U.S. patent application number 10/442780 was filed with the patent office on 2003-10-30 for efficient algorithm for pcr testing of blood samples.
Invention is credited to Conrad, Andrew J., Heldebrant, Charles M., Peddada, Lorraine B., Schmid, Peter.
Application Number | 20030204321 10/442780 |
Document ID | / |
Family ID | 25113899 |
Filed Date | 2003-10-30 |
United States Patent
Application |
20030204321 |
Kind Code |
A1 |
Peddada, Lorraine B. ; et
al. |
October 30, 2003 |
Efficient algorithm for PCR testing of blood samples
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.sub.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.
Inventors: |
Peddada, Lorraine B.;
(Arcadia, CA) ; Heldebrant, Charles M.; (Arcadia,
CA) ; Conrad, Andrew J.; (Malibu, CA) ;
Schmid, Peter; (Los Angeles, CA) |
Correspondence
Address: |
CHRISTIE, PARKER & HALE, LLP
P.O. BOX 7068
PASADENA
CA
91109-7068
US
|
Family ID: |
25113899 |
Appl. No.: |
10/442780 |
Filed: |
May 20, 2003 |
Related U.S. Patent Documents
|
|
|
|
|
|
Application
Number |
Filing Date |
Patent Number |
|
|
10442780 |
May 20, 2003 |
|
|
|
09549477 |
Apr 14, 2000 |
|
|
|
6566052 |
|
|
|
|
09549477 |
Apr 14, 2000 |
|
|
|
09081926 |
May 20, 1998 |
|
|
|
6063563 |
|
|
|
|
09081926 |
May 20, 1998 |
|
|
|
08778610 |
Jan 6, 1997 |
|
|
|
5780222 |
|
|
|
|
08778610 |
Jan 6, 1997 |
|
|
|
08683784 |
Jul 16, 1996 |
|
|
|
5834660 |
|
|
|
|
08683784 |
Jul 16, 1996 |
|
|
|
08419620 |
Apr 10, 1995 |
|
|
|
5591573 |
|
|
|
|
Current U.S.
Class: |
702/20 ;
435/287.2; 435/6.11 |
Current CPC
Class: |
B01L 3/0296 20130101;
C12Q 1/701 20130101; C12Q 1/686 20130101; B01L 3/505 20130101; C12Q
1/686 20130101; C12Q 2537/165 20130101 |
Class at
Publication: |
702/20 ;
435/6 |
International
Class: |
C12Q 001/68; G06F
019/00; G01N 033/48; G01N 033/50 |
Claims
What is claimed is:
1. A method for forming subpools for use in testing for presence of
a specified agent in a set of fluid samples, the method comprising
the operations of: (a) associating each of the fluid samples with a
distinct matrix element of an N-dimensional matrix, each of the
matrix elements being identified by values of N indices, the N
indices corresponding one-to-one to the N dimensions of the matrix;
(b) forming N aliquots from each of the fluid samples; and (c)
combining aliquots to form the subpools such that each of the
subpools comprises one aliquot from each of the fluid samples
associated with matrix elements that have an identical value for
one of the N indices.
2. The method of claim 1, further comprising the operations of: (d)
testing each of the subpools to detect presence of the specified
agent; and (e) producing a positive indication for one of the
subpools when the presence of the specified agent is detected in
said subpool.
3. The method of claim 2, further comprising the operations of: (f)
combining identities of all subpools that have a positive
indication to identify at least one corresponding positively
indicated fluid sample.
4. The method of claim 1, wherein the N indices have an identical
range of values.
5. The method of claim 1, wherein N is equal to 3.
6. The method of claim 2, wherein operation (d) is a
high-sensitivity test.
7. The method of claim 6, wherein the high-sensitivity test is a
polymerase chain reaction (PCR) test.
8. The method of claim 3, wherein N is equal to 3 and there are 3
sets of said subpools corresponding to the 3 indices, each subpool
of the 3 sets of subpools being identified by a specific value of a
corresponding one of the N indices.
9. The method of claim 8, wherein operation (f) results in
identifying K positively indicated fluid samples, K being an
integer greater or equal to 1, when one of the N sets of subpools
has K subpools having positive indication and each of the remaining
N-1 sets of subpools has only one subpool having positive
indication.
10. The method of claim 8, wherein operation (f) results in
identifying a set of candidates for positively indicated fluid
samples when more than one of the N sets of subpools has more than
one subpool having positive indication.
11. The method of claim 10, further comprising the operation of:
forming a new aliquot from each of the candidates for positively
indicated fluid samples; testing each of the new aliquots to detect
presence of the specified agent; and producing a positive
indication for one of the new aliquots when presence of the
specified agent is detected in said new aliquot, said new aliquot
identifying a corresponding positively indicated fluid sample.
Description
CROSS-REFERENCE TO RELATED APPLICATIONS
[0001] This application is a division of patent application No.
09/549,477, filed Apr. 14, 2000, now U.S. Pat. No. 6,566,052, which
is a division of patent application No. 09/081,926, filed May 20,
1998, now U.S. Pat. No. 6,063,563, which is a division of patent
application No. 08/778,610, filed Jan. 6, 1997, now U.S. Pat. No.
5,780,222; which is a continuation-in-part of patent application
No. 08/683,784, filed Jul. 16, 1996, now U.S. Pat. No. 5,834,660;
which is a division of patent application No. 08/419,620, filed
Apr. 10, 1995, now U.S. Pat. No. 5,591,573.
FIELD OF THE INVENTION
[0002] The present invention relates generally to systems and
processes for preparing and analyzing samples taken from plasma
donations to uniquely identify donations which are virus
contaminated. In particular, the invention relates to an apparatus
and process for forming individual, separately sealed, and
connected containers holding samples of the same plasma as is
contained in the donation. The invention also relates to an
apparatus and process for forming initial screening test pools from
the containers and testing the pools for the presence of a virus in
accordance with an algorithm to identify individual contaminated
donations in the fewest number of testing cycles.
BACKGROUND OF THE INVENTION
[0003] Blood, plasma, and biological fluid donation programs are
essential first steps in the manufacture of pharmaceutical and
blood products that improve the quality of life and that are used
to save lives in a variety of traumatic situations. Such products
are used for the treatment of immunologic disorders, for the
treatment of hemophilia, and are also used in maintaining and
restoring blood volume in surgical procedures and other treatment
protocols. The therapeutic uses of blood, plasma, and biological
fluids require that donations of these materials be as free as
possible from viral contamination. Typically, a serology test
sample from each individual blood, plasma, or other fluid donation
is tested for various antibodies, which are elicited in response to
specific viruses, such as hepatitis C (HCV) and two forms of the
human immunodeficiency virus (HIV-1 and HIV-2). In addition, the
serology test sample may be tested for antigens designated for
specific viruses such as hepatitis B (HBV), as well as antibodies
elicited in response to such viruses. If the sample is serology
positive for the presence of either specific antibodies or
antigens, the donation is excluded from further use.
[0004] Whereas an antigen test for certain viruses, such as
hepatitis B, is thought to be closely correlated with infectivity,
antibody tests are not. It has long been known that a blood plasma
donor may, in fact, be infected with a virus while testing serology
negative for antibodies related to that virus. For example, a
window exists between the time that a donor may become infected
with a virus and the appearance of antibodies, elicited in response
to that virus, in the donor's system. The time period between the
first occurrence of a virus in the blood and the presence of
detectable antibodies elicited in response to that virus is known
as the "window period." In the case of HIV, the average window
period is approximately 22 days, while for HCV, the average window
period has been estimated at approximately 98 days. Therefore,
tests directed to the detection of antibodies, may give a false
indication for an infected donor if performed during the window
period, i.e., the period between viral infection and the production
of antibodies. Moreover, even though conventional testing for HBV
includes tests for both antibodies and antigens, testing by more
sensitive methods have confirmed the presence of the HBV virus in
samples which were negative in the HBV antigen test.
[0005] One method of testing donations, which have passed available
antibody and antigen tests, in order to further ensure their
freedom from incipient viral contamination, involves testing the
donations by a polymerase chain reaction (PCR) method. PCR is a
highly sensitive method for detecting the presence of specific DNA
or RNA sequences related to a virus of interest in a biological
material by amplifying the viral genome. Because the PCR test is
directed to detecting the presence of an essential component of the
virus itself, its presence in a donor may be found almost
immediately after infection. There is, theoretically therefore, no
window period during which a test may give a false indication of
freedom of infectivity. A suitable description of the methodology
and practical application of PCR testing is contained in U.S. Pat.
No. 5,176,995, the disclosure of which is expressly incorporated
herein by reference.
[0006] PCR testing is, however, very expensive and since the
general donor population includes a relatively small number of PCR
positive donors, individual testing of each donation is not cost
effective or economically feasible. Hence, an efficient and
cost-effective method of testing large numbers of blood or plasma
donations to eliminate units having a viral contamination above a
predetermined level is required.
[0007] One method of testing a large number of plasma donations is
to pool a number of individual plasma donations. The pool is then
PCR tested and the individual donations comprising the pool are
either retained or disposed of, depending on the outcome of the PCR
test. While reducing the number of PCR tests, and the costs
associated therewith, this method results in a substantial waste of
a significant portion of virus free donations. Since only a single
donation with a viral contamination above a pre-determined level
will cause a pool to test PCR positive, the remaining donations
that contribute to a pool may well be individually PCR negative.
This result is highly probable given that a relatively small number
of PCR positive donors exist in the general donor population. In
the conventional pooling approach, all donations comprising the
pool are disposed of upon a PCR positive result, including those
donations that are individually PCR negative.
[0008] In addition, plasma donations are often frozen soon after
they are received. When samples of individual plasma donations are
needed for pooling, each donation must be thawed, an aliquot of the
blood or plasma removed from the donation, and the donation must
then be refrozen for preservation. Multiple freeze-thaw cycles may
adversely affect the recovery of the RNA or DNA of interest as well
as the proteins contained within the plasma, thus adversely
affecting the integrity of the PCR test. Moreover, each time an
aliquot of individual plasma donations is withdrawn to form a pool,
the donation is subject to contamination, both from the surrounding
environment, and from the apparatus used to withdraw the aliquot.
Further, if the donation contains a virus, it can contaminate other
donations. In order to avoid introducing viral contaminants into an
otherwise viral free donation, the sample taking apparatus must be
either sterilized after each individual use, or used for taking
only a single aliquot from a single individual donation and a new
sample taking apparatus used for taking an aliquot from a
subsequent individual donation. Either of these methods involves
considerable expense and is quite time consuming.
[0009] Accordingly, there is a need for a process and system for
obtaining multiple blood or plasma samples from individual
donations such that particular samples may be pooled without
contaminating the remaining samples. It is also desirable that the
process and system is able to form such pools in a fast and
efficient manner, without contaminating either a clinical testing
lab technician or the testing laboratory environment.
[0010] In addition, it is desirable that the process and system
provide for efficient and cost-effective testing of the blood or
plasma donations to identify only uniquely PCR positive donations
in the fewest possible number of testing cycles.
