Anti-E - 3 Anti-C - 4 anti-e + 5 Anti-K - 6 Control (diluent) -

When using the ABE cassette, _A1rr cells were used as samples. When using the RHK cassette, R ₁ R ₁ K(-) cells were used as samples. Pipetting proceeds from the column of the leftmost box all the way to the right. Cassettes were centrifuged at 794 +/- 16 xg for 2 minutes in an Ortho BIOVUE ^™ centrifuge (Ortho Diagnostic Systems Inc., Raritan NJ). Then centrifuge again at 1510+/30xg for 3 minutes. Evaluate each column with a negative reaction (red blood cells not agglutinated) to see if the red blood cells have passed through the entire column. If, for example, an anti-H antibody is transferred from column 1 of the ABE cassette to column 2 and only then reacts in column 2, a false positive reaction can occur.

The results are shown in Table 11, all expected positive reactions were positive in the column with the O-ring. However, not all expected negative reactions were negative. The cause, although not identified, was not siphoning of the reagents. Table 11 shows the number and percentage of boxes and columns with false positive reactions out of the total number of boxes used for each test. The method of inserting the bushing did not affect the results using bushings with O-rings, but indirectly affected the results using bushings without O-rings, as all false positive reactions occurred on bushes with visible siphons. in the reagent box.

Table 11 Bushing Insertion Method Bushing without O-ring Bushing with O-ring tool (vertical) Box 1/96(1%) 1/96(1%) column 1/384(0.3%) 1/384(0.3%) Manual (reverse) Box 9/96 (10%) 0/96 column 10/384 (2.6%) 0/384 total Box 10/192 (5.2%) 1/192(0.5%) column 11/768 (1.4%) 1/768(0.1%)

Claims (26)

1 one kinds of containers that are used to carry out CA comprise:

A) one has a last chamber that is used to receive the opening of fluid reaction agent;

B) one is used to receive from the fluid of last chamber and contains the following chamber that is useful on the matrix of separating agglutinator; And

C) will go up the barrier that chamber and following chamber separate for one, this barrier can remain on fluid in the chamber under normal gravity and atmospheric pressure, and can make under greater than the pressure of atmospheric pressure fluid from chamber under the inflow of chamber, this barrier comprises a kind of helical structure.

The container of 2 claims 1, wherein said helical structure have a path that is limited by screw thread, central shaft and post jamb, and this path is little as to be enough under normal gravity and atmospheric pressure in the chamber in the fluid maintenance.

The container of 3 claims 2, wherein helical structure comprises a spirality plug-in unit.

4 one kinds of containers that are used to carry out CA comprise:

A) one has one and is used to receive the opening of fluid reagent and the last reaction chamber of a perforation, and this perforation is limited by a plug-in unit with a helical geometry, thus under gravity and atmospheric pressure, fluid can be remained on described in the reaction chamber; And

B) following chamber that communicates with last chamber by the spirality plug-in unit, described chamber down contains the isolation medium that is useful on the separation agglutinator.

5 containers according to claim 4, wherein the spirality plug-in unit is polypropylene or rexolite.

6 one kinds of containers that are used to carry out CA comprise:

A) first Room that is used to receive and keep fluid sample and reagent;

B) second Room that communicates, is used for receiving fluid with first Room from first Room, described second Room contains the isolation medium that is useful on the separation agglutinator; And

C) barrier that described first and second Room are separated, this barrier can stop fluid to enter second Room from first Room under normal gravity or atmospheric pressure, and under the pressure bigger, can make fluid flow into second Room from first Room than atmospheric pressure, wherein said barrier comprises a spirality plug-in unit.

7 containers according to claim 6, wherein the spirality plug-in unit comprises that diameter is little as to be enough under normal gravity and atmospheric pressure fluid remained on path in first Room.

8 containers according to claim 7, wherein the diameter of spirality plug-in unit is in about 0.110 to 0.140 inch scope.

9 according to Claim 8 containers, wherein the diameter of spirality inserter shaft is approximately 0.030 to 0.090 inch.

10 containers, wherein nearly 6 to 30 screw threads of spirality plug-in unit per inch according to claim 9.

11 1 kinds of containers that are used to carry out CA comprise:

A) one has a last chamber that is used to receive the opening of fluid reaction agent;

B) one is used for the following chamber that receives fluid from last chamber and contain the matrix of separating agglutinator; And

C) will go up the barrier that chamber and following chamber separate for one, this barrier can remain on fluid in the chamber under normal gravity and atmospheric pressure, and can make under greater than the pressure of atmospheric pressure fluid from the chamber flow under the chamber, wherein said barrier comprises a dividing plate.

The container of 12 claims 11, wherein said dividing plate have little a center pit that is enough under normal gravity and atmospheric pressure fluid remained in the chamber.

The container of 13 claims 12, its median septum is a silicon rubber.

14 1 containers that are used to carry out CA comprise:

A) one has a last chamber that is used to receive the opening of fluid reaction agent;

B) one is used for the following chamber that receives fluid from last chamber and contain the matrix of separating agglutinator; And

C) will go up the barrier that chamber and following chamber separate for one, this barrier can remain on fluid in the chamber under normal gravity and atmospheric pressure, and can make under greater than the pressure of atmospheric pressure fluid from the chamber enter chamber down, wherein said barrier comprises a perforated piston.

The container of 15 claims 14, wherein said perforated piston has the path that passes therethrough, and these paths are little as to be enough under normal gravity and atmospheric pressure fluid be remained in the chamber.

The container of 16 claims 15, wherein perforated piston is polyacrylic.

17 1 kinds are used for preventing that reagent or solution from transferring to another post of described box from a post of CA box and cause the lining of cross pollution, wherein said lining comprises a main body and at least one conical part that stretches out from it, described conical part has a hole on its narrow top, and described conical part has packoff on the position that separates with described narrow top.

The lining of 18 claims 17, wherein conical part comprises first end and near second end from separating the described main body with described narrow top, wherein said packoff is positioned on described second end or in its vicinity.

The lining of 19 claims 18, wherein packoff comprises an O shape ring around described conical part.

The lining of 20 claims 19, wherein packoff comprises an O shape ring that is integrated with described conical part.

The lining of 21 claims 20, wherein six conical parts linearly stretch out on described main body.

The lining of 22 claims 21, its size is complementary with the reaction vessel of box.

The lining of 23 claims 22, wherein lining is acrylic acid.

24 1 kinds of linings, comprise a main body and six conical parts that stretch out from it, wherein each conical part comprise one spaced apart and have first end of a perforation and one and isolated second end of described first end on its narrow top with described main body, described second end has an O shape ring and becomes one around described conical part and with it.

The box that comprises six reaction vessels that are arranged in a linear within it of 25 1 kinds of metal foil seals, and a kind of lining that comprises a main body and six conical parts that linearly stretch out from it, wherein conical part comprises a narrow tip that is suitable for piercing through described metal foil seal, and this lining that be inserted into this moment frictionally is installed in the described box, so that the bonding part between seal box and the lining.

The box of the metal foil seal of 26 claims 25 and lining, the device that wherein is used to seal described bonding part are around described each conical part and the O shape ring that becomes one with it.

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