HK1025361B - Method for pcr testing of pooled blood samples - Google Patents

Description

PCR test method for collecting blood samples

Technical Field

This application is a continuation-in-part application having application number 08/683,784 filed on 16/7/1996, and application number 08/683,784 is a divisional application of application number 08/419,620 filed on 10/4/1995, the contents of which are expressly incorporated herein by reference.

The present invention relates generally to methods and systems for preparing and analyzing samples taken from donated plasma to uniquely identify virus-infected donated plasma. More particularly, the present invention relates to an apparatus and method for forming separate, separately sealed, connected containers containing samples of the same plasma as that contained in a donation. The present invention also relates to an apparatus and method for creating an initial screening test library from containers and examining the library for the presence of viruses according to an algorithm that identifies individual infected donations in a minimum number of test cycles.

Background

Blood, plasma and biological fluid donation procedures are very important first steps in the manufacture of drugs and blood products that can improve quality of life and be used to save lives in various traumatic situations. Such products are used to treat immune system disorders, treat hemophilia, and are used in surgery to maintain and store blood volume and other treatment protocols. The therapeutic use of blood, plasma and biological fluids requires that the donor of these substances be as free as possible from viral infections. Typically, serum samples from each blood, plasma or other donated fluid are tested for various antibodies caused by particular viruses such as Hepatitis C Virus (HCV) and two human immunodeficiency viruses (HIV-1 and HIV-2). In addition, serum samples may be tested for antigens against particular viruses, such as Hepatitis B Virus (HBV), as well as for antibodies caused by such viruses. If the sample serum is positive for the presence of a particular antibody or antigen, the donation is not used further.

However, antigen testing of certain viruses, such as hepatitis B virus, is considered to be associated with infectivity, whereas antibody testing is not. It has long been known that plasma donors may have actually become infected with a virus and that serological tests for antibodies associated with the virus are negative. For example, there is a period of time between the possibility that the donor is infected with the virus and the presence of antibodies within the donor system caused by the virus. The time period from the first appearance of the virus in the blood to the presence of detectable antibodies caused by the virus is defined as the "observation period" (window period). In the case where the virus is HIV, the mean observation period is about 22 days, whereas for HCV, the mean observation period is estimated to be about 98 days. Thus, if the test is performed during the observation period, i.e., the time period between viral infection and antibody production, the test aimed at detecting the antibody may give the infected donor an erroneous indication. In addition, even though the conventional HBV test includes tests for antibodies and antigens, the test conducted in a more sensitive manner has confirmed the presence of HBV virus in the specimen, which is negative in the HBV antigen test.

To further ensure that it is not infected with the primary virus, one method of testing donations that has undergone prior antibody and antigen testing involves testing donations by the Polymerase Chain Reaction (PCR) method. PCR is a very sensitive method for verifying the presence of specific DNA or RNA sequences associated with the virus of interest in biological material by amplifying the viral genome. Since the objective of the PCR assay is to detect the presence or absence of the basic components of the virus itself, the presence of the virus in the donor can be found almost immediately after infection. Thus, there is theoretically no test that could falsely indicate an observation period without infection. The method and practical application of the PCR assay is described in U.S. Pat. No.5,176,995, which is expressly incorporated herein by reference.

However, PCR testing is expensive and individual testing of individual donors is irrevocable or economically unreasonable as less PCR positive donors are included in the entire donor population. Thus, there is a need for an economical and efficient method for testing large blood or plasma donations to exclude units in which viral infection exceeds a predetermined level.

One method of testing large amounts of donated plasma is to pool a number of individual donated plasma. The plasma pool is then subjected to a PCR assay, and depending on the results of the PCR assay, the individual donations that make up the plasma pool are either left behind or discarded. Despite the reduction in the number of PCR assays and the costs associated therewith, this approach results in significant waste of large virus-free donations. Since only one donation whose viral infection exceeds a predetermined level will render one specimen pool PCR positive, the remaining donations that make up the specimen pool may each render PCR negative. This result is most likely to occur in cases where there are a small number of PCR positive donors in the entire donor. In the conventional pooling method, all the donations constituting the specimen pool are discarded on the basis of a positive result of PCR, including those donations that are negative for each PCR.

In addition, donated plasma is usually frozen immediately after collection. When individual plasma donation samples are to be mixed, the individual donations must be thawed, aliquots of blood or plasma must be removed from the donations, and the donations must be stored frozen again. Many freeze-thaw cycles may adversely affect the recovery of the RNA or DNA of interest and the proteins contained in the plasma, thereby adversely affecting the uniformity of the PCR assay. In addition, each time an aliquot of each donated plasma is withdrawn to form a collection, the donations are contaminated by the surrounding environment and the aliquot withdrawal device. In addition, if a donation contains a virus, it may infect other donations. To avoid introducing viral contaminants into other virus-free donations, the sampling device must be sterilized after each use, or the device can only be used to draw an aliquot from each individual donation and a new sampling device used to draw an aliquot from a subsequent individual donation. Such methods all involve significant costs and are rather time consuming.

Therefore, there is a need for a method and system for obtaining a plurality of blood or plasma samples from individual donations so that a particular sample can be pooled without contaminating the other samples. It would also be desirable to be able to form such sample libraries from the methods and systems quickly and efficiently without contaminating the medical laboratory's laboratory or laboratory environment.

It is also desirable that the systems and methods ensure efficient and economical testing of blood or plasma donations to distinguish between only one PCR positive donation in a minimum of test cycles.

Disclosure of Invention

Thus, in the practice of the present invention, a method for economically and efficiently preparing and testing a plurality of blood or plasma donation samples to uniquely identify virus-infected donations, as well as a system and apparatus for practicing the method, are provided.

The present invention provides a method for uniquely identifying a donor biological fluid that is positive for a viral test in a minimum number of high sensitivity test cycles, the method comprising the steps of: providing a plurality of donated biological fluids; defining an n-dimensional matrix, where n is an integer greater than 1, the matrix further having a plurality of elements, each element being defined by an intersection of the n dimensions of the matrix, each individual element being represented by a respective matrix symbol comprising at least one index for each dimension of the array; sampling each donated biological fluid; marking each sample according to a corresponding special matrix element in each matrix element, wherein each independent sample is represented by a corresponding matrix symbol of the corresponding matrix element; taking aliquots from each sample, the number of aliquots taken from each sample being defined by the dimension representing the matrix characteristic; forming a sub-sample library from an aliquot of each sample, each sub-sample library comprising an aliquot of all samples represented by matrix symbols having a dimension label fixed therein, each respective sub-sample library being defined by said fixed dimension label; sending the sub-sample pools to a high sensitivity assay device, wherein all sub-sample pools are subjected to a virus display assay in a single high sensitivity assay cycle; determining each fixed dimension mark of the sub-sample library with positive virus test; and incorporating said fixed dimension code into a matrix symbol, thereby clearly identifying the unique matrix element defined by the matrix symbol and thereby clearly identifying the unique specimen positive for the viral test.

Preferably, the matrix is designed as a regular matrix, the n dimensions of which are each characterized by an identical integer of the matrix elements. The regular matrix comprises a three-dimensional matrix which is further subdivided into rows, columns and layers, each element of the matrix being represented by a matrix symbol X_rcsAnd (4) representing, wherein dimension labels r, c and s respectively represent matrix elements forming a row, a column and a layer of the matrix. The sub-sample library forming step further comprises: forming a sub-library of aliquots from samples represented by the same r-label and different c, s-labels; forming a sub-library of aliquots from samples represented by the same c-label and different r, s-labels; forming a sub-library of aliquots from samples represented by the same s-label and different r, c-labels; and determining each r, c, s subsample pool based on a positive display of the virus obtained by the high sensitivity assay. The method may further comprise the steps of: determining the integer label of each r-sub sample library with positive virus test result; determining the integer label of each c-subsample library with positive virus test results; and determining the integer label of each s-subsample library with positive virus test results. The method can also comprise the following step of converting the integer label of each r, c and s sub-sample library which has positive virus test results into the dimension label r, c and s of the matrix symbol, thereby distinguishing the unique matrix element limited by the matrix symbol and uniquely identifying the corresponding virus positive sample. The three-dimensional matrix comprises an 8 multiplied by 8 regular array, and dimension labels r, c and s respectively take integer values between 1 and 8. Three aliquots were extracted from each respective donated biological fluid sample. Each of the steps may further comprise: forming 8 line subsample banks, wherein each line subsample bank is composed of a whole number between 1 and 8Numerical representations, each column subsample library was composed of 64 aliquots; forming 8 column subsamples, wherein each column subsample is uniquely represented by an integer between 1 and 8, and each column subsample is composed of 64 equal parts of samples; and forming 8 layer sub-sample libraries, wherein each layer sub-sample library is uniquely represented by an integer between 1 and 8, and each layer sub-sample library is composed of 64 aliquots.

The present invention also provides a method for uniquely identifying a donor biological fluid that is positive for a viral test in a minimum number of high sensitivity test cycles, comprising: providing a plurality of donated biological fluids; defining an N-dimensional matrix, where N is an integer greater than 1, the matrix further comprising a plurality of elements, each element being defined by an intersection of N dimensions of the matrix, wherein each individual element is represented by a respective matrix symbol, wherein the subscripts of the matrix symbols define the dimension of the matrix; taking a plurality of aliquots from each sample of each donated biological fluid, the number of aliquots taken from each sample being determined by the number of dimensions comprising the matrix; preparing an aliquot of each sample into a sub-sample library, each sub-sample library comprising an aliquot from all samples represented by a matrix symbol in which one dimension is fixed; sending the sub-sample pools to a high sensitivity assay device, wherein all sub-sample pools are subjected to a virus display assay in a first high sensitivity assay cycle; calculating the dimension standard of each subsample library which is positive in the virus test in the first high-sensitivity test cycle, and if the virus test result of only a single subsample library representing each dimension standard is positive, determining a unique matrix element represented by the dimension standard of each positive subsample library by the calculation of the dimension standard, thereby clearly identifying the sample which is positive in the virus.