SUMMARY OF THE INVENTION
[0011] There is, therefore, provided in the practice of this
invention a cost-effective and efficient process for preparing and
testing samples from a multiplicity of blood or plasma donations to
uniquely identify donations which are infected with virus as well
as systems and devices for practicing the process.
[0012] The process of the present invention results in blood and
plasma products being substantially safer because one can readily
test for virus contamination in the blood or plasma supply
directly. Cost-effective, high-sensitivity testing can be performed
immediately, and contaminated donations identified, without regard
to an infectivity window period.
[0013] In one embodiment of practice of the present invention, the
process comprises the steps of providing a blood or plasma donation
in a collection container. A flexible collection segment is
connected to the container and is open to the inside of the
container. The collection segment is filled with blood or plasma
from the collection container, and a portion of the collection
segment is sealed at both ends. The sealed portion of the
collection segment is removed from the container and, either before
or after the sealed collection segment portion is removed, a
plurality of spaced-apart seals are provided at intervals along the
length of the collection segment between the sealed ends. The
segment portions in the intervals between adjacent seals define
containers, wherein each such container contains a plasma or blood
sample, and wherein the intervals between the seals provide a
sufficient volume in each such container for the planned
testing.
[0014] In a more detailed embodiment of the present invention,
individual plasma donations are collected in a plasma collection
bottle which has a testing container connected thereto by a
flexible hollow tubing segment. After being filled with a donor's
plasma the plasma bottle is tipped so as to transfer plasma to the
testing container and the flexible tubing segment, thereby filling
the tubing segment. The tubing segment is sealed at spaced-apart
intervals along its length, the tubing segment portions in the
intervals between the seals define pouches each of which contains a
sample of the plasma donation. The tubing segment, which has been
converted into a series of pouches, is then disconnected from the
plasma collection bottle and frozen until needed for testing.
[0015] In an additional aspect of the present invention, the hollow
tubing segment comprises a series of linked-together Y-sites,
including an injection site provided on one leg of the Y, and where
each branch leg of a particular Y-site which does not include an
injection site is connected to the base of the next Y-site in the
chain by a flexible plastic tubing segment. Spaced-apart heat seals
are formed along the length of each flexible plastic tubing segment
separating the Y-sites.
[0016] In a further aspect of the present invention a device for
providing multiple heat seals along the length of the tubing
segment filled with the blood or plasma donation comprises first
and second opposed seal platens. Each seal platen includes a
plurality of spaced-apart raised portions along its length
alternating with recessed portions. The raised and recessed
portions on the first platen are in registry with corresponding
raised and recessed portions on the second platen. The opposed seal
platens are moved together onto a plastic tubing segment filled
with the blood or plasma donation to form heat seals on those
portions of the tubing segment compressed between the raised
portions and to form chambers defined by opposed recessed portions.
The heat seals define a plurality of individual and sequential
pouches therebetween and each chamber, defined by each closed pair
of recessed portions, is configured to house a pouch.
[0017] In particular, a device for providing multiple heat seals
along the length of the tubing segment filled with a blood or
plasma donation is configured to be mounted on a commercially
available heat seal apparatus, as an after-market modification.
[0018] In yet a further embodiment of the invention, a system for
collecting and preparing plasma samples for testing comprises a
plasma collection container and a hollow plastic tube connected to
the container, each of which are constructed of plastic and each of
which contain a coded indicia molded into the plastic. The coded
indicia is disposed along the major axis of the tubing segment and
the code repeats at spaced-apart intervals so that the tubing
segment can be provided with a plurality of spaced apart seals
along its length to thereby define pouches between the seals. The
code intervals of the indicia correspond to the intervals of the
pouches, so that each pouch will contain at least one cycle of the
code.
[0019] To begin the testing process of the present invention, a
first pouch is removed from each of a group of tubing segments
corresponding to a plurality of separate plasma donations. A
portion of the contents of each such first pouch is withdrawn and
the contents formed into a pool in a container.
[0020] In an exemplary embodiment of the present invention, the
first pool is tested for a viral indication. When the first pool
tests positive for a viral indication, a next, or second,
sequential pouch is removed from each of the tubing segments that
were used to form the first pool. The second pouches are divided
into two approximately equal subgroups, and the contents of one of
the subgroup pools is tested for the presence of a specific virus.
When the tested subgroup pool tests negative for the virus, a
further sequential pouch is removed from corresponding tubing
segments used to form the untested subgroup. The pouches are
divided into two approximately equal next generation subgroups, and
the contents of the subgroup pouches are formed into pools. One of
the next generation subgroup pools is tested for a viral
indication.
[0021] When the tested subgroup pool tests positive for such viral
indication, a pouch is removed from corresponding tubing segments
used to form the tested subgroup. The process is iterated, with
each positive pool being further subdivided into successively
smaller subgroups, with each of the successive subgroups comprising
a fraction of the samples of the preceding positive subgroup, until
the final pouch corresponding to a single plasma donation is
identified.
[0022] In a further embodiment of the present invention, an
additional process for testing a multiplicity of plasma donations
to uniquely identify donations having a positive viral indication
in a single PCR testing cycle includes the steps of defining an
n-dimensional grid which defines internal elements at the
intersections of each of the n-dimensions of the grid. A sample
from each of a number of plasma donations is mapped to a
corresponding element of the grid, with each sample being defined
by a matrix notation, X.sub.rcs, where the subscript of the matrix
element notation defines dimensional indices of the grid. Aliquots
are taken from each sample of each of the plasma donations and
formed into subpools. Each subpool includes an aliquot of all
plasma donation samples in which one of the dimensional indices is
fixed. The subpools are all tested at once, in a single PCR testing
cycle, and the dimensional indicia of each subpool which tests
positive is evaluated 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, and
thus unambiguously identifying a uniquely positive sample.
BRIEF DESCRIPTION OF THE DRAWINGS
[0023] These and other features, aspects, and advantages of the
present invention will be more fully understood when considered
with respect to the following detailed description, appended
claims, and accompanying drawings, wherein:
[0024] FIG. 1 is a semi-schematic perspective view of one example
of a plasma donation bottle and sample container attached by a
tubing segment useful in the practice of the present invention;
[0025] FIG. 2 is a semi-schematic perspective view of a tubing
segment connected between a plasma donation bottle and sample
container and divided into pouches in accordance with the present
invention;
[0026] FIG. 2a is a semi-schematic perspective view of a tubing
segment connected between a plasma donation bottle and sample
container and including a series of linked-together Y-sites in
accordance with the present invention;
[0027] FIG. 3a is an enlarged top plan view of a portion of the
tubing segment shown in FIG. 2 showing additional details of the
seals which separate the pouches;
[0028] FIG. 3b is a semi-schematic cross-sectional view of a tubing
segment seal;
[0029] FIG. 4 is a semi-schematic perspective view of a device
provided in accordance with practice of the present invention for
sealing a tubing into individual pouches;
[0030] FIG. 4a is a semi-schematic perspective view of a top and
bottom platens of a heat sealing device provided in accordance with
practice of the present invention for mounting onto a commercially
available heat sealer;
[0031] FIG. 5 is a semi-schematic perspective view of a sampling
plate and cover provided in accordance with the present
invention;
[0032] FIG. 6 is a semi-schematic partial cross-sectional view of a
plasma pouch contained in a sampling plate sample well provided in
accordance with the present invention;
[0033] FIG. 7 is a semi-schematic perspective view of a device
provided in accordance with the present invention for crushing
sample pouches and expressing the fluid samples container therein
into a pool;
[0034] FIG. 8 is a semi-schematic cross-sectional view of a
crushing cylinder of the device of FIG. 7;
[0035] FIG. 9a is a semi-schematic partial cross-sectional view of
a screen plate against which sample-containing packets are
crushed;
[0036] FIG. 9b is a semi-schematic top view of a screen plate
showing radial and concentric fluid gutters for collecting sample
fluid from crushed sample containers;
[0037] FIG. 10 is a semi-schematic partial cross-sectional view of
a crushing piston of the device of FIG. 7;
[0038] FIG. 11 is a flow chart depicting the test methodology
according to the invention for determining PCR positive donors from
a donation pool;
[0039] FIG. 12 is a flow chart depicting a test sequence according
to the invention for identifying a single PCR positive donation
from a 512 donation pool;
[0040] FIG. 13 is a flow chart depicting a second test methodology
according to the invention for determining PCR positive donors from
a donation pool; and
[0041] FIG. 14 is a representation of a 3-dimensional grid
according to the invention showing the definition of r, c, and s
indices.
DETAILED DESCRIPTION
[0042] The present invention relates to systems, processes and
devices useful for testing blood or plasma donations to detect
those specific donations which have a viral contamination above a
predetermined level. Such contaminated donations are then disposed
of to thereby prevent their incorporation into the raw material
stream for pharmaceutical products or their transfusion into human
patients. The viral detection tests used in accordance with
practice of the present invention can be any that directly detect a
virus instead of antibodies elicited in response to the virus. The
tests include polymerase chain reaction (PCR) tests and other tests
which are sufficiently sensitive to directly detect a virus even
after pooling samples from multiple donations.
[0043] In one embodiment of practice of the present invention, a
plurality of separate blood or plasma donations are provided. A
blood or plasma sample is drawn from each donation into a
corresponding flexible, hollow tubing segment. A plurality of
spaced-apart seals are provided at intervals along the length of
the tubing segment, so that segment portions in the intervals
between seals define pouches where each pouch contains a blood or
plasma sample. As is discussed below in greater detail, a unique
methodology is provided in accordance with the present invention
for testing plasma samples from the pouches after the samples are
formed into pools to thereby efficiently and effectively detect and
isolate any such blood or plasma donation which is contaminated
with virus.
[0044] Turning to FIG. 1, an exemplary embodiment of a system
provided in accordance with practice of the present invention for
effecting the sampling process is shown. The system includes a
standard plasma donation container 20, constructed of a nonreactive
material such as polyvinyl chloride (PVC). The donation container
20 includes a cap 22 having two hollow elbow shaped fittings 23 and
24, respectively, attached to the top surface thereof. The fittings
communicate with the interior of the donation bottle through
orifices provided in cap 22 for such purpose. A flexible hollow
filler tube 26, constructed of a biologically neutral material,
such as PVC plastic, is connected at one end to the elbow fitting
23 and connected at the other end to, for example, a needle which
is inserted into a donor in order to procure a donation. In the
illustrated embodiment, a test container 28, is also provided, for
collecting a sample from the donation to be serology tested. The
test container 28 is generally test tube shaped and is also
constructed of a biologically nonreactive material. The test
container 28 includes an integral cap member 30 through which
orifices are provided in order to communicate with the interior of
the test container.
[0045] A flexible hollow tubing segment 32, constructed of a
biologically nonreactive plastic material, is connected between the
cap member 30 of the test container 28 and the hollow elbow fitting
24 of the plasma donation container cap. The tubing segment 32 is
connected to the cap member 30 in a manner such that fluid passing
through the tubing segment will enter the test container 28 through
an orifice provided in the cap member 30 for such purpose. The
tubing segment 32 may be friction fit into said orifice, sonically
welded thereto, or otherwise attached in a coaxial relationship
with the orifice by techniques well understood by those skilled in
the art.
[0046] A second orifice may also be provided in the cap member 30,
to which a vent tube 34 is connected in a manner similar to tubing
segment 32. The vent tube 34 is typically no more than one to two
inches in length, and is typically terminated with an inserted,
friction fit bacteria excluding filter 36.
[0047] In an exemplary embodiment, a blood or plasma donation is
withdrawn from a donor and collected in the plasma donation
container 20 for subsequent storage until needed. In the case of a
plasma donation, blood is typically withdrawn from a donor and
passed through a continuous centrifuge apparatus, wherein red blood
cells are centrifuged out from the supporting plasma fluid and
returned to the donor. The plasma is then collected.
[0048] After a plasma donation is taken from a donor and the
donation container 20 is filled, the donation container is tilted
so as to raise the fluid level over the elbow fitting 24 connected
to the tubing segment 32. Plasma enters the tubing segment, flows
through the tubing segment, and fills the test container 28. During
filling, air trapped within test container 28 escapes through the
vent tube 34, allowing the test container to be filled completely.
The bacteria excluding filter 36 filters out any bacteria in the
returning air, thus preventing contamination of the sample by the
surrounding environment. After the test container is filled, plasma
from the donation is allowed to fill the tubing segment 32.
[0049] Turning now to FIG. 2, after the plasma sample from the
donation is drawn into the tubing segment 32, the tubing segment is
sealed by a heat weld 38 or other suitable sealing means such as a
sonic weld, at a location proximate to the tubing segment's
connection to the plasma donation container. A further heat seal 40
is applied to the tubing segment at a location proximate to the
segment's connection to the test container 28. An elongated hollow
tube, closed off at both ends, and containing a quantity of the
plasma donation is thus provided.
[0050] The filled portion of the tubing segment 32 is removed from
the plasma donation and test containers by cutting the tubing
segment away through the center of the seals, 38 and 40. The
separate plasma donation container is then removed for freezing and
storage, while the separated test container is removed to a
laboratory for serology testing. Typically, the contents are tested
for various antibodies, which are elicited in response to specific
viruses, such as hepatitis C (HCV), or HIV-1 and HIV-2.
[0051] Additional seals 42 are also provided at spaced-apart
intervals along the length of the tubing segment, to define
sequential individual and connected pouches, each suitably
comprising a hollow tubing segment portion 44. Each such portion 44
contains a particular quantity of blood or plasma needed for the
specific generation pool to be formed. For example, for pouches to
be formed for PCR testing, approximately 0.02 to 0.5 ml of blood or
plasma from the host donation may be sealed.
[0052] The tubing segment is sealed in a manner to provide from 5
to 15, individual and connected pouches. Sealing, to define the
pouches, may be done either after the tubing segment has been
removed from between the plasma donation container and the serology
test container or preferably is done while the tubing segment is
still attached to the plasma donation container, in order to avoid
hydrostatic pressure build-up. Sealing may be done by any known
method, such as thermo-compression sealing (heat sealing), sonic
welding or the like, so long as the length of the region compressed
and sealed is sufficient to permit the connected pouches to be
separated from one another by cutting through the center of the
seal without violating the integrity of the pouch on either side,
as indicated more clearly in FIGS. 3a and 3b. A second embodiment
of the tubing segment adapted to be subdivided into blood or plasma
sample-containing aliquot portions is depicted in FIG. 2a which is
a semi-schematic perspective view of a collection tubing segment
embodiment connected between a plasma donation bottle 20 and sample
container 28 and divided into aliquot-containing portions in
accordance with the present invention.
[0053] The collection tubing segment 50 is connected between the
cap member 30 of the test container 28 and the hollow elbow fitting
24 of the plasma donation container cap. The tubing segment 50
suitably comprises a plurality of Y-sites 51 connected together in
series by flexible, hollow, medical-grade plastic tubing segments
52. The Y-sites 51 are of the type commonly adapted for connection
to an intravenous infusion set and include a cylindrical body
portion 53 with a flow path defined therethrough, having an outlet
54 at one end of the flow path and an access site 55 at the other
end. A branch port 56 is provided along the body 53 of the Y-site
and includes a fluid path which is in communication with the fluid
path through the body 53.
[0054] One Y-site is connected to the next by solvent bonding a
flexible, hollow, medical-grade plastic tube 52 between the outlet
port 54 on the bottom of one Y-site to the branch port 56 of the
next Y-site in the series. An initial hollow entry tube 57 is
solvent bonded to the branch port of the initial Y-site in the
series. The initial entry tube 57 is connected, in turn, to the
elbow fitting 24 of the plasma donation container cap. Connection
may be made by friction-fitting the initial entry tube 57 onto the
elbow fitting 24, sonically welding the tube thereto, or otherwise
attaching the tube in a coaxial relationship with the fitting by
techniques well understood by those skilled in the art. Moreover,
the initial entry tube 57 may terminate in a standard luer-type
fitting 58 which would allow the series-connected Y-sites to be
removably connected to a donation container which was provided with
a mating luer-type connector at the end of the elbow 24.
[0055] In like manner, the terminal Y-site is fitted with a
flexible, hollow terminal exit tube 59 which is solvent-bonded to
the terminal Y-site at its outlet port. This tube may also be
connected to a standard luer-type fitting at its distal end.
[0056] In a manner similar to that described in connection with the
first embodiment, after a plasma donation is taken from a donor and
the donation container 20 is filled, the donation container is
tilted so as to raise the fluid level over the elbow fitting 24
connected to the entry tube segment 57. Plasma enters the tubing
segment and flows through the series-connected Y-sites, entering
each Y-site through its branch port 56 and flowing into the next
Y-site from the preceding Y-site's outlet port 54. Plasma is
decanted until the test container 28 is filled. After the test
container is filled, the donation is further decanted until the
series-connected Y-sites comprising the tubing segment 50 are also
filled.
[0057] After the plasma sample from the donation is drawn into the
tubing segment 50, the terminal exit tubing segment 59 is closed
off by a heat seal or weld 40a or other suitable sealing means such
as a sonic weld at a suitable location along its length proximate
to the terminal exit tubing segment's connection to the test
container 28.
[0058] The filled tubing segment 50 is removed from the test
container by cutting the terminal exit tubing segment 59 away from
the test container through the center of the seal 40a.
Alternatively, if the tubing segment 50 terminates in a luer-type
connector, the tubing segment 50 is removed from the test container
28 by disconnecting the luer. A second heat seal 38a is applied to
the initial entry tubing segment 57 at a location along its length
proximate to the initial segment's connection to the donation
container 20. The filled portion of the tubing segment 50 is
removed from the plasma donation by cutting the initial entry
segment 57 away through the center of the seal 38a, or by
disconnecting the luer-type fitting 58, if such is provided. An
elongated, hollow, articulated tube, closed off at both ends and
comprising a plurality of Y-sites linked-together in series, is
thus provided. Each of the linked-together Y-sites contains an
aliquot of the blood or plasma donation.
[0059] As will be described in greater detail below, the tubing
segments connecting a preceding Y-site's outlet port to a
subsequent Y-site's branch port are also provided with heat seals
42a to define sequential, individual, and connected sample
aliquots, each suitably comprising an individual Y-site. Each such
Y-site contains a particular quantity of blood or plasma needed for
a specific generation pool to be formed. Sealing to isolate each
Y-site may be performed either after the tubing segment 50 has been
removed from the plasma donation container or may be performed
while the tubing segment is still attached. Preferably, sealing to
isolate the Y-sites is performed while the tubing segment 50 still
attached to the plasma donation container so that the volume
reduction caused flattening a portion of the tubing during the
sealing process does not cause a build-up in the internal
hydrostatic pressure of the sample. When the tubing segment 50
remains connected to the plasma donation container, excess fluid
created by the volume reduction of the tubing created by the heat
seals is allowed to be expressed back into the donation container.
Excess hydrostatic pressure, which may lead to dangerous spurting
during sample extraction, is thus safely relieved.
[0060] Sealing may be performed by any known method, such as
thermo-compression sealing (heat sealing), sonic welding or the
like, so long as the length of the region which is compressed and
sealed is sufficient to permit the connected Y-sites to be
separated from one another by cutting through the center of the
seal without violating the integrity of the tubing segment on
either side of the seal.
[0061] Turning now to FIGS. 3a and 3b, in a preferred embodiment,
the seal between pouches (42 of FIG. 2) and/or Y-sites (51 of FIG.
2a) includes a flat pad area 46, including a central narrow portion
47 through which the seal is cut or torn in order to separate the
connected pouches. Cutting is done through the central portion in
order to insure that each separated pouch remains sealed at
compressed tab portions 48 at either end after separation. The
length of the seal pad may be made greater or smaller, depending on
the chosen separation method. Separation may be done by use of a
scalpel, a guillotine cutter, or a simple pair of scissors.
[0062] Turning to FIG. 4, an exemplary embodiment of a sealing
device 60, useful for providing pouches of specific desired sizes,
including means to easily separate the pouches and identify their
sequence number along a segment, is shown. The sealing device 60
suitably comprises opposed first and second platens 61 and 62,
respectively, each including a plurality of raised, seal head
portions 63, arranged in a spaced apart relationship on the
opposing surfaces of the platen. The sealing device 60 is
preferably constructed such that the raised seal head portions 63
are movable along their respective platens such that the spacing
from one raised seal head portion to another may be varied. The
raised seal heads 63 may be arranged along the platen such that the
distance between successive seal heads is made progressively
smaller so that sealing is performed along the length of a tubing
segment at progressively closer spaced intervals. Thus, sample
pouches of progressively smaller size and, therefore, progressively
smaller volume content may be formed by moving pairs of opposed
seal heads along their respective platens to a desired
location.
[0063] In order to form multiple heat seals along the length of the
plastic tubing segment filled with a blood or plasma sample, the
tubing segment is placed within the sealing device 60 between the
upper and lower sealing platens 61 and 62, respectively. The
opposed platens are brought into proximity with one another, thus
compressing and sealing the tubing segments. As depicted in FIG. 4,
the plurality of spaced-apart, extended or raised seal head
portions 63 along the length of each platen alternate with recessed
portions 64. As the opposed platens are moved together to form heat
seals on those portions of a plastic tubing segment filled with a
blood or plasma sample compressed between the raised seal head
portions 63, chambers are formed by the opposed recessed portions
64. The chambers are provided in order to accommodate those
portions of the tubing segment which are not to be compressed but,
rather, to be formed into pouches. Each chamber defined by each
closed pair of recessed portions is configured to house a
pouch.
[0064] A heater 65 is configured to heat each of the seal head
portions of the platen in order for opposed raised portions to form
a heat seal on the tubing segment when the sealing device is
closed. The heater 65 may be any one of well known heater types
such as radiant heaters, induction or resistance heaters, or the
like. The heater 65 is preferably connected directly to each of the
raised seal heads 63 to heat the raised portions without unduly
heating the recesses. If desired, insulation can be provided to
reduce heat transfer between the raised portions and the recesses.
In an exemplary embodiment, a cooling device 66, such as cooling or
radiator fins, a moving air flow, or a cold finger, may also be
connected to the sealing device 60. The cooling device 66 is
connected directly to each of the recessed portions 64 so that the
chambers defined when opposed recessed portions move together are
maintained at a low temperature. Blood or plasma samples contained
in pouches formed within the chamber during the seal process are
thus not damaged by the high temperatures of the heat seal.
[0065] The narrow area (47 of FIG. 3b) through approximately the
center of the seal is formed by an elongated ridge structure 67
provided down the center of the extended seal head portion 64 of
the seal platens. As the tubing segment is squeezed between the
upper and lower sealing heads, the ridge 67 forces an indentation
on the top and bottom surface of the seal portion. The indentations
narrow the plastic material comprising the center the seal, thus
making it easy to separate.
[0066] In one embodiment of the invention, the ridge 67 may be
serrated in order to provide perforations disposed in a direction
orthogonal to the major axis of the tubing segments. The
perforations allow the individual and connected pouches to be
removed from one another without the danger inherent with cutting
with a sharp object of violating the integrity of a pouch by
inadvertently cutting through to the sample containing area. The
perforations are preferably provided during the seal process by
providing the seal heads with serrations. Alternatively,
perforations may be provided shortly thereafter by use of a
separate perforating jig or die.
[0067] Means 68 are also provided to open and close the sealing
device 60 in order to compress the seal platens together and thus
form seals along the length of the tubing segment. Such means are
well known in the art and may suitably comprise a manual apparatus
which opens and closes, such as a lever handle attached to one
support frame and which moves the frame against, for example, a
hinge. Other suitable arrangements may include vertical guides,
springs, or hydraulically operated piston presses, or other common
mechanical, electrical, or hydraulic presses.
[0068] Turning now to FIG. 4a, there is depicted in semi-schematic
view, a specific embodiment of a sealing device 70, useful for
providing thermo-compression heat seals at uniform, spaced-apart
intervals, so as to form pouches of specific desired sizes, or to
isolate linked-together Y-sites into individual sample-containing
aliquots. The sealing device 70 suitably comprises top and bottom
platens 71 and 72, respectively, adapted to be mounted along the
pressure lever and seal band, respectively, of a commercially
available impulse sealer, such as one of the ALINE M-series impulse
sealers, manufactured and sold by the ALINE Company of Santa Fe
Springs, Calif. The specific embodiment depicted in FIG. 4a is a
two-part heat sealing head adapted to be attached to an ALINE MC-15
Impulse heat sealer as an after market modification, and allows the
MC-15 to produce pre-filled pouches of plasma for further
processing in accordance with the system and method of the present
invention.
[0069] The bottom platen 72 of the heat sealing head 70 is
constructed of a suitable rigid, heat resistant material such as
laminated Kevlar.RTM. manufactured and sold by the DuPont
Corporation. In the illustrated embodiment, the bottom platen 72 is
preferably about 15 inches in length in order to fit on the
mounting surface of the MC-15 Impulse heat sealer. The bottom
platen 72 includes a longitudinal slot 73 which is centrally
disposed and runs along the entire length of the bottom platen 72.
The width of the longitudinal slot 73 is approximately 0.2 inches
in order to accommodate standard medical tubing, which typically
has an outer diameter of approximately 0.1875 ({fraction (3/16)})
inches, in nested fashion along the length of the slot.
[0070] A plurality of transverse slots 74 are provided at
spaced-apart intervals along the length of the bottom platen 72
which are disposed in a direction orthogonal to that of the central
slot 73. The transverse slots 74 have a width of approximately 0.5
inches and are located on 1.125 (11/8) inch centers. Each
transverse slot is, therefore, separated from its neighbors by a
residual block of platen material centrally divided by the central
longitudinal slot 73 which is about 0.625 (5/8) inches in
width.
[0071] Both the longitudinal and transverse slots 73 and 74,
respectively, are cut only partially through the material of the
bottom platen 72, thereby forming a substantially flat bed 75 which
defines the bottom surface of both the longitudinal and transverse
slots. When the apparatus is used to form heat seals, a length of
0.1875 ({fraction (3/16)}) standard medical tubing is nested in
position along the longitudinal slot 73 and rests on the bed 75 of
the bottom platen which functions as a bearing surface during the
heat seal process.
[0072] A heating element 76, such as a nickel-chromium (NiCr)
resistive wire, is provided in a snake-fashion from slot to slot
and is disposed lengthwise along each transverse slot comprising
the bottom platen in about the center of the slot. Where the
heating element 76 traverses the center of the transverse slots 74,
the NiCr wire is protected from contacting the thermo-sensitive
plastic tubing by covering the wire with a piece of, for example,
Teflon.RTM. tape. Blood or plasma samples contained in the pouches
formed within the sealing device during the seal process are thus
not damaged by the high temperatures of the heat seal.
[0073] The top platen 71 is also approximately 15 inches in length
and is suspended over the bottom platen 72 by the pressure lever of
the MC-15 heat sealer. The top platen 71 is constructed from a
heat-resistant plastic material such as Lexan.RTM. or milled
Kevlar.RTM. and comprises a set of equally spaced-apart, generally
rectangular teeth protruding from its bottom surface, and extending
in a direction toward the bottom platen. The teeth 77 are about 0.5
inches in length and are spaced-apart on 1.125 (11/8) inch centers.
Accordingly, it can be seen that each of the teeth 77 is
dimensioned to fit into the cavity defined by the transverse slots
74 of the bottom platen 72. Each of the teeth 72 of the top platen
71 is positioned to be suspended over a corresponding intersection
of a transverse sot 74 and the longitudinal slot 73 of the bottom
platen 72. Thus, each tooth 77 is configured to fit into the cavity
thus defined when the heat seal platens are closed together by
removal operation of the MC-15 device.
[0074] After a flexible tubing segment is placed within the
longitudinal slot 73, the top platen 71 is pushed into contact with
the bottom platen 72, by lowering the lid of the MC-15 heat seal
apparatus. As the lid is lowered, the teeth 77 of the top platen 71
enter the cavity defined by the transverse slots 74 of the bottom
platen 72 and contact that portion of the tubing segment which lies
exposed on the bed 75 at the intersection of each transverse slot
74 with the central longitudinal slot 73. Current is provided to
the nickel-chromium resistive heating wire which causes the plastic
material of the tubing segment to soften. At the same time, the top
platen 71 is compressed onto the bottom platen, thus applying
pressure to the plastic material being softened by the heating
element 76.
[0075] After sealing, the tubing segment is labeled on at least one
end with a unique identifier that corresponds to the original
plasma donation. This may be achieved by, for example, gluing a
label onto the segment or by imprinting a bar coded emblem directly
onto the tubing material. A prepared recess 78 is suitably provided
on the heat sealer 70 for holding and aligning a pre-printed bar
code identifier tag. Such a tag is formed from a suitable
heat-sealable material and is heat sealed to the tubing segment at
the first seal position for identification purposes. The tubing
segment, including the sample containing pouches, is then frozen
for preservation.
[0076] Returning to FIG. 2, it is important to be able to
unambiguously identify all of the various parts of the system that
comprise an individual plasma donation. Thus, unique identifiers
such as coded threads, coded dots, bar codes, or other structure
coded with the unique identifier, may be placed in the physical
structure of the plasma collection system For example, in one
embodiment, a coded thread 37 is molded into the donation container
20, a coded thread 39 is molded along the edge of the bottle cap
22, a coded thread 41 is molded along the side of the test
container 28, and a coded thread 43 is molded into the tubing
segments at spaced-apart intervals. The unique identifier in the
tubing segment runs along the length of the tubing segments and the
code is repeated in order to permit segmentation of the tubing
segments while maintaining identification integrity of each segment
so prepared. Furthermore, each portion of the donation system is
identified with the same code so that donation identity is
maintained for all parts of the system.
[0077] Returning now to FIGS. 3a, 3b, and 4, it may be further
desirable to have each individual pouch along a segment identified
by an alpha or numeric code equal to the position of the pouch
along the linear length of the original tubing segment. Such code
may be imprinted, for example, on the compressed portion of the
seal pad located between adjacent pouches by use of a stamping die.
Such a stamping die may comprise an integral part of the sealing
device as depicted in FIGS. 4 and 4a, so that sealing, forming
pouches of variable sizes, and providing narrow or perforated areas
for easy separation, as well as identification numbers, are all
accomplished in a single efficient step. Alternatively, the alpha
or numeric identifier could comprise part of a perforating jig or
die. Stamping dies are known which include means for advancing the
alpha or numeric character to a next sequential one such that
sequential pouches in a tubing segment are each identified by a
corresponding sequential string of alpha (a, b, c .... .) or
numeric (1, 2, 3, . . . ) characters.
[0078] Therefore, if a first testing pool is being prepared from
pouches from several donations, a quality control check may be
performed by confirming that all pouches to be pooled from each
tubing segment have the same location code, for example, number 1.
Likewise, when preparing a second testing pool from samples of the
same donations, a quality control check may be performed by
confirming that all pouches to be pooled from each tubing segment
have, for example, the number 2 imprinted at some point on the
compressed portion of the pouch.
[0079] In order to effect efficient PCR testing of a donation, the
serology test sample taken from each individual donation in test
container 28 is tested for various known antigens and/or antibodies
which are designated for specific viruses. If a sample is positive
for one or more known antigen or antibody tests, the individual
donation and its corresponding tubing segment are excluded from
further testing and both may be disposed of in an appropriate
manner.
[0080] Tubing segments corresponding to the remaining serology
negative donations are divided into identified groups, each group
comprising a selected number of donations. As will be described
further below, the number of donations per group is determined by
the sensitivity of the specific high-sensitivity tests, such as a
PCR test, the anticipated concentration of the viral RNA or DNA of
interest in the plasma sample, and the anticipated frequency of a
PCR positive sample occurring within the general donor population.
For example, for the detection of the hepatitis C virus, containing
the RNA of interest, in a population of repeat plasmapheresis
donors, it is appropriate to pool samples of between 100 and 700
individual donations. For a population in which viral contamination
occurs more often, smaller pools of between 50 and 100 individual
donations may be appropriate.
[0081] One embodiment of a process of preparing a PCR testing pool
in accordance with the present invention will now be described in
connection with FIGS. 5 and 6. A sampling plate 80, generally
similar in application to a titer plate but configured in
accordance with practice of the invention, is provided. The
sampling plate 80 is configured to contain generally
hemi-cylindrical sample wells 81 disposed horizontally on the plate
in a generally regular array. A suitable sampling plate used to
practice the method of the invention has 64 such sample wells
arranged in a 8.times.8, row/column, rectangular fashion. A cover
plate 82 having approximately the same exterior dimensions as the
sampling plate 80 is also provided. The cover plate 82 is adapted
to cover the surface of the sampling plate 80 in close-fit
attachment. Through-holes 83 are arranged on the cover plate in the
same array fashion as the sample wells of the sampling plate 80.
When the cover 82 is placed over the surface of the sampling plate
80, through-holes 83 line up vertically over the sample wells 81,
thereby allowing communication with the sample wells through the
through-holes. The diameter of the through-holes is substantially
smaller than the surface area of the test sample pouches and the
corresponding sample wells. However, the through-hole diameter is
sufficiently large to permit a needle or other cannula like object
to pass through the holes and enter the sample wells beneath.
[0082] As shown in connection with FIG. 6, a terminal (first
generation, "number 1") pouch 84 is removed from each tubing
segment that has been identified as belonging to a particular PCR
group to be tested. Each terminal pouch 84 is washed, but not
opened, and placed in a corresponding sample well 81 of the
sampling plate 80. The cover plate 82 is secured over the top of
the sampling plate 80 and the plate, cover, and pouches are thawed
at an appropriate temperature.
[0083] An equal volume of between about 0.02 to 0.5 ml of plasma is
removed from each pouch and pooled in a testing container. A needle
85 or other cannula like device is inserted through the
through-hole in the cover plate and into the sampling plate sample
well directly below, thereby piercing the tubing material of the
side wall of the pouch and gaining access to the plasma sample
therein. In an exemplary embodiment, the needle is connected to a
device that provides a continuous vacuum or suction to extract all
of the blood or plasma contained in the pouch and minimize any
leakage of fluid into the surrounding tray. The needle may be held
in a device which allows the needle to move through the
through-hole and top wall of the pouch, but restricts its downward
progress so that the needle is prevented from touching or piercing
the bottom wall of the pouch as the pouch sits in the sample well.
When the cannula is withdrawn after extracting a sample, the cover
plate material 86 surrounding the through-hole prevents accidental
withdrawal of the pouch along with the cannula, as depicted in FIG.
6.
[0084] While the method of preparing a PCR test pool has been
described in terms of manually extracting a sample by inserting a
cannula individually into each sample well, the method may equally
be practiced using an automated process. The sampling plate
containing pouches in each well may be held so as to allow an array
of cannulas, arranged in a manner corresponding to the arrangement
of through-holes in the cover plate, to be pressed down onto the
sampling plate, thereby allowing all of the sample pouches to be
pierced and samples extracted therefrom at the same time.
Alternatively, a single cannula or cannula holding device may be
automated or programmed to successively pierce and withdraw fluid
from each pouch. In order to prevent carryover contamination, a
clean cannula is used to withdraw samples for each pool.
[0085] In addition, it will be evident to one having skill in the
art that the combination of sampling plate, sample wells, cover,
through-holes, and cannula, while described in connection with
extracting sample fluid from a sample packet, is equally applicable
to extracting sample fluid from the Y-site sample containers of
FIG. 2a. The configuration of the sample wells of FIGS. 5 and 6 are
determined by the shape of the fluid-holding container, and only
minor modifications are required to reconfigure them for Y-sites.
For example, the sample wells may comprise an elongated cylinder,
oriented vertically, into which each Y-site is inserted. A notch
may be provided at some appropriate location about the upper
periphery of each sample well which functions as a detent into
which the Y-site's branch port may be positioned. This would also
function to orient each Y-site and provide additional positional
security. In the same manner as described in connection with FIGS.
5 and 6, fluid may be extracted from each Y-site by inserting a
cannula through each Y-site's access port and into fluid
communication with the sample. As the cannula is removed from the
access port, the cover plate material surrounding each through hole
acts as a stop and prevents the Y-site from being withdrawn from
the sample well.
[0086] It will be further evident to one having skill in the art
that this configuration is equally suitable for practice of the
invention using an automated process. An array of cannulas may be
arranged in a manner corresponding to the arrangement of
through-holes in the cover plate, thereby allowing all of the
access ports of the Y-sites to be pierced and samples extracted
therefrom at the same time. Alternatively, a single cannula or
cannula holding device may be automated or programmed to
successively pierce each access port and withdraw fluid from each
Y-site.
[0087] An additional embodiment of an apparatus and method suitable
for preparing a PCR testing pool in accordance with the present
invention will now be described in connection with FIGS. 7, 8, 9a,
9b, and 10. Turning first to FIG. 7, a plasma donation pool
comprising expressed fluids from a multiplicity of plasma samples
is prepared from a number of plasma donation sample packets in an
electrically powered hydraulic press 90. The hydraulic press 90
suitably comprises a crushing cylinder 91 in which sample packets
are placed, and a hydraulically operated piston 92 which crushes
the sample packets. The samples contained within the packets are
expressed from the crushing cylinder 91 by a suitable compressed
gas, such as compressed air or nitrogen, and collected in a pooling
container as a pool.
[0088] Initially, a generational pouch (for example, pouch # 1) is
removed from each tubing segment that has been identified as
belonging to a particular PCR group to be tested. Each generational
pouch is washed, but not opened, and placed within the crushing
cylinder 91 of the press 90. Loading of the crushing cylinder is
performed within the environment of a class II biosafety hood and
air-flow path so as to ensure against inadvertent contamination of
the surrounding environment by a packet which has lost structural
integrity. In a manner to be described in greater detail below, the
crushing piston 92 is firmly seated into the open throat 91 a of
the crushing cylinder 91 in such a manner that containment of the
contents of the crushing cylinder 91 is assured and that the
cylinder 91 and piston 92 combination completely encloses the
sample packets. The manner in which the crushing piston 92 engages
the crushing cylinder 91 is designed to ensure that the environment
outside of the cylinder 91 is protected from contamination by any
harmful viruses that may be present in any of the samples contained
by the sample packets.
[0089] The crushing cylinder 91 is next mounted on a cylinder seat
93 which aligns the cylinder in correct position on the hydraulic
press 90 and further allows a hydraulic shaft 94, operatively
connected to a hydraulic cylinder 95, to align with and mate to the
crushing piston 92. In a manner that will be described in greater
detail below, the crushing piston 92 is releasably connected to the
hydraulic shaft 94, such that the piston 92 can be both raised and
lowered by operation of the hydraulic cylinder 95.
[0090] After the cylinder 91 and piston 92 have been properly
aligned on the cylinder seat 93 and connected to the hydraulic
cylinder 95 through the shaft 94, a control valve 96 is operated so
as to cause the hydraulic cylinder to exert a force on the shaft 94
and piston 92 which, in turn, crushes the sample packets within the
crushing cylinder 91. The hydraulic cylinder 95 operates in
conjunction with a four horsepower 240 volt AC electric motor 97
which operates a hydraulic reciprocating pump 98 which pumps
hydraulic fluid in conjunction with a fluid reservoir 99 to thereby
operate the cylinder 95. About 4,000 lbs of force is point loaded
at the hydraulic shaft 94 which develops a pressure of about 800 to
900 psi applied to the sample packets by the piston 92.
[0091] After the sample packets have been crushed, the fluid
donation samples contained therein are expressed from the crushing
cylinder 91 by a compressed gas supplied by, for example, a
compressed air cylinder 100 which is connected through a pressure
regulator 101 to a pop-off valve 102 provided in the crushing
piston 92. In order to allow the pop-off valve 102 to operate
correctly, the piston 92 is first raised slightly from its fully
extended crushing position. Compressed air is vented into the
cylinder 91 through the pressure regulator 101 until the threshold
pressure of the pop-off valve is reached. The valve 102 then opens,
allowing the compressed gas to pressurize the interior of the
cylinder which forces the plasma pool out of the crushing cylinder
91 through a collection port 103 provided in the bottom of the
cylinder. The plasma pool is then collected in a pooling container
connected to the collection port 103 by an express line or tube as
the fluid is forced out of the cylinder by the compressed air. The
compressed air is exhausted into a class II biological safety hood,
after passage through a bleach trap.
[0092] Turning now to FIG. 8, there is depicted a cross-sectional,
partially cut-away view of a crushing cylinder 91 constructed in
accordance with principles of the invention. The crushing cylinder
91 suitably comprises a generally circular base plate 105 having a
top and bottom surface and a circumferential lip 106 extending in
an upwardly direction from the top surface, with threads cut into
its interior face. A cylindrical cylinder wall 107, open at both
ends, is threaded on the exterior face of its bottom end. A rabbet
or notch 108 is cut into the interior face of the bottom end of the
cylinder wall 107 so as to define an annular lip 109 which is
disposed parallel to the top surface of the base plate 105 and
presents an opposing face thereto. As the cylinder wall 107 is
screwed into the base plate 105, a screen plate 110 disposed on the
surface of the base plate 105 is engaged by the annular lip 109 of
the cylinder wall 107 and compressed between the annular lip 109
and the top surface of the base plate.
[0093] Turning now to FIGS. 9a and 9b, the screen plate 110 is a
generally circular, disc-shaped plate against which the sample
containing packets are forced when they are crushed by the crushing
piston 92. As is depicted in FIG. 9b, the screen plate 110 includes
fluid gutters comprising radial slots 111 and concentric circular
slots 112, all approximately {fraction (1/32)} inches in width,
which are cut in the top surface of the screen plate. The radial
slots 111 are cut at an angle which slopes toward the center of the
screen plate 110 where they terminate into an axially located drain
or sump 113 which drains through a 1/4 inch drain pipe 114 (best
seen in FIG. 8) drilled through the base plate 105.
[0094] Returning now to FIG. 8, a seal is formed between the
cylinder wall 107 and the screen plate 110 by engaging and
compressing an O-ring 115, provided in a seal race 116 cut into the
base plate 105 for such purpose. The seal race 116 is located in
the base plate such that the O-ring 115 lies beneath the vertical
intersection of the screen plate 110 and the cylinder wall 107. A
step 117 is cut into the base plate 105, and a mating groove 118 is
cut into the screen plate, so that a positive detent is able to
precisely locate the screen plate onto the base plate for proper
alignment with the O-ring such that the cylinder wall 107 will
properly engage the screen plate and their intersection will
properly engage the O-ring 115.
[0095] Turning now to FIG. 10, there is depicted, in
cross-sectional partial cut-away view a crushing piston 92 provided
in accordance with principles of the invention. The crushing piston
92 comprises a generally cylindrical piston head 120, having an
axially extending, centrally located cup 121 protruding therefrom,
the cup 121 having generally cylindrical walls and one open end to
define thereby a socket 123 for receiving a generally cylindrical
hydraulic cylinder shaft 94.
[0096] An annular flange 122 is provided around the circumference
of the cylindrical cup 121, and surrounds the cup's open mouth. The
outer surface of the flange 122 is beveled, such that the beveled
surface increases in diameter in the direction towards the bulk
body of the piston head 120. As the hydraulic shaft 94 is advanced
into the socket 123, a pair of spring-loaded retaining clips 124
are advanced over the beveled surface of the annular flange 122
until they detent into position and grip the underside of the
annular flange.
[0097] To accommodate mating with the socket 123, each retaining
clip 124 includes a beveled tooth 125 which rides along the beveled
surface of the piston head's annular retaining ring 122, thereby
spreading open the jaws of the spring loaded retaining clips 124.
As the hydraulic shaft 94 continues to advance, the beveled teeth
125 of the retaining clips 124 are eventually advanced past the
beveled surface of the annular retaining ring 122. The spring
loading of the retaining clips forces the beveled teeth into
contact with the outer surface of the cup side wall. The teeth of
the retaining clips 124 are thus engaged with the underside surface
of the annular retaining collar 122, thereby gripping the crushing
piston 92 and providing means for causing the piston to move in
both directions.
[0098] In addition, it will be evident to one having skill in the
art that the spring loaded retaining clips 124 may be easily
disengaged from the annular retaining ring 122 by a simple
squeezing together of the ends of the clips opposite the beveled
retaining teeth 125. Accordingly, it will be seen that the piston
head 120, the cylindrical, axially mounted cup 121, the annular
retaining ring 122 and the retaining clips 124, in combination,
provide means for quickly and easily disconnecting the hydraulic
shaft 94 from the crushing piston 92. This quick-disconnect feature
allows the piston 92 and cylinder 91 combination to be easily
removed from the cylinder seat 93 of the hydraulic pressure 110 for
cleaning, sterilization, refilling with additional sample packets,
and the like.
[0099] As is shown in FIG. 10, the crushing piston 92 further
includes several O-rings 126 disposed in seal races 178 provided
about the periphery of the piston head 120. The O-rings are
provided in order to form a tight pressure seal between the
exterior circumferential surface of the piston head 120 and the
inner circumferential surface of the cylinder wall 107 of the
crushing cylinder 91. Multiple O-ring seals provide a measure of
safety and security, in order to ensure containment of potentially
contaminated sample fluid within the confines of the cylinder 91.
While three O-rings 126 are depicted in the illustrated embodiment
of FIG. 10, it will be evident that a greater or lesser number of
O-ring seals may be provided in accordance with the invention. All
that is required is that a seal be formed between the crushing
piston 92 and the crushing cylinder 91 so as to ensure containment
of potentially contaminated fluid within the cylinder.
[0100] Returning now to FIG. 8, the crushing cylinder side wall
includes a 0.020 inch beveled step 130 which is machined into the
interior surface of the side wall. The first approximately 1.0
inches, from the top, of the cylinder side wall 107 is thus,
machined to have an inside diameter (ID) approximately 0.040 inches
larger than the ID of the remaining portion of the cylinder side
wall 107 which extends downwardly towards the screen plate 110 and
base 105. The interface between the step and the remaining side
wall portion is beveled, so as to provide a relatively smooth,
angled transition from the slightly larger upper ID, to the
slightly smaller lower ID.
[0101] The step on the cylinder side wall 107 is provided so that
the crushing piston 92 may be manually inserted into the open
throat of the crushing cylinder 91 with only slight contact being
made between the O-rings (126 of FIG. 10) and the ID surface of the
cylinder. Once the manually assembled piston and cylinder
combination is placed on the cylinder seat (93 of FIG. 7) the
hydraulic shaft 94 is advanced to mate with the socket 123 of the
piston and is extended until the retaining clips 124 detent against
the underside surface of the piston head's annular retaining collar
122. The hydraulic shaft 94 is then further advanced so as to push
the piston further into the cylinder, thereby pushing the O-rings
beyond the step 130 on the ID of the cylinder wall. When pushed
beyond the step, the O-rings fully compress between the ID of the
cylinder side wall 107 and the piston seal races 127, forming
thereby a tight seal.
[0102] In operation, the crushing piston 92 develops pressure of
about 800 to 900 psi (4,000 lbs of force point loaded at the
hydraulic shaft) which is a sufficient pressure to crush the sample
packets contained within the cylinder. Blood or plasma sample fluid
flows along the fluid gutter provided in the screen plate and into
the central sump, where it is collected and allowed to flow out the
extraction port and into a pooling container. Following the
crushing operation, the hydraulic cylinder 95 is operated to raise
the crushing piston 92 a small distance (approximately 1/2 to 1
inches) above the mass of crushed sample packets, thereby creating
a chamber within the cylinder. A pressurized gas, such as
compressed air, is forced into the chamber through the pop-off
valve 102 in the piston 92. Pressurizing the chamber causes any
remaining blood or plasma sample fluid to be expressed out of the
cylinder through the outlet port 103 into the pooling
container.
[0103] Once the crushing and pooling operation is completed, the
express line connected to the outlet port 103, is clamped, to
prevent any additional sample fluid from exiting the cylinder. The
express line is placed into a bleach container, and the hydraulic
cylinder 95 is caused to raise the piston further in the cylinder,
thereby creating a suction which siphons bleach from the container
into the cylinder. Preferably, the crushing and bleach siphoning
steps are repeated two additional times, in order to ensure that
any blood or plasma sample "flash back" fluid is fully expressed
from the crushing cylinder 91 and that the bleach has ample
opportunity to fill the interior volume of the crushing chamber,
thereby reducing any gross viral contamination that may be found
within.
[0104] Next, the quick release clamps are operated and the
piston/cylinder combination is removed from the hydraulic press 90
and subjected to sterilization procedures in, for example, an
autoclave. The piston and cylinder may be subsequently chemically
cleaned by soaking them in a 10% bleach solution for fifteen
minutes, followed by a rinse cycle of H.sub.2O, 1% SDS (sodium
dodecyl sulfate) surfactant, and H.sub.2O again, prior to
autoclaving. If there is insufficient time for autoclave
sterilization, the chemical clean may be concluded with a 70% ETOH
and sterile H.sub.2O solution. If such additional chemical cleaning
is desired, it is performed in a class II biosafety hood which
exhausts through a HEPA filter. While under the hood, the crushing
cylinder is loaded with a next group of sample packets to be
crushed and the crushing piston 92 is manually inserted into the
open mouth of the crushing cylinder 91 and forced down until the
piston's O-rings make contact with the beveled step formed in the
side wall of the cylinder. The newly reloaded cylinder/piston
combination is now ready to be placed on the cylinder seat 93 of
the crusher 90. The hydraulic cylinder 95 is operated to cause the
hydraulic shaft 94 to lower onto the piston 92 such that the quick
release clamps engage the annular retaining ring on the piston. The
crushing, expressing, and bleach-cleaning process is now
repeated.
[0105] From the foregoing, it will be evident to one having skill
in the art that the electrically operated hydraulic press (the
crusher) 90 allows harvesting of blood or plasma samples from a
great number of sample packets in a minimal amount of time. The
number of sample packets able to be crushed by such an apparatus is
limited primarily by the scale of the device and the pressure able
to be developed by the crushing piston against the mass of sample
packets contained in the cylinder. The 800 to 900 psi of pressure
developed by the hydraulic press of the illustrated embodiment is
sufficient to completely crush up to 64 sample containing packets
of the type described in connection with FIG. 2. Accordingly, large
scale pools comprising up to 512 samples, can be formed by 8
operation cycles of the crusher of the present invention. This
would provide a significant reduction in pool formation time over a
method in which 512 sample packets were individually accessed by a
cannula to harvest the samples therefrom.
[0106] In addition, it will be apparent to one having skill in the
art that a single large scale pool, comprising up to 512 samples or
more, can be formed from a crusher apparatus made sufficiently
large enough to accommodate the greater number of sample pockets in
the cylinder. The hydraulic press portion would also be increased
in size to provide greater crushing power to overcome the greater
resistance of the increased number of pockets. As was mentioned
above, the pool size would only be limited by the desired scale of
the crusher.
[0107] Referring now to FIG. 11, there is shown a flow chart of a
PCR test methodology according to the invention, which allows for
the identification of a unique PCR positive donation with the
fewest number of individual tests.
[0108] The process begins at block 200 with the definition of an
appropriate initial pool size which, in turn, depends on various
factors such as the frequency of occurrence of the virus of
interest in the general donor population, the likely final
concentration of viral DNA or RNA after dilution in the pool, and
the like.
[0109] Although the PCR test is highly sensitive and is capable of
detecting a single virus in a contaminated sample, a virus must
necessarily be present in the sample for the PCR test to provide a
positive result. If, for example, a sample from a contaminated
donation having a relatively low virus concentration is pooled
together with a large number of uncontaminated samples, the
concentration of virus in the resulting pool may be so low that
there is a statistical probability that no virus is present in a
sample taken from the pool for PCR testing. Such pools may, indeed,
falsely test negative for viral contamination.
[0110] For example, if a 0.02 ml sample was prepared from a plasma
donation contaminated with viruses at a concentration of 500
viruses per ml of sample, the 0.02 ml sample would comprise, on
average, 10 viruses. If this 0.02 ml contaminated sample were
pooled with approximately 500 other 0.02 ml samples from
uncontaminated donations, the resulting 10 ml pool would comprises
viruses at a concentration of 1 per ml. Accordingly, if a 1 ml
sample were taken from the pool for PCR testing, there is a
significant statistical probability that the PCR sample will
contain no viruses.
[0111] Such low concentrations of virus contamination pose little
threat for products produced from plasma, because several methods
are available for inactivating viruses present in such low
concentration donations. Such viral inactivation methods include
the use of solvent/detergent or heating at over 60.degree. C. for
an appropriate time or the like. These methods, generally, are
described as being capable of reducing the concentration of viruses
by a number of "log units." For example, the solvent detergent
method is capable of reducing the viral contamination of hepatitis
C by at least 10.sup.7 per ml or "7 log units." Thus, plasma
products such as factor VIII, factor IX or prothrombin complex may
be prepared from plasma donations routinely treated by, for
example, the solvent detergent method after having been PCR tested
negative.
[0112] For blood products, routinely transfused directly to a
recipient, there remains some small risk of low concentration viral
contamination, after such donations have PCR tested negative.
[0113] In the embodiment illustrated in connection with FIG. 11,
the factors discussed above, such as the frequency of occurrence of
the virus of interest in the donor population and the likely
concentration of the virus after dilution, are evaluated. An
appropriately sized first level PCR testing pool is designed which
minimizes the statistical probability that viruses present in low
concentrations will go undetected. The pool is prepared at block
201 by pooling the contents of terminal pouches of identified
tubing sections, in the manner described above. At block 202, a PCR
test is performed on the first level PCR pool.
[0114] Block 203 represents a decision point in the methodology of
the invention which depends on the results of the PCR test
performed in block 202. In the event of a negative result on the
test, all of the donations corresponding to samples used to make up
the first level PCR pool are presumed to be free of viral
contamination and released for further processing into
pharmaceutical products. The methodology thus exits on receipt of a
negative PCR test result. When the PCR test returns a positive
indication, this indicates that a viral contaminant is present in
one, or more than one, of the donations which made up the original
PCR first level pool. At block 204, an additional sample pouch, the
pouch next to the one first removed, is taken from tubing segments
which correspond to donations comprising the original PCR first
level pool. These additional sample pouches are divided into two
approximately equal subgroups, designated A and B herein for
purposes of clarity.
[0115] These subgroups are then separately pooled using a separate,
clean cannula to form each subgroup pool in the same manner as
described above, and only one of the subgroup pools is PCR tested,
as indicated at block 205. It is immaterial for purposes of the
invention which of the two subgroups is tested. In block 205,
subgroup A is identified as the subgroup to be tested, but subgroup
B could just as easily have been designated without disturbing the
methodology of the invention.
[0116] At block 206, a decision is made depending on the outcome of
the PCR test of subgroup pool A. In the event that subgroup pool A
tests negative for a PCR viral indication, no further testing is
performed on samples from donations that comprised subgroup A.
Rather, as indicated at block 207, the next sample pouches in
sequence are taken from tubing segments that comprised subgroup B
which are then, in turn, divided into two approximately equal
subgroups A' and B'. Each subgroup in this step comprises
approximately half the number of samples as comprised the
immediately preceding subgroup. The contents of the subgroup sample
pouches are again pooled separately in the same manner as described
above. In the event that subgroup A tested PCR positive, indicating
at least one of its component donations was virus contaminated, the
other untested subgroup (subgroup B in the example of FIG. 11) is
now PCR tested at block 108 to confirm that it is not also PCR
positive. Subgroup A now becomes the subgroup further subdivided
into two approximately equal subgroups (A' and B'), as indicated at
block 209.
[0117] At block 210, PCR testing is performed on only one of the
subgroup pools, A' or B', defined in preceding step 207 or 209. The
method now iterates and returns to block 206, wherein the decision
step is applied to the results of the PCR test performed at block
210. Again, if the PCR test results prove negative for the tested
subgroup, the untested subgroup would be further subdivided into
two approximately equal subgroups, each comprising approximately
half the samples of the preceding subgroup. If the tested subgroup
returned a PCR positive result, the tested subgroup would be
further subdivided into two approximately equal subgroups, each of
which would comprise one half of the samples of the preceding:
subgroup. In this case, the untested subgroup would again be PCR
tested in order to confirm that it was not also PCR positive.
[0118] The test methodology continues iterating from block 206
through block 210 until testing is determined to be complete. Test
completion is defined as when a subgroup division results in the
creation of two subgroups, each containing only one sample pouch
corresponding to a single donation. One of the samples is PCR
tested at block 210 and, if the test results are negative, the
other sample is identified as belonging to a virally contaminated
plasma donation. If the tested sample tests positive, the remaining
sample is then also PCR tested in order to confirm that it is not
also PCR positive.
[0119] Upon completion of all testing, the methodology of the
invention ends at block 211. It should be clear from the flow chart
of FIG. 11, that the testing methodology of the invention only
requires that two PCR tests be performed at each test level when
the initially tested pool is positive: One initial test for one of
the two subgroups, and one subsequent test to confirm that the
corresponding initially untested pool is indeed negative. The test
methodology requires only a single PCR test at each test level when
the initially tested pool is negative.
[0120] Application of the system and method for sample testing of
the invention will now be described in connection with a particular
PCR test pool size, as depicted in FIG. 12. In FIG. 12, the
terminal pouches of 512 individual donations are formed into an
initial PCR testing pool at 212. For purposes of illustration, it
will be assumed that only one of the 512 samples was taken from a
donation which was contaminated by a virus of interest. The tubing
segment depicted in FIG. 12 which comprises 10 individual and
connected pouches represents the tubing segments originally
connected to and taken from the contaminated plasma donation
container.
[0121] The initial 512 sample pool is PCR tested and because of the
presence of the contaminated sample, returns a positive viral
indication. At step 213, two 256 donation pools (256A and 256B) are
prepared from the next sequential pouches taken from segments that
made up the prior positive pool. Pool 256B is now PCR tested and,
as depicted in FIG. 12, returns a negative viral indication, thus
indicating that pool 256A contains a sample from the contaminated
donation.
[0122] At step 214, two 128 donation pools are prepared from the
next sequential pouches of tubing segments that made up pool 256A.
Thus, according to the invention, pool 256A has been subdivided
without having been PCR tested. At step 203, pool 128A is now PCR
tested and, since it returns a negative viral indication, pool 128B
is now known to include a sample pouch from the contaminated
donation. Pool 128B is then subdivided into two 64 donation pools
(64A and 64B) by removing the next sequential pouch from those
tubing segments whose preceding pouches made up pool 128B.
[0123] Next, pool 64B is PCR tested and, in the example of FIG. 12,
returns a positive viral indication. In this case, PCR testing is
performed on pool 64A in order to verify that it is, indeed,
negative and that no additional contaminated samples are present
beyond those in pool 64B. At step 216, pool 64B is further
subdivided into two 32 donation pools, 32A and 32B, by removing the
next sequential pouch from tubing segments used to make up
preceding pool 64B. Pool 32B is PCR tested, returns a negative
viral indication, as indicated, and pool 32A is therefore further
subdivided into two 16 donation pools, 16A and 16B. Again, the 16
donation pools are prepared by removing the next sequential sample
pouch from tubing segments that made up the preceding positive
pool, 32A.
[0124] At step 217, pool 16B is PCR tested and returns a positive
viral indication. Pool 16A, therefore, is PCR tested in order to
confirm that it is negative, and that all contaminated samples are
present in pool 16B.
[0125] At 218, pool 16B is subdivided into two 8 donation pools, 8A
and 8B, by removing the next sequential sample pouch from tubing
segments that made up the preceding positive pool 16B. Pool 8B is
then PCR tested and, as illustrated, returns a negative viral
indication, indicating that pool 8A contains a sample from a
contaminated donation. Pool 8A is then further subdivided into two
4 donation pools, 4A and 4B, at step 219. PCR testing is performed
on pool 4B, which returns a negative indication, thus indicating
that pool 4A contains a sample from a contaminated donation. Pool
4A is then subdivided, at 220, into pools 2A and 2B in the same
manner, as described above. Upon PCR testing, pool 2A returns a
negative viral indication indicating that one of the two samples
comprising group 2B was taken from a tubing segment of a
corresponding contaminated donation.
[0126] At step 221, the individual donations are tested by removing
the final pouch from the tubing segments that made up group 2B. The
final individual donations are PCR tested in order to identify the
specific positive donation, which is then removed from storage and
appropriately disposed of. The remaining 511 viral free donations
are retained for further processing into pharmaceutical
products.
[0127] In the above example, a single contaminated donation has
been uniquely identified from a group of 512 such donations, by
performing only 13 separate PCR tests, including the primary PCR
test on the original 512 donation pool. The method of the
invention, allows for skipping a PCR test on a particular subpool,
so long as the corresponding tested subpool returns a negative
viral indication. By thus skipping certain PCR tests, the method of
the invention reduces the number of PCR tests that must be
performed in order to identify a specific positive donation,
without sacrificing the resolution of the PCR test methodology.
Under the method of the invention, all positive donations will be
identified but without requiring that all donations be tested.
[0128] From the exemplary embodiment of FIG. 12, it will be clear
that either one of the successively smaller subgroups may be PCR
tested and that the arbitrary position of the positive sample may
be varied. Thus, if a sample from the positive donation were
present in each initially tested subpool, 18 tests would be
required to uniquely identify the positive donation (one initial
test which returns a positive indication and one additional test to
assure that the corresponding subpool is negative).
[0129] By the same token, if each initially tested subpool returns
a negative indication, 10 tests would be required to identify the
positive donation. In practice, positive and negative test results
on the subpools would tend to distribute equally, thus, 14 tests on
average would be required to identify a uniquely positive donation
from an initial donation pool for 512 units.
[0130] It is therefore clear from the foregoing that the system and
method of the present invention, including the provision of tubing
segments comprising individual and connected pouches each
containing a sample of a plasma donation, is advantageous in
providing a multiplicity of PCR test pools. Unlike conventional
pool preparation, in which a sequence of initial and subsequent
pools are formed from a single sample of each donation at the same
time, the present invention allows for formation of a test pool
immediately prior to testing. This manner of "just-in-time" pool
formation permits construction of test pools from individual
pouches only as needed. The possibility of contamination is
eliminated since the pools are constructed at different times, each
from sealed sample pouches. Moreover, sample pouches remain frozen
until needed to develop a test pool. Multiple freeze-thaw cycles
which may adversely affect the recovery of the DNA or RNA of
interest are avoided, thus insuring the integrity of the PCR
test.
[0131] While the above-described method is effective for
identifying a viral positive donation with the fewest number of
relatively expensive PCR tests, other methods for identifying
individual positive donations are also provided in accordance with
practice of the present invention. In particular, one such method
has the property of being able to identify individual positive
donations within two to three PCR testing cycles, thus
significantly reducing the amount of time and administrative
overhead required to screen a large number of donations.
[0132] For example, in the above-described method, once a
particular subpool has been identified as containing a positive
donation, a technician must identify those donations which
contributed samples to form that particular subpool. Those
donations must then be revisited, and an additional sample packet
must be harvested from each corresponding tubing segment. Two
next-generation subpools must then be formed, and PCR testing
repeated. This harvesting, subgroup pool formation, and PCR testing
process is repeated for smaller and smaller generational subpools
until the method uniquely identifies the virally contaminated
donation.
[0133] However, a significant amount of time is consumed in each
PCR testing cycle (harvesting, subgroup pool formation, and PCR
testing). Taking the 512 sample first generational pool as an
exemplar, it will be evident that at least 10 PCR testing cycles
will be required to identify a unique viral contaminated donation.
While highly cost-effective, the above-described method may present
challenges to a PCR testing laboratory when time is of the
essence.
[0134] A methodology for uniquely identifying viral positive blood
or plasma donations in the fewest number of PCR testing cycles will
now be described in connection with FIGS. 13 and 14.
[0135] Turning now to FIG. 13, there is depicted a flow chart of a
PCR testing methodology, in accordance with the invention, for
efficiently detecting a PCR positive individual donation in a pool
with the minimum number of PCR analysis cycles. As was the case
with the prior-described PCR testing method, the method of FIG. 13
assumes that the PCR test has sufficient sensitivity to detect the
presence of a positive sample in a pool of the appropriate size.
For purposes of illustration only, the initial grouping has been
chosen to represent 512 blood or plasma donations. It will be
understood by those having ordinary skill in the art that the
initial grouping size may be larger or smaller depending on the
particular genome marker being evaluated, the sensitivity of the
PCR test procedure used, the expectation value of the genome marker
concentration within a sample aliquot, and the sample aliquot
size.
[0136] The method begins in block 301 by defining an N-dimensional
sample matrix or grid. The matrix may be of any size and comprise
any number of dimensions from 2 to N, but preferably is a
3-dimensional regular matrix, organized as a square.
[0137] An example of such a matrix is depicted in FIG. 14, which is
a graphical illustration of square matrix, characterized by
3-dimensional; row, column, and slice (r,c,s). In the exemplary
matrix of FIG. 14, there are 3 rows, 3 columns, and 3 slices,
thereby defining 3.sup.3, or 27, elements. In the exemplary
embodiment, a row is considered as comprising all of the elements
defined by taking an imaginary vertical section through the square
regular matrix. In the embodiment of FIG. 14, the elements
comprising, for example, row 3 of the matrix are identified by the
letter r.sub.3 on their row faces.
[0138] Likewise, a column comprises all of the matrix elements
defined by taking a second imaginary vertical section through the
matrix, in a direction orthogonal to the direction of a row. In the
exemplary embodiment of FIG. 14, the elements that comprise, for
example, column 1 have the letter c.sub.1 on their column faces. A
slice is defined as all elements comprising a horizontal section
taken through the exemplary matrix of FIG. 14. In like manner to
the row, column, definition, the elements comprising slice 1 are
identified with the letter s.sub.1 on their slice faces.
[0139] It can be seen, therefore, that each of the 27 elements in
the matrix of FIG. 14 uniquely belongs to 1 of the 3 rows, 1 of the
3 columns, and 1 of the 3 slices. Mathematically, this may be
expressed by the relationship X.sub.rcs, where X denotes an
element, and rcs is a dimensional index, where each of the indices
may take on a value from 1 to 3. The specific element X.sub.113 may
be identified as that element at the intersection of row 1, column
1, and slice 3.
[0140] From the foregoing, it will be apparent that although the
exemplary matrix of FIG. 14 is a 3.times.3.times.3 matrix, the
principles of matrix definition and element formation will hold for
matrices with a much greater number of rows, columns, and slices.
In particular, an 8 row, 8 column, 8 slice matrix may still be
represented mathematically as X.sub.rcs, where rc and s may now
take on values from 1 to 8. Thus, a 3-dimensional 8.times.8.times.8
matrix is able to accommodate identifiers for 512 elements.
[0141] Returning now to the method flow diagram of FIG. 13,
following definition of an N-dimensional sample matrix, particular
blood or plasma donation samples are mapped to each of the elements
defined by the matrix. In an exemplary 3-dimensional
8.times.8.times.8 matrix, a sample from each of 512 individual
donations is associated with a matrix element, and identified by a
corresponding, unique X.sub.rcs indicator.
[0142] Next, an aliquot is taken from each sample, and a
multiplicity of minor sub-group pools are formed. Each minor pool
comprises the aliquots of all of the samples (X.sub.rcs) in which 1
of the dimensional indices is fixed. In other words, in accordance
with the above-described exemplary matrix, all of the samples
(X.sub.rcs) which have r=1, regardless of the column or slice
value, are formed into a minor pool; likewise for r=2, r=3 . . .
r=N; likewise for c=1, regardless of row or slice value, c=2, . . .
c=N; likewise for s=1, regardless of row or column value, s=2 . . .
s=N. Each minor pool thus represents each row, column, layer, or
other dimensional index, such that if an N-dimensional matrix has
been defined, there will be N-dimensions times the (total number of
samples).sup.1/n minor pools. For the exemplary 3-dimensional
8.times.8.times.8 matrix containing 512 samples, there will be 24
minor subgroup pools (8 row pools, 8 column pools, and 8 layer
pools). The creation of minor pools, in accordance with the
invention, may be viewed as being similar to the mathematical
method of reducing a determinant by the method of minors. In like
manner, each sample will be understood to be represented in N minor
pools, 1 for each dimension of the matrix.
[0143] In addition to forming the minor pools, an aliquot of each
sample, or an aliquot of each of the minor pools, is combined to
form a single master pool which contains a sample from all of the
512 donations comprising the present donation space. After all of
the pools are formed, any remaining samples and the minor pools and
master pool may be refrozen and stored until such time as PCR
testing is desired.
[0144] When PCR testing is desired, a PCR test is first performed
on the master pool which represents an aliquot of each sample
comprising the matrix. If the test results for the master pool are
negative, there are, at least to the sensitivity level of the PCR
test, no viral positive donations represented by samples forming
the matrix. The blood or plasma donations which have contributed
samples to the matrix may be released for further use. However, if
the PCR test of the master pool is positive for a particular genome
marker, a second PCR testing cycle is entered, at 300, in which
each of the minor pools are now tested.
[0145] In a manner similar to that described above, the major pool
sizes chosen such that the statistical probability of their being
more than one positive sample in the major pool (the 512 samples)
is small, preferable less than 1 to 2%. This can be done by
evaluating the frequency of occurrence of the virus of interest in
the general donor population to a 98% to 99% confidence level. For
example, if it is determined that only 1 donor out of a general
donor population of 1,000 is contaminated with the virus of
interest to a 98% confidence level, there is a 2% probability of
finding more than 1 contaminated donor in the next 1,000 donors
being evaluated. This assures that the algorithm will, in general,
be able to identify the single reactive unit in a pool of
appropriate size within the PCR testing cycle. In accordance with
the invention, given a single positive sample within the matrix, 3
of the minor pools will contain an aliquot of the positive
donation, 1 in each dimension. In the exemplary embodiment (the
matrix of 512 samples), there are 8 minor row pools, 8 minor column
pools, and 8 minor layer pools. If the master pool tests positive,
then 1 row, 1 column, and 1 layer pool will test positive during
the second PCR testing cycle as shown at 307. The intersection of
the row, column, and layer element index unambiguously identifies
the reactive donation as shown at 309.
[0146] As an example, if the reactive sample has been mapped to
matrix element X.sub.113, the row 1 minor pool will return a
positive PCR test result, while the row 2, and subsequent row minor
pools will test negative. Further, the column 1 minor pool will
return a positive test result, while the column 2 and subsequent
column pools will test negative. Likewise, the layer 1 and 2 minor
pools will return a negative result, the layer 3 pool will test
positive, and subsequent layer minor pools will test negative. The
3 positive minor pools (row 1, column 1, and layer 3) have only a
single element in common, .sub.X113. Thus, the positive donation is
uniquely identified as represented by the sample mapped to element
X.sub.113.
[0147] If there is more than 1 reactive donation in the matrix, the
reactive donations may still be unambiguously identified by the
method of the invention, by no more than 1 additional PCR testing
cycle. If it is observed that more than 1 minor pool of a single
dimensional index returns a positive test result, while only a
single minor pool representing each of the remaining dimensional
indices returns a positive test result, the more than 1 positive
donations may be unambiguously identified by mathematically
evaluating the test results without the need for a third PCR
testing cycle.
[0148] For example, if a row 1 minor pool, and none other, tests
positive; a column 1 minor pool, and no other, tests positive; and
a layer 1 minor pool and layer 3 minor pool both test positive,
there are only two positive donations comprising the matrix, and
they are able to be unambiguously identified as X.sub.111 and
X.sub.113. No further testing is required to arrive at this
result.
[0149] If, on the other hand, it is observed that multiple minor
pools test positive and their identities indicate changes along 2
dimensional indices as shown at 310, it will be apparent that there
will be z.sup.2 elements identified as potentially mapped to a
positive donation, where z is the actual number of positive
donations comprising the grid.
[0150] For example, if the row 1 minor pool, and no other, tests
positive; the column 1 and column 3 minor pools test positive; and
the layer 1 and layer 3 minor pools test positive, this suggests
that the potentially positive candidate elements are X.sub.111,
X.sub.113, X.sub.131, and X.sub.133. Since there is a multiple in
only two of the dimensional indices (column and layer), and for
candidate elements, it will be seen that there are only two actual
positive donations comprising the matrix. In this circumstance, all
4 donations may be arbitrarily identified as being positive, and
disposed of or, alternatively, an aliquot may be taken from each of
the 4 candidate elements and individually PCR tested during a third
PCR testing cycle at 311, in order to uniquely identify which 2 of
the 4 comprise the actual positive donations.
[0151] In like manner, it is mathematically evident that if there
are more than 2 positive donations in the matrix, and their
identifiers vary in more than 2 dimensions, there will be, at most,
z.sup.n potentially positive candidate elements identified, where z
is the actual number of positive donations, and where n is the
number of dimensions which vary. In this circumstance, aliquots are
taken of all suspect elements in the matrix and directly
tested.
[0152] Thus, it can be seen that the method of the invention
permits unambiguous identification of donations which are reactive
for a particular genome marker within a single PCR testing cycle
for an initially positive master pool, within 2 PCR testing cycles
for all matrices which contain a single reactive donation or a
multiplicity of reactive donations which vary along only a single
dimensional index, and within 3 PCR testing cycles for any other
situation.
[0153] Accordingly, the practice of the present invention results
in the blood supply, and blood or plasma products prepared
therefrom, being substantially safer by virtue of its being as free
as possible from viral contamination. Advantageously,
cost-effective, high-sensitivity testing is readily performed for
the presence of a virus directly. Thus, false indications of virus
contamination usually associated with antibody testing during the
infectivity window period is avoided. Moreover, the present
invention allows cost-effective use of high-sensitivity tests which
are capable of detecting the presence of a single virus in the test
sample, thus helping to insure the freedom of the blood supply from
incipient viral contamination.
[0154] Those skilled in the art will appreciate that the foregoing
examples and descriptions of various preferred embodiments of the
present invention are merely illustrative of the invention as a
whole, and that variations in the shape, size, and number of the
various components of the present invention, as well as the types
of tests implemented, may be made within the spirit and scope of
the invention. For example, it will be clear to one skilled in the
art that the length of the individual and connected pouches, and
therefore their volumetric content, may be progressively increased
along the length of the tubing segment. As successive testing
subpools are formed from a smaller and smaller number of samples,
the volume of plasma comprising the pool necessarily decreases. It
should be clear that in order to maintain a sufficient volume of
plasma in each successive subpool, successive sample pouches may
contain a larger volume in order to accommodate a desired final
pool volume. In order to accommodate pools ranging in size from
about 1 ml to about 10 ml, it will be clear that the volumes of
successive sample pouches will increase from about 0.02 ml to about
0.5 ml, in progressive steps. In one exemplary embodiment, the
pouch volume is 0.02 ml in the first pouch to be used in the
largest pool and is 0.2 ml in the final pouch.
[0155] It will also be clear to those skilled in the art that the
system of the invention is not limited to the exemplary plasma
collection container and an associated tubing segment. Blood bags
or other biological fluid containers may be used with equal
facility and suitable tubing segments may be attached thereto both
prior to fluid collection and after fluid collection is completed.
All that is required is that sample quantities of biological fluids
be transferred to a tubing segment which is then formed into
pouches in accordance with practice of the invention.
[0156] Accordingly, the present invention is not limited to the
specific embodiments described herein but, rather, is defined by
the scope of the appended claims.
* * * * *