Method of PCR testing of pooled blood samples

• US 5,780,222 A
• Filed: 01/06/1997
• Issued: 07/14/1998
• 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): Horlick, Kenneth R.
• Assistant Examiner(s): Tung, Joyce
• Application Number:
  US08/778,610
• Time in Patent Office: 554 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 testing a multiplicity of plasma donations to uniquely identify donations having a positive viral indication, in a single PCR testing cycle, the method comprising the steps of:

providing a plurality of plasma donations, wherein a portion of each donation is contained in a tubing segment divided along its length by spaced-apart seals, the tubing segment portions between the seals defining sequential containers, wherein each container contains a plasma sample of said donation;

defining an n-dimensional grid, wherein n is 2 or 3 and, wherein the grid elements are each defined by the intersecting coordinates of the n-dimensions of the grid;

mapping a sample from particular ones of the plurality of plasma donations to a corresponding one of each element of the grid, each sample being defined by a matrix notation X_rcs, wherein the subscript of the matrix notation defines the dimensional indices of the grid;

taking n aliquots from each plasma sample of each of the plurality of plasma donations, the number of aliquots taken from each sample defined by the number of dimensional indices comprising the grid;

forming subpools from the aliquots of each sample, wherein each subpool comprises an aliquot of all samples in which one dimensional index is fixed;

testing all of the subpools, in a single PCR testing cycle, for a viral indication; and

evaluating the dimensional indicia of each subpool which tested positive in the single PCR testing cycle, in accordance with a reduction by the method of minors, thereby unambiguously identifying a unique element defined by the dimensional indicia of each positive subpool, thus unambiguously identifying a uniquely positive sample.

2. The method according to claim 1, wherein the grid is a 3-dimensional grid configured as a cube, and wherein each sample is characterized by a matrix element notation identified as X_rcs, where the dimensional indicia r, c, and s identify a row, column, and slice of said grid.

3. The method according to claim 2, wherein three aliquots are taken from each plasma sample of a donation.

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

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

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

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

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

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 indicating.

6. The method according to claim 5, wherein the integer indices of the r, c, and s subpools, which returned a positive viral indication, uniquely identify the positive sample when the integer designations are substituted for the dimensional indices r, c, and s to thereby identify the sample from the mapping X_rcs.

7. The method according to claim 6, wherein all candidate samples are individually tested if more than one indices identify more than one subpools as having a positive viral indication.

8. The method according to claim 2, wherein the dimensional indicia r, c, and s may each take on any value from 0 to N, where N is an integer.

9. The method according to claim 8, wherein the dimensional indicia r, c, and s each have the same maximum value N so as to form a regular 3-dimensional grid.

10. A method for testing a multiplicity of plasma donations to uniquely identify donations having a positive viral indication in a single PCR testing cycle, the method comprising the steps of:

providing a plurality of plasma donations, wherein a portion of each donation is contained in a tubing segment divided along its length by spaced-apart seals, the tubing segment portions between the seals defining sequential containers, wherein each container contains a plasma sample of said donations;

providing and defining an N-dimensional grid, wherein the grid elements are each defined by the intersection of the N-dimensions of the grid;

mapping a sample from particular ones of the plurality of plasma donations to a corresponding one of each element of the grid, each sample being defined by a matrix notation X_i . .N, wherein the subscript of the matrix notation defines the dimensional indices of the grid;

taking n aliquots from each plasma sample of each of the plurality of plasma donations, the number of aliquots taken from each sample defined by the number of dimensional indices comprising the grid;

forming subpools from the aliquots of each sample, wherein each subpool comprises an aliquot of all samples in which one dimensional index is fixed;

testing all of the subpools, in a single PCR testing cycle, for a viral indication; and

evaluating the dimensional indicia of each subpool which tested positive in the single PCR testing cycle, in accordance with a reduction by the method of minors, thereby unambiguously identifying a unique element defined by the dimensional indicia of each positive subpool, thus unambiguously identifying a uniquely positive sample.