The matrix is preferably designed as a regular three-dimensional matrix which is further subdivided into rows, columns and layers, wherein each matrix element is represented by a matrix symbol X_rcsAnd characterizing, wherein dimension labels r, c and s respectively represent matrix elements forming a row, a column and a layer of the matrix. If more than one subsample of a single dimension target is positive, the virus test results are positive, and only one subsample represents each of the remaining dimension targetsLibrary virus tests are positive and calculation of the dimension labels determines a number of matrix elements defined by the dimension labels of each positive subsample library, thereby clearly identifying more than one sample positive for a particular virus. If many of the subsample library virus test results representing each dimension target are positive, then the calculation of the dimension target determines zⁿThe number of test matrix elements for which each virus is positive, where z represents the actual number of virus positive samples and n represents the number of dimensions of the pool of many positive subsamples. The method may further comprise: from being determined as zⁿAdditionally taking an aliquot from each sample of each virus positive detection matrix element; sending the aliquots to a high sensitivity testing apparatus, wherein all aliquots receive a virus display test within a second high sensitivity testing cycle; and clearly identify all virus positive samples. The sub-sample library forming step further comprises: forming a sub-library of aliquots from samples represented by the same r-label and different c, s-labels; forming a sub-sample library of aliquots of samples represented by the same c-label and different r, s-labels; forming a sub-sample library of aliquots of samples represented by the same s-label and different r, c-labels; and judging each r, c and s subsample bank which proves the virus to be positive by a high-sensitivity test. The method further comprises the following steps: determining the integer label of each r-sub sample library with positive virus test result; determining the integer label of each c-subsample library with positive virus test results; and determining the integer label of each s-subsample library with positive virus test results. The method may further comprise: the integer labels of each r, c, s sub-sample library that are positive for a virus test are converted to the dimensional labels r, c, s of the matrix symbols, thereby identifying the unique matrix element defined by the matrix symbol and thus uniquely identifying the corresponding virus-positive sample. The three-dimensional matrix comprises an 8 multiplied by 8 regular array, and dimension labels r, c and s respectively take integer values between 1 and 8. The method may further comprise: forming 8 line sub-sample banks, wherein each line sub-sample bank is uniquely represented by an integer between 1 and 8, and each line sub-sample bank is composed of 64 equal parts of samples; forming 8 column subsamples, wherein each column subsample is uniquely represented by an integer between 1 and 8, and each column subsample isIs made up of 64 aliquots; and forming 8 layer sub-sample libraries, wherein each layer sub-sample library is uniquely represented by an integer between 1 and 8, and each layer sub-sample library is composed of 64 aliquots. The high sensitivity assay is a PCR assay.

The method of the invention results in blood and plasma products that are substantially safer because one can conveniently detect viral infections directly in the blood or plasma supplied. Highly sensitive, highly economical tests can be performed immediately and infected donations identified regardless of the observation period of the infection.

In one embodiment of the practice of the present invention, the method includes the step of providing the donated plasma or blood in a collection container. A flexible collecting tube section is connected to the container and opens towards the interior of the container. The tube segments are filled with plasma or blood from a collection container, and a portion of the collection tube segments are sealed at both ends. The sealed portion of the collection tube segment is disengaged from the container and a plurality of spaced apart seals are formed at intervals along the length of the collection tube segment between the sealed ends, either before or after the sealed collection tube segment is partially disengaged. The portions of the tube segments located in the spaces between adjacent seals constitute containers in each of which a plasma or blood sample is contained, and the spaces between the seals provide a sufficiently large volume in each such container for the intended test.

In a more specific embodiment of the invention, the individual donated plasma is collected in a plasma collection bottle having a test container connected thereto by a hollow flexible tube segment. After filling the donor's plasma, the plasma bottle is tilted to transfer the plasma into the test container and the hose section, thereby filling the tube section. The tube segment is sealed at spaced intervals along its length, with the portions of the tube segment in the intervals between the seals forming pouches, each of which contains a donated plasma sample. The tube sections that had been converted into a series of pouches were then removed from the plasma collection bottle and frozen until they were used in the test.

In another aspect of the invention, the hollow tube section comprises a series of connected Y-shaped portions including an injection portion on one leg of the Y-shape, each leg of a particular Y-shaped portion not including an injection portion being connected to the base of the next Y-shaped portion in the series by a flexible plastic tube section. Spaced apart heat seals are formed along the length of the respective plastic hose segments separating the Y-shaped portions.

In yet another aspect of the invention, an apparatus for forming a plurality of heat seals along the length of a tube segment filled with donated plasma or blood includes first and second opposed sealing platens. Each sealing platen is provided with a plurality of spaced apart projections and recesses spaced apart along its length. The protrusions and depressions on the first platen are aligned with the corresponding protrusions and depressions on the second platen. The opposed sealing jaws are brought together on a plastic tube segment filled with donated plasma or blood to form heat seals at those portions of the tube segment pressed between the protuberances and the cavities formed by the opposed depressions. The heat seals define a plurality of individual continuous pouches therebetween, and each cavity formed by each pair of folded depressions is designed to receive one pouch.

In particular, devices that form a plurality of heat seals along the length of a tube segment filled with donated plasma or blood are designed to be mounted as an after-market retrofit on a commercially available heat sealing device.

In another embodiment of the present invention, a system for collecting and preparing a plasma sample for testing includes a plasma collection container and a hollow plastic tube connected to the container, each of which is constructed of plastic and includes a coded indicia impregnated into the plastic. The coded indicia are disposed along the major axis of the tube segment and the code is repeated at spaced intervals so that the tube segment can be provided with a plurality of spaced seals along its length, thereby defining pouches between the seals. The coded spacing of the indicia corresponds to the spacing of the pouches such that each pouch will contain at least one set of codes.

To begin the testing method of the present invention, the first pouch is detached from each of a set of tubing segments corresponding to a number of separate plasma donations. A portion of the donated plasma from each first pouch is withdrawn and made into a sample pool in a container.

In an exemplary embodiment of the invention, a virus assay is performed on the first library. When the first pool is positive for a positive test, the next or second successive pouch is detached from the tube segments comprising the first pool. The second pouch was divided into two approximately equal panels and plasma from one of the panel pools was examined to determine the presence of a particular virus. When the test group pool shows negative results, the next pouch is detached from the corresponding tube segment constituting the non-test group. The pouches were divided into two approximately equal offspring groups and the plasma in the group pouches was pooled into a sample pool. One of the next generation groups of pools was subjected to a virus test.

When the test group pool is positive for a positive test, one pouch is removed from the corresponding tube segment that forms the test group. The above process is repeated and each positive pool is subdivided into smaller groups, each group comprising a portion of the previous positive group of samples until the last pouch corresponding to a single donated plasma is identified.

In another embodiment of the present invention, an additional method for testing a plurality of donated plasma within a single PCR test cycle to uniquely identify donations that are positive for a viral test result includes the step of defining an n-dimensional grid of coordinates that defines internal network elements at each n-dimensional intersection of the grid. Samples from each of a plurality of donated plasma are plotted on a corresponding grid element of coordinates, each sample being represented by a matrix symbol X_rcsAnd defining, wherein the subscripts of the matrix element symbols define the dimension labels of the coordinate network. Aliquots of plasma were removed from each sample of each donated plasma and a subsample pool was formed. Each subsample pool comprises an aliquot of all the donated plasma with a dimension fixed therein. All sub-pools were immediately examined within a single PCR test cycle and each sub-pool with a positive test result was judged by induction based on sorting using the determinant methodThereby clearly identifying the unique matrix element represented by the dimension mark of each positive sub-sample library and thus the unique positive sample.

Drawings

These and other advantages, features and aspects of the present invention will become better understood when the invention is considered in conjunction with the following drawings, wherein:

FIG. 1 is a semi-schematic perspective view of one example of a sample container and a plasma donor bottle connected by a tubing segment for use in the practice of the present invention;

FIG. 2 is a semi-schematic perspective view of a tube segment connected between a plasma donor bottle and a sample container and divided into pouches according to the present invention;

FIG. 2a is a semi-schematic perspective view of a tube segment connected between a sample container and a plasma donor bottle and including a series of Y-shaped portions connected together in accordance with the present invention;

FIG. 3a is an enlarged top view of a portion of the tube segment shown in FIG. 2 showing additional details of the seal separating the pouches;

FIG. 3b is a semi-schematic cross-sectional view of a tube segment seal;

FIG. 4 is a semi-schematic perspective view of an apparatus for sealing tubes into individual pouches provided in accordance with the present invention;

FIG. 4a is a semi-schematic perspective view of upper and lower platens of a heat sealing mechanism arranged in accordance with the present invention and adapted to be mounted on a commercially available heat sealing device;

FIG. 5 is a semi-schematic perspective view of a sampling plate and cover arranged in accordance with the present invention;

FIG. 6 is a semi-schematic partial cross-sectional view of a plasma bag mounted in a sample well of a sampling plate provided in accordance with the present invention;

FIG. 7 is a semi-schematic perspective view of an apparatus arranged in accordance with the present invention for squeezing a sample bag and pressing a liquid sample container therein into a sample reservoir;

FIG. 8 is a semi-schematic cross-sectional view of the container of the apparatus shown in FIG. 7;

FIG. 9a is a semi-schematic partial cross-sectional view of a screening sheet compressing packets containing test samples;

FIG. 9b is a semi-schematic top view of the screening plate showing radial concentric flumes for collecting sample liquid from crushed sample containers;

FIG. 10 is a semi-schematic partial cross-sectional view of a crushing piston of the apparatus shown in FIG. 7;

FIG. 11 is a flow chart showing an assay method of the invention for determining PCR positive donors from a donor pool;

FIG. 12 is a flow chart showing the test procedure of the present invention for distinguishing a PCR positive donation from a 512 donation pool;

FIG. 13 is a flow chart showing a second testing method according to the present invention for determining PCR positive donors from a donor pool; and

FIG. 14 is a view of the three-dimensional coordinate network of the present invention showing the definition of the symbols r, c, s.

Detailed Description

The present invention relates to systems, methods and devices for testing donated blood or plasma to detect those specific donations that have a viral infection level above a predetermined level. Such contaminated donations are then disposed of, thereby preventing them from being mixed into the flow of pharmaceutical raw material or being infused into the patient. The virus test assay employed in accordance with the general practice of the present invention may be any assay that tests for viruses directly rather than for antibodies raised by the virus. Such assays include Polymerase Chain Reaction (PCR) assays and other assays that detect viruses directly with sufficient acuity even after collection of samples from many donations.

In one embodiment of the invention, a plurality of separate donated blood or plasma fractions are provided. Blood or plasma samples are drawn from each donation into a corresponding hollow tube segment. A plurality of spaced seals are provided at intervals along the length of the tube segment so that the portions of the tube segment in the spaces between the seals form a pouch for holding a sample of blood or plasma. As will be described in detail below, a unique method of testing plasma samples of pouches after the samples are made into a sample library is provided according to the present invention, thereby effectively and efficiently detecting and isolating those donated blood or plasma that has been contaminated with a virus.

Referring to fig. 1, there is shown an exemplary embodiment of a system for implementing a sampling process provided in accordance with the present invention. The system includes a standard donor plasma container 20 made of a non-reactive material such as polyvinyl chloride (PVC). The donor container 20 includes a cap 22 having two hollow elbow fittings 23, 24, each secured to a top surface of the cap. The elbow communicates with the interior of the donor bottle through a hole provided in the cap 22. A hollow fill hose 26 constructed of a bio-neutral material such as PVC plastic is connected at one end to elbow fitting 23 and at the other end to, for example, a needle that is inserted into the donor's body to obtain blood or plasma donations. In the illustrated embodiment, a test receptacle 28 is provided for collecting blood samples from donors for serological testing. Test container 28 is generally in the shape of a test tube and is also constructed of a biologically inactive material. The test vessel 28 includes an integral lid 30 which is perforated to facilitate communication with the interior of the test vessel.

A hollow flexible tube section 32 of biologically inert plastic material is connected between the cap 30 of the test container 28 and the hollow elbow 24 of the donor plasma container lid. The tube segment 32 is connected to the cap 30 in such a way that liquid flowing through the segment will flow into the test container 28 through the aperture provided in the cap 30. The tube segment 32 may be friction fit into the bore or may be coaxially connected to the bore by ultrasonic welding or by other means known to those skilled in the art.

A second opening may also be provided in the cap 30, and an air escape tube 34 may be connected to the cap in a manner similar to the tube section 32. The air escape tube 34 is typically no longer than one or two inches and typically terminates in an inserted, friction-fit sterilizing filter 36.

In one embodiment, blood or plasma is withdrawn from the donor and collected in the donor plasma container 20 for subsequent storage until needed. In the case of plasma donation, blood is typically withdrawn from the donor and passed through a continuous centrifuge where red blood cells are centrifuged off of the plasma fluid carrier and returned to the donor. Plasma is then collected.

After collection of the plasma donor from the donor and filling of the plasma donor container 20, the plasma donor container is tilted to raise the fluid level above the elbow 24 connected to the tube segment 32. Plasma enters the tube segment, flows through the tube segment and fills the test container 28. During filling, air trapped in test container 28 is vented through air escape tube 34, thereby allowing the test container to fill. The sterilizing filter 36 filters out any bacteria in the return air, thus preventing contamination of the sample from the surrounding environment. After filling the test container, the donated plasma is filled into the tubing section 32.

Referring now to fig. 2, after the donated plasma sample is drawn into tube segment 32, the tube segment is sealed adjacent to the connection of the tube segment to the donated plasma container by a heat weld 38 or by other suitable sealing means, such as ultrasonic welding. Another heat seal 40 is also provided to the tube segment adjacent to the connection of the tube segment to the test container 28. Thereby forming an elongated hollow tube closed at both ends and containing a quantity of donated plasma.

The filled portion of the tubing segment 32 is separated from the donor plasma container and the test container by cutting the tubing segment through the center of the seals 38, 40. The individual donated plasma containers are then removed for freezing and storage, and the separated test containers are sent to a serological laboratory. In general, various antibody tests are performed on donated plasma, which antibodies are caused by specific viruses such as Hepatitis C Virus (HCV) or HIV-1 and HIV-2.

Additional seals 42 are also provided at spaced intervals along the length of the tube segment to define, in sequence, individually connected pouches, each bag suitably including a hollow tube segment portion 44. Each tubing section portion 44 contains a specific amount of blood or plasma necessary to form a specific sample pool. For example, for a sachet used for PCR testing, approximately 0.02ml to 0.05ml of blood or plasma from a primary donor may be sealed.

The tube sections are sealed in a manner that provides 5 to 15 individually connected pouches. The seal defining the pouch may be performed after the tubing segments have been removed from the plasma donation container and the serological test container or, preferably, while the tubing segments are still connected to the plasma donation container, thereby avoiding hydrostatic pressure buildup. The sealing may be performed by any known means such as heat and pressure sealing (heat sealing), ultrasonic welding, etc., as long as the length of the compression and sealing zones is sufficient to allow the connected pouches to be separated from each other by cutting through the center of the seal on either side without destroying the pouch integrity as described in detail in figures 3a, 3 b. In fig. 2a second embodiment of a tube segment adapted to be subdivided into aliquots containing blood samples or plasma samples is shown, fig. 2a being a semi-schematic perspective view of an embodiment of a collection tube segment connected between a donor plasma bottle 20 and a test container 28 and divided into aliquots containing portions according to the invention.

The collection tube segment 50 is connected between the cap 30 of the test container 28 and the hollow elbow 24 of the donor plasma container lid. The tube section 50 suitably comprises a plurality of Y-shaped portions 51 which are connected in series by hollow medical grade plastic tube sections 52. The Y-shaped portion 51 is of the type generally adapted for connection to an iv set and includes a cylindrical body portion 53 with a flow passage having an outlet 54 at one end of the flow passage and an inlet portion 55 at the other end. A branch port 56 is provided along the main body portion 53 of the Y-shaped portion and has a flow passage communicating with the flow passage through the main body portion 53.

One Y-portion is connected to the next by a solvent connecting a hollow medical plastic hose section 52 between an outlet 54 on the base of one Y-portion and a branch 56 of the next Y-portion in the series. An initially hollow inlet tube 57 is connected by solvent to the branching mouth of the first Y-shaped part in a string. This initial inlet tube 57 is in turn connected to the donor plasma container cap elbow 24. The connection may be accomplished by friction fitting the initial inlet tube 57 to the elbow fitting 24 and connecting the tube to the fitting coaxially by ultrasonic welding or by other means known to those skilled in the art. Alternatively, the input tube 57 may initially terminate in a standard luer fitting 58 which will allow the serial Y-shaped portion to be removably connected to a donor container fitted with a mating luer fitting at the end of the elbow 24.

Similarly, the terminal Y-section is provided with a hollow terminal outlet hose 59 which is connected at its outlet to the final Y-section by solvent. The tube may also be connected at its end to a standard luer fitting.

In a manner similar to that described in connection with the first embodiment, after the donated plasma is withdrawn from the donor human body and the donated containers 20 are filled, the donated containers are tilted so as to raise the fluid level above the elbow 24 connected to the inlet tube section 57. Plasma enters the tubing segments and flows through the series Y-sections, into each Y-section through its branch port 56 and into the next Y-section from the outlet 54 of the previous Y-section. The plasma was decanted until the test container 28 was filled. After filling the test vessel, the donation is decanted until the serial Y-shaped portion including the tube segment 50 is also filled.

After the donor plasma sample is drawn into the tube segment 50, the final output tube segment 59 is sealed along its length by a heat seal or weld 40a or by other suitable sealing means such as ultrasonic welding, as appropriate near the connection of the final output tube segment to the test container 28.

The filled tube section 50 is removed from the test container by cutting the final output tube section 59 centrally from the test container through the seal 40 a. Alternatively, if the end of tubing section 50 is a luer fitting, tubing section 50 is removed from test container 28 by disconnecting the luer fitting. The initial input tube segment 57 is provided with a second heat seal 38a along its length adjacent the connection of the initial segment to the donor container 20. The fill portion of tubing segment 50 is removed from the donor plasma container by either cutting the initial input tubing segment 57 centrally through seal 38a or by disconnecting the connector in the case of luer-type connector 58. An elongated hollow articulated tube is thus formed which is closed at both ends and comprises a plurality of Y-shaped portions connected to one another in series. Each connected Y-shaped portion contains an equal amount of donated blood or plasma.

As will be described in more detail below, the tube segment connecting the exit of the previous Y-site and the branch of the next Y-site is also provided with a heat seal 42a, thereby sequentially forming separate and connected aliquot halves, each of which suitably has a separate Y-site. Each Y-shaped portion contains a specific amount of blood or plasma necessary for the formation of a particular sample library. The seal may be made to isolate each Y-shaped portion after the tubing segment 50 has been disconnected from the donor plasma container or while the tubing segment is still connected thereto. The operation of sealing to block the Y-shaped portion is preferably performed while the tubing segment is still connected to the plasma donation container, so that the reduction in volume caused by flattening portions of the tubing during the sealing process does not result in the accumulation of hydrostatic pressure within the specimen. While the tubing segment 50 is still connected to the plasma donor container, excess fluid resulting from the reduction in tube volume caused by the heat seal can be forced back into the donor container. Thus, the excessive hydrostatic pressure, which may cause dangerous splashing during sample withdrawal, is safely relieved.

The sealing may be performed by any known method such as heat and pressure sealing (heat sealing), ultrasonic welding, etc., as long as the length of the compression and sealing zones is sufficient to allow the joined Y-shaped sections to be separated from each other integrally by cutting through the center of the seal on either side of the seal without damaging the tube segments.

Referring to fig. 3a, 3b, in a preferred embodiment, the seal between the pouch (42 of fig. 2) and/or the Y-shaped portion (51 of fig. 2 a) comprises a land area 46 comprising a central narrow portion 47 for cutting or tearing the seal to separate the connected pouches. The cut is made centrally to ensure that the separate pouches remain sealed after separation at the compressed tab portions 48 at either end. The length of the gasket can be large or small depending on the separation method chosen. Separation can be performed with a scalpel, a guillotine, or a simple pair of scissors.

Referring to fig. 4, an exemplary embodiment of a sealing device 60 for forming a pouch having a particular desired size is shown, which includes a device that simply separates the pouches and determines the serial number of the pouches along the length of the tube. The sealing device 60 suitably comprises opposed first and second pressure plates 61, 62 each comprising a plurality of raised sealing heads 63 spaced apart on opposite faces of the pressure plates. The sealing device 60 is preferably formed such that the protruding sealing heads 63 can be moved along the respective pressure plate, so that the distance between the sealing heads of adjacent protrusions is variable. Raised sealing heads 63 may be disposed along the pressure plate so that the distance between the front and rear sealing heads may be progressively reduced so as to progressively seal along the length of the pipe section. Thus, a reduced size sample bag and reduced volume bag contents can be formed by moving the opposed pairs of sealing heads along the respective platens to desired positions.

To form a plurality of heat seals along the length of a plastic tubing segment filled with a blood or plasma sample, the tubing segment is placed in the sealing device 60 and correspondingly between the upper sealing platen 61 and the lower sealing platen 62. The opposed platens are brought closer together, thereby compressing and sealing the tube segment. As shown in fig. 4, a plurality of elongated or raised sealing heads 63 spaced along the length of each platen are arranged alternately with depressions 64. The cavity is formed by the opposed concave portions 64 when the opposed platens are moved together to form a heat seal at those locations of the plastic tube segment filled with the blood or plasma sample that are compressed between the raised sealing heads 63. . The cavity is arranged to accommodate those portions of the tube section which should not be compressed but which are to be formed into a pouch. Each cavity defined by each pair of closed depressions is designed to receive a pouch.

A heater 65 is provided to heat each sealing head of the platen so that the opposed projections form heat seals on the tube segments when the sealing device is closed. The heater 65 may be any known type of heating device such as a radiant heating device, an induction heater, or a resistive heater, among others. A heater 65 is preferably connected directly to each raised sealing head 63 to heat the raised portion without unduly heating the recessed portion. If desired, thermal insulation may be provided to inhibit heat transfer between the protrusions and the depressions. In an exemplary embodiment, a cooling device 66, such as a cooling fin or heat sink fin, motive air flow, or cooling fingers, may also be coupled to the sealing device 60. A cooling device 66 is directly connected to each depression 64 so that the cavity defined when the opposed depressions are moved together remains cold. Therefore, the blood sample or plasma sample contained in the pouch formed in the cavity during the sealing process is not damaged by the high temperature of the heat-sealed portion.

The constriction (47 of figure 3 b) passing approximately through the centre of the seal is formed by an elongate ridge structure 67 formed at the centre of the elongate sealing head 64 of the sealing platen. The ridge 67 presses a concave depression on the upper and lower surfaces of the seal portion when the tube section is compressed between the upper and lower seal heads. The gravure thins the plastic material constituting the center of the seal portion, thereby making it easy to separate.

In one embodiment of the invention, the ridges 67 may be serrated, thereby forming perforations arranged in a direction perpendicular to the major axis of the pipe segment. These perforations allow the connected pouches to be detached from each other without the risk of damaging the pouch integrity by inadvertently cutting into the specimen-containing area by cutting with a sharp object. The aperture is preferably formed during the sealing process by providing the sealing head with serrations. Alternatively, the eyelet may be formed immediately thereafter using a separate piercing jig or die.

Means 68 are also provided for opening and closing the sealing means 60 to compress the sealing platen and thereby form a seal along the length of the pipe section. Such means are well known in the art and may suitably comprise a manually operable tool which can be opened and closed, such as a lever handle which is secured to a support and moves the support towards the hinge. Other suitable arrangements may include vertical guides, spring or hydraulically operated piston presses, or other common mechanical, electrical or hydraulic presses.

Referring to fig. 4a, a particular embodiment of a sealing device 70 is shown in semi-schematic form that can be used to form heat-pressure seals at uniform intervals to form pouches of a particular size or to separate connected wye into individual sample-containing aliquots. The sealing device 70 suitably includes upper and lower press plates 71, 72 adapted to be mounted along the sealing bands and press bars, respectively, of a commercially available impulse sealer, such as the ALINE M series of impulse sealers, manufactured and sold by ALINE corporation of Santa Fe Springs, Calif. The particular embodiment shown in fig. 4a is a two-piece heat sealing head adapted to be installed as an after-market retrofit on an ALINE MC-15 type pulse heat sealer and to allow the MC-15 to produce pre-filled bagged plasma for further processing in accordance with the system and method of the present invention.

The base plate 72 of the heat sealing head 70 is constructed of a suitable rigid heat resistant material such as the laminated Kevlar ® product manufactured and sold by dupont. In the illustrated embodiment, the base plate 72 is preferably about 15 inches long to fit on the mounting face of an MC-15 pulse heat sealer. The base plate 72 includes a longitudinal slot 73 which is centrally located and extends along the entire length of the base plate 72. The longitudinal slot 73 has a width of about 0.2 inches to nestingly receive a standard medical tube having an outer diameter of typically about 0.1875(3/16) inches along the length of the slot.

A plurality of transverse slots 74 are provided at intervals along the length of the base plate 72 and are arranged in a direction perpendicular to the central slot 73. The transverse slots 74 are approximately 0.5 inches wide and 1.125(1-1/8) inches apart at their centers. Thus, the transverse slots are separated from each other by the remaining segment material separated by the center of the central longitudinal slot 73 and have a width of about 0.625(5/8) inches.

The longitudinal channels 73 and the transverse channels 74, respectively, only partially penetrate the material of the bottom plate 72, thereby forming a substantially flat base 75 which constitutes the bottom surface of the longitudinal and transverse channels. When the device is used to form a heat seal, a standard medical tube 0.1875(3/16) inch in length is fitted in place along longitudinal slot 73 and pressed against base 75 of the base plate, which acts as a bearing surface during the heat sealing process.

The heating element 76, for example a nickel-chromium resistance wire, is arranged in a curved manner from slot to slot and is arranged approximately centrally in the slot in the longitudinal direction of the transverse slots forming the base plate. In the central portion of the heating element 76 spanning the transverse slot 74, the wire is covered with a strip of, for example, teflon tape so that the Ni-Cr wire does not contact the heat sensitive plastic tube. Therefore, the blood sample or plasma sample contained in the pouch formed in the sealing device during the sealing process is not damaged by the high temperature of the heat-sealed portion.

The top plate 71 is approximately 15 inches in length and is suspended above the bottom plate 72 by the struts of an MC-15 type heat sealer. The top plate 71 is constructed of a heat resistant plastic such as Lexan ® or processed Kevlar ® and includes a set of equally spaced and generally rectangular teeth 77 projecting from its bottom surface and projecting toward the bottom plate. The teeth are about 0.5 inches long and spaced 1.125 inches apart on center. Thus, it can be seen that each tooth 77 is dimensioned to fit within the cavity defined by the transverse slot 74 of the base plate 72. Each tooth 77 of the top plate 71 is suspended above the respective intersection of the transverse slot 74 and the longitudinal slot 73 of the bottom plate 72. Thus, each tooth 77 is designed to nest within the cavity defined thereby when the heat seal platens are brought together by the moving operation of the MC-15 type apparatus.

After the hose section is placed in the longitudinal groove 73, the top plate 71 is pushed into contact with the bottom plate 72 by lowering the cover plate of the MC-15 type heat sealing machine. When the cover plate is lowered, the teeth 77 of the top plate 71 enter the cavities defined by the transverse slots 74 of the bottom plate 72 and contact the portion of the tube segment exposed on the base 75 at the intersection of each transverse slot 74 and the central longitudinal slot 73. The nickel-chromium resistance wire is energized, which softens the plastic of the pipe section. At the same time, the top plate 71 is pressed against the bottom plate, thereby applying pressure to the plastic material softened by the heating member 76.

After sealing, the tubing segment is marked at least at one end with a unique identifying marking that corresponds to the original donated plasma. This may be achieved, for example, by adhering a label to the tube segment or by embossing bar code markings directly on the tube. A pre-formed recess 78 is suitably provided in the heat sealer 70 to receive and condition the pre-printed bar code identification label. Such a label is made of a suitable heat-sealable material and it is heat-sealed to the first sealed location of the tube segment for identification. The tube section, including the pouch containing the sample, is then stored frozen.

With reference to fig. 2, it is important to be able to clearly identify all the different system components, including the individual donated plasma. Thus, unique identification marks such as coded lines, coded dots, bar codes or other structures coded with unique identification means can be placed into the physical structure of the plasma collection system. For example, in one embodiment, the encoded thread 37 is molded into the donor container 20, the encoded thread 39 is molded along the edge of the cap 22, the encoded thread 41 is molded along the side of the test container 28, and the encoded thread 43 is molded into the tube segment at intervals. Unique pipe segment identification markings are distributed along the length of the pipe segment and the coding is repeated to separate the pipe segments while maintaining the marking uniformity of each pipe segment so prepared. In addition, portions of the donation system are identified with the same code, and thus, the donation identity remains the same for all portions of the system.

Referring to figures 3a, 3b, 4, it may also be desirable to have each individual pouch along a segment marked with an alphabetic or numeric symbol equal to the pouch position along the straight length of the original tube segment. Such a code may be embossed on the compressed portion of the seal between adjacent pouches, for example, by a stamping die. The die may thus comprise an integral part of the sealing device as shown in figures 4 and 4a, so that sealing, forming, thinning or perforation areas for separation and identification numbers can be achieved in a single efficient step for pouches of various sizes. Alternatively, the alphabetic or numeric symbol may comprise part of a punch die or punch holder. Stamping dies are well known and include means for advancing an alphabetic or numeric symbol to the next sequential symbol such that successive pouches in a tube segment are identified by a corresponding sequential string of letters (a, b, c …) or numeric symbols (1, 2, 3 …), respectively.

Thus, if a first test library is being constructed from several donated pouches, a quality control check can be performed by confirming that all pouches to be assembled from each tube segment have the same location number, numeral 1. Similarly, when a second test library is formed from specimens of the same donation, a quality control check can be performed by identifying all pouches to be assembled from each tube segment, such as with the number 2 imprinted on a point on the compressed portion of the pouch.

To achieve an efficient donation PCR assay, serological test samples from each individual donation in test container 28 are subjected to various known antigen and/or antibody assays representative of the particular virus. If the specimen is positive according to one or more known antibody or antigen tests, the individual donation and its corresponding tube segment are blocked from further testing and can be disposed of appropriately.

The segments corresponding to the donations that were negative for the other serological tests were divided into identification groups, each group containing a predetermined number of donations. As will be described below, the number of donations per group is determined by the sensitivity of a particular high sensitivity test, such as a PCR test, the expected concentration of the relevant viral RNA or DNA in the plasma sample, and the expected probability of PCR positive samples appearing in the entire donated population. For example, in a group of donors who repeatedly draw blood by plasmapheresis, in order to test hepatitis C virus containing RNA of interest, a collection of 100 to 700 samples of human donations is suitable. For populations where viral infections occur more frequently, a small scale collection of 50 to 100 individual donations may be appropriate.

One embodiment of a method for preparing a PCR assay library according to the present invention is described below with reference to fig. 5 and 6. A sampling plate 80, generally identical to that used for the titer plate but designed according to the present invention, is provided. The sampling plate 80 has generally semi-cylindrical sampling recesses 81 arranged horizontally in a generally rectangular array on the plate. A sampling plate suitable for carrying out the method of the invention has 64 such sampling recesses arranged in an 8 x 8 (row/column) rectangular matrix. A cover plate 82 having substantially the same outer dimensions as the sampling plate 80 is also provided. The cover plate 82 is adapted to closely fittingly cover the surface of the sampling plate 80. The cover plate 82 is provided with perforations 83 in the same array as the sampling grooves of the sampling plate 80. When the cover plate 82 is put on the surface of the sampling plate 80, the hole 83 is vertically aligned with the sampling groove 81 from above, thereby allowing communication with the sampling groove through the through hole. The diameter of the aperture is significantly smaller than the surface area of the sample pouch and the corresponding sampling recess. However, the diameter of the aperture is large enough to allow a needle or other cannula-like object to pass through the aperture and into the sampling recess below.

As shown in fig. 6, the terminal pouch 84 (first generation, No. 1) is detached from the tube sections that have been identified as belonging to the particular PCR set to be tested. Each terminal pouch 84 is flushed, but not opened, and placed in a corresponding sampling recess 81 of the sampling plate 80. A cover plate 82 is secured to and covers the top of the sampling plate 80 and the pouch is then thawed at the appropriate temperature.

An equivalent amount of plasma equal to about 0.02ml to about 0.5ml was removed from each pouch and pooled in a single test container. A needle 85 or other cannula-like device is passed through the aperture in the cover plate and into the sampling recess of the sampling plate directly below, thereby puncturing the tubing in the sidewall of the pouch and obtaining a plasma sample therein. In an exemplary embodiment, the needle is connected to a device that continuously creates a vacuum or suction to draw all of the blood or plasma contained in the pouch and minimize any fluid leakage into the surrounding sink. The needle may be retained in a means which allows the needle to pass through the aperture and the top wall of the pouch, but which restricts further downward movement of the needle, thereby preventing the needle from contacting or penetrating the bottom wall of the pouch when the pouch is pressed against the sampling recess. When the cannula is withdrawn after sampling, as shown in fig. 6, the cover material 86 around the eyelet prevents accidental withdrawal of the pouch with the cannula.

Although the preparation method of the PCR test library has been described in terms of manual sampling by inserting a cannula into each sampling recess separately, the above method may also be automatically performed. The array of cannulas, which may be arranged in the same manner as the layout of the perforations of the cover plate, may be allowed to be pressed down onto the sampling plate to secure the sampling plate containing the pouches contained in each sampling recess, thereby allowing all of the sample pouches to be pierced and samples to be drawn simultaneously. Alternatively, a single cannula or cannula fixation device may be automated or programmed to penetrate and withdraw fluid from each pouch in succession. To prevent carryover infection, clean cannulas were used to draw samples from each bank.

In addition, it will be apparent to those skilled in the art that the combination of sampling plate, sampling recess, cover plate, aperture and cannula (although described in connection with the extraction of sample fluid from a sample bag) may also be used to extract sample fluid from the sample container having the Y-shaped portion of FIG. 2. The configuration of the sampling recess of fig. 5 and 6 is determined by the shape of the liquid-containing vessel and can be designed for the Y-shaped portion with only minor modifications. For example, the sampling recess may comprise a vertically oriented elongate cylinder into which each Y-shaped portion is inserted. A notch may be provided at a suitable location along the upper periphery of each sampling groove which acts as a latching mechanism for the branch mouth in which the Y-shaped portion is located. This will also serve to orient the respective Y-shaped portions and provide additional security of positioning. In a manner similar to that described in connection with fig. 5 and 6, liquid may be withdrawn from each Y-shape by inserting a cannula into the inlet of each Y-shape and into fluid communication with the sample. When the cannula is withdrawn from the inlet, the material of the cover around each hole acts as a stop and prevents the Y-shaped portion from being withdrawn from the sampling recess.

This configuration is also suitable for automated implementation of the present invention, as will be apparent to those skilled in the art. A series of cannulas may be arranged in the same manner as the placement of the lid eyelets, thereby allowing all of the inlets of the Y-shaped portion to be pierced and sample fluid to be simultaneously withdrawn therefrom. Alternatively, a single cannula or cannula fixation device may be automated or programmed to sequentially penetrate each portal and draw fluid from each Y-site.

Another embodiment of a method and apparatus suitable for preparing a PCR assay library of the present invention will be described below with reference to FIGS. 7, 8, 9a, 9b, and 10. Referring to fig. 7, a plasma donation bank including fluid expressed from a plurality of plasma samples is formed from a plurality of plasma donation sample bags in an electro-hydraulic press 90. The hydraulic machine 90 suitably includes a container 91 in which the sample bag is placed, and a hydraulically operated piston 92 which squeezes the sample bag. The sample in the bag is forced out of the container 91 by a suitable compressed gas such as compressed air or compressed nitrogen and collected in a collection container such as a blood pool.

First, a generation of pouches (e.g., bag # 1) is detached from each tube segment that has been considered to belong to a particular PCR set to be tested. The generations of pouches are flushed, but not opened, and placed in the press 90 in the press barrel 91. The loading of the container is carried out in the environment of the secondary biosafety shield and airflow channel, thereby ensuring that pouches that lose structural integrity do not inadvertently contaminate the surrounding environment. In a manner described in detail below, the squeeze piston 92 is tightly seated in the open throat 91a of the squeeze tube 91 in a manner that ensures that the squeeze tube 91 is filled and that the combination of the tube 91 and piston 92 completely encloses the sample bag. The manner in which the squeeze piston 92 engages the squeeze tube 91 is designed to ensure that the environment outside the tube 91 is not contaminated by any harmful viruses that may be present in the sample contained in the sample bag.

Next, the container 91 is mounted on a seat 93 which positions the container in the correct position on the hydraulic machine 90 and further allows a hydraulic rod 94 operatively connected to a hydraulic cylinder 95 to align and engage the squeeze piston 92. In a manner to be described in greater detail below, the squeeze piston 92 is removably connected to a hydraulic rod 94 so that the piston 92 can be raised and lowered by operation of the hydraulic cylinder 95.

After the barrel 91 and piston 92 have been properly aligned on the barrel seat 93 and connected to the hydraulic cylinder 95 by the rod 94, the control valve 96 is operated so that the hydraulic cylinder applies a force to the rod 94 and piston 92, which in turn squeezes the sample bag in the container 91. The hydraulic cylinder 95 is operated in conjunction with a 4 horsepower 240V ac motor 97 which operates a hydraulic reciprocating pump 98 which, in conjunction with a fluid reservoir 99, pumps hydraulic fluid, thereby operating the hydraulic cylinder 95. About 4000lbs of force is concentrated on hydraulic ram 94, which generates about 800-900 psi of pressure applied by piston 92 to the sample bag.

After the specimen bag has been crushed, the donated sample fluid contained therein is forced out of the container 91 by compressed gas, such as supplied by a compression cylinder 100, which is connected by a pressure regulating mechanism 101 to a quick-release safety valve 102 mounted in the squeeze piston 92. To allow the quick relief valve 102 to function properly, the piston 92 is first lifted slightly from its fully extended, depressed position. The compressed air flows into the container 91 through the pressure regulating mechanism 101 until the critical pressure of the quick-release safety valve is reached. The valve 102 is then opened, thereby allowing the pressurized gas to pressurize the interior chamber of the cartridge, which forces the plasma pool out of the container 91 through a collection port 103 disposed at the bottom of the cartridge. Thus, when the compressed air forces the fluid out of the cartridge, the plasma pool is collected in a collection container that is connected to the collection port 103 by an expression line. After passing through the bleach trap, the compressed air is discharged into a secondary biosafety hood.

Referring to fig. 8, a partial cross-sectional view of a container 91 constructed in accordance with the principles of the present invention is shown. The container 91 suitably includes a generally circular base plate 105 having upper and lower surfaces and a peripheral flange 106 extending upwardly from the upper surface with threads cut into the inner surface. The cylindrical barrel wall 107, which is open at both ends, is threaded on the outer surface of its bottom end. A socket or receptacle 108 is cut into the inner surface of the bottom end of the cartridge wall 107 to define an annular flange 109 parallel to the upper surface of the base plate 105 and having an opposed surface. As the cartridge wall 107 is threaded into the base plate 105, the screening plate 110, which rests on the surface of the base plate 105, engages the annular flange 109 of the cartridge wall 107 and is compressed between the annular flange 109 and the upper surface of the base plate.

Referring to fig. 9a and 9b, the screening plate 110 is a generally circular disk-shaped plate against which the sample bag is pressed when pressed by the pressing piston 92. As shown in fig. 9b, the screen plate 110 includes fluid slots having radial slots 111 and concentric ring slots 112, both of which are approximately 1/32 inches wide and which are open on the top surface of the screen plate. Where the radial slots terminate in an axially disposed drain or sump 113, the radial slots 111 are cut at an angle oblique to the center of the screen plate 110, the drain 113 draining through an 1/4 inch drain 114 (best seen in fig. 8) penetrating the bottom plate 105.

Referring to fig. 8, a seal is formed between the cartridge wall 107 and the screening plate 110 by press fitting an O-ring 115 disposed in a seal channel cut in the base plate 105. The sealing channel 116 is located in the floor so that the O-ring 115 is located below the vertical intersection of the screen plate 110 and the drum wall 107. A step 117 is cut into the base plate 105 and a matching groove 118 is cut into the screening plate so that the advantageous dimples precisely locate the screening plate onto the base plate for proper alignment of the O-rings whereby the cartridge walls 107 will accurately engage the screening plate and their junctions will properly engage the O-rings 115.

Referring to fig. 10, there is shown in a partially cut away cross sectional view an extrusion piston 92 configured in accordance with the principles of the present invention. The squeeze piston 92 includes a generally cylindrical piston head 120 having an axially extending centrally disposed raised cup portion 121, the cup portion 121 having a generally cylindrical wall and an open end, thereby defining a recess 123 for receiving the generally cylindrical cylinder rod 94.

An annular flange 122 is provided around the circumference of the cylindrical cup portion 121, surrounding the opening of the cup portion. The outer surface of the flange 122 is beveled so that the diameter of the bevel increases in a direction toward the body of the piston head 120. As the hydraulic rod 94 enters the recess 123, a pair of spring-loaded retaining clips 124 advance over the ramps of the annular flange 122 until they snap into place and grip the underside of the annular flange.

To accommodate the fit with the recess 123, each retaining clip 124 includes a ramped tooth 125 that rides along the ramp of the annular retaining ring 122 of the piston head, thereby spreading the jaws of the spring-loaded retaining clip 124. As the hydraulic ram 94 continues to advance, the angled teeth 125 of the retaining clip 124 eventually ride up the angled surface of the annular retaining ring 122. The spring loading of the retaining clip forces the helical teeth into contact with the outer surface of the cup-shaped portion side wall. The teeth of the retaining clip 124 then engage the underside of the annular retaining ring 122, thereby gripping the squeeze piston 92 and forming a mechanism for causing the piston to move bi-directionally.

In addition, the spring-loaded retaining clip 124 can be easily disengaged from the annular retaining ring 122 by simply squeezing the clip ends opposite the retaining helical teeth 125 together, as will be apparent to those skilled in the art. Thus, it will be appreciated that the combination of the piston head 120, the axially mounted cylindrical cup portion 121, the annular retaining ring 122 and the retaining clip 124 form a mechanism for quickly and easily disengaging the hydraulic rod 94 from the squeeze piston 92. This quick release feature allows the combination of the piston 92 and the cartridge 91 to be easily disengaged from the cartridge seat 93 of the hydraulic press 110 for cleaning, sterilization, refilling with additional sample bags, etc.

As shown in FIG. 10, the squeeze piston 92 also includes several 0-rings 126 disposed in a seal channel 178 disposed around the periphery of the piston head 120. The O-ring 126 is provided to form a tight pressure seal between the outer circumferential surface of the piston head 120 and the inner circumferential surface of the barrel wall 107 of the container 91. The plurality of O-rings provides a safety measure to ensure that potentially infectious sample fluid is contained within the cartridge 91. Although three O-rings 126 are shown in the embodiment of fig. 10, it will be apparent that more or fewer O-rings may be provided in accordance with the present invention. All that is required is to form a seal between the squeeze piston 92 and the squeeze cylinder 91 to ensure that potentially infectious liquid is contained within the cylinder.

Referring to fig. 8, the container sidewall includes a 0.020 inch sloped step 130 machined into the inner surface of the sidewall. Thus, the first approximately 1.0 inch of the cartridge sidewall portion 107 from the top surface is machined to have an Inner Diameter (ID) approximately 0.040 inches greater than the inner diameter of the remaining cartridge sidewall portion 107 extending downwardly toward the screen plate 110 and the bottom plate 105. The interface of the step and the other sidewall portion is beveled to provide a relatively smooth transition that slopes from a slightly larger upper inner diameter to a slightly smaller lower inner diameter.

The step on the barrel sidewall 107 is provided so that the squeeze piston 92 can be manually loaded into the open throat of the squeeze barrel 91 with slight contact between the inner diameter surface of the barrel and the O-ring (126 of fig. 10). Once the manually assembled piston and cylinder combination is seated on the cylinder seat (93 of fig. 7), the hydraulic rod 94 is immediately advanced to engage the recess 123 of the piston and extend until the retaining clip 124 snaps over the bottom surface of the annular retaining ring 122 of the piston head. The hydraulic rod 94 continues to advance pushing the piston further into the barrel, thereby pushing the O-ring past the step 130 on the inner diameter of the barrel wall. When pushed over the step, the O-ring is fully compressed between the inner surface of the barrel sidewall 107 and the piston seal channel 127, thereby forming a tight seal.

In operation, the squeeze piston 92 generates a pressure of about 800psi to 900psi (4000 lbs of force applied locally to the hydraulic ram) that is sufficient to rupture the sample bag in the cartridge. The fluid of the blood sample or plasma specimen flows through a fluid channel opening in the screening plate and into a central drainage port where it is collected and allowed to flow out of the withdrawal port and into a collection container. After the squeeze operation, the hydraulic cylinder 95 is operated to raise the squeeze piston 92 a short distance (about 1/2 inches to 1 inch) above the crushed sample bag body, thereby forming a cavity within the barrel. Forcing compressed gas, such as compressed air, into the cavity through the blow-by relief valve 102 in the piston 92. Pressurizing the cavity causes the remaining blood sample or plasma sample to be forced out of the cartridge through the outlet 103 and into the collection container.

Once the squeezing and pooling operations are completed, the expression line connected to the outlet 103 is clamped to prevent additional sample fluid from flowing out of the cartridge. The extrusion line is placed into a bleach vessel and hydraulic cylinder 95 is caused to further lift the piston within the barrel, thereby creating suction that siphons bleach from the vessel into the barrel. The squeezing and bleach siphoning steps are preferably performed twice more to ensure that any blood sample or plasma sample is forced completely out of the squeeze container 91 in reverse flow and that the bleach has sufficient opportunity to fill the interior space of the squeeze chamber, thereby reducing significant viral infections that may be found therein.

The quick release clamp is then operated and the piston/cylinder combination is disengaged from the hydraulic press 90 and sterilized, for example, in an autoclave. The piston and cylinder may then be chemically cleaned by immersion in a 10% bleach solution for 15 minutes and then subjected to a rinse cycle of rinsing with water, 1% SDS (sodium dodecyl sulfate 12 alkyl) surfactant and water in that order before being autoclaved. If the autoclaving time is not sufficient, the chemical cleaning step can be performed with 70% ETOH and a sterile aqueous solution. If such additional chemical cleaning is required, it is performed in a secondary biosafety hood that expels waste through a HEPA filter. When inside the safety shield, the container is filled with the next set of sample bags to be crushed, and the squeeze piston 92 is manually inserted into the opening of the container 91 and forced downward until the O-ring of the piston contacts the angled step formed on the side wall of the container. The reloaded barrel/piston combination can now be placed onto the barrel seat 93 of the hydraulic machine 90. The hydraulic cylinder 95 is operated to lower the hydraulic rod 94 onto the piston 92 to quickly release the clamp from the annular retaining ring on the piston. At present, the processes of extrusion, bleaching and cleaning are repeatedly carried out.

As will be apparent to those of ordinary skill in the art, the electro-hydraulic machine 90 allows for the time-consuming obtaining of blood or plasma samples from a number of sample bags, as described above. The number of sample bags that can be crushed by such machines is limited mainly by the scale of the equipment and the pressure that can be generated by the squeeze piston on the sample bag body contained in the cartridge. The 800psi to 900psi pressure generated by the hydraulic press of the illustrated embodiment is sufficient to completely crush up to 64 sample bags of the type shown in FIG. 2. Thus, a large sample library of 512 samples can be formed over 8 cycles of operation of the hydraulic press of the present invention. This will significantly reduce the time to library formation compared to a method in which 512 sample bags are accessed one by a sample collection cannula and samples are obtained.

In addition, it will be apparent to those skilled in the art that a single large sample library of at least 512 samples can be made by a hydraulic press large enough to accommodate more sample bags within the cartridge. The size of the hydraulic section will also increase to provide more squeezing power to overcome the greater resistance of more sample bags. As mentioned above, the sample reservoir size will only be limited by the size of the hydraulic press required.

Referring to FIG. 11, there is shown a flow chart of the PCR assay method of the present invention, which allows for the identification of unique PCR positive donations with a minimum number of individual assays.

The process begins at block 200 where an appropriate primary sample pool size is defined, which in turn determines factors such as the probability of occurrence of the relevant virus in the total donor population, the ultimate concentration of viral DNA or RNA that may occur after dilution of the sample pool, and the like.

Although PCR assays are sensitive and can detect a single virus in an infected sample, the virus must be present in the PCR sample to provide a positive result. For example, if a sample of infected donations with a relatively low virus concentration is mixed with a large number of uninfected samples, the concentration of virus in the resulting consortium may be so low that statistics may show no virus in one sample from the PCR library. Such a library may indeed falsely test a viral infection as negative.

For example, if a 0.02ml sample is prepared from virus-infected donor plasma at a concentration of 500 viruses per ml of sample, then the 0.02ml sample will have an average of 10 viruses. If the 0.02ml infected specimen is mixed with approximately 500 0.02ml specimens from uninfected donations, the resulting 10ml specimen pool will contain viruses at a concentration of 1 virus per ml. Thus, if a 1ml sample is drawn from the sample pool for a PCR assay, statistics may clearly indicate that the PCR sample does not contain virus.

Such low concentrations of viral contaminants pose little threat to plasma preparations, since several methods are known to inactivate viruses in such low concentrations of donations. Such methods of virus inactivation include the use of solvents/detergents or heating above 60 ℃ and holding for a suitable period of time, etc. These methods are generally considered to be able to reduce virus concentration by some log units. For example, the solvent/detergent approach can reduce the viral concentration of hepatitis C virus by at least 10⁷Or 7 log units, so that plasma preparations such as factor VIII, factor IX or prothrombin complexes can be prepared from donated plasma which is often processed after a PCR test has been negative, for example by the solvent/detergent method.

For blood products that are conventionally delivered directly to the container, there is also a small risk of low-intensity viral infection after such donations have been shown to be negative by PCR testing.

In the example shown in connection with FIG. 11, factors such as the probability of the occurrence of the relevant virus in the donor population and the virus concentration that may occur after dilution were determined. A first-level PCR assay library of appropriate size is designed which greatly reduces the statistical likelihood that viruses present at low concentrations will not be detected. At block 201, a sample library is made by mixing the contents of the identified tubing segment terminal pouches in the manner described above. At block 202, a PCR assay is performed on the first stage PCR sample library.

Block 203 represents the decision point of the method of the invention, which depends on the results of the PCR test carried out in block 202. In the case of negative test results, all donations corresponding to the specimens constituting the primary PCR pool are considered to be free from viral infection and are delivered to further processing for pharmaceutical preparation. The method then ends with a negative result for accepting a PCR test.

When the PCR assay reflects a positive indication, this indicates a viral infection in at least one of the donations that make up the initial PCR primary sample pool. At block 204, the next specimen bag adjacent to the first removed pouch is removed from the tube segment corresponding to the donation constituting the initial PCR first stage sample pool. These additional specimen bags are divided into two substantially identical sub-groups, defined herein as group a and group B for clarity.

The groups were then individually pooled with a single clean cannula to form groups of pools, and PCR was performed on only one of the groups, in the same manner as described above, as shown in block 205. It is not important for the purpose of the present invention which small set of sample libraries are examined. In block 205, group a is designated as the panel to be examined, while group B may likewise be simply designated as the panel to be examined, without this disturbing the method of the invention.

At block 206, a decision is made based on the results of the PCR assay of the panel library a. If the panel library A was tested negative by PCR virus display, no further testing was performed on the donated samples that make up panel A. Instead, as indicated by block 207, the next sample bag is removed from the tube segments comprising group B in sequence, which are then divided into approximately equal groups a ', B'. Each subgroup in this step had approximately half the number of samples constituting the preceding subgroup. The contents of the small sample bags were mixed again in the same manner as described above. If the PCR test of group A is positive, indicating that at least one of the donations was infected with the virus, the other untested group (group B in the example of FIG. 11) now receives a PCR test at block 208 to confirm that it is not PCR positive. The subgroup a will now be further subdivided into two roughly equal groups of subgroups (a ', B'), as indicated by block 209.

At block 210, PCR tests are performed on only one subset of the pools A 'or B' defined in the previous step 207 or 209. The method is now repeated and returns to block 206 where the results of the PCR assay performed at block 210 are determined. If the PCR test results prove that the tested group is negative, the untested group is subdivided into two roughly equal groups, each comprising approximately half of the sample of the superior group. If the test panel shows a positive PCR test, the test panel will be further subdivided into two approximately equal panels, each having approximately half the sample of the superior panel. In this case, the untested panel would again undergo a PCR test to confirm that it was also not PCR positive.

The test method is repeated between block 206 and block 210 until the test is deemed to be complete. The end of the test is defined when subdividing the groups results in two groups, where each group has only one specimen bag corresponding to a single donation. One of the samples is subjected to a PCR test at block 210 and if the test is negative, the other sample is determined to be a donated plasma that has been infected with the virus. If the test result of the test sample is positive, another sample is then also subjected to a PCR test to confirm that it is not PCR positive.

Upon completion of all tests, the method of the present invention ends at block 211. It should be clear from the flow chart of FIG. 11 that the test method of the present invention requires only two PCR tests in each test stage when the initial test sample pool is positive, wherein the first test is performed on one of the two sub-groups and the subsequent test is performed to confirm that the corresponding initially untested sample pool is truly negative. This test method requires only one PCR test in each test stage when the initially tested sample pool is negative.

The implementation of the sample testing method and system of the present invention will be described below in conjunction with the particular PCR test sample library size shown in fig. 12. In fig. 12, an initial PCR test sample library is prepared in step 212 from the terminal pouches of 512 individual donations. For ease of description, it is assumed that only one of the 512 samples is taken from a single donor infected with the virus of interest. The tubing segment shown in fig. 12 having 10 separate and connected pouches represents the tubing segment that was initially connected to and from the infected donated plasma container.

The initial 512 pools were subjected to a PCR assay and the virus showed a positive due to the presence of infected samples. In step 213, two 216-up donation sample pools (256A and 256B) are formed from the next succeeding sachet taken from the tube segment forming the previously positive sample pool. Now, a PCR test is performed on sample pool 256B, and as shown in fig. 12, the viral test results appear negative, thus indicating that sample pool 256A contains a sample from an infected donation.

In step 214, two 128-up donation sample pools are made from the next successive pouch comprising the tube segment of sample pool 256A. Thus, according to the present invention, the sample library 256 is subdivided and not subjected to PCR assays. At step 214, pool 128A is subjected to a PCR assay, and since its viral assay results appear negative, pool 128B is now known to contain a single specimen bag from an infected donation. The sample collection 128B is then subdivided into two 64-pack donor sample collections (64A and 64B) by removing the next successive pouch from those tube sections whose previous pouches constituted the sample collection 128B.

Subsequently, the library 64B is subjected to a PCR test, and its virus test result shows positive in the example of fig. 12. In this case, a PCR test was performed on pool 64A to prove that it was truly negative and that there were no additional infected samples other than those in pool 64B. In step 216, the sample pool 64B is further subdivided into two 32-up donation sample pools 32A and 32B by removing the next successive pouch from the tube segment comprising the previous sample pool 64B. Sample pool 32B was subjected to PCR tests, as shown, for which the viral test results were negative, and therefore, sample pool 32A was subdivided into two 16-pool donor sample pools 16A and 16B. A 16-part sample pool is also made by removing the next successive sample bag from the tube segment that constitutes the previous positive sample pool 32A.

In step 217, the sample pool 16B is subjected to a PCR test, the results of which are positive for the viral test. Thus, a PCR test is performed on pool 16A to confirm that it is negative and that all infected specimens are in pool 16B.

In step 218, the collection 16B is subdivided into two 8-up donated sample collections 8A and 8B by removing the next successive specimen bag from the tube segment constituting the previously positive collection 16B. Next, pool 8B was subjected to a PCR assay and its viral assay results were shown to be negative, indicating that pool 8A contained a sample from an infected donation. The sample pool 8A is then further subdivided into two 4-up donation sample pools 4A and 4B in step 219. The result of the PCR assay performed on pool 4B was negative, indicating that pool 4A contained a sample from an infected donation. Thereafter, the sample pool 4A is subdivided into two sample pools 2A and 2B in the same manner as described above in step 220. When the PCR assay was performed, the results of the virus assay for pool 2A were negative, indicating that one of the two samples comprising pool 2B was from a corresponding tube segment of infected donation.

In step 221, the individual donations are tested by removing the final specimen bag from the tube segment comprising the specimen pool 2B. The final individual donations are subjected to a PCR test to confirm a positive specific donation, which is then removed from the refrigerator and disposed of appropriately. The remaining 511 virus-free donations are retained for continued pharmaceutical preparation.

In the above example, a single infected donation was uniquely identified from a 512 pool of donations by performing only 13 separate PCR trials, including a primary PCR trial on the original 512 pool of donations. The method of the invention allows omitting PCR tests on a particular sub-set of sample pools, as long as the corresponding tested sub-set of sample pools shows negative results in viral tests. Because certain PCR tests are skipped in this manner, the method of the present invention reduces the number of PCR tests that must be performed to identify a particular positive donation without sacrificing the discriminatory power of the PCR test method. In the methods of the invention, all positive donations will be identified without the need to test all donations.

It is clear from the exemplary embodiment of FIG. 12 that PCR tests can be performed on any of the subordinate subgroups and that the random position of the positive samples can vary. Thus, if a specimen from a positive donation is present in each of the initially tested sub-pools, 18 tests will need to be performed in order to uniquely identify the positive donation (the initial test results are positive and an additional test is performed to ensure that the corresponding pool is negative).

Similarly, if each initially tested subsample pool is negative, only 10 trials are required to identify positive donations. In fact, the positive and negative test results for the sub-pools are equally likely, and therefore an average of 14 tests will be required to identify a single positive donor from the initial 512-pool group of donor samples.

Thus, it is apparent from the above description that the method and system of the present invention, which includes forming tubing segments having separate and connected pouches each containing a donated plasma sample, is advantageous in providing a plurality of PCR test sample libraries. Unlike traditional sample library preparation methods, which simultaneously make a series of primary and subsample libraries from a single specimen of each donation, the present invention allows test sample libraries to be formed just prior to testing. This "temporary" library formation method allows only the individual sample bags that are needed to be made into the test library. Since the sample reservoir is made at different times and each time consists of a sealed sample bag, the possibility of contamination is eliminated. Alternatively, the sample bag may be kept frozen until a test sample library is to be formed. Multiple freeze-thaw cycles that may adversely affect recovery of the relevant DNA or RNA are avoided, thus ensuring uniformity of the PCR assay.

Although the above method is effective for identifying a positive donation from a viral test in a minimal number of relatively expensive PCR tests, other methods for identifying each positive donation may be provided in accordance with the practice of the present invention. In particular, there is a method that has the capability of identifying a single positive donation in two to three test cycles, thereby significantly reducing the time and overhead required to screen a large number of donations.

For example, in the above-described methods, once a particular subsample pool is identified as containing a positive donation, the technician must identify those donations whose samples constitute that particular subsample pool. Then, the donations must be returned and another specimen bag must be obtained from each respective tube segment. Subsequently, two next generation pools of daughter samples must be formed and the PCR assay repeated. This process, including harvesting, sub-pool profiling and PCR testing, is repeated for smaller and smaller sub-pool generations until the method uniquely identifies the virus-infected donation.

However, it takes a lot of time in each cycle of PCR assay (acquisition, sub-pool shaping and PCR assay). Taking the first generation of the 512 pools as an example, it is clear that at least 10 test cycles are required to uniquely identify a single virus-infected donation. Although the above method is very economical, it can be a challenge for a laboratory to run PCR tests where time is important.

The method of uniquely identifying a donated blood or donated plasma that is positive for a viral test with a minimum of cycles of PCR testing is now described in connection with fig. 13 and 14.

Referring to FIG. 13, a flow chart of a PCR assay method according to the present invention is shown that efficiently detects single donations positive to a PCR assay in a sample pool with a minimum number of cycles of PCR analysis. As in the case of the PCR assay methods described above, the method of FIG. 13 assumes that the PCR assay is sufficiently sensitive to detect the presence of a positive sample in an appropriately sized sample pool. For ease of description, the primary group was selected to represent 512 donated blood or donated plasma. One of ordinary skill in the art will appreciate that the size of the primary set can be larger or smaller depending on the particular genomic marker being determined, the sensitivity of the PCR assay method being employed, the expected concentration of the genomic marker in the aliquot, and the size of the aliquot.

The method begins in block 301 where an N-dimensional sample matrix or grid is defined. The size of the matrix may be arbitrary and it includes any dimension from 2 to N, but is preferably a regular three-dimensional square matrix.

An example of such a matrix is shown in fig. 14, which is a schematic diagram of a square matrix featuring three dimensional labels: rows, columns and layers (r, c, s). In the exemplary matrix of FIG. 14, there are three rows, three columns and three layers, thus forming 3³Or 27 elements. In the exemplary embodiment, a row is defined to include all elements of the matrix obtained from an imaginary vertical section taken through a regular square matrix. In the embodiment of fig. 14, for example, the element comprising the third row of the matrix is marked r on its row plane₃。

Similarly, a column comprises all matrix elements obtained by a second imaginary vertical section through the matrix in a direction perpendicular to the direction of the rows. In the exemplary embodiment of fig. 14, for example, the matrix element comprising column 1 is labeled c on its column side₁. A layer is defined to include all the elements of the matrix obtained by a horizontal section through the exemplary matrix of fig. 14. Similar to the definition of rows and columns, the elements forming the first layer are denoted s at their level₁。

It can thus be seen that the moments in FIG. 14In the matrix, each of the 27 matrix elements belongs to only one of three rows, three columns and three layers. From a mathematical point of view, this can be represented by the relation X_rcsWherein X represents a matrix element, rcs is a dimension index, and each dimension index can be one value of 1-3. Specific matrix element X₁₁₃May be defined as the element of the matrix where the first row, the first column and the third layer meet.

From the above, although the exemplary matrix of fig. 14 is a 3 × 3 × 3 matrix, it is obvious that the principles of matrix element composition and matrix definition are also applicable to matrices with a larger number of rows and columns. In particular, a matrix of eight rows, eight columns, eight layers may still be mathematically represented by X_rcsWherein the values of r, c and s can be one of 1-8. Thus, a three-dimensional 8 x 8 matrix can accommodate a label for 512 elements.

Referring to the method flow diagram of fig. 13, after defining an N-dimensional sample matrix, specific donated blood or donated plasma is mapped according to each matrix element. In an exemplary three-dimensional 8X 8 matrix, a sample from each of 512 donations is associated with a matrix element and a corresponding unique X_rcsAnd marking.

Next, an aliquot is taken from each sample and a plurality of sub-libraries are formed. Each subsample library includes all samples (X) with a dimension index number of 1_rcs) An aliquot of (a). In other words, according to the above exemplary matrix, all samples (X) in which r is 1 (regardless of the number of columns and the number of layers) are obtained_rcs) Merging into a sub-sample library; when r is 2, r is 3 … r is N, the same applies. Similarly, regardless of the number of rows and layers, c is 1, c is 2, and … c is N; this is similar to the case where s is 1, s is 2, and … s is N, regardless of the number of rows and columns. Each sub-sample library then represents rows, columns, layers or other dimensional labels, so that if an N-dimensional matrix has been defined, there will be N times (total number of samples)^1/nA sub-sample library of (a). For the exemplary three-dimensional 8 x 8 matrix comprising 512 samples, there will be 24 lower sub-pools (8 row pools, 8 column pools, 8 level pools). According toIn the present invention, the formation of a sub-sample library can be viewed as analogous to a mathematical approach to determinant reduction by a determinant approach. Similarly, each sample will be understood to be represented in N sub-sample banks, with 1 representing each dimension of the matrix.

In addition to forming the child pools, an aliquot of each sample or an aliquot of each child pool is combined into a single parent pool, which contains a single aliquot from all 512 donations that make up the donation lattice (space). After all the sample pools have been formed, the remaining sample and child and parent pools may be refreeze until needed, for example, for PCR testing.

When a PCR test is required, a PCR test is first performed on a stock sample library representing an aliquot of each sample constituting the matrix. If the test result of the master library is negative, then the absence of a donation represented by the test sample forming the matrix that is positive for the viral test is determined based at least on the sensitivity level of the PCR test. The donated blood or plasma, the samples of which form the matrix, can be delivered for further use. However, if the PCR test of the master library is positive for a particular genomic marker, then a second cycle of PCR tests is entered in step 300, wherein each of the child libraries is tested.

In a similar manner to that described above, the size of the stock is chosen such that the statistical probability of at least one positive sample appearing in the stock (512 samples) is low, preferably less than 1% to 2%. This can be achieved by estimating the probability of the virus in question to appear in the whole donor population based on a 98% to 99% confidence level. For example, if only one of the 1000 donors is infected with the relevant virus, as determined by a 98% confidence level, then the probability of at least one infected donor being found among the next 1000 donors calculated is 2%. This ensures that the algorithm is generally able to identify individual reactions in a well-scaled pool of samples during a PCR assay cycle. According to the present invention, assuming a single positive specimen is present in the matrix, then 3 of the subsamples will contain an aliquot of the positive donation, one in each dimension. In the exemplary embodiment (matrix of 512 samples), there are 8 sub-column sample pools, and 8 sub-layer sample pools. If the master library test result is positive, then in the second PCR test cycle as shown in step 307, the 1-row, 1-column, 1-tier library test is positive. The intersections of the row, column, layer element labels clearly identify reactive donations, as shown in step 309.

As an example, if the reactive sample is organized as an element X of a matrix₁₁₃Then the results of the PCR test for the first row of the subsamples will be positive, while the subsamples of the second and subsequent rows will be negative. In addition, the test results for the first column of the sub-pools will be positive, while the test results for the second and subsequent columns of the pools will be negative. Similarly, the first and second layer of the sub-pools will be negative, while the third layer will be positive, and the subsequent layer will be negative. Three positive sub-sample pools (first row, first column, third layer) have only one common matrix element X₁₁₃. So, since it is organized as an element X₁₁₃The specimen of (a) represents, the positive donation is uniquely identified.

If there is more than one reactive donation in the matrix, the reactive donations can still be clearly identified by the method of the present invention without going over 1 additional cycle of PCR experiments. If more than one single dimensional subset of samples is observed to be positive, and only the single subset representing each of the remaining dimensions is positive, then the test results can be mathematically evaluated without requiring a third cycle of PCR testing to clearly identify more than one positive donation.

For example, if the test results of the first row of the sub-sample pools (others not) are positive, the test results of the first column of the sub-sample pools (others not) are positive, and the test results of the first and third tier of the sub-sample pools are positive, then there are only two positive donations that make up the matrix, which can be clearly designated as X₁₁₁And X₁₁₃. No further testing was required to obtain such results.

On the other hand, if it is viewedBy observing that many of the subsamples are positive for the test result and show identity changes along the two-dimensional label as shown in step 310, it is apparent that z is present²Individual elements, potentially designated as positive contributions, where z is the actual number of positive contributions comprising the array.

For example, if the test results of the first row of the sub-sample library (none of the others) are positive, the test results of the first and third columns of the sub-sample library are positive, and the test results of the first and third layers of the sub-sample library are positive, this suggests that the potential positive test matrix element is X₁₁₁、X₁₁₃、X₁₃₁、X₁₃₃. Since the multiples of the matrix elements to be tested are only present on the two dimensions (columns and layers), it can be seen that there are actually only two positive contributions that make up the matrix. In this case, all four donations may be randomly identified as positive and removed, or an aliquot may be taken from each of the four test matrix elements and subjected individually to a PCR test in the third PCR test cycle of step 311 to uniquely identify which two of the four constitute true positive donations.

Similarly, mathematically, if there are more than two positive donations in the matrix, it is clear that their identity will vary in more than two dimensions, and at most there will be zⁿThe identified potentially positive test subject matrix elements, where z is the actual number of positive donations and n is the dimension of variation. In this case, aliquots are taken from all suspect matrix elements of the matrix and identified directly.

Thus, it can be seen that the method of the present invention allows the clear identification of donors that are reactive to a certain genomic marker in a single PCR test cycle against a primary positive stock, and that for a matrix comprising a single reactive donor or a plurality of reactive donors, positive donors can be tested in two PCR test cycles, wherein these donors vary along a single dimension only, and for any other case in three PCR test cycles.

The practice of the invention thus results in blood transfusions and blood or plasma products prepared therefrom that are substantially safe in that they are as free as possible from viral infections. Advantageously, cost effective and sensitive assays can be easily performed to directly detect the presence of a virus. Thus, false indications of viral infection, which are typically associated with antibody testing during the observation period of infection, are avoided. In addition, the present invention allows the economical use of highly sensitive tests that are able to detect the presence of a single virus in a sample, thus helping to ensure that a blood transfusion is not infected with an incipient virus.

Those skilled in the art will recognize that the foregoing examples, and descriptions of preferred embodiments of the invention, are provided solely for the purpose of illustrating the invention as a whole, and that the number, size, and shape of the various elements of the invention, as well as the type of experiments performed, can be varied within the scope and spirit of the invention. For example, the length of the individual and connected pouches and their volume may increase progressively along the length of the tube segment, as will be apparent to those skilled in the art. Since successive test sub-pools are made up of fewer and fewer samples, the amount of plasma making up the pool must be reduced. It should be understood that in order to maintain a sufficient amount of plasma in each successive sub-sample library, successive sample bags may have a larger volume to provide the desired final sample library volume. To provide a library of sizes from about 1ml to about 10ml, it is apparent that the volume of successive sample bags will gradually increase from about 0.02ml to 0.5 ml. In one exemplary embodiment, the pouch volume is 0.02ml in the first pouch for the largest sample pool and 0.2ml in the last sample pouch.

It will be appreciated that the system of the present invention is not limited to the exemplary plasma collection container and tubing segment associated therewith. The same device may be provided for blood bags or other biological fluid containers and any suitable tubing segment may be connected thereto either before collection of the fluid or after collection of the fluid is completed. It is only necessary to note that the sample amount of biological fluid should be transferred to a tube section which is then made into a pouch according to the present invention.

Accordingly, the invention is not limited to the specific embodiments described above, but is to be defined by the scope of the following claims.

Claims (19)

1. A method for identifying a donor biological fluid that is positive for a viral test, the method comprising the steps of:

providing a plurality of donated biological fluids;

defining an n-dimensional matrix, where n is an integer greater than 1, the matrix further having a plurality of elements, each element being defined by an intersection of the n dimensions of the matrix, each individual element being represented by a respective matrix symbol, the matrix symbol including a label for each dimension of the array;

sampling each donated biological fluid;

marking each sample according to a corresponding matrix element in each matrix element, wherein each independent sample is represented by a corresponding matrix symbol of the corresponding matrix element;

taking aliquots from each sample, the number of aliquots taken from each sample being defined by the dimension representing the matrix characteristic;

forming a sub-sample library from an aliquot of each sample, each sub-sample library comprising an aliquot of all samples represented by matrix symbols having a dimension label fixed therein, each respective sub-sample library being defined by said fixed dimension label;

feeding the subsamples to a PCR assay device, wherein all of the subsamples are subjected to a virus display assay during a PCR assay cycle;

determining each fixed dimension mark of the sub-sample library with positive virus test; and

the fixed dimension labels are combined into a matrix symbol, thereby clearly identifying the unique matrix element defined by the matrix symbol and thus clearly identifying the specimen that is positive for the virus test.

2. The method of claim 1, wherein the matrix is designed as a regular matrix, and the n-dimensions of the matrix are each characterized by a same integer number of matrix elements.

3. A method as claimed in claim 2, characterized in that the regular matrix comprises a three-dimensional matrix which is further subdivided into rows, columns and layers, each element of the matrix being represented by a matrix symbol X_rcsAnd (4) representing, wherein dimension labels r, c and s respectively represent matrix elements forming a row, a column and a layer of the matrix.

4. The method of claim 3, wherein the sub-sample library forming step further comprises:

forming a pool of r subsamples from aliquots from samples represented by the same r label and different c, s labels;

forming a c-subsample library from aliquots from samples represented by the same c-label and different r, s-labels;

forming a library of s subsamples from aliquots from samples represented by the same s label and different r, c labels; and

the r, c, s sub-pools were judged from the positive display of the virus by PCR assay.

5. The method of claim 4, further comprising the steps of:

determining the integer label of each r-sub sample library with positive virus test result;

determining the integer label of each c-subsample library with positive virus test results; and

and determining the integer label of each s-subsample library with positive virus test results.

6. The method of claim 5, further comprising the step of converting the integer label of each r, c, s sub-sample library which is positive for a viral test result into a dimensional label r, c, s of a matrix symbol, thereby identifying the unique matrix element defined by the matrix symbol and thereby uniquely identifying the corresponding sample which is positive for the virus.

7. The method of claim 6, wherein the three-dimensional matrix comprises an 8 x 8 regular array of numbers, and the dimension labels r, c, and s respectively take integer values between 1 and 8.

8. The method of claim 7, wherein three aliquots are extracted from each respective donated biological fluid sample.

9. The method of claim 8, further comprising the steps of:

forming 8 line sub-sample banks, wherein each line sub-sample bank is uniquely represented by an integer between 1 and 8, and each line sub-sample bank is composed of 64 equal parts of samples;

forming 8 column subsamples, wherein each column subsample is uniquely represented by an integer between 1 and 8, and each column subsample is composed of 64 equal parts of samples; and

forming 8 layer sub-sample libraries, wherein each layer sub-sample library is uniquely represented by an integer between 1 and 8, and each layer sub-sample library is composed of 64 aliquots.

10. A method for identifying a donor biological fluid that is positive for a viral test, comprising:

providing a plurality of donated biological fluids;

sampling each donated biological fluid;

defining an N-dimensional matrix, wherein N is an integer greater than 1, the matrix further comprising a plurality of elements, each element being defined by an intersection of N dimensions of the matrix, wherein each individual element is represented by a corresponding matrix symbol, wherein the matrix symbols define the dimensions of the matrix, and where a sample from each donated biological fluid is assigned to one of said elements;

taking a plurality of aliquots from each of the donated biological fluids, the number of aliquots taken from each sample being determined by the number of dimensions comprising the matrix;

preparing an aliquot of each sample into a sub-sample library, each sub-sample library comprising an aliquot from all samples represented by a matrix symbol in which one dimension is fixed;

feeding the subsamples to a PCR assay device, wherein all of the subsamples are subjected to a virus display assay in a first PCR assay cycle;

calculating the dimension mark of each subsample library which is positive in the virus test in the first PCR test period, and if the virus test result of only a single subsample library representing each dimension mark is positive, determining a unique matrix element represented by the dimension mark of each positive subsample library by calculating the dimension mark, thereby clearly identifying the sample which is positive in the virus.

11. The method of claim 10, wherein the matrix is designed as a regular three-dimensional matrix further subdivided into rows,Columns and layers, in which each element is represented by a matrix symbol X_rcsAnd characterizing, wherein dimension labels r, c and s respectively represent matrix elements forming a row, a column and a layer of the matrix.

12. The method of claim 11 wherein if more than one subspecies library virus test of a single dimension label is positive and only one subspecies library virus test representing each of the remaining dimension labels is positive, the calculation of the dimension label determines a plurality of matrix elements defined by the dimension label of each positive subspecies library, thereby clearly identifying more than one sample that is positive for the particular virus.

13. The method of claim 12, wherein if a plurality of subsample library virus test results representing each dimension label are positive, calculating the dimension label determines zⁿThe number of test matrix elements for which each virus is positive, where z represents the actual number of virus positive samples and n represents the number of dimensions of the pool of many positive subsamples.

14. The method of claim 13, further comprising: from being determined as zⁿAdditionally taking an aliquot from each sample of each virus positive detection matrix element;

sending the aliquots to a PCR test apparatus, wherein all aliquots receive a virus display test in a second PCR test cycle; and

all virus positive samples were clearly identified.

15. The method of claim 11, wherein the sub-sample library forming step further comprises:

forming a pool of r subsamples from aliquots from samples represented by the same r label and different c, s labels;

forming a c-subsample library from aliquots from samples represented by the same c-label and different r, s-labels;

forming a library of s subsamples from aliquots from samples represented by the same s label and different r, c labels; and

judging each r, c and s sub-sample library which is proved to be positive by the PCR test.

16. The method of claim 15, further comprising: determining the integer label of each r-sub sample library with positive virus test result;

determining the integer label of each c-subsample library with positive virus test results; and

and determining the integer label of each s-subsample library with positive virus test results.

17. The method of claim 16, further comprising: the integer labels of each r, c, s sub-sample library that are positive for a virus test are converted to the dimensional labels r, c, s of the matrix symbols, thereby identifying the unique matrix element defined by the matrix symbol and thereby clearly identifying the corresponding virus-positive specimen.

18. The method of claim 17, wherein the three-dimensional matrix comprises an 8 x 8 regular array of numbers, and the dimension labels r, c, and s respectively take integer values between 1 and 8.

19. The method of claim 18, further comprising:

forming 8 line sub-sample banks, wherein each line sub-sample bank is uniquely represented by an integer between 1 and 8, and each line sub-sample bank is composed of 64 equal parts of samples;

forming 8 column subsamples, wherein each column subsample is uniquely represented by an integer between 1 and 8, and each column subsample is composed of 64 equal parts of samples; and

forming 8 layer sub-sample libraries, wherein each layer sub-sample library is uniquely represented by an integer between 1 and 8, and each layer sub-sample library is composed of 64 aliquots.