Method of PCR testing of pooled blood samples

• US 6,063,563 A
• Filed: 05/20/1998
• Issued: 05/16/2000
• Est. Priority Date: 04/10/1995
• Status: Expired due to Term

• Current Assignee: Baxalta GmbH (Shire plc), Baxalta Incorporated (Shire plc)
• Original Assignee: Alpha Therapeutic Corporation (Grifols SA /Grifols Family/)
• Inventors: Conrad, Andrew J., Peddada, Lorraine B., Heldebrant, Charles M.
• Primary Examiner(s): Marschel, Ardin H.
• Assistant Examiner(s): Tung, Joyce
• Application Number:
  US09/081,926
• Time in Patent Office: 727 Days
• Field of Search: 435/5, 435/6, 435/91.2, 536/25.3
• US Class Current: 435/5
• CPC Class Codes: B01L 3/0296 from piercable tubing, e.g. in extracorporeal blood sampling
• CPC Class Codes: B01L 3/505 flexible containers not provided for above
• CPC Class Codes: C12Q 1/686 Polymerase chain reaction [PCR]
• CPC Class Codes: C12Q 1/701 Specific hybridization probes
• CPC Class Codes: C12Q 2537/165 Mathematical modelling, e.g. logarithm, ratio

Abstract

Systems, processes, and devices are provided which are useful for testing blood or plasma donations to detect those specific donations which are contaminated by a virus above a predetermined level. An apparatus and process is described which forms individual, separately sealed and connected sample containers from a flexible hollow tubing segment connected to a fluid donation container. The tubing segment is sealed at spaced-apart intervals along its length, with tubing segment portions in the intervals between the seals defining containers, each of which holds a portion of a plasma sample. The contents of the containers are formed into pools which are subsequently tested for virus contamination by a high-sensitivity test such as PCR The pools are tested in accordance with an algorithm by which a sample from each donation is mapped to each element of an N-dimensional matrix or grid. Each element of the matrix is identified by a matrix identifier, X_rcs, where rcs defines the dimensional index. An aliquot is taken from each sample, and subpools are formed, each subpool comprising aliquots of samples in which one dimensional index is fixed. All of the subpools are tested in one PCR test cycle. The dimensional indicia of each positive subpool is evaluated mathematically in accordance with a reduction by the method of minors, thereby unambiguously identifying a unique element in the grid, thereby unambiguously identifying a uniquely positive blood or plasma donation.

Claims

1. A method for uniquely identifying viral positive biological fluid donations in the fewest number of high-sensitivity test cycles, the method comprising:

providing a multiplicity of biological fluid donations;

defining an n-dimensional matrix, where n is an integer, the matrix further comprising a multiplicity of elements, each element defined by an intersection of the n-dimensions of the matrix, each individual element identified by a respective matrix notation, the matrix notation comprising at least an index for each dimension of the array;

taking a sample from each of the multiplicity of biological fluid donations;

mapping each sample to a respective particular one of each element of the matrix, each individual sample identified by its corresponding element'"'"'s respective matrix notation;

taking aliquots from each sample, the number of aliquots taken from each sample defined by the number of dimensions characterizing the matrix;

forming subpools from the aliquots of each sample, each subpool containing an aliquot from all samples identified by a matrix notation in which one dimensional index is fixed, each respective subpool identified by said fixed dimensional index;

providing the subpools to a high-sensitivity testing facility, wherein all of the subpools are tested for viral indication in a single high-sensitivity test cycle;

determining the respective fixed dimensional indices of subpools which return a positive viral indication; and

combining said fixed dimensional indices into a matrix notation, thereby unambiguously identifying a unique matrix element defined by the matrix notation, thus unambiguously identifying a uniquely viral positive sample.

2. The method according to claim 1, wherein the matrix is constructed as a regular array, each of the array'"'"'s n-dimensions characterized by an equal, integer number of elements.

3. The method according to claim 2, wherein the regular array comprises a 3-dimensional array, the array further subdivided into rows, columns, and layers, and wherein each element is characterized by a matrix notation X_rcs, where the dimensional indices r, c, and s, respectively, identify elements comprising a row, a column, and a layer of the array.

4. The method according to claim 3, wherein the subpool formation step further comprises:

forming subpools of aliquots from samples identified by identical r indices but different c and s indices;

forming subpools of aliquots from samples identified by identical c indices but different r and s indices;

forming subpools of aliquots from samples identified by identical s indices but different r and c indices; and

evaluating each of the r, c, and g subpools for a viral positive indication returned by high-sensitivity testing.

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

determining the integer index of each r subpool which returned a positive viral indication;

determining the integer index of each c subpool which returned a positive viral indication; and

determining the integer index of each s subpool which returned a positive viral indication.

6. The method according to claim 5, further comprising the step of substituting the integer indices of each r, c, and s subpool which returned a positive viral indication for the dimensional indices r, c, and s of the matrix notation, thereby identifying a unique matrix element defined by said matrix notation, thus uniquely identifying the corresponding viral positive sample.

7. The method according to claim 6, wherein the 3-dimensional array comprises an 8×

8×

8 regular array, the dimensional indices r, c, and s each taking on integer values from 1 to 8.

8. The method according to claim 7, wherein three aliquots are taken from each respective sample of the biological fluid donations.

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

forming eight row subpools, each row subpool uniquely identified by an integer from 1 to 8, each row subpool formed from 64 sample aliquots;

forming eight column subpools, each column subpool uniquely identified by an integer from 1 to 8, each column subpool formed from 64 sample aliquots; and

forming eight layer subpools, each layer subpool uniquely identified by an integer from 1 to 8, each layer subpool formed from 64 sample aliquots.

20. The method according to claim 1 wherein the high-sensitivity test is a PCR test.

21. the method according to claim 3 wherein the high-sensitivity test is a PCR test.

22. The method according to claim 9 wherein the high-sensitivity test is a PCR test.

10. A method for uniquely identifying viral positive biological fluid donations in the fewest number of high-sensitivity test cycles, the method comprising:

providing a multiplicity of biological fluid donations;

defining an n-dimensional matrix, where n is an integer, the matrix further comprising a multiplicity of elements, each element defined by an intersection of the n-dimensions of the matrix, where each individual element identified by a respective matrix notation X_i . . . N, wherein the subscript of the matrix notation defines the dimensional indices of the array;

taking N aliquots from each sample of each of the multiplicity of biological fluid donations, the number of aliquots taken from each sample defined by the number of dimensional indices comprising the array;

forming subpools from the aliquots of each sample, each subpool comprising an aliquot from all of the samples identified by a matrix notation in which one dimensional index is fixed;

providing the subpools to a high-sensitivity testing facility, wherein all of the subpools are tested for viral indication in a first high-sensitivity test cycle; and

evaluating the dimensional indicia of each subpool which returned a viral positive indication in the first high-sensitivity test cycle, in accordance with a reduction by the method of minors, the evaluation identifying a unique element defined by the dimensional indicia of each positive subpool if only a single subpool representing each dimensional index returns a positive viral indication,, thus unambiguously identifying a viral positive sample.

11. The method according to claim 10, wherein the matrix is constructed as a regular, 3-dimensional array, the array further subdivided into rows, columns, and layers, and wherein each element is characterized by a matrix notation X_rcs, where the dimensional indices r, c, and s, respectively, identify elements comprising a row, a column, and a layer of the array.

12. The method according to claim 11, wherein the dimensional indicia evaluation identifies a multiplicity of elements defined by the dimensional indicia of each positive subpool, if more than one subpool of a single dimensional index returns a positive viral indication while only a single subpool representing each of the remaining dimensional indices returns a positive viral indication, thus unambiguously identifying more than one unique viral positive samples.

13. The method according to claim 12, wherein the dimensional indicia evaluation identifies zⁿ power viral positive candidate elements if multiple subpools representing each dimensional index return a positive viral indication, where z represents the actual number of viral positive samples and where n represents the number of dimensions having multiple positive subpools.

14. The method according to claim 13, further comprising the step of taking an additional aliquot from each sample identified to each of the zⁿ viral positive candidate elements:

providing the aliquots to a high-sensitivity testing facility, wherein all of the aliquots are tested for viral indication in a second high-sensitivity test cycle; and

unambiguously identifying all viral positive samples.

15. The method according to claim 11, wherein the subpool formation step further comprises:

forming subpools of aliquots from samples identified by identical r indices but different c and s indices;

forming subpools of aliquots from samples identified by identical c indices but different r and s indices;

forming subpools of aliquots from samples identified by identical s indices but different r and c indices; and

evaluating each of the r, c, and s subpools for a viral positive indication returned by PCR testing.

16. The method according to claim 15, further comprising the steps of:

determining the integer index of each r subpool which returned a positive viral indication;

determining the integer index of each c subpool which returned a positive viral indication; and

determining the integer index of each s subpool which returned a positive viral indication.

17. The method according to claim 16, further comprising the step of substituting the integer indices of each r, c, and s subpool which returned a positive viral indication for the dimensional indices r, c, and s of the matrix notation, thereby identifying a unique matrix element defined by said matrix notation, thus uniquely identifying the corresponding viral positive sample.

18. The method according to claim 17, wherein the 3-dimensional array comprises an 8×

8×

8 regular array, the dimensional indices r, c, and s each taking on integer values from 1 to 8.

19. The method according to claim 18, further comprising the steps of:

forming eight row subpools, each row subpool uniquely identified by an integer from 1 to 8, each row subpool formed from 64 sample aliquots;

forming eight column subpools, each column subpool uniquely identified by an integer from 1 to 8, each column subpool formed from 64 sample aliquots; and

forming eight layer subpools, each layer subpool uniquely identified by an integer from 1 to 8, each layer subpool formed from 64 sample aliquots.

23. The method according to claim 10 wherein the high-sensitivity test is a PCR test.

24. The method according to claim 19 wherein the high-sensitivity test is a PCR test